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Research report

Npas4 deficiency increases vulnerability to juvenile stress in mice Laurence Coutellier ∗ , Valerie Gilbert, Ryan Shepard Department of Psychology and Neuroscience, The Ohio State University, Columbus, OH, USA

h i g h l i g h t s • Npas4 deficient mice show long-term cognitive deficits after exposure to juvenile chronic stress. • Juvenile chronic stress induces long-lasting reduction of Npas4 expression in the prefrontal cortex. • Juvenile chronic stress reduces the number of migrating neuroblasts toward cortical regions in Npas-deficient mice.

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Article history: Received 20 January 2015 Received in revised form 30 March 2015 Accepted 15 April 2015 Available online xxx Keywords: Chronic stress Adolescence Npas4 Cognitive function Neuroblast

a b s t r a c t During specific windows of postnatal brain development, individuals are particularly susceptible to developing mental illnesses in adulthood. Adolescence is such a window during which environmental stress can have long-lasting consequences on social and cognitive functions. In individuals, highly vulnerable to stress, a relatively mild stressful situation can trigger the onset of psychiatric conditions. The genetic factors and mechanisms underlying vulnerability to stress are not well understood. Here, we show that variations in expression of the brain-specific transcription factor Npas4 contributes to the long-term consequences of juvenile stress on cognitive abilities. We observed that transgenic Npas4-deficient mice exposed to chronic mild stress during adolescence (but not during adulthood) develop prefrontal cortexdependent cognitive deficits in adulthood, while the same stress did not affect Npas4 wild-type mice. These cognitive deficits were accompanied by fewer neuroblasts in the subventricular zone, and reduced ability of these immature neuronal cells to migrate away from this neurogenic zone toward cortical regions. These findings suggest for the first time that the transcription factor Npas4 could play a significant role in coping with juvenile stress. They also suggest that Npas4 could modulate resilience or vulnerability to stress by mediating the effects of stress on neurogenesis. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Repeated exposure to stressful events is strongly associated with the development of psychiatric disorders [1,2]. More specifically, stress during particular windows of brain development (such as adolescence) can lead to cognitive deficits, social impairments and depressive and anxious behavior in adulthood [3]. A recent imaging study in humans revealed that the developing brain is more vulnerable to the cognitive and neurobiological effects of daily stress than the adult one [4]. However, not everyone exposed to stressful conditions during adolescence will develop a pathological condition later on. It is now well recognized that the development of neuropsychiatric disorders is the result of interactions between specific environmental conditions (such as stress) and individual genetic predisposition [5]. The genetic factors that

∗ Corresponding author. Tel.: +1 614 688 2270; fax: +1 614 292 6798. E-mail address: [email protected] (L. Coutellier).

predispose individuals to stress-related psychiatric conditions, as well as the mechanisms by which they confer resilience or vulnerability to stress, remain largely unknown. The question of why some people are susceptible to stress, while other are resilient, is fundamental to the understanding, diagnosis and treatment of stress-related disorders. The adolescent brain is characterized by an extremely high rate of neurogenesis [6,7], and by the ability of newly born neurons to integrate into cortical regions such as the prefrontal cortex (PFC) [8]. Recent studies have demonstrated that juvenile neurogenesis contributes to the postnatal maturation of the brain and is necessary for important adult functions, such as affiliative behaviors and cognitive abilities [7,9]. Disturbance of this very important cellular mechanism might thus contribute to the development of social and cognitive dysfunctions in adulthood. It is now believed that stress effects on neural plasticity and neurogenesis in limbic regions (such as the hippocampus or the PFC) contribute to some neuropsychiatric conditions such as depression or schizophrenia [10–12]. Recently, it has been suggested that high levels of forebrain

http://dx.doi.org/10.1016/j.bbr.2015.04.027 0166-4328/© 2015 Elsevier B.V. All rights reserved.

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adult neurogenesis could confer resilience to stress and to its negative consequences on cognition and emotional behaviors. Animal models have supported this idea, as neurogenesis-deficient mice showed impaired hypothalamic−pituitary−adrenal axis negative feedback, and transient reduction in cell proliferation in adolescence has been associated with susceptibility to social stress in adulthood [13–16]. Others have suggested that postnatal neurogenesis could be involved in recovery from stress [17], supported by the fact that a chronic treatment with fluoxetine, which increases forebrain postnatal neurogenesis, reduces significantly the negative consequences of exposure to chronic stress on behaviors in mice [18,19]. Altogether, these findings highlight a predominant role of postnatal neurogenesis in resilience to stress and its negative consequences. We thus suggest that genetic factors that regulate postnatal neurogenesis, especially during windows of vulnerability such as adolescence, could confer resilience to stress. A possible candidate gene is the one coding for the transcription factor Npas4. Npas4 is a helix−loop−helix transcription factor, which plays a role in the development of inhibitory synapses by regulating the expression of activity-dependent genes in hippocampal neurons [20]. It is highly expressed in the adult hippocampus and cortex [20] and regulates in a dose-dependent manner cognitive performances and social functions [20–24]. Recently, it has been suggested that stress-induced Npas4 expression reduction could mediate the effects of stress on cognitive functions. Indeed, it has been shown that the memory deficits induced by social isolation, chronic restraint stress or acute corticosterone treatment in mice are associated with reduced expression of Npas4 [25,26]. Furthermore, this stress-induced reduction of Npas4 expression correlates with changes in hippocampal neurogenesis, suggesting a possible mechanism by which the effects of stress on behavior are due to reduced level of Npas4 leading to abnormal neurogenesis. Other findings support the role of Npas4 in postnatal neurogenesis; for instance, Npas4 regulates the expression of brain-derived neurotrophic factor (BDNF) [20], which is known to promote cell survival in the subgranular zone (SGZ) of the dentate gyrus of the hippocampus [27] and to control adult subventricular zone (SVZ) neurogenesis [28]). In this study, we therefore aimed to investigate the role of Npas4 in susceptibility to stress-related disorders. More precisely, we hypothesized that stress-induced modulation of Npas4 expression may shape the behavioral response as well as vulnerability to chronic stress by its effect on neurogenesis. We also hypothesized that this effect would be more important in situations of stress exposure during the juvenile period when the rate of neurogenesis needs to remain high to allow proper development of social and cognitive functions. To test these hypotheses, we assessed the long-term consequences of juvenile and adult chronic stress on the cognitive functions and on forebrain neuroblasts formation of transgenic Npas4-deficient mice expressing only 50% of the normal level of Npas4. We hypothesized, therefore, that mice with reduced expression of Npas4 subjected to juvenile chronic stress will show long-term cognitive deficits and that these deficits will be associated with a reduced number of neuroblasts in neurogenic zones.

2. Materials and methods 2.1. Npas4 expression in the juvenile and adult brain To assess the level of Npas4 mRNA expression in the PFC and hippocampus of juvenile and adult mice, C57BL/6 male mice were used. Four 5-week-old and four 24-week-old C57BL/6 male mice were ordered from Jackson Laboratory (Maine, USA). Mice were group housed per age and allowed to habituate to our colony

room for 1 week. After habituation, mice were anesthetized with isoflurane and rapidly decapitated. Brains were collected and flashfrozen on dry ice. All brains were stored at −80 ◦ C until dissection. Brains were dissected in a cold room on dry ice: the PFC and dorsal hippocampus (dHipp) were collected according to the Mouse Brain Atlas of Franklin and Paxinos [29]. RNA was extracted from tissue using PureZOL RNA Isolation Reagent (Bio-Rad, Hercules CA, USA) and NucleoSpin RNA II (Machery-Nagel, Allentown PA, USA). cDNA templates were generated using iScript Reverse Transcription kit (Bio-Rad, Hercules CA, USA). The target cDNA (Npas4) and the reference target glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified simultaneously with SsoAdvanced SYBR Green Supermix in a CFX96 Real-Time PCR Detection System (BioRad, Hercules CA, USA). Primer sequences for Npas4 were the same as those used in Ramamoorthi et al. [22]. Assays were run in triplicate. Amplification conditions were: 95 ◦ C for 30 s and 40 cycles of PCR (denaturation: 95 ◦ C for 5 s, annealing and/or extension: 60 ◦ C for 30 s). Data were analyzed using the comparative Ct (cycle threshold) method. 2.2. Effects of chronic mild stress (CMS) during the juvenile or adult period on PFC-dependent cognitive functions in Npas4-deficient mice 2.2.1. Animals To determine the role of Npas4 in stress resilience and vulnerability, we used Npas4 transgenic mice [20]. Male Npas4 wild-type (WT) mice and heterozygous (HET) mice, expressing only 50% of the Npas4 WT level, were obtained by HET×HET breeding in our facility. Mice were group-housed (two to five mice per cage–unless specified otherwise) and maintained on a 12 h reverse light–dark cycle with access to food and water ad libitum. Three groups of WT and HET male mice were formed (the number of mice in each group is indicated in the legend of each figure – see Fig. 1 for a schematic diagram representing the different experimental groups). The first group of mice was subjected to CMS during the juvenile period (from PND28 to PND42). After PND42, mice were left undisturbed for 2 weeks until adulthood when they were tested for PFC-dependent cognitive functions. A second group of mice was subjected to CMS during the adult period (from PND145 – 20 weeks – to PND159). After PND159, mice were left undisturbed for 2 weeks, and then tested for PFCdependent cognitive functions. A third group of mice was used as control (standard rearing – SR). They remained group-housed and undisturbed (aside from standard facility procedures) during adolescence and early adulthood, until tested for PFC-dependent cognitive functions in adulthood. 2.2.2. CMS procedure Npas4 WT and HET mice were bred in our facility. The day of birth was recorded as PND0. Mice were weaned at PND21 and group-housed by sex (up to five mice per cage). The CMS procedure consisted of social isolation associated with forced swim stress every 2 days for 2 weeks. On the first day of CMS, mice were single-housed. Every 2 days, they were placed in a glass cylinder (height: 30 cm; diameter: 15 cm) filled with water (24−25 ◦ C) for 4 min. After the swim stress, mice were placed back in their home cage on a heated pad until dry. After 2 weeks of this procedure, mice were left undisturbed (aside from standard facility procedures) until behavioral testing. 2.2.3. Assessment of PFC-dependent cognitive functions All behavioral testing was conducted during the dark phase of the day/night cycle by an experimenter blind to the group

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Fig. 1. Schematic diagram representing the different experimental groups of mice used to study the short- and long-term effects of juvenile stress on cognitive functions and neuroblasts formation. White triangles represent cognitive assays; Black triangles represent brain collection; horizontal black arrows represent CMS. Groups 1, 2 and 3 were used to assess the effects of juvenile (group1) or adult (group 2) CMS on cognitive functions when compared with a non-stress (control) group (group 3). Groups 1, 2, 4 and 5 were used to assess the short- (groups 4 and 5) and long- (groups 1 and 2) term effects of CMS on neuroblast formation.

and genotype of the mice. Three days prior testing, mice were habituated to be handled by an experimenter (1 min per day for 3 days). On the day of testing, mice were allowed to habituate to the testing room for at least 1 h prior to testing. Two tests were used to assess PFC-dependent cognitive functions in the three groups of mice described previously (Section 2.2.1).

of time spent sniffing and with head orientation within 1 cm of the objects was scored as exploration of the objects. A discrimination ratio (DR) was calculated as: (time exploring mismatch-time exploring match)/(total time of exploration). A DR of zero indicates chance level. At the end of each trial, the arena and the objects were cleaned using 70% ethanol.

2.2.3.1. Spontaneous alternation test. Spontaneous alternation, as an indicator of working memory, was assessed using a modified version of the procedure developed by Lalonde [30]. The apparatus consists of a gray plastic maze formed by three arms (A, B and C) so as to form a Y shape (arm length: 40 cm; width: 8 cm; height: 15 cm). Each animal was placed in the apparatus for 8 min during which it was allowed to explore the entire maze. This test is based on a strong tendency in rodents to alternate arm choices, explained by their natural propensity to explore a novel environment over a recently explored one. The series of arm entries (e.g. ACBCABCBCA) was recorded using an overhead camera, and videos were scored by an observer unaware of the animal’s genotype. Alternation was defined as successive entry into the three arms, on overlapping triplet sets (e.g. in the sequence ACBCABCBCA, five alternations were recorded). Percent alternation was calculated as the ratio of actual alternations to possible alternations (defined as the total number of arm entries minus two), multiplied by 100. At the end of each trial, the arena was cleaned using 70% ethanol.

2.3. Short and long-term effects of juvenile CMS on the level of neuroblasts

2.2.3.2. Object/context mismatch test. The object/context mismatch test assesses contextual information processing and requires integrity of the medial PFC [31]. The first step of the test consists in habituating mice to two different contexts for 10 min per day for 2 days: a square arena with white walls and rough floor, and a circular arena with transparent walls and smooth floor. The contexts were placed in separate rooms with different lighting conditions (bright white light vs. dim white light). After habituation (on day 3), each context housed a unique pair of identical objects. Each mouse was placed in one context with a set of objects and allowed to explore them for 8 min. After this first learning session, each mouse was directly placed in the second context with the second set of objects for another 8 min learning session. At the end of these two learning sessions, each mouse was placed back in its home cage for a delay of 10 min. After these 10 min, each mouse was placed in one of the two contexts with one object that was congruent with that context and one that was not. Each mouse was allowed to explore the objects for 5 min. A mouse that successfully associated an object with a context will spend more time exploring the mismatch (non-congruent) object than the match object (congruent). Each session was recorded using an overhead camera. The amount

The short- and long-term effects of juvenile CMS on the postnatal level of neuroblasts were assessed on four groups of mice (see Fig. 1 for a schematic diagram representing the different experimental groups). 2.3.1. Short-term effects of juvenile CMS The first group of WT and HET mice was exposed to CMS during the juvenile period (PND28 to 42) as described earlier. Twenty-four hours after the last forced-swim stress, mice were euthanized via perfusion with 4% paraformaldehyde (PFA) and their brains were collected. A second group of WT and HET mice was used as control. Mice were group-housed during the juvenile period and left undisturbed (aside from standard facility procedures). At PND43 mice were euthanized via perfusion with 4% PFA and their brains were collected. 2.3.2. Long-term effects of juvenile CMS The mice from the first and third groups described in Section 2.2.1 were used to assess the long-term effects of juvenile CMS on neuroblasts. After behavioral testing, mice were euthanized via perfusion with 4% PFA and their brains were collected. 2.3.3. Immunohistochemistry for doublecortin (DCX) expression Animals were anesthetized with isoflurane and perfused transcardially with 50 ml of 0.1 M phosphate-buffered saline (pH 7.4) followed by 50 ml of fresh 4% PFA. Brains were removed and postfixed overnight in PFA at 4 ◦ C, placed in 30% sucrose until they sank, then frozen on dry ice and sectioned at 50 ␮m using a cryostat. All sections containing the subventricular zone (SVZ – +1.10 to +0.02 mm from Bregma), and subgranular zones (SGZ) of the dorsal hippocampus (dHipp) and ventral hippocampus (vHipp) (−1.8 to −5.2 mm and −5.2 to −6.7 mm from Bregma, respectively – as indicated in de Andrade et al. [32]) were collected so as to obtain three sets of each region. Sections were stored in cryoprotectant solution at −20 ◦ C until immunohistochemistry on free-floating sections was performed.

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The evaluation of the number of neuroblasts was based on the expression level of doublecortin (DCX) in the three adult neurogenic zones: the SVZ and the SGZ of the dHipp and vHipp. DCX is commonly used to estimate postnatal neurogenesis as it reveals a mixed population of 1–14-day-old newly born neurons [33], and has been validated as a reliable and specific marker of neuroblasts formation [34]. A set of SVZ and SGZ sections were stained using a rabbit anti-DCX primary antibody (ABCam, Cambridge, MA, USA; dilution 1:2000), detected using Vectastain Elite ABC reagents (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine. The quantitative analysis of DCX expression in the SVZ and SGZ of the dHipp and vHipp was achieved using the unbiased stereology method by an experimenter blind to the genotype and treatment of the mice. The total number of DCX-positive cells in each region was quantified using the optical fractionator method [35], with assistance from the StereoInvestigator software from MBF Bioscience (Williston, VT, USA). Cells were counted in every three sections. A total of six sections per animal were analyzed for the SVZ, eight sections for the SGZ in the dHipp and four sections for the SGZ in the vHipp. The sections containing the SVZ were also used to assess the number of DCX-positive cells in the corpus callosum. Regions of interest were outlined under low magnification (5×) according to the Mouse Brain Atlas of Franklin and Paxinos [28], and positive cells counted at high magnification (63× oil immersion). The counting criteria were determined so as to obtain a mean coefficient of error (CE) of Gundersen [36] below 0.10. To assess the number of immature neurons migrating through the corpus callosum toward the cortex, DCX-positive cells were also counted in the corpus callosum (above the dorsal part of the SVZ). In addition to DCX immunohistochemistry, cell proliferation in the SVZ was also assessed using the Ki67 marker of proliferative cells. A set of SVZ sections were stained using a rabbit anti-Ki67 primary antibody (ABCam, Cambridge, MA, USA; dilution 1:500), detected using Vectastain Elite ABC reagents (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine. The number of Ki67 positive cells was assessed using the unbiased stereology method as described earlier. 2.4. Effects of juvenile CMS on Npas4 expression To assess the effects of juvenile CMS on Npas4 expression, WT and HET male mice were exposed to CMS during adolescence as previously described or were maintained under standard rearing conditions (control) (n = 4–5 per genotype per group). Animals were anesthetized 24 h after the last forced-swim (or at the matching age for controls) and rapidly decapitated. Brains were collected, dissected so as to obtain the PFC, dHipp and vHipp, and treated as described in Section 2.1. Data were analyzed using the comparative Ct (cycle threshold) method. 2.5. Statistical analyses All data are presented as mean ± standard error of the mean (SEM). Data were analyzed using the software Prism 5.01 (GraphPad Software Inc., CA, USA). Normality of all data was tested and verified using the Shapiro–Wilk test. Npas4 mRNA expression in juvenile and adult PFC and hippocampus was analyzed using a 2 × 2 ANOVA with age and brain region as independent variables, followed by Tukey’s post hoc test when appropriate. Behavioral data were analyzed using a 2 × 2 ANOVA with genotype (WT and HET) and stress exposure (SR, CMS juvenile and CMS adult) as independent variables, followed by Bonferroni’s post hoc tests when appropriate. Neuroblast level data were analyzed using a 2 × 2 ANOVA with genotype (WT and HET) and stress exposure (SR and CMS juvenile) as independent variables, followed by Bonferroni’s post hoc tests when appropriate. Npas4 expression after exposure

Fig. 2. mRNA expression of Npas4 in the prefrontal cortex (PFC) and hippocampus (Hipp) in juvenile (J) and adult (A) C57BL/6 male mice (n = 4 per group). Significant main effect of age (p = 0.0056), main effect of brain region (p = 0.0008) and interaction (p = 0.043). **p ≤ 0.01.

to juvenile CMS was analyzed separately for each brain region (PFC, dHipp and vHipp) using a 2 × 2 ANOVA with genotype (WT and HET) and stress exposure (SR and CMS juvenile) as independent variables. Statistical significance was set at p ≤ 0.05. 3. Results 3.1. Npas4 expression in the juvenile and adult brain Npas4 mRNA expression was assessed in the PFC and hippocampus of juvenile and adult C57BL/6 mice (Fig. 2). We observed a significant main age effect (F1,12 = 11.31; p = 0.0056) and main brain region effect (F1,12 = 19.83; p = 0.0008), as well as a significant interaction (F1,12 = 5.047; p = 0.043). Post hoc analyses showed that Npas4 mRNA expression in the juvenile PFC was significantly higher than in the adult PFC (p = 0.009) and in the juvenile hippocampus (p = 0.002). 3.2. PFC-dependent cognitive abilities after juvenile or adult CMS in Npas4 WT and HET mice In the spontaneous alternation test, we did not observe any genotype or stress effect on the percent of alternation (Fig. 3a). However, we observed a significant interaction effect for the total arm entries (Fig. 3b – F2,47 = 3.195; p = 0.05). Post hoc analyses showed that WT mice were not affected by CMS during the juvenile period, but exposure to CMS during adulthood leads to a reduced number of arm entries when compared with their controls (WT-SR – p = 0.0004). We also found that HET mice exposed to CMS during the juvenile period have a significant increase in the number of arm entries when compared with their control (HET-SR – p = 0.040), while CMS in adulthood did not affect their activity level. In the Object/Context mismatch test, we observed that CMS during the juvenile period or during adulthood induces an increase in the time spent sniffing the object during the first and second learning sessions in both WT and HET mice (main stress exposure effect: learning 1: F2,36 = 17.86; p < 0.001; learning 2: F2,36 = 21.36; p < 0.001 – Fig. 4a and b). Post hoc analyses showed that the control groups differ from both the group exposed to CMS during the juvenile period (p = 0.01 for both learning sessions) and the group exposed to CMS during adulthood (p < 0.001 for both learning sessions). Analysis of the discrimination ratio (DR – Fig. 4c) shows a significant genotype effect (F1,36 = 11.37, p = 0.0018) and a significant interaction (F2,36 = 3.315, p = 0.047). Post hoc analyses show that the DR of Npas4 HET exposed to CMS during the juvenile period was significantly lower than the DR of Npas4 HET raised in standard

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Fig. 3. Performances in the spontaneous alternation Y-maze test of Npas4 wild-type (WT) and heterozygous (HET) mice reared in standard conditions (SR–WT n = 11; HET n = 16), exposed to chronic mild stress (CMS) during the juvenile period (WT n = 8; HET n = 5), or during adulthood (WT n = 7; HET n = 6). (a) Percent of alternation: no effect of genotype or of rearing condition. (b) Number of arm entries: adult CMS affects WT mice (***p < 0.0001) while juvenile CMS affects HET mice (*p < 0.05).

Fig. 4. Performances in the object/context mismatch test of Npas4 wild-type (WT) and heterozygous (HET) mice reared in standard conditions (SR – WT n = 11; HET n = 8), exposed to chronic mild stress (CMS) during the juvenile period (WT n = 6; HET n = 5) or during adulthood (WT n = 7; HET n = 5). (a,b) Learning session 1 and 2, respectively: CMS affects sniffing *p < 0.05; ***p < 0.0001. (c) Discrimination ratio: juvenile CMS affects HET mice (*p < 0.05).

conditions (p = 0.050), while no significant difference was observed in WT animals or between control Npas4 HET mice and Npas4 HET exposed to CMS during adulthood. 3.3. Postnatal level of neuroblasts Short-term effects of juvenile CMS on the number of DCX-positive cells. In the SGZ of the dHipp, no main effect of genotype or stress condition was observed (Fig. 5a). However, a significant interaction was noticed (F1,13 = 5.07; p = 0.04). No significant differences were revealed by post hoc analyses. In the SGZ of the vHipp, no main effect of genotype or stress condition, as well as no interaction between the two independent variables was observed (Fig. 5b). In the SVZ (Fig. 5c), a significant genotype effect was observed (F1,14 = 6.659; p = 0.022). However, this main genotype effect is associated with a significant interaction between genotype and stress condition (F1,14 = 9.387; p = 0.008). Post hoc analyses show that while WT and HET mice did not differ when reared under standard conditions, HET mice exposed to CMS have significantly fewer DCX-positive cells than WT-CMS mice (p = 0.017). Long-term effects of juvenile CMS on the number of DCX-positive cells. Analysis of the number of DCX-positive cells in the SGZ of the dHipp showed no significant genotype or stress condition effect (Fig. 6a). In the SGZ of the vHipp, we observed a significant stress condition effect (F1,13 = 9.218; p = 0.009 – Fig. 6b), with exposure to CMS during the juvenile period increasing the number of DCX-positive cells. No effect of genotype or significant interaction was noticed. In the SVZ, a significant genotype effect was found (F1,14 = 6.288; p = 0.025) with HET mice having fewer DCXpositive cells than WT mice (Fig. 6c). We then assessed the number of DCX-positive neurons migrating from the SVZ through the corpus callosum. An increased number of studies highlight such a migration in the rodent brain (i.e. [37]); indeed, we observed a significant number of DCX-positive cells in the corpus callosum that present a migratory morphology: oval cell body and long processes, while other DCX-positive cells appear more mature with larger, rounder cell bodies as described by Lalonde [30]. We observed a significant genotype effect (F1,12 = 4.840; p = 0.048) associated with

a significant interaction between genotype and stress condition (F1,12 = 6.968; p = 0.022). Post hoc analyses show that the number of DCX positive cells in the corpus callosum of HET mice exposed to CMS was significantly lower than that of WT mice exposed to CMS (p = 0.04 – Fig. 6d). Because these data seem to indicate a genotype-dependent effect of juvenile CMS on migration of immature neurons born in the SVZ, we chose to determine whether other aspects of neurogenesis (such as proliferation) showed a similar effect. This supplemental analysis was also performed because we observed that the number of DCX-positive cells in the SVZ of HET–CMS seem to increase when compared with HET–SR controls (even if no statistical difference was noted – Fig. 5c). We thus stained SVZ-containing sections with a marker of cell proliferation (Ki67) and counted the number of positive cells as previously described. We were unable to find any genotype, stress condition or interaction effects (Fig. 6e). 3.4. Npas4 expression after juvenile CMS The effect of juvenile CMS on Npas4 expression in the PFC, dHipp and vHipp of WT and HET was assessed via RT-PCR. Data are presented in Fig. 7. We observed a main effect of genotype in all our brain regions, which was expected, since HET mice carry only one copy of the Npas4 gene and express only 50% of the WT levels. A main effect of CMS was observed only in the PFC (F1,13 = 9.263; p = 0.009 – Fig. 7a). No interaction between genotype and CMS was observed in any of the brain regions analyzed. 4. Discussion In this study, we provide evidence for the involvement of Npas4 in mediating cognitive deficits induced by chronic stress exposure during adolescence. First, we showed that Npas4 is significantly more expressed in the juvenile brain than the adult brain. Then, we showed that a genetic deficiency in Npas4 expression in the mouse brain accompanied by chronic mild stress during adolescence leads to long-lasting PFC-dependent cognitive deficits, which are not observed when chronic stress is applied during adulthood.

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Fig. 5. Short-term consequences of CMS during adolescence on the number of neuroblasts in Npas4 wild-type (WT) and heterozygous (HET) mice. The number of doublecortin (DCX)-positive cells was counted using the unbiased stereological method in Npas4 WT and HET mice reared in standard conditions (SR) or exposed to CMS during adolescence. (a) SGZ of the dorsal hippocampus: SR: WT n = 4; HET n = 5; CMS WT n = 4; HET n = 4. Significant interaction p = 0.042; post hoc test: T p = 0.084. (b) SGZ of the ventral hippocampus: SR: WT n = 4; HET n = 4; CMS WT n = 4; HET n = 4. (c) Subventricular zone (SVZ): SR: WT n = 5; HET n = 4; CMS WT n = 5; HET n = 4. Genotype effect p = 0.022; interaction p = 0.008; post hoc test *p < 0.05.

Furthermore, we demonstrated that Npas4-deficient mice exposed to juvenile chronic stress have long-lasting abnormalities in neuroblast formation, which were not observed in Npas4 wild-type mice. These findings suggest that Npas4 may be a genetic factor contributing to stress resilience or vulnerability during the juvenile period by its action on neurogenesis. Adolescence is a critical period for the development of the brain. Many studies showed that exposure to negative environmental factors, such as chronic social stress or psychological stress, during this specific period of maturation can lead to long-lasting social and cognitive deficits (i.e. [38–40]). However, the effects of stress during adolescence on adult functions depends on the

type of stressors, and some studies indicated that a predictable chronic mild stress might have beneficial consequences in adulthood such as reduced anxiety, resilience to stress and improved cognitive functions [33,41]. Our present results indicate that the effects of juvenile stress on cognitive functions depend also on specific genetic components such as the level of expression of the transcription factor Npas4. We observed that mice with normal levels of Npas4 expression were moderately affected by juvenile and adult chronic stress. More specifically, their general level of activity was affected by exposure to adult chronic stress, while cognitive functions remained intact. Indeed, we observed a decrease in locomotor activity in the spontaneous alternation test after exposure

Fig. 6. Long-term consequences of CMS during adolescence on the number of neuroblasts in Npas4 wild-type (WT) and heterozygous (HET) mice. The number of doublecortin (DCX)-positive cells and Ki67-positive cells was counted using the unbiased stereological method in Npas4 WT and HET mice reared in standard conditions (SR) or exposed to CMS during adolescence. (a) SGZ of the dorsal hippocampus: SR: WT n = 4; HET n = 4; CMS WT n = 4; HET n = 4. (b) SGZ of the ventral hippocampus: SR: WT n = 4; HET n = 5; CMS WT n = 4; HET n = 4. CMS effect **p < 0.01. (c) Subventricular zone (SVZ) – DCX: SR: WT n = 4; HET n = 4; CMS WT n = 6; HET n = 4. Genotype effect *p < 0.05. (d) Corpus callosum: SR: WT n = 5; HET n = 4; CMS WT n = 4; HET n = 4. Genotype p = 0.048; Interaction p = 0.022; post hoc test *p < 0.05. (e) Subventricular zone (SVZ) – Ki67: SR: WT n = 3; HET n = 4; CMS WT n = 3; HET n = 3.

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Fig. 7. Npas4 mRNA expression in the brain of Npas4 wild-type (WT) and heterozygous (HET) mice reared in standard conditions (SR – WT n = 4; HET n = 5) or exposed to chronic mild stress (CMS–WT n = 4; HET n = 4) during the juvenile period. (a) Prefrontal cortex: genotype effect **p < 0.01; CMS effect p = 0.0035. (b) Dorsal hippocampus: Genotype effect **p < 0.01. (c) Ventral hippocampus: Genotype effect *p < 0.05.

to adult chronic stress and an increase in exploratory behavior in the learning phases of the object/context mismatch test after both juvenile and adult stress. Chronic-stress-induced hypoactivity in an unfamiliar environment, as the one we noticed in the spontaneous alternation test, has been previously observed in rodents and has been interpreted as anxiety-like behavior [42,43]. We would need to use behavioral assays specific to the measure of anxiety-like behavior in mice (i.e. open field test or elevated-plus maze test) to determine whether the hypoactivity observed in our Npas4 wild-type mice in the spontaneous alternation test reflects a stress-induced anxious phenotype. However, this hypoactivity in the Y-maze was associated with an increased activity in the object/context mismatch test after adult stress. While these results seem to be contradictory, the differences between the conditions of the two tests are likely to explain them. The object/context mismatch test exposes mice to unfamiliar objects in each of the learning phases. Novelty-seeking in rodents is a reward-driven behavior with novel stimuli causing the release of dopamine in the brain [44]. Chronic stress has been shown to increase dopamine levels in several brain regions [45], which can explain the hyperactivity observed in the object/context mismatch test. Finally, the lack of cognitive deficit in Npas4 wild-type mice following our chronic stress procedure is probably associated with the predictability of the stressors we used. Indeed, mice were carried every 2 days in the same room and exposed to the same 4 min forced swim stress. In addition to the swim stress, mice were socially isolated; chronic social stress has been associated previously with impaired hippocampal-dependent long-term memory [46] but not with deficits in working or short-term memory [47], which is supported by our findings. Contrary to what we observed in Npas4 wild-type mice, Npas4deficient mice appeared to be more affected by juvenile stress, not only regarding their general activity level, but also their cognitive abilities. We first observed that exposure to stress during the juvenile period induces hyperactivity in the spontaneous alternation test, and a similar increase in exploratory behavior in the object/context mismatch test to the one observed in Npas4 wildtype mice. We then observed impaired PFC-dependent cognitive performance in the object/context mismatch test. In comparison, exposure to adult stress did not have any significant effects, aside from the increase in exploratory behavior in the object/context mismatch test. It is interesting to observe that the phenotype of Npas4 heterozygous mice exposed to juvenile stress resembles that of Npas4 knockout mice [23]. We found in our previous work a dose-dependent effect of Npas4 expression on locomotor activity and object recognition. Npas4 knockout mice were reported to have hyperactivity and cognitive deficits, while the phenotype

of Npas4 heterozygous mice was intermediate to that of Npas4 wild-type and knockout mice. Our current results further support this dose-dependent effect of Npas4 on general activity and cognitive functions. Indeed, we showed here that exposure to chronic mild stress decreases significantly, the level of Npas4 mRNA in both Npas4 wild-type and heterozygous mice, especially in the prefrontal cortex. We noticed that the Npas4 level in wild-type mice exposed to stress reached the level observed in non-stressed heterozygous mice (which are known to display normal locomotor activity and cognitive functions, [23]). While this stress-induced reduction of Npas4 expression in wild-type mice is significant, it might not be severe enough to induce a change in behavioral phenotype. Furthermore, our conclusions are based on mRNA levels and not protein levels. It is possible that the level of Npas4 protein is not affected by stress, since post-translational processes could be put in place to maintain high level of Npas4 in the system in situation of stress. On the contrary to wild-type mice, the same effect of stress in heterozygous mice might reduce the Npas4 expression below the minimal amount necessary to maintain a normal phenotype and thus, induces behavioral impairments (as observed in Npas4 knockout mice, [23]). Interestingly, increased locomotor activity and impaired contextual information processing (as assessed by the object/context mismatch test) are both known to require an intact prefrontal cortex and to be independent of the hippocampus [31,48]. Our molecular data support the idea of long-term effects of chronic stress on the prefrontal cortex but not hippocampus: at least 24 h after the end of the chronic stress, Npas4 expression levels were reduced in the prefrontal cortex but not in the hippocampus. Our findings support only partially previous findings, where stress was reported to induce Npas4 expression reduction in both the frontal cortex and hippocampus [26,49]. The stress procedure, as well as the timing of tissue collection, described in these studies was significantly different than ours: the authors used restraint and corticosterone injection as stressors and measured Npas4 expression 4 h after these manipulations, while we waited 24 h after the last forced swim stress to collect brain tissue. These results suggest that the prefrontal cortex might be more susceptible to the long-term consequences of juvenile stress, while the hippocampus might be able to recover faster. Such a fast recovery of the hippocampus after exposure to chronic juvenile stress was also observed in regards to the number of neuroblasts. In the short term, no significant effect of stress was found in the SGZ of both the dorsal hippocampus and ventral hippocampus. In the long-term, juvenile chronic stress did not affect the dorsal hippocampus, and induced an increase in the number of immature (DCX) neurons in the ventral hippocampus in both Npas4 wild-type and Npas4-deficient mice. This finding supports

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previous studies showing that in the case of a predictable chronic stress, increased hippocampal neurogenesis can occur [33]. This increase in hippocampal neurogenesis has been previously associated with increased mood and hippocampal-dependent memory; however, in our current work, we have not tested for these functions to determine whether they were improved, and if so, in a genotype-dependent way. In the SVZ, significant differences in the number of immature neurons were found. First, we observed a significant genotype effect in brains collected 2 weeks after cessation of the juvenile chronic stress procedure. Npas4 heterozygous mice have significantly fewer neuroblasts than Npas4 wild-type, independent of stress exposure. This result supports previous findings where a loss of Npas4 function was associated with reduced DCX expression in the postnatal olfactory bulb [50]. However, we were not able to observe such a significant genotype effect in the brains of mice collected directly at the end of the stress procedure. This might reflect an age effect; indeed this group was younger than the previously described group (PND42 vs. PND70), which reflects the transition from adolescence to adulthood. Juvenile neurogenesis is known to be significantly more important than adult neurogenesis [6,7]; the high level of juvenile neurogenesis might hide any possible genotype effects. Another explanation could be that the brains collected 2 weeks after cessation of the chronic stress were from animals tested in the cognitive functions assays, while brains collected directly after stress exposure were from behavioral assay-naïve animals. Exposure to behavioral testing might have affected neuroblast formation in a genotype-dependent manner. The contribution of Npas4 expression to experience-induced neuroblast formation, and neurogenesis in general, would require more investigation. Our data also indicate that juvenile chronic stress strongly affects neuroblast level in the SVZ, and does so in a genotype-dependent way. First, juvenile chronic stress did not affect the number of DCX cells in Npas4 wild-type mice in either short- or long-term. This lack of stress effect on the formation of neuroblasts in the SVZ might be due to the rather predictable nature of the chronic stress procedure we used. Indeed, in the case of more severe stressors, such as unpredictable chronic stress or daily forced swim stress for 3 weeks, a decrease in survival of newly-generated cells in the SVZ and delayed maturation of newly born neurons were previously found [17,51]. Contrary to Npas4 wild-type mice, Npas4-deficient mice exposed to juvenile chronic stress had fewer neuroblasts in the SVZ directly after the end of the stress procedure. In the long-term, this effect disappeared and we observed a nonsignificant increase number of DCX cells in the SVZ. This increase was associated with significantly fewer neuroblasts with a migratory phenotype in the corpus callosum. These results suggest that in Npas4-deficient mice, juvenile chronic stress does not prevent the formation of new neuroblasts in the SVZ, but rather prevents their migration toward cortical regions. This is supported by the absence of genotype and stress effects on the number of Ki67 positive cells, a marker of cell proliferation. However, the absence of stress effects on cell proliferation might need to be confirmed, since a small number of animals (n = 3 per group) was used in the current study, which might limit the interpretation of the data. Altogether, our results indicate that the continuous formation of neuroblasts in the SVZ associated with poor migration could explain the accumulation of immature neurons in this neurogenic zone. Continuous migration of neuronal precursor cells from the SVZ to the cortex has been observed in the adult rodent [52,53] and monkey brains [54]. While the molecular mechanisms regulating this migration remain to be determined, our results suggest that Npas4 could act as a chemo-attractant and contribute to the migration of these neuroblasts toward cortical regions such as the PFC. Our data indeed indicate that Npas4 is preferentially expressed in the PFC during the juvenile period, when high level of migration from the SVZ toward the PFC is observed [8]. While the function

of this particular population of migrating neuroblasts generated during adolescence and reaching the PFC is still unclear, increasing evidence shows that juvenile neurogenesis is necessary for important adult functions such as affiliative behaviors and cognitive abilities [7,9]. The stress-induced decrease of Npas4 in the PFC of heterozygous mice might thus prevent migration of neuroblasts from the SVZ and their maturation into neurons, leading to improper postnatal maturation of the PFC and PFC-dependent behavioral impairments in adulthood. In the current study, the use of a neuroblast marker (DCX) does not allow us to conclude on an effect of Npas4 expression and stress on neurogenesis. The use of a BrdU approach will help answering this question. Furthermore, further studies involving conditional Npas4 gene inactivation during the juvenile period will help clarifying the contribution of Npas4 to the survival of neuroblasts and their migration toward, and their integration into PFC circuits. Additional studies are also needed to determine whether the effects of stress on the migration of newlygenerated neurons in the juvenile SVZ contribute directly to the behavioral deficits we observed here. 5. Conclusion While the present experiments do not allow us to conclude a causal relationship between stress-induced altered neuroblast formation in Npas4-deficient mice and their adult PFC cognitive deficits, they clearly demonstrate that reduced expression of Npas4 in the brain can lead to long-lasting stress-related behavioral impairments. Interestingly, a recent study in humans reveals that Npas4 shows naturally occurring variance in the brain [55]. Some of these natural variants significantly reduced Npas4 transcriptional activity. The reduced activity of Npas4 in these individuals might put them at risk for stress-related disorders, especially after stress exposure in adolescence. Acknowledgments We would like to thank Dr. M. Shamloo from Stanford University for providing the original breeders for our Npas4 mouse colony. References [1] McEwen BS. Early life influences on life-long patterns of behavior and health. Ment Retard Dev Dis Res Rev 2003;9:149–54. [2] Buwalda B, Geerdink M, Vidal J, Koolhaas JM. Social behavior and social stress in adolescence: a focus on animal models. Neurosci Biobehav Rev 2011;35:1713–21. [3] Heim C, Nemeroff CB. The role of childhood trauma in the neurobiology of mood and anxiety disorders: preclinical and clinical studies. Biol Psychiatry 2001;49(12):1023–39. [4] Rahdar A, Galván A. The cognitive and neurobiological effects of daily stress in adolescents. Neuroimage 2014;92:267–73. [5] Caspi A, Moffitt TE. Gene-environment interactions in psychiatry: joining forces with neuroscience. Nat Rev Neurosci 2006;7(7):583–90. [6] He J, Crews FT. Neurogenesis decreases during brain maturation from adolescence to adulthood. Pharmacol Biochem Behav 2007;86(2):327–33. [7] Cushman JD, Maldonado J, Kwon EE, Garcia AD, Fan G, Imura T, Sofroniew MV, Fanselow MS. Juvenile neurogenesis makes essential contributions to adult brain structure and plays a sex-dependent role in fear memories. Front Behav Neurosci 2012;6:3. [8] Inta D, Alfonso J, von Engelhardt J, Kreuzberg MM, Meyer AH, van Hooft JA, Monyer H. Neurogenesis and widespread forebrain migration of distinct GABAergic neurons from the postnatal subventricular zone. Proc Natl Acad Sci U S A 2008;105(52):20994–9. [9] Wei L, Meaney MJ, Duman RS, Kaffman A. Affiliative behavior requires juvenile, but not adult neurogenesis. J Neurosci 2011;31(40):14335–45. [10] McEwen BS. The neurobiology of stress: from serendipity to clinical relevance. Brain Res 2000;886:172–89. [11] Czéh B, Müller-Keuker JI, Rygula R, Abumaria N, Hiemke C, Domenici E, Fuchs E. Chronic social stress inhibits cell proliferation in the adult medial prefrontal cortex: hemispheric asymmetry and reversal by fluoxetine treatment. Neuropsychopharmacology 2007;32(7):1490–503. [12] Duman RS. Depression: a case of neuronal life and death. Biol Psychiatry 2004;56:140–5.

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