Behavioural Brain Research 281 (2015) 276–282

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Effects of Npas4 deficiency on anxiety, depression-like, cognition and sociability behaviour Emily J. Jaehne a,1 , Thomas S. Klaric´ b,1,3 , Simon A. Koblar b , Bernhard T. Baune a , Martin D. Lewis c,∗,2 a

Discipline of Psychiatry, University of Adelaide, Adelaide, South Australia, Australia Stroke Research Programme, School of Medicine, University of Adelaide, Adelaide, South Australia, Australia c Discipline of Genetics, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia b

h i g h l i g h t s • Npas4−/− mice show decreased anxiety behaviour in the elevated zero maze. • Npas4−/+ mice show increased depression-like behaviour in forced swim test. • Npas4−/+ mice show impaired spatial recognition memory in the Y-maze.

a r t i c l e

i n f o

Article history: Received 4 November 2014 Received in revised form 15 December 2014 Accepted 19 December 2014 Available online 27 December 2014 Keywords: Neuronal PAS domain-containing protein 4 Anxiety Depression Sociability Memory

a b s t r a c t The transcription factor neuronal PAS domain-containing protein 4 (Npas4), which regulates the formation of inhibitory synapses on excitatory neurons, has been suggested as a candidate gene for neurological and psychiatric conditions such as bipolar depression, autism spectrum and cognitive disorders. A mouse model of Npas4 deficiency has been developed to investigate any role in these disorders. Behavioural characterisation of Npas4−/− , Npas4+/− and Npas4+/+ mice has been conducted using the open field, elevated zero maze (EZM), Y-maze, sociability test and forced swim test (FST) to investigate a range of behaviours. Npas4−/− mice spent more time in the open arm of the EZM than other genotypes, suggesting decreased anxiety-like behaviour. Npas4+/− mice, however, were more immobile in the FST than other genotypes, suggesting increased depression-like behaviour, and also showed impaired spatial recognition memory in the Y-maze. There were no differences between genotype in social behaviour. These results suggest that differential levels of Npas4 expression in the brain may regulate anxiety, depression and cognition related disorders. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Homeostasis of neuronal networks is essential for normal behaviour and cognition. A major player in this process is the

Abbreviations: Npas4, neuronal PAS domain-containing protein 4; BDNF, brainderived neurotrophic factor; SERT, serotonin reuptake transporter; KO, knockout; PFC, prefrontal cortex; uCMS, unpredictable chronic mild stress; DG, dentate gyrus; EZM, elevated zero maze; FST, forced swim test; EPM, elevated plus maze; NORT, novel object recognition test; HET, heterozygous. ∗ Corresponding author at: South Australian Health & Medical Research Institute, PO Box 11060, Adelaide, SA 5001, Australia. Tel.: +61 8 8128 4703. E-mail address: [email protected] (M.D. Lewis). 1 Joint first author. 2 Present address: SAHMRI Mind and Brain Theme, North Terrace, Adelaide, SA 5001, Australia. 3 Present address: Genos d.o.o., Hondlova 2/11, 10000 Zagreb, Croatia. http://dx.doi.org/10.1016/j.bbr.2014.12.044 0166-4328/© 2014 Elsevier B.V. All rights reserved.

activity-dependent transcription factor neuronal PAS domaincontaining protein 4 (Npas4) which promotes excitatory/inhibitory homeostasis by regulating the formation of inhibitory synapses on excitatory neurons [1,2]. It does so by driving a genetic programme that is responsible for effecting changes at synapses via activation of a number of plasticity-related immediate-early genes, such as Arc, Fos and Egr1 [3], as well as other genes that are known to play a role in synaptic plasticity such as brain-derived neurotrophic factor (BDNF) [2,4] and the actin-binding protein developmentally regulated brain protein (Drebrin) [5]. Its central role in neuronal connectivity makes Npas4 a candidate gene for neurological conditions related to improper circuit function and there is already growing evidence that dysregulation of Npas4 may be implicated in a host of psychiatric conditions such as bipolar disorder [6], autism spectrum disorder [7,8] and cognitive disorders [9]. Npas4 has been shown to be associated with behavioural changes in many different mood disorder models. In a serotonin

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reuptake transporter (SERT) knockout (KO) rat model of anxiety and depression-like symptoms [10], reduced levels of Npas4 mRNA have been shown in the hippocampus and prefrontal cortex (PFC) [11,12]. Data from other models, in which socio-environmental stressors are used to induce depression, are not as clear. In rats, 4 weeks of unpredictable chronic mild stress (uCMS) resulted in decreased Npas4 mRNA expression in the hippocampus [13], yet no change in Npas4 expression was seen in mice subjected to 7 weeks of uCMS in either the amygdala, cingulate cortex or dentate gyrus (DG) [14]. This may indicate that there are species-specific differences underlying the uCMS paradigm or, alternatively, it is possible that changes in Npas4 expression occur specifically in regions of the hippocampus other than the DG and hence they were not detected in the latter study. Nevertheless, Npas4−/− mice [2] do not appear to show signs of anxiety [3,15] though no investigations have been carried out to explicitly determine whether they exhibit a depression-like phenotype. Further to its possible role in emotion regulation, there have been a number of recent studies that have highlighted the importance of Npas4 in cognition and memory. First, Ploski et al. showed that Npas4 is required for amygdala-dependent fear memory formation and retrieval [16]. Subsequently, Ramamoorthi et al. identified Npas4 as a ‘master regulator’ of activity-dependent learning that regulates a transcriptional programme of immediate-early genes required for contextual memory formation in the CA3 region of the hippocampus [3]. More recently, Npas4 was implicated in the process of extinction learning when it was shown that expression of Npas4 is transcriptionally suppressed in Tet1−/− mice which display specific impairments in memory extinction [17]. Indeed, Npas4−/− mice themselves show deficits in several types of memory including short-term and long-term hippocampal memory [3], spatial working memory and object recognition memory [15]. So far the only clinical evidence of a connection between Npas4 and cognitive processes in humans comes from a patient diagnosed with mild intellectual disability who had a 1 Mb microdeletion on chromosome 11 which included the Npas4 locus [9]. It is also becoming increasingly clear that Npas4 plays a role in modulating neuroplasticity in response to social experiences and that dysregulation of Npas4 is associated with abnormal behavioural phenotypes. For example, in mice Npas4 expression is up-regulated in the CA1 and CA3 regions of the hippocampus 45 min after a social interaction [15]. Coutellier et al. have also shown using a variety of different tests of social behaviour that NPAS4−/− mice are less social and more aggressive and dominant, although they do show normal discrimination between familiar and novel mice [15]. Together, the evidence described above, particularly from Coutellier et al. [15] and Ramamoorthi et al. [3], outlines a clear role for Npas4 in behaviour and cognition. We therefore aimed to replicate these initial findings and to expand the investigations to additional domains of behaviours including cognition, social, anxiety and depression-like behaviour. To test this, we compared the performance of Npas4+/+ , Npas4+/− and Npas4−/− mice across a range of behavioural tests designed to assess their memory, sociability and predisposition to anxiety and depression-like behaviour. We hypothesised, therefore, that Npas4 may play a role in maintaining normal cognitive and social behaviours and that mice lacking Npas4 would show specific deficits in the areas of mood, memory and/or social interaction. 2. Materials and methods

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Heterozygotes were crossed to produce Npas4−/− , Npas4+/+ , and Npas4+/− littermates. A total of 56 mice, were used for testing, aged 2–9 months, with both male and female mice in each genotype group. All experimental mice were housed in groups of 2–6 in individually ventilated cages. At all stages mice had food and water available ad libitum and ambient temperature of the housing and testing rooms was 22 ± 1 ◦ C. Mice were housed under a 12-h light-dark cycle, lights on at 07:00 h, and all behavioural testing was conducted between 08:00 and 16:00 h. All animals were housed and treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The University of Adelaide Animal Ethics Committee approved all experiments prior to commencement (Animal Ethics #M-2011-58 and #M-2013-122). 2.2. Experimental plan Behavioural characterisation of the Npas4−/− , Npas4+/− and Npas4+/+ mice (17 Npas4+/+ , 9 male + 8 female; 15 Npas4+/− , 10 male + 5 female; 24 Npas4−/− , 15 male + 5 female) used a battery of behavioural tests conducted in the following order (least to most stressful): open field, elevated zero maze (EZM), Y-maze, sociability and forced swim test (FST), conducted over a period of 8 days. Saccharin preference test was also conducted in a subset of mice prior to other testing (17 Npas4+/+ , 15 Npas4+/− , 10 Npas4−/− ). An imaging program (ANY-maze, USA) was used to track movements, and was used for all behavioural testing. All behavioural equipment was purchased from Stoelting Co (USA). All behavioural tests have been used previously in this laboratory [18,19] and experimenters were blinded to genotype during behavioural testing. 2.3. Behavioural testing procedures 2.3.1. Exploratory behaviour 2.3.1.1. Open field. Mice were placed into a brightly lit square arena, 40 cm × 40 cm, with opaque black walls 35 cm high for 5 min according to published protocols [20–22]. The floor was divided into inner and outer zones. Time spent in each zone was measured as an indication of anxiety-like behaviour, and total distance travelled was measured as an indication of baseline locomotor activity. 2.3.2. Emotion-like behaviour 2.3.2.1. Elevated zero maze: anxiety-like behaviour. Mice were placed in an open quadrant of the EZM, which is an elevated circular platform, 50 cm diameter, with a 5 cm wide platform 40 cm above ground. Two quadrants are open, and two are enclosed with walls 15 cm high. The advantage of the zero maze over the plus maze is that this design solves the issue of the ambiguity of the time spent in the central square. Mice were allowed to explore the apparatus for 5 min according to published protocols [23], and time spent in the open quadrants was measured as an indication of anxiety-like behaviour (time in open arms is calculated as: time in open armlatency to enter closed arm). Mice which spend more time in the open arms are considered to be less anxious. 2.3.2.2. Forced swim test: despair (depression-like) behaviour. The FST consists of a circular container, 45 cm high, with a diameter of 20 cm, which is filled with water to approximately half-way. Mice were placed in the water for 6 min and immobility time was measured as an indication of despair and depression-like behaviour according to published protocols [24,25].

2.1. Mice The transgenic mouse Npas4tm1Meg (MGI:3828099), established on a C57BL/6J background was a gift from Prof. Yingxi Lin [2].

2.3.2.3. Saccharin preference test: anhedonia (depression-like) behaviour. Mice were first trained for the saccharin preference test with a continuous two-bottle choice of water and 0.1% saccharin

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for 64 h. For this period, mice were moved as a group to new cages which could hold two bottles. Immediately after this training period, the test was conducted. The test period involved mice being moved to individual cages and given the two-bottle choice of water and 0.1% saccharin for 48 h. Bottles were weighed and total water and saccharin consumption (cons.) were measured. This protocol has been adapted from several previously published protocols [26–28]. Preference was calculated as: saccharin preference % = [saccharin cons./(saccharin cons. + water cons.)] × 100.

as significant. Comparisons were performed using 1 way ANOVA with Tukey’s post hoc test for comparisons between genotypes or repeated measures 2-way ANOVA with Bonferroni’s post hoc test when comparisons within genotype groups were required, as stated throughout the results. Post hoc tests were used only when a significant ANOVA effect was seen. 3. Results 3.1. Exploratory and anxiety behaviour

2.3.3. Cognition-like behaviour 2.3.3.1. Y-maze: spatial recognition memory. Mice were placed in the start arm of the Y-maze, which is a Y-shaped apparatus, with three arms (start arm and two test arms), each 35 cm long and 5 cm wide, with walls 10 cm high. The arms are at a 120◦ angle from each other, and the two test arms have different coloured pieces of tape on the inside walls. Testing was conducted according to published protocols [18,29]. During the training phase, one of the test arms was blocked off, and mice were allowed to explore the start arm and open test arm for 10 min. 30 min later, they were placed back in the Y-maze for 5 min, with all three arms open. Time spent in the novel arm compared to the familiar arm was used as a measure of spatial recognition memory. Number of entries in to each arm was also measured. 2.3.4. Social behaviour 2.3.4.1. Sociability test. The sociability test apparatus consists of a rectangular three-chambered enclosure, 60 cm × 40 cm, with clear walls 22 cm high. Removable doors block access from the centre chamber to the outer chambers. Two stranger cages, diameter 7 cm, height 15 cm, are placed in the two outer chambers. The test consists of three stages, which were conducted immediately after one another according to published protocols [30,31]. Between each stage, mice were confined to the centre compartment. Mice were first habituated to the apparatus for 5 min, with no mice in the stranger cages. During the second, sociability, stage a stranger mouse was placed in one of the stranger cages, and time spent interacting with the stranger mouse, and the empty cage, was measured. During the final stage, called preference for social novelty, a second stranger mouse was placed in the empty stranger cage, and time spent interacting with each mouse was measured. Interaction was measured manually and defined as time spent sniffing the stranger mouse or cage, or climbing the cage. Preference for the stranger mouse over the empty cage was used as a measure of sociability. Preference for the novel stranger mouse over the familiar stranger mouse was used as a measure of preference for social novelty. 2.4. Statistical analysis All analysis was done using Graphpad Prism statistical software. All results are presented as mean ± SEM and p < 0.05 taken

To analyse locomotor activity at baseline, 1-way ANOVA of distance travelled in the open field was performed showing no difference between the three genotypes (p = 0.10, Fig. 1A). Analysis of time spent in the centre of the open field also shows no difference in anxiety levels in this apparatus (p = 0.96, Fig. 1B). In contrast, 1way ANOVA (p = 0.013) with Tukey’s post hoc test of time spent in open arms of the elevated zero maze shows that Npas4−/− mice are less anxious than both Npas4+/+ (p = 0.044) and Npas4+/− mice (p = 0.027) in this test (Fig. 1C), spending more time in the open arms. 3.2. Depression-like behaviour 1-way ANOVA (p = 0.0078) with Tukey’s post hoc test indicates that Npas4+/− mice exert increased immobility (despair, depression-like behaviour in the FST, Fig. 2A) compared to both Npas4+/+ (p = 0.011) and Npas4−/− mice (p = 0.017). Analysis of total water and saccharin consumption showed that all genotypes consumed significantly more saccharin than water, with no effect of genotype showing there was no difference in consumption of either water of saccharin (repeated measures 2-way ANOVA Interaction p = 0.35, genotype p = 0.52, water/saccharin p < 0.0001, subjects (matching) p = 0.49) (anhedonia, depression-like behaviour in the saccharin preference test, Fig. 2B). Genotype groups also did not show any differences in saccharin preference (Npas4+/+ 94.2 ± 1.2%, Npas4+/− 92.4 ± 1.5%, Npas4−/− 91.3 ± 2.0; 1-way ANOVA p = 0.90). 3.3. Spatial recognition memory Repeated measures 2-way ANOVA comparing time spent in the three arms of the Y-maze (Interaction p = 0.0019, Genotype p = 0.59, Arm p = 0.11, Subjects (matching) p > 0.99) with Bonferroni post hoc test of Genotype effect shows that Npas4+/− mice spend more time in the familiar test arm than both Npas4+/+ (p = 0.0035) and Npas4−/− mice (p = 0.0009), while also spending less time in the start arm than Npas4−/− mice (p = 0.012). Bonferroni post hoc test of Arm effect shows that both Npas4+/+ (p = 0.034) and Npas4−/− mice (p = 0.038) spend more time in the novel compared to the familiar arm, indicating normal spatial recognition memory, whereas Npas4+/− mice appear to have impaired cognition, spending the

Fig. 1. (A) Distance travelled and (B) time spent in the centre of the open field. (C) Time spent in open arms of the elevated zero maze. Data were analysed using 1-way ANOVA with Tukey’s post hoc test. * cf. Npas4+/+ , # Npas4+/− cf. Npas4−/− . *p < 0.05 (n = 17 NPAS4+/+ , 15 NPAS4+/− , 24 NPAS4−/− ).

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Fig. 2. (A) Immobility time in the Forced Swim Test. Data analysed by 1-way ANOVA, * cf. Npas4+/+ , # Npas4+/− cf. Npas4− /− . *p < 0.05 (n = 17 NPAS4+/+ , 15 NPAS4+/− , 24 NPAS4− /− ). (B) Water and saccharin consumption in the saccharin preference test. Data analysed by repeated measures 2-way ANOVA, ****p < 0.0001 cf. water consumed (n = 17 NPAS4+/+ , 15 NPAS4+/− , 10 NPAS4− /− ).

same amount of time in both arms (p = 0.58). Npas4+/− mice also spent more time in the familiar arm compared to the start arm (p = 0.038), while Npas4−/− mice spent less time in the familiar compared to start arm (p = 0.013) (Fig. 3). These results are confirmed by analysing the preference index (PI) of mice for the novel arm over the familiar arm. Npas4+/+ (PI: 0.62 ± 0.04) and Npas4−/− mice (PI: 0.61 ± 0.04) both have a PI > 0.5, indicating preference for the novel arm, while Npas4+/− mice (PI: 0.45 ± 0.04) have a PI around 0.5, indicating no preference for either arm. 1-way ANOVA shows that Npas4+/− mice have a significantly lower PI than both Npas4+/+ (p = 0.0096) and Npas4−/− mice (p = 0.012), confirming their characterisation of impaired memory in this test.

Additionally, analysis of the preference for social novelty phase showed comparable results across strains (2-way ANOVA Interaction p = 0.28, Genotype p = 0.18, Stranger mouse p < 0.0001, Subjects (matching) p = 0.77). Bonferroni post hoc tests of Stranger mouse effect again confirmed normal social behaviour, as all genotypes spent more time interacting with the novel compared to the familiar stranger mouse (Npas4+/+ p = 0.0010, Npas4+/− p = 0.0092, Npas4−/− p < 0.0001, Fig. 4B). Preference index data again confirms these results, with all groups of mice having PI values > 0.5, indicating preference for the novel stranger mouse (Npas4+/+ PI: 0.67 ± 0.06, Npas4+/− PI: 0.67 ± 0.05, Npas4−/− PI: 0.76 ± 0.03), with no significant differences between genotypes (1-way ANOVA p = 0.26).

3.4. Social behaviour Repeated measures 2-way ANOVA (Interaction p = 0.97, Genotype p = 0.34, Cage/Stranger mouse p < 0.0001, Subjects (matching) p = 0.81) with Bonferroni post hoc test of Cage/Stranger mouse effect shows that all strains spent more time interacting with the stranger mouse compared to the empty cage in the sociability phase (Npas4+/+ p < 0.0001, Npas4+/− p < 0.0001, Npas4−/− p < 0.0001), indicating normal sociability behaviour (Fig. 4A). Preference index data confirms these results, with all groups of mice having PI values > 0.5, indicating preference for the stranger mouse (Npas4+/+ PI: 0.82 ± 0.03, Npas4+/− PI: 0.87 ± 0.02, Npas4−/− PI: 0.78 ± 0.05), with no significant differences between genotypes (1way ANOVA p = 0.36).

Fig. 3. Time spent in arms during the retention phase of the Y-maze test. Data analysed by repeated measures 2-way ANOVA with Bonferroni post hoc test. * Novel cf. familiar arm, # familiar cf. start arm *p < 0.05 (n = 17 Npas4+/+ , 15 Npas4+/− , 24 Npas4− /− ).

4. Discussion Previous studies have examined the role of Npas4 in cognition and behaviour and indicated that Npas4 may be involved in regulating aspects of mood, social interaction and memory. Here, we extend these findings using a more comprehensive battery of behavioural tests including cognition-, social- and anxiety-like behaviours not previously studied in Npas4 transgenic mice. Our results show that Npas4−/− mice have lower levels of anxiety on the EZM while Npas4+/− mice have increased depression-like behaviour and impaired spatial recognition memory. These findings are only partially consistent with those reported by other groups, though differences in the types of tests used and the specific parameters applied during testing may account for these seemingly incongruous observations. For example, Coutellier et al. [15] used only male mice which were housed on a reverse light-dark cycle, so that all behavioural testing was conducted in the dark phase of the light cycle, which is likely to contribute to some of the differences seen. Other possible reasons are discussed below. In our study, Npas4 does not affect baseline locomotor activity as measured by distance travelled in the open field test, which is consistent with the findings of Ramamoorthi et al. [3], but differs to Coutellier et al. [15] who showed that Npas4−/− mice are more hyperactive in the novel environment of the open field test. This may suggest that different cohorts of Npas4 mice may differ in baseline behaviours between labs, possibly due to different housing and handling environments, such as the use of a reverse light-dark cycle. The open field test protocols used by the previous studies were also longer than in the present study (10 min cf. 5 min) and Coutellier et al. also used a larger open field area [3,15], almost twice the size of our open field, which may also lead to the differences in results seen between papers.

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Fig. 4. (A) Time spent interacting with an empty cage compared to a stranger mouse and (B) preference for a novel compared to a familiar stranger mouse, during the Sociability test. Data analysed by repeated measures 2-way ANOVA with Bonferroni post hoc test. * Empty cage cf. stranger mouse (A) or familiar cf. novel stranger mouse (B). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (n = 17 Npas4+/+ , 15 Npas4+/− , 24 Npas4−/− ).

In the current study, we found that Npas4−/− mice were less anxious than Npas4+/− and Npas4+/+ mice, as shown by increased time spent in the open arm of the EZM. This is in contrast to other studies conducted in a SERT−/− rat model of anxiety and depression-like symptoms [10], where reduced levels of Npas4 mRNA have been shown in the hippocampus and PFC [11,12], suggesting a complex association between decreased Npas4 and anxiety. While Coutellier et al. [15] did not test Npas4−/− mice in the EZM, they compared only Npas4+/− and Npas4+/+ mice and found no difference between the genotypes. Ramamoorthi et al. used the elevated plus maze (EPM) to measure anxiety though they did not see any differences in time spent in the open arm between Npas4−/− and Npas4+/+ mice [3]. The EZM and EPM apparatus both measure the same behavioural phenotype, however the EZM removes the issue of ambiguity with time spent in the centre of the EPM [23], which may lead to different anxiety results being seen in some studies. Increased time in the centre of the open field can also be a measure of anxiety behaviour and in this test we showed similar results to Coutellier et al. [15], where none of the genotypes tested showed a difference in time spent in the centre. It has been suggested that anxiety-related behaviour can be different depending on the paradigms tested [32], which may also explain differences seen in the open field compared to EZM tests of our study. It has also been suggested that anxiety may exacerbate poor memory [33], while an alternative explanation from Jurgenson et al. [34] suggests that animals with poor cognition may spend more time on the open arms on the elevated plus maze as they do not recognise and learn the danger associated with the open arms, and hence does not actually indicate a change in anxiety. As Npas4−/− mice showed normal memory in the Y-maze neither suggestion is likely in this study. In the FST we found that Npas4+/− mice had higher despair type depression-like symptoms, measured as more time spent immobile compared to Npas4+/+ and Npas4−/− mice. However, baseline saccharin preference, a measure of anhedonia type depression-like behaviour, showed no differences between genotypes. It has been suggested that different aspects of depression may be mediated by different brain regions, which may explain the differing results seen in these two tests. Areas such as the striatum and amygdala, involved in emotional memory, could mediate anhedonia, while the neocortex and hippocampus may mediate cognitive aspects of depression, including feelings of hopelessness [35]. Depressionlike behaviour has not been investigated in Npas4−/− or Npas4+/− mice previously, however as stated previously, the SERT−/− rat model of anxiety and depression-like symptoms, including both increased immobility time in FST and decreased sucrose preference [10], has reduced levels of Npas4 mRNA [11,12]. This suggests reduced levels of Npas4 may also be associated with depressionlike behaviour, as well as anxiety. This link is not clear though, as

uCMS paradigms which lead to depression-like behaviour have led to either decreased Npas4 mRNA expression in the hippocampus associated with decreased sucrose consumption [13], or no change in Npas4 expression in the cingulate cortex, with no direct measure of depression-like behaviour used [14]. Our results, as well as these previous studies, suggest that decreased levels of Npas4 may be associated with increased depression-like behaviour, although this depends on the level of Npas4 decrease, i.e. full or partial KO, and the type of depression-like behaviour studies, i.e. despair or anhedonia. In the Y-maze, Npas4+/+ and Npas4−/− mice showed a significant preference for spending time in the novel test arm compared to the familiar test arm, while Npas4+/− mice did not, indicating they had impaired spatial recognition memory or cognition. This differs to the findings of Coutellier et al. [15] who showed Npas4−/− to have a deficit in working memory and recognition memory, while Npas4+/− mice had a deficit in reversal memory but not spatial learning. However, while these results seem incongruous, it is difficult to make direct comparisons between studies because of the different tests used to assess cognition/memory performance. The study by Coutellier et al. [15] employed the Y-maze alternation task, novel object recognition test (NORT) and a water based Ymaze reversal learning test that measure slightly different types of memory and cognition. That is, the Y-maze task used in the current study measures short-term recognition memory (30-min inter-trial interval), while the NORT used by Coutellier et al. [15] measured long-term recognition memory (24-h inter-trial interval) and their Y-maze tests measure working memory or learning behaviour. The differences in results suggest that different levels of Npas4 may be involved in different types of memory and cognition which could be explored further with more extensive tests of learning such as the Barnes maze. Other studies looking at the role of Npas4 in memory and cognition have only looked at fear based memory tasks. Impaired memory has been shown using a contextual fear conditioning paradigm in mice with global KO of Npas4 and selective deletion in the CA3 region of the hippocampus [3], while Npas4 knockdown in the lateral nucleus of the amygdala led to impaired fear memory formation and impairs retention of a reactivated fear memory [16]. Npas4 has also been shown to be downregulated in Tet1−/− mice which display impaired memory extinction in a fear memory paradigm [17]. Mice generally have a preference for social interactions with other mice [36]. We had hypothesised that we would see differences in social behaviour between phenotypes in this test, as Npas4 has been linked to neurodevelopmental disorders such as schizophrenia and autism in animal models of social, cognition and sensorimotor gating impairments [15] and of rearing in social isolation [37,38]. Npas4 has also been linked to attention-deficit

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hyperactivity disorder [39–41] and autism comorbid with epilepsy [42] in patient populations, through mutations affecting the SLC9A9 gene, a putative target of Npas4 [42]. However, the social behaviour of all genotypes was found to be normal, with all mice preferring to socialise in the first phase of the sociability test, and spend more time with the novel stranger mouse in the preference for social novelty stage. Coutellier et al. [15] used various different social tests to our 1-day, 2-trial sociability and preference for social novelty test. They showed that Npas4−/− mice are less social than Npas4+/+ and Npas4+/− in a 6-trial social test and more aggressive/dominant in the tube dominance test, but do show normal discrimination between familiar and novel mouse in a 2-day social test. The 6trial and 2-day social tests use similar methods to the test we used, with never before met stranger mice being used, as well as subsequent addition of a 2nd, novel stranger mouse. However, differing numbers of trials and inter-trial intervals could contribute to differences seen between studies. Impaired social recognition, which was not seen in either the current study or Coutellier et al. [15], can indicate a type of schizophrenia or autism type behaviour [30,43], suggesting this mouse may not be a model of these types of schizophrenia-like behaviour. It is interesting that Npas4+/− mice showed the strongest phenotypic differences in cognition and depression-like behaviour. It could be that if Npas4 is completely knocked out, then alternative pathways are activated, perhaps during development, which compensate for the lack of Npas4. If, however, mice have partial Npas4 activity (i.e. heterozygous (HET)), then alterative pathways may not be activated but mice still would have a deficit in Npas4 preventing normal function. Interestingly, this assumption is supported by previous studies. For example, Ploski et al. [16] used Npas4 knockdown mice, not KO mice, to show impaired fear memory formation, and Coutellier et al. [15] also showed reversal learning and other behavioural changes in only Npas4+/− mice, however Npas4−/− mice were not always tested. A similar phenomenon was observed in the study of Lin et al. [2] in slice cultures, where acute knockdown of Npas4 in culture lead to reduced number of inhibitory synapses and decreased amplitude of mini inhibitory postsynaptic currents, whereas global knockout in the whole mouse had no effect on these measures. Knockout homozygosity induces the development of alternative compensatory pathways resulting in an apparent normal phenotype. A heterozygote does not develop the compensatory pathways due to existing albeit reduced functional expression of Npas4, however this reduced expression alone exhibits a measurable phenotype. Further work should be done to determine pathways involved in the possible compensatory mechanisms leading to normal cognition and depression-like behaviour in Npas4−/− mice, including development of inducible KO mice. In conclusion, we have shown in this study that Npas4 appears to play a role in anxiety, as well as memory and depression-like behaviour, which is potentially relevant to various neurological and psychiatric conditions. This role appears to be related to different levels of Npas4 deficiency, with Npas4+/− mice, which have partial expression of Npas4, showing impaired behaviour compared to both Npas4+/+ and Npas4−/− mice in the Y-maze and FST. The use of a more extensive memory and learning test, such as the Barnes maze, as well as other tests of emotion, such as the noveltysuppressed feeding test would be useful to confirm behavioural differences seen in this study, and the role Npas4 play in memory, anxiety and depression-like behaviour. Pathways which may have compensated for the total KO of Npas4 in the Npas4−/− mice in these tests should be investigated in future studies, including the use of antidepressant or other drugs which could reverse the behavioural phenotype of Npas4+/− mice, and the measurement of related molecules in different regions of the brain involved with different types of behaviour.

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Conflict of interest statement The authors declare no conflict of interest. Acknowledgements This study was supported by the National Health and Medical Research Council of Australia (APP1003417). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We would also like to thank Sarah Cohen-Woods for assistance with statistical analysis. References [1] Bloodgood BL, Sharma N, Browne HA, Trepman AZ, Greenberg ME. The activitydependent transcription factor NPAS4 regulates domain-specific inhibition. Nature 2013;503:121–5. [2] Lin Y, Bloodgood BL, Hauser JL, Lapan AD, Koon AC, Kim TK, et al. Activitydependent regulation of inhibitory synapse development by Npas4. Nature 2008;455:1198–204. [3] Ramamoorthi K, Fropf R, Belfort GM, Fitzmaurice HL, McKinney RM, Neve RL, et al. Npas4 regulates a transcriptional program in CA3 required for contextual memory formation. Science 2011;334:1669–75. [4] Pruunsild P, Sepp M, Orav E, Koppel I, Timmusk T. Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene. J Neurosci 2011;31:3295–308. [5] Ooe N, Saito K, Mikami N, Nakatuka I, Kaneko H. Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression. Mol Cell Biol 2004;24:608–16. [6] Large-scale genome-wide association analysis of bipolar disorder identifies a new susceptibility locus near ODZ4. Nat Genet 2011;43:977–83. [7] Bersten DC, Bruning JB, Peet DJ, Whitelaw ML. Human variants in the neuronal basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) transcription factor complex NPAS4/ARNT2 disrupt function. PLOS ONE 2014;9:e85768. [8] Ebert DH, Greenberg ME. Activity-dependent neuronal signalling and autism spectrum disorder. Nature 2013;493:327–37. [9] Floor K, Baroy T, Misceo D, Kanavin OJ, Fannemel M, Frengen E. A 1 Mb de novo deletion within 11q13.1q13.2 in a boy with mild intellectual disability and minor dysmorphic features. Eur J Med Genet 2012;55:695–9. [10] Olivier JD, Van Der Hart MG, Van Swelm RP, Dederen PJ, Homberg JR, Cremers T, et al. A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders. Neuroscience 2008;152:573–84. [11] Calabrese F, Guidotti G, Middelman A, Racagni G, Homberg J, Riva MA. Lack of serotonin transporter alters BDNF expression in the rat brain during early postnatal development. Mol Neurobiol 2013;48:244–56. [12] Guidotti G, Calabrese F, Auletta F, Olivier J, Racagni G, Homberg J, et al. Developmental influence of the serotonin transporter on the expression of npas4 and GABAergic markers: modulation by antidepressant treatment. Neuropsychopharmacology 2012;37:746–58. [13] Zhang Z, Fei P, Mu J, Li W, Song J. Hippocampal expression of aryl hydrocarbon receptor nuclear translocator 2 and neuronal PAS domain protein 4 in a rat model of depression. Neurol Sci 2013. [14] Surget A, Wang Y, Leman S, Ibarguen-Vargas Y, Edgar N, Griebel G, et al. Corticolimbic transcriptome changes are state-dependent and region-specific in a rodent model of depression and of antidepressant reversal. Neuropsychopharmacology 2009;34:1363–80. [15] Coutellier L, Beraki S, Ardestani PM, Saw NL, Shamloo M. Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders. PLoS ONE 2012;7:e46604. [16] Ploski JE, Monsey MS, Nguyen T, DiLeone RJ, Schafe GE. The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories. PLoS ONE 2011;6:e23760. [17] Rudenko A, Dawlaty MM, Seo J, Cheng AW, Meng J, Le T, et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 2013;79:1109–22. [18] Camara ML, Corrigan F, Jaehne EJ, Jawahar MC, Anscomb H, Koerner H, et al. TNF-alpha and its receptors modulate complex behaviours and neurotrophins in transgenic mice. Psychoneuroendocrinology 2013;38:3102–14. [19] Jaehne EJ, Baune BT. Effects of chemokine receptor signalling on cognitionlike, emotion-like and sociability behaviours of CCR6 and CCR7 knockout mice. Behav Brain Res 2014;261:31–9. [20] Baune BT, Wiede F, Braun A, Golledge J, Arolt V, Koerner H. Cognitive dysfunction in mice deficient for TNF- and its receptors. Am J Med Genet B: Neuropsychiatr Genet 2008;147B:1056–64. [21] Gould TD, Dao DT, Kovacsics CE. The Open Field Test; 2009. p. 1–20. [22] Hart PC, Bergner CL, Smolinsky AN, Dufour BD, Egan RJ, Laporte JL, et al. Experimental models of anxiety for drug discovery and brain research. Methods Mol Biol 2010;602:299–321. [23] Shepherd JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT. Behavioural and pharmacological characterisation of the elevated zero-maze as an animal model of anxiety. Psychopharmacology (Berl) 1994;116:56–64.

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Effects of Npas4 deficiency on anxiety, depression-like, cognition and sociability behaviour.

The transcription factor neuronal PAS domain-containing protein 4 (Npas4), which regulates the formation of inhibitory synapses on excitatory neurons,...
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