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Article history: In Parkinson's disease (PD), there is a significant loss of noradrenergic neurons in the locus
Accepted 4 December 2010 coeruleus (LC) in addition to the loss of dopaminergic neurons in the substantia nigra (SN).
Available online 11 December 2010 The goal of this study was to determine if the surviving LC noradrenergic neurons in PD
demonstrate compensatory changes in response to the neuronal loss, as observed in
Keywords: Alzheimer's disease (AD). Tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH)
Parkinson's disease mRNA expression in postmortem LC tissue of control and age-matched PD subjects
Alzheimer's disease demonstrated a significant reduction in the number of noradrenergic neurons in the LC of
Locus coeruleus PD subjects. TH mRNA expression/neuron did not differ between control and PD subjects,
Tyrosine hydroxylase but DBH mRNA expression/neuron was significantly elevated in PD subjects compared to
Dopamine β-hydroxylase control. This increase in DBH mRNA expression in PD subjects is not a response to neuronal
Norepinephrine transporter loss because the amount of DBH mRNA expression/neuron in AD subjects was not
mRNA significantly different from control. Norepinephrine transporter (NET) binding site
Immunohistochemistry concentration in the LC of PD subjects was significantly reduced over the cell body region
as well as the peri-LC dendritic zone. In PD subjects, the loss of dendrites from surviving
noradrenergic neurons was also apparent with TH-immunoreactivity (IR). This loss of LC
dendritic innervation in PD subjects as measured by TH-IR was not due to LC neuronal loss
because TH-IR in AD subjects was robust, despite a similar loss of LC neurons. These data
suggest that there is a differential response of the noradrenergic nervous system in PD
compared to AD in response to the loss of LC neurons.
Published by Elsevier B.V.
⁎ Corresponding author. Northwest Network for Mental Illness Research, Education, and Clinical Center (116), Puget Sound Health Care
System, 1660 S. Columbian Way, Seattle, WA 98108, USA. Fax: +1 206 768 5456.
E-mail address: szot@u.washington.edu (P. Szot).
Fig. 1 – TH mRNA expression in the LC of age-matched control (n = 8) and PD (n = 7) subjects. (A) The number of TH positively
labeled neurons at the 50% and 70% level of the LC of age-matched control and PD subjects. In PD subjects, there is a significant
decrease in the number of TH positively labeled neurons at both levels of the LC (50% level p = 0.009, 70% level p = 0.008).
(B) Expression of TH mRNA/neuron in the LC of control and PD subjects at the 50% and 70% levels. All labeled neurons that were
counted as positively labeled were also quantitated for the amount of TH mRNA expression/neuron. There is not a difference in
the amount of TH mRNA expression/neuron in PD subjects as compared to control age-matched subjects. The bottom two
images are dark-field photomicrographs of TH mRNA-labeled neurons in age-matched control (bottom left) and PD (bottom
right) at the 50% level of LC. Asterisk (*) indicates significant difference from age-matched control subjects. Arrow indicates a TH
mRNA-labeled neuron. Scale bar = 100 μm.
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then dopamine is converted to NE by DBH, which is specific Therefore, TH mRNA expression was analyzed only at the 50
to adrenergic neurons. NE transporter (NET) binding sites and 70% levels of the LC in both control and PD subjects (Fig. 1).
were measured in the LC of PD subjects and age-matched Although limited, these measurements are critical and
control subjects as a marker of dendritic innervation, to illuminating because the mid to caudal portion of the LC is
determine if it is altered in the LC of PD subjects. In addition, thought to innervate regions in the midbrain area, including
TH-immunoreactivity (IR) was measured in the LC of PD, AD, the SN/VTA (Fallon and Loughlin, 1982; Loughlin et al., 1982).
DLB and control subjects to support data generated with TH TH-positive labeled cell numbers from control subjects in this
mRNA and NET binding. These measurements would study yielded values at each level within the range that was
indicate if the surviving LC noradrenergic neurons in PD observed in the control subjects of previously published work
subjects result in a similar response as observed in AD (Szot et al., 2006). Fig. 1A (and photomicrographs) shows that
subjects (Szot et al., 2006). in the LC there was a significant loss of TH-positive neurons in
PD subjects at both the 50% level (88% reduced) (p = 0.009) and
70% level (71% reduced) (p = 0.008). Fig. 1B (and photomicro-
2. Results graphs) shows that in the surviving LC neurons in PD subjects
TH mRNA expression (grain count)/neuron did not differ
2.1. In the LC of PD subjects there is a significant loss of between control and PD subjects at the two different levels.
TH positively labeled neurons with no compensatory response
2.2. In the LC of PD subjects there is a significant loss of
TH is considered the rate-limiting enzyme in the synthesis of DBH positively labeled neurons but the surviving neurons
NE; changes in the expression of this protein will alter the level demonstrate a compensatory increase
of NE at terminal regions. Atlas matching of tissue specimens
of PD and control subjects indicated that many of the PD DBH is the synthetic enzyme responsible for the conversion of
subjects lacked the rostral portion of the LC (30% level). This dopamine (DA) to NE and is localized to noradrenergic neurons.
portion of the LC may be missing from PD subjects because the DBH mRNA expression serves as another marker to examine the
most caudal portion of the SN overlaps anatomically with the noradrenergic neuronal loss in PD subjects. The number of DBH-
most rostral portion of the LC. Since the SN is such an positive neurons in both control and PD subjects (Fig. 2A and
important region in PD, studies would require the entire SN. photomicrographs) was very similar to the number of TH-positive
Fig. 2 – DBH mRNA expression in the LC of age-matched control (n = 8) and PD (n = 7) subjects. (A) The number of DBH positively
labeled neurons at the 50 and 70% level of the LC of age-matched control and PD subjects. In PD subjects, there is a significant
decrease in the number of DBH positively labeled neurons at both levels of the LC (50% level p = 0.007, 70% level p = 0.03).
(B) Expression of DBH mRNA/neuron in the LC of age-matched control and PD subjects at the 50% and 70% levels. All labeled
neurons that were counted as positively labeled were also quantitated for the amount of DBH mRNA expression/neuron. There
is a significant increase in the amount of DBH mRNA expression/neuron at the 50% (p = 0.03) level of the LC in PD subjects,as
compared to control age-matched subjects. The bottom two images are dark-field photomicrographs of DBH mRNA-labeled
neurons in age-matched control (bottom left) and PD (bottom right) at the 50% level of LC. Asterisk (*) indicates significant
difference from age-matched control subjects. Arrow indicates a DBH mRNA-labeled neuron. Scale bar = 100 μm.
BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 2 4 0 –2 52 243
50
2.3. In the LC of AD subjects there is a significant loss of
DBH-positive neurons and no compensatory response 25
DBH Grains/cell
increase in DBH mRNA expression in response to the loss of LC
neurons should also be detectable in AD subjects, who also 300
exhibit a severe loss of LC neurons and compensatory
adaptations in response to this loss (Szot et al., 2006). Thus, 200
we measured DBH mRNA expression in the LC of the AD
subjects, plus DLB, that were previously studied (Szot et al., 100
2006). Quantitation of the number of DBH mRNA-labeled
neurons in AD and DLB subjects indicate a significant 0
30% 50% 70%
reduction from control subjects at the 30% (F(2, 38, 40)=17.1, Level of LC
p < 0.0001), 50% (F(2,37,39) = 38.0; p < 0.0001) and 70% (F(2,39,41) =
29.7; p < 0.0001) levels of the LC (Fig. 3A). Two-way ANOVA Fig. 3 – DBH mRNA expression in the LC of age-matched control
indicated a significant difference in TH-positive-labeled (n = 15), AD (n = 15) and DLB (n = 13) subjects. (A) The number of
neurons between the 30% level to the 50% (F(4,114) = 67.9, DBH positively labeled neurons at the 30 (F(2,38,40) =17.1,
p < 0.001) and 70% level (F(4,114) = 86.2, p <0.001) in control p < 0.0001), 50 (F(2,37,39) = 38.0, p < 0.0001) and 70% (F(2,39,41) =29.7,
subjects; between the 30% level and 70% level (F(4,114) = 46.0, p < 0.0001) level of the LC of age-matched control, AD, and DLB
p < 0.001) and between the 50% level and 70% level (F(4,114) =31.5, subjects. In AD and DLB subjects, there is a significant decrease
p < 0.05) in AD subjects; and between 30% level and the 70% in the number of DBH positively labeled neurons at all three
level (F(4,114) = 30.4, p < 0.05) in DLB subjects. The loss of DBH levels of the LC. (B) Expression of DBH mRNA/neuron in the LC
mRNA-positive labeled neurons in AD subjects was similar to of age-matched control, AD, and DLB subjects at the 30%, 50%
DLB subjects at the all three levels of the LC (approximately and 70% levels. All labeled neurons that were counted as
70–80%). This assessment of LC noradrenergic neuronal loss in positively labeled were also quantitated for the amount of DBH
AD and DLB subjects with DBH mRNA is similar to what was mRNA expression/neuron. There is not a difference in the
observed using TH-positive labeled cells (Szot et al., 2006). amount of DBH mRNA expression/neuron in the surviving LC
However, DBH mRNA expression (grain count)/neuron was not neurons in AD and DLB subjects as compared to control
significantly different between control, AD and DLB subjects at age-matched subjects. Asterisk (*) indicates significant
any level of the LC, indicating that DBH mRNA expression does difference from age-matched control subjects.
not demonstrate compensatory changes in response to LC
neuronal loss (Fig. 3B).
Fig. 4 – NET binding site concentrations over the LC region in age-matched control (n = 8) and PD (n = 7) subjects. Top panel: NET
binding site concentrations over LC cell body region and over the peri-LC dendritic zone at the 50% and 70% level of the LC in
age-matched control and PD subjects. NET binding site concentrations are significantly reduced in PD subjects at both levels of
the LC and in both regions of the LC as compared to age-matched control subjects (cell body region 50% level p = 0.006, 70% level
p = 0.03; dendritic region 50% level p = 0.012, 70% level p = 0.015). Bottom panel: Autoradiographic image of NET in the LC of
age-matched control and PD subjects. Asterisk (*) indicates significant difference from age-matched control subjects. The dark
circle represents NET binding over the peri-LC dendritic zone and the gray oblong represents NET binding over LC body region.
Fig. 6 – Photomicrographs of TH-IR labeling in the LC at the 70% level in control (A and C), AD (B) and PD (D) subjects. White
arrowhead indicates TH-IR labeled cell body and dark arrowhead indicates a TH-IR labeled fiber tract. Scale bar = 100 μm.
Studies in postmortem human tissue do not indicate the example, when NE levels are reduced, the neurotoxic damage
order of dopaminergic and noradrenergic neuronal loss in PD. of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and
However, clinical studies of PD subjects indicate that the methamphetamine on dopaminergic neurons in the SNpc is
noradrenergic system may be affected before the dopaminer- enhanced (Bing et al., 1994; Fornai et al., 1995, 1996, 1997, 1999;
gic system. In the progression of this disorder, the loss of Marien et al., 1993; Mavridis et al., 1991). In dopamine β-
dopaminergic neurons is not observed until Stage 3 (out of six hydroxylase (DBH) knockout mice that cannot synthesize NE
stages) (Braak et al., 2003a,b, 2006), and approximately a 70% from DA, motor deficits develop with age that mimic PD
loss of dopaminergic neurons is required to observe PD (Rommelfanger et al., 2007). Conversely, increased NE levels
symptoms. During the earlier stages of the disorder, non- reduce the neurotoxic effect but do not completely protect the
motor preclinical symptoms of PD are observed. These dopaminergic neurons (Rommelfanger et al., 2004; Srinivasan
preclinical PD symptoms include hyposmia (Berendse et al., and Schmidt, 2004). These animal studies suggest that
2001; Ponsen et al., 2004), REM-sleep disorder (Boeve et al., reduced NE input results in a greater loss of dopaminergic
2003; Scheneck et al., 2003) or autonomic dysfunction (Boeve et neurons to neurotoxic damage; reduction in NE input after a
al., 2003), which can be attributed to neuropathological dopaminergic neurotoxin does not affect the recovery of the
changes in the noradrenergic nervous system. dopaminergic system (Bing et al., 1994; Fornai et al., 1997),
There is no clear evidence in human subjects to indicate a suggesting that a loss of noradrenergic innervation can
role for the CNS noradrenergic nervous system in the support enhance the loss of dopaminergic neurons.
of the dopaminergic system. Human studies suggest that the The lack of compensatory changes in the surviving LC
noradrenergic nervous system is affected in the early stages of neurons of PD subjects as shown by TH mRNA, NET binding
PD, as indicated by the non-dopaminergic symptoms de- sites and TH-IR in the LC region are in contrast to what was
scribed above, but the consequence for dopaminergic neurons observed in the surviving LC neurons of AD (Szot et al., 2006).
is unknown. Increasing synaptic NE with an α2-adrenorecep- In AD, there is a significant loss of LC noradrenergic neurons;
tor antagonist reduces the detrimental side effects of L-DOPA however, the surviving LC neurons demonstrate a significant
treatment without affecting improved motor function increase in the expression of TH mRNA (grain count)/neuron
(Gomez-Mancilla and Bedard, 1993; Henry et al., 1999; Grondin and dendritic sprouting to the surrounding peri-LC dendritic
et al., 2000; Rascol et al., 2001; Savola et al., 2003; Fornai et al., zone (Szot et al., 2006). Also as indicated above, the response
2007; Fulceri et al., 2007). Animal research suggests that the of DBH mRNA expression (grain count)/neuron in PD sub-
noradrenergic nervous system may protect dopaminergic jects is in contrast to what is observed in the surviving LC
neurons from damage by neurotoxins (Gesi et al., 2000). For neurons in subjects with dementia. These results indicate
BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 2 4 0 –2 52 247
that noradrenergic neuronal loss alone does not necessarily (grain count)/neuron. The data are expressed as the average
result in a compensatory response of the surviving neurons. of grains per cell ± SEM at each level of the LC for each PD
Mechanisms of neuronal loss (either directly at the LC or subject.
degeneration from terminals) may have a major role in the
response of the surviving neurons. However, in PD subjects,
the data indicate that the surviving LC noradrenergic 5. DBH mRNA
neurons do not appear to be compensating as measured by
immunohistochemistry for synthesizing enzymes for nor- Tissue preparation and labeling of the DBH oligonucleotide
epinephrine (NE) (Gasper et al., 1991). Our data would probes was performed as described previously for oligonu-
suggest noradrenergic function is reduced in PD. cleotide labeling (Szot et al., 1997). For each subject, three
In summary, our studies indicate that surviving noradren- consecutive slides at each level of the LC were labeled with
ergic neurons in the LC of PD subjects respond differently from DBH mRNA oligonucleotide probes. Three different oligonu-
those in AD subjects. This suggests that external circum- cleotides (regions 478–529, 928–979 and 1339–1390) of the
stances surrounding each of these disorders may affect how human DBH sequence (Kobayashi et al., 1989) were used and
surviving LC noradrenergic neurons respond to neuronal loss. labeled as described above for TH mRNA. The oligonucleo-
Chan-Palan (1989) and German et al. (1992) drew similar tide probes were 3′ end labeled with [33P]dATP (PerkinElmer,
conclusions when examining the pattern of LC neuronal loss Boston, MA) using terminal deoxyribonucleotidyl transferase
in PD and AD subjects. (Invitrogen, Piscataway, NJ). The DBH probe contained
1.38 × 106 cpm/50 μl for the PD subjects and 1.17 × 106 cpm/
50 μl for the AD subjects. Slides were washed as described in
4. TH mRNA in situ hybridization detail in previously published work for oligonucleotides (Szot
et al., 1997) and apposed to film (Eastman Kodak, Rochester,
Tissue preparation and labeling of the TH oligonucleotide NY) for 4 days at room temperature, and then the slides
probes was performed as described previously for oligonu- were coated with NTB2 Nuclear Track Emulsion (undiluted)
cleotide labeling (Szot et al., 1997). For each subject, three (Eastman Kodak) and stored at −20 °C for 7 days. Films and
consecutive slides at each level of the LC of PD subjects slides were developed as described previously (Szot et al.,
were labeled with TH mRNA oligonucleotide probe. The TH 1997). Quantitation of DBH mRNA expression was similar to
probe consisted of three separate oligonucleotide probes to that performed by Szot et al. (2000) and described above for
the following nucleotides of the published human sequence TH mRNA.
(O'Malley et al., 1987) (GenBank accession number X05290): Due to the variability of labeling of the oligonucleotide
326–377, 520–571, and 1309–1360. The oligonucleotide probes and parameter for analysis of the labeling, the
probes were 3′ end labeled with [33P]dATP (PerkinElmer, quantitative amount of DBH mRNA expression (grain count)/
Boston, MA) using terminal deoxyribonucleotidyl transfer- neuron in the control group for AD/DLB subjects cannot be
ase (Invitrogen, Piscataway, NJ). The TH probe contained compared to the control group for PD subjects. Because these
1.04 × 106 cpm/50 μl and was washed as described in detail variables can affect labeling, all experimental groups are run
in previously published work with oligonucleotides (Szot et in the same assay and analyzed at the same time. DBH mRNA
al., 1997). Slides were apposed to film (Eastman Kodak, expression in PD subjects was a separate experiment from
Rochester, NY) for 5 days at room temperature, and then DBH mRNA expression in AD/DLB subjects; comparison of
the slides were coated with NTB2 Nuclear Track Emulsion expression/neuron cannot be made between control subjects.
(undiluted) (Eastman Kodak) and stored at −20 °C for 7 days.
Films and slides were developed as described previously
(Szot et al., 1997). 6. NET binding sites
Quantitation of TH mRNA expression in control and PD
subjects was similar to that performed by Szot et al. (2000). NET binding sites were measured in the LC of PD subjects with
3
All quantitation was done blind to the subject's condition H-nisoxetine (American Radiolabeled Chemicals, St. Louis,
and by a single individual to reduce variability. The number MO). From each subject, four consecutive slides from each LC
of cells that achieved labeling threefold higher than back- level were run: three slides for total binding and the fourth
ground were counted on the three slides at each level of the slide for nonspecific binding. Slides were thawed at room
LC for each subject and expressed as TH positively labeled temperature for 10 min, and then 600 μl/slide of incubation
cell ± SEM. Experimental data were analyzed using the buffer (~3 nM 3H-nisoxetine in 50 mM Tris buffer with 300 mM
computer program GraphPad Prism (v. 5.0, GraphPad Soft- NaCl and 5 mM KCl, pH 7.7) was placed over the tissue. Slides
ware Inc.). The density of TH mRNA expression (grain count)/ were incubated for 90 min at room temperature and then
neuron in PD subjects was determined by measuring the washed twice for 2 min in ice-cold 50 mM Tris buffer, pH 7.4,
amount of silver grains over the cell bodies of labeled dipped in ice-cold distilled water to remove salts, and then
neurons that were threefold higher than background under rapidly dried under a stream of cool air. Nonspecific binding
20X dark-field illumination with a side-mounted light using was defined in the presence of 1 μM mazindol. Slides were
the MicroComputer Imaging Device system (MCID) (Imaging apposed to Biomax MR film (Eastman Kodak) for 8 weeks.
Research, St. Catharines, Ontario, Canada). Therefore, all Films were developed and analyzed as described previously
labeled neurons that were counted as positively labeled were (Szot et al., 1997). Specific binding was obtained by taking the
also quantitated for the amount of TH mRNA expression total average value minus nonspecific value in the same
248 BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 2 4 0 –25 2
region. Specific binding for 3H-nisoxetine constituted 60–70% Statistical difference between control and PD subjects was
of total binding. assessed by two-tailed unpaired Student's t-test. Statistical
Quantitation of NET binding sites was performed over the difference between control, AD and DLB subjects was assessed
cell body region and over the peri-LC dendritic zone of each by one-way ANOVA followed by a post hoc Tukey's test. Two-
subject and expressed as optical density as determined by way ANOVA was used to compare the effect of different LC
MCID. All quantitation was done blind to the subject's levels in the different subject groups. Bonferroni tests were
condition and by a single individual to reduce variability. used to compare the different subject groups. p value less than
Cell body region was defined by comparing NET binding 0.05 was considered significant.
labeling on film to TH and DBH mRNA labeling on each Simple linear regressions were performed between NET
section, while labeling beside the cell body region was binding in the cell body region and peri-LC dendritic zone to
considered the peri-LC dendritic zone. The peri-LC dendritic the number of TH positively labeled neurons in the LC of
zone is the region where the vast majority of LC afferents control and PD subjects using GraphPad Prism, with signifi-
affect the activity of LC neurons; LC afferents rarely go cance at p < 0.05. This analysis determined if the surviving LC
directly to the LC cell bodies (Aston-Jones et al., 2004). This neurons in PD are sprouting.
method has been used previously to demonstrate compen-
satory changes in the surviving LC noradrenergic neurons in
AD subjects (Szot et al., 2006). 7. Experimental procedures
TH-IHC was performed in each PD (n = 7) and age-matched Postmortem tissue was obtained from several sources: NICHD
control (n = 8) subject, as well as in control subjects (n = 15) age- Brain and Tissue Bank for Developmental Disorders (BTBDD),
matched for AD (n = 15) and DBL (n = 13) subjects that were used Alzheimer's disease Research Center (ADRC) at the University
to measure DBH mRNA expression. For PD and control of Washington, Emory Tissue Bank and University of Califor-
subjects, two consecutive frozen LC sections from the 70% nia San Diego (SD), where permission for use of tissue in
level were used, while a single frozen LC section from each scientific experiments was obtained. PD is commonly charac-
control, AD and DLB subjects at the 70% level were used. The terized by various motor deficits including tremor, rigidity and
slides were air dried and fixed in 4% paraformaldehyde/PBS for bradykinesia (Singh et al., 2007). All PD subjects (n = 7) in this
8 min at room temperature. Sections were permeabilized in study had exhibited these symptoms, though it is unknown if
1% Triton X-100/PBS for 30 min before the melanin in LC was any PD subjects in this study received pharmacological
bleached by incubating slides in 10% H2O2/PBS for 22 h. intervention (L-DOPA) to alleviate the symptoms. Medical
Sections were blocked in 5% non-fat milk in PBS supplemented records of control subjects (n = 8) indicated no clinical,
with 10% normal goat serum and 1% BSA for 1 h at room neurological, or psychiatric illness or evidence of cognitive or
temperature, followed by incubation with rabbit polyclonal functional decline and had no obvious neuropathology during
anti-tyrosine hydroxylase (AB152, Millipore, Temecula, CA) at autopsy. Table 1 contains information on age, sex, postmor-
1:500 for 2 h and biotinylated anti-rabbit secondary antibody tem interval (PMI) and duration of PD (when known). Subjects
at 1:200 for 30 min at room temperature. Finally, sections were (control and PD) that had been diagnosed with depression
incubated in an avidin–biotin complex (Vector's Vectastain before death or had taken any antidepressant medication
Elite ABC kit, Burlingame, CA), and the reaction product was were excluded because depression and antidepressants can
visualized with 0.05% diaminobenzidine (DAB)/0.01% hydro- alter the noradrenergic nervous system (Bauer and Tejani-
gen peroxide in PBS. Negative controls with secondary Butt, 1992; Lacroix et al., 1991; Harro and Oreland, 2001;
antibody alone did not immunostain tissue sections (data Ordway et al., 2003). Subjects with a history of alcohol or drug
not shown). abuse were also excluded. The original goal was to exclude PD
The number of TH-IR positively labeled cell bodies were subjects with impaired cognition (Boller et al., 1980; Gasper
counted in the LC of control, AD, DLB and PD subjects and and Gray, 1984; Hakim and Mathieson, 1979; Mayeux et al.,
expressed as the average ± SEM. TH-IR labeled fibers in the LC 1992), but because of the high prevalence of cognitive
was assessed by a semi-qualitative method, the amount of TH- impairment in PD, some of the PD subjects used exhibited
IR fibers was scored as light (1), moderate (2) or intense (3) for mild cognitive impairment (MCI) prior to death (Table 1).
each subject. Scoring was done blind to the subjects condition
and a single individual to reduce variability. Scores were 7.2. PD tissue
averaged for each group and presented as the mean ± SEM.
Photomicrographs were taken with a digital camera and The LC tissue blocks obtained from the sources described
imported into Adobe Photoshop for mounting. To optimize above were stored at −70 °C until sectioned. Serial coronal
visualization of staining, photomicrographs were modified, sections (20 μm) were cut on a cryostat, thaw mounted onto
when necessary, by adjusting brightness and contrast. Fisher Super Frost slides, and stored at −70 °C. The rostral-to-
caudal distance of the LC was determined for each case by
6.2. Statistical analysis examining sections stained with thionin every 500 μm. The
rostral (or 0%) was defined as the beginning of the trochlear
Experimental data were analyzed using the computer pro- nucleus, and the caudal pole (100%) ended at the rostral level
gram GraphPad Prism (v. 5.0, GraphPad Software Inc.). of the trigeminal motor nucleus (Hoogendijk et al., 1999). After
BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 2 4 0 –2 52 249
Table 1 – Specifics on age, PMI, sex and duration of PD for each subject.
Source Disease Age/sex PMI (h) Dementia Duration (y)
BTBDD = Brain and Tissue Bank for Development Disorder; ADRC = Alzheimer's Disease Research Center at UW; SD = UC San Diego. Tissue from
SD has not been disclosed to us until the studies are done.
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