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Three-dimensional spatial selectivity of hippocampal neurons during
space flight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
JJ Knierim, BL McNaughton and GR Poe
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contents
articles
NMDA receptor-mediated control of protein synthesis at developing
synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
AJ Scheetz, AC Nairn and M Constantine-Paton
Imaging synapse
A multimodal cortical network for the detection of changes in the
formation in vivo. sensory environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Page 231. J Downar, AP Crawley, DJ Mikulis and KD Davis
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editorial
Mysterianism lite
A philosophical view known as mysterianism holds that even biochemistry to computation) tend to go unreported, because they
though there is nothing supernatural about how consciousness aris- are very difficult to explain to lay people.
es from neural activity, the human brain is simply not equipped to The problem goes deeper than this, though. Even where mecha-
2000 Nature America Inc. http://neurosci.nature.com
understand it. The reason we find the mindbrain problem so baf- nistic explanations of brain function have been possible, they do not
fling, the argument goes, is that humans did not evolve sufficient feel like explanations of mental processes. Consider the paper by
cognitive abilities to solve it, just as armadillos did not evolve the Treue et al. on page 270 of this issue, which presents a striking exam-
ability to understand arithmetic. This argument has been advocated ple of how far our understanding of perception has progressed. Based
by philosophers such as Colin McGinn and cognitive scientists such on knowledge of how motion is represented by populations of neu-
as Steven Pinker. Now it has been taken up by a prominent science rons in the visual cortex, the authors were able to predict an entire-
journalist, John Horgan, whose new book The Undiscovered Mind ly unexpected visual illusion; two different patterns of moving dots
offers a view of brain science that might best be described as mys- are perceptually indistinguishable, apparently because they both
terianism lite. It is not just consciousness that is beyond our grasp, he evoke the same pattern of activity in a cortical area called MT. Of
says; neuroscience as a whole is failing, because the brain is too com- course a graph showing the distribution of neuronal firing rates in
plicated for human understanding. MT doesnt feel like an explanation of perception. But why should
Horgan attracted attention, even notoriety, for his 1996 book The it? The criterion for a good theory is not that it feels right, but that it
End of Science, in which he argued that the age of great scientific dis- can successfully predict unexpected results. If a physical theory of
coveries is coming to an end because most of the big questions have neural processing can predict an unexpected mental phenomenon,
been answered. The brain is an obvious exception, but Horgan now that is surely a substantial achievement.
argues that neuroscience too is reaching its limits, not because it has It goes without saying that Treues study raises many further
succeeded in its aims but because those aims are unachievable. The issueshow is the population activity decoded, what other areas
subtitle of his new book is How the human brain defies replication, are involved in representing the stimuli, and so forthbut there is no
medication, and explanation; its thesis is that the achievements of reason why questions of this type should not eventually be answered.
neuroscience (along with psychology, psychiatry and other related Certainly, it will be a challenge to understand how (say) a moving
areas) are being oversold, that the supposed practical benefits have red bar is perceived as a unitary stimulus if its orientation, motion
been exaggerated, and that the field is now confronting an explana- and color are each represented in different cortical areas. Horgan
tory gap that may never be bridged. Unlike particle physicists or mol- may well be right that existing hypotheses to solve this so-called
ecular biologists, says Horgan, neuroscientists have yet to achieve binding problem (such as synchronous oscillations) will prove incor-
their reductionist epiphany. Instead of finding a great unifying insight, rect. But to deny the possibility of further progress seems perverse. A
they just keep uncovering more and more complexity. Neurosciences deeper understanding of the mechanisms underlying mental process-
progress is really a kind of anti-progress. As researchers learn more es should follow from greater knowledge of anatomical and func-
about the brain, it becomes increasingly difficult to imagine how all tional connectivity, better methods of recording and manipulating
the disparate data can be organized into a cohesive, coherent whole. neural activity, and more realistic computational models, all of which
It is tempting to dismiss this as another example of what Richard should be achievable with enough time and effort. For ethical rea-
Dawkins once called the argument from personal incredulity, but sons, we may never know as much as we would like about human
Horgan is surely not alone in finding neuroscience difficult to brain activity, but it seems reasonable to expect that insights from
approach. The brain is immensely complicated, and in the absence other organisms will some day provide good models for much of
of a grand unifying theory for how it works, researchers tend to study what happens inside our own heads.
very diverse problems that often seem unconnected to each other. The mysterians might turn out to be correct in claiming that we
It is therefore understandable that their achievements do not always will never fully understand how brain activity leads to subjective
seem intellectually satisfying to nonspecialists. experience: why the firing of MT neurons feels like visual motion,
Part of Horgans critique may reflect how neuroscience is report- why dopamine release in the nucleus accumbens feels pleasurable, or
ed in the media. Among the stories that attract the most attention why electrical stimulation of the anterior supplementary motor area
are the identification of genes or brain areas that are associated with feels amusing. But that is very different from claiming that these
particular behaviors (think of fosB, the gene for maternal behav- phenomena can never be explained in physical terms, or that neu-
ior, or the orbitofrontal cortex, the brains moral compass), but roscience is, as Horgan puts it, bumping up against fundamental
typically such findings are only the first steps on a long road toward limits of science. Most neuroscientists, fortunately, take a more opti-
mechanistic understanding. In contrast, many of the most impor- mistic view than Horgan; the explanatory gap may not be closed at
tant mechanistic insights into how the brain works (at all levels, from a single stroke, but it is getting narrower by the day.
When neuroscientists can consistently for each. In contrast, if the center of grav- What do monkeys see when this hap-
predict what people perceive by studying ity is important, then the location and pens? Salzman and colleagues trained
their neural activity, we will have achieved number of peaks should not matter. Under monkeys to indicate the perceived direc-
a remarkable level of understanding of the latter mechanism, subjects would per- tion of motion, and found that they alter-
brain function. A notable advance toward ceive a single stimulus intermediate nated between reporting the real motion
2000 Nature America Inc. http://neurosci.nature.com
this goal is presented by Treue, Hol and between the two actual stimuli, regardless direction and the stimulation-induced
Rauber 1 in this issue of Nature Neuro- of whether the population response has motion direction, as if perhaps they could
science. These authors have used the two separate peaks of activity. see both and were simply picking one of
response profiles of neurons in a motion- Visual motion processing is one the two on each individual trial3. Howev-
sensitive area of the monkey brain, area domain where these issues have been er, we trained monkeys to track the
MT, to predict how humans will perceive explored fairly extensively. Motion per- motion using eye movements, and found
moving stimuli. ception is thought to rely on the popula- that the animals responded as if they saw
The brain encodes many kinds of sen- tion responses in visual area MT, which is only the vector average of the two direc-
sory stimuli using maps of neurons that specialized for processing moving stimuli tions4. Both of these experiments likely
are tuned to the properties of those stim- and contains a columnar organization for involved a population response composed
uli. How does the neural activity in these motion direction (for review, see ref. 2). of two peaks of activity: the neurons
maps subserve perception and sensory- Because of this topographical organiza- whose preferred direction of motion
guided action? Because neurons are tion, microstimulation can be used to acti- matched the visual stimulus and the neu-
broadly tuned, a single stimulus typically vate a population of neurons with similar rons at the tip of the microstimulating
activates a large population of neurons motion preferences, thereby simulating the electrode. Perceptual judgments were cor-
the so-called population response. Sever- response to real motion. Microstimulation related with the locations of these peaks,
al different theories have been proposed in concert with an actual moving visual whereas eye movements were correlated
for how population responses in turn stimulus is presumed to cause a popula- with the vector average of activity in MT.
mediate perception and action. The most tion response in MT that corresponds to Microstimulation is artificial, of
obvious possibilities are that perceptual two different directions of visual motion course. What happens when real stimuli
outcome is determined either by the peak the actual direction of motion of the visu- moving in two directions are presented?
of the population response or by its center al stimulus and the preferred direction of When two patches of moving dots are
of gravity (also known as the vector aver- the cells being stimulated electrically. superimposed on each other (a situation
age of the response).
When only one stimulus is present, the
peak and the center of gravity of the pop-
ulation response are the same. But what
happens when two stimuli with different
features occur at the same place and time?
Both stimuli influence the population
response, but are they perceived as inde-
pendent? Do they both contribute to
behavioral responses? How do the two
stimuli interact? If the peak of the popu-
lation response is the most important fea-
ture, then both stimuli would be perceived
so long as the two stimuli are sufficiently Bob Crimi
different from one another that the pop-
ulation response contains a separate peak Fig. 1. Electrophysiological recordings in visual area MT of rhesus monkeys by Treue and col-
leagues1 suggest that the population response to a transparent motion stimulus with two compo-
nents separated by 40 degrees is probably the same as the population response to a transparent
Jennifer Groh is at the Department of motion stimulus with three components (+50, 0, 50 degrees). Treue and colleagues predicted
Psychological and Brain Sciences, Center for that human observers would therefore perceive the two stimuli as containing identical motion.
Cognitive Neuroscience, Dartmouth College, This prediction was confirmed: human observers judged that both stimuli contained the same
Hanover, New Hampshire 03755, USA. upward and rightward component, even though in one case this component had an angle of 40
e-mail: jennifer.m.groh@dartmouth.edu degrees and in the other case it had an angle of 50 degrees.
known as transparent motion), humans population responses as certain two-com- Perhaps the most intriguing aspect of
can perceive the two stimuli as distinct ponent stimuli. For example, the popula- this work is the notion that the shape of the
provided the directions of motion are sep- tion response to a transparent motion population response in MT can be impor-
arated by at least 10 degrees5. Clearly, the stimulus consisting of two components 80 tant for motion perception. There are well-
center of gravity could not subserve this degrees apart should be the same as the defined algorithms for identifying peaks of
perceptor else we would always perceive response to a motion stimulus with 3 activity (winner-take-all), or computing
transparent motion as containing only components each 50 degrees apart (see the center of gravity (for example, via vec-
one component of motion at a direction Fig. 3 of ref. 1). If so, and if motion per- tor averaging) to arrive at a perceptual
intermediate between the actual direc- ception relies on this population of neu- judgment or behavioral response, and it is
tionsbut what about the peak(s) of the rons, then the direction of motion of these comparatively easy to imagine how neur-
population response? Does the popula- two stimuli should be indistinguishable. al circuits might perform these calculations
tion response in MT contain separate They tested this hypothesis in human (for example, see J.M. Groh, Soc. Neurosci.
peaks for each component of a transpar- observers, and found it was indeed the Abstr. 23, 1560, 1997). Yet the findings of
ent motion stimulus? Do these peaks case: these two very different motion stim- Treue and collegues suggest that percep-
merge together into one broad peak at the uli appear perceptually to have the same tion can be affected by details of the shape
point where the two directions are too components (Fig. 1). of the active population, details that are lost
close to be resolved? A number of issues remain to be through either of these calculations. There-
In an elegant series of experiments, resolved. For example, do MT cells actual- fore, we need to explore new algorithms
2000 Nature America Inc. http://neurosci.nature.com
Treue and colleagues1 tested this hypoth- ly respond identically to the two- and for reading population codes.
esis. Although the responses of MT neu- three-component stimuli? Do the demands
1. Treue, S., Hol, K. & Rauber, H.-J. Nat. Neurosci.
rons to both single and multiple stimuli of the psychophysical task affect how MT 3, 270276 (2000).
have been well characterized (for review, represents motion information? Monkeys 2. Albright, T. D. in Visual Motion and its Role in the
see ref. 6), it is less clear how the popula- can certainly be trained to perform motion Stabilization of Gaze (eds. Miles, F. A. & Wallman,
tion response varies as a function of the tasks like the one used by Treue and col- J.) 177201 (Elsevier, New York, 1993).
relative directions of the components of leagues in humans, but there is reason to 3. Salzman, C. D. & Newsome, W. T. Science 264,
multiple stimuli. Treue and colleagues first think that the task itself might influence 231237 (1994).
studied the responses of monkey MT neu- population responses in MT. In particular, 4. Groh, J.M., Born, R.T. & Newsome, W.T.
rons to transparent motion stimuli. Their previous work by Treue and others has J. Neurosci. 17, 43124330 (1997).
results show that because these neurons demonstrated that when an animal is 5. Mather, G. & Moulden, B. Q. J. Exp. Psychol. 32,
325333 (1980).
are broadly tuned for direction, the pop- attending to only one of two directions of
6. Britten, K. H. & Heuer, H. W. J. Neurosci. 19,
ulations of neurons responding to each motion, neurons in MT represent the 50745084 (1999).
component of motion overlap quite exten- attended direction much more strongly79.
7. Treue, S. & Maunsell, J. H. Nature 382, 539541
sively. For directions separated by less than Thus, if MT neurons were studied while (1996).
about 90 degrees, only a single broad peak monkeys performed the psychophysical 8. Groh, J. M., Seidemann, E. & Newsome, W. T.
exists (although when the directions are task used here in human observers, the Curr. Biol. 6, 14061409 (1996).
farther apart, two separate peaks do presence and/or location of peaks in the 9. Seidemann, E. & Newsome, W. T.
appear). Importantly, this single peak population response might be different. J. Neurophysiol. 81, 17831794 (1999).
occurs in monkey MT even when the
directions are sufficiently different to be
readily distinguishable to human observers
(and presumably to the monkeys).
Thus, the relationship between neur-
al activity and perception of the compo-
ChIPping away at potassium
nents of transparent motion does not
seem to be based on the presence or channel regulation
absence of segregated peaks of activity, as Min Li and John P. Adelman
would have been predicted by algorithms
that identify peaks of activity (for exam-
Kv4 subunits form A-type potassium channels. To replicate
ple, winner-take-all). Rather, the transi-
tion from perception of two directions of native currents, these subunits require additional factors,
transparent motion to perception of a sin- now shown to be a family of calcium-binding proteins.
gle direction of motion must depend on
some as-yet unidentified aspect of the In a recent issue of Nature, Kenneth concerning the molecular identity of A-
shape of the population response in MT. Rhodes and colleagues1 present results type potassium channels. They describe
If the overall shape of the population that resolve long-standing questions the isolation and characterization of a
response is critical to motion perception, family of calcium-binding proteins, the
then Treue and colleagues reasoned that John Adelman is in the Vollum Institute, Oregon KChIPs (K + channel interacting pro-
stimuli that produce population respons- Health Sciences University, 3181 S.W. Sam teins; Fig. 1), that bind to the intracel-
es having the same shape should produce Jackson Park Road, Portland, Oregon 97201- lular amino (N)-terminal domain of
the same percepts. Based on their record- 3098, USA. Min Li is in the Department of cloned Kv4 channels and endow them
ings using two-component stimuli, Treue Physiology, Johns Hopkins University School of with many of the properties ascribed to
and colleagues designed three-component Medicine, Baltimore, Maryland 21205, USA. native A-type potassium channels. Co-
stimuli that should produce the same e-mail: adelman@ohsu.edu expression of the KChIPs and cloned
Kv4 subunits increased current densi- tion 6 , increasing their similarity to immunoprecipitated with Kv4 subunits
ties, shifted the voltage dependence of native channels. In addition, a role for from transfected cells and rat brain
activation and speeded their recovery calcium in modulating A-type channels membrane preparations. Immunocyto-
from inactivation. Mutagenesis experi- has been suggested from recordings of chemistry confirmed that the two pro-
ments that eliminated the ability of the cholinergic neostriatal neurons, where teins are colocalized at cellular and
KChIPs to bind calcium showed that blocking voltage-dependent calcium subcellular levels. Remarkably, co-expres-
their channel-modulating effects require channels with cadmium shifts the volt- sion of KChIPs with Kv4 subunits recon-
calcium binding, although the physical age dependence of A-type current acti- stituted the functional characteristics of
association between the interacting pro- vation and inactivation to more native A-type channels, as well as repli-
teins was calcium independent. depolarized potentials3. Until now, how- cating the effects of co-expressing rat
A-type channels are voltage-depen- ever, the relationship between these brain mRNA on cloned Kv4 subunits.
dent potassium channels that activate in observations has remained obscure. Current densities were increased, the
the subthreshold range of membrane The new report from Rhodes and col- voltage dependence of activation was
potentials and completely inactivate leagues 1 reconciles these differences shifted to more hyperpolarized poten-
during depolarizing pulses, while other between native A-type potassium chan- tials, and the channels recovered from
voltage-dependent potassium channels nels and cloned Kv4 channels at the mol- inactivation much more rapidly.
are just beginning to activate. As a ecular level. Using the intracellular The KChIPs range in size from 216
result, A-type channels influence the N-terminal region of Kv4.3 as bait in a to 256 amino acids. The N-terminal
2000 Nature America Inc. http://neurosci.nature.com
time required for membrane depolar- yeast two-hybrid hunt through a rat mid- domains, 50 amino acids, vary consid-
ization to reach threshold for action brain cDNA library, they captured and erably, but throughout their carboxyl
potential generation as well as the time characterized three members of a family (C)-terminal domains, they share 70%
between action potential spikes. Thus of Kv4 channel interacting proteins sequence identity. The conserved
they are important determinants of the (KChIPs). The KChIPs interact selective- regions of the molecules contain four
firing frequency in excitable cells such ly with Kv4 subunits, and they are E-F hand calcium-binding motifs. Cal-
as neurons and cardiac myocytes. expressed in tissues that also express Kv4 cium binding to the KChIPs was con-
Sequence homologies among cloned subunits. When co-expressed in heterol- firmed by calcium-dependent mobility
subunits distinguish several subfamilies ogous cells, KChIPs were localized with shifts. Interestingly, a mutant KChIP-1
of voltage-dependent potassium chan- Kv4 subunits. KChIPs were selectively that could not bind calcium still inter-
nels, the Kv subfamilies. The Kv chan-
nels are tetramers, and heteromeric
channels are assembled only from sub-
units in the same subfamily. Heterolo-
gous expression studies show that K+
members of the Kv4 subfamily form K+
channels similar (but not identical) to
native A-type potassium channels 2. In
particular, they demonstrate inactiva-
tion mediated by a specialized intracel- KChIP KChIP
lular domain at the extreme N terminus, KChIP
the ball, which physically occludes the
pore when the channel is open. The
expression profiles of Kv4 subunits in Ca2+
the CNS and heart are consistent with KChIP
this role, and Kv4 subunits have been
identified as the components of A-type
potassium channels in rat neostriatal
cholinergic interneurons 3 and cardiac
ventricular myocytes4. KChIP KChIP
The functional profiles of Kv4 chan-
nels expressed in different heterologous Golgi Ca2+ Ca2+
cells are variable5, suggesting a contri-
bution by factors present in the host
cell. This is further supported by differ-
ences between Kv4 channels expressed
in Xenopus oocytes with or without rat
brain mRNA. Co-expression of the low-
molecular-weight mRNA fraction
Fig. 1. KChIPs are integral components of A-type potassium channels. The KChIPs bind to the
increases current density, most likely
N-terminal domain of Kv4 subunits, close to the membrane, and close to the ball domain that
reflecting an increased number of A- mediates channel inactivation. The physical association between KChIPs and Kv4 subunits does
type channels. This co-expression also not require calcium binding, but the effects on channel gating are calcium dependent. The KChIPs
shifts the activation voltage of the chan- are also present in the Golgi, where the association with Kv4 channels during their biosynthetic
nels to more negative potentials and development may regulate the levels of functional A-type potassium channels present in the sur-
allows faster recovery from inactiva- face membrane.
acted with Kv4 subunits, suggesting that KChIPs for the various Kv4 subunits? ing associations to members of the same
the association between channels and What other proteins might KChIPs subfamily. The N-terminal region also
KChIPs is calcium independent and may bring into the A-type channel complex? interacts with the Kv proteins, some of
be constitutive. However, the mutant This is the latest in a series of find- which endow inactivation that is mod-
KChIP-1 no longer modulated Kv4 ings suggesting that intracellular calci- ulated by protein kinases13. More recent-
channel function. um signaling can modulate membrane ly, two alternatively spliced proteins,
Sequence alignments show that potential. Fluctuations in intracellular ZIP1 and ZIP2, have been identified
KChIPs belong to the recoverin family calcium levels have long been appreci- that act as molecular bridges, linking the
of calcium-binding proteins that ated as an important modulator of ion subunits to protein kinase C
includes the Drosophila protein fre- channel activity. A generally accepted (ref. 14). At their C termini, many volt-
quenin, which regulates transmitter model posits that second messenger sys- age-dependent potassium channels bind
release, and mammalian homologs tems, such as calcium-sensitive protein to PDZ-containing proteins, such as
NCS-1 (neuronal calcium sensor-1) and kinases or phosphatases, alter the phos- PSD-95/SAP90 family members, to
hippocalcin7. Although KChIPs 1 and 2 phorylation status of the channel, affect- modulate the distribution and surface
have not been previously described, ing channel activity and cellular expression of the potassium channels,
KChIP-3 is identical to DREAM (down- excitability10. These are relatively slow which is affected by the presence of a
stream regulatory element antagonist processes that require calcium interac- subunit at the N-terminal domain in
modulator), which is reported to regu- tion with the signaling molecule and some cases 15 . These PDZ-containing
2000 Nature America Inc. http://neurosci.nature.com
late transcription in a calcium-depen- subsequent interaction with the ion proteins, in turn, interact with other
dent manner8. KChIP-3 is also identical channel. putative regulatory molecules. The
to calsenilin, a protein that resides in the Recently, several reports have sug- emerging picture suggests that the
ER and Golgi and interacts with the gested a faster calcium signaling process subunits of potassium channels are
C-terminal domains of the presenilins, by demonstrating that calmodulin is embedded in large, multimeric protein
transmembrane proteins found in the constitutively bound to the ion-con- complexes with components that sense a
same subcellular compartments. Muta- ducting subunits of voltage-depen- wide range of metabolic signals. Indeed,
tions in the presenilin genes account for dent calcium channels (VDCCs) and the ZIP proteins, which do not them-
40 percent of early-onset familial small conductance calcium-activated selves interact with the subunits,
Alzheimer disease and sensitize neu- potassium channels (SK channels). In begin to define a larger microdomain, a
ronal cells to apoptosis, possibly by dis- both cases, compelling evidence sup- molecular neighborhood, in which the
rupting intracellular calcium levels9. In ports a model in which calmodulin is an channels reside. The two-hybrid screen
this regard, it is tempting to speculate integral part of the channel complex, can be used with each newly identified
that the increased current density and calcium binding to calmodulin resident to determine the next nearest
(thought to reflect an increased number induces structural alterations in neighbor.
of channels in the membrane) induced calmodulin, which are transduced into
by co-expression of KChIPs with Kv4 conformational changes in the channel 1. An, W. F. et al. Nature 403, 553556 (2000).
subunits may reflect effects on mem- proteins that alter their function 11,12 .
2. Serodio, P., Vega-Saenz de Miera, E. & Rudy,
brane trafficking, which could be relat- Local signaling induced by calcium B. J. Neurophysiol. 75, 21742179 (1996).
ed to the interactions between KChIP-3 entry may be much more rapid than 3. Song, W.-J. et al. J. Neurosci. 18, 31243137
and presenilins in the trans Golgi. second-messenger-mediated processes, (1998).
Taken together, the results suggest and can respond rapidly to discrete, 4. Yeola, S. W. & Snyders, D. J. Cardiovasc. Res.
that KChIPs are integral parts of Kv4 localized alterations in intracellular cal- 33, 540547 (1997).
channels, both in the plasma membrane cium. For example, VDCCs are the like- 5. Petersen, K. R. & Nerbonne, J. M. Pflugers
and during their biosynthetic journey ly source of the calcium ions that would Arch. 437, 381392 (1999).
through the trans Golgi. On elevation of bind to channel-associated calmodulin, 6. Serodio, P., Kentros, C. & Rudy, B. J.
intracellular calcium levels, such as dur- and the precise distance between SK Neurophysiol. 72, 15161529 (1994).
ing an action potential, calcium bind- channels and VDCCs or intracellular 7. Pawlowski, K., Bierzynski, A. & Godzik, A. J.
Mol. Biol. 258, 349366 (1996).
ing to KChIPs induces a conformational calcium release sites strongly affects the
alteration that is rapidly transduced to dynamics of burst frequency. The work 8. Carrion, A. M., Link, W. A., Ledo, F.,
Mellstrom, B. & Naranjo, J. R. Nature 398,
the channel, resulting in altered gating. by Rhodes and colleagues1 suggests that 8084 (1999).
The previous localization of KChIP-3 to KChIPs are an integral component of A- 9. Buxbaum, J. D. et al. Nat. Med. 4,
the trans Golgi suggests that the type potassium channels, acting as 11771181 (1998).
increased current densities may result direct calcium sensors that affect chan- 10. Levitan, I. B. Annu. Rev. Physiol. 56,
from regulated cell-surface expression nel gating properties, analogous to the 193212 (1994).
of the channel complexes. role of calmodulin for SK channels and 11. Zuhlke, R. D., Pitt, G. S., Deisseroth, K.,
Several questions await further VDCCs. Tsien, R. W. & Reuter, H. Nature 399,
159162 (1999).
experiments. For example, just how The KChIPs join an expanding list of
does calcium binding to the KChIP proteins that bind to potassium chan- 12. Keen, J. E. et al. J. Neurosci. 19, 88308838
(1999).
result in altered channel activity? Which nels and influence their activity, but do
13. Sheng, M. & Kim, E. Curr. Opin. Neurobiol.
amino acids in the N-terminal domain not contribute to ion conduction. 6, 602608 (1996).
of Kv4 subunits interact with KChIPs? Among the voltage-dependent potassi- 14. Gong, J. et al. Science 285, 15651569
Does the interacting domain of Kv4 um channels, the intracellular N-termi- (1999).
overlap with binding sites for other pro- nal domain has been shown to mediate 15. Tiffany, A. M. et al. J. Cell Biol. 148, 147158
teins? How selective are the different heteromeric subunit assembly, restrict- (2000).
(William James1, p. 659) ronment, in which they could presumably hippocampus might use processes that do
establish many diverse associations not require NMDA receptors after expo-
In this two-factor view of the biological through exploration. By electron sure to environmental enrichment. In CA1,
basis of memory, James characterized the microscopy, environmental enrichment LTP can be induced by strong patterned
persistence factor as a physiological prop- was shown to increase the number of stimulation, even when NMDA receptors
erty of ones brain tissue. He envisioned synaptic connections within hippocam- are pharmacologically blocked5. This form
persistence as a native tenacity, except for pal area CA1 in normal mice, and sur- of LTP is NMDA receptor independent.
natural variability among individuals and prisingly also in the mutant mice, even Tsien and colleagues2 show that many new
decline with illness or aging. By contrast, without NMDA receptors. Furthermore, dendritic spines form, and robust synap-
James characterized the number of paths enrichment improved learning perfor- togenesis occurs within CA1 after enrich-
factor as very much modifiable with expe- mance in control mice and almost elimi- ment experience (Fig. 1b) in both mutant
rience. He argued that memory could be nated the memory deficits observed in the and control mice. This synaptogenesis does
improved substantially by establishing a CA1 NMDA receptor-knockout mice. not depend on NMDA receptors because
large network of linkages through which How is it possible that enriched expe- an equal number of new dendritic spines
one could readily associate, and later access, rience can support new memory even and synapses formed in mutant and con-
a new memory. As an example, James
described the college athlete who was a
dunce at his books but could astonish Standard Enriched
with his ability to remember sports statis-
tics precisely because he had worked at cre-
ating a rich knowledge framework for this Cortex
kind of information. James may have been
prescient in proposing that enriching ones
memory network can make up for a lesser
native persistence, as in findings from Joe
Tsien and colleagues2 in the current issue of Hippocampus
Nature Neuroscience.
This report extends a recent study that
used state-of-the-art molecular genetics
to knock out the N-methyl-D-aspartate
(NMDA) receptor beginning at postnatal
weeks three and four selectively within the
CA1 region of the hippocampus3. These
mutant mice lack NMDA-receptor-depen-
Bob Crimi
dent long-term potentiation (LTP), a type
of physiological plasticity thought to be a Fig. 1. Environmental enrichment increases the connections within the hippocampus and neocor-
cellular substrate of memory. Corre- tex. (a) Under control conditions, there are fewer synapses within both the hippocampus and cor-
spondingly, they have severely impaired tex, as well as between these areas. The pathway through the hippocampus could be required to
spatial learning and memory4. Now Tsien connect distinct representations in the neocortex (red and blue), and this capacity could be medi-
ated by strengthening the existing connections within the hippocampus using NMDA receptors
(yellow). (b) After exposure to an enriched environment, more connections are formed within
Howard Eichenbaum is in the Department of both the hippocampus and neocortex, and perhaps between these areas. Strengthening of addi-
Psychology, and Kristen Harris is in the tional non-NMDA receptor connections (orange) within the hippocampus, or between the hip-
Department of Biology, Boston University, pocampus and cortex, may suffice to improve memory. Alternatively, the additional connections
Boston, Massachusetts 02215, USA. within the neocortex (green) may suffice to link distinct neocortical representations and thereby
e-mail: hbe@bu.edu or harrisk@bio.bu.edu short circuit the hippocampal contribution.
trol mice following environmental enrich- can induce enough connectivity outside hypothesis discussed above would be to
ment. Other studies have shown robust the hippocampus, specifically within the determine whether the CA1-NMDA
synaptogenesis in the adult brain when neocortex. Tsien and colleagues did not knockout mice after enrichment can tol-
synaptic activity is silenced pharmacologi- examine the cortex, but previous evi- erate loss of hippocampal area CA1 and
cally6,7. The new spines form either when dence indicates that enriched experience still enjoy improved learning and memo-
presynaptic release of neurotransmitter is increases intrinsic connectivity within the ry. An early study15 found that enriched
blocked or when postsynaptic glutamate neocortex12. It is clear that memory is not experience reduced, but did not eliminate,
receptors are blocked, and new spines can mediated solely by CA1, or even by the the effects of hippocampal damage on
last for at least eight hours without subse- entire hippocampus alone. Rather, the spatial learning. These findings are con-
quent activation. Furthermore, induction hippocampus is part of a memory system sistent with the possibility that both puta-
of NMDA-receptor-dependent LTP in hip- that prominently involves its bidirection- tive mechanisms contribute to the effects
pocampal area CA1 does not require the al connections with diverse and inter- of enrichment.
formation of new synapses8,9. Together connected regions of the cerebral cortex13
with the findings from Tsien and col- (Fig. 1). Within this system, memories are 1. James, W. The Principles of Psychology (Holt,
leagues, these studies show that dendritic likely stored among large cell assemblies New York, 1890).
spines can form in the mature brain with- widespread across the cortex, and the 2. Rampon, C. et al. Nat. Neurosci. 3, 238244
out NMDA-receptor-dependent processes organization of associations is mediated (2000).
like LTP, and even without synaptic activi- by the formation of links between the cell 3. Tsien, J. Z. et al. Cell 87, 13171326 (1996).
2000 Nature America Inc. http://neurosci.nature.com
ty. Perhaps the new spine synapses can assemblies10. The role of the hippocam- 4. Tsien, J. Z., Huerta, P. T. & Tonegawa, S. Cell
facilitate NMDA-receptor-independent pus may be to facilitate the consolidation 87, 13271338 (1996).
processes within the hippocampus to of these cortical linkages by storing 5. Morgan, S. L. & Teyler, T. J. J. Neurophysiol. 82,
736740 (1999).
enhance subsequent learning and memo- aspects of new information, or indices
ry in CA1-NMDA knockout mice. pointing to cortical loci of new represen- 6. Bravin, M., Morando, L., Vercelli, A., Rossi, F.
& Strata, P. Proc. Natl. Acad. Sci. USA 96,
How might the enrichment-induced tations, and using these to temporarily 17041709 (1999).
dendritic spines within the hippocampus link otherwise separated cortical memo- 7. Kirov, S. A. & Harris, K. M. Nat. Neurosci. 2,
facilitate learning and memory? Hebb10 ries (Fig. 1a). We know that the role of 878883 (1999).
originally suggested that learning and the hippocampus is temporary because it 8. Muller, D. Rev. Neurosci. 8, 7793 (1997).
memory occurs by strengthening some is not necessary for the recall of long- 9. Sorra, K. E. & Harris, K. M. J. Neurosci. 18,
connections and weakening other, inap- established memories, suggesting that 658671 (1998).
propriate connections. Tsien and collegues eventually new intracortical connections 10. Hebb, D. O. The Organization of Behavior
show that the enrichment effects are spe- form to mediate permanent links14. The (Wiley, New York, 1949).
cific to a particular type of spine synapse, increase in synaptic connectivity in neo- 11. Toni, N., Buchs, P. A., Nikonenko, I., Bron,
causing an increase only in those with a cortex, likely to have occurred as a result C. R. & Muller, D. Nature 402, 421425
(1999).
continuous (that is, non-perforated) of enriched training experience12, might
12. Klintsova, A. V. & Greenough, W. T. Curr.
postsynaptic surface. There was no change be so effective that lasting plasticity with- Opin. Neurobiol. 9, 203208 (1999)
in the frequency of large irregularly shaped in the hippocampus is not required (Fig.
13. Eichenbaum, H. Annu. Rev. Psychol. 48,
synapses, those with perforated postsy- 1b), at least for the relatively simple types 547572 (1997).
naptic surfaces. Thus, the non-perforated of learning examined by Tsien and col- 14. Squire, L. R. & Alvarez, P. Curr. Opin.
synapses might enhance some forms of leagues2. Neurobiol. 5, 169177 (1995).
learning and memory via NMDA-recep- One way to distinguish the cortical 15. Hughes, K. R. Can. J. Psychol. 19, 325332
tor-independent mechanisms. Other stud- hypothesis from the hippocampal (1965).
ies have shown a transient elaboration of
a subset of perforated synapses with
NMDA-receptor-dependent LTP 11. An
open question is whether NMDA-recep-
tor-dependent changes at perforated
synapses might be involved in refinement
Attention - brains at work!
of synaptic connections during more com- Roger B.H. Tootell and Nouchine Hadjikhani
plex learning protocols than those tested
by Tsien and colleagues2. Either way, these Two new studies use event-related fMRI to reveal a network
findings are among the first to demon- of brain regions that are activated during different steps in
strate a possible role for non-perforated the control of visual spatial attention.
synapses in learning and memory. Under-
standing the function of the small non-
perforated synapses is especially important The amount of information that is handle. Much of this information must
because these are normally the most abun- potentially available through our sense therefore be discarded, and the brain
dant synapse type (> 75%) in both hip- organs is far greater than our brains can must select only those stimuli that are
pocampus and neocortex. of greatest relevance for further pro-
The second possible explanation for The authors are at the Magnetic Resonance cessing. Understanding how this occurs
the findings of Tsien and colleagues2 is Imaging Center, Department of Radiology, is a major challenge for cognitive neu-
that the hippocampus can be short-cir- Massachusetts General Hospital, 149 13th Street, roscience, and two papers1,2 in the cur-
cuited altogether during learning and Charlestown, Massaschusetts 02129, USA. rent issue of Nature Neuroscience
memory if environmental enrichment e-mail: tootell@nmr.mgh.harvard.edu provide the most detailed spatio-tem-
LO
Attention has some- ferent components of the task.
times been likened to a Both groups used covert attention
Cue
a
spotlight, and func- tasks, thus avoiding any complications
tional neuroimaging due to eye movements. The subjects were
has recently allowed instructed to fixate on the center of a
researchers to see the screen and then shift their focus of atten-
M
beam directly38. Sev- tion to either the left or the right, as indi-
SPL
IPL
eral studies have con- cated by a cue at the fixation point. A few
TPJ
firmed that attention is seconds later, a target appeared either at
VIP
mapped topographical- the cued location (valid cue condition)1,2
ly in all the early or on the opposite side (invalid condi-
Target (valid)
b
(retinotopic) visual tion)2, and subjects had to respond to it.
cortical areas, and that Both groups confirmed that their subjects
2000 Nature America Inc. http://neurosci.nature.com
SPL
location, the part of the their subjects reaction times were faster
IPL
TPJ
cortex that represents after valid than invalid cues, and Hopfin-
VIP
that location becomes ger et al.1 showed that the neural activity
increasingly responsive. evoked by the arrow cue (which, being in
But where and how are the center, could activate both hemi-
Target (invalid)
c attention signals first spheres) was greater in the hemisphere
Bob Crimi
generatedin other that represents the cued side.
Fig. 1. Brain areas activated in a 'naked' eye and brain, from a subject words, how is the Despite their similar techniques, the
who was facing a display screen and doing a covert attention task, spotlight controlled? two studies addressed different questions
similar that used in Hopfinger et al.1 and Corbetta et al.2 (a) A cue Answering this ques- and yielded complementary results.
instructs the subject to attend to a given location: here to the sub- tion is now an impor- Hopfinger et al.1 made few prior assump-
ject's left. Then the attention target appears (here a checkerboard tant goal for the field9. tions, and simply asked which brain
stimulus, but usually a more subtle stimulus change), at either the One problem with regions were activated in response to the
expected location (b) prompted by the 'valid' cue, or at an unex-
most previous neu- cues (reflecting an attentional shift) and
pected location (c), misdirected by the 'invalid' cue in (a). The acti-
vated areas described in Hopfinger et al. and Corbetta et al. include
roimaging studies of which were activated by the subsequent
DLPF (dorsolateral prefrontal cortex), IPL (inferior parietal lobule), attention (as well as target presentation (reflecting processing
LO (lateral occipital region of visual cortex), M (supplementary many other cognitive of the attended stimulus). Cues and tar-
motor region), PS (peri-sylvian), SPL (superior parietal lobule), TPJ processes) is that they gets both activated a number of different
(temporal-parietal junction) and VP (ventral parietal region). have used a block regions; the surprising finding was that
Additional areas were activated but are not visible from this vantage design, in which the there was relatively little overlap between
point (see refs. 1 and 2). High levels of activity are shown in red, and hemodynamic signal is the two sets of responses, suggesting that
lower levels of activity are shown in green. averaged over many the brain structures that control spatial
similar trials. This gen- attention are largely distinct from those
erates a static activation that participate in the processing of the
map that represents the attended stimulus.
poral views yet of the brain structures average activation for a particular task, In a more hypothesis-driven approach,
that control the deployment of visual without giving any information about Corbetta et al.2 tested two specific propos-
spatial attention. the individual steps involved. Yet spatial als regarding the role of parietal cortex in
Our focus of attention is constantly attention is inherently dynamic, and our attention. Based on studies of brain-dam-
shifting, either automaticallyin response brains are constantly choosing new loca- aged patients, it has been suggested that
to an attention-grabbing stimulusor tions of interest, disengaging attention the region around the temporal-parietal
voluntarily. Usually, an attentional shift is and (often) eye position from previously junction (TPJ) is involved in reorienting
followed by an eye movement to the attended locations, and shifting attention attention toward stimuli at unexpected
newly attended location, but it is also pos- and eye position to new targets. It is dif- locations, and that the region around the
sible to attend to a location without ficult to resolve these different steps using intraparietal sulcus (IPs) is involved in vol-
looking at it; we are sometimes forced to a block design. untary orientation and maintenance of
do this during demanding visual tasks The new studies1,2 avoid this problem attention at cued locations. As described
(such as driving on a busy road), where it by using event-related designs. Event- below, their data support both these ideas,
is impossible to fixate all items of interest related fMRI is a relatively new analytical and provide a view of parietal function
simultaneously. In the laboratory, these method, in which the hemodynamic sig- that is complementary to, and largely con-
so-called covert attentional shifts nal is analyzed on a trial-by-trial basis to sistent with, that of the other study.
can be detected because reaction identify patterns that occur at fixed times Both groups agree on the role of a pos-
times are shorter for trials in which after a given event, such as cue or target terior parietal region in and around the
intraparietal sulcus; this region is activat- could happen except through top-down tion fits very well with the clinical litera-
ed in response to the cue and remains signals, and the challenge now will be to ture on parietal neglect, and the link
active as attention is maintained, but identify the anatomical connections that seems even more compelling given that
shows a reduced response once the target underlie these effects. the other activations seen in these stud-
is presented (regardless of whether it The Corbetta et al. 2 study has some ies were not lateralized.
appears at an expected or unexpected interesting clinical implications. For many In conclusion, these two papers demon-
location)2. Similar responses have been years, it has been known that damage to the strate the power of new imaging techniques
observed in electrophysiological record- right parietal cortex, particularly the tem- to resolve complex cognitive operations
ings from alert monkeys (see ref. 2 for ref- poral-parietal junction, causes a complex into their component steps, and to reveal
erences), and the combined evidence from syndrome known as unilateral visual the neural structures involved in each step.
physiology and neuroimaging strongly neglect (reviewed in ref. 12). Parietal neglect They are likely to stimulate many future
suggests that the posterior parietal cortex patients have problems attending to and studies, and by combining ever-better
is involved in selecting a location and responding to objects on in the left visual imaging methods with other approaches
retaining it in working memory (although field; for instance, they often bump into such as patient studies and physiology of
other areas may also be involvedsee objects on their left, and when asked to non-human primates, we can hope to gain
below). Interestingly, the greater region of draw what they see, they tend to neglect a new depth of understanding of how the
posterior parietal activation may include what is in the left visual field. The syndrome brain controls attention.
the visual cortical area V7, which is retino- has attracted a great deal of interest, not
2000 Nature America Inc. http://neurosci.nature.com
topically organized (albeit crudely), sug- only for its clinical importance but also 1. Hopfinger, J. B., Buonocore, M. H. & Mangun,
gesting a possible role in the spatial because of its implications for normal per- G. R. Nat. Neurosci. 3, 284291 (2000).
allocation of attention3,9,10. ceptual mechanisms. One interpretation of 2. Corbetta, M., Kincade, J.M., Ollinger, J.M.,
Another popular candidate for storing parietal neglect is that the TPJ is responsible McAvoy, M.P. & Shulman, G.L. Nat. Neurosci. 3,
292297 (2000).
spatial cues in working memory is the for disengaging attention from its present
dorsolateral prefrontal cortex (see ref. 1 focus and redirecting it to a new target. 3. Tootell, R. B. H. et al. Neuron 21, 14091422 (1998).
and references cited therein). Hopfinger Corbetta et al.2 now provide elegant 4. Brefczynski, J. & DeYoe, E. Nat. Neurosci. 2,
370374 (1999).
et al.1 observed activation of this region support for this hypothesis. Unlike other
during the cueing period, but Corbetta parts of the parietal cortex, the TPJ 5. Somers, D. C. et al. Proc. Natl. Acad. Sci. USA
96, 16631668 (1999).
et al.2using a better analytical method showed little or no response to the initial
6. Ghandi, S., Heeger, D. & Boynton, G. Proc.
that avoided prior assumptions about the cue, but it responded strongly to the sub- Natl. Acad. Sci. USA 96, 33143319 (1999).
time course of the hemodynamic sequent presentation of the target. More-
7. Martinez, A. et al. Nat. Neurosci. 2, 364369
responsefound that the activation in the over, the TPJ response was much stronger (1999).
prefrontal cortex was more transient than for invalid than for valid targets, suggest- 8. Kastner, S. et al. Science 282, 108111 (1998).
that observed in the intraparietal cortex. ing that it is specifically involved in reori- 9. Corbetta, M. Proc. Natl. Acad. Sci. USA 95,
Thus, the intraparietal cortex seems to be enting of attention in cases where the 831838 (1998).
the stronger candidate for storing spatial target appears at an unexpected location. 10. Culham, J. C. et al. J. Neurophysiol. 80,
working memories, although it is possi- Finally, the TPJ response was always 26572670 (1998).
ble that both regions are involved, partic- stronger in the right hemisphere than the 11. Desimone, R. & Duncan, J. Annu. Rev.
ularly as they are known to be left, regardless of the side where the tar- Neurosci. 18, 193222 (1995).
interconnected in monkeys (and presum- get was presented. This right lateraliza- 12. Mesulam, M. M. Ann. Neurol. 10, 309325 (1981).
ably in humans too).
Most previous models of visual atten-
tion have assumed that it is controlled by
higher cortical regions, which regulate the Signaling dendritic growth in vivo
processing of sensory inputs in lower
regions via top-down projections. This Small GTPases of the Rho family affect cell morphology by regulating the cytoskeleton,
makes sense because decisions about allo- and they have been implicated in neurite outgrowth. On page 217 of this issue, Holly
cating attention are often based on high- Cline and colleagues (Cold Spring Harbor Laboratory, New York) report that RhoA, Rac
level features that are not represented at and Cdc42 regulate different aspects of dendritic growth in vivo. The authors used
the earliest stages of the cortical hierar- vaccinia virus to express constitutively active or dominant-
chy. However, alternative, bottom-up negative forms of these GTPases in albino Xenopus tadpoles.
models have also been proposed 11 , in Time-lapse imaging of DiI-labeled neurons showed that
which attention arises as an emergent constitutively active Rac and, to a lesser extent, Cdc42
property from competitive interactions increased branch addition and retraction. Activation of
at each level in the hierarchy. The new endogenous RhoA promoted the elongation of existing
findings do not completely exclude the branches. Cline has previously shown that blocking NMDA
latter model, but they are more consistent receptors reduces dendritic growth, and the dominant-
with a top-down model. In particular, negative form of RhoA prevented this effect, suggesting that
both groups found that a cue instructing RhoA may act downstream of NMDA receptors to control
subjects to attend to a particular location dendritic development.
caused increased activation of the corre-
sponding parts of the early retinotopic Sandra Aamodt
visual areas, even before any stimulus
appeared. It is difficult to see how this
brief communications
Three-dimensional spatial during space flight10, it was of interest to determine whether the
hippocampus can create a stable representation of space during
selectivity of three-dimensional movement in the absence of gravity.
During the Neurolab Space Shuttle mission of April, 1998,
hippocampal neurons ensembles of place cells were recorded from three rats implanted
with a multi-electrode recording array11,12. The rats were trained
during space flight to negotiate a three-dimensional track (the Escher staircase) in
which 3 turns of 90 in yaw were interleaved with 3 turns of 90 in
pitch (Fig. 1). As a result, the rat completed a full circuit of the
James J. Knierim1,2, Bruce L. McNaughton1 and track and returned to its starting location/direction after having
Gina R. Poe1 made only 3 right turns (270 total yaw). The spatial information
provided by external landmarks was thus presumably in conflict
1 University of Arizona, Arizona Research Laboratories Division of Neural with the direction information from HD cells, which under nor-
Systems, Memory & Aging, 384 Life Sciences North Bldg., Tucson, Arizona,
85724, USA
mal conditions require a fourth 90 turn (360 total yaw) to signal
2 a return to the starting direction. Nonetheless, place cells even-
University of Texas-Houston Medical School, Department of Neurobiology &
Anatomy and the W. M. Keck Center for the Neurobiology of Learning and tually demonstrated normal, spatially specific firing properties.
Memory, P.O. Box 20708, Houston, Texas 77225, USA Spatial firing patterns of 16 active place cells from rat 2 were
recorded on the ninth day of flight (FD9; Fig. 2a), which was the
2000 Nature America Inc. http://neurosci.nature.com
brief communications
ACKNOWLEDGEMENTS
We thank the crew of STS-90 (Scott Altman, Jay Buckey, Alex Dunlap, Kay
Hire, Rick Linnehan, Chiaki Mukai, Jim Pawelczyk, Rick Searfoss and Dave
Fig. 2. Representative place fields. (a) Normal place fields from rat 2 Williams), Bryan Roberts, Lisa Baer, Mike Eodice, Laurie Dubrovin, Tom
recorded on FD9. The number inside each map indicates the firing rate Howerton, Louis Ostrach, Chris Maese, Justine Grove, Ali Werner, Dave
coded by red (for instance, > 1 spike per s for the first cell). Bergner, Tom McCarthy, George Swaiss, Steve Carmen, Jim Cockrell and
(b) Preflight place fields from rat 2 as the rat ran on a rectangular track. others at NASA-Ames. We also thank Casey Stengel (who designed and built
(c) Normal place fields from rat 1 on FD9. the recording system), Krzystof Jagiello (who designed and wrote the data
acquisition software), Kathy Dillon, Shanda Roberts, Shane Smith, Vince
Pawlowski, Carol Barnes, Katalin Gothard, Veronica Fedor-Duys, Mark
Bower, Karen Reinke, Chris Duffield, Luann Snyder, Doug Wellington and
recorded during baseline and behavioral sessions in all rats on others at the University of Arizona. Additionally, we thank Bill Skaggs and
FD4 and FD9. Although the small number of subjects pre- Matt Wilson, who wrote much of the data analysis software, and science and
cluded a statistical analysis, they showed normal theta rhythm engineering support and management teams at Johnson Space Center and
during active locomotion and normal sharp waves and rip- Kennedy Space Center. Supported by grants NAG 2-949 from NASA;
ples15 during quiet inactivity in the sleeping pouch on both NS33471 and NS20331 from NIH; and N0014-98-1-0180 and
flight days. N0014-96-1-1082 from ONR.
It is interesting to note that on the first experience on the
Escher staircase on FD4, firing of place cells showed abnormal
patterns of spatial selectivity that differed between rats 1 and RECEIVED 3 SEPTEMBER 1999; ACCEPTED 1 FEBRUARY 2000
2 (J.J.K., B.L.M. and G.R.P., unpublished observations). Thus
hippocampal cells can form unique, reliable representations of
position on three orthogonal surfaces in microgravity, but they 1. Redish, A. D. Beyond the Cognitive Map (MIT Press, Cambridge,
Massachusetts, 1999).
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articles
1 Department of Molecular, Cellular and Developmental Biology, Yale University, Kline Biology Tower, P.O. Box 208103, New Haven, Connecticut 06520-8103, USA
2 Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, Box 296, 1230 York Avenue, New York, New York 10021, USA
3 Present address: Massachusetts Institute of Technology, Department of Biology, Building 68, Rm 380, 77 Massachusetts Ave., Cambridge, Massachusetts 02139-4307, USA
4 Present address: Department of Molecular Biophysics & Biochemistry, Yale University, 333 Cedar St., P.O. Box 208024, New Haven, Connecticut 06520-8024, USA
Correspondence should be addressed to A.J.S. (alfred.scheetz@yale.edu)
synaptic protein synthesis in the superior colliculi of young rats. Within minutes of receptor activation,
translation of alpha Ca2+/calmodulin dependent kinase II (CamK II) was increased, whereas total pro-
tein synthesis was reduced. NMDAR activation also increased phosphorylation of eukaryotic elongation
factor 2 (eEF2), a process known to inhibit protein translation by reducing peptide chain elongation.
Low doses of cycloheximide, which reduce elongation rate independently of eEF2 phosphorylation,
decreased overall protein synthesis but increased CaMK II synthesis. These observations suggest that
regulation of peptide elongation via eEF2 phosphorylation can link NMDAR activation to local
increases in the synthesis of specific proteins during activity-dependent synaptic change.
Activity-dependent synaptic plasticity often involves changing the isolated, functional pre- and postsynaptic elements) prepared
function of a subset of a neurons synapses in response to activa- from the superficial visual layers of the developing rat sSC8. By
tion of neurotransmitter receptors. Dendritic synthesis of specific postnatal day (P) 13, the majority of synapses are formed by reti-
proteins is implicated in many of these modifications1,2. Activity- nal ganglion cell axons on sSC neurons15. This property makes
dependent translation of mRNA into protein in dendrites may the sSC well suited for the preparation of relatively homogeneous
occur during synaptogenesis, when young neurons must selectively synaptic fractions for studies of NMDAR-linked signaling path-
stabilize subsets of inputs and eliminate others based on their pat- ways during this period of synaptogenesis.
tern of activity3. The presence of polyribosomes at young synaps- The effect of NMDAR activation on protein synthesis in sSC
es supports this idea4,5. Furthermore, activation of NMDARs is synaptoneurosomes was examined using a 30-second pulse of
associated with synaptic phosphorylation of eEF2 (ref. 6), and plas- 10 M glutamate and 50 M NMDA (referred to as NMDAR
ticity of young contacts requires activation of NMDARs. stimulation)16. NMDAR stimulation was terminated by the addi-
In the rat retinocollicular pathway, blocking NMDAR activa- tion of AP-5 to a concentration of 120 M. For each interval, we
tion during development disrupts normal synaptogenesis7 and incubated an additional set of samples in 120 M AP-5 for 5 min-
reduces levels of CamK II (ref. 8), a protein widely implicated in utes before NMDAR stimulation and continuously thereafter to
synaptic plasticity9. To examine the role of local protein synthesis serve as the NMDAR-inactivated controls. Protein synthesis in
in this pathway, we used isolated synaptic preparations from the these AP-5 controls showed no consistent variation and remained
superficial layers of the postnatal rat superior colliculus (sSC). relatively constant throughout the incubation period. Newly syn-
We found that brief NMDAR stimulation reduced overall pro- thesized proteins were pulse labeled with 35S-methionine at stag-
tein synthesis but rapidly increased CaMK II synthesis. The fast gered intervals after NMDAR stimulation (Fig. 1a) using a
NMDAR-mediated regulation of protein synthesis was tempo- pulsechase protocol. One-minute pulses of 35S-methionine were
rally correlated with increased eEF2 phosphorylation, a modifi- followed by 10-minute chases in the excess non-radioactive
cation that inhibits protein synthesis by reducing peptide chain methionine to allow complete synthesis of proteins labeled dur-
elongation1013. We also found that inhibition of protein synthe- ing the pulse period. All data on protein synthesis following
sis elongation independent of eEF2 phosphorylation similarly NMDAR activation is presented as a percentage of synthesis mea-
increased CaMK II synthesis. Taken together, these results sug- sured in these matched AP-5 control samples.
gest that the dendritic translation of CaMK II transcripts is dif- Overall protein synthesis was maximally decreased within five
ferentially increased by transient blockade of elongation and that minutes of NMDAR stimulation (Fig. 1b). Protein synthesis lev-
phosphorylation of eEF2 represents a rapid, local and selective els subsequently increased above baseline and remained elevated
mechanism that may increase CaMK II synthesis in response from 15 minutes after stimulation until the end of the experi-
to NMDAR activation at developing synaptic contacts. ment. To monitor the effects of prolonged incubation and AP-5
exposure independent of any exposure to the NMDA-stimula-
RESULTS tion cocktail, some samples received either no treatment or
To study the effects of NMDAR activation on synaptic protein AP-5 treatment for one hour. No changes in protein synthesis
synthesis, we used synaptoneurosomes14 (fractions enriched in were observed in these samples (Fig. 1b, boxes).
articles
35S-methionine
Fig. 1. NMDAR activation dynamically regulates
a NMDAR activation AP-5 Cold methionine
protein synthesis in synaptic preparations.
Unstimulated (a) Schematic description of the pulse-labeling
AP-5 Alone procedure used to label newly synthesized pro-
tein in synaptoneurosomes. All samples (17)
3 min. were incubated for the same total time. At the
5 min. designated times, samples received NMDAR acti-
Sample sets
10 min. Time between vation (top) or AP-5 for five min before NMDAR
stimulation and
15 min. 35S-methionine activation (bottom). At the designated times after
30 min. pulse NMDAR stimulation, both pairs of samples were
45 min. pulse-labeled with 35S-methionine for one min
60 min. (black) followed by a ten-min chase with non-
radioactive methionine (gray) For 60 minutes pre-
Total duration, 75 min ceding the 35S-methionine pulse in control
experiments, samples either were untreated or
35S-methionine incorporation
protein (percent AP-5 control)
To resolve a large number of proteins, we used two-dimen- vation on CaMK II synthesis. The level of newly synthesized
sional gel electrophoresis. Under basal conditions, only a few pro- CaMK II was measured by densitometry after immunoprecip-
teins were 35S-methionine labeled (Fig. 1c, panel 1). During the itation and subsequent gel electrophoresis. We detected two radi-
period of maximal inhibition of protein synthesis, incorporation olabeled bands corresponding to molecular weights of 50 and
of 35S-methionine into these proteins was qualitatively reduced 48 kDa. In a western blot, the 50 kDa protein comigrated with
(Fig. 1c, panel 2). A larger array of proteins in samples were 35S- the CaMK II protein detected with a different monoclonal anti-
methionine labeled following 60 minutes of NMDAR stimula- body (Fig. 2b, inset). The other band may correspond to a mid-
tion. Although we cannot rule out the possibility that brain-specific CaMK II isoform 18. An analysis of variance
posttranslational modifications account for some of the observed showed that NMDAR stimulation produced statistically signifi-
changes in distribution of spots, it seems probable that these cant changes in CaMK II synthesis (p < 0.05, F6 = 15.627). In
changes were primarily due to changes in synthesis of individual contrast to overall protein synthesis levels, 3 minutes after
proteins for two reasons. First, the two-dimensional gel elec- NMDAR stimulation CaMK II synthesis was increased (p < 0.05
trophoresis analysis correlates well with our measures of NMDAR- on a planned post-hoc comparison). Following this rapid increase,
stimulated changes in overall protein synthesis. Second, we find 15 minutes after the cessation of NMDAR activation, CaMK II
no evidence of large-scale changes in protein phosphorylation synthesis was reduced to 40% of the AP-5 control level and sub-
that could account for dramatic shifts in protein migration with sequently showed a longer-latency increase that lasted for the
NMDAR stimulation16. We conclude that, even though each of duration of the experiment (Fig. 2a). Western blots of material
the 35S-methionine-labeled spots observed 60 minutes after from samples taken five minutes after stimulation of NMDARs
NMDAR stimulation does not necessarily reflect new translation, (sample set #3 in Fig. 1a) and immunoprecipitated 16.5 minutes
35S-methionine incorporation into postsynaptic protein shows a after NMDAR stimulation confirmed an increase in total synap-
complex response following brief NMDAR activation. tic CaMK II levels (Fig. 2b). Thus, the synthesis as well as the
The mRNA for CaMK II is present at high concentrations overall expression level of CaMK II in sSC synaptoneurosomes
in dendrites17. We therefore examined the effect of NMDAR acti- rapidly increased in response to brief NMDAR stimulation. Both
articles
Optical density
after NMDAR stimulation). (b) Steady-state
35S-methionine
the rapid increase in 35S-methionine-labeled CaMK II (Fig. 2a, tent qualitative difference in the composition of these complex-
three minutes) and the increase in total CaMK II protein mea- es was observed between P8 and P13 synaptoneurosomes
sured 16.5 minutes after NMDAR stimulation (Fig. 2b) are con- (Fig. 2d). Newly synthesized proteins from P8 sSC synaptoneu-
sistent with the interpretation that synaptic activation of rosomes that co-immunoprecipitated with CaMK II included
NMDARs increases CaMK II levels via local protein synthesis. two proteins with masses of approximately 25 and 30 kDa
To assess the selectivity of NMDAR-induced protein transla- (arrows on left). Immuncomplexes derived from P13 synap-
tion, we immunoprecipitated several other synaptic proteins from toneurosomes contained a different set of labeled proteins
35S-methionine-labeled synaptoneurosomes, including GluR2/3 (arrows on right), including proteins of 10, 15, 35 and 42 kDa.
(Fig. 2c), NR1, calcineurin, spinophilin and eEF2 kinase (data NMDAR activation in the developing tadpole retinotectal pro-
not shown). None of these proteins showed any NMDAR-depen- jection induces an increase in synaptic phosphorylation of eEF2
dent increase in 35S incorporation. that is localized to the immediate postsynaptic cytoplasm 6.
At P11, the retinocollicular map within the developing rat Because eEF2 phosphorylation is both mediated by a Ca2+-depen-
sSC completes its refinement19,20 and NMDA receptor currents dent kinase and is associated with decreased protein synthesis via
are downregulated en masse 21 . To determine if changes in inhibition of peptide elongation1013, it is possible that NMDAR-
NMDAR-stimulated CaMK II synthesis correlated with in vivo induced eEF2 phosphorylation in sSC synaptoneurosomes could
alterations in synaptic organization and NMDAR function, we account for the observed rapid depression of overall synaptic pro-
analyzed sSC preparations from both P8 and P13 rats. NMDAR- tein synthesis. NMDAR activation of sSC synaptoneurosomes
mediated changes in CaMK II synthesis showed the same tem- resulted in a fivefold increase in phospho-eEF2 levels within one
poral pattern at both ages. However, studies suggest that minute of stimulation. Phospho-eEF2 levels remained elevated
Ca associates with other key synaptic proteins such as at least threefold for 5 minutes and then decreased, reaching base-
actin22 and the 2B subunit of the NMDAR23,24. Non-denaturing line levels after 15 minutes (Fig. 3a). Phospho-eEF2 levels were
immunoprecipitation five minutes after NMDAR stimulation not increased by NMDAR stimulation in samples treated before
revealed that CaMK II complexes in the developing sSC con- and during NMDA stimulation with either AP-5 or EGTA (data
tain multiple newly synthesized proteins. Furthermore, a consis- not shown). Thus, the rapid NMDAR-induced depression of
articles
50 g/ml cycloheximide
overall protein synthesis via eEF2 phosphorylation could account
for the observed increase in CaMK II synthesis if CaMK II trans-
lation shared this paradoxical response to mild elongation inhibi-
tion. We therefore treated synaptoneurosomes with low doses of
cycloheximide, an agent that reduces elongation rate via a mecha-
35S-methionine
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articles
consequently become the rate-limiting step in protein synthesis. in ice-cold oxygenated buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM
MgSO4, 2.5 mM CaCl2, 1.53 mM KH2PO4 212.7 mM glucose) supple-
Such a shift should favor upregulation of translation of abun-
mented with 0.002 l per ml of Complete protease inhibitor cocktail
dant but poorly initiated transcripts such as CaMK II in den- (Boehringer-Mannheim, Indianapolis, Indiana), 0.04 units per ml of
drites. Other proteins are probably selectively translated, but their human placental RNase inhibitor (Ambion, Austin, Texas) and 200 g
identities as well as the downstream implications of such signal- per ml of chloramphenicol (Sigma). All subsequent steps were carried
ing remain unexplored. We propose that this pathway constitutes out at 4C. Samples were passed through a series of nylon filters of
a mechanistic link between the activation of a neurotransmitter descending pore size. The final pass was through a MLCWP 047 Milli-
receptor and the rapid and local control of protein production. pore filter with a 10-m pore size. Samples were then centrifuged for
A number of other studies suggest that dendritic protein syn- 15 min at 1,000 g. The supernatant was discarded and the pellet resus-
thesis may be regulated locally by activation of receptors on den- pended to a final protein concentration of 0.5 mg per ml.
drites. In the hippocampus, dendritic protein synthesis is increased A protocol with characterized specificity6,16 for NMDAR stimulation
consisted of a 30-s exposure to a cocktail of 10 M glutamate and 50 M
by simultaneous stimulation of afferents and activation of acetyl-
NMDA added from a concentrated stock solution. Synaptoneurosomes
choline receptors30; isolated processes of Aplysia sensory neurons (500 l, containing 0.5 mg per ml protein) were maintained at 37C for
stimulated with serotonin respond by increasing protein synthe- at least 10 min before NMDAR stimulation. Stimulation was terminated
sis levels1 and stimulation of metabotropic glutamate receptors by addition of AP-5 from a concentrated stock to a final concentration of
in isolated synaptic contacts from neonatal rat brain increases 120 M. Five min before NMDAR stimulation, negative control samples
protein synthesis31 as well as the synthesis of proteins from exoge- received AP-5.
nous mRNA in isolated neurites32. In addition, local application of
35S-Methionine pulsechase labeling. Synaptoneurosomes were first
neurotrophins to isolated hippocampal dendrites produces an
enhancement of synaptic transmission that is blocked by inhibitors warmed to 37C for 10 min. For each time point, two samples were pre-
pared, one sample was treated with AP-5 for five min before NMDAR
of protein synthesis2. Earlier data also show that regulation of
stimulation, and another sample received NMDAR stimulation followed
translation can be exerted on specific proteins. For example, syn- by AP-5 treatment. At designated times after NMDAR stimulation,
thesis of the fragile X protein is increased by activation of 50 Ci of 35S-methionine was added to each sample. After 1 min, non-
metabotropic glutamate receptors in synaptoneurosomes33, and radioactive methionine was added to a final concentration of 200 M.
several unidentified proteins are synthesized in response to depo- Samples were incubated for an additional 10 min to allow completion
larization of isolated synaptic fractions34. Rapid increases in den- of synthesis of proteins labeled during the pulse period.
dritic CaMK II levels have also been demonstrated after synaptic
activation, both in vitro35,36 and in vivo37, suggesting that local syn- Assessment of overall protein synthesis. To measure overall protein syn-
thesis of CaMK II in dendrites could have an appreciable effect thesis, synaptoneurosome samples were treated with an equal volume of
on the synaptic concentration of CaMK II. ice-cold 10% trichloroacetic acid (TCA) for 1 h. Insoluble material was
then pelleted by centrifugation and washed 3 times in ice-cold 5% TCA
Despite these compelling observations, the mechanism(s)
(for 1 h total). Pellets were washed 3 times with ice-cold methanol and
involved in the regulation of dendritic protein synthesis remain allowed to dry at room temperature. Samples were solubilized in either
unknown. Here we provide a plausible explanation for the speed 0.1N NaOH for scintillation counting or a buffer (8.0 M urea, 0.5% SDS
and selectivity of responses in dendritic translation, particular- and 0.4% 2-mercaptoethanol) for two-dimensional isoelectric focus-
ly in relationship to NMDARs, Ca2+ and CaMK II. However, ing24. Gels were treated with Amplify (Amersham Life Sciences, Piscat-
there are probably additional mechanisms of synaptic transla- away, New Jersey) before drying and exposure. Radioactive proteins were
tional control in neurons. For example, cytoplasmic polyadeny- detected either using X-ray film (Kodak, Rochester, New York) or phos-
lation of CaMK II mRNA in rat visual cortex is increased by 30 phorimager plates (Fuji, Chicago, Illinois).
minutes of light exposure after dark rearing38, and this polyadeny-
CaMK II immunoprecipitation. CaMK II was immunoprecipitated under
lation is correlated with an increase in CaMK II protein levels at
non-denaturing conditions as previously described40. Synaptoneurosome
synapses, suggesting that, as in oocytes39, polyadenylation increas- samples were labeled as described above before immunoprecipitation. Sam-
es translational efficiency. Interestingly, the time course observed ples were pelleted and solubilized in cold immunoprecipitation buffer
with a dark rearing/light exposure protocol correlates well with (20 mM Tris-HCl, pH 7.4, 5.0 mM EDTA, 1.0% Triton X-100, 153 mM NaCl,
the long-latency increase in CaMK II we observed in isolated 20 mg per ml BSA, 0.03% sodium azide and Complete protease inhibitors;
neonatal sSC synapses. Boehringer Mannheim). Samples were mixed with either 1 l of the beta sub-
articles
unit-specific antibody CB1 (Gibco, Rockville, Maryland) or IgG and incu- 13. Ryazanov, A. G., Shestakova, E. A. & Natapov, P. G. Phosphorylation of
bated overnight at 4oC. Immuncomplexes were collected by absorption with elongation factor 2 by EF-2 kinase affects rate of translation. Nature 334, 170173
50 l of a 1:1 slurry of protein A sepharose beads (Pharmacia, Piscataway, (1988).
14. Hollingsworth, E. B. et al. Biochemical characterization of a filtered
New Jersey) in immunoprecipitation buffer. Pellets were washed 3 times in synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine
wash buffer (PBS, 0.25% NP-40, 0.05% sodium azide) before addition of 4 3:5-monophosphate-generating systems, receptors and enzymes. J. Neurosci. 5,
sample buffer. Samples were boiled and centrifuged. Samples (5 l) were 22402253 (1985).
removed for scintillation counting, and the remainder was loaded on 8%18% 15. Lund, R. & Lund, J. Development of synaptic patterns in the superior colliculus of
the rat. Brain Res. 42, 120 (1972).
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counting or densitometric scanning of phosphorimager plates generated com- phosphoproteins in the developing tectum. J. Neurosci. 15, 14601469 (1996).
parable results. Western blots using a monoclonal antibody (6G9, Boehringer 17. Burgin, K. E. et al. In situ hybridization histochemistry of Ca++/calmodulin-
Mannheim) were used to detect CaMK II protein. dependent protein kinase II in developing brain. J. Neurosci 10, 17881798
(1990).
18. Brocke, L., Srinivasan, M. & Schulman, H. Developmental and regional
Measurement of phospho-eEF2 levels. At designated times after NMDAR expression of multifunctional Ca2+/calmodulin-dependent protein kinase in rat
activation, samples were centrifuged and the supernatant removed. Pel- brain. J. Neurosci 15, 67976808 (1995).
lets were suspended in 4 sample buffer and applied directly to 8%18% 19. Simon, D. K. & OLeary, D. D. M. Limited topographic specificity in the targeting
gradient acrylamide gels. Separated protein was transferred to nitrocel- and branching of mammalian retinal axons. Dev. Biol. 137, 125134 (1990).
lulose and phospho-eEF2 detected with the polyclonal antibody cc81 20. Simon, D. K. & OLeary, D. D. M. Development of topographic order in the
mammalian retinocollicular projection. J. Neurosci 12, 12121232 (1992).
(refs. 6, 41) visualized using SuperSignal chemiluminescence detection 21. Shi, J., Aamodt, S. M. & Constantine-Paton, M. Temporal correlations between
(Pierce, Rockford, Illinois). Total levels of eEF2 were estimated by strip- functional and molecular changes in NMDA receptors and GABA
ping the blot and reprobing it using an affinity-purified anti-eEF2 anti- neurotransmission in the superior colliculus. J. Neurosci. 17, 62646276 (1997).
2000 Nature America Inc. http://neurosci.nature.com
body that does not discriminate between phospho- and dephospho-eEF2. 22. Shen, K. & Meyer, T. Dynamic control of CaMKII translocation and localization
in hippocampal neurons by NMDA receptor stimulation. Science 284, 162166
Signal was quantitated as previously described8 using NIH Image 1.60. (1999).
23. Strack, S. & Colbran, R. J. Autophosphorylation-dependent targeting of calcium/
Cycloheximide treatment. A concentrated stock of cycloheximide (50 mg calmodulin- dependent protein kinase II by the NR2B subunit of the N-methyl-
per ml) was prepared in 0.2% DMSO. Synaptoneurosomes prepared from the D- aspartate receptor. J. Biol. Chem 273, 2068920692 (1998).
sSC of P13 rat pups were treated with cycloheximide at final concentrations 24. Leonard, A. S., Lim, I. A., Hemsworth, D. E., Horne, M. C. & Hell, J. W.
Calcium/calmodulin-dependent protein kinase II is associated with the
of 0.5 g per ml, 5.0 g per ml and 50.0 g per ml. At designated times, sam- N- methyl-D-aspartate receptor Proc. Natl. Acad. Sci. USA 96, 32393244 (1999).
ples were pulse-labeled as above for one min and chased with non-radioac- 25. Brendler, T., Godefroy-Colburn, T., Carhill, R. D. & Thach, R. E. The role of
tive methionine for ten min. Samples were then subjected to either TCA mRNA competition in regulating translation II. Development of a quantitative in
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26. Brendler, T., Godefroy-Colburn, T., Yu, S. & Thach, R. E. The role of mRNA
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ACKNOWLEDGEMENTS 27. Godefroy-Colburn, T. & Thach, R. E. The role of mRNA competition in
This work was supported by U.S. Public Health Service Grants EY 06039 to regulating translation IV. Kinetic model. J. Biol. Chem. 256, 1176211773
M.C.P and GM 50402 to A.C.N. (1981).
28. Walden, W. E., Godefroy-Colburn, T. & Thach, R. E. The role of mRNA
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motor synapses: a function for local protein synthesis in memory storage. Cell 91, schaffer collateral stimulation initiates protein synthesis in hippocampal
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3. Constantine-Paton, M., Cline, H. T. & Debski, E. A. Patterned activity, synaptic postsynaptic protein synthesis. Proc. Natl. Acad. Sci. USA 90, 71687171 (1993).
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Neurosci. 13, 129154 (1990). cone: regulated mRNA transport and local protein synthesis. Neuron 17,
4. Steward, O. & Falk, P. M. Protein-synthetic machinery at postsynaptic sites 11731187 (1996).
during synaptogenesis: a quantitative study of the association between 33. Weiler, I. J. et al. Fragile X mental retardation protein is translated near synapses
polyribosomes and developing synapses. J. Neurosci. 6, 412423 (1986). in response to neurotransmitter activation. Proc. Natl. Acad. Sci. USA 94,
5. Steward, O. & Falk, P. M. Selective localization of polyribosomes beneath 53955400 (1997).
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J. Comp. Neurol. 314, 545557 (1991). characterized by gel electrophoresis. Neurochem. Res. 21, 681690 (1996).
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receptor activation and visual activity induce elongation factor-2 Tetanic stimulation leads to increased accumulation of Ca2+/calmodulin-
phosphorylation in amphibia tecta: a role for N-methyl-D-aspartate receptors in dependent protein kinase II via dendritic protein synthesis in hippocampal
controlling protein synthesis. Proc. Natl. Acad. Sci. USA 94, 1477014775 (1997). neurons. J. Neurosci. 19, 78237833 (1999).
7. Simon, D. K., Prusky, G. T., OLeary, D. D. M. & Constantine-Paton, M. NMDA 36. Ouyang, Y., Kantor, D., Harris, K. M., Schuman, E. M. & Kennedy, M. B.
receptor antagonists disrupt the formation of a mammalian neural map Proc. Visualization of the distribution of autophosphorylated calcium/calmodulin-
Natl. Acad. Sci. USA 89, 1059310597 (1992). dependent protein kinase II after tetanic stimulation in the CA1 area of the
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9. Kelly, P. T. Calmodulin-dependent protein kinase II. Multifunctional roles in J. Neurosci. 19, 78347845 (1999).
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articles
1 Cold Spring Harbor Laboratory, Beckman Bldg., 1 Bungtown Rd., Cold Spring Harbor, New York 11724, USA
2 Department of Neurobiology and Behavior, SUNY Stony Brook, New York 11794, USA
Correspondence should be addressed to H.T.C. (cline@cshl.org)
The development and structural plasticity of dendritic arbors are governed by several factors, includ-
2000 Nature America Inc. http://neurosci.nature.com
ing synaptic activity, neurotrophins and other growth-regulating molecules. The signal transduction
pathways leading to dendritic structural changes are unknown, but likely include cytoskeleton regu-
latory components. To test whether GTPases regulate dendritic arbor development, we collected
time-lapse images of single optic tectal neurons in albino Xenopus tadpoles expressing dominant
negative or constitutively active forms of Rac, Cdc42 or RhoA. Analysis of images collected at two-
hour intervals over eight hours indicated that enhanced Rac activity selectively increased branch
additions and retractions, as did Cdc42 to a lesser extent. Activation of endogenous RhoA decreased
branch extension without affecting branch additions and retractions, whereas dominant-negative
RhoA increased branch extension. Finally, we provide data suggesting that RhoA mediates the
promotion of normal dendritic arbor development by NMDA receptor activation.
The structure of the dendritic arbor critically determines what regulating the cytoskeleton. Investigations of their function have
synaptic inputs a neuron receives and how they are integrated1. been aided by mutations that result in a constitutively active,
For instance, a neuron within the visual system whose dendritic GTP-bound form or a dominant-negative, GDP-bound form.
arbor has a large tangential spread can receive inputs from more Studies of neurite outgrowth from cultured neurons have given
visual afferents, leading to a larger receptive field over which us the first insights into the role of the Rho family of GTPases in
information is processed. Similarly, neurons that extend their regulating neuronal process growth911. Because neurites in cell
dendritic arbors into superficial laminae can receive inputs and culture often fail to take on the characteristics of dendrites and
process information from afferents in those laminae2. Conse- axons, few such studies have been able to distinguish effects of
quently, factors that regulate development and plasticity of the Rho GTPases on axonal and dendritic outgrowth. Rac, Cdc42
dendritic arbor control both the neurons structure and function and RhoA influence dendrite number in dissociated cortical neu-
and may ultimately affect circuit properties. rons12. Given that neuronal arbor elaboration in vivo is governed
The molecular mechanisms that underlie development of the by activity-dependent and activity-independent factors13, which
dendritic arbor are not clear yet. In vivo time-lapse imaging per- may operate through GTPases14, we wanted to determine whether
mits direct observation of dendritic arbor development. Time- Rho GTPases regulate dendritic arbor formation in the live ani-
lapse images of optic tectal neurons collected at intervals ranging mal with the normal pattern of synaptic inputs and local
from minutes to days in living Xenopus tadpoles show that den- environment. Our previous studies showed that NMDA recep-
dritic branches are very dynamic during arbor formation36. The tor activity is required for the initial phase of dendritic arbor
dynamic processes include addition of new branches, retraction growth in tectal neurons5,6. A potential link between synaptic
of branches, and selective extension or shortening of existing activity and the Rho GTPases remains to be defined.
branches. These events can be observed and quantified by col- As opposed to cultured cells, studies in intact animals pro-
lecting repeated images over several hours36. The data suggest vide the opportunity to investigate dendritic and axonal devel-
that the net growth of dendritic arbor occurs as a result of sev- opment in their normal complex environment. In vivo
eral distinct events: emergence of a new branch, selective main- experiments in transgenic flies, worms, Xenopus and mice all
tenance of the new branch, and extension of branch length. Each point to a crucial role of Rac and Cdc42 in axonal growth and
of these events may be individually regulated. Furthermore, it is target recognition1519. The roles of the Rho GTPases in regulat-
very likely that machinery controlling the actin cytoskeleton is ing dendritic arbor structural plasticity in vivo are relatively unex-
involved, because cytoskeleton reorganization accompanies struc- plored20. Although the dendrites of Purkinje cells in transgenic
tural changes in cells. mice expressing constitutively active Rac in the cerebellum branch
Members of the Rho family of small GTPases, Rac, Cdc42 and normally, dendritic spines are reduced in size and increased in
RhoA, are required components of signal transduction pathways number16. In Xenopus, retinal ganglion cell dendritic arbor elab-
through which extracellular signals cause morphological changes oration is inhibited by expression of constitutively active RhoA
in various cell types79. Rho GTPases mediate these changes by and constitutively active Cdc42, but promoted by constitutively
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present at the first image, but retracted over the eight-hour peri-
od. Any branches that were added after the first image and were
still there at the last time point were categorized as new branch-
es. The branches that were added after the first image and then
retracted before the last image were categorized as transient
branches. This analysis showed that among all the branches ever
present during imaging, 34.8 1.2% of them were transient in
control cells. This number was significantly increased by the
expression of constitutively active Rac, and to a lesser extent by
constitutively active Cdc42 (Table 1; Fig. 4). Notably, dominant-
Fig. 3. Rac activity promotes dendritic branch dynamics. (a) Drawings negative Rac triggered an increase in transient branches (Fig. 4).
of 2 representative tectal cells from control, constitutively active Rac In addition, dominant-negative Rac significantly decreased the
virus-infected and dominant-negative Rac virus-infected animals imaged relative number of new branches, that is, those that were added to
over 20 h. Note the rapid changes in fine branch tips in cells expressing the arbor and maintained to the end of the observation period.
constitutively active Rac. (b, c) Total branch additions (b) and retrac- When we evaluated the fraction of stable branches, we noticed
tions (c) for neurons imaged every two hours over eight hours. For each that only constitutively active Rac significantly affected this cat-
condition, 1847 cells were analyzed. Scale bar, 50 m. *p < 0.05; egory, causing a 50% reduction of stable branches. No effects on
**p < 0.002; ***p < 0.001.
arbor dynamics were observed when we expressed dominant-
negative Cdc42 or dominant-negative RhoA or added LPA. Taken
together, the analysis of arbor dynamics indicated that Rac and
to a lesser extent Cdc42, but not RhoA are crucial in regulating
of the arbor is very dynamic, characterized by continuous branch arbor dynamics.
additions and retractions (Fig. 3). In animals infected with con- To test whether constitutively active Rac increases arbor dynam-
stitutively active Rac or dominant-negative Rac vaccinia virus, ics in the time frame of minutes, we imaged single labeled neurons
the dendritic arbor was more dynamic than in control neurons. every 3 minutes over periods up to 30 minutes. Six cells were imaged
As a result of these dynamics, the arbor structure was quite dif- from animals infected with either EGFP vaccinia virus (control) or
ferent from one time point to the next. Images of neurons infect- constitutively active Rac vaccinia virus. The rapid branch dynamics
ed with vaccinia virus expressing constitutively active Cdc42, are most easily recognized in a time-lapse movie of the cells (see
dominant-negative Cdc42 and dominant-negative RhoA or neu- http://neurosci.nature.com/web_specials/). The movie also demon-
rons exposed to LPA did not show obvious changes in arbor strates that dendritic arbors of neurons from animals infected with
dynamics during the eight-hour imaging period (see Fig. 3). constitutively active Rac vaccinia virus appear more dynamic.
To quantify whether an increase or decrease in RhoA, Rac or Branches within an arbor can have a variety of lifetimes, ranging
Cdc42 activity had significant effects on arbor dynamics, we from several minutes to several days3,4,23,24. The lifetimes of branch-
counted the numbers of newly added branches and retracted es can be shifted to longer or shorter times during development4,
branches that were imaged during the eight-hour period. All by increased CaMKII activity3 or by blocking NMDA receptor activ-
groups had a comparable number of branch tips and total den- ity6. Quantitation of the lifetimes of each branch in the arbors show
dritic branch length at the first imaging observation. Neurons that 67% of branches in constitutively active Rac neurons have life-
from animals infected with constitutively active Rac vaccinia virus times less than 9 minutes, whereas only 31% of branches in con-
added significantly more dendritic branches than control cells trol neurons have similarly short lifetimes. The shift toward shorter
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Transient branch
dendritic arbors were significantly less complex than in controls.
Fig. 4. Rac and Cdc42 increase transient branches. (a) Schematic drawing
Sholl analysis also confirmed the impression from the drawings in
of the types of branch categories analyzed (see text for details). (b) Plots
Fig. 3 that decreased RhoA activity enhanced arbor complexity, of the fraction of stable, transient, new and lost branches in arbors from
whereas LPA treatment caused simpler dendritic arbors. each of the designated treatments. *p < 0.05; **p < 0.01; ***p < 0.001.
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(m per 20 h)
TDBL growth
adhesion molecules32 and neurotrophins33. Indeed,
GTPase activity has been suggested to differentially reg-
ulate the elaboration of basal and apical dendrites in
dissociated cortical neurons12. Because the Rho fami-
ly GTPases are regulated by extracellular stimuli9, and
also seem to mediate neuronal responses to substrate-
bound guidance cues, including myelin and inte-
grins3436, normal dendritic arbor elaboration may
Fig. 6. NMDA receptor-mediated arbor growth operates through RhoA. Drawings of
reflect the complex composition of environmental cues two representative neurons from controls (a), APV-treated animals (b) and APV-
in intact animals. Consequently, in our experiments, treated animals expressing dominant-negative Rho (c). (d) Change in total dendritic
introduction of constitutively active Rac may have had branch length (TDBL) for neurons from the designated groups. For each condition,
different outcomes in distal and proximal dendrites 1224 neurons were analyzed. Scale bar, 50 m. *p < 0.05.
because of local variations in endogenous GTPase activ-
ity within the arbor.
Increased Cdc42 activity in tectal neurons increased
the proportion of transient dendrites. Active Cdc42 induces mechanisms, with respect to the cytoskeleton. The cytoskeleton
filopodia in growth cones37, which serve a sensory role in axon of transient dendritic branches may be entirely actin-based38
pathfinding. Cdc42 may serve a similar function to promote and their addition likely due to actin polymerization39. The
filopodia in the dendritic arbor, but does not seem to regulate maintenance of a newly added branch may be due to the inva-
either the stabilization or extension of dendritic branches. sion of the new branch by microtubules 39, as described for
growth cones40. Finally branch extension may be due to assem-
Interaction of GTPases with the cytoskeleton bly of microtubules. Although all three GTPases are reported to
Our data suggest that the GTPases modify specific aspects of regulate the actin cytoskeleton, RhoA may also regulate tubu-
dendritic arbor morphology. Rac seems to govern the rates of lin assembly41, supporting the idea that RhoAs principal effect
additions and retractions of new branches without affecting sub- on dendritic arbor elaboration is through the extension or
sequent regulatory events that determine whether the branch retraction of existing dendritic branches, which are enriched in
will extend. The increased actin filaments we observed by phal- microtubules38. In addition, GTPases can regulate cellcell con-
loidin staining in constitutively active Rac and constitutively tacts through presentation and clustering of cadherins and inte-
active Cdc42 cells suggest that these GTPases likely mediate their grins on the cell surface7. Evidence that cadherins can function
effects on the dendritic arbor dynamics by affecting the actin in synapse stabilization42 suggests an intriguing connection
cytoskeleton. Although constitutively active Cdc42 expression between GTPases and synaptogenesis.
increased filamentous actin according to the phalloidin stain- The GTPases are positioned in the midst of several signal
ing, it had only a modest effect on the parameters of dendritic transduction pathways, which govern cell shape and polarity7,9.
dynamics assayed here. It is possible that Cdc42 affects differ- The activity of each GTPase may be altered downstream of cell
ent aspects of cytoskeletal structure in neurons that were not surface receptors, including growth factors, cytokines and neu-
assessed in this study. Neither LPA nor dominant-negative RhoA rotransmitters7,14,37. We provide evidence that the NMDA-type
had a significant effect on branch dynamics, although they clear- glutamate receptor may be upstream of the RhoA GTPase in reg-
ly regulated branch shortening and extension, respectively. This ulating dendritic arbor elaboration. Indeed, it seems that NMDA
suggests that RhoA activity influences the extension or retrac- receptor activity decreases RhoA activity and thereby increases
tion of existing branches but does not govern the emergence of branch elongation. This observation is surprisingly comple-
new branches. Addition and retraction of short branches, main- mentary to a report that activation of the p75 neurotrophin
tenance of branches and their extension are distinct cell biolog- receptor can increase retinal axon elongation by decreasing
ical events that are likely controlled by different regulatory endogenous RhoA activity14. In addition, each of the GTPases
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affects the cytoskeleton through downstream effectors. It would Each optical section was an average of 816 frames. Animals were anes-
be of interest to identify the interacting partners through which thetized with 0.02% MS222 during DiI labeling, screening and imag-
the GTPases regulate dendritic arbor growth. Potential down- ing. Animals recovered from anesthesia between imaging sessions,
stream candidates for RhoA are the serine/threonine kinase, except for the three-minute imaging experiment, where animals were
ROCK, and mDia, which regulate the formation of different types anesthetized throughout the imaging protocol. In experiments using
LPA to activate RhoA or APV to block NMDA receptors, the drug was
of actin fibers43. Rac may act through LIM kinase and cofilin44,45.
added to the rearing solution immediately after the first image was
Filopodia formation induced by Cdc42 seems to depend on collected.
N-WASP, a ubiquitously expressed Cdc42 binding protein46.
Finally, Rac and Cdc42 share common effectors, including the Image analysis. Dendritic arbors were reconstructed by tracing the por-
serine/threonine kinase PAK, which is involved in the morpho- tion of the neuron in each optical section onto an acetate sheet until the
logical changes mediated by Rac and Cdc42 (refs. 9, 47). entire neuron was drawn. This method provides a more detailed repre-
sentation of the morphology than the three-dimensional reconstruction
generated by computer, because fine processes visible in the optical sec-
METHODS tions are lost in the computer-generated reconstruction. Total dendrit-
Construction of recombinant vaccinia virus. Human RacV12, RacN17
ic branch length was measured from scanned drawings of cells with NIH
and Cdc42V12 were myc tagged at the amino (N) terminal. Myc-RacV12,
Image 1.61. The number of branch tips was counted manually. To analyze
myc-RacN17 and myc-Cdc42V12 were cloned into the Sal I/Spe I site
the arbor dynamics, drawings of cells from sequential time points were
upstream of IRES-EGFP in the pBluescript vector. The myc-GTPase-
superimposed to identify added and retracted branches. The magnitude
IRES-EGFP constructs were cut out from pBluescript vector and cloned
of arbor dynamics determined in this and previous studies3,4, in terms
2000 Nature America Inc. http://neurosci.nature.com
articles
9. Van Aelst, L. & DSouza-Schorey, C. Rho GTPases and signaling networks. 29. Kim, J. H., Liao, D., Lau, L.-F. & Huganir, R. L. SynGAP: a synaptic RasGAP that
Genes Dev. 11, 22952322 (1997). associates with the PSD-95/SAP90 protein family. Neuron 20, 683691 (1998).
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12. Threadgill, R., Bobb, K. & Ghosh, A. Regulation of dendritic growth and cadherins and catenins in the chick optic tectum. Mol. Cell Neurosci. 4,
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13. Katz, L. C. & Shatz, C. J. Synaptic activity and the construction of cortical 33. McAllister, A. K., Lo, D. C. & Katz, L. C. Neurotrophins regulate dendritic
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articles
1 Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Childrens Hospital, Q2:09, 171 76 Stockholm, Sweden
2 Department of Neurology, Emory University School of Medicine, Atlanta, Georgia, Georgia 30322, USA
3 Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021-6399, USA
Correspondence should be addressed to A.A. (aniap@child.ks.se)
2000 Nature America Inc. http://neurosci.nature.com
Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of
dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal
microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained
dopamine receptors of both classes. We also provide functional evidence for such colocalization: in
essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited
both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist
of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of lig-
ands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.
Of the vast number of biogenic amines and peptide neurotrans- protein (Fig. 1c). DIC imaging verified the absence of unlabeled
mitters acting in the mammalian brain, dopamine receives by far (glial) cells. Cultured neurons were labeled using antibodies
the most attention1. This is partly explained because abnormal- against either D1-subclass or D2-subclass receptors, each raised in
ities of dopaminergic neurotransmission are implicated in the two species (Fig. 1dl). The cells were examined by confocal
etiology of several major neurological and psychiatric disorders, microscopy. The improved signal-to-noise ratio for the deter-
including Parkinsons disease2,3, schizophrenia4,5, attention deficit mination of dopamine receptors, made possible by the smaller
hyperactivity disorder6,7 and drug abuse8,9. There is considerable depth of focus and the high lateral resolution, permits detection
evidence from immunocytochemical as well as in situ hybridiza- of substances present in low abundance. In all cases, we detected
tion studies that the two major subclasses of dopamine receptors immunofluorescence from virtually all cells. With both anti-
are enriched in different projection neurons in the neostria- bodies against the D1 receptor subclass, the signal was random-
tum1015. Thus, the D1 subclass of dopamine receptors is enriched ly distributed in the cytoplasm and in the vicinity of the plasma
in striatal neurons containing substance P and dynorphin that membrane; with both antibodies against the D2 receptor sub-
project to the substantia nigra, pars reticulata and entopedun- class, the signal was more distinct in the region of the plasma
cular nucleus16,17. Conversely, the D2 subclass of dopamine recep- membrane and in the perinuclear region. Both D1 and D2 sig-
tors is enriched in enkaphalin-containing striatal neurons nals were present in dendrites. Double labeling confirmed the
projecting to the external segment of the globus pallidus16,17. In co-existence of D1- and D2- like receptors in virtually all cells.
contrast to these anatomical data, electrophysiological measure- DARPP-32 (dopamine- and cyclic AMP-regulated phospho-
ments as well as measurements of mRNA in single cells suggest protein, 32 kDa) is present in all medium spiny neurons and only
that many striatal neurons contain both classes of dopamine in medium spiny neurons in the striatum and nucleus accum-
receptors18. The advent of confocal microscopy led us to re-exam- bens19,20. We used this selective distribution to identify cells con-
ine the possible colocalization of D1 and D2 subclasses of recep- taining both D 1 and D 2 subclasses of receptors. DARPP-32
tors. Moreover, on-line recording of intracellular sodium immunofluorescence was observed in virtually all cultured neu-
concentration in these cells permitted functional evaluation of rons. Double labeling of cultured neurons for D1-subclass recep-
the co-expression of the two subclasses of dopamine receptors. tors and DARPP-32 (Fig. 2ac) or D2-subclass receptors and
DARPP-32 (Fig. 2df) indicated colocalization of both subclass-
RESULTS es of receptors with DARPP-32 in virtually all cells; no neurons
After two to three weeks in culture, virtually all cells from embry- were singly labeled for D1- or D2-subclass receptors. These results
onic rat striatum seemed to be mature neurons, as they resem- identify the dopamine receptor-labeled cultured cells as medi-
bled typical medium spiny neurons and stained positively for um spiny neurons.
three neuronal markers: -tubulin III (Fig. 1a), the alpha 3 iso- To evaluate the possibility that the high degree of colocalization
form of Na+/K+-ATPase (Fig. 1b) and neuron-specific nuclear of D1 and D2 subclasses of dopamine receptors resulted from an
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DISCUSSION
Our immunocytochemical studies indicated that virtually all stri-
atal projection neurons contained both D1 and D2 subclasses of
dopamine receptors. Moreover, electrophysiological measure-
ments of Ca2+ and Na+ currents18,22, together with our
a b 0
60
0
50
0
40
[Na+]i (mM)
Ratio/min
0
30
0
2000 Nature America Inc. http://neurosci.nature.com
20 0
10 0
0 0
Fig. 3. D1-subclass receptors colocalize with D2-subclass receptors in neurons of
0.0 5.0 10.0 15.0 20.0
rat neostriatal slices. (ac) Neostriatum, high magnification. (df) Neostriatum,
140 mM Na+ min
low magnification. (gi) Accumbens shell, low magnification. (a, d, g) D1 receptor
labeling. (b, e, h). D2 receptor labeling. (c, f, i) D1 and D2 receptor labeling. 4 mM K+
Antibodies were as described in Fig. 1d and e.
b 0.140
influx (Fig. 4b, upper panel). The effect was reversed in the pres-
0.120 Influx
ence of the D1 subclass antagonist, SCH 23390. Fenoldopam also
significantly inhibited Na+/K+-ATPase-dependent Na+ efflux, and Efflux
0.100
this effect of fenoldopam was also abolished by SCH23390. The
Ratio/min
Fenoldopam
Fenoldopam
Fenoldopam
+SCH23390
+SCH23390
Control
Control
0.0
(Fig. 4b, lower panel). Virtually all cells also showed a slow min
increase in intracellular Na+ in response to fenoldopam (Fig. 5a),
indicating that the effect of this agonist of D1-subclass receptors
on pump inhibition masked its effect on channel inhibition 0.200
(Fig. 4b, upper panel). The finding that sodium levels were altered *
both by stimulation of D1 and by stimulation of D2 receptors in 0.180
0.160
0.140 Influx
Fig. 4. Effects of D1 and D2 agonists and antagonists on initial rates of Efflux
Ratio/min
0.120
Na+ influx and efflux in primary cultures of striatal cells.
0.100
(a) Experimental design to study the initial rates of Na+ influx and efflux
under Vmax conditions21. (b) Fenoldopam (105 M; a D1 agonist), 0.080
SCH23390 (105 M; a D1 antagonist), quinpirole (105 M; a D2 agonist) 0.060
and sulpiride (105 M; a D2 antagonist) were present in the incubation
0.040
mixture as indicated. Images were taken every 20 s. From each cover-
slip, a field of view containing 515 cells was examined. Values (means 0.020
s.e.) for each coverslip were calculated as rate of change in sodium 0.000
Quinpirole
Quinpirole
+ sulpiride
Quinpirole
Quinpirole
+ sulpiride
(340:380 ratio) per min. Each group included 612 experiments. For
Control
Control
both influx and efflux studies, 50120 cells were used. The control and
treatment experiments were carried out on the same day on paired cov-
erslips from the same batch of cells. *Significant differences between
control and treated cells, p < 0.01, Students t-test.
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METHODS
Cell culture. Cultures of striatal neurons were prepared from 1819 day-
old rat embryos as described35, with modifications. The cells were plated
Fig. 5. Effects of D1 and D2 agonists on [Na+]i in primary cultures of rat on poly-D-lysine-coated glass coverslips and cultured in Eagles minimal
striatal cells. At time 0, SBFI-loaded cells were incubated with essential medium (MEM) plus F-12 (1:1). The cultures were grown in
5 105 M fenoldopam (a) or 5 105 M quinpirole (b), and the ratio the presence of 5% FBS. To suppress the growth of glial cells, FBS was
images were recorded every 30 s. Values represent ratio changes from exchanged with N2 (1%) two days after plating, and cytosine arabinoside
the basal level. The results were corrected for SBFI bleaching and leak- (5 M) was added to the culture medium from day 5 to day 7. Cells were
age. Curves were fit by using the method of least squares. Digital images maintained in culture for two to three weeks before experiments.
of SBFI fluorescence under control conditions and eight min after
fenoldopam (a) or quinpirole (b) treatment are shown in insets. Intracellular Na+ measurement with SBFI. Cells were loaded with the
Changes in color from blue to red on the pseudo-color scale used in sodium-sensitive fluorescent probe, SBFI-AM (sodium-binding benzo-
these images indicate increases in [Na+]i. furan isophthalate; 10 M). Coverslips were then transferred to an exper-
imental chamber (FCS2, Bioptechs, Butler, Pennsylvania) with laminar
flow that allowed instantaneous exposure to the indicated drugs. The
cells were excited at wavelength 340/10 nm and 380/10 nm every
2030 s with a 400-ms exposure time. Emission fluorescence was select-
results on Na+ influx and efflux, provide physiological evidence ed and collected with a 510/30 nm band-pass filter. Recordings were made
for a high degree of colocalization of D1- and D2-subclass recep- from cell soma. Collected data were then processed by an image acquisi-
tors. Contradictory evidence suggesting a low degree of overlap of tion program from Inovision Corporation, Raleigh, North Carolina. Cal-
D1 and D2 subclasses of receptors1013 can be reconciled with our ibration of intracellular [Na+] was carried out in living cells.
conclusion by assuming that the striatonigral projection neurons
contain high levels of D1-subclass receptors and low levels of D2- Immunostaining. Cells were fixed for 20 min in ice-cold
subclass receptors, and that the converse is true for the stri- 2% paraformaldehyde, permeabilized with 0.1% saponin, blocked with
atopallidal pathway. Thus, previous failure to substantially 7% normal goat (NGS) or donkey (NDS) serum and then incubated
colocalize the two subclasses using immunocytochemistry23 and overnight at 4C with primary antibody in PBS containing 2.8% serum
and 0.1% saponin. The cells were washed, incubated with fluorescent
in situ hybridization24 is probably attributable to inadequate sen- secondary antibody in PBS with 2.8% serum and 0.1% saponin and
sitivity of these analytical procedures. Conversely, the high degree washed again. For double labeling, the cells were incubated again
of overlap of D1 and D2 subclasses of receptor mRNA seen using overnight at 4C with the second primary antibody, followed by incuba-
PCR25 is attributable to the extremely high sensitivity of this tech- tion with the second fluorescent secondary antibody. The slides were
nique. Furthermore, our data suggest that almost undetectable subsequently mounted in Prolong antifade (Molecular Probes, Eugene,
levels of D1-subclass receptors in striatopallidal neurons and of Oregon). Similar protocols were used for the immunostaining of adult
D2-subclass receptors in striatonigral neurons were adequate to rat striatal slices with minor modifications. D1 subclass dopamine recep-
produce physiological responses. tors were probed with an affinity-purified rabbit polyclonal antibody
Intracellular Na+ in cultured striatal neurons was 16 mM, con- against human D1 dopamine receptor (1:100; ref. 36) or with a rat mon-
oclonal antibody against the human D1 dopamine receptor (1:100;
siderably higher than in non-neuronal cells, in which [Na+]i is
ref. 23). D2-subclass dopamine receptors were probed with polyclonal
generally 37 mM2628. We attribute the high [Na+]i in neurons goat affinity-purified anti-human D2 dopamine-receptor antibody (1:200;
to the presence of the neuron-specific catalytic subunit of the Santa Cruz Biotechnology, Santa Cruz, California) or with an affinity-
sodium pump, 3. Most non-neuronal cells express only the purified rabbit polyclonal antibody against human D2 (1:100; ref. 23).
more widespread 1 isoform29,30. Studies on purified Na+/K+- The two D1 antibodies used are both reported to be specific for the D1
ATPase and on cells transfected with different isoforms of receptor in the D1 subclass of dopamine receptors and not to crossreact
articles
with the D5 receptor23,36. Likewise, the two D2 antibodies are both report- 13. Surmeier, D. J., Reiner, A., Levine, M. S. & Ariano, M. A. Are neostriatal
ed to be specific for the D2 receptor in the D2 subclass of dopamine recep- dopamine receptors co-localized? Trends Neurosci. 16, 299305 (1993).
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mouse antibody against bovine DARPP-32 (1:1000; ref. 37). The -tubu- the rat striatum: sensitive cRNA probes demonstrate prominent segregation
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articles
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5435, USA
Correspondence should be addressed to S.J.S. (sjsmith@leland.stanford.edu)
We used time-lapse fluorescence microscopy to observe the growth of Mauthner cell axons and their
postsynaptic targets, the primary motor neurons, in spinal cords of developing zebrafish embryos.
2000 Nature America Inc. http://neurosci.nature.com
Upon reaching successive motor neurons, the Mauthner growth cone paused briefly before continu-
ing along its path. Varicosities formed at regular intervals and were preferentially associated with
the target regions of the primary motor neurons. In addition, the postsynaptic motor neurons
showed highly dynamic filopodia, which transiently interacted with both the growth cone and the
axon. Both Mauthner cell and motor neurons were highly active, each showing motility sufficient to
initiate synaptogenesis.
During development of the central nervous system, a mass of Analysis of brain development in vivo involving mainly electron
undifferentiated cells develops into a precisely organized system microscopy (EM) reveals many synapses on dendritic filopo-
that acquires information, processes that information and uses it dia2527; however, such static studies do not reveal cellular dynam-
to generate appropriate behavior. Formation of central chemical ics or detailed interactions between cells.
synapses, an important part of this self-organization, is poorly We used time-lapse confocal laser-scanning microscopy and
understood. Here we take advantage of the small size, optical two-photon laser-scanning microscopy of neurons labeled in liv-
transparency and rapid development of the zebrafish (Danio rerio) ing zebrafish embryos to characterize the cell motility events
to study central synaptogenesis in an intact vertebrate embryo. involved in making initial contacts. We found that the Mauthn-
The Mauthner cells (M-cells) are large, paired, identified hind- er growth cone moved rapidly and relatively smoothly down the
brain neurons that are well characterized in the teleost fishes17 and spinal cord and seemed to interact transiently with successive
can be identified on the basis of size, position and morphology4,5 target cells. Primary motor neurons interacted with the nearby
(Fig. 1a and b). The M-cells mediate the escape response, a rapid axon via dynamic filopodia. These findings are consistent with
movement away from auditory, vibrational or tactile stimuli1,2,7. an active involvement of both pre- and postsynaptic elements in
Each M-cell axon crosses the ventral midline to extend down the establishing synaptic contacts. In addition, regularly spaced vari-
contralateral spinal cord to synapse on primary motor neurons cosities formed along the Mauthner axon in close proximity to
(Pmns) in each spinal hemisegment2,7 (Fig. 1a and c). Serially motor neurons; these varicosities may represent nascent presy-
repeating, bilaterally paired sets of three primary motor neurons, naptic boutons.
the RoP (rostral primary), MiP (middle primary) and CaP (cau-
dal primary)811, are activated during the escape reflex and fast RESULTS
swimming12. In goldfish and tench, the M-cellPmn synapse forms Growth of the Mauthner cell axon
between a dorsal collateral of the M-cell axon and a spine-like struc- To observe migration of the M-cell growth cone down the spinal
ture on the ventral dendrite of the Pmn2,13,14 (Fig. 1d). cord, lipophilic dye, DiD (confocal imaging) or DiO (two-pho-
Time-lapse imaging in vivo is used to study growth cone ton imaging)28, was iontophoretically applied to the cell bod-
dynamics and axonal pathfinding of the retinotectal projections ies of the M-cell 1820 hours after fertilization (Fig. 1b).
in Xenopus laevis1519 and zebrafish20, innervation of axial muscle Consistent with estimations from fixed embryos5,9, the Mauth-
by the Pmns11 and embryonic olfactory neuron pathfinding in ner growth cone moved at a mean rate of 99.6 22.7 m per
zebrafish21, as well as the effect of collapsin 1 on sensory neuron hour (n = 12, 28C; Fig. 2). In general, the Mauthner growth
growth cones22; however, these studies do not address the dynam- cone moved smoothly and steadily, but time-lapse sequences
ics of pre- or postsynaptic cells as they contact one another. variably showed slowing or brief pauses every 1015 m; these
During synapse formation in vitro, dendritic filopodia of cen- were more pronounced in some cases (Fig. 2b, top and mid-
tral neurons show rapid and extensive dynamics23,24, suggesting an dle) than in others (Fig. 2b, bottom). In all sequences exam-
active role in synaptogenesis. The number of filopodia gradual- ined, varicosities formed with variable timing along the nascent
ly decreases as the number of dendritic spines increases, sug- axon (Fig. 2c), often just behind the growth cone but not always
gesting that filopodia may represent precursors to the more stable in sequence. Thus, as it moved, the growth cone seemed to leave
spines23,24. However, even in a tissue slice, the similarity of events a trail of axonal thickenings spaced at 13.5 4.3 m (67 vari-
in culture to those in an intact, developing organism is unknown. cosities from 6 axons).
articles
Because the varicosities we observed may have been nascent od investigated (data not shown). However, we cannot rule out
presynaptic termini, we investigated their structure using EM. short-term changes corresponding to the growth cone pauses we
Micrographs of cross-sectioned embryonic spinal cord revealed observed.
long, narrow (0.6 m) segments of Mauthner axon largely
devoid of synaptic vesicles (Fig. 2d). In addition, short areas of Double-label time-lapse analysis
the axon were of larger diameter (1.2 m) and contained small To examine initial contact and possible responses between cells
(40 nm), clear synaptic vesicles (Fig. 2e). Accumulation of during synaptogenesis, we labeled both Mauthner axons and
synaptic vesicles in Mauthner axon swellings suggests that these motor neurons and collected time-lapse sequences. Both the
varicosities were sites of early synaptic contact. Mauthner growth cone and the motor neurons behaved essen-
tially the same as in single-label experiments; motor neurons
Labeling of primary motor neurons showed dynamic filopodia, and growth cones moved rapidly and
We injected the ventrolateral myotomes of segments 1215 smoothly.
with DiD or DiO to label motor neurons. An injection was gen- As the Mauthner growth cone first approached and became
erally confined to a single hemisegment, labeling only the neu- apposed to a primary motor neuron, its path crossed close to
rons innervating it. Because of the injection placement, the the motor neurons ventral dendrites. In some cases, the growth
RoP and CaP motor neurons were preferentially labeled, cones seemed to transiently interact with the target regions of
although occasionally the MiP motor neuron could be identi- the motor neurons (Fig. 4a). As in single-label experiments,
fied (Fig. 1c). Before each time-lapse sequence, we used migra- analysis of motility of the M-cell growth cone revealed brief and
tion of the lateral line primordium to assess embryonic stage periodic pauses. Such behavior could either represent the spe-
to prim-stages 518 (ref. 29), roughly corresponding to 2433 cific recognition of targets, or it could simply represent the sto-
hours post-fertilization (hpf; at 28.5C). chastic nature of growth cone progress. To investigate this, we
Labeled motor neurons consistently showed variable numbers analyzed growth cone dynamics relative to labeled motor neu-
of characteristic filopodia extending from the cell bodies and ven- rons (Fig. 4bd). Averaging the growth cone motilities from nine
tral dendrites (Figs. 1c and 3). Time-lapse analysis revealed suitable time-lapse data sets smoothed away much of the tem-
extremely dynamic and variable protrusive/retractive behavior of poral variability, leaving only a marked discontinuity when the
filopodia, occurring on a time scale of seconds to minutes (Fig. 3). growth cone approached a motor neuron (Fig. 4d). This sup-
Filopodia were analyzed in 19 primary motor neurons for sev- ports the conclusion that the Mauthner growth cone interacts
eral basic properties, length, lifetime, density and rates of exten- transiently with each successive motor neuron, and that these
sion and of retraction. The lifetimes of individual filopodia interactions have a subtle but detectable effect on growth cone
seemed exponentially distributed with a time constant, , of dynamics. However, we observed no overt change in growth cone
approximately three minutes (Fig. 3b). The filopodium length structure, such as collapse or loss of filopodia.
(mean, 2.7 0.7 m; n = 571; Fig. 3c) typically varied from 1.3 to The axonal varicosities observed in time-lapse analysis of
4.0 m, but could reach lengths of 13 m. Density of filopodia labeled M-cell axons were also observed in double-label time-
on the ventral dendrites was highly variable, both over time in lapse experiments. Importantly, they formed preferentially near
the same cell and from cell to cell, and varied from 0.1 to 0.7 the ventral dendrites of motor neurons, over time associating
filopodia per m of dendrite. Rates of extension essentially with them very closely (Figs. 5 and 6). In 5 of 13 observations,
matched those of retraction (1.2 0.3 m per min). These para- an axonal varicosity formed within 5 minutes of the passing of
meters did not change significantly over the developmental peri- the growth cone, suggesting that the varicosity had arisen from
articles
Distance (m)
This sequence shows the emergence of an axonal
varicosity that occurred well after the growth cone
had moved on. Each displayed image is a projection
from a stack of 16 optical sections. (b) Quantitative
analysis of the growth cones reveals transient
pauses not readily apparent from direct observa-
tion of time-lapse sequences. The occurrence and
periodicity of these pauses is variable, as shown in
this analysis of three different growth cones.
Changes in velocity of two growth cones are indi-
Distance (m)
cated by an apparent staircase pattern (b, top and
middle), whereas another growth cone seemed to
move more smoothly (b, bottom). (c) A dorsal
view of a DiD labeled M-cell axon imaged with a
confocal microscope showing periodicity of the
varicosities. (d) An electron micrograph of a
2000 Nature America Inc. http://neurosci.nature.com
Distance (m)
varicosity in the same Mauthner axon. The axon
has a larger diameter and contains many synaptic
vesicles (arrow), as well as a dense core vesicle
(arrowhead) and a mitochondrion (M). Dorsal is to
the right, and scale bar is 0.25 m in (d, e).
c
articles
Percent filopodia
Fit to a single exponential reveals a filopodia life-
time of 3 min. (c) Distribution of filopodium
lengths for the same data set as in (b). The mean
length is 2.7 0.7 m.
Percent filopodia
very rapidly and without significant disrup-
tion of growth cone structure or dynamics.
In time-lapse images, we consistently
observed formation of varicosities on Mau-
thner axons in the wake of growth cone
migration (Figs. 2, 5 and 6). Electron micro-
graphs showing synaptic vesicles within vari-
cosities along the Mauthner axon (Fig. 2d)
support the idea that varicosities represent Length (m)
nascent synapses. Similar swellings are
observed on developing corticorubral neu-
rons; EM reveals these varicosities as sites of
synaptic contact25. The formation of varicosities along the Mau- ily from motility of the growth cone or motility of the dendrite.
thner axon may, therefore, represent the early organization of Synaptogenesis, even in what seems a hardwired system like the
presynaptic boutons. Mauthnermotor neuron synapse, is probably a stochastic
Perhaps our most provocative and striking observation was process, depending on neither the growth cone nor dendritic
the presence of highly dynamic filopodia extending from the ven- filopodia exclusively. Moreover, possible redundancy in capaci-
tral dendrites of primary motor neurons, which interact with the ties of both the pre- and postsynaptic cell to initiate synaptoge-
M-cell axon (Fig. 3). Present throughout the period of synapto- nesis may be important for ensuring the formation of such a
genesis, these filopodia made contact with Mauthner axons and stereotyped synapse. Thus, further investigations may reveal
were enriched in regions of the motor neurons that ultimately examples of synaptogenesis resulting from action of both growth
receive large numbers of synaptic inputs. The varicosity shown in cones and dendritic filopodia.
Fig. 6 formed after filopodiaaxon interactions and was frequently
contacted by dendritic filopodia during the time-lapse sequence. Timing of synaptogenesis
However, it is unclear whether varicosity formation was actually Currently, our only marker for the presence of a nascent synapse
induced by contact with filopodia. Our observations are consis- is the formation of an axonal varicosity. By this criterion, there
tent with proposals that dendritic filopodia may be involved in can be a considerable and variable lag between the interaction of
initiating synaptogenesis and might represent developmental pre- the growth cone with the motor neuron and synapse formation.
cursors of spines23,24. Electron microscopy demonstrates dendrit- Zebrafish embryos respond to touch by 21 hpf; hindbrain
ic filopodia as frequent sites of early synaptic contact25,26, although lesions destroy this touch sensitivity32. Based on the timing of
spine synapses may develop primarily from shaft synapses26. axon extension by the reticulospinal neurons, the Mauthner cell
If synaptogenesis does not result directly from growth cone was suggested to participate in this early behavior32. The Mau-
motility, then dendritic filopodia may actively help to bridge the thner growth cone reaches the rostral spinal cord by 20 hpf,
gap between the two cells and to initiate contact. However, it may indicating a lag of 1 h between growth cone passage and initia-
be unrealistically simplistic to assume that synaptogenesis results tion of synaptic function. The time frame for synapse formation
exclusively from the actions of either the growth cone or of the established by the behavioral studies is consistent with our obser-
dendritic filopodia. Contact between the cells may be the criti- vations of formation of axonal varicosities with a variable lag fol-
cal event, and it may be irrelevant if this contact results primar- lowing passage of Mauthner growth cones.
articles
Distance (m)
was highly active and transiently interacted with the CaP
cell, one of its synaptic partners. The growth cone
moved past the CaP cell without collapsing, stalling or
significantly altering its morphology. (bd) Analysis of
growth cone migration with respect to primary motor
neurons. To assess whether growth cone deceleration
was related to growth conetarget interactions, we mea-
sured rate of growth cone motility relative to the posi-
tions of labeled motor neurons. In single-label
experiments, growth cone motility varied in overall
c
speed as well as the degree of saltation. (b) This growth
cone distinctly paused as it passed the labeled motor
Distance (m)
neuron. (c) The growth cone represented in this graph
(shown in a) moved without clear periodic changes in
rate and did not pause near the motor neuron. (d) To fil-
2000 Nature America Inc. http://neurosci.nature.com
Distance (m)
METHODS
Labeling of neurons. Zebrafish embryos were collected from
a laboratory colony33 and were allowed to develop at 28.5C
in a beaker of fresh tank water. At times ranging from 2435
hpf (motor neuron labeling) or 1821 hpf (Mauthner label-
ing), embryos were de-chorionated and mounted on glass
depression slides in 12% agarose with 0.05% tricaine (3-
amino benzoic acid ethyl ester; Sigma). DiD (1,1-dioctade-
cyl-3,3,3,3-tetramethylindocarbocyanine; Molecular Probes, Time (min)
Eugene, Oregon; 0.51% in dimethylformamide) or DiO
Imaging. Labeled embryos were remounted on cover slips in 12%
(3,3-dioctadecyloxacarbocyanine perchorate; Molecular Probes; 0.51%
agarose with 0.05% tricaine and imaged by confocal or two-photon
in dimethylformamide) was pressure injected into the ventral myotome
microscopy. The embryos were imaged either on a confocal laser-scan-
of anesthetized embryos or iontophoretically applied (0.51% in 50%
ning microscope using a Zeiss 20/NA 0.75 dry objective, or on a two-
dimethylformamide/50% ethanol) to the cell bodies of Mauthner cells.
photon laser-scanning microscope using a Zeiss 63/NA 0.9
Embryos were then allowed to develop for several hours, allowing time for
water-immersion objective. (Both microscopes were designed and built
growth cones to invade the spinal cord. Well-labeled growth cones were
by S.J.S., software by N.E. Ziv.) The 633-nm line of a helium-neon laser
further selected either for single-label time-lapse analysis, or for motor
(0.51 mW at the specimen) was used to excite the DiD, and fluores-
neuron labeling and double-label time-lapse analysis.
cence passed through a 650-nm long-pass filter before detection. Z-stacks
Electron microscopy. Embryos (2430 hpf) were fixed for 1 h at room
temperature in 3% glutaraldehyde, 2% formaldehyde, 1% acrolein and Fig. 5. Projections of the
1% DMSO in PBS and then further processed for electron microscopy Mauthner axon and sets of
in a Pelco 3450 Laboratory Microwave Processor (Ted Pella, Redding, primary motor neurons. In
California). Embryos were washed with 0.1 M cacodylate buffer for 5 both panels, an image stack
min at room temperature and post-fixed with 1% osmium tetroxide in was collected a few seg-
0.1 M cacodylate buffer containing 0.8% potassium ferricyanide on the ments rostral to the caudally
bench for 5 min on ice, then given 2 short (10 s) pulses in the microwave. directed growth cone. In
The embryos were washed in distilled water for 5 min, stained en bloc each case, distinct varicosi-
with 2% aqueous uranyl acetate for 20 min and given another short pulse ties were associated with the
(10 s) in the microwave. They were then dehydrated in an ascending ventral dendrites of primary
ethanol series (50%, 70%, 95%, 100% 3) in the microwave oven for 10 motor neurons. Secondary
s each. The embryos were infiltrated in the microwave in 1:1, 2:1 (resin: motor neurons (asterisks;
ethanol) for 5 min each, and in 100% resin for 5 min using Embed 812 characterized by their small
resin (Electron Microscopy Sciences, Fort Washington, Pennsylvania). size, round cell bodies and
The embryos were flat embedded and polymerized in a 60C oven ventral position) are present
overnight. Thin sections were then cut using a Reichert-Jung Ultracut E in both panels. In the bottom
ultramicrotome (Leica, Deerfield, Illinois). After staining with 6% uranyl panel, one varicosity does
acetate and 1% lead citrate, the sections were imaged in a Philips CM12 not seem to be associated
transmission electron microscope (FEI/Philips, Hillsboro, Oregon) oper- with a primary motor neuron. However, it is in the correct position
ating at an accelerating voltage of 80 kV. to be associated with an unlabeled MiP motor neuron.
articles
2000 Nature America Inc. http://neurosci.nature.com
articles
Filopodia lengths. Lengths were defined as the distance from the base of 6. Metcalfe, W. K., Mendelson, B. & Kimmel, C. B. Segmental homologies
the filopodium to its tip. Only filopodia 3 pixels (0.7 m) were scored, among reticulospinal neurons in the hindbrain of zebrafish larva. J. Comp.
Neurol. 251, 147159 (1986).
as shorter filopodia could not be identified reliably. Lengths were mea- 7. Fetcho, J. R. & Faber, D. S. Identification of motoneurons and interneurons
sured from maximum-intensity projections. Thus the values we present in the spinal network for escapes initiated by the Mauthner cell in goldfish.
slightly underestimate (by 13%) the actual three-dimensional lengths34. J. Neurosci. 8, 41924213 (1988).
Rates. The rate of extension/retraction for a given filopodium was defined 8. Myers, P. Z. Spinal motoneurons of the larval zebrafish. J. Comp. Neurol. 236,
as the change in unit length between successive frames. Filopodial exten- 555561 (1985).
9. Myers, P. Z., Westerfield, M. & Eisen, J. S. Development and axonal
sion/retraction rate for a given motor neuron was calculated as the mean outgrowth of identified motoneurons in the zebrafish. J. Neurosci. 6,
rate of extension or retraction for all filopodia observed on that neuron. 22782289 (1986).
10. Westerfield, M., McMurray, J. V. & Eisen, J. S. Identified motoneurons and
Lifetimes. The lifetime of an individual filopodium was defined as length their innervation of axial muscles in the zebrafish. J. Neurosci. 6, 22672277
(1986).
of time for which its length was measureable. A filopodium extending 11. Eisen, J. S., Myers, P. Z. & Westerfield, M. Pathway selection by growth cones
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togram (bin size, 2 min). Population lifetime was calculated from the 12. Liu, D. W. & Westerfield, M. Function of identified motoneurones and co-
time constant, , of a single-exponential fit to the histogram. ordination of primary and secondary motor systems during zebra fish
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time-lapse sequence. 14. Yasargil, G. M. & Sandri, C. Topography and ultrastructure of commisural
2000 Nature America Inc. http://neurosci.nature.com
articles
Department of Molecular Biology, Princeton University, Washington Road, Princeton, New Jersey 08540-1014, USA
The first two authors contributed equally to this work.
Correspondence should be addressed to J.Z.T. (jtsien@molbio.princeton.edu)
Comparative anatomical studies of hippocampal cytoarchitecture ing in adult rats16. Based on findings of NMDA receptor function
reveal a selective expansion of the CA1 region in the hippocampus in the developing brain17,18, it is postulated that NMDA receptors
in primates with respect to rodents and in humans with respect might be required for behavioral experience-induced structural
to monkeys, suggesting that this subregion is important in human plasticity in adult brain. However, new dendritic spines can form
hippocampal function1. Damage to the hippocampus or the CA1 on mature hippocampal neurons in vitro in the absence of synap-
subregion in humans leads to deficits in memories of people, tic activity19, and LTP has no effect on synapse number in the CA1
objects, places and events (declarative memory)24. Similar spa- region20. These findings imply that the role of the NMDA recep-
tial and nonspatial memory deficits are also observed in a variety tor in structural changes in adult brain might differ from that in
of laboratory animals with hippocampal lesions4,5. Recordings of the developing brain. By using unbiased stereological electron
neuronal activity in the CA1 subregion of rodents during behav- microscopy, we examined the structural changes in our CA1-KO
ioral tests show the importance of this subregion in the encoding mice before and after enriched experience.
of nonspatial information6. However, the molecular mechanisms
underlying these processes remain unknown. RESULTS
N-methyl-D-aspartate (NMDA) receptors are widely distrib- NMDAR1 knockout in the CA1 region
uted in the brain7 and are essential for the induction of major We confirmed the complete deletion of the NMDAR1 gene in
forms of long-term potentiation (LTP) and long-term depres- CA1-KO mice using in-situ hybridization histochemistry. No
sion (LTD)8,9. Enhanced NMDA receptor function in the fore- NMDAR1 mRNA was detected in the CA1 subregion (Fig. 1),
brain improves learning and memory10, indicating its crucial role indicating that the genetic deletion of NMDAR1 in young adult
in these processes. Using the Cre/loxP-mediated recombination mice was heritable after three years of breeding and was complete.
system, we developed a region-specific gene-knockout technique
to generate conditional-knockout mice in which NMDAR1, a key Nonspatial learning and memory deficits
subunit of NMDA receptor, was selectively deleted in the CA1 CA1-KO and control mice used in all behavioral tests were 24
subregion11. These CA1-specific NMDAR1 knockout mice lacked month-old littermates. We used three hippocampus-dependent
NMDA-mediated currents and plasticity in the CA1 region and behavioral tasks to assess associative, nonspatial memory functions.
were profoundly impaired in spatial memory tasks12,13. The hippocampus is important in the formation of recognition
In this study, our first goal was to examine the role of CA1 memory in both human patients and animals 35. However, little is
NMDA receptor activity in the formation of nonspatial memory known about its anatomical basis and its molecular and cellular
and the effects of enriched experience on these memory func- mechanisms21. We evaluated recognition memory with a novel-
tions. Our second goal was to examine whether enriched experi- object recognition-memory task10. During the training session, the
ence during adulthood could lead to structural changes in the total amount of time spent exploring two objects was 28.62 3.94
CA1 region, and whether CA1 NMDA receptor-mediated respons- seconds in control mice (n = 17) and 34.71 3.56 seconds in CA1-
es are required for such structural modifications. Synaptic struc- KO mice (n = 12), and no significant exploratory preference was
tural changes are assumed to be the anatomical substrate of found between control and CA1-KO mice (data not shown). These
long-term storage of learned experience14,15. For example, numbers observations indicate that these two types of mice have the same
of dendritic spines in the hippocampus increase after spatial learn- levels of motivation, curiosity, and interest in exploring novel objects.
articles
For retention tests, at 30 minutes, 2 hours and 24 hours after Control and CA1-KO mice significantly differed in contextual
the training (Fig. 2), one object was replaced by a novel object. freezing, but not in freezing responses immediately after the
As expected, control mice showed a significant preference for shocks, in retention tests at 2 hours and 24 hours (p < 0.05,
exploring the novel object during each retention test. In contrast, p < 0.01, respectively; Fig. 4a), indicating that CA1-KO mice were
CA1-KO mice showed only marginal preference for the novel impaired in contextual fear memory. However, in a cued-condi-
object (Fig. 2). A self-ANOVA between training and retention tioning test, which is hippocampus independent 25, freezing
tests revealed a significant difference in control mice (F3,64 = 7.744, responses of CA1-KO mice to a tone were similar to those of con-
p < 0.001) but not in CA1-KO mice. Repeated measures of trol mice in retention tests at 2 hours (data not shown) and 24
ANOVA on retention tests showed a highly significant difference hours (Fig. 4b). In addition, no abnormal nociceptive responses
between control and CA1-KO mice (F2,27 = 19.435, p < 0.001). A were found in CA1-KO mice: current required to elicit flinch-
post-hoc analysis using Dunnetts test demonstrated significance ing/running, jumping or vocalization in the CA1-KO mice was
of this difference in retention at 30 minutes, 2 hours and 24 hours the same as in control mice (data not shown). These data clear-
(p < 0.05, p < 0.01 and p < 0.01, respectively; Fig. 2). These results ly showed that hippocampus-dependent, but not hippocampus-
indicate profound deficits in novel object recognition memory independent, fear memory was impaired in CA1-KO mice.
in CA1-KO.
We then examined olfactory discrimination memory using a
social transmission of food preference 22. Rodents develop a
Exploratory preference (%)
preference for foods they have recently smelled on the breath of Control
other individuals 23. Lesions restricted to the hippocampus CA1 - KO
impair this kind of memory 24 hours after social interaction
(training)24. However, the molecular mechanisms underlying
this kind of memory remain unknown22,24. The task used here
consisted of three stages. First, animals became accustomed to
eating from a food cup placed on the cage floor. Second, in
training sessions, observer mice were allowed to interact with
demonstrator mice fed with either cinnamon-scented (1% per
weight) or cocoa-scented (2% per weight) food. Third, observ-
er mice were then tested by presentation of both scented foods, Training session
and consumption of each food was recorded. Control mice
showed a significantly higher preference for food smelled dur-
ing the training session over unsmelled food than did CA1-KO b
mice (F1,26 = 5.291; Fig. 3), indicating that olfactory-discrimi-
nation memory was impaired in CA1-KO mice. To ensure that
Exploratory preference (%)
Control
this observation was not due to a difference in feeding behav- CA1 - KO
iors, we measured the total amount of food taken during the ** **
retention session; this quantity did not significantly differ *
articles
articles
Enrichment-induced anatomical changes CA1 pyramidal cells of enriched animals. We observed a signifi-
What are the molecular and structural mechanisms underlying cantly higher density of dendritic spines in enriched contol mice
the enrichment-induced effects? Enriched experience promotes compared with naive animals (10.8 1.1 versus 8.1 0.8;
various biochemical and morphological changes in several brain p < 0.05; Fig. 6c). This is consistent with a previous report of
regions26,2831. Synaptic structural changes are believed to be the increased spine density on CA1 neurons in rats after enrich-
anatomical substrate of long-term storage of learned experience, ment16. Surprisingly, enrichment similarly increased spine density
and it is assumed that NMDA receptors are required for experi- in CA1-KO mice (10.0 0.3 versus 8.6 0.6; p < 0.05). Statistical
ence-dependent anatomical changes in the adult brain. As the analysis revealed no significant difference between enriched con-
first step toward the genetic dissection of the molecular mecha- tol and enriched CA1-KO animals (Fig. 6c).
nisms of experience-induced structural plasticity in the adult hip-
pocampal CA1 region, we used light and electron microscopy to
examine the effects of enrichment on anatomical changes in the a b
CA1 region and the role of the NMDA receptor in this effect.
Nissl-stained tissue of CA1-KO mice shows no gross anatomical
abnormality12. We used the Golgi-impregnation technique32 to ini-
tially assess dendritic structures. Golgi-impregnated CA1 pyrami-
dal neurons of control and CA1-KO naive animals presented similar
dendritic morphology (Fig. 6a and b). Quantitative analysis of den-
dritic-spine density on CA1 pyramidal cells revealed no significant
difference between naive control (8.1 0.8 spines per 10 m of api-
cal dendrite, mean s.e.) and naive CA1-KO mice (8.6 0.6 spines;
Fig. 6c). This suggests that conditional knockout of the NMDAR1
gene in the CA1 region, which occurs in the postnatal third and
fourth weeks, did not result in abnormal spine density. c
To characterize the effects of enrichment on dendritic spine
density, we then counted the number of spines on dendrites of
Dendritic spines per 10 m
articles
Perforated
naive or reared in an enriched environ- Non-perforated
** *
Shaft
ment. Synaptic densities were calculated
per surface area and then expressed as
the number of synapses per 100 m3 by
using the stereological coefficient
obtained with the disector method. The
data are expressed as mean s.e.;
Naive control Enriched control Naive CA1-KO Enriched CA1-KO
*p < 0.05, Mann-Whitney U-test.
articles
and were counted separately from those on dendritic spines (Fig. 8b). unit 2B of the NMDA receptor also leads to overall enhancement
We found a significantly higher density of non-perforated synapses in of learning and memory10. Thus, changes in the composition (for
control mice after enrichment (77.1 5.2, mean s.e.) than in naive instance, ratio of NR2A to NR2B subunits) of the NMDA recep-
control animals (62.6 2.3, p < 0.05; Fig. 8c). Remarkably, enrich- tor complex may be a possible mechanism for the enrichment-
ment also significantly increased the density of non-perforated induced effects. Taken together, these findings indicate that
synapses in CA1-KO mice (78.3 5.4 versus 60.7 5.1; p < 0.05; Fig. learning and memory might be enhanced in mammals by genet-
8d), whereas the densities of perforated and shaft synapses remained ic factors as well as behavioral experience.
unchanged after enrichment in both control and CA1-KO mice. For
better observation of the synaptic structural changes, a sophisticat-
ed approach using three-dimensional reconstruction of synapses METHODS
Animals. The CA1-KO and control mice were produced as described11.
viewed through serial electron micrographs would be valuable33.
Throughout the behavioral and histological experiments, observers were
Possible tissue shrinkage during the embedding process can blind to the genotype of an individual animal.
be ruled out as explanation for these findings. First, the effect is
synapse specific. If there were a generalized shrinkage in the In situ hybridization. Described procedures10 were used. Briefly, an anti-
enriched animals with no other change, then all types of synaps- sense 42-mer oligonucleotide probe (5-ACC ACT CTT TCT ATC
es should be affected uniformly. Second, the mitochondrial cross- CTG CAG GTT CTT CCT CCA CAC GTT-3), which recognizes NR1
sectional diameters were uniform across the naive and enriched exon 20, was end labeled with [35S]-dATP. After hybridizing with the
conditions, showing that no generalized shrinkage of individual oligonucleotide probe (5 105 cpm per slide) at 47C for 24 h, brain sec-
2000 Nature America Inc. http://neurosci.nature.com
process occurred (data not shown). tions (20 m) were washed in 2 SSC at room temperature (RT) fol-
lowed by two washes in 0.2 SSC at 60C and one wash in 0.1 SSC at
RT. Interestingly, preliminary observations indicated that the deletion
DISCUSSION of the NR1 gene in six-month-old knockouts seemed to spread to other
Here we examined three kinds of nonspatial memory in mice forebrain regions such as the cortex, probably reflecting the recombina-
lacking NMDAR1 in the CA1 region. Our analysis provides evi- tion threshold effect as the CaMKII promoter-driven Cre accumulated.
dence supporting the important role of NMDA receptor activity
in the CA1 region for the formation of hippocampus-dependent Enriched environment training. Adult littermates (1.52 months old) were
nonspatial memory. Taking into account the deficit in spatial randomly distributed into two experimental groups. One group was kept
learning in the CA1-KO mice12, we conclude that NMDA recep- in standard cages (naive group) and the other group trained in an enriched
tor activity in CA1 is essential for the formation of both spatial environment for three hours daily for two months (enriched group). The
and nonspatial memory. enrichment training was carried out in specially designed boxes (1.5 m
0.8 m 0.8 m), in which various toys, running wheels and small houses were
More interestingly, we found that the memory deficits associ-
changed every other day to encourage exploration. Food and water were
ated with the disruption of the hippocampal NMDA receptor could also available in the boxes. Because electrophysiological, behavioral and
be rescued by daily training in an enriched environment. We show anatomical experiments showed similar results for wild-type and Cre trans-
that behavioral experience can enhance learning and memory in genic mice, these two genotypes of mice were pooled together as controls.
the mutant animals. Thus, the memory deficits observed in gen-
tic mutant animals are not necessarily irreversible, and enriched Novel-object recognition task and fear-conditioning tasks. The experi-
experience could promote recovery of some of the deficits. mental protocol was the same as described previously10, except that the
To investigate further the possible cellular mechanisms under- duration of the training period in the object-recognition task was 15 min.
lying these enrichment effects, we systematically evaluated the
Social transmission of food preference task. In this experiment, observ-
anatomical changes using light and electron microscopy. We
er mice were tested for memory and demonstrator wild-type mice were
showed that enriched experience promotes a significant increase used to interact with observers according to a described procedure22.
in synapse density in the CA1 hippocampal field. Because there is After interaction, the observers were deprived of food for 24 h and then
no neurogenesis in the CA1 region26, the increased synapse den- tested for memory. During testing, observers were offered both cinna-
sity is likely to reflect a higher number of synapses per neuron mon- and cocoa-scented food for 2 h, and consumption of each food
rather than an increased number of pyramidal cells in CA1. At the was subsequently measured. The preferred food had the same scent as
ultrastructural level, we showed that mice lacking the NMDA food eaten by the demonstrator with whom the observer interacted. Pref-
receptor in the CA1 region also increase synaptic density follow- erence was calculated as percentage ratio of the amount of preferred food
ing exposure to an enriched environment. This suggests that consumed over 50% of the total amount of food consumed.
NMDA-receptor activity is not required for structural plasticity in
Histology. For electron microscopy, contol groups were composed of males,
the CA1 region of adult animals induced by behavioral experience,
3.55.5 months old. The CA1-KO naive mice were 3 males (4.5, 4 and 4
and thus the molecular mechanism underlying adult activity- months) and 1 female (3.5 months), and the CA1-KO enriched mice were
dependent structural plasticity is very different from mechanisms 3 males (5, 4.5 and 3.5 months) and 1 female (4 months). Because CA1
active in the developing brain17,18. As synaptic plasticity can also dendritic spine density in rats fluctuates over the estrous cycle39, females
be induced in the CA1 region by an NMDA receptor-independent were killed during proestrus, the stage at which the spine numbers of
process37,38 such as LTP dependent on voltage-gated calcium chan- female rats approach those of male rats (although it is not known whether
nels, other mechanisms might serve as putative candidates for expe- the same phenomenon exists in mice). Animals were anesthetized and per-
rience-induced structural plasticity in adult brain. fused with 2% paraformaldehyde, 2% glutaraldehyde and 1.5% saturated
It remains to be determined whether the enrichment-induced picric acid in 0.1 M phosphate buffer (pH 7.4) and postfixed overnight in
the same solution. Following a modified version of the single-section Golgi
increase in CA1 synaptic density has any functional role in the
impregnation technique32, 100 m-thick coronal sections were cut with a
concomitant behavioral effects. It is likely that other brain regions vibratome into a bath of 3% potassium dichromate in distilled water and
may undergo similar biochemical and structural changes after incubated overnight in this solution. The slide assemblies were incubated
enrichment, and also might participate in the enrichment-induced in the dark in a solution of 1.5% silver nitrate for 2 days.
behavioral improvement. Enhanced synaptic coincidence detec- For electron microscopy, we used a vibratome to cut 250 m-thick coro-
tion in the forebrain of transgenic mice via upregulation of sub- nal sections into a bath of 0.1 M sodium cacodylate buffer. Hippocampal
articles
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articles
1 Department of Physiology and Biophysics, Mt. Sinai Medical School, Box 1218, 1 Gustave L. Levy Place, New York, New York 10029, USA
2 Neuroscience Program, Department of Biological Sciences, Irvine Hall, Ohio University, Athens, Ohio 45701, USA
The first two authors contributed equally to this work.
Correspondence should be addressed to S.L.H. (Hooper@ohio.edu)
2000 Nature America Inc. http://neurosci.nature.com
Neurons receive input across an extremely wide frequency range output is modulated by two other, much slower rhythmic neur-
(from high-frequency sound at tens of kHz to circadian rhythms al networks. The muscles responded to these modulations and
at 10 Hz). Muscles receive input across a narrower but still followed the slow network activity, even though no neuron of
wide range (10 Hz to several hundred Hz, the maximum firing the slow networks innervates the muscles. These results extend
rate of neurons). Neurons and muscles cannot accurately follow the known range of functional low-frequency response some
the higher portions of these ranges. For instance, the refractory two orders of magnitude, and provide an example of a signal
period limits neuron firing to a few hundred Hz, and relaxation carried partially or exclusively in a slow modulation imposed
kinetics prevent muscles from accurately following motor neu- on the targets driver from elsewhere in the nervous system.
ron bursts faster than 1 Hz1. Neuronal and muscle intracellu- Furthermore, two of these muscles are innervated by the same
lar processes (for example, Ca 2+ and second messenger motor neurons and, hence, receive identical neuronal input.
concentration, protein kinase activation, protein phosphoryla- However, because of differences in their contractile properties,
tion and gene expression) are also slow, with time courses rang- one muscle followed both slow networks, whereas the other fol-
ing from tens of milliseconds (10100 Hz) to minutes (0.01 Hz) lowed only one. These two muscles responded to different fre-
or longer215, and therefore would also be expected to be unable quency domains of one signal, and thus provide a clear example
to accurately follow high-frequency input. of both encoding of information by the nervous system and its
Restriction of responsiveness to a particular frequency range decoding by neuronal followers, on the basis of frequency. Pre-
might be thought to limit function. Indeed, the inhibitory and liminary accounts of this work appeared in abstract form
neuromodulatory muscle innervation in several invertebrates (L.G.M. et al., Soc. Neurosci. Abstr. 25, 1642, 1999; J.B.T. &
may be present to increase the frequency range over which the S.L.H., Soc. Neurosci. Abstr. 25, 1641, 1999).
muscles can accurately follow their inputs1619. However, selec-
tivity for low frequencies may also be advantageous because it RESULTS
allows receivers to demodulate, and hence respond to, low-fre- We studied the pyloric neuromuscular system of the lobster
quency signals carried in a modulated high-frequency (carrier (Panulirus interruptus), a part of the stomatogastric system that
wave) input. The wide range of kinetics present in the respons- is driven by the pyloric neural network. Every 0.5 to 2 seconds,
es of neurons and muscles to input suggests that these systems the motor neurons of this network produce 510 action poten-
might have the cellular mechanisms necessary to perform such tial bursts of 100500 millisecond duration25. Pyloric muscles
demodulation, and different patterns of temporal input can dif- are non-spiking muscles that contract as a graded function of
ferentially alter various intracellular processes12,2024. However, their neuronal input26. Based on the pyloric networks cycle
except for the exclusion of inputs with frequencies of 1 Hz or period and bursting nature, it was thought that each motor
greater in vertebrate skeletal muscle1, no examples in which the neuron burst induces a single muscle contraction that fully
signal of interest is carried in the low-frequency component of relaxes between bursts, and that the muscles thus temporally
a broad-band input have been described in biological systems mirror the bursting activity of their input 25,2730. Work on
on the cellular level. pyloric muscle contractions evoked by nerve stimulation in the
We describe here three slow muscles driven by a bursting crab, Cancer borealis, and the shrimp, Palaemon serratus, sup-
neural network with a cycle frequency of 1 Hz. This networks port this belief3133.
articles
amplitude (m)
Contraction
After intercontraction summation had stabilized, overall spike frequency
(spike number per burst divided by cycle period) coded average con-
traction amplitude (one half phasic plus tonic contraction) for this mus-
cle. All data are from the cpv1b muscle39.
Time (s)
2000 Nature America Inc. http://neurosci.nature.com
amplitude (mm)
ly34,35 (Fig. 1a). The top trace shows the characteristic rhythmic
Average
action potential bursts of a pyloric dilator (PD) neuron. The sec-
ond trace shows an isotonic contraction (muscle shortening
against constant load) of an extremely slow muscle innervated
by the PD neurons (the dorsal PD muscle). This contraction was
induced by stimulation of the motor nerve with parameters
matching the first burst in the neuron trace. If the neuron were Overall spike freq. (Hz)
driving this muscle (in all experiments the motor nerve was cut
to prevent spontaneous neuron input to the muscle), the next summation had stabilized, the muscles response therefore con-
contraction would have occurred before the contraction induced sisted of a sustained, tonic baseline contraction on which rode
by the previous burst had ended (arrow; angled to account for phasic contractions that matched the stimulation cycle period.
the long delay between the neuron burst and the contraction Here the average contraction amplitude (one half the phasic con-
beginning). traction amplitude plus the tonic contraction amplitude) of the
When the motor nerve was rhythmically stimulated with muscle is particularly important, and after muscle summation
bursts of shocks using physiologically relevant parameters, the has stabilized, this average amplitude is well predicted by the
muscle contractions temporally summated (Fig. 1b). After the overall spike frequency (spike number per burst divided by cycle
period) of the motor neuron (Fig. 1c; ref. 34 and L.G.M. & S.L.H.,
Soc. Neurosci. Abstr. 24, 1891, 1998). These data suggest that if
1 the overall spike frequencies of pyloric motor neurons were slow-
PY ly modulated by extrinsic influences, average contraction ampli-
neuron
tude of pyloric muscles might similarly vary.
The stomatogastric nervous system also contains the gastric
mill and cardiac sac networks, which have much slower cycle
10 mV
periods (510 seconds and 1 minute, respectively) than the
pyloric network25. Gastric mill and cardiac sac activity modu-
2 lates pyloric activity25,28,3638, and the overall spike frequency of
aln
several pyloric neurons varies with gastric mill and cardiac sac
activity (J.B.T. & S.L.H., Soc. Neurosci. Abstr. 25, 1641, 1999).
Using stimulation parameters determined from previously mea-
sured effects of gastric mill and cardiac sac network activity on
3 10
Overall 7.5
spike 5 Fig. 2. A muscle innervated by PY neurons expressed a gastric mill net-
freq. (Hz) work contraction pattern even though no gastric mill neurons innervate
2.5
the muscle. Trace 1, intracellular recording of the PY neuron whose
0 activity was used to stimulate the muscles motor nerve. Most hyperpo-
larized PY neuron membrane potential, 55 mV. Trace 2, extracellular
4 0.15 mm recording from the aln of the activity of the GM neurons of the gastric
mill network. Trace 3, PY neuron overall spike frequency. Trace 4, iso-
PY tonic contractions of a PY neuron-innervated muscle. Gray shading
muscle
shows the tonic component of the muscle contraction; the muscle
almost fully relaxed only at the end of each gastric mill cycle (dashed
lines mark one cycle period). Solid horizontal line, fully relaxed muscle
2s length. Recording is from p8 muscle39.
articles
Fig. 3. Although no cardiac sac neurons innervate it, the extremely slow
1 Cardiac dorsal PD muscle primarily contracted in a pattern matching the very
sac
slow cardiac sac rhythm. Trace 1, schematic representation of cardiac sac
network activity; rectangles correspond to cardiac sac bursts, dashed
lines mark one cardiac sac cycle period. Trace 2, intracellular recording of
2 PD
neuron
the PD neuron whose activity was used to stimulate the muscles motor
nerve. Trace 3, time expansion of PD neuron activity immediately before,
during and after a cardiac sac burst. Most hyperpolarized PD neuron
membrane potential, 65 mV. Trace 4, PD neuron overall spike fre-
quency. Trace 5, isotonic contractions of a dorsal PD muscle (inset is a
time expansion showing small contractions in pyloric time). Scale bars,
3 20 s (traces 1, 2, 4 and 5) or 2 s (trace 3) and 5 mV (traces 2,3) or 60 m
(trace 5). Recording is from the cpv1b muscle39.
5 mV
2s
20
4 priate for this new spike frequency. If overall spike frequency
Overall 15 remained at the new value, the muscle would stabilize at the aver-
spike 10 age contraction amplitude predicted by the equivalent of Fig. 1c
2000 Nature America Inc. http://neurosci.nature.com
freq. (Hz)
5 for the PY muscle. However, overall spike frequency does not sta-
0 bilize; consequently, the muscle is driven toward different regions
of this curve each pyloric cycle, which generates the slow varia-
5 tions in average contraction amplitude (Fig. 2).
For the relatively fast PY muscle, the contractions in pyloric
Dorsal PD time were a large percentage of total contraction amplitude.
muscle
baseline
In contrast, the extremely slow dorsal PD muscle responded
(full relaxation) 60 m to PD neuron input modulated by the very slow, 0.01 Hz, car-
20 s
diac sac network primarily with a slow, rhythmic variation in
tonic contraction amplitude (Fig. 3). During cardiac sac net-
pyloric network output, we explored the effects of altering pyloric work bursts (trace 1; each rectangle represents one cardiac sac
neuron activity on pyloric muscles by stimulating motor nerves burst, dashed lines mark one cardiac sac cycle), PD neuron
innervating various pyloric muscles (Fig. 2). The first trace shows activity was markedly altered, but the neuron continued to
an intracellular recording from a pyloric (PY) motor neuron; the burst in pyloric time (trace 2; expanded over one cardiac sac
second trace is an extracellular recording from the anterior lat- burst in trace 3). PD neuron overall spike frequency (trace 4)
eral nerve (aln), which carries the axons of the gastric mill (GM) showed a triphasic increasedecreaseincrease pattern with
neurons of the gastric mill network39. PY neuron firing waxed each cardiac sac burst. When the motor nerve innervating the
and waned as a function of gastric mill network activity (the extremely slow dorsal PD muscle was stimulated with this
dashed lines mark one gastric mill network cycle), and the third activity pattern, the muscles contractions in pyloric time
trace shows that this change in PY neuron activity resulted in (shown on expanded time scale in inset) were only 8% of the
corresponding changes in PY neuron overall spike frequency. maximum contraction amplitude, whereas its contractions in
The fourth trace shows the contractions of a muscle inner- cardiac sac time were 50% of maximum contraction ampli-
vated by the PY neurons induced by motor nerve stimulation tude (trace 5). Thus, even though no motor neurons of the
exactly matching the PY neuron spiking pattern shown in the cardiac sac network innervate it39, the muscle primarily con-
first trace. As overall spike frequency increased at the beginning tracted in cardiac sac time.
of each GM neuron burst, the phasic amplitude and summation These data show that the PD and PY muscles can respond to
of the pyloric-timed PY muscle contractions increased and, con- slow modulations of PD and PY neuron activity. However,
sequently, the muscles tonic contraction component (gray shad- because the activity patterns of the PD and PY neurons are dif-
ing) increased. As overall spike frequency decreased during the ferent, the PD and PY muscles receive different input; these data,
GM neuron interburst interval, the muscles phasic contraction therefore, do not show how different muscles respond to the same
amplitude and summation and, thus, its tonic contraction, input. As well as innervating the extremely slow dorsal PD mus-
decreased, allowing it to return nearly to rest length before the cle, the PD neurons also innervate the faster ventral PD muscle,
next GM neuron burst. Thus, although no gastric mill neuron which allowed us to investigate whether the two muscles selec-
innervates this muscle, it nonetheless displayed both a pyloric tively respond to different frequency domains within an identical
(the rapid rhythmic contractions) and a gastric mill (the tonic neural input.
contraction component) motor pattern. In addition to showing large (15 Hz) variations in overall
The changes in the PY muscle tonic contraction can be qual- spike frequency in time with the slow cardiac sac network
itatively understood by considering Fig. 1b and c. Contractions of (0.01 Hz; Fig. 3), the PD neurons often also showed small
the extremely slow dorsal PD muscle took 1015 pyloric cycles (2.5 Hz) variations in overall spike frequency (Fig. 4, trace 1)
to stabilize (Fig. 1b). The PY muscle is faster than the dorsal PD in time with the faster gastric mill network (0.10.2 Hz; trace
muscle, but it nonetheless took 23 cycles to stabilize in response 2; dashed lines mark one gastric mill cycle). The extremely slow
to rhythmic stimulation and shows similar coding dependence dorsal PD muscles showed much smaller variations in gastric
(M. Rehn, L.G.M. & S.L.H., unpublished observations). When mill time than did the faster ventral PD muscles and, depend-
PY neuron overall spike frequency changes, the muscles con- ing on gastric mill cycle period, sometimes completely exclud-
traction amplitude changes toward an average contraction appro- ed the gastric mill signal. In these cases (Fig. 4, trace 3), the
articles
3 Dorsal
PD
input. These results have implications not only for interpreting
muscle studies of the pyloric system, but also, more generally, for
0.2 mm
baseline (full relaxation) understanding how and to what extent biological systems can
respond to input signals carried in different frequency domains.
2000 Nature America Inc. http://neurosci.nature.com
articles
articles
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articles
Dept. of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA
Correspondence should be addressed to S.M.-C. (smart@hms.harvard.edu)
When viewing a stationary object, we unconsciously make small, involuntary eye movements or
microsaccades. If displacements of the retinal image are prevented, the image quickly fades from
2000 Nature America Inc. http://neurosci.nature.com
Our interest in microsaccades and burst firing grew out of an became clear that the cells firing and the monkeys microsac-
attempt to study the activity of single cortical cells in an awake cades were correlated, and that the eye movements tended to
monkey during free viewing of a visual scene. We made con- be associated with bursts of spikes.
tinuous recordings of eye position while recording spikes from This paper is devoted to analyzing this relationship between
a cortical cell. We marked on a video screen the position of the microsaccades and spikes. The results are based on recordings
eyes a suitable length of time before each spike (eye-position- from 258 cells in 3 monkeys. We asked how often, with or with-
corrected reverse correlation). If the scene viewed by the mon- out a stimulus, a microsaccade was followed by spike activity and
key consisted, for example, of a large bright circle on a dark whether any suppression of activity accompanied or followed a
background, we expected the cell to fire whenever its receptive microsaccade. We also asked the converse question: how often
field was crossed by an appropriately oriented contour 1. To were spikes preceded by microsaccades, and how would the cor-
sample the scene evenly, we had the animal fixate on a spot relation be influenced by whether one looks at single spikes,
whose position changed at random every few seconds. For instantaneous firing rates or bursts of spikes. To ask these ques-
some cells, we could see a clear correlation between the stim- tions meaningfully, we began by defining the terms microsac-
ulus and the eye positions that were spike related, but for other cade and burst.
cells, the constellation of spots marking spike-associated eye
positions showed little or no relationship to the scene Analysis of microsaccades
(Fig. 1a and b). During our recordings, we noticed that for cer- In most studies of longer-range eye movements (movements out-
tain gaze locations, cells tended to fire in bursts rather than side the scope of this study), determining length, direction and
trains of random spikes. When we filtered our records to exam- velocity of the movements is relatively simple. Large saccades
ine only burst firing, we saw a much clearer correlation between reach velocities in the hundreds of degrees per second, and so a
the cells activity and the stimulus (Fig. 1c and d). Evidently, straightforward velocity detector can be used to determine when
any tendency for cells to fire in high-frequency bursts was and where each saccade begins and ends. Moreover, the starting
enhanced by the contours of the stimulus. This project and end positions of the eye movements are generally determined
stemmed in part from our suspicion that the bursts were pro- in advance. With microsaccades, starting and end positions and
duced largely in response to microsaccades that occurred dur- timing are relatively random, and their velocities are low. Previ-
ing the intermittent periods of visual fixation. Here we examine ous studies identify microsaccades with instantaneous velocity
the relationship between microsaccades and various firing pat- thresholds2,3 set to fairly high levels (10 per second) to protect
terns of V1 cells, specifically, single spikes, instantaneous fir- the analysis from noise. By using the fastest portion of the
ing rates and spike bursts of various lengths. microsaccade to categorize them in terms of size and speed, these
studies neglected portions of the microsaccade at speeds of less
RESULTS than 10 per second, a considerable portion of a microsaccade
We stimulated each V1 cell with an optimally oriented bright, because of its slow speed and small size. In addition, these stud-
stationary bar centered over the cells receptive field while the ies measured either the horizontal or vertical directional com-
monkey fixated on a spot of light on another part of the ponent of microsaccades, but not both. Thus the size of obliquely
screen. From simply listening to the cells firing while watch- directional microsaccades was underestimated by as much as 2
ing the eye position on the computer screen, it immediately (or 41% of the actual size).
articles
eye position associated with a single spike. (c, d) The same data, filtered so that each pixel rep-
second and never varied from a
resents a burst of 4 or more spikes occurring within an interval of 20 ms at a delay of 35 ms.
straight trajectory by more than 15 (b, d) Same data as (a, c), except that the circle is removed to reveal the pixels. Duration of the
in any direction. Microsaccades were recording was about 40 min.
recorded from two monkeys during
several two-second intervals (Fig. 2).
Probability of discharges following microsaccades records to see if the effect of microsaccadic suppression had
We set out first to determine whether microsaccades were corre- been drowned out by the averaging process, and found that
lated with modulations in neural activity, and if the modulations only 6 of the 246 cells showed clear suppression of firing asso-
were stimulus driven or caused by the microsaccades themselves ciated with microsaccade onset. In all six cells, the suppression
and unrelated to the visual stimulus. preceded a period of excitation.
In the main portion of our analysis, we examined 246 cells Average probability of spikes recorded from 45 cells after
from 2 monkeys. After the beginning of a microsaccade when microsaccades without any visual stimulus (the monitor was
an optimal stimulus was centered over the cells receptive field, black except for the fixation point) revealed no correlation
average spike probability showed a clear but small increase of between the microsaccades and modulation of neural activity
about 0.5%, peaking at about 70 ms (Fig. 3a, upper curve). (Fig. 3a, lower curve), indicating that microsaccade-induced
The average did not show suppression of activity associated activity in V1 was caused by microsaccades driving V1 neu-
with the onset of microsaccades. We examined the individual rons by moving their receptive fields over stationary stimuli,
and not by direct excitation from
the motor system.
We found similar increases in
neural firing correlated with the
end (as opposed to the beginning)
articles
Spike probability
(vertical line). The probability of spikes after microsac-
cades increased in the presence of the stimulus,
whereas it did not change if the stimulus was absent.
(b) Probability that a microsaccade preceded any given
Without stimulus
spike. (All spikes were aligned at the vertical line.)
b
Time after start of microsaccade (ms)
Microsaccade probability
of microsaccades. The result held true even
when we correlated neural activity with each
individual millisecond of each microsaccade in Spike
flight (data not shown).
We ruled out the possibility that the
observed excitation after microsaccades was
caused by flicker on the stimulus monitor by
recording from 12 additional neurons from a
2000 Nature America Inc. http://neurosci.nature.com
third monkey using a bar projected from a Time before spike (ms)
tungsten incandescent bulb as a visual stimu-
lus. We again found excitation after microsac-
cades, ruling out the possibility that microsaccade-related activity perpendicular to the orientation of the cells receptive field might
was due to flicker in our video display. then preferentially activate the cell. We found, however, no clear
relationship between microsaccade direction and response mag-
Probability that a spike was preceded by a microsaccade nitude. This finding may have been due to the aperture problem:
We next computed the reverse correlation between spikes and a V1 cell cannot distinguish between a slow microsaccade that
preceding microsaccades, that is, the probability of a microsac- moves the stimulus perpendicularly across its receptive field and
cade preceding the discharge for the 246 cells from the main por- a fast microsaccade that moves the stimulus in an oblique direc-
tion of our study (Fig. 3b). The peak probability of spikes tion to the receptive field. Thus two unlikely events would need
correlated with previous microsaccades was enhanced by 9%, to occur simultaneously for microsaccade direction to be
many times the peak probability seen with the forward correlation involved: the stimulus would need to be perfectly aligned with
(compare with Fig. 3a). the angle of the receptive field, and the microsaccade direction
We presume that the difference arose because the reverse cor- would need to be perfectly parallel to the angle of the receptive
releation (Fig. 3b) considered only those microsaccades that were field. In addition, it may be that only a fraction of microsaccades
effective, whereas the forward correlation (Fig. 3a) included all activated area V1 cells effectively because of imperfectly main-
microsaccades, regardless of
their effectiveness. We con-
sidered whether a microsac- a b
cade would be rendered
Probability of microsaccade
orientation. Microsaccades
articles
Probability of microsaccade
burst was plotted as a function of latency (time
before the first spike in each burst) and ISI.
(d, e) Normalized contour plots of the data in
before burst
before burst
Bursts of spikes
ISI Time before burst (ms) ISI Because previous experiments (Fig. 1) sug-
Time before burst (ms)
gested that bursts may convey more reliable
d e information about the visual scene than
single spikes1, we were especially interested
s )
s)
in studying the relationship between bursts
m (m
s t( t (of one or more spikes) and preceding
r rs
bu bu microsaccades. We assigned each spike
r e r e
IS fo fo
I be IS
I be uniquely to a burst by determining the
e e
m
Ti Ti
m interval between it and the spikes preced-
ing and following: two spikes with a given
ISI (or less) were thus considered members
Probability of microsaccade before burst Probability of microsaccade before burst
of a single burst, and two spikes with an
interval greater than the given ISI were con-
tained fixation: some microsaccades may have begun and ended sidered elements of separate bursts (S. M. Smirnakis, D. K. War-
with the stimulus outside the receptive field, without ever cross- land, M.J. Berry and M. Meister, Neural Information Coding
ing it. Such occurrences would diminish the average enhance- Conference, Snowbird, Utah, 1997; Fig. 5a). The ISI chosen is cru-
ment of firing by microsaccades but not affect the incidence of cial in determining the overall distribution of burst lengths (num-
microsaccades preceding cell firing. ber of spikes per burst). Because we could not know in advance
the most appropriate ISI for any given cell, we compared, for all
Instantaneous firing rate the records and for all the cells, all ISIs between 1 and 100 ms and
We examined the relationship between the interspike intervals measured the probability of a microsaccade occurring some time
(ISIs) that separated pairs of successive spikes (within the range (latency; 200 ms) before the first spike in each burst. We then
of 1100 ms) and the microsaccades that preceded these spike sorted all bursts according to length (numbers of spikes per burst),
pairs by up to 200 ms (Fig. 4a). We define instantaneous fir- which ranged from 1 (lone spike) to 20. We then constructed 20
ing rate as the inverse of interspike interval (1/ISI)4. Plots of (for each burst length) three-dimensional arrays; from each of
the probability of a microsaccade preceding the first of two these we plotted the probability of a microsaccade preceding a burst
successive spikes with a given ISI (Fig. 4b and d) demonstrate against latency ( 200 ms) and ISIs ( 100 ms). These plots allowed
that microsaccades were more closely linked to high instanta- us to assess the probability of a microsaccade as a function of burst
neous firing rates (small ISIs) than to slow rates. The proba- size, latency and ISI.
bility of a microsaccade was maximal about 44 ms before a We show three-dimensional plots and normalized contour
spike pair. At higher ISIs, the probability of a microsaccade plots for bursts of one (Fig. 5b) and five (Fig. 5c) spikes for the
before a pair of spikes did not drop to zero, presumably because same cell as in Figs. 4b and d. Burst of one, or lone spikes, were
of spontaneous firing. relatively poor indicators of previous microsaccades. Bursts of 5
We plotted average microsaccade probability against ISI were more closely correlated with previous microsaccades in this
for the cell population by averaging all three-dimensional cell, and had a peak correlation of almost one. Peak probability
graphs and slicing the result parallel to the probabilityISI varied with latency in all cells, whereas peak latencies remained
plane at the average peak latency, 65 ms (Fig. 4c). Pairs of stable as burst size varied and were consistent with V1 responses.
spikes were most closely related to preceding microsaccades For this cell, bursts of 5 spikes were the best indicators that a
when the interspike interval was short. Averaged across all microsaccade had occured about 44 ms before.
cells, the peak probability of a microsaccade preceding a pair We next compared the strengths of the associations for all the
of spikes was 0.42. different burst lengths examined, regardless of ISI or latency
articles
(Fig. 6a; same cell). The optimal burst size was clearly five, with a
fall-off for shorter and longer bursts. But why did the correlation
decrease for bursts longer than five? The occurrence of longer bursts
during the experiment that were not well correlated with microsac-
cades led us to wonder if larger eye movements omitted from our
analysis were associated with longer bursts. To test this, we examined
all eye movements longer than three minutes of arc and found a
clear correlation with all bursts measured (data not shown).
Burst size (number of spikes)
For the same cell, we then plotted probability against ISI for
bursts of 1 and 5 spikes at a latency of 44 ms (the cells optimal
2000 Nature America Inc. http://neurosci.nature.com
Probability of a microsaccade
the probability variations were presumably explained by the ten-
dency of spikes to occur in clumps separated by periods of rela-
tive quiescence, often lasting hundreds of milliseconds: when ISIs
that defined bursts were small, they tended to cut these clumps
into fragments that were poorly correlated with microsaccades. Single spikes
ISIs greater than the optimum value did not affect the probabil-
ities unless sufficiently large that successive clumps coalesced. To
sum up, burst size and latency were highly correlated with pre-
vious microsaccades, whereas the ISI used to define bursts was Tighter bursts
less crucial, at least for values greater than 2030 ms.
The correlation with preceding microsaccades was always
higher for bursts, defined in terms of ISIs, than for pairs of spikes
ISI used when assigning spikes to bursts
that determined instantaneous firing rates. This is demonstrated
by plotting for all ISIs and latencies the peak probability of a
microsaccade before the optimum burst size against the peak for all ISIs and latencies the distribution of burst sizes associated
probability of a microsaccade before the optimum instantaneous with peak probabilities (Fig. 8). Peak burst sizes showed a wide
firing rate (Fig. 7). The difference in the probabilities obtained spread, with an average of 11 ( 5, s.d.) spikes per burst and a
by the two methods was significant (p < 0.001; t-test). Thus for substantial fall-off for short bursts. The average latency associ-
every cell tested, burst configuration indicated a previous ated with microsaccade activity was 65 46 ms, and the average
microsaccade with more reliability than the best possible instan- peak ISI was 56 29 ms.
taneous spike rate.
From Fig. 6a, it is clear that a burst size of five supplied by far DISCUSSION
the best indicator of a previous microsaccade for the cell under The phenomenon of fading of a stabilized image comes as a great
consideration. We were curious to learn the variability of such a surprise to someone who learns of it for the first time; when first
preference across our sample population.We therefore plotted discovered5,6 in the early 1950s, it was little short of astonishing.
Determined attempts to reproduce the fading by careful fixation
often fail, especially for objects in or near the fovea, and observers
are quite unaware of the microsaccades whose occurrence large-
Peak probability from burst analysis
articles
ulation of cells. Optimal burst sizes tended to be greater than three spikes. saccadic velocities (typically, over 300 per s). Hypothetically, sup-
pression associated with microsaccades could block awareness of
fixational eye movements, which produce displacements of images
systems seem to be organized on the principle that, whether we across the retina sufficient to be easily seen if produced by a mov-
are predators or prey, changing stimuli are essential for survival ing object. We saw microsaccade-related suppression in only 7 of
and that sensory adaptationthe decline with time of a response the 258 cells we studied, and the suppression was always followed
to a maintained stimulusguarantees that a novel stimulus will by excitation. It is possible that our experimental protocol made
stand out, especially if it moves. The normal inability to see ones excitation easier to detect than inhibition. In any case, we found no
own retinal blood vessels or optic disc is presumably also related conclusive mechanism to explain unawareness of microsaccades,
to this process9. Ironically, the process works so well that some at least at the level of the striate cortex.
further mechanism seems to be required to overcome the fading, The enhancement of firing in V1 cells by microsaccades that
so that we can see a stationary scene despite adaptation. we observed is at odds with similar recordings of cells in mon-
If involuntary eye movements during fixation are crucial role key V1 during fixation with a small grating covering the recep-
in maintaining the visibility of stationary scenes, we should be tive field 3. This study revealed enhancement of firing after
able to observe the relationship in awake alert animals by record- microsaccades in only 6 of 35 cells, suppression of firing in 13
ing eye movements while observing the firing of single cells in cells and no changes in the remaining 16 (but enhancement of
the presence of an adequate visual stimulus. This was the pur- firing downstream, in areas V2, V4 and IT). The failure to see
pose of the present study. microsaccade-related activation in V1 surprised us, given the
Quantitatively correlating microsaccades and bursts of spikes marked elevation of firing rate we consistently saw whenever a
has been a challenging problem. Under the term bursts, we stationary bar was placed in the receptive field and the clear asso-
included both clustered activity
(short, high-frequency trains of two to Single session 239 sessions
eight or more spikes) and bursty fir-
ing, consisting of highly irregular
longer-lasting periods of rapid activity
separated by irregular silent periods.
Both types of firing were described in
Microsaccade velocity
articles
ciation, to simple inspection, between eye movements and fir- ability that a microsaccade precedes a single burst of arbitrary size
ing. The discrepancy in results may be due to differences in meth- (say of three spikes) is greater than the sum of the probabilities
ods. We stimulated our cells with stationary bars of light, whereas associated with three spikes considered separately. In our results,
they used circular patches of oriented gratings that sometimes this seemed, on average, not to be so. For example, if a given spike
were binocularly rivalrous, with orthogonal orientations in the was related to a previous microsaccade with a probability of over
two eyes. Their cell population in V1 was much smaller, and they 0.3 (Fig. 3b), simple addition should predict that a rapid succes-
sampled eye position at 200 Hz, interpolating 80% of their eye sion of three spikes should give a probability of three times 0.3,
movement data to simulate a sampling rate of 1 KHz. In addi- and on average this is roughly what we found. There may thus be
tion, our monkeys fixated continuously during the entire record- nothing magic about bursts: by definition, several spikes in quick
ing session (nothing in our experimental protocol signaled the succession necessarily constitute a burst.
beginning or end of a trial to the monkey), so the visual stimulus We cannot, on the other hand, rule out the existence of presy-
remained on the screen throughout. Their monkeys, on the other naptic facilitation in some synapses. At a presynaptic level, both
hand, performed a visual discrimination task with discrete trials synaptic facilitation and depression are described in vitro21,22.
that were separated by blank screens. Regardless of these effects, however, rapidly sequential EPSPs should
In the present analysis, we did not look for effects of slow drifts sum postsynaptically, so that even when synaptic efficacy is reduced
in eye position on the firing of cells, and cannot rule out a possible in size, as in presynaptic depression, multiple EPSPs should improve
role for them in the prevention of image fading. The relative role of the chances of postsynaptic signaling through classical temporal
microsaccades and drifts has been a subject of considerable dis- summation. For a pair of spikes, a clear example of paired synaptic
2000 Nature America Inc. http://neurosci.nature.com
cussion in the literature. Several studies17,18 address both types of facilitation has been shown in vivo for retinogeniculate synapses23.
movement but do not isolate microsaccades and drifts from each Thus, although we have no direct evidence pertaining to cortical
other. The strongest claim against a role of microsaccades in pre- synapses, given the simplest assumptions of temporal summation,
serving visual perception19 is mainly based on two ideas. First, visu- it seems reasonable to expect a burst of spikes to be more reliable
al thresholds may be elevated during saccades. This is true of than single spikes in influencing a postsynaptic cell.
long-range saccades, but the evidence for suppression during the Equally interesting is the expectation that each microsaccade
much slower microsaccades is controversial, and we found little will produce synchronous bursts in many cells. Receptive fields of
physiological evidence for it in the present study. Second, subjects neighboring cells in striate cortex overlap extensively and share
can be trained to suppress their microsaccades without experi- the same orientation preference. Consequently, as a stimulus is
encing fading of images. The possibility that drifts alone can pre- swept across their receptive fields, many cells should fire more or
vent fading does not prove that microsaccades play no part. less synchronously, in bursts that can be expected to overlap in
Moreover, if one fixates steadily on a point while paying attention time. The result should be strong spatial summation of messages
to a small (and, preferably, low contrast) object in the periphery carried by axons converging at the next stage. Microsaccades would
of the visual field, the object will disappear almost immediately20. thus seem to represent an ingenious mechanism for enforcing and
If such precise fixation eliminates microsaccades, this would seem refreshing information coming from stationary visual stimuli.
to support their role in preventing fading. A careful analysis of the It is furthermore possible that microsaccades may be impor-
physiological effects of slow drifts in eye position would be useful, tant for allowing us to use latency in our visual discriminations.
and we are currently attempting such an analysis. Latency differences in neural responses are proposed as an encod-
Having determined the probability that a microsaccade is fol- ing mechanism for changes in the magnitude of contrast2426. But
lowed by spike activity, our next step was to ask whether, con- it has remained unclear how the brain could use latency infor-
versely, neural discharges were associated with an increased mation, as the brain cannot know the latency a priori. Microsac-
probability of previous microsaccades. By doing so, we could cades could theoretically be used by the visual system to measure
concentrate on those microsaccades that had been effective in latency, because the brain knows both when the microsaccade
driving the cells but ignore ones that were ineffective. (The stim- motor signal starts and when the visual signal arrives. Relative
ulus can at times drift away from the receptive field, among other latencies of responses to stimuli could then be used to indicate
possibilities.) We found that the probability of microsaccades contrast.
before a spike (Fig. 3b) was much higher than the probability of
a spike after microsaccades (Fig. 3a). The instantaneous firing
rate of each cell, defined as the inverse of the interval between METHODS
the two members of a pair of successive spikes, was still better We used three rhesus monkeys, Macaca mulatta. A midline stainless-steel
head post was mounted by screws over the skull, and a steel recording
than single spikes in indicating a preceding microsaccade.
chamber was mounted over the occipital operculum. We recorded from
Furthermore, bursts of spikes were better correlated with pre- single neurons in area V1 with lacquer-coated electropolished tungsten
ceding microsaccades than single spikes or instantaneous firing electrodes. A visual search coil attached to the sclera of one eye recorded
rates. For most cells, bursts of fewer than four spikes were less the animals eye movements. Standard sterile surgical techniques and
effective than longer bursts, and on average there was no sharp animal care methods are described1,27. The Harvard Medical Area Stand-
optimum size. ing Committee on Animals (protocol #02935) approved all electrophys-
Why did V1 cells show such a marked tendency to fire in bursts iological experimentation.
when microsaccades made their receptive fields move across a
visual stimulus? Here we will not discuss the intrinsic properties of Stimuli and receptive-field mapping. Monkeys were trained to fixate on
a small spot displayed on a monitor at a distance of 58 cm. A monkey
neurons that cause them to fire in bursts, but will instead consid-
was required to keep fixation within a 2 window to receive a drop of
er the teleological significance of such firing. Are bursts useful in juice; eye movements exceeding the windows limits were also recorded.
terms of enhancing the information conveyed by one cell to a The stimuli displayed on the monitor had a luminance of 24.3 cd
postsynaptic cell? Specifically, are spikes arriving at a synaptic ter- per m2, and the monitor background was 3.84 cd per m2. We recorded V1
minal more or less likely to evoke spikes in a postsynaptic cell if over the occipital operculum (eccentricity, 68) or from the folds of
they occur in bursts? One can begin by asking whether the prob- calcarine cortex immediately beneath. Receptive fields were about
articles
0.33 0.5, and thus the same size or larger than many eye movements if a particular burst size and ISI occurred only once and followed a
during fixation. We first mapped the receptive field roughly by hand, microsaccade, the probability of such a unique event was thus 1. We
using computer-generated moving slits whose orientation, size and rate therefore smoothed every three-dimensional array with a boxcar filter
of movement were controlled with a computer mouse. We then used a whose width was 15 elements in the ISI-latency plane; thus every proba-
bar of optimum length, width and orientation that flashed in quick suc- bility was averaged with those corresponding to 7 ms latency and 7
cession in various parts of the visual field to more precisely determine ms ISI. (At the array edges, this boxcar was reduced to fewer elements.)
the position and size by reverse correlation. A stationary bar with these This smoothing removed virtually all the random peaks of probability
optimal parameters of width, length and orientation was then positioned associated with rare bursts.
over the center of the receptive field, and collected spikes were subse-
quently correlated with the monkeys microsaccades. The bar turned on
before data collection and remained on throughout the recording ses-
ACKNOWLEDGEMENTS
We thank Gail Robertson, David Freeman, Michael Lafratta and Frederic Russo
sion for each cell. Within total recording times of 100800 s (mean, 334
s per cell), we recorded eye movements and spikes in consecutive 2-s tri- for technical assistance and Margaret Livingstone, Clay Reid, Richard Born and
als. The beginning and end of each trial were unknown to the animal, Max Snodderly for reading the manuscript and making comments. We also thank
and the fixation point persisted between trials. Murray Sherman, Jonathan Victor, John Maunsell and Guy Orban for their
advice on the analysis and design of the project. This work was supported by
Analysis of microsaccades. We developed an algorithm for determining research (to D.H.H.) and training grants (to S.L.M.) from the National Eye
when the eye was stopped and when it was moving. We regarded an eye Institute. S.M.-C. is a fellow from the MEC-FPI program (Spain).
movement as ended when either its speed dropped below some thresh-
old or its direction changed by more than some predetermined angle.
2000 Nature America Inc. http://neurosci.nature.com
articles
1 Institute of Neurophysiology, Area Ricerca CNR, 1 via Alfieri, 56100 Pisa, Italy
2 Institute of Developmental Neurology and Psychiatry, University of Pisa and Institute for Medical Research IRCCS Stella Maris, 2 via Giacinti, 56018
Calambrone, Pisa, Italy
3 Division of Neurosciences, Kings College Hospital, University of London, Denmark Hill, London SE5 9RS, UK
Correspondence should be addressed to V.P. (porciatt@in.pi.cnr.it)
2000 Nature America Inc. http://neurosci.nature.com
Television and video games may be powerful triggers for visually induced epileptic seizures. To
better understand the triggering elements of visual stimuli and cortical mechanisms of
hyperexcitability, we examined eleven patients with idiopathic photosensitive epilepsy by recording
visually evoked potentials (VEPs) in response to temporally modulated patterns of different contrast.
For stimuli of lowmedium, but not high, temporal frequency, the contrast dependence of VEP
amplitude and latency is remarkably abnormal for luminance contrast (blackwhite), but not so for
chromatic contrast (equiluminant redgreen) stimuli. We conclude that cortical mechanisms of con-
trast gain control for pattern stimuli of relatively low temporal frequency and high luminance
contrast are lacking or severely impaired in photosensitive subjects.
Photosensitive epilepsy (PSE) is the most common form of stim- We studied both control subjects and patients with pure idio-
ulus-induced epilepsy1. Its prevalence in children 414 years old pathic PSE whose seizures originated from the occipital lobe9,10.
is substantial (0.5%0.8%), and its incidence is increasing as a VEPs were recorded in response to simple visual patterns
result of the proliferation of television display units and video (black/white and red/green sinusoidal gratings of different con-
games, which may act as triggers2. During a recent television trast), sinusoidally contrast-reversed at various temporal fre-
showing of the Pocket Monsters cartoon in Japan, 685 children quencies. The EEG activity remained in a physiological state
experienced epileptic seizures3,4. The degree of danger inherent throughout the experiment. In patients, the dependence of VEP
in such seizures ranges from nil to potentially life threatening, in amplitude on luminance-contrast for stimuli of relatively low
exceptional cases5. Epileptic seizures induced by photic stimula- temporal frequency (410 Hz) was dramatically altered. Specif-
tion may be primarily generalized (tonic-clonic, myoclonic, ically, at increasing contrast, the VEPs saturated in amplitude
absence seizures) or focal (occipital, with or without spreading and shortened in phase in controls, whereas in patients there was
to other cortical areas). PSE is defined as pure when seizures are little saturation or phase shift. These results suggest that cortical
exclusively photic induced, and is usually idiopathic (with no mechanisms of contrast gain control were lacking or severely
other etiology than a genetic background)6. impaired in PSE.
Because of the complexity and heterogeneity of video-gener-
ated patterns, reports of pattern-induced epilepsies are mainly RESULTS
anecdotal. Oriented lines are considered more powerful than Luminance gratings: effect of temporal frequency
checkerboards in inducing epileptiform EEG changes7, and oscil- In both controls and patients, steady-state VEPs were recorded as a
lating patterns more epileptogenic than static patterns8. Howev- function of temporal frequency of luminance-contrast gratings.
er, the characteristics of the visual stimulus and the cortical Stimulus contrast (90%) and spatial frequency (2 cycles per degree)
dynamics leading to the hypersynchronous neuronal response were chosen to maximize response amplitude11,12. In agreement with
underlying PSE are poorly understood. A better knowledge of previous studies11,12, the form of the temporal function consistent-
the triggering elements of visual stimuli might help in devising ly showed two amplitude peaks (at 38 Hz and 1620 Hz) and a
safer video-generated patterns. local minimum at 1012 Hz in normal subjects (Fig. 1a and c). In
The temporal frequency of an intermittent photic stimulus is PSE patients (Fig. 1b and c), the high temporal-frequency cutoff
crucial for arousing epileptic activity. This suggests that, even for was comparable to that of controls. However, the shape of the tem-
patterned stimuli, the temporal frequency may be critical. How- poral function was more variable. In particular, low- and high-fre-
ever, there is no systematic study on the visually evoked potentials quency amplitude peaks were less defined, and in some subjects,
to temporally modulated patterned stimuli in PSE patients. Anoth- substantial activity was present at intermediate frequencies (1012
er crucial parameter of pattern stimuli is spatial contrast (related to Hz), at which controls responded poorly. Overall, VEP amplitude
the spatial variations in brightness). It is conceivable that contrast was significantly larger in patients than controls
is also a critical parameter for cortical excitability. (Fig. 1c; two-way ANOVA, F1,271 = 6.2, p = 0.012).
articles
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effective frequencies2,18. Photoparoxysmal activity may also be gesting that a mechanism normally responsible for contrast gain
provoked by viewing a striped black/white pattern19. Contrast control has been suppressed25. Our VEP data do not allow us to
values above 40%, a spatial frequency of 24 cycles per degree advance suggestions about the site and nature of the gain-control
and 1020 Hz reversal frequency are most epileptogenic19. mechanism1416,26. Note, however, that the pattern ERG in response
Human photosensitivity is a form of reflex epilepsy arising in to both luminance and chromatic gratings does not show signif-
the visual cortex, with high tendency toward generalization19. icant saturation and phase shift with increasing contrast27. The
The efficacy of the triggering stimulus depends on its ability to average saturation index of VEP amplitudes and the average laten-
elicit action potentials in a hyperexcitable visual cortex, on syn- cy decrement with increasing stimulus contrast in controls are in
chronizing effects and on the size of the neural population acti- agreement with data obtained from simple cortical cells under
vated8. similar stimulus conditions14. This encourages us to interpret the
The present findings, obtained with patterned stimuli of high control data as indicating a mechanism of contrast gain control
contrast sinusoidally modulated in time, show that a range of rel- and to speculate that such a mechanism is defective or absent in
atively low temporal frequencies may be critical for cortical hyper- the visual system of PSE patients. VEP responses at the higher
excitability. Indeed, the VEP responses of the patients in this temporal frequencies, at which normal subjects did not show
temporal frequency range tend to be higher in amplitude in com- amplitude saturation, were unaffected in PSE.
parison with controls, in spite of most patients being treated with VEPs to high-contrast red/green equiluminant gratings tend-
antiepileptic drugs, which may reduce the size of the VEPs2,21. ed to be larger than normal and showed a smaller phase advance
To investigate the origin of the different VEP responses of in PSE, although saturation indices were not different from those
2000 Nature America Inc. http://neurosci.nature.com
patients and controls, we measured the amplitude and phase for controls. The present findings do not contradict the previ-
dependence of VEPs on stimulus contrast at two temporal fre- ous report that stationary equiluminant patterns are ineffective in
quency ranges, 410 Hz and 1622 Hz. Whereas the VEP depen- inducing photoparoxysmal EEG activity7.
dence on contrast is comparable for patients and controls at In conclusion, our results indicate that pattern stimuli of rel-
higher temporal frequency, there is a clear difference at the lower atively low temporal frequency and high contrast may be partic-
temporal frequency. The amplitude saturation at high contrasts ularly effective in uncovering cortical hyperexcitability in PSE,
and the phase advance with increasing contrast typically found possibly because of an impairment of contrast gain-control mech-
in normal subjects22 were absent or much reduced in patients. anisms normally present at these temporal frequencies. High-
Interestingly, the contrast threshold and the contrast dependence contrast stimuli endowed with such temporal characteristics are
in the low contrast range were not affected in PSE. The critical common in TV images and in video games, and may be impor-
range of reversal of square-wave patterns reported to elicit epilep- tant in triggering the abnormal cortical response underlying visu-
tic EEG activity (1020 reversals per second)19corresponds to a ally induced epileptic seizures.
fundamental temporal frequency of 510 Hz, in agreement with
our findings of sine-wave temporal modulation. METHODS
Single cells of the primary visual cortex of the cat and mon- Subjects. Subjects were 12 normal volunteers (mean age, 15.2 3.2 years;
key show response saturation and phase advance in response to n = 6 males) and 11 adolescents and young adults (mean age, 18 3 years;
drifting sinusoidal gratings of increasing contrast14,23. These non- n = 3 males) with PSE9 (Table 1). All patients showed high-amplitude
VEPs (with normal waveform) to both bright flashes and checkerboard
linear response properties could result from a contrast gain con-
patterns21. Nine patients were being treated with antiepileptic drugs dur-
trol stage that scales the input contrast taking into account the ing the study. Antiepileptic drugs (especially VPA) attenuate the pho-
average contrast in the surround16 or the average activity of a large toparoxysmal response and may reduce the VEP amplitude2,21. These
population of surrounding cells14,24. In the cat visual cortex, effects are interpreted as resulting from reduced spread of cortical activ-
removing inhibitory effects by local application of bicuculline ity rather than alteration of mechanisms responsible for its generation28.
abolishes VEP response saturation and reduces phase shift, sug- EEG monitoring throughout the VEP sessions did not show epileptiform
articles
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Informed consent was obtained after the nature of the technique and the Japan after seizures from viewing cartoon. The Herald International Tribune
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articles
The Rockefeller University, 1230 York Avenue, New York, New York 10021-6399, USA
Correspondence should be addressed to C.D.G. (gilbert@rockvax.rockefeller.edu)
We studied the transition of stimuli from novel to familiar in visual search and in the guidance of atten-
tion to a particular object. Ability to identify an object improved dramatically over several days of train-
ing. The learning was specific for the objects position in the visual field, orientation and configuration.
Improvement was initially localized to one or two positions near the fixation spot and then expanded
radially to include the full area of the stimulus array. Characteristics of this learning process may reflect
a shift in the cortical representation of complex features toward earlier stages in the visual pathway.
2000 Nature America Inc. http://neurosci.nature.com
In a visual search task, a target must be detected within a field of trally positioned fixation spot. A screen, in which the target was
distractors. The target can be defined by various attributes, such either present in a randomized position or absent, was presented
as color, orientation or form. Depending on the combination of every 3 seconds for a duration of 300 milliseconds (Fig. 1a). The
target and distractors, search efficiency may be influenced by the subjects task was to report whether the target was present. We mea-
number of distractors14. sured the percent of correct responses for a fixed presentation time.
According to feature integration theory, the difficulty of visu- To compensate for guessing, 20% of the trials were a null condition
al search is determined by a targets uniqueness in the map of in which no target was present. A separate false-positive rate was
some elementary feature1. Visual input is processed in two stages. calculated for each experiment. This false-positive rate, fp, was used
The first stage uses a set of retinotopically organized maps coding to adjust the percentage of positive responses, p, according to the
for an elementary attribute such as color or orientation. This formula p = (p fp)/(1 fp) to yield p, the true-positive rate, which
stage operates in parallel across visual space, but it produces no we averaged for all subjects performing each experiment. The false-
information about conjunctions of elementary features. Detec- positive rate was below 3% for all subjects. The tests of significance
tion of conjunctions represents a second stage that operates seri- were carried out using a two-tailed t-test over the data collected from
ally to produce the percept of a whole object. all subjects; error bars correspond to standard deviations.
This theory considers a shape to be a conjunction of elemen- In certain types of search tasks, the target attracts attention
tary features or strokes3. Identifying a shape would therefore require even when the observer has no knowledge about its characteris-
serial search, and the ability to identify it should diminish with the tics. For example, even without a previous cue, a red object
number of distractors. However, this degradation of performance embedded in an array of blue distractors will draw the viewers
can be strongly counteracted by familiarity, even when the low- attention6. Our search task, however, required that the viewer
level features of targets and distractors are held constant. For have explicit knowledge of the object sought. Subjects could not
instance, recognizing the digit 2 among an array of the digit 5 perform the task unless they were instructed to find a triangle of
becomes much harder when rotating the entire image by 90 ren- a particular orientation.
ders the characters less familiar5. Experiments were run in blocks of 150 trials with a target of
Under some circumstances, performance in a visual search a single orientation. Before each block, subjects were informed
task can also be improved by priming. The effect of priming is of the orientation of the target. Each session consisted of eight
thought to be limited to simple visual attributes and is passive different blocks. In two sessions before training, performance
and automatic6,7. Here we show that perceptual learning extends levels on detecting triangles of the four different orientations
the range of priming effects and is important in the ability to were tested. Naive subjects showed an average performance below
guide attention to a particular object. 20% for all different orientations.
We chose a visual search task that involved searching for a tri-
angle (a target defined by form) among an array of distractors, Effects of training
in this case triangles of other orientations (Fig. 1a). We show that After having measured performance levels before training, we
perceptual learning dramatically increases the ability to find a chose a single orientation, and the subject was trained by
shape. Moreover, we show specificity of this learning for visuo- repeating blocks in this particular orientation. Different orien-
topic position, object orientation and object configuration. tations were used as targets for different subjects. All subjects
substantially improved performance over the training period.
RESULTS Training stopped when subjects reached threshold, which we
Performance before training arbitrarily set in the range of 7080% correct responses
We used a visual search task in which the observer was required to (Fig. 1b). For different subjects, the time to reach threshold var-
find a target embedded in an array of distractors. The target con- ied between 4 and 6 days, corresponding to 50007000 trials.
sisted of a triangle of one of four possible orientations (up, left, right To measure the change in performance for the trained and
or down), surrounded by triangles of the other three orientations. untrained orientations, we then repeated the two test sessions in
The triangles were presented in a 5 5 stimulus array with a cen- which subjects were tested on triangles of the four orientations.
articles
Percent correct
specific to the object at the
trained orientation. (a) A
stimulus consisting of a
5 5 array composed of
triangles of 4 possible ori-
entations (right, left, up or
down) was presented for
300 ms. The target, a trian-
Days trained gle of particular orienta-
tion, was present in 80% of
Trained orientation Untrained orientation
c the cases. The distractors
were triangles in the three
remaining orientations. (b)
One subjects progress
Target (not present)
through the course of
Percent correct
Subjects showed an average 5-fold increase of performance in ty of the training effect by comparing the change in performance
detecting triangles of the trained orientation (p = 15.4 5.3% at specific locations within the array. In particular, we wanted to
before training; p = 74.0 2.9% after training; significance, determine if the learning occurred sequentially in different loca-
p < 106) but no significant increase in detection of triangles of tions of the visual field or if the improvement resulted from a
the untrained orientations (p = 19.5 4.7% before training, globally and uniformly increased ability in all the locations of the
p = 21.3 5.0% after training; significance, p > 0.3, average array. Learning tended to occur sequentially in different loca-
over 4 subjects; Fig. 1c). tions of the visual field, expanding from the fovea to the periph-
Once training for one orientation was completed, we waited ery, and the spatial pattern of performance levels was very similar
one month and repeated the test session to examine retention of for consecutive or nearly consecutive blocks (Fig. 2a). Further-
the improvement. There was no change in the training effect after more, the expansion in the spatial coverage of the learning tend-
the 1-month hiatus (p = 74 2.5% after learning and ed to occur between adjacent sites in the array. This spatial
p = 77 1.9% after hiatus, average over 2 subjects, p > 0.2), indi- correlation was not exclusively a function of eccentricity. That is,
cating that the improvement showed no extinction over time if a subject was more likely to detect a target in one particular
(Fig. 1d). After training on triangles of a second orientation, how- position in the array than in another, he would more easily detect
ever, performance on the initially trained orientation declined, drop- a target in the same and neighboring locations in the subsequent
ping from p = 74.0 2.9% after the first training to p = 57.0 3.5% trials. To quantify this, we measured the Euclidean distance
after subjects were trained for 7 days in a second orientation, aver- between the blocks. Put simply, distance is a quantitative mea-
aged over 2 subjects (significance, p < 0.05; Fig. 1d). sure of the spatial differences in performance level between con-
secutive blocks, D1, or blocks separated by greater intervals (D2,
Spatial dependence of the learning D3,..., Dn). We plotted Euclidean distances between positions as a
The results above were averaged over all spatial locations at which function of block separation under three different conditions,
the stimuli appeared. One can examine the visuotopic specifici- either in the real data, in shuffled data (in which the responses
articles
articles
Trained orientation
Untrained orientation the failure of training effects to trans-
Trained region fer to arrowheads suggests that this
Percent correct
articles
articles
location within the array. After each presentation, the subject indicated 10. Karni, A. & Sagi, D. Where practice makes perfect in texture discrimination:
whether or not a target shape was present by pressing the appropriate evidence for primary visual cortex plasticity. Proc. Natl. Acad. Sci. USA 88,
button of a computer mouse. 49664970 (1991).
11. Schneider, W. & Shiffrin, R. M. Controlled and automatic human
The array subtended 4.2 4.2, and a small fixation spot of one information processing: I. detection, search and attention. Psychol. Rev. 84,
arcmin radius (1) was positioned in its center. Figures used as target or 166 (1977).
distractors in the different experiments were equilateral triangles, squares, 12. Shiffrin, R. M. & Schneider, W. Controlled and automatic human
diamonds or arrowheads. The sides of all shapes were 27 in length and information processing: II. perceptual learning, automatic attending and a
general theory. Psychol. Rev. 84, 127191 (1977).
their centers were separated by 54. 13. Treisman, A., Verira, A. & Hayes, A. Automaticity and preattentive
Average distance as a function of block separation Dn, was calculated as processing. Annu. Rev. Neurosci. 105, 341362 (1992).
follows. From each block we calculated a 5 5 matrix (Ma, is the corre- 14. Ahissar, M. & Hochstein, S. Learning pop-out detection: specificities to
sponding matrix to block a) where each position of the matrix (mai,j) is stimulus characteristics. Vision Res. 36, 34873500 (1996).
defined as the level of performance in the corresponding location of the visu- 15. Braun, J. Vision and attention: the role of training. Nature 393, 424425
(1998).
al field in this block (Fig. 2a). We then have an ordered array of matrixes, 16. Ahissar, M. & Hochstein, S. Attentional control of early perceptual learning.
and we can consider the distances between any two of those matrixes. Proc. Natl. Acad. Sci. USA 90, 57185722 (1993).
17. Ito, M., Westheimer, G. & Gilbert, C. D. Attention and perceptual learning
5 5 modulate contextual influences on visual perception. Neuron 20, 11911197
d(Ma, Mb) = (m
i=1 j=1
a
i,j mbi,j)2 (1998).
18. Fahle, M. & Morgan, M. No transfer of perceptual learning between similar
stimuli in the same retinal position. Curr. Biol. 6, 292297 (1996).
We then define the average distance as block separation to be 19. Crist, R. E, Kapadia, M., Westheimer, G. & Gilbert, C. D. Perceptual learning
of spatial localization: specificity for orientation, position and context. J.
2000 Nature America Inc. http://neurosci.nature.com
articles
Cognitive Neuroscience Laboratory, Dept. of Neurology, University of Tbingen, Auf der Morgenstelle 15, 72076 Tbingen, Germany
Correspondence should be addressed to S.T. (treue@uni-tuebingen.de)
Dot patterns sliding transparently across one another are normally perceived as independently mov-
ing surfaces. Recordings from direction-selective neurons in area MT of the macaque suggested that
2000 Nature America Inc. http://neurosci.nature.com
this perceptual segregation did not depend on the presence of two peaks in the population activity.
Rather, the visual system seemed to use overall shape of the population response to determine the
number and directions of motion components. This approach explained a number of perceptual
phenomena, including susceptibility of the motion system to direction metamers, motion patterns
combining three or five directions incorrectly perceived by subjects as comprising only two
directions. Our findings offer insights into the coding of multi-valued sensory signals and provide
constraints for biologically based computational models.
Analysis of visual motion is an important aspect of processing fore use the terms tuning curve (which describes a single neu-
visual information. It is therefore not surprising that primates rons response to many directions of motion) and population
have cells and cortical areas specialized for visual motion pro- response (which describes the response of a population of neu-
cessing. The specialized neurons are direction selective: each rons tuned to different directions of motion to one stimulus)
responds only to a particular subset of directions and speeds interchangeably.
of motion within its receptive field. Cortical areas along the The bell-shaped distribution of population activity suggests
dorsal pathway of the primate visual system are specialized for that the visual system might recover the direction of a stimulus by
the analysis of visual motion; most notably, these include area determining the most active neurons. Such an approach of taking
MT, which is dominated by direction-selective cells, and area the preferred direction of the most active of a population of direc-
MST, which also contains cells encoding more complex tion-tuned neurons as the perceived direction may explain the
motions like rotation or expansion1. MT and MST are arguably ability of the visual system to extract the overall direction of
the most intensively studied extrastriate cortical areas. The motion even for displays combining more than one direction5,6.
curve facing out and left in Fig. 1a is an idealized tuning curve Even for these displays, activity is greatest for neurons whose pre-
of an MT cell to a random dot pattern moving in a single direc- ferred directions match the center of the distribution of stimulus
tion. The response distribution is bell shaped and is well fit by directions.
a Gaussian curve. As is characteristic of direction-selective neu- Not all visual motion displays create a perception of unidi-
rons throughout the visual system, responses are broadly tuned, rectional motion, though. Combination of two patterns sliding
with a tuning width of approximately 90, on average 24 . past each other often creates the impression of two separate sur-
Although neurons differ in their preferred directions, their tun- faces, each moving in a distinct direction. If the percept of a dis-
ing curves are otherwise very similar. If typical tuning curves tinct direction is based on the existence of a peak in the
are combined and plotted as a function of direction preference, population activity, then the perception of transparent motion
we obtain a three-dimensional graph of population responses implies the presence of two peaks. This is either assumed in or a
as a function of both stimulus direction and preferred direc- feature of several models of motion perception79 and cortical
tions of the neurons (Fig. 1a). Experimentally, this three- motion processing10,11.
dimensional plot of activity can be derived by determining If we hypothesize that the response to a transparent stimulus
responses of individual cells to movement in different direc- is simply the scaled sum of the activities evoked by the individual
tions (contour lines in Fig. 1a). The brain, however, must cal- components12,13, we can generate a hypothetical curve for pop-
culate this function from information derived from the ulation activity in response to movement in two directions at a
opposite scenario: a single stimulus simultaneously activates a large angle (Fig. 1c). (Several studies suggest that the response
population of neurons, each preferring a different direction. to two motions is the average of the individual responses, giving
We can predict this population response to a single stimulus a scaling factor of 0.5. Note, however, that the exact value is not
from the three-dimensional plot by taking neural responses along critical, as multiplication by any factor gives the same shape for
a line parallel to the axis denoting direction preference (the curve the population activity as the unscaled sum of the two individual
facing right in Fig. 1a). This population response has the same responses.) However, if the two directions of motion form such
shape as the tuning curve for an individual cell. We will there- a small angle that the two peaks merge (Fig. 1d), the percept
articles
articles
30 transparent
stimulus
30 transparent
Normalized response
60 transparent
60 transparent
2000 Nature America Inc. http://neurosci.nature.com
stimulus
stimulus
90 transparent 90 transparent
stimulus stimulus
Fig. 2. Population activity profiles for transparent motion of various angles. (a) Response profiles for an example cell to the single-direction stimulus
(top curve) and the bidirectional stimuli at 30, 60, 90 and 120 (bottom 4 curves). The x axis plots the stimulus direction relative to the cells pre-
ferred direction. For the transparent motion patterns, the stimulus direction plotted is the average of the two direction components. (b) Response
profiles averaged across all cells after normalizing the firing rates to the response to the preferred single direction (determined by fitting with a
Gaussian curve). (c) Contour plot of the responses across all cells. The data from each neuron are normalized to the highest response in each
response profile. The resulting data points form a horizontal line in the graph at the y position defined by the angle between the two directions pre-
sent in the stimulus normalized by the width of the neurons tuning curve for single directions. The x axis similarly expresses the angle of the stimulus
direction relative to the neurons single-direction tuning width. The four horizontal dashed lines indicate data for the example cell from the left col-
umn. From top to bottom, they show data points for the single direction, 30, 60 and 90 stimulus conditions on the contour plot. Because of the
narrow tuning of the example cell, the data from the 120 stimulus condition fell below the scale. The data are in agreement with our prediction that
the population activity splits into two peaks when the stimulus angle exceeds the bandwidth (when y > 1).
normalized firing rate. The graph forms an inverted Y, clearly at the tuning width of 90 or smaller. Nevertheless, the visual
showing that for ratios less than one (when the angle between system can recover the directions underlying transparent motion
stimuli is smaller than the neurons tuning width), the response with much smaller directional separation. How is this remark-
profile is single-peaked, whereas there are two peaks for larger able ability achieved? Note that, although the population activi-
angles. ty was not multi-peaked, it nevertheless contained information
This demonstrates that the simple scaled sum of the respons- about the underlying directions. Single-lobed activity profiles
es to individual components accounts for responses to multidi- generated by bidirectional stimuli are notably broader than tun-
rectional stimuli very well. Thus, the response profile was not ing curves for a single direction (Figs. 1d and 2). This change in
multi-peaked when the angle between component directions was overall shape of the activity profile might be used by the visual
articles
Fig. 3. Psychophysical stimuli. We created two stimuli with three (b1) and five (b2) directions that
should be directional metamers to the 40 bidirectional stimulus (b). The three and five directions mally a robust method of recovering
and the corresponding dot densities were chosen such that the sum of the individual curves (shown the underlying direction(s), it should
along the right) was virtually identical to the activity evoked by the 40 bidirectional stimulus. If the fail to distinguish between different
population activity was indeed the one proposed here and the only information available to the stimuli when they evoke the same
motion system, these three stimuli (b, b1, b2) should appear to contain the same direction compo- activity profile. Such metamers, stim-
nents. In addition to the potentially metameric bidirectional stimulus (b), we created two other bidi- uli that are perceptually indistin-
rectional stimuli (a and c). These cause population activities different from the three potential guishable despite actual differences,
metamers (as shown in the left graph) and should therefore be perceptually distinguishable from the can be used to psychophysically esti-
other stimuli. Note that the directions in the 50 bidirectional stimulus (c) are present as the
mate direction-tuning bandwidth16.
steepest directional components in stimuli b1 and b2. Because the subjects were asked to judge the
most upward component in all stimuli, stimuli b1 and b2 should result in the same responses as the If the visual system indeed recovers
50 stimulus if the motion system correctly encoded the individual motion components. Tuning direction components of transparent
width for the response profiles used here was assumed to be 90, although note that the width of motion by assessing the width or
the summed activity for the three- and five-direction stimuli (b1 and b2) remains close to the activ- overall shape of the population
ity width evoked by the two-directional stimulus (b) for a large range of tuning-curve widths. response, it should perceive only two
Further material and demonstrations of our stimuli can be viewed at directions in patterns containing three
http://neurosci.nature.com/web_specials/. or five directions, as long as the result-
ing profile of the population activity
matches that expected for two direc-
tions of motion (Fig. 3).
system to identify direction(s) evoking the activity. Component In a series of psychophysical experiments, human subjects were
directions of a bidirectional stimulus can even be approximated asked to report the number of directions perceived in these pat-
(to within 10) by simply subtracting a cells tuning width for terns under eccentric fixation. In more than 94% of the trials with
a single-direction stimulus from the width of the response profile stimuli containing 3 directions, subjects reported perceiving two
to the given bidirectional stimulus. directions. Even the stimulus with 5 directions was reported to
Note that such an approach is different from vector averag- contain just 2 directions in more than 73% of the trials. We pro-
ing, which recovers a direction by calculating the vector sum of ceeded to perform a more direct test of our hypothesis. We pre-
the preferred directions of all activated neurons, weighted by the sented the 3 control stimuli (bidirectional stimuli with
respective activities of those neurons. Such an approach would components at 30, 40 or 50) together with a stimulus com-
yield an intermediate direction whenever two directions are com- prising 3 directions that contained components moving at 0 and
bined. In contrast, a winner-take-all approach predicts perceived 50 (b1 in Fig. 3). If the visual system indeed finds the best-fitting
direction as the preferred direction of the most active neurons15. sum of two Gaussians for this stimulus, subjects should perceive
Note that, again, transparently moving patterns would be per- two directions of motion moving in the same directions as in the
ceived as having a single direction of motion, and, as our data 40 stimulus rather than the 50 components actually present.
show, this direction would be equivalent to that predicted by the Subjects could reliably discriminate between stimuli with 2 direc-
vector-averaging model if the two actual directions of motion tions at 30, 40 and 50, but the 2 direction components per-
formed an acute angle. At larger angles, the population activity ceived in the stimulus containing 3 directions were indeed the
shows two peaks, and the winner-take-all model would pick one same as for the 40 stimulus with only 2 directions (Fig. 4, left).
of the two peaks. Clearly, this cannot account for the perception We wondered if this inability of the visual system to exclude
of two directions of motion when they form either a large angle the influence of movement in the horizontal direction could be
or a perceptually separable acute angle (larger than 10). overcome if we supplied additional segregation cues. We colored
We suggest that the visual system uses another approach to upward- and downward-moving dots red and the horizontally
recover the direction(s) underlying a given population activity moving ones green. Subjects were asked to judge the direction of
across direction-selective neurons. The perceived direction(s) the red, upward-moving component. Subjects still judged the
represent the position(s) of the smallest number of Gaussian- perceived upward motion in the stimulus comprising 3 direc-
shaped activity profiles (with widths equal to that for the aver- tions to be the same as the one in the 40 stimulus (Fig. 4, cen-
articles
Perceived directions
subjects veridically perceived the upward
component, and the responses for the
stimulus with 3 directions did not statisti-
cally differ from those for the 50 stimu-
lus.
DISCUSSION
In summary, we show that the response of
direction-selective neurons in MT to trans- Color Disparity
parent motion was well approximated by
the scaled sum of their responses to the Fig. 4. Psychophysical results. The filled bars show the upward directional components reported
individual motion components. Most for the 30 (bar a), 40 (bar b) and 50 (bar c) stimuli. Error bars indicate standard errors. The
white bar indicates the reported upward direction in the three-direction stimulus (bar b ). Left, in
importantly in the context of this study, the standard condition, the difference between the two middle bars was not significant:1 subjects
population activity profiles were single- reported the same upward component in the 40 stimulus as in the 3-direction stimulus (which
2000 Nature America Inc. http://neurosci.nature.com
peaked for transparent-motion compo- contains a 50 upward component). Center, when oblique pattern components consisting of red
nents moving at acute angles. Our ability dots and the horizontally moving components consisting of green dots were used, the difference
to perceive multidirectional motion is, between the two middle bars was not significant. Right, here the oblique pattern components were
therefore, not dependent on multiple presented with crossed and the horizontal component with uncrossed disparity. In contrast with
peaks in activity profiles across MT neu- the other 2 conditions, subjects reported the same upward component in the 3-direction stimulus
rons; rather, it seems to be based on an as in the 50 stimulus.
analysis of the overall shape of the popu-
lation response, effectively recovering the
Gaussian-shaped profiles of responses to
the individual components underlying the population activity. parent motion patterns, which is strongest for angles of 3040.
This is supported by our demonstration of direction metamers, The population responses to such patterns are somewhat wider
showing an inability of the visual system to recover motion com- than expected from our simple model, but more data and a more
ponents if identical activity profiles could be created by alterna- formal analysis are required to determine whether this could
tive stimuli with fewer directional components. Our finding that account for the perceptual errors observed in motion repulsion.
stereoscopic disparity information but not color information The demonstration of direction metamers also suggests that
helps the visual system to disambiguate these stimuli supports independent access to the direction components in transparent
the proposal that perception of direction in our stimuli involves motion is lost (in the absence of additional cues such as stereo-
signals from area MT, which contains both disparity-tuned and scopic disparity or speed30), even though the front end of the
direction-tuned neurons 1719 . Such neurons would allow segre- motion system seems to require local imbalance of motion sig-
gation of direction components if they lay at different depths, nals for perception of transparency31,32. Stereoscopic disparity,
effectively creating independent population activities for the dif- but not color differences, also help to create perceptual trans-
ferent planes. Some color information is processed in the con- parency in paired random-dot displays33.
text of motion processing20,21, but it seems to be insufficient for The inability to use color as a segregation aid does not seem to
the segregation of motion components in our displays, presum- hold under all circumstances, though. The influence of noise dots
ably because MT neurons are not color-selective enough to sup- on perception by human subjects and monkeys of coherently
port two distinct surface representations based on color moving random dot patterns seems to be reduced if they are of a
information22,23. different color20,21. A number of differences between previous
Our data show that, contrary to previous proposals, the vec- studies and ours might account for these discrepancies; notably,
tors in multidirectional motion do not seem to be encoded indi- these include the requirement of subjects to make precise direc-
vidually by separate populations of neurons. Rather, the broad tion judgments in our experiment and the use of long stimulus
tuning of direction-selective neurons in areas such as MT leads durations and larger dot sizes in the previous experiments, pos-
to a population code in which the overall shape of the popula- sibly allowing eccentric tracking of individual points.
tion activity, and not (at least for acute angles) separable peaks, It is indeed possible to devise a computational model that can
encodes directions of motion. Using the complete population correctly predict the perceived directions34,35, including the two
activity probably also increases signal-to-noise ratios by using all directions perceived when viewing our three-direction stimuli,
available information4,24. from population activities such as those we report here for MT
Such an encoding scheme accounts for a number of motion neurons. Similarly, in some models of motion processing in
phenomena, especially single-direction9 and bidirectional10 after- MT36,37, acute-angled transparent motion results in single-peaked
effects produced by adaptation of subjects with narrow- and activity profiles.
wide-angled bidirectional patterns, respectively. Our data also In summary, using a combination of single-neuron record-
might help account for some results of psychophysical studies ings in monkeys and psychophysics in humans, we demonstrat-
using stimuli that combine many directions or speeds5,2527. Sim- ed a neural code that supports perception of multiple values
ilarly, our data might contribute to an explanation of the per- without requiring separate neuronal populations to encode the
ceptual phenomenon of motion repulsion14,28,29, a tendency of different stimulus values. Given the straightforwardness of this
subjects to overestimate the angle between directions in trans- coding scheme, it is probably not restricted to the encoding of
articles
motion directions. Rather, it might represent a more general In experiment 1 and 3, all dots were black; in experiment 2, the hori-
approach, suggesting that metamers similar to the ones demon- zontally moving dots were either green or an approximately isoluminant
strated here for the domain of visual motion direction might exist red. In experiment 3, RDPs were viewed through a mirror stereoscope.
for other sensory domains. The patterns moving obliquely were presented with 0.25 crossed dis-
parity, whereas the patterns moving horizontally were presented with
0.25 uncrossed disparity.
METHODS
Physiology. All animal procedures were approved by the local animal Subjects. The same twelve subjects served in all experiments. Nine were
research committee and complied with relevant laws and institutional paid volunteers naive to the research aim, and three were from within
guidelines. Standard preparation techniques were used for electrophysi- the laboratory. All had normal or corrected-to-normal visual acuity.
ological recordings from single neurons in area MT38 of three male
macaque monkeys. Cells were characterized as MT neurons based on Task. Psychophysical testing protocols were approved by the ethics com-
their receptive field locations, directional tuning and position in the cor- mission of the University of Tbingen Medical School. The different pat-
tex. MT cells were analyzed if they showed directional tuning (respons- terns were presented in random order in blocks of 160 trials. Subjects
es as a function of stimulus direction were better fit by a Gaussian than by had to report the upward direction component in each pattern. Imme-
a horizontal line, and responses to the preferred direction were at least diately after the moving dot patterns, a line was presented on the screen
fivefold larger than to the null direction). until the subject made a decision. In a two-alternative forced choice pro-
The monkey initiated each trial by depressing a lever, and had been tocol, subjects were required to indicate whether the perceived steepness
trained to respond to a change in luminance of a small spot at the top of of the upward moving component in the RDP was smaller or larger than
the fixation cross by releasing the lever. Throughout a trial, the animals the slope of the line.
2000 Nature America Inc. http://neurosci.nature.com
gaze had to stay within 11.3 of visual angle from the fixation cross. If
the animal broke fixation or did not respond to the spots luminance Data analysis. After each trial, in a 50%-staircase procedure, the line was
change within the reaction-time window (200500 ms after the change), brought toward the perceived direction of motion. For the value of per-
the trial was terminated without reward. Only data from correctly com- ceived direction, the results of leftward and rightward motion of all sub-
pleted trials were analyzed. jects for each of the four stimuli were averaged.
During a trial, a moving random dot pattern was presented inside the Note: further material and demonstrations of our stimuli can be viewed
classical receptive field. Its speed and size was matched to the preferred on the Nature Neuroscience web (http://neurosci.nature.com/web_specials/).
properties of the cell under study. Dots were bright white squares of 0.1
width presented at a density of 5 dots per square degree on an otherwise
dark computer monitor positioned 57 cm from the animal. For single- ACKNOWLEDGEMENTS
directional patterns, all dots moved coherently in the same direction; for
This work was supported by a grant from the MWF Baden-Wrttemberg. We
bidirectional stimuli, half of the dots were assigned to each direction. Dur-
ing stimulus presentation, direction(s) continuously changed at a rate of are grateful to O. Braddick, N. Qian and R.S. Zemel for comments on previous
100 per s (ref. 39). For our analysis, the value of the spike density profile versions of the manuscript.
of the cells responses (kernel sigma, 30 ms) was taken every 50 ms. Con-
trol measurements were taken to ensure that our results, especially the
absence of two peaks in the population response to acute angled trans- RECEIVED 29 DECEMBER 1999; ACCEPTED 25 JANUARY 2000
parent motion, could be replicated using non-changing patterns.
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articles
1 Institute of Medical Science, University of Toronto, and Toronto Western Research Institute, MP14-322, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Canada
2 Department of Medical Imaging, University of Toronto, and Toronto Western Research Institute, MP3-404, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Canada,
3 Department of Surgery, University of Toronto, Division of Neurosurgery, Toronto Western Hospital, and Toronto Western Research Institute, MP14-322, 399
Bathurst Street, Toronto, Ontario, M5T 2S8, Canada
Correspondence should be addressed to K.D.D. (kdavis@playfair.utoronto.ca)
2000 Nature America Inc. http://neurosci.nature.com
Sensory stimuli undergoing sudden changes draw attention and preferentially enter our awareness.
We used event-related functional magnetic-resonance imaging (fMRI) to identify brain regions
responsive to changes in visual, auditory and tactile stimuli. Unimodally responsive areas included
visual, auditory and somatosensory association cortex. Multimodally responsive areas comprised a
right-lateralized network including the temporoparietal junction, inferior frontal gyrus, insula and
left cingulate and supplementary motor areas. These results reveal a distributed, multimodal
network for involuntary attention to events in the sensory environment. This network contains areas
thought to underlie the P300 event-related potential and closely corresponds to the set of cortical
regions damaged in patients with hemineglect syndromes.
The ability to detect changes in the sensory environment is cru- oddball protocols, in which subjects are presented with a train of
cial to survival. It is necessary to attend to such changes to eval- repeating, standard stimuli occasionally punctuated by a different
uate and modify behavior in the face of developing obstructions, oddball stimulus. Our study used a modified version of this
opportunities or threats. For this reason, changes in the sensory approach. In our protocol, the stimuli in each modality were pre-
environment, especially abrupt changes, tend to draw attention sented continuously, but alternated independently between two
involuntarily. A sensory element undergoing a change also inserts different states, A and B. Each alternation was termed a transi-
itself preferentially into awareness. For example, a hiker might tion regardless of whether the direction of the change was from
not notice the constant sound of birds chirping unless they were A to B or B to A (Fig. 1). We used these counterbalanced transi-
to stop abruptly, at which point the hiker would become aware tions between stimulus states, rather than a stereotypical oddball
of both the birds and the sudden absence of noise. When the abil- stimulus, to ensure that activations were due to a general change in
ity to attend to stimuli in the sensory world is lost, as in patients the quality of the stimulus and were not merely due to a difference
suffering from neglect syndromes, awareness of the stimuli is lost in some specific feature of the oddball stimulus as compared with
as well1,2. Understanding the mechanisms by which the brain the standard. A transition occurred in one of the 3 sensory modal-
detects changes in the sensory environment will provide us with ities every 14 seconds in a randomized sequence to minimize the
a better understanding of the mechanisms of both involuntary effects of expectancy and habituation. To identify activations, we
attention and awareness. treated transitions as the stimulus events. This approach enabled us
We used event-related fMRI to identify the network of neu- to identify cortical areas responsive to transitions within a single
roanatomical structures underlying the detection of changes in sensory modality, as well as a cortical network responsive to tran-
the sensory environment. Visual, auditory and tactile stimuli sitions in multiple sensory modalities.
were used to identify areas responding to changes in multiple
sensory modalities. These multimodal areas are of particular RESULTS
relevance to the understanding of higher-order cognitive process- Unimodally responsive areas
es such as the construction of an integrated, multisensory per- To identify unimodally responsive areas, we used a voxelwise t-
ceptual environment, the directing of attention to salient features test to compare the blood oxygenation level-dependent (BOLD)
of that environment and the selection of those features for entry signal two to eight seconds following a transition within a given
into awareness3. modality with the signal two to eight seconds after a transition
Subjects underwent fMRI while being presented with visual, in the other modalities. Based on previous studies, these time
auditory and tactile stimuli. To avoid activation due to response periods encompassed the peaks of the hemodynamic respons-
selection, planning or working memory, subjects were not required es to brief stimuli46. Brain regions satisfying the criteria for uni-
to make any sort of response during the experiment. Instead, they modal activation (see Methods) are therefore maximally
were merely instructed to observe the stimuli passively. Studies responsive to transitions in one modality as compared with the
concerning the detection of stimulus events frequently involve others (Table 1; Figs. 2 and 3).
articles
2000 Nature America Inc. http://neurosci.nature.com
Fig. 1. Excerpt of stimulus-presentation protocol. Visual, auditory and tactile stimuli were presented to subjects simultaneously during imaging. The
visual stimulus was either the blue or the red abstract figure shown; the auditory stimulus was either the sound of running water or the sound of
croaking frogs (actual waveforms shown); the tactile stimulus was either circular brushing or rapid tapping of the lower right leg, using a 6 10 cm
shower brush. At 14-s intervals, in pseudo-random order, 1 of the 3 stimulus modalities underwent a transition from one stimulus type to the other.
A total of 44 transitions (15 visual, 15 auditory, 14 tactile) occurred during the course of the experiment. V, visual; A, auditory; T, tactile.
Visual-specific areas comprised bilateral visual association these areas during the 6 seconds before and after each transi-
cortices of both the dorsal and ventral visual-processing streams, tion, with a 2-second delay to accommodate the hemodynamic
including the fusiform gyrus and middle occipital gyrus. The for- response. Transition-related intermodal inhibition thus appeared
mer area includes inferior temporal regions known to be involved to affect only the visual modality.
in the identification of complex figures such as objects and faces7,
whereas the latter corresponds to area V3 of visual association Multimodally responsive areas
cortex. In addition, there was a locus of activation in the right To identify areas responsive to transitions in all three modalities,
superior parietal lobule, on the superior bank of the intrapari- we used a voxelwise correlation to the predicted hemodynamic
etal sulcus. This area activates for shifts of attention in the visu- response to brief stimuli spaced 14 seconds apart, based on pre-
al modality8. vious studies46. Brain regions satisfying the criteria for multi-
Auditory-specific areas comprised Brodmann areas 41, 42 and modal activation (see Methods) are presented in Table 1 and Figs.
22 of the right and left superior temporal gyri. These areas cor- 2, 3 and 4.
respond to primary and secondary auditory cortex. No other Areas activated by transitions in all three sensory modalities
auditory-specific loci of significant activation were noted. comprised a distributed network of frontal, medial, temporopari-
Tactile-specific areas comprised the secondary somatosenso- etal and insular cortical areas (Fig. 4). The network showed a
ry cortex (S2). Although stimulation was applied only to the right greater overall volume of activated voxels in the right hemisphere
leg (Fig. 1), activation was observed bilaterally, consistent with (5596 mm3) as compared with the left (1244 mm3; Table 1). The
the bilateral receptive fields of many S2 neurons911. No activa- most prominent area of activation was located at the right tem-
tion was observed in the primary somatosensory cortex (S1) for poroparietal junction (TPJ), immediately posterior to the sec-
the right leg. ondary auditory and somatosensory cortices (Figs. 2 and 4). A
Inhibitory mechanisms for attention, both within and much smaller activation was identified in left TPJ. Ventral and pos-
between modalities, are topics of considerable interest. An fMRI terior to the activation in the right TPJ was a small activation in
study of visual cortical activity during binocular rivalry found the right middle temporal gyrus (Figs. 2 and 4). Frontal areas
that extrastriate areas responsive to a particular stimulus show responsive to transitions in all modalities were located bilaterally in
deactivations when a competing stimulus becomes perceptual- the inferior frontal gyrus and frontal operculum. As in the TPJ,
ly dominant12. Intermodal inhibition of visual cortical activity frontal activation was more extensive in the right hemisphere than
during a tactile discrimination task is also reported13. To assess in the left. There were also foci of activation in an anterior and a
the possibility of intermodal inhibition in our study, we exam- posterior region of the right insula. Medial multimodal activations
ined the averaged responses of unimodal-specific areas to tran- were located in the left anterior cingulate and the supplementary
sitions in their own versus the other modalities (Fig. 3). motor areas (CMA and SMA).
Although all unimodal areas showed consistent activation for
their particular modalities, only the visual-specific areas showed DISCUSSION
significant deactivation in response to transitions in other The results of our study reveal a distributed cortical network for
modalities (auditory, t88 = 3.69, p < 0.001; tactile, paired t82 = the detection of changes in the sensory environment, with both
6.04, p < 107), as measured by comparing the BOLD signal of unimodal and multimodal components.
articles
The unimodal components of this network corresponded to Visual unimodal areas showed a significant deactivation in
visual association cortex, primary and secondary auditory cor- response to transitions in other modalities. This finding is con-
tex, and S2. It is noteworthy that V1, V2 and S1 did not show a sistent with a study showing that attention to tactile stimuli deac-
significant response to transitions. Because all stimuli were tivates visual association areas 13. Our study indicates that
applied continuously throughout the experiment, it is unlikely attention-drawing transitions in the auditory modality can also
that these areas were simply inactive during scanning. It is more deactivate visual association cortex. Such intermodal inhibition
likely that their overall level of activation did not change signifi- did not seem to affect auditory or somatosensory association
cantly during or following transitions. The activity of these areas areas, however. The reasons for the unique vulnerability of visu-
may thus reflect a more literal representation of sensory input, al association cortex to transition-related intermodal inhibition
less influenced by habituation or attentional modulation than remain to be investigated.
that of secondary and association cortices. The largest of the multimodal nodes of activation was found
The unimodal auditory activations observed in primary and in the right TPJ. The next highest volumes of activation were in
secondary auditory cortex are consistent with recent findings the right inferior frontal cortex, the left SMA and CMA and right
concerning the detection of sensory events in the auditory modal- anterior insula, with smaller activations in the left TPJ and infe-
ity. Deviant or oddball auditory stimuli embedded in a train of rior frontal cortex, the right posterior insula and the right mid-
standard, repeating stimuli normally evoke a mismatch negativ- dle temporal gyrus. Nonetheless, the total volume of activated
ity (MMN) observable in electroencephalographic (EEG) record- voxels in the two hemispheres, the specific areas activated and
ings. An EEG study of patients with focal cortical lesions suggests their order of prominence show remarkable agreement with neu-
2000 Nature America Inc. http://neurosci.nature.com
auditory association cortex as a major generator of the MMN for roanatomical correlates of sensory neglect. In this syndrome, the
oddballs presented to the contralateral ear14. Our data supports ability to attend to salient stimuli on one side of the body (usu-
this hypothesis by demonstrating the activation of auditory cor- ally the left), whether voluntarily or involuntarily, is lost as a
tex in response to changes in continuous auditory input. result of neurological damage. Patients suffering from neglect
Table 1. Brain regions showing significant activation in response to transitions in visual, auditory, tactile and all three
sensory modalities.
Brain region Brodmann area Coordinates Volume (mm3) t
x y z
Unimodal activations
Visual
R fusiform gyrus 19/37 29 60 14 5835 16.97
L fusiform gyrus 19/37 31 63 15 5689 15.75
R middle occipital gyrus 18/19 31 81 4 2371 16.97
L middle occipital gyrus 18/19 29 81 7 2912 17.53
R superior parietal lobule 18/19 25 62 47 801 12.56
Auditory
R superior temporal gyrus 22/41/42 53 21 5 2185 9.66
L superior temporal gyrus 22/42 51 37 10 1858 10.32
L superior temporal gyrus 41/42 53 15 3 403 8.97
Tactile
R postcentral gyrus (S2) 40 50 28 25 2177 9.15
L postcentral gyrus (S2) 40 59 28 27 1259 8.34
Multimodal activations
articles
Tactile transitions
followed by lesions of the CMA, SMA or prefrontal cortex, espe-
cially in the vicinity of Brodmann area 44 (ref. 1). It should be
remembered that fMRI signal provides only an indirect measure involved in the planning of motor responses, not in the shifting of
of the neural activity of a given area. Nonetheless, the remark- attention or the detection of sensory events. Previously report-
able degree of overlap between the known anatomical correlates ed CMA and SMA activation during spatial shifts of attention
of hemineglect and the network identified in our study empha- might be attributed to the planning and execution of motor
sizes the close relationship between the detection of salient events responses during task performance8. However, we observed SMA
in the sensory environment and the selection of these salient stim- and CMA activation even though our subjects made no such
uli for entry into awareness. Furthermore, it underscores the responses during the course of the experiment. The activations
importance of the identified cortical regions to the performance we observed in CMA and SMA could be attributed to the plan-
of both functions across multiple sensory modalities. ning of unexecuted, involuntary motor orienting responses to
The observation of SMA and CMA activation in this study is the changing sensory input. A more interesting possibility, how-
of particular interest. These areas are generally considered to be ever, is that these regions may have an attentional role in senso-
Fig. 3. Responses to visual, auditory and tactile transitions. Averaged event-related hemodynamic responses to transitions in each stimulus modality
are shown for representative unimodally and multimodally responsive areas. (a) Bilateral fusiform gyrus. (b) Bilateral superior temporal gyrus.
(c) Bilateral secondary somatosensory cortex (S2). (d) Bilateral TPJ. Unimodal areas (ac) respond only to transition events within visual, auditory
and tactile modalities, respectively, whereas the TPJ (d) responds bilaterally to transitions in all three modalities. A significant (p = 0.01) deactivation
of the fusiform gyrus in response to auditory and tactile transitions is visible in (a). Multimodal activation in the right anterior insula, not shown in Fig.
2, is visible in (d). The Talairach z coordinate of each slice is indicated in the upper left-hand corner. Error bars indicate s.e.
a c
Change in signal (%)
articles
Fig. 4. Multimodally responsive brain regions. The plane coordinate of each slice is indicated in the upper left-hand corner. SMA, supplementary
motor area; CMA, cingulate motor area.
ry processing as well as their more generally accepted role in the affected by lesions of the parietal cortex immediately dorsal to
planning and execution of movements. This view is supported the TPJ21,23,24. These EEG findings support the results of our study
by the observation that lesions of the SMA and CMA can give in suggesting the importance of TPJ and prefrontal cortex in the
rise to attentional deficits such as hemineglect1. In addition, detection of salient stimuli across multiple modalities.
recordings of epicortical field potentials in humans demonstrate There remains the possibility that additional multimodal areas
the involvement of the SMA, CMA and other premotor areas in were not detected in our study because their responses habitu-
the anticipation of and attention to forthcoming stimuli18. Our ated rapidly over the first few transitions. For example, although
study cannot conclusively demonstrate the involvement of pre- activation in the dorsolateral prefrontal cortex was not observed
motor areas in attentional processes independent of response in our study, evidence from EEG studies of patients with focal
planning; however, the possibility bears further investigation. cortical lesions suggests that this area may be involved in detect-
The involvement of the TPJ and inferior frontal gyrus in ing novel sensory events in visual, auditory and tactile modali-
detecting changes in continuous streams of sensory input is con- ties14,21,24. Similarly, a study using fMRI indicates that novel
sistent with EEG studies of the effects of focal cortical lesions on auditory events activate the right prefrontal cortex25. In addition,
P300 event-related potentials (ERPs), which are widely used as
markers of attention to salient sensory stimuli19. Lesions of the
TPJ produce a marked reduction in the amplitude of both the
P3a ERP, which is normally elicited by unexpected, task-irrele-
vant stimuli, and the P3b ERP, which is normally elicited by
expected, task-relevant stimuli20,21. Such reductions are observed
Change in signal (%)
articles
a number of studies implicate a set of rapidly habituating areas, Data processing and analysis. Preprocessing, volumetry and analysis
including prefrontal, posterior parietal and hippocampal areas, in were performed using BrainVoyager 3.5 (Brain Innovation, Frankfurt,
the detection of novel events and their subsequent encoding into Germany), implemented on a 450 MHz Pentium II platform. Function-
memory26,27. Furthermore, evidence from EEG studies using task- al data were corrected for within-frame time of acquisition, high-pass
filtered with a cut-off at 0.0175 Hz to remove slow drifts in signal inten-
relevant target and task-irrelevant nontarget oddballs suggests
sity (period >1 min), coregistered with the 3-dimensional anatomical
that the context in which task-irrelevant stimuli are presented, images, transformed into standard stereotactic space31 and resampled at
more generally than their novelty, may affect the physiological a resolution of 3 3 3 mm. Data were smoothed using a Gaussian ker-
response28. Our study was not designed to identify areas respon- nel of 4 mm full width at half maximum to accommodate anatomical
sive only to novel events. This issue will be the focus of future and functional-anatomical variation between subjects. Individual sub-
investigations. jects data were averaged together for group analysis. During the gener-
A widespread, multimodal cortical and subcortical network ation of statistical maps, data were further interpolated to 1 1 1 mm
for the shifting of attention to salient features of the sensory envi- resolution to facilitate estimation of the volume of activation in each
ronment was first proposed on the basis of neuroanatomical and area. To identify unimodally responsive areas, we used a voxelwise t-test
lesion studies29. The results of our study provide an illustration of to compare the BOLD signal two to eight s after a transition within a
given modality to the signal two to eight s after transitions in the other
the unimodal and multimodal elements of such a network in
modalities. We used an initial threshold t > 3.70 for individual 1-mm3
action. The agreement of these results with previous EEG and voxels (n = 130 frames, uncorrected p = 0.00032). To identify multi-
lesion studies provides converging evidence for a close relation- modally responsive areas, we used a voxelwise correlation to the pre-
ship between the detection of salient stimuli in the multisensory
2000 Nature America Inc. http://neurosci.nature.com
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articles
1 Center for Neuroscience and Department of Psychology, One Shields Ave.,University of California, Davis, Davis, California 95616, USA
2 Department of Radiology, University of California, Davis Medical Center, 4701 X St., Sacramento, California 95817, USA
3 Center for Cognitive Neuroscience, LSRC Bldg., Rm. B203, Duke University, Durham, North Carolina 27708, USA
Correspondence should be addressed to G.R.M. (mangun@duke.edu)
Selective visual attention involves dynamic interplay between attentional control systems and sensory brain
structures. We used event-related functional magnetic resonance imaging (fMRI) during a cued spatial-
2000 Nature America Inc. http://neurosci.nature.com
attention task to dissociate brain activity related to attentional control from that related to selective process-
ing of target stimuli. Distinct networks were engaged by attention-directing cues versus subsequent targets.
Superior frontal, inferior parietal and superior temporal cortex were selectively activated by cues, indicating
that these structures are part of a network for voluntary attentional control. This control biased activity in
multiple visual cortical areas, resulting in selective sensory processing of relevant visual targets.
Selective attention enables us to focus awareness on objects and analysis13,14 allowed us to combine the spatial resolution necessary
events that are relevant to our immediate goals. Spatial attention, for localization of neural activity, which this technique provides,
the selective direction of visual attention toward a location, can with neuroimaging methods that selectively extract components
occur covertly, without overt movements of the head or eyes. of hemodynamic activity15 correlated with distinct aspects of com-
Theoretically, mechanisms of covert, voluntary spatial attention plex-task performance.
can be decomposed into elementary mental operations: disen- Here we used event-related fMRI methods to determine which
gaging attention from the current focus, orienting attention to a brain regions were involved in attentional-control processes and
new locus and selectively modulating new stimulus inputs are to distinguish these from areas involved in subsequent selective
three stages proposed in current models1,2. Attentional disen- processing of target stimuli. This was accomplished by separate-
gagement and voluntary orienting can be considered aspects of ly convolving the onsets of the cue and target stimuli with syn-
top-down attentional control, whereas subsequent selective mod- thetic hemodynamic response functions. Subjects oriented
ulation of sensory inputs reflects the result of this top-down con- attention covertly (without moving their eyes) to spatial locations
trol on sensory information processing. based on instructive cues shown at fixation (Fig. 1). Subsequent-
Studies in neurological patients and physiological studies in ly, reversing checkerboard stimuli were presented at both sides of
humans and animals implicate a network of cortical and subcor- the visual field, and subjects were required to discriminate whether
tical regions that support visual selective attention 36. Neu- the checkerboard at the cued location contained some gray checks
roimaging studies identify areas involved in spatial attention in the or only black and white checks and to respond accordingly. All
frontal, parietal, temporal and occipital lobes as well as in subcor- stimuli in the opposite (uncued) hemifield were to be ignored.
tical structures711. However, although neuroimaging studies impli- Based on previous studies implicating various brain regions in
cate various brain regions in selective attention, inherent limitations attentional control5,7,8,11, we hypothesized that attentional-control
of classical neuroimaging analysis impair efforts to relate neural processes in response to an instructive cue directing spatial atten-
structures/networks to specific attentional operations. Specifical- tion would activate a network of regions in frontal and parietal
ly, such methodologies use averaging of hemodynamic responses cortex. Additionally, based on ERP studies in humans12, regions
over seconds or minutes, a time course that is too long to permit of occipital cortex corresponding to portions of visual space indi-
direct viewing of brain activity related to the subcomponents of cated by the cue should show spatially specific changes in activity
task performance. As a result, human neuroimaging studies to date in anticipation of selective processing of target stimuli. We also
are only partly successful in distinguishing between neural activi- expected lateralized changes in activity of occipitotemporal regions
ty related to top-down attentional-control processes and activity in response to the bilateral target stimuli to reflect spatial atten-
related to selective sensory and motor processing. tion711. In line with these predictions, we found distinct patterns
Recordings of event-related neuroelectric potentials (ERPs) allow of neural activation for attention-directing cues as compared with
the selective averaging of responses to different classes of events those for subsequent target stimuli, thereby distinguishing top-
and, thus, serve to index mechanisms of attentional control sepa- down attentional control from selective modulations of target pro-
rately from selective stimulus processing during spatial attention12. cessing in sensory structures.
However, although ERPs provide information about the timing of
neural processes, the limited spatial resolution of this approach hin- RESULTS
ders identification of the neural structures involved in attentional Subjects discriminated black and white checkerboards from those
control. Advances in functional magnetic resonance imaging (fMRI) containing some gray checks at the cued location with a mean
articles
Fig. 1. Stimuli and timing. For simplicity, we show here the inverse
black/white contrast of the actual stimulus screen; subjects saw a black Cue
background with white outline boxes and fixation cross, and the cues were 500 ms
overlapping isoluminant yellow and blue arrows (pointing in opposite direc- +
tions). Subjects were told which color arrow to attend for the entire ses-
sion and were required to covertly orient their attention to the visual field ISI
location indicated by that arrow. After a variable interstimulus interval 1000 ms (17%) or
8160 ms (83%)
(1000 or 8160 ms), the target checkerboards appeared bilaterally, and sub- +
jects were required to perform a discrimination of the checkerboard stim-
uli at the attended location only.
Time Target
750 ms
4 Hz reversal
+
articles
be activated in response to both targets and cues. Additionally, selective attentional activations in response to the direction of
bilateral targets activated several regions of the ventral and dorsal the instructive cue, but before target presentation, using the atten-
occipital cortex of both hemispheres. Cue and target responses tion-related activations to targets as regions of interest. Direct
were directly statistically compared (Tables 1 and 2; Fig. 4). statistical evaluation of the cue-left versus cue-right effects
To investigate the consequences of lateralized spatial attention revealed relative increases of activity in extrastriate cortex con-
on stimulus processing, the event-related neural activity evoked tralateral to the direction of attention. These contralateral cue-
by the bilateral target stimuli was evaluated for attention to the related activations overlapped closely with the extrastriate regions
left versus right. In line with previous neuroimaging studies in showing attentional modulations in response to the subsequent
humans9,10,1619 and related findings in animals3, attending to the targets (Fig. 6). That is, there was a relative increase in activity
left visual field increased activity in the right ventral occipital cor- in the visual cortical regions representing the spatial locations of
tex, whereas attending to the right increased activity in the left the expected target stimuli before the target stimuli were actual-
occipital cortex (Fig. 5a). In addition, activity in a more dorsal ly presented. These differential activations were not related to
region of visual cortex was also modulated by spatial attention. inherent sensory features of the cues that activated separate cor-
To relate these attention-related activations to specific visu- tical regions in control sessions.
al-field areas, the vertical and horizontal meridian were mapped
in two subjects to identify the borders of the early cortical visual DISCUSSION
areas20,21. Although the spatial smoothing used in the present This study used event-related fMRI methods to distinguish
analysis tended to blend together the attention-related activa- between neural networks involved in top-down attentional-
2000 Nature America Inc. http://neurosci.nature.com
tions in adjacent visual cortical areas, the contralateral attention- control processes and those participating in the subsequent spa-
related activations in occipital cortex can be seen to cross multiple tially selective attentional processing of target stimuli. A network
visual area borders from V2 through V4 (Fig. 5b), an observa- of cortical areas including superior frontal, inferior parietal and
tion in line with other reports10,18,22. superior temporal brain regions were implicated in top-down
Models of spatial attention propose that attention effects on attentional control because they were found to be active only
target processing result from a gain-control mechanism that in response to instructive cues. In contrast, other regions of the
enhances the excitability of extrastriate neurons coding attend- cortex, including the ventrolateral prefrontal cortex, anterior
ed regions of visual space4,9,23. To investigate this possibility, we cingulate and supplementary motor area, were found to be
further examined extrastriate cortex for evidence of spatially selectively activated by the target stimuli, suggesting that these
articles
articles
the cue stimuli when one might expect working memory process- coding to-be-attended locations were differentially activated in
es to be engaged. Rather, these cortical regions were activated response to the instruction to orient attention by the cue. Single-
upon presentation of the target stimuli. This finding may be con- unit studies in non-human primates show an increase in back-
sistent with a role in inhibitory filtering of information from the ground firing rates of neurons coding an attended region of space
ignored hemifield when the bilateral targets appeared3638. in the absence of stimuli45, and proposals based on neuroimag-
Previously, the supplementary motor area and the anterior ing data suggest that increased background firing rates may medi-
cingulate cortex were hypothesized to be part of attentional con- ate attentional facilitation in visual cortex46,47. The present data
trol systems2,8. The present finding that these areas were activat- show a precise spatial correspondence between areas of visual cor-
ed in response to the target stimuli but not the attention-directing tex activated by top-down processes and those showing subse-
cues, however, suggests a role in either the selective analysis of quent selective modulations of target processing. Moreover, these
target features or decisional processes engaged once the relevant priming effects in visual cortex were shown to follow the retino-
stimulus is presented, such as selecting appropriate motor actions topic mapping of the visual fields onto visual cortex as attention
based on target features39,40. was shifted from left to right locations by the cue instructions.
As predicted by the previous literature, target-evoked activity In summary, a top-down attentional control system was isolat-
in posterior lingual and fusiform regions was enhanced in the ed using event-related fMRI methods. This network included the
hemisphere contralateral to the direction of attention. This sup- superior frontal cortex, inferior parietal cortex, superior temporal
ports the idea that spatial attention-related increases in regional cortex and portions of the posterior cingulate cortex and insula.
blood flow in extrastriate cortex are related to enhanced process- The present evidence indicates that this top-down control system
2000 Nature America Inc. http://neurosci.nature.com
ing of target stimuli falling within the attentional spotlight, as sug- modulated activity in extrastriate cortex as a function of where spa-
gested by prior ERP4,41,42 and functional imaging studies 9,10,1619 tial attention was directed in the visual field. These spatially selective
in humans and single-unit studies in monkeys3,43. In addition, a effects in extrastriate cortex, observed before the presentation of
more dorsal region of visual cortex was also enhanced by the direc- the target stimuli, resulted in modulations in the activity evoked
tion of attention. This region may be area V3a, which has an upper by subsequent target stimuli in precisely corresponding early cortical
visual field representation in dorsal visual cortex44 and which is areas. Hence, it seems that effects of spatial attention in visual cor-
activated in response to flashing checkerboard patterns18. tex before target onset may reflect changes in sensory-neural
Priming of visual neurons by top-down attentional control excitability as a function of top-down control. These findings pro-
signals is suggested by the finding that regions of visual cortex vide direct evidence for clear distinctions between the neural mech-
articles
anisms of attentional control and the resulting modulation of sen- Stimuli and parameters: stimulation of meridia and control sessions. In
sory signals. The challenge for the future is to relate activity in each two of the subjects, visual field mapping and cue control sessions were per-
of these identified brain regions to the specific neurocomputation- formed. Meridian stimuli for visual area mapping were 9.0 2.7 checker-
board patterns aligned on the vertical or horizontal meridia and composed
al operations they subserve during visual spatial attention.
of alternating 0.9 square black and white checks, reversing at 15 Hz. Stim-
uli were presented in alternating 22-s blocks of right versus left meridia
METHODS and in a separate run, upper versus lower meridia. The alternating pattern
Stimuli and task parameters: cueing study. Each trial began with a cue of vertical and horizontal meridia activations in visual cortex were used to
that consisted of two overlapping isoluminant arrows presented at fixa- identify the borders between V1, V2, VP and V4 (refs. 44, 47). For the cue
tion (one blue and one yellow, pointing in opposite directions, each 1.9 sensory-control session, the cues used in the main experiment were flashed
tall 0.96 wide; duration, 500 ms). To eliminate differential activations every 750 ms (duration, 500 ms) for 22 s alternating with 22-s no-stimu-
based on the physical differences in right versus left cues, half the subjects lus blocks. A synthetic hemodynamic response function was convolved
were instructed to orient attention based on the direction of the blue with the box-car function that represented the periodic alternation in the
arrow, and the other half were required to use the yellow arrow. The rel- experimental conditions to yield the model response function that includ-
evant arrow pointed randomly to the left or right. Target locations were ed a temporal derivative. Low-frequency drifts were modeled and filtered
demarcated by white outline rectangles (8.1 wide 7.2 tall, centered 5.6 with a high-pass filter cutoff of 88 s, and the data were temporally smoothed
above the horizontal meridian and 7.7 lateral to the vertical meridian) by convolving the time series with a 4-s full width at half maximum
that remained on the screen throughout all runs. (FWHM) Gaussian kernel. In another cue control, the cue and target stim-
Target stimuli appeared within the white outline rectangles and consisted uli were identical to that used in the main attention study except that the
of black and white checkerboards (8.1 7.2, composed of 0.9 square cues were not instructive as to where to orient attention. (Subjects had to
articles
Fig. 5. Selective processing of target stimuli and retinotopy (single sub- Selective attention (targets)
ject). (a) A T2-weighted coronal slice (y = 72) showing the activations
due to selective visual attention on target processing for one subject.
(single subject)
The left column shows the regions showing greater activity when atten- a
tion was focused on the left visual field. The right column shows those Att. left > att. right Att. right > att. left
areas showing greater activity when attention was focused on the right. L R 6 5 L R
Attention-related activations in the contralateral fusiform and lingual
4
gyri are outlined in black (coordinates of left hemisphere maximum, 4
32, 60, 8; Z = 5.26; right hemisphere maximum, 20, 68, 8; Z = 5.39). 3
A more dorsal region in the cuneus (not outlined in black) also showed 2
2
enhanced activity contralateral to the direction of attention (left hemi-
1
sphere maximum = 32, 64, 28, Z = 4.02; right hemisphere
maximum = 24, 72, 28, Z = 6.27). (b) Left panel, ventral visual areas for 0 0
the same subject shown at top, determined using the meridia scans Z value
(see Methods) and traced onto contrast-inverted, T2-weighted MRI scans
(y = 72). Right panel, effect of attention to target stimuli on responses in
lingual and fusiform gyri (shown as thick black outlines) overlaid onto the b
subjects retinotopically mapped visual areas. Attention-related target pro- Visual areas Selective attention
cessing extends from V2 through V4 in the ventral visual-processing stream. L R L R
2000 Nature America Inc. http://neurosci.nature.com
V1
V2
detect gray checks in both hemifields). During these sessions, target stim- VP
uli (250 ms) were presented on only 22% of trials with an ISI of 300 ms,
whereas the other 78% of trials (catch trials) consisted only of the cue stim-
V4
uli; only these catch trials were analyzed, using event-related methods (see
below). Attend Attend
right left
Scanning procedures. Functional images were acquired with a Signa resolution proton density and T2-weighted fast spin echo (FSE) images were
Advantage 1.5 Tesla whole-body MRI system (General Electric, Wauke- also obtained in the coronal plane during the same scanning session. The
sha, Wisconsin), with an elliptical end-capped quadrature radio fre- anatomical scans were acquired with the following parameters: TR = 4.2 s;
quency and local gradient head coil (Medical Advances, Milwaukee, TE = 17 and 136 ms; echo train = 8; FOV = 22 cm, matrix = 512 256 (inter-
Wisconsin). Images were acquired using T2*-weighted gradient-recalled polated to 512 512 for display), slice thickness of 6 mm, with an interslice gap
echo, echo planar imaging (EPI) in the coronal plane with a repetition of 2 mm. In each EPI acquisition, 144 images were acquired at each of the
time (TR) of 2.75 seconds, an effective echo time (TE) of 40 ms and a 22 slice locations, for a total acquisition time of 396 s (6 min, 36 s).
flip angle (FA) of 90. Twenty-two interleaved slices were collected with
a 22-cm field of view (FOV), 64 64 matrix, slice thickness of 6 mm and Image processing. The first 16 images at each slice location (initial 44 s of
an interslice gap of 2 mm, yielding a voxel size of 3.43 3.43 6.00 mm3. acquisition) were discarded from the analysis. The remaining 128 images at
Fourier image reconstruction included N/2 ghost correction using image each slice were used for the time-series analyses. Images were realigned and
phase correction48. Neural activation was detected via the blood oxygenation corrected for movement artifacts. The anatomical T2 images were coregis-
level dependent (BOLD) contrast mechanism15, which provided differences tered with the proton density images and the functional images for each
in signal intensity based upon differences in tissue T2* and perfusion. High- subject. Each subjects T2 (and coregistered proton density scans) were then
Cuneus
Z value
Cuneus
2 2 activations were 01 cm from those in response to
Lingual Fusiform targets and were all significant at p < 0.001, uncor-
1 1
rected. (Because regions of interest analyzed for cue
0 L R L R 0 responses were specified a priori by analyses for atten-
tion effects on target responses, we give uncorrected
p values for cue responses.)
articles
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articles
1 Department of Neurology and Neurological Surgery, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, Missouri 63110, USA
2 Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, Missouri 63110, USA
3 Department of Anatomy and Neurobiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, Missouri 63110, USA
4 Department of Psychology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, Missouri 63110, USA
2000 Nature America Inc. http://neurosci.nature.com
Human ability to attend to visual stimuli based on their spatial locations requires the parietal cortex.
One hypothesis maintains that parietal cortex controls the voluntary orienting of attention toward a
location of interest. Another hypothesis emphasizes its role in reorienting attention toward visual
targets appearing at unattended locations. Here, using event-related functional magnetic resonance
(ER-fMRI), we show that distinct parietal regions mediated these different attentional processes.
Cortical activation occurred primarily in the intraparietal sulcus when a location was attended before
visual-target presentation, but in the right temporoparietal junction when the target was detected,
particularly at an unattended location.
Acute structural damage to the right temporoparietal cortical involved in voluntary orienting, then it should be activated when
junction (TPJ; inferior parietal lobule and superior temporal an observer attends to a location before presentation/detection
gyrus) in humans produces a complex clinical syndrome charac- of a visual target. If TPJ is necessary for reorienting to a visual
terized by the inability to attend and respond to objects positioned target, then its activation should follow the presentation/detec-
in the left visual field (unilateral visual neglect)13. Some symp- tion of the target, particularly when it is presented at an unat-
toms of neglect may reflect a deficit in reorienting attention toward tended location. We tested these predictions using ER-fMRI and
new stimuli in the visual field opposite to the lesion (contrale- an ANOVA-based procedure17. This method has two important
sional field)47. Patients with parietal lesions can detect visual stim- characteristics: it can separate the responses to events presented
uli in the contralesional field when correctly cued to their within the same cognitive trial, and it is sensitive to differences
locations, but they are slow or fail to detect the same stimuli when in both magnitude and timing of responses. Unlike other pub-
attending to other locations4. This deficit is more severe after right lished ER-fMRI methods1821, this method makes no assump-
than left parietal lesions5, and is localized to the TPJ7. These find- tions about the shape of the underlying response function.
ings suggest that the posterior parietal cortex near TPJ may be
critical for reorienting the focus of attention toward visual stimuli RESULTS
appearing at unattended locations (reorienting hypothesis). Normal observers were given a cue indicating the most likely
Other data suggest that posterior parietal cortex near/along location of a subsequent target stimulus they were required
the intraparietal sulcus (IPs), which separates the superior from to detect, according to a protocol modified from a published
the inferior parietal lobule, is involved in voluntarily directing procedure4. The stimulus display consisted of a central fixa-
attention to a spatial location (voluntary orienting hypothesis). tion cross flanked on either side by square boxes. The length of
Neurons in the IPs increase firing rate when a monkey attends each arm of the central fixation cross subtended 16 minutes
to a location while preparing a response811. Human functional of visual angle. The boxes (size, 1) were placed at 3.3 of visu-
brain imaging shows activations in the IPs (and superior pari- al angle to either side of the fixation spot. Accurate fixation
etal lobule) when observers voluntarily pay attention to and detect of the central cross-hair was emphasized throughout the
peripheral visual stimuli, with or without concurrent eye move- experiment. At the beginning of a trial, a cue arrow pointing
ments1216. It is unknown to what extent areas in human and to the left or right box was superimposed on the fixation cross.
monkey IPs are homologous. The arrow indicated the most likely location of a subsequent
These complementary functional anatomical theories (reori- target stimulus, and leftward or rightward arrows were equal-
enting to targets in the TPJ and voluntary orienting in the IPs) ly probable. The cue arrow remained on the screen for one
make specific predictions about which regions should be acti- MR frame (2360 ms; cue period).
vated while attending to a spatial location and, subsequently, The sequence of events following presentation of the cue
when detecting a visual target there. If IPs is preferentially arrow depended on the type of trial. On a cue trial (20% of the
articles
trials), the trial ended immediately after cue presentation. The We used this protocol during whole-brain measurements of
end of the trial was signaled by a change in the color of the fix- blood oxygenation level dependent (BOLD) responses on a
ation cross from green to red. During a cue trial, a subject shift- Siemens Vision 1.5 T magnet. The time course of the BOLD
ed attention toward the location indicated by the cue and response for each trial type and each trial period (cue period, test
maintained it there until the end of the trial. On a noise trial period/noise, test period/valid, test period/invalid) was estimat-
(20% of the trials), the cue period was followed by a test period ed in each subject using linear regression. Pixel-wise and region-
lasting 2 MR frames (4720 ms) in which no target was present- al ANOVAs were used for appropriate statistical contrasts17 (see
ed. During noise trials, the subject presumably shifted and Methods).
maintained attention on the cued location for a longer time
than during a cue trial (7080 versus 2360 ms; Fig. 1). On a valid Behavior
trial (44% of the trials), the cue period was followed by a test Reaction times for target detection were faster on valid than
period during which a target appeared at the location indicated invalid trials (380 ms versus 426 ms, F1,11 = 21.92, p = 0.0007),
by the cue. The target was a white asterisk that appeared in one indicating that subjects used the cue arrow to attend to the loca-
of the square boxes for 100 ms. On an invalid trial (16% of the tion of the target. No responses were recorded during noise and
trials), a target appeared during the test period at the uncued cue trials.
location. The cue arrow correctly predicted the target location
on 73% of the trials in which a target was presented. These four Imaging
trial types (cue, noise, valid, invalid) were randomly intermixed. During the cue period, a series of ventral and dorsal visual
Subjects were instructed to press a button as quickly as possi- regions were active; these included bilateral anterior fusiform
ble upon detection of the target and to withhold responses on (Fus; x, y, z atlas coordinates, 35, 57, 20, right; 31, 55, 16,
cue or noise trials. left), lateral occipital (LO;31, 83, 0, left; 27, 87, 0, right)22,23,
Table 1. List of parietal regions during cue and target periods (averaged over valid and invalid targets) and showing
significant validity effect.
Cue Target Valid invalid
Regions x y z Z-score x y z Z-score x y z Z-score
L pos IPS 25 67 48 7.58 25 65 48 6.94
L ant IPs 25 57 46 7.55 25 57 42 7.27
L vIPS 23 67 32 7.42
27 75 26 7.23 27 77 20 7.16
R ant IPs 27 59 52 6.75 33 51 48 7.84 39 47 48 4.09
R pos IPs 21 65 52 6.62
R vIPS 29 71 22 6.01 27 71 30 6.63
R IPL 53 45 20 8.53 53 49 30 5.12
R STG 51 55 4 5.41 57 45 12 4.44
R PC 7 73 32 7.71 7 75 34 4.28
L PC 9 71 40 7.37 5 71 34 4.27
See Figs. 2 and 3 for anatomical labels. Coordinates (x, y, z) correspond to the Talairach atlas.
articles
a b Cue period
c d MR frame number
cue ITI
MR frame number
Fig. 2. BOLD responses during cue and target periods. (a) ANOVA F map transformed to Z map for the cue period (averaged over subjects, cue
direction). Left, sagittal slice 25 mm left of midline. Right, coronal slice 45 mm posterior to center of atlas space. Yellow lines indicate corresponding
planes of section. Parietal regions with sustained responses to cues are labeled in red: posIPs, posterior intraparietal sulcus; antIPs, anterior intrapari-
etal sulcus; vIPs, ventral intraparietal sulcus. The lateral occipital region LO (labeled in white) showed a transient response to the cue. (b) BOLD time
courses in different regions (averaged over subjects, cue direction and hemispheres) during the cue period. Response typically peaked two frames
after onset of cue arrow on frame 1. ITI began on frame 4. (c) Target period (averaged over subjects, valid and invalid targets). Parietal regions with
prevalent responses to targets are shown in red label: TPJ, temporoparietal junction; Precun, precuneus. Several motor-related responses are also
shown: SMcx, sensory-motor cortex; Put, putamen; Cbl, cerebellum. (d) BOLD time courses in IPs and TPJ (averaged over subjects, cue direction,
target field and hemispheres) during cue and target (valid) periods. Target was randomly presented around frame 3. IPs/cue, IPs response during cue
period; IPs/valid, IPs response during valid-target period; TPJ/cue, TPJ response during cue period; TPJ/valid, TPJ response during valid-target period.
MT+ (45, 69, 2, left; 45, 69, 4, right) 24,25 and ventral reflect longer times required for processing cues related to ori-
(vIPs), anterior (ant IPs) and posterior intraparietal sulcus (pos enting toward and maintaining attention at the cued location.
IPs; Fig. 2a, left; see Table 1 for coordinates of parietal foci). No To further test this idea, we compared BOLD responses of these
significant activation was detected in the TPJ during the cue regions during the noise period, in which subjects maintained
period (Fig. 2a, right). The IPs activity did not reach the sur- attention at a peripheral location for 4.72 seconds after the offset
face of the inferior parietal lobule, but spread into the superior of the cue arrow. Across all regions active during the noise peri-
parietal lobule. The vIPs region was located at the junction of od, only antIPs and vIPs showed sustained activity (ANOVA
dorsal occipital and parietal cortex, just dorsal and anterior to regions frame, F77,924 = 7.80, p = 0.0001), with responses of the
the V3A representation26. left hemisphere more sustained than those of the right hemi-
The time course of the BOLD response during the cue peri- sphere (Ant IPs, F 7,84 = 3.76 p = 0.0014; vIPs, F 7,84 = 5.40,
od was more sustained within the intraparietal sulcus (posIPs, p = 0.0001). Hence, after the presentation of the cue arrow, some
antIPs, vIPs) than in occipital regions (Fus, LO, MT+; ANOVA IPs regions (antIPs, vIPs) showed a sustained BOLD response
regions frame, F56,672 = 5.18, p = 0.0001). We compared a tran- that was maintained during the noise period, during which sub-
sient time course in two occipital regions (LO, MT+) with more jects attended to the cued location for almost five seconds while
sustained time courses in antIPs and posIPs (Fig. 2b). The dif- waiting for the target stimulus.
ference in response duration cannot be explained by a differ- During the target period, many visual and motor regions
ence in the peak magnitude. LO and posIPs, for example, were active for both valid and invalid targets (Fig. 2c). All occip-
showed similar peak magnitudes on frame 3, but different ital regions that were active during the cue period also respond-
response duration (ANOVA regions frames 3 and 4 only, ed to target presentation, and these responses were significantly
F1,12 = 24.3, p = 0.0003). Similarly, antIPs and MT+ had similar stronger for targets in the contralateral visual field. In parietal
magnitudes, but the response was more sustained in antIPs cortex, significant responses were recorded in antIPs, posIPs,
(ANOVA regions frames 3 and 4 only, F1,12 = 7.22, p = 0.02). vIPs, precuneus (Precun) and the TPJ, where the activation was
Whereas transient time courses in occipital regions probably much stronger in the right than the left hemisphere (Fig. 2c;
reflect visual processes related to the presentation of the foveal Table 1). BOLD responses in these parietal regions during the
cue, the more sustained time courses in intraparietal cortex may cue and valid-target periods were compared by ANOVAs. Data
articles
L valid
a b c L valid
L invalid L invalid
Fig. 3. BOLD responses for valid and invalid targets. (a) Coronal section through TPJ cortex (~47 mm posterior). ANOVA (validity frame) F map
transformed to Z map. Voxels that show significant validity effect (different BOLD responses for valid and invalid targets) independent of the visual
field of the target. BOLD time courses (averaged over subjects) were estimated in right inferior parietal lobule (R IPL; b) and right superior temporal
gyrus (R STG; c) during the target period for valid and invalid targets in left and right visual field.
2000 Nature America Inc. http://neurosci.nature.com
for antIPs and posIPs were collapsed, as the borders between the location was voluntarily attended, independent of processes relat-
two regions could not be readily identified. Responses of IPs ed to target detection (for instance, visual responses and their
(Ant + Pos) and vIPs regions were stronger during the cue peri- attentional modulation or motor responses). In contrast, the right
od, whereas those of the TPJ and precuneus region were stronger TPJ responded to target presentation more strongly when the
during the target period (F21,252 = 4.29 p = 0.0001; Fig. 2d). These target occurred at an unattended location.
findings indicate that the voluntary orienting and maintenance
of attention to a location primarily recruited the cortex within Voluntary orienting of attention
the IPs. In contrast, target detection recruited the TPJ (and pre- Two findings support a role for the IPs in the voluntary orient-
cuneus), although a significant target response was also evident ing and maintenance of attention to a target location. First, the
in IPs (Fig. 2d). presentation of a cue arrow indicating the most likely location
To test whether TPJ was preferentially involved in reorient- of a subsequent visual target triggered transient responses in
ing attention toward novel unattended stimuli, the time cours- occipital cortex, but more sustained responses in IPs. Transient
es of the BOLD responses during the target periods for valid responses in occipital cortex may reflect the encoding of the cue,
and invalid trials were compared. The strongest validity effect which was probably completed within a half second27. In con-
(difference in the BOLD response for valid and invalid trials) trast, sustained parietal responses may reflect the required shift
across the whole brain was localized in the right TPJ cortex, toward and maintenance of attention at the cued location for
with separate foci in the inferior parietal lobule (IPL) and supe- the entire cue period (2360 ms). Second, when the delay after
rior temporal gyrus (STG; pixel-wise ANOVA Z-score = 5.12; the cue offset (noise period) was extended to 4.72 seconds, forc-
Fig. 3a; Table 1). A regional ANOVA confirmed that the valid- ing subjects to maintain attention at the cued location for longer,
ity effect was significant and independent of the visual field of IPs was the only brain region that showed a sustained response
the target (ANOVA frame validity, R IPL, F 7,84 = 8.13, during the delay.
p < 0.0001; R STG, F7,84 = 7.53, p < 0.0001). The response in Our results extend findings of increased activity in extras-
right IPL and STG was more sustained for valid than invalid triate, frontal and IPs cortex without sensory stimulation when
targets in each visual field (Fig. 3b and c; ANOVA, frames 5 and subjects attend to a specific object at a specific location during
6 only validity, R IPL, F 1,12 = 5.212, p = 0.041; R STG, an identification task20. Those activations are thought to reflect
F1,12 = 6.51, p < 0.025). Significant validity effects were also an attentional signal-biasing activity in visual cortex before
localized bilaterally in the precuneus and near the intersection stimulus presentation28. Here we show, in a simpler detection
of the right IPs and postcentral sulcus. This latter region did protocol, that IPs was uniquely active when attention was ori-
not overlap with the IPs regions active during the cue period ented toward and maintained at a relevant location, suggesting
(vector distance, 17 mm; Table 1). Significant validity effects that IPs is the source of spatial biases observed in extrastriate
not discussed here were also observed in other regions outside visual cortex.
of parietal cortex. Cue-related activity in IPs may underlie an attentional sig-
nal that marks a location of interest9,11 or an intentional signal
DISCUSSION that prepares a response (eye movement or hand reaching)
We tested two functional-anatomical theories about the role of toward that location 10. However, attending to direction of
posterior parietal cortex in visuospatial attention. One theory, motion also drives IPs regions before the presentation of any
supported by studies of neglect patients with parietal lesions, motion target17. Activations are found in IPs during shifts in an
proposes that the TPJ cortex is necessary for reorienting toward object feature (for instance, color or shape)29 or between per-
visual targets appearing at unattended locations. Another theory, cepts during binocular rivalry30. These results suggest that the
based on single unit and imaging data, proposes that cortex along intraparietal cortex may be involved in visual selection beyond
the IPs is involved in the voluntary orienting of attention toward the selection of locations.
a location. Our results provide direct confirmation of both views, The BOLD response in IPs was time locked to different
showing that IPs was active before target presentation when a processes in the course of a trial. Early in the trial (cue and noise
articles
period), the response was controlled by voluntary orienting critical for visual reorienting, and dissociates this region from
processes, whereas the response was controlled by detection voluntary orienting in nearby IPs.
processes later in the trial (target period; Fig. 2d). The coexis-
tence of different processes in human IPs resembled that observed METHODS
in monkey IPs, where neurons also manifest activity time locked Subjects. Thirteen subjects (6 female, 7 male; aged 1838) were recruit-
to different components of a task (for instance, visual, attentional, ed from the Washington University community following procedures
memory, oculomotor or reaching)911,31,32. Although spatial over- approved by the local human studies committee. All subjects were strong-
lap between regions of activation during different protocols (visu- ly right-handed as measured by the Edinburgh handedness inventory44
and had normal or corrected-to-normal visual acuity and no significant
al attention versus eye movements) in previous imaging studies abnormal neurological history. Informed consent was obtained from
was used to examine colocalization of processes16,33, the enhanced each subject. Before the MR session, subjects participated in one behav-
temporal resolution of new ER-fMRI procedures17,20,34,35 allows ioral session during which they were trained to perform the task while
a richer and more realistic view of the temporal dynamics of acti- maintaining central fixation. Eye movements were monitored with elec-
vation in the human brain. tro-oculography, and all subjects were able to perform the task without
breaks of fixation (resolution, 1.5). Eye movements were not recorded
Target detection during the fMRI session. Although we cannot rule out the occurrence
The right TPJ (and precuneus) was specifically engaged during of small eye movements during the fMRI session, several arguments
target detection. Unlike other parietal regions that showed both diminish the likelihood of this possibility. The visual set-up was identi-
cal to the one used in the psychophysical session, in which eye move-
cue and target responses (antIPs, vIPs), right TPJ and precuneus
2000 Nature America Inc. http://neurosci.nature.com
ments were monitored and found negligible. The detection task was not
showed little if any response to the cue. demanding in terms of acuity. Subjects reported no problems in main-
The pattern of activation in the TPJ cortex fits two main fea- taining fixation, in agreement with many studies involving detection
tures of unilateral spatial neglect. The much stronger activation tasks27. Additionally, there was no activity in the frontal eye field during
on the right than the left TPJ agrees with the clinical2,3,36 and neu- the noise period, when the tendency to look at the peripheral box was
ropsychological findings5 that neglect is more severe following strongest. Reaction times were not collected from one subject because
right TPJ lesions. The stronger TPJ response following the pre- of equipment malfunction.
sentation of a target at an invalid location is also consistent with
clinical and experimental4,7 observations that neglect is worse for Apparatus. Stimuli were generated by an Apple Power Macintosh com-
objects presented at unattended locations. puter and projected onto a screen at the head of the bore by a Sharp LCD
projector. Subjects viewed the stimuli through a mirror attached to the
Activation of the right TPJ may underlie the process of spa- head coil. Subjects recorded behavioral responses by pressing an MRI-
tially redirecting the focus of attention toward the location of compatible fiber-optic key held in the right hand.
unattended stimuli. This reorienting was indexed by longer reac-
tion times for invalid targets than for valid targets; thus, the sen- Data analysis and fMRI scan acquisition. An asymmetric spin-echo,
sitivity of the TPJ activation to target validity implies that the echoplanar imaging sequence was used to measure blood oxygenation-
BOLD signal, despite its slow temporal resolution (seconds), can level-dependent (BOLD) contrast (TR = 2.36 s, TE = 50 ms, flip
track neuronal processes that yield behavioral differences of a angle = 90). Each scan consisted of 128 frames during which 16 con-
few tens of milliseconds (the difference in reaction time between tiguous 8 mm axial slices were acquired (3.75 3.75 mm in-plane res-
valid and invalid trials). olution). Anatomical images were acquired using a sagittal MP-RAGE
sequence (TR = 97 ms, TE = 4 ms, flip angle = 12, inversion time
Another possibility, however, is that activity in right TPJ cor-
T1 = 300 ms). Functional data were realigned within and across runs to
tex is related to spatially nonselective neural processes triggered correct for head movement and coregistered with anatomical data.
by reorienting to an unattended target. Interestingly, TPJ dam- Whole-brain normalization was applied to equalize signal intensity
age reduces the amplitude of P300 scalp electrical potentials, across subjects. In each subject, hemodynamic responses (8 frames
which are commonly elicited by the detection of infrequent visu- long) were estimated at the voxel level using the general linear model.
al, auditory and somatosensory targets during spatial and non- The design matrix was defined using impulse-basis functions such that
spatial tasks37. The right TPJ is also selectively activated when at each frame, the data were modeled as the sum of the overlapping
observers monitor the environment for infrequent targets (for hemodynamic response produced by each task effect and a linear trend.
example, vigilance38), and is the region most densely innervated The use of catch trials (trials in which only the cue stimulus was pre-
by noradrenergic projections from the locus coeruleus39 that are sent) made it possible to estimate unique responses for the cue, noise,
target-valid, and target-invalid periods even though the cue response
thought to mediate vigilance and arousal. Damage to the right overlaps noise and target responses in each full trial17 (J.M.O. et al.,
TPJ can cause vigilance problems in patients with unilateral Soc. Neurosci. Abstr. 24, 1178, 1998). A random-effects analysis was
neglect40. Changes in the level of vigilance have a slower time performed by entering the individual time points of each hemodynamic
course than shifts of attention41 and, therefore, might produce response into voxel-level ANOVAs45. These ANOVAs had two main
stronger and more sustained right TPJ responses. effects, time and task. The main effect of time was used to determine
A dissociation of function between intraparietal and tem- which voxels were activated. The resulting F-maps were corrected for
poroparietal cortex may explain why a verbal cue directing atten- multiple comparisons using a Gaussian random fields approach46. F-
tion toward the contralesional field can transiently reduce neglect statistics at each voxel were converted to equivalent Z-statistics. These
in some neglect patients, who typically have damage in the right Z-maps were used to delineate regions of interest. Separate ANOVAs
were then run at the regional level to determine the task effect of cue
TPJ region. This effect, extensively used by therapists to amelio-
direction (left, right), noise direction (left, right), visual field of the
rate unilateral visual neglect42,43, may reflect the preserved acti- target (left, right) and target validity (valid, invalid).
vation of the IPs, which mediates the allocation of attention by
cognitive cues. Normal orienting, however, can be maintained ACKNOWLEDGEMENTS
only for a short time in neglect patients based on voluntary strate- This research was supported by NIH EY00379 and EY12148 (M.C.). We thank
gies. Typically, orienting involves both cognitive and sensory-dri- Thomas Conturo, Avi Snyder and Erbil Akbudak for technical support.
ven mechanisms 27. This study, along with lesion analyses of
patients with TPJ damage, indicates that the right TPJ region is RECEIVED 27 SEPTEMBER 1999; ACCEPTED 6 JANUARY 2000
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