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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Age-dependent measures of anxiety and cognition in male histidine decarboxylase knockout (Hdc / ) mice
Summer F. Acevedo a , Hiroshi Ohtsu b , Theodore S. Benice a , Angela Rizk-Jackson a , Jacob Raber a,b,c,
a

Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USA Department of Engineering, Tohoku University, School of Medicine, Aoba-ku, Sendai 980-8775, Japan c Departments of Neurology, Division of Neuroscience, ONPRC, Oregon Health and Science University, Portland, OR 97239, USA
b

A R T I C LE I N FO Article history: Accepted 22 November 2005 Available online 17 January 2006 Keywords: Histamine Open field Lightdark Elevated plus maze Elevated zero maze Rotorod Spatial learning and memory Water maze Passive avoidance

AB S T R A C T Histidine decarboxylase deficient (Hdc/) and wild-type male mice on the C57Bl6/J background were used to determine the role of histamine in brain function. 35 (Y) and 1214 (MA) month-old Hdc/ mice showed hypoactivity and increased measures of anxiety in the open field, lightdark, elevated plus-maze, and elevated zero maze tests. Y Hdc/ mice showed superior performance in the hidden sessions of the water maze and passive avoidance memory retention. In contrast, Y Hdc/ mice were impaired in novel location recognition, spent less time searching in the target quadrant and more time searching in the outer zone of the water maze during the probe trials. These behaviors are likely due to increased measures of anxiety and are not found in MA Hdc/ mice. These data support a role for histamine in anxiety and cognition and underline the importance of considering age and potential effects on measures of anxiety in the interpretation of the role of histaminergic neurotransmission in cognitive function. 2005 Elsevier B.V. All rights reserved.

Abbreviation: Hdc, histidine decarboxylase

1.

Introduction

In the nervous system, histamine production results from a catalytic reaction involving the enzyme histidine decarboxyl-

ase (HDC), which converts L-histidine to histamine (Ohtsu et al., 2001). Neuronal histamine is normally produced within the histaminergic neurons in the tuberomammillary nucleus of the posterior hypothalamus (Miklos and Kovacs, 2003; Panula

Corresponding author. Department of Behavioral Neuroscience, L470, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA. Fax: +1 503 494 6877. E-mail address: raberj@ohsu.edu (J. Raber). 0006-8993/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.11.067

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et al., 1988). From these neurons, there is an extensive network of HDC fibers projecting to various regions of the central nervous system, including the cerebral cortex, amygdala, and hippocampus (Haas and Panula, 2003; Hill et al., 1997; Panula et al., 1988). Neuronal histamine functions through the interaction with pre-synaptic (H3) and post-synaptic (H1, H2, and H4) receptors (Haas and Panula, 2003). H3 receptors also mediate levels of arginine vasopressin (AVP) and corticotrophinreleasing factor (CRF), which are involved in the regulation of the hypothalamicpituitaryadrenal axis, anxiety, and cognition (Haas and Panula, 2003; Hill et al., 1997; Rizk et

al., 2004). In the amgydala of H3 receptor knockout mice, levels of AVP were increased and correlated with measures of anxiety in the elevated zero maze and the acoustic startle response (Rizk et al., 2004). Depending on their expression pattern in the brain, the different histamine receptors may regulate various behavioral processes. There is evidence that more than one histamine receptor type is involved in regulating specific behavioral processes (Passani et al., 2000; Toyota et al., 2002). Therefore, blockade of only one of the histamine receptors might not be sufficient to examine the overall role of histamine in brain function. To study this role, inhibitors of HDC and mice lacking

Table 1 Open field, lightdark, elevated zero maze, and elevated plus maze performance of Hdc/ and Hdc+/+ mice Genotype Age Distance (cm) Center a
A) Open field Hdc+/+ Hdc/ Hdc+/+ Hdc/ Y Y MA MA 1135 315 1171 405 106 66*** 154 142***

Activity (s) Center c

83.9 33.1 122.2 50.3 9 7** 15 19***

Rest (s) Center b, d

5.9 2.6 32.4 16.2 1 1 10 7

Periphery b, c
4020 2580 2821 1947 192 116** 281 208**

Periphery b, d
459.5 431.5 365.2 351.4 8 13 15 24

Periphery c
50.5 132.7 80.0 181.9 10 17** 13 31**

Light b, e B) Lightdark Hdc+/+ Hdc/ Hdc+/+ Hdc/

Dark f, g

Light b, e

Dark b, e

Light

Dark h

Y Y MA MA

1923 808 2627 1662

209 202*** 212 179**

2626 2462 1816 1982

240 196 230 183

249.4 112.3 359.5 248.1

26 30*** 23 28**

281.0 358.9 167.7 255.1

26 31* 21 27*

40.9 33.7 64.5 52.9

16 14 11 11

28.6 68.1 8.2 43.8

12 19* 4 13*

Time (s) Open C) Zero maze Hdc+/+ Hdc/ Hdc+/+ Hdc/ Closed
b

Mean velocity (cm/s) Closed

Open

Y Y MA MA

1234 175 519 97

444 118* 92 66*

551 792 1665 1302

169 203 112 78

67.8 16.4 74.5 38.1

5 7* 25 16

532.2 582.9 525.5 561.7

5 7* 25 16

3.41 1.98 2.86 2.80

0.18 0.20** 0.32 0.39

Open D) Plus maze Hdc+/+ Hdc/ Hdc+/+ Hdc/

Closed

Open

Closed

Open

Closed

Y Y MA MA

423 99 564 173

82 63** 80 27***

2404 2120 2673 2449

160 120 115 117

55.0 13.3 60.7 22.3

9 8** 9 4***

329.8 314.2 317.4 307.2

14 12 13 11

8.9 0.5 9.3 2.9

0 0.1*** 2 1**

156.3 265 174.9 247.3

8 14*** 13 16*

Distance, active times, rest time, velocity, and edges are shown standard error of the mean (SEM). Subsequent Dunnett's tests indicate SD (*P b 0.05, **P b 0.01, and ***P b 0.001) between genotypes Hdc+/+ and Hdc/ with the appropriate agematch controls. a There were effects of genotype and age for distance moved in the center of the open field (F(3,40) = 15.43, genotype: P b 0.001; age: P b 0.001). b P b 0.05 between age groups for both Hdc+/+ and Hdc/ mice. c There was an effect of genotype on distance moved in the periphery (F(3,40) = 19.43, P b 0.001), rest time in the periphery (F(3,40) = 7.00, P b 0.001), and active times in the center (F(3,40) = 7.85, P b 0.001). d There was an effect of age on active times in the periphery (F(3,40) = 8.47, P b 0.001) and rest time in the center (F(3,40) = 4.98, P b 0.005). e There were effects of genotypes and age on distance moved in the light (F(3,40) = 12.92, genotype: P b 0.001; age: P b 0.001), and active times in the light (F(3,40) = 12.28, genotype: P b 0.001; age: P b 0.001) and dark (F(3,40) = 7.79, genotype: P b 0.001; age: P b 0.001). f P b 0.05 between age groups for the Hdc+/+ mice only. g There was an effect of age on distance moved in the dark (F(3,40) = 3.20, P b 0.03). h There was an effect of genotype on rest times in the dark (F(3,40) = 3.26, P b 0.03).

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HDC (Hdc/) can be used. The irreversible inhibitor of HDC fluoromethyl-[S]-histidine (FMH), which produces histamine loss in a tissue-dependent fashion (Watanabe et al., 1990), reduces locomotor activity in open field. Compared to age- and sex-matched 129/Sv wild-type mice, 5-month-old male Hdc/ mice on the 129/Sv genetic background mice also showed reduced exploratory behavior in the open field, as well as increased measures of anxiety in plus-maze, impairments in long-term object recognition memory and superior motor coordination on the rotorod (Cacabelos and Alvarez, 1991; Dere et al., 2004; Dere et al., 2003; Toyota et al., 2002). They also showed reduced locomotor activity in the dark in the home cage and facilitated methamphetamine-induced locomotor activity in the open field (Abe et al., 2004; Kubota et al., 2002; Toyota et al., 2002). As the 129/Sv strain is known to have problems in sensorimotor coordination and to have increased levels of anxiety in the open field compared to C57Bl6 mice (Kelly et al., 1998; Paulus et al., 1999), the behavioral alterations in Hdc/ mice might be strain-dependent. It is also not known whether these alterations are age-dependent. In this study, 3 5 and 1214 month-old Hdc/ and wild-type mice on the C57Bl6/J background were used to determine if the behavioral alterations in Hdc/ mice are strain- and age-dependent.

1A). In addition, MA mice showed less anxiety like behavior in the open field, assessed as percent time spent in the center, than Y mice (Fig. 1A). In the lightdark test, Hdc/ mice also moved less and showed more anxiety-like behavior (Table 1B and Fig. 1B). Similar as in the open field, MA mice sowed less anxiety-like behavior than Y mice (Fig. 1B). These data indicate age-related reduced anxiety-like behavior that is genotype-independent. In Hdc/ mice, decreased locomotion and increased anxiety-like behavior were also revealed in the elevated zero maze (Table 1C and Fig. 1C). Hdc/ mice spent less time (F(3,37) = 4.08, P b 0.001) and moved less (F(3,39) = 3.18, P b 0.05) in the open areas of the maze than Hdc+/+ mice. Consistent with increased anxiety-like behavior in the elevated zero maze, increased anxiety-like behavior was also revealed in the elevated plus maze (Table 1D and Fig. 1D). Hdc/ mice spent less time (F(3,39) = 5.58, P b 0.003), moved less (F(3,39) = 9.89, P b 0.001), and showed reduced active times (F(3,39) = 15.16, P b 0.001) and rest times (F(3,39) = 5.82, P b 0.003) in the open arms of the elevated plus maze than Hdc+/+ mice (Fig. 1D). Thus, Hdc/ mice show increased anxiety-like behavior and decreased locomotor activity in various anxiety tests.

2.

Results

2.2. Hdc/ mice show age-dependent impairments in novel location recognition

Y and MA Hdc/ mice spent less time exploring all objects over the five trials than Hdc+/+ mice. Y Hdc/ mice spent less time exploring all objects in all trials than Y Hdc+/+ mice (F(9,79) = 6.02, P b 0.001) and MA Hdc/ mice spent less time exploring all objects in trial 5 than MA Hdc+/+ mice (F(9,89) = 2.71,

2.1. Hdc/ mice show decreased locomotion and increased anxiety-like behavior
In the open field, Hdc/ mice moved less and showed more anxiety-like behavior than wild-type mice (Table 1A and Fig.

Fig. 1 Anxiety-like behavior of Hdc/ and Hdc+/+ mice. Hdc/ mice showed increased anxiety-like behavior, as they spent less time in the center of the open field (A), less time in the light compartment in the lightdark test (B), less time in the open areas of the zero maze (C), and less time in the open arms of the plus maze (D). There were effects of genotype and age on % time spent in center of the open field (F(3,40) = 8.64, genotype: P b 0.001; age: P b 0.001), the % time in the light compartment of the lightdark test (F(3,38) = 9.51, genotype: P b 0.001; age: P b 0.001). *P b 0.05, **P b 0.01, and ***P b 0.001 versus age-matched Hdc+/+. o P b 0.05 and ooP b 0.01 between ages. n = 810 mice per genotype per age.

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Fig. 2 Novel location and novel object recognition of Hdc/ and Hdc+/+ mice. Y Hdc/ mice spent less time exploring all objects than age-matched controls over the five trials (A). Y Hdc/ mice did not show novel location recognition (B). In both genotypes, there was an age-related decline in novel object recognition (C). **P b 0.01; ***P b 0.001 versus old location. ooP b 0.01 and oooP b 0.001 between ages. n = 810 mice per genotype per age.

P b 0.008) (Fig. 2A). In contrast to Y Hdc+/+ mice and MA Hdc/ and Hdc+/+ mice, Y Hdc/ mice did not show novel location recognition (Fig. 2B). While MA mice showed reduced novel object recognition compared to Y mice (two-way ANOVA: F(3,33) = 13.18, P b 0.001), there was no genotype difference at either age (Fig. 2C).

2.3. mice

Age-dependent enhanced spatial learning in Hdc/

During the visible sessions, Y Hdc/ mice swam faster than age-matched Hdc+/+ mice (Table 2). Therefore, latency was not used as a performance measure. During the visible sessions, all groups improved their performance with training (distance moved: (F(39,418) = 4.86, P b 0.001); cumulative distance to platform: (F(39,123) = 4.89, P b 0.001) but there was no genotype difference in distance moved (Figs. 3A and B) or cumulative

distance (Figs. 3C and D) to platform. Overall, Y mice moved more than the MA mice during the visible sessions (distance moved: F(3,418) = 2.49, P b 0.05; cumulative distance to the platform: F(3,418) = 8.45, P b 0.001). During the hidden sessions, all groups improved their performance with training (distance moved: F(23,555) = 5.21, P b 0.001; cumulative distance to platform: F(23,555) = 6.81, P b 0.001) and Hdc/ mice performed better than age-matched controls (distance moved: F(23,555) = 5.21, P b 0.03; cumulative distance to platform: F(23,555) = 6.81, P b 0.001) (Fig. 3). This overall genotype difference was driven by the Y mice. Y, but not MA, Hdc/ mice performed better than age-matched controls (Figs. 3A and C). Thigmotaxis decreased with training in both the visible (F(15,123) = 8.00, P b 0.001) and hidden (F(31,185) = 2.22, P b 0.001) sessions (Figs. 3E and F). However, there was no effect of genotype on thigmotaxis during either the visible or hidden sessions.

Table 2 Swim speeds of Hdc/ and Hdc+/+ mice in the water-maze Genotype
Hdc+/+ Hdc/ Hdc+/+ Hdc/

2.4. Hdc/ mice show age-dependent impaired spatial memory retention

During the second and third probe trials, Y Hdc/ mice spent less time in the target quadrant than age-matched controls (F(11,100) = 5.06, P b 0.03, Figs. 4C and D). In contrast to the other groups, Y Hdc/ mice did not preferentially search the target quadrant (data not shown). These impairments can be attributed to increased thigmotaxis within the last 30 s of the probe trails. During all probe trials, Y Hdc/ mice spent more time in

Age
Y Y MA MA

Visible
15.02 15.96 14.83 14.24 0.29 0.26 a 0.32 0.26

Hidden
15.22 15.65 14.08 15.41 0.36 0.32 0.39 0.32 a

Average swim speeds cm/sec are shown SEM. a P b 0.01 versus age-matched Hdc+/+ mice.

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Fig. 3 Spatial learning and memory of Hdc/ and Hdc+/+ mice in the water-maze. Average distance moved to the platform (A) and cumulative distance to the platform (C) during hidden (bar graphs), but not during visible sessions, were lower in Y Hdc/ than that age-matched control mice. In contrast, there were no genotype differences in MA mice in average distance moved to the platform (B) or cumulative distance to the platform (D) during the visible or hidden sessions (bar graphs). There was no genotype difference in the % time spent in the outer zone (EF) of the water maze in Y or MA mice. *P b 0.05 and **P b 0.01 versus Hdc+/+ mice. n = 810 mice per genotype per age.

the outer zone than age-matched controls (F(11,100) = 2.24, P b 0.05, Figs. 4E and F). Within the first 30 s of each probe trial, there was no effect of genotype in the time spent in the outer zone (F(11,100) = 0.55, P = 0.7303, not shown). However, in the last 30 s of the probe trials, Y Hdc/ mice spent more time in the outer zone than age-matched controls (F(11,100) = 4.48, P b 0.01, not shown). Thigmotaxis during the 60 s probe trials correlated with the % time in the light compartment during the lightdark test (r = 0.4592, P b 0.009, Fig. 4G), the % time spent in the center of the open field (r = 0.3702, P b 0.04, not shown), and the % time

the mice explored the object in a novel location (r = 0.4438, P b 0.01, Fig. 4H).

2.5.

Sensorimotor coordination

When sensorimotor function was assessed on the rotorod, there was improved performance with training and an agerelated decline in performance (F(35,352) = 5.98, trial: P b 0.001; age: P b 0.001, Fig. 5). However, there was no genotype difference in rotorod performance.

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Fig. 4 Spatial memory retention of Hdc/ and Hdc+/+ mice during the water maze probe trials. A representative search pattern of a Hdc+/+ (A) and Hdc/ (B) mouse. The lines define the quadrants used to divide the maze. The drop location is designated by a circle in the quadrant opposite to the one containing the platform during the hidden sessions. The circles illustrate how the tank is divided into an outer (020 cm), middle (2040 cm), and center zone. The platform location was in the center of the target quadrant and in the middle circle. There were three probe trials, one at the end of each day of hidden platform training. Y Hdc/ mice spent less time searching the target quadrant (C) and more time searching in the outer zone of the maze than Y Hdc+/+ mice (E). In contrast, no genotype difference was observed in MA mice in the time spent searching for the platform in the target quadrant (D) or outer zone (F). *P b 0.05 versus age-matched Hdc+/+ mice. The % time in the light compartment during the light dark test (G) and the % time exploring the object in the novel location in the object recognition test (H) correlated with the % time spent in the outer zone during the water maze probe trials. n = 8 mice per genotype per age.

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Fig. 5 Sensorimotor function in Hdc/ and Hdc+/+ mice. Sensorimotor performance was assessed on the rotorod. There was no genotype difference in rotorod performance. n = 810 mice per genotype per age.

2.6.

Passive avoidance learning and memory

There was no genotype or age difference in trials to criterion during passive avoidance training (Fig. 6A). However, Y Hdc/ mice showed improved memory retention 24 h later as compared to Y Hdc+/+ mice. In the Y mice, Hdc/ mice showed higher latencies than Hdc+/+ mice to re-enter the dark compartment (F(3,32) = 2.87, genotype: P b 0.05) (Fig. 6B), and less Hdc/ than Hdc+/+ mice entered the dark compartment within the 300 s trial time (chi-square, P b 0.01) (Fig. 6C).

3.

Discussion

Our data indicate that HDC deficiency throughout development alters measures of anxiety and cognition. Y and MA Hdc/ mice showed hypoactivity and increased anxiety-like behavior in all anxiety tests used, consistent with increased anxiety-like behavior in young Hdc/ mice on the 129/Sv genetic background. As mice on the 129/Sv strain show reduced locomotor activity and more anxiety-like behavior than mice on the C57BL/6 background (Holmes et al., 2002; Paulus et al., 1999; Ralph et al., 2001), these data indicate that the hypoactivity and

increased anxiety-like behavior in Hdc/ mice are not straindependent. In both genotypes, there was an age-related decline in locomotor activity in the open field and in measures of anxiety in the open field and lightdark tests. Y Hdc/ mice were impaired in novel location recognition, but showed superior performance in the hidden sessions of the water maze, both consistent with cognitive performance in 5month-old Hdc/ mice on the 129/Sv genetic background (Dere et al., 2003). However, MA Hdc/ mice and age-matched controls showed no longer any performance difference in these tests, indicating that these alterations in cognitive performance may be age-dependent. In open field, lightdark, elevated zero maze, and elevated plus maze, Hdc/ mice showed increased measures of anxiety and hypoactivity. These data are consistent with previous studies indicating that Y Hdc/ show reduced locomotion in the open field (Dere et al., 2004). This hypoactivity might reflect heightened anxiety-like behavior. Both the Y and MA Hdc/ mice moved less in the light and dark compartments during the lightdark test. MA mice spent more time in the center of the open field and lightdark tests than the Y mice and the genotype difference in time spent in the center was less pronounced in MA than Y mice. In the open field and lightdark tests, both the MA Hdc+/+ and Hdc/ mice exhibited less anxiety-like behaviors than genotype-matched Y mice. This is consistent with lower anxiety levels in the elevated plus maze in older wild-type and Ames Dwarf mice compared to their younger counterparts (Kinney et al., 2001). However, as the MA mice were tested at a younger age, we cannot exclude that the early handling contributed to their reduced anxiety levels. During the object recognition test, Y Hdc/ mice spent less time exploring all the objects than age-matched controls during all trials. In addition, they did not show novel location recognition. These data are consistent with previous studies showing that Y Hdc/ mice are impaired in long-term memory based novel location discrimination, but have normal longterm novel object discrimination (Dere et al., 2004). In contrast, MA Hdc/ mice showed novel location recognition, indicating that this impairment is age-dependent. While novel object recognition was weaker in MA than Y mice, all groups of mice showed novel object recognition and there was no genotype difference at either time point. Y Hdc/ mice spent less time

Fig. 6 Passive avoidance learning and memory in Hdc/ and Hdc+/+ mice. There was no genotype difference in trials to criterion (A). However, in the Y mice, there was a genotype difference 24 h later in test trial latency (B) and in the % mice of mice re-entering the light compartment (C). *P b 0.05 and **P b 0.01 versus age-matched Hdc+/+ mice. n = 810 mice per genotype per age.

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exploring all objects, but they showed novel object recognition. This suggests that total time spent exploring all objects is not a factor in their inability to form temporal inter-object relationships. While the MA Hdc/ mice spent less time exploring all objects in trial 3, they showed similar novel location and novel object recognition as age-matched controls. The fact that the deficit in the formation of temporal inter-object relationships is not seen in the MA Hdc/ mice that show lower anxiety levels than Y Hdc/ mice suggests that the heightened anxiety levels in Y Hdc/ mice may contribute to this deficit. During the hidden water maze sessions, Y Hdc/ performed better than age-matched controls. As Y Hdc/ mice have elevated plasma concentrations of testosterone and other androgenic steroids (Pap et al., 2002) and testosterone enhances spatial learning (Cherrier et al., 2001; Flood et al., 1995; Janowsky et al., 1994; Raber et al., 2002), the elevated plasma testosterone levels might contribute to their superior performance in the hidden sessions of the water maze. Interestingly, Y Hdc/ mice were impaired in novel location recognition. While both tests are considered spatial, the role of testosterone in novel location recognition is not clear. In addition, it is hard to compare these two tests as they contain a different number of training and testing trials and involve different motivational factors. During the visible, but not hidden, sessions, Y Hdc/ mice swam faster than age-matched control. In contrast to these data, Y Hdc/ on a 129/Sv background appeared to swim slower than control mice in the visible component of the water maze (Dere et al., 2003). Genetic strain differences in sensorimotor ability might contribute to these divergent findings (Holmes et al., 2002; Paulus et al., 1999; Schimanski and Nguyen, 2004). To assess if Y Hdc/ mice have superior ability to learn the spatial cues during the water maze test, the loss of the thigmotaxic searching associated with increased allocentric navigation (spatial searching) was assessed (Champagne et al., 2002). There was no genotype difference in the % time spent in the outer zone of the water maze during visible or hidden sessions, suggesting that both genotypes have similar abilities with respect to spatial strategies to find the hidden platform. Y Hdc/ mice may remember the location of the hidden platform better or might be more motivated to find it. In contrast to their enhanced acquisition in the sessions with the hidden platform, Y Hdc/ mice spent less time searching in the target quadrant and more time searching in the outer zone of the water maze during the probe trials. This wall seeking or thigmotaxic behavior in Y Hdc/ mice supports heightened anxiety-like behavior and increased motivation to get out of the water maze (Champagne et al., 2002; Schulz et al., 2002). The thigmotaxic behavioral appears compounded in the last 30 s of the probe trial. Consistent with this interpretation, the % time in the center of the open field and the % time in the light compartment of the lightdark test correlated with the % time spent in the outer zone in the water maze probe trials. In addition, the enhanced thigmotaxic behavior was no longer present in MA Hdc/ mice, which show lower measures of anxiety. Finally, poor performance in water maze probe trials was shown to be affected by stress and not necessarily spatial learning ability (Holscher, 1999). The lack of novel location recognition in Y Hdc/ mice might also relate to

altered anxiety levels and be due to increased neophobia of the object in a novel location. This interpretation is supported by the correlation between the % time spent in the outer zone in the water maze probe trials and the % time spent exploring the object in a novel location. However, an alternative explanation for the water maze probe trial data could be that in Y Hdc/ mice there is a fast relearning process whereby the mice conclude that there is no safe place any more and therefore spent more time close the wall. Consistent with this alternative explanation is the fact that Y Hdc/ mice also show improved memory retention 24 h following training in the aversive passive avoidance test. In this scenario, thigmotaxis would be the effect rather than the cause. In contrast to superior rotorod performance observed in Hdc/ mice on the 129/Sv genetic background (Dere et al., 2004), Hdc/ mice on the C57Bl/6 background showed comparable rotorod performance as controls. As the 129/Sv strain is known to have deficits in sensorimotor coordination compared to C57BL/6 background (Paulus et al., 1999; Ralph et al., 2001), these data suggest that the superior rotorod performance on the 129/Sv genetic background might be subtle and not revealed on a background strain with better rotorod performance than the 129/Sv strain. Compared to age-matched controls, Y Hdc/ showed improved passive avoidance memory retention. This is consistent with previous studies in rats suggesting that histamine is involved in passive avoidance memory (Eidi et al., 2003; Giovannini et al., 1999; Zarrindast et al., 2002a,b). This genotype difference in memory retention was not detected in MA mice, suggesting that it is age-dependent. The higher latency of Y Hdc/ mice in the passive avoidance test trial could be due to heightened levels of anxiety to re-enter the dark compartment. Consistent with this possibility, there was a significant negative correlation between the passive avoidance test trial latency and the % time the mice explored the center of the open field (r = 0.5969, P b 0.001, not shown). Further, lack of neuronal histamine increases emotional reactivity when mice are placed in unfamiliar or aversive environments (Dere et al., 2004). In addition, MA Hdc/ mice showed lower levels of anxiety than Y Hdc/ mice and showed similar passive avoidance memory retention as age-matched wild-type mice. The higher latency of Y Hdc/ mice in the passive avoidance test trial may also be explained by their apparent hypoactivity. However, this is unlikely as they show similar trials to criterion and there is no genotype difference in latency to enter the dark chamber in the first trial (data not shown). Although in Y Hdc/ mice heightened anxiety-like behavior might contribute to the deficits in novel object recognition and enhanced passive avoidance memory retention, histamine may still be involved in memory aspects of these tests as supported by studies using classical histamine receptor pharmacological compounds (Cangioli et al., 2002; Flood et al., 1998; Giovannini et al., 1999; Passani et al., 2000; Zarrindast et al., 2002a). In summary, this study supports an important role for histamine in anxiety-like behavior and cognition and underlines the importance of considering potential effects on anxiety-like behavior in the interpretation of the potential role of histaminergic neurotransmission in cognitive function.

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4.
4.1.

Experimental procedures
Animals

Wild-type (Hdc+/+) and Hdc/ male mice generated as described (Ohtsu et al., 2001) were backcrossed onto the C57Bl6/J background for 6 generations. The mice were kept on a 12:12 h lightdark schedule (lights on at 6AM) with lab chow (PicoLab Rodent Diet 20, #5053; PMI Nutrition International, St. Louis, MO) and water given ad libitum. Animals were housed singly beginning 48 h prior to the first behavioral test. All procedures were according to the standards of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Oregon Health and Science University. 4.2. Behavioral testing

min session. Breaks in the photo beams were used to calculate path length, active times and rest time in the open, and closed compartments of the enclosure. Mice with increased measure of anxiety in this enclosure spend less time in the lighted compartment of the maze (Bhatnagar et al., 2004). 4.6. Elevated plus-maze

The elevated plus maze consisted of two open arms and two closed arms equipped with infrared photocells interfaced with a computer (Hamilton-Kinder, Poway, CA; luminescence: 200 lx). Active times, distance moved and rest time were recorded for a single 10min session. Recorded beam brakes were used to calculate path lengths and time spent within each arm of the maze. Mice with increased measures of anxiety in this maze spend less time in the open arms of the elevated plus maze (Pellow et al., 1985). 4.7. Novel object and novel location recognition

Mice were tested at 25 months of age (Y) or 1214 months of age (MA). All experiments were performed during the light phase. The sequence of behavioral testing was such that tests were administered in the order of increasing stress level. Mice were tested in the open field, lightdark, and elevated plus maze tested between 8 and 10 a.m. in week 1, for novel location and novel object recognition between 8 a.m. and 3 p.m. in week 2, for water maze performance between 8 and 10 a.m. and 1 and 3 p.m. in week 3, and for rotorod between 10 and 11 a.m. and for passive avoidance between 8 a.m. and 3 p.m. in week 4. 4.3. Open field

To evaluate exploratory behavior and measures of anxiety, mice were placed in a 40.64 cm 40.64 cm brightly lit (luminescence: 200 lx) open arena equipped with infrared photocells interfaced with a computer (HamiltonKinder, Poway, CA). Active times (single beam breaks within 1 s), distance moved, and rest times (no beam breaks within 1 s) were recorded for a single 10-min session. In the open field, the center zone (8 8 in.) is more anxiety-provoking than the peripheral zone. Mice that are more anxious in the open field spend less time in the center (Choleris et al., 2001). Therefore, to assess measures of anxiety in the open field, the data were also analyzed for only the center or peripheral zone. 4.4. Elevated zero maze

For three consecutive days, mice were individually habituated, for 5 min, to the open field described above (luminescence: 200 lx). On the fourth day, the mice were first given three 10-min trials with three plastic objects (monkey, horse, man) placed in different corners of the enclosure and occupying an area of 6 cm2. Objects were chosen based on preliminary data showing that they were of equal interest to naive C57B6/J wild-type mice (data not shown). There was a 3-min inter-trial interval during which mice were placed back in their home cage and the maze and the objects were cleaned with 5% acetic acid to remove odors. All familiar objects were exchanged with replicas in subsequent trials. For the fourth 10-min trial, the monkey was moved from one corner of the enclosure to another to evaluate novel location recognition. For the fifth 10-min trial, the horse was replaced by a cow to assess novel object recognition. For each trial, the total time the mouse spent exploring each object within 2 cm was recorded by an observer blinded to the genotype of the mice. The time spent exploring all objects over the first three trials assessed the familiarization with the objects. The difference between the percentage of time spent exploring the object in the familiar (trial 3) and novel location (trial 4) was calculated to assess novel location recognition. The percentage of time spent exploring the novel object during trial 5 was calculated to assess novel object recognition. 4.8. Water maze

To assess measures of anxiety, the elevated zero maze, elevated plus maze, and lightdark tests were also used. The custom built elevated zero maze (HamiltonKinder) consisted of two enclosed areas and two open areas, identical in length to the open and closed arms in the elevated plus maze (35.5 cm) (HamiltonKinder, Poway, CA). Mice were placed in the closed part of the maze and allowed free access for 10 min (luminescence: 200 lx). They could spend their time either in a closed safe area or in an open area. A video tracking system (Noldus Information Technology, Sterling, VA, set at six samples/second) was used to calculate the velocity, time spent, distance moved, and entries into the open and closed areas. Mice that are more anxious in the zero maze spend less time in the open areas (Rizk et al., 2004; Shephard et al., 1994). 4.5. Lightdark

In the lightdark test, mice were placed in the open field enclosure (described above) containing black plastic inserts covering from the sides and the top 50% of the open field (HamiltonKinder, Poway, CA). A single opening in the wall of the insert adjacent to the open area allowed the mice to enter or exit the more anxietyprovoking light area (luminescence: 200 lx) of the maze. Active times, distance moved and rest time were recorded for a single 10-

A modified version of the Morris water maze (Morris, 1984; Raber et al., 2004) was used to assess spatial learning and memory. A circular pool (diameter 140 cm) was filled with opaque water (24C) and mice were trained to locate a submerged platform in order to escape from the water (luminescence: 40 lx). First they were trained to locate a clearly marked platform (days 1 and 2). Subsequently, they were trained to locate a platform hidden beneath the surface of water made opaque using a white chalk (days 35). During the training with the hidden platform, the mice had to navigate to it using the available spatial cues. There were two daily sessions 3.5 h apart, each consisting of three 60 s trials (with 1015 min inter-trial intervals). Mice that failed to find the platform within 60 s were led to the platform by the experimenter and allowed to stay on the platform for 3 s. During the visible platform training, the platform was moved to a different quadrant of the pool for each session. For the hidden platform training, the platform location was kept constant for each group of mice. The mice were assigned to four groups using a randomized block design to avoid any potential quadrant bias. Mice were placed into the water facing the wall at the side of the pool in 9 different locations around the pool circumference, and the starting location was changed for each trial. The swimming patterns of the mice were recorded with the Noldus Ethovision video tracking system, set at 6 samples/s.

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Time to locate the platform (latency), distance moved, swim speeds, and cumulative distance to platform (distance to the platform location summed over all samples) (Gallagher et al., 1993) were analyzed. Probe trials assessed spatial memory retention 1 h after the last hidden platform training trial of each day of hidden training (total of 3 probe trials). The time that mice spent swimming in the target quadrant (where the platform was located during hidden platform training), and in the three non-target quadrants was measured over a 60 s trial. For the probe trials, mice were dropped in the quadrant opposite from the target quadrant (Fig. 4A). Thigmotaxis was measured as the percentage of time spent in the outer zone of the maze. The outer zone was defined as 20 cm from the edge of the tank, the middle zone as 2040 cm from the edge of the tank, and the remaining area was defined as the center zone (Fig. 4B). 4.9. Rotorod

To assess sensorimotor ability, mice were tested using a rotorod apparatus (Hamilton-Kinder, Poway, CA). Mice were placed on an elevated rod (7.0 mm in diameter) initially rotating at 4rpm. Every 15 s, the rod was accelerated by 15 rpm. Fall latency was recorded by timers, which stopped when the mouse broke the photobeams at the bottom of the chamber. 4.10. Passive avoidance

To assess emotional learning and memory, the passive avoidance (PA) test was used. Each mouse was placed in a lighted compartment of a chamber also containing a dark compartment (Hamilton-Kinder, Poway, CA). After 5 s of acclimation, the bright house light turned on and the connecting gate to the dark compartment opened. The mouse preferring the darkened left side, steps quickly through the gate to enter the dark compartment. Upon doing so, the mouse received a brief and slight foot shock (0.3 mA for 3 s). Each mouse was trained until it met a learning criterion of three consecutive 120-s trials without entering the dark compartment, or up to 10 trials, whichever came first. After 24 h, the mouse was again placed in the light compartment, and the time to re-enter the dark compartment measured up to 300 seconds. 4.11. Statistical analyses

Untransformed (raw) data were analyzed with the JMP3.1 statistical software package (SAS Institute Inc., Cary, NC). Following initial multi-measures or two-way ANOVAs as indicated, the data were analyzed by planned comparisons to an age-matched control group using the Dunnett's or TukeyKramer post hoc tests. In addition to effects of age and genotype, interactions between effects of age and genotype were also assessed. To assess potential correlations, Spearman's correlations were performed. A probability value of less than 0.05 was considered significant.

Acknowledgments
This work was supported by NIH Grant R01 AG20904, EMF grant AG-NS-0201-02, and NIDA training grant T32-DA07262.

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