Region-specific impairments in parvalbumin interneurons in social isolation-reared mice

Accepted Manuscript Region-Specific Impairments in Parvalbumin Interneurons in Social IsolationReared Mice Hiroshi Ueno, Shunsuke Suemitsu, Shinji Murakami, Naoya Kitamura, Kenta Wani, Motoi Okamoto, Yosuke Matsumoto, Takeshi Ishihara PII: DOI: Reference:

S0306-4522(17)30486-4 http://dx.doi.org/10.1016/j.neuroscience.2017.07.016 NSC 17893

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

19 February 2017 17 May 2017 9 July 2017

Please cite this article as: H. Ueno, S. Suemitsu, S. Murakami, N. Kitamura, K. Wani, M. Okamoto, Y. Matsumoto, T. Ishihara, Region-Specific Impairments in Parvalbumin Interneurons in Social Isolation-Reared Mice, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.07.016

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Hiroshi Ueno et al.

REGION-SPECIFIC IMPAIRMENTS IN PARVALBUMIN INTERNEURONS IN SOCIAL ISOLATION-REARED MICE

Hiroshi Ueno1, 2*, Shunsuke Suemitsu3, Shinji Murakami3, Naoya Kitamura3, Kenta Wani3, Motoi Okamoto2, Yosuke Matsumoto4, Takeshi Ishihara3

1

Department of Medical Technology, Kawasaki University of Medical Welfare, Okayama, 701-0193, Japan

2

Department of Medical Technology, Graduate School of Health Sciences, Okayama University, Okayama, 700-8558, Japan

3

Department of Psychiatry, Kawasaki Medical School, Kurashiki, 701-0192, Japan

4

Department of Neuropsychiatry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8558, Japan

*Corresponding author. Hiroshi Ueno, PhD Address: Department of Medical Technology, Kawasaki University of Medical Welfare, 288, Matsushima, Kurashiki, Okayama, 701-0193, Japan Phone: +81-86-462-1111, Fax: +81-86-462-1193 E-mail: [email protected] (H. Ueno) 1

Hiroshi Ueno et al.

Abbreviations

PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; S1, primary somatosensory cortex; M1, primary motor cortex; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; S1BF, primary somatosensory cortex – barrel field; S1Tr, primary somatosensory cortex – trunk region; CA1, cornu ammonis area 1; CA3, cornu ammonis area 3; DG, dentate gyrus; Ect, ectorhinal cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex

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Abstract Many neuropsychiatric disorders show localized dysfunction in specific cortical regions. The mechanisms underlying such region-specific vulnerabilities are unknown. Post-mortem analyses have demonstrated a selective reduction in the expression of parvalbumin (PV) in GABAergic interneurons in the frontal rather than the sensory cortex of patients with neuropsychiatric disorders such as schizophrenia, autism spectrum disorders, and bipolar disorders. PV neurons are surrounded by perineuronal nets (PNNs), and are protected from oxidative stress. Previous studies have shown that the characteristics of PNNs are brain region-specific. Therefore, we hypothesized that PV neurons and PNNs may be targeted in region-specific lesions in the brain. Oxidative stress was induced in mice by rearing them in socially isolated conditions. We systemically examined the distribution of PV neurons and PNNs in the brains of these mice as well as a control group. Our results show that the regions

frequently

affected

in

neuropsychiatric

disorders

show

significantly lower PV expression and a lower percentage of PV neurons surrounded by PNNs in the brains of socially isolated mice. These results indicate that PV neurons and PNNs exhibit region-specific vulnerabilities. Our findings may be useful for elucidating the mechanisms underlying region-specific disruption of the brain in neuropsychiatric disorders.

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Key words: brain region-specific, mouse, parvalbumin, perineuronal nets, social isolation, vulnerability

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INTRODUCTION A balance between excitation and inhibition is important for normal brain activity. The main components of cortical neural circuits are excitatory glutamate pyramidal

neurons

and

inhibitory

GABAergic

interneurons. GABAergic

interneurons regulate excessive excitation of pyramidal neurons (Dichter, 2006), and it has been shown that the impairment of GABAergic interneurons leads to excessive excitation of pyramidal neurons, thereby causing epileptic seizures (Galanopoulou, 2010; Poduri and Lowenstein, 2011). In addition, recent human and animal studies have suggested that many psychiatric disorders are characterized by an imbalance in the excitation - inhibition pattern in the brain (Marín, 2012). GABAergic interneurons are classified based on morphology, connectivity pattern, synaptic properties, marker expression, and intrinsic firing properties (Kepecs and Fishel, 2014). When these interneurons are simply classified based on

neuronal

interneurons

markers, (PV

calbindin-expressing

they

are

neurons),

grouped

into

parvalbumin-expressing

somatostatin-expressing

interneurons,

calretinin-expressing

interneurons,

interneurons,

vasointestinal peptide-expressing interneurons, and the like. Parvalbumin, calbindin, and calretinin proteins are Ca2+-binding proteins (CaBPs) (McDonald and Mascagni, 2001). Of the GABAergic interneurons, PV neurons are the most abundant (Cowan et al., 1990; Kubota and Kawaguchi, 1993). PV neurons synapse to the cell bodies and proximal dendrites of excitatory pyramidal neurons and regulate the output of pyramidal neurons (Williams et al., 1992; Markram et al., 2004). As each PV neuron can form synapses with hundreds of 5

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pyramidal neurons, these GABAergic interneurons play an important role in controlling the synchronous firing of pyramidal neurons (Bartos et al., 2007; Sohal et al., 2009). This is essential for the generation of γ-oscillations related to working memory (Bartos et al., 2007; Sohal et al., 2009). Many PV neurons in the cortex are surrounded by extracellular matrix molecules called perineuronal nets (PNNs) (Berretta et al., 2015). PNNs mainly consist of chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid, and link proteins (Bandtlow and Zimmermann, 2000; Dityatev and Schachner, 2003). The details of the functions of PNNs are still unclear, but they are known to be involved in synaptic plasticity and neuronal protection (Wang and Fawcett, 2012). PNNs protect the PV neurons surrounded by them from oxidative stress (Cabungcal et al., 2013; Suttkus et al., 2014); hence, PV neurons with immature PNNs or those that lack PNNs are susceptible to oxidative stress PNNs protect the PV neurons that are surrounded by them from oxidative stress; hence, PV neurons that are surrounded by immature PNNs and those that lack PNNs are susceptible to oxidative stress. (Cabungcal et al., 2013; Suttkus et al., 2014; Do et al., 2015). In order to carry out their neuroprotective function, PNNs require aggrecan and link proteins as important component elements (Suttkus et al., 2014).

It

has

been

suggested

that

aggrecan-based

PNN-associated

interneurons are devoid of amyloid β-protein neurotoxicity (Morawski et al.,2004; 2010). In recent years, abnormalities of PV neurons have attracted attention as one of the potential causes underlying neuropsychiatric disorders (Marín, 2012). Studies on postmortem brains of patients with schizophrenia and autism have 6

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revealed that the number of PV neurons is lower than those in the frontal cortex, the entorhinal cortex, and the hippocampus of typically developing control individuals (Beasley and Reynolds, 1997; Lawrence et al., 2010; Lewis, 2014; Chung et al., 2016; Hashemi et al., 2016). Reductions in γ power (amplitude) associated with PV neurons are also found in patients with schizophrenia (Williams and Boksa, 2010; Uhlhaas and Singer, 2010). Interestingly, in neuropsychiatric disorders, PV neuronal abnormalities have been observed in selected brain regions and not in the somatosensory cortex and the visual cortex. This suggests that the vulnerability of PV neurons to neuropsychiatric conditions varies depending on the brain region involved. It is important to identify the factors that determine this region-specific vulnerability, in order to establish preventive and therapeutic methods for the management of neuropsychiatric disorders. In a previous study, we showed that PV neuron density, the proportion of PV neurons surrounded by PNNs, PNN structure, and PNN components are region-specific in the mouse brain (Ueno et al., 2017). Many PV neurons in the frontal cortex and the entorhinal cortex are not surrounded by PNNs, and the PV expression level in these areas is low, possibly causing structural vulnerability. However, it is unknown whether the PV neurons in these areas are in fact vulnerable. In this study, we investigated whether the vulnerability of PV neurons is higher in the frontal cortex, the entorhinal cortex, and the hippocampus than in other cortical regions of the mouse brain. Mice were raised in a socially isolated environment during adolescence, since many studies have shown that social 7

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isolation stress induces oxidative damage in the rodent brain (Huong et al., 2005; Schiavone et al., 2009; Cacioppo et al., 2011; Shao et al., 2015; Haj-Mirzaian et al., 2016). We found that PV neurons were affected by the oxidative stress caused by social isolation breeding. Social isolation is a type of chronic psychosocial stressor (Barratt et al., 2011; O’Keefe et al., 2014). The social isolation mouse model has been used in many studies as one of the neurodevelopmental animal models of schizophrenia (Fabricius et al., 2011; Kaalund et al., 2013; Shao et al., 2015). This model demonstrates abnormal neuropsychiatric behaviors (Cilia et al., 2001; Fabricius et al., 2011). The motor cortex on the same section as the frontal cortex, the visual cortex on the same section as the entorhinal cortex, and the somatosensory cortex on the same section as the dorsal hippocampus were examined simultaneously. The results of this study suggest that PV neurons possess brain region-specific vulnerability.

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EXPERIMENTAL PROCEDURES Animals Only male mice (C57BL/6N) were used for these experiments. The study was carried out in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications number 80-23, revised in 1996) and approved by the Committee for Animal Experiments at Okayama University Advanced Research Center. All efforts were made to minimize the number of animals used. The animals were purchased from Charles River Laboratories (Kanagawa, Japan) and housed in cages with food and water provided ad libitum, under 12 h/12 h light/dark conditions and at 23-26 °C.

Experimental Procedure The day of birth was considered postnatal day 0 (P0). Male P21 mice (10 males) were randomly divided into two groups, a socially isolated (one mouse per cage; five males) and a control (five mice per cage) group. The mice in the control group were group-housed in standard transparent plastic cages (22 × 34 × 15 cm) with wire tops, while the mice in the social isolation group were housed separately in opaque plastic cages (22 × 34 × 15 cm) with wire tops for 5 weeks, starting at P21.

Tissue Preparation On P56, the animals were deeply anesthetized with a lethal dose of sodium pentobarbital (120 mg/kg, intraperitoneally administered) and transcardially perfused with ice-cold phosphate-buffered saline (PBS) for 2 min and 4% 9

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paraformaldehyde in PBS (pH 7.4) for 10 min. The brains were dissected and post-fixed overnight in the same fixative at 4 °C. They were cryoprotected in 15% sucrose for 12 h followed by 30% sucrose for 20 h, at 4 °C and then frozen in OCT (optimum cutting temperature) compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan), using dry ice-cold normal hexane. Serial coronal sections with a thickness of 40 µm were obtained at -20 °C using a cryostat (CM3050S; Leica Wetzlar, Germany). The sections were collected in ice-cold PBS containing 0.05% sodium azide and stored at 4 °C.

Immunohistochemistry The cryostat sections were treated with 0.1% Triton X-100 in 0.1 M PBS at room temperature for 15 min. After three washes in 0.1 M PBS, the sections were incubated with 10% normal goat serum (ImmunoBioScience Co., WA, USA) in 0.1 M PBS at room temperature for 1 h. After three washes in 0.1 M PBS, the sections were incubated with biotinylated Wisteria floribunda agglutinin (WFA) (B-1355, Vector Laboratories, Funakoshi Co., Tokyo, Japan; 1:200) and primary antibody directed against PV (clone PARV-19, P3088, Sigma-Aldrich Japan, Tokyo, Japan; 1:1,000) in 0.1 M PBS overnight at 4 °C. After washing in PBS, the sections were incubated with streptavidin-conjugated TexasRed (SA-5006, Vector

Laboratories,

Funakoshi

Co.,

Tokyo,

Japan)

and

Alexa

Fluor488-conjugated goat anti-mouse IgG (ab150113, Abcam, Tokyo, Japan; 1:1,000) in 0.1 M PBS at room temperature for 2 h. Streptavidin is used to bind biotinylated WFA. Next, the sections were rinsed again with 0.1 M PBS and mounted onto glass slides with Vectashield mounting medium (H-1400, Vector 10

Hiroshi Ueno et al.

Laboratories, Funakoshi Co., Tokyo, Japan). The prepared slides were either imaged immediately or stored at 4 °C until imaging.

Microscopy Imaging To quantify the number and immunoreactive intensity of PV- and WFA-positive neurons, confocal laser scanning microscopy (LSM700; Carl Zeiss, Oberkochen, Germany) was used to obtain images of the stained sections. Images (1024 × 1024 pixels) were saved as TIFF files using the Zeiss ZEN software package (Carl Zeiss). Briefly, the analysis was performed using a 10× or 20× objective lens and a pinhole setting that corresponded to a focal plane thickness of less than 1 µm. Images from whole sections were obtained using a ×10 objective of the fluorescence microscope (BZ-X, KEYENCE, Tokyo, Japan) and merged using the KEYENCE BZ-X Analyzer software (KEYENCE).

Quantification of labeled neurons Quantification was performed as previously reported (Ueno et al., 2017). The number of neurons was quantified for at least three coronal sections per animal. All confocal images were converted to TIFF files and analyzed with the ImageJ software (NIH, Bethesda, MD; http://rsb.info.nih.gov/nih-image/). Neuronal density estimates (cells/mm2) were also calculated. Data are presented as mean ± standard error of the mean (SEM). For each mouse, three coronal sections were randomly selected at the level of the prefrontal cortex, the dorsal hippocampus, and the temporal cortex, and were processed for staining. Next, 8-bit grayscale images were captured using a 11

Hiroshi Ueno et al.

digital camera. The ellipse circumscribing the PV-positive soma and WFA-positive PNNs were traced manually, and the gray levels for PV and WFA labeling were measured using the ImageJ software. We avoided fluorescence saturation by adjusting the exposure time and gain. The same capture conditions were used for all sections. The background intensity was subtracted using unstained portions of each section. The slides were coded and quantified by a blinded independent observer.

Data Analysis Data are expressed as the mean ± SEM of five animals per group. Statistical analyses were carried out using SPSS Statistics (IBM Corp., Armonk, NY, USA). Statistical significance was determined by a two-way ANOVA followed by Bonferroni t-test. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.0001.

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RESULTS The primary somatosensory cortex on the same section as the dorsal hippocampus, the primary visual cortex on the same section as the temporal cortex, and the primary motor cortex on the same section as the prefrontal cortex were analyzed for comparison.

The Effect of Adolescent Social Isolation Stress on the Number of PV Neurons in Each Brain Area We first examined the effect of social isolation on the number of PV neurons in each brain region. PV neurons were observed in all the brain regions analyzed in this study (Fig. 1, 2). The number of PV neurons was separately counted in each layer of the somatosensory cortex, the primary visual cortex, the motor cortex, and the prefrontal cortex sub-regions (Fig. 3A). In the dentate gyrus (DG) of the hippocampus, the density of PV neurons was lower in socially isolated mice than in control mice. In other areas, there was no difference in the density of PV neurons between the social isolation and the control mice group.

The Effect of Adolescent Social Isolation Stress on the Number of WFA-positive PNNs in Each Brain Area To determine the effect of adolescent social isolation stress on WFA-positive PNN formation, we quantified the density of WFA-positive PNNs in each brain region (Fig. 3B). We found a difference between the control and the socially isolated mice group in the density of WFA-positive PNNs in L2/3 of the prelimbic sub-region (PL) of the frontal cortex. There was no significant difference in the 13

Hiroshi Ueno et al.

WFA-positive PNN density between the control and the socially isolated mice groups in the somatosensory cortex, the visual cortex, the motor cortex, the temporal cortex, and the hippocampus.

The Effect of Adolescent Social Isolation Stress on WFA-positive PNN Formation around PV Neurons in Each Brain Area We examined whether social isolation stress affects WFA-positive PNN formation around PV neurons in each brain region (Fig. 2, 4). The percentage of PV neurons enveloped by WFA-positive PNNs was significantly different in each brain region and each cortical layer (Fig. 4). There was no difference in the percentage of PV neurons enveloped by WFA-positive PNNs in the somatosensory cortex, the motor cortex, and the temporal cortex, between the control and the socially isolated mice groups. In L2/3 of the visual cortex and the dorsal anterior cingulate cortex (dAC), the percentage of PV neurons enveloped by WFA-positive PNNs was lower in socially isolated mice than in control mice. Moreover, in the CA1 and DG regions of the hippocampus, the percentage of PV neurons surrounded by WFA-positive PNNs was lower in socially isolated mice than in control mice.

Figure 5 shows an enlarged image of PV neurons and WFA-positive PNNs under the same conditions, revealing that PV fluorescence intensity and WFA fluorescence intensity were different in each brain region. In addition, PV fluorescence intensity and WFA fluorescence intensity differed between the control mice and socially isolated mice. In the hippocampus of socially isolated 14

Hiroshi Ueno et al.

mice, PV fluorescence intensity was especially low (Fig. 1).

The Effect of Adolescent Social Isolation Stress on PV Protein Expression in Each Brain Area Analysis of fluorescence intensity revealed that the PV fluorescence intensity was different in each brain region and each cortical layer (Fig. 6A). There were no differences in PV fluorescence intensity in the somatosensory cortex, the visual cortex, and the motor cortex between control mice and socially isolated mice. In the dAC sector of the frontal cortex, the PV fluorescence intensity was lower in socially isolated mice than in control mice. In all analyzed regions of the temporal cortex and the hippocampus, the PV fluorescence intensity was lower in socially isolated mice than in control mice.

The Effect of Adolescent Social Isolation Stress on WFA-positive PNN Formation in Each Brain Area Analysis of fluorescence intensity revealed that the WFA fluorescence intensity differed in each region and each cortical layer of the mouse brain (Fig. 6B). There was no difference in the WFA fluorescence intensity between control mice and socially isolated mice in the somatosensory cortex, the visual cortex, the motor cortex, and the hippocampus. In the PL part of the frontal cortex in socially isolated mice, the PV fluorescence intensity was lower than in the same region in control mice. In the perirhinal (PRh) and the dorsolateral entorhinal (DLEnt) parts of the temporal cortex, the PV fluorescence intensity was lower in socially isolated mice than in control mice. 15

Hiroshi Ueno et al.

The Effect of Adolescent Social Isolation Stress on the Soma of PV Neurons in Each Brain Area As the PV fluorescence intensity was lower in the PV neurons of control mice than in the PV neurons of socially isolated mice in many brain regions, we also analyzed the soma of PV neurons (Fig. 7). In the PL part of the frontal cortex and CA3 and DG regions of the hippocampus, the area of the soma of PV neurons was smaller in socially isolated mice than in control mice. No significant differences in soma size were found in the PV neurons of the somatosensory cortex, the visual cortex, the motor cortex, and the temporal cortex between the control and the socially isolated mice group.

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DISCUSSION In this study, we showed that social isolation stress during adolescence affects PV neurons in specific brain regions. Social isolation stress mainly affected the PV protein expression level, the soma area of PV neurons, and the degree of condensation of WFA-positive PNN components. To our knowledge, this study is the first to show the brain region-selective vulnerability of PV neurons to environmental factors. The regions in which this PV neuron abnormality was observed in this study coincide with brain regions in which abnormalities of PV neurons have been reported in patients with schizophrenia and autism. For social animals, adolescent social behavior is important for normal brain development (Casey et al., 2008; Eiland and Romeo, 2013). Many studies have shown that social isolation stress increases chronic sympathetic tonus, oxidative stress, hypothalamic pituitary-adrenal axis activation, and causes alterations of neurotransmitters, such as the brain-derived neurotrophic factor (BDNF) (Dronjak 2004; Huong et al., 2005; Sandstrom and Hart, 2005; Shao et al., 2015; Pisu et al., 2016; Ieraci et al., 2016). However, the detailed mechanisms underlying neuropsychiatric disorder-like behavior due to social isolation stress are unknown (Cacioppo et al., 2009; Yusufishaq and Rosenkranz, 2013; White et al., 2015). Furthermore, it is not clear whether social isolation stress affects all neurons in the brain or only subsets of neurons selectively. A number of recent studies have shown that the functional deterioration of PV neurons is involved in the onset of schizophrenia and autism spectrum disorders (Beasley and Reynolds, 1997; Lawrence et al., 2010; Lewis, 2014; Chung et al., 2016; 17

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Hashemi et al., 2016). In addition, it has been reported that social isolation stress decreases the number of PV neurons in the rodent hippocampus as well as the number of synaptic terminals of PV neurons in the prefrontal cortex (Harte et al., 2007; Bloomfield et al., 2008). PV neurons are fast spiking interneurons involved in the generation of cortical γ-oscillations that synchronize cortical activity during cognitive processing (Bartos et al., 2007; Sohal et al., 2009). In order to fire frequently, PV neurons require high levels of energy. Mice with impaired mitochondrial antioxidant function in PV neurons show autism-like or schizophrenia-like abnormal behavior, despite no changes in the number of PV neurons (Inan et al., 2016). PV neurons are structurally highly vulnerable to oxidative stress, and are more vulnerable to oxidative stress than other GABAergic interneurons (Sullivan and O'Donnell, 2012; Cabungcal et al., 2013). Many PV neurons in the cortex are surrounded by PNNs and it has been shown that PNNs function to protect PV neurons from oxidative stress (Morawski et al., 2004; Jiang et al., 2013; Do et al., 2015). The removal of PNNs by chondroitinase ABC increases their vulnerability to oxidative stress (Cabungcal et al., 2013). Furthermore, the activity of PV neurons is regulated by BDNF expression (Berghuis et al., 2006; Nieto-Gonzalez and Jensen, 2013). We did not observe a decrease in PV neuron density in many regions of the frontal cortex, the temporal cortex, and the hippocampus of socially isolated mice. On the other hand, the PV fluorescence intensity in these regions was significantly lower in socially isolated mice than in control mice. Previous studies have shown that the number of PV neurons in the hippocampus is lower in 18

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socially isolated rodents than in control animals (Czeh et al., 2005; Harte et al., 2007; Hu et al., 2010), but it is unclear whether the reduction in the number of PV neurons corresponds to a loss of neurons or a decrease in the expression level of PV protein in GABAergic interneurons. In addition, it is known that the total number of neurons remains unchanged in socially isolated mouse models (Kaalund et al., 2013). Postmortem studies of patients with schizophrenia have shown that the expression levels of PV protein and mRNA in the frontal cortex and the hippocampus, and not the number of PV neurons, are decreased (Hashimoto et al., 2003; Mellios et al., 2009; Fung et al., 2010; Volk et al., 2012). Reduced PV expression in the frontal cortex and the hippocampus of patients with autism and bipolar disorder has been reported (Zikopoulos and Barbas, 2013; Berridge et al., 2013; Sohalr et al., 2014;). Therefore, both the unaltered PV neuron density and the decreased PV protein expression in socially isolated mice shown in this study are consistent with the pathological findings in patients with neuropsychiatric disorders such as schizophrenia. The PV protein is a Ca2+-binding protein (CaBP). CaBPs have neuroprotective roles and are resistant to excitotoxicity, mechanical lesions, and neurodegenerative disorders (Sloviter, 1989; Leranth et al., 1991; German et al., 1992; Lukas and Jones, 1994). The main role of CaBP is that of a Ca2+ transporter and buffer (Heizmann, 1993). Therefore, a decrease in PV protein level leads to an impairment in the frequent firing of PV neurons and a loss of γ-oscillatory activity (Sohal et al., 2009; Volman et al., 2011). A decrease in PV protein levels observed in socially isolated mice thus suggests that the vulnerability of PV neurons is increased in these animals. The decrease in PV protein levels presumably leads to cognitive 19

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impairment, which is one of the core symptoms of schizophrenia (Lewis et al., 2012; Murray et al., 2015). It has indeed been reported that working memory is impaired in socially isolated mice (Kercmar et al., 2011). Mice with genetically deficient PV protein expression show autism-like abnormal behavior (Wöhr et al., 2015). However, further studies are necessary to clarify whether the decrease in PV protein expression in socially isolated mice is due to an increase in oxidative stress or a decrease in BDNF expression. In this study, the areas of the soma of PV neurons in the frontal cortex (PL sub-region) and in the hippocampal CA3 and DG sub-regions of socially isolated mice were smaller than those of the control mice. It has been reported that the PV neuronal soma area is smaller in mice with genetically encoded developmental abnormalities in PV neurons (Smith et al., 2014; Rietveld et al., 2015; Sampathkumar et al., 2016). In other words, the decreased PV neuronal soma area in socially isolated mice seems to cause functional deterioration. In the mouse sensory cortex, PV neurons form PNNs during the course of postnatal development, and neural plasticity decreases (Wang and Fawcett, 2012). PNN formation corresponds to the end of the critical period in the development of the sensory cortex. Treatment with chondroitinase ABC after mouse maturation restores neural plasticity (Pizzorusso et al., 2002). An increase in the number of PV neurons without WFA-positive PNNs observed in socially isolated mice suggests an increase in neuronal plasticity. In mice lacking matrix metalloproteinase-9, an extracellular matrix-degrading enzyme necessary for PNN formation, WFA fluorescence intensity decreases and neural plasticity increases (Kelly et al., 2015). In rats placed in an enriched environment, WFA 20

Hiroshi Ueno et al.

fluorescence intensity decreases and neural plasticity increases (Slaker et al., 2016). A decrease in the WFA fluorescence intensity seen in socially isolated mice reflects an impairment in PNN formation, suggesting an increase in neural plasticity. The neural circuit in the entorhinal cortex of socially isolated mice has been reported to change during adolescence (Liu et al., 2016). In addition, a number of studies have shown that social isolation alters neural plasticity (Djordjevic et al., 2009; Quan et al., 2010; Djordjevic et al., 2012; Ieraci et al., 2016). The results of this study are therefore consistent with those from previous studies. The influence of oxidative stress on PNNs has been extensively studied (Morawski et al., 2004; Cabungcal et al., 2013; Suttkus et al., 2014);however, the influence of social isolation stress on PNNs has not been investigated to date. The function of PV neurons surrounded by immature or no PNNs is impaired by oxidative stress (Do et al., 2015). In socially isolated mice, the WFA fluorescence intensity in the frontal cortex and the temporal cortex was lower, and the number of PV neurons without WFA-positive PNNs was higher than in control mice. These results suggest that the vulnerability of PV neurons is higher in the observed regions. Further studies are needed to clarify whether the vulnerability is further increased by social isolation stress. In this study, the percentage of PV neurons without PNNs in the visual cortex was higher in socially isolated mice than in control mice. This is an unexpected result. However, reduced GABA concentrations and reduced GAD67 mRNA levels in the visual cortex of patients with schizophrenia have been reported (Hashimoto et al., 2008; Yoon et al., 2010). This suggests that the 21

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decrease in neuroprotective PNN formation increases the vulnerability of PV neurons in the visual cortex of socially isolated mice, and the present results support previous reports. In the hippocampus, both PV neuron density and WFA-positive PNN density did not differ between socially isolated mice and control mice. However, the percentage of PV neurons without PNNs was higher in the hippocampus of socially isolated mice. These results indicate that WFA-positive PNNs cover the surface of other interneurons. Indeed, WFA-positive PNNs in the hippocampus surround inhibitory interneurons other than PV neurons (Yamada et al., 2014). The increased numbers of PV neurons without PNNs observed in the hippocampus of socially isolated mice suggest an increase in the plasticity and vulnerability of PV neurons. In a previous study, we systematically examined PV neurons and WFA-positive PNNs in the mouse cortex (Ueno et al., 2017), and speculated that PV neurons in the frontal cortex, the temporal cortex, and the secondary sensory cortex are morphologically more vulnerable to psychosocial stress than those in the primary sensory cortex and the motor cortex. In the former areas, many PV neurons were not surrounded by WFA-positive PNNs, the PV fluorescence intensity was low, and there was no aggrecan expression, which is essential for neuroprotection. The proportion of PV neurons surrounded by WFA-positive PNNs is higher in the motor cortex and the sensory cortex than in the frontal cortex and the entorhinal cortex. Other studies have shown that most of the PV neurons in the motor cortex, the somatosensory cortex, and the visual cortex are surrounded by WFA-positive PNNs (McRae et al., 2007; Nowicka et al., 2009; Ye and Miao, 2013). It has also been shown that PNNs in the sensory cortex 22

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contain aggrecan as a PNN component (McRae et al., 2007; Nowicka et al., 2009; Ye and Miao, 2013). The present study indicates that PV neurons in the frontal cortex and the entorhinal cortex, which are morphologically vulnerable, are affected in a region-selective manner. PV neurons in the frontal cortex, the entorhinal cortex, and the hippocampus could be highly vulnerable to social isolation stress, a type of psychosocial stress.

CONCLUSION These results indicate that social isolation stress affects PV neurons in specific brain regions where abnormalities have been reported in patients with psychiatric disorders. Importantly, these alterations are not observed in the sensory cortex and the motor cortex, where abnormalities are not frequently reported in psychiatric disorders, suggesting that social isolation stress further increases the vulnerability in these areas. Overall, our results suggest region-selective

vulnerability

in

the

mouse

brain.

Understanding

the

mechanisms by which highly vulnerable brain regions containing PV neurons and WFA-positive PNNs develop abnormalities, and the differences in the structure of these regions compared to other regions of the cortex as well as the components involved in the processes leading to these abnormalities, will help preserve and treat PV neuronal function in patients with neuropsychiatric disorders.

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AUTHOR CONTRIBUTIONS All authors had full access to all the data in the study and take full responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: H.U., M.O., T.I. Acquisition of data: H.U., S.S. Analysis and interpretation of data: H.U., S.S. Drafting of the manuscript: H.U., M.O. Critical revision of the manuscript for important intellectual content: S.M., N.K., K.W., Y.M., T.I. Statistical analysis: H.U., S.S. Study supervision: M.O., T.I.

CONFLICT OF INTEREST The authors declare they have no competing financial interests.

Acknowledgements: We thank S. Shimamura and M. Shimizu for technical assistance. We thank the Kawasaki Medical School Central Research Institute for providing the instruments that supported this work. The authors would like to thank Editage (www.editage.jp) for English language review.

Funding Sources This research did not receive any specific grant from funding agencies in the public commercial or not-for-profit sectors.

Author Information Hiroshi Ueno, E-mail: [email protected] Shunsuke Suemitsu, E-mail: [email protected] Shinji Murakami, E-mail: [email protected] 24

Hiroshi Ueno et al.

Naoya Kitamura, E-mail: [email protected] Kenta Wani, E-mail: [email protected] Motoi Okamoto, E-mail: [email protected] Yosuke Matsumoto, E-mail: [email protected] Takeshi Ishihara, E-mail: [email protected]

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FIGURE LEGENDS Figure 1. PV neurons and WFA-positive PNNs in control or socially isolated mice. Whole-brain sections double-stained with PV and WFA at the level of the prefrontal cortex (A), the dorsal hippocampus (B), and the temporal cortex (C) are shown. PV neurons are indicated by green fluorescence (Alexa 488) and WFA-positive PNNs are indicated by red fluorescence (TexasRed). There is no apparent difference in the density of PV neurons and WFA-positive PNNs in each brain region between control and socially isolated mice. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; S1, primary somatosensory cortex; M1, primary motor cortex; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; S1BF,

primary

somatosensory

cortex

–

barrel

field;

S1Tr,

primary

somatosensory cortex – trunk region; CA1, cornu ammonis area 1; CA3, cornu ammonis area 3; DG, dentate gyrus; Ect, ectorhinal cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; Scale bar = 1 mm in A, B, and C.

Figure 2. PV neurons and WFA-positive PNNs in each brain region of control or socially isolated mice. Representative

double

immunofluorescence

images

of

the

primary

somatosensory cortex - barrel field (A), the primary visual cortex (B), the primary motor cortex (C), the prefrontal cortex - prelimbic section (D), the temporal cortex - dorsintermed entorhinal section (E), and the hippocampus - CA1 (F) are 35

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shown. PV neurons are indicated by green fluorescence (Alexa 488) and WFA-positive PNNs are indicated by red fluorescence (TexasRed). The same capture conditions were used for all images. Scale bar = 100 µm in E (applies to A-E), and 50 µm in F. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net.

Figure 3. Density of PV neurons and WFA-positive PNNs in each brain region of control or socially isolated mice. Region-specific pattern of PV neuron density (A) and WFA-positive PNN density (B) in the primary somatosensory cortex, the primary visual cortex, the primary motor cortex, the prefrontal cortex (dAC, PL, and IL), the temporal cortex (PRh, DLEnt, and DIEnt), and the hippocampus (CA1, CA3, and DG) of control or socially isolated mice. Data are expressed as the mean ± SEM (n = 5 mice per group). Statistical significance using a two-way ANOVA followed by Bonferroni t-test. (A) somatosensory cortex; group: F(1,60) = 0.4, layer: F(2,60) = 47.1, group × layer: F(2,60) = 0.2. visual cortex; group: F(1,60) = 1.2, layer: F(2,60) = 45.5, group × layer: F(2,60) = 0.9. motor cortex; group: F(1,40) = 3.6, layer: F(1,40) = 1.0, group × layer: F(1,40) = 1.0. prefrontal cortex (dAC); group: F(1,84) = 0.8, layer: F(1,84) = 25.3, group × layer: F(1,84) = 0.1. prefrontal cortex (PL); group: F(1,84) = 0.1, layer: F(1,84) = 120.5, group × layer: F(1,84) = 0.1. prefrontal cortex (IL); group: F(1,84) = 0.1, layer: F(1,84) = 225.3, group × layer: F(1,84) = 0.3. temporal cortex; group: F(1,51) = 0.1, region: F(2,51) = 54.5, group × region: F(2,51) = 0.8. hippocampus; group: F(1,123) = 5.6, region: F(1,123) = 16.1, group × region: F(2,123) = 1.6. (B) 36

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somatosensory cortex; group: F(1,60) = 3.1, layer: F(2,60) = 135.8, group × layer: F(2,60) = 0.8. visual cortex; group: F(1,60) = 0.2, layer: F(2,60) = 41.5, group × layer: F(2,60) = 0.2. motor cortex; group: F(1,40) = 16.9, layer: F(1,40) = 0.1, group × layer: F(1,40) = 0.1. prefrontal cortex (dAC); group: F(1,84) = 6.3, layer: F(1,84) = 2.3, group × layer: F(1,84) = 0.1. prefrontal cortex (PL); group: F(1,84) = 4.6, layer: F(1,84) = 0.8, group × layer: F(1,84) = 0.6. prefrontal cortex (IL); group: F(1,84) = 4.3, layer: F(1,84) = 1.8, group × layer: F(1,84) = 0.1. temporal cortex; group: F(1,51) = 4.3, region: F(2,51) = 46.3, group × region: F(2,51) = 1.5. hippocampus; group: F(1,123) = 3.5, region: F(2,123) = 49.5, group × region: F(2,123) = 0.1. Statistical significance is represented by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.0001. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; DG, dentate gyrus; SEM, standard error of the mean.

Figure 4. The percentage of PV neurons surrounded by WFA-positive PNNs in each brain region of control or socially isolated mice. Region-specific pattern of the percentage of PV neurons surrounded by WFA-positive PNNs in the primary somatosensory cortex, the primary visual cortex, the primary motor cortex, the prefrontal cortex (dAC, PL, and IL), the temporal cortex (PRh, DLEnt, and DIEnt), and the hippocampus (CA1, CA3, and DG) of control or socially isolated mice. Data are expressed as the mean ± SEM (n = 5 mice per group). Statistical significance using a two-way ANOVA followed 37

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by Bonferroni t-test. somatosensory cortex; group: F(1,60) = 6.4, layer: F(2,60) = 30.8, group × layer: F(2,60) = 1.2. visual cortex; group: F(1,60) = 8.7, layer: F(2,60) = 14.8, group × layer: F(2,60) = 4.0. motor cortex; group: F(1,40) = 6.1, layer: F(1,40) = 5.3, group × layer: F(1,40) = 0.3. prefrontal cortex (dAC); group: F(1,83) = 14.0, layer: F(1,83) = 0.1, group × layer: F(1,83) = 3.9. prefrontal cortex (PL); group: F(1,82) = 1.1, layer: F(1,82) = 0.6, group × layer: F(1,82) = 2.3. prefrontal cortex (IL); group: F(1,66) = 0.1, layer: F(1,66) = 0.9, group × layer: F(1,66) = 0.1. temporal cortex; group: F(1,48) = 0.4, region: F(2,48) = 8.3, group × region: F(2,48) = 0.2. hippocampus; group: F(1,123) = 9.8, region: F(2,123) = 14.6, group × region: F(2,123) = 0.9. Statistical significance is represented by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.0001. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; DG, dentate gyrus; SEM, standard error of the mean.

Figure 5. PV protein expression and WFA-positive PNN formation in each brain region of control or socially isolated mice. Representative

double

immunofluorescence

images

of

the

primary

somatosensory cortex - barrel field (A, A’, B, B’), the prefrontal cortex - dAC (C, C’, D, D’), the temporal cortex - PRh (E, E’, F, F’), and the hippocampus - CA1 (G, G’, H, H’) are shown at high magnification. Double confocal images of PV and WFA reactivity in control (A, A’, C, C’, E, E’, G, G’) and socially isolated mice (B, B’, D, D’, F, F’, H, H’) are shown. PV neurons are indicated by green 38

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fluorescence (Alexa 488) and WFA-positive PNNs are indicated by red fluorescence (TexasRed). The same capture conditions were used for all images. Scale bar = 20 µm in H’ (applies to A-H’). Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; dAC, dorsal anterior cingulate cortex; PRh, perirhinal cortex.

Figure 6. Changes in the fluorescence intensity of PV neurons and WFA-positive PNNs in each brain region of control or socially isolated mice. Region-specific pattern of the mean fluorescence intensity of PV neurons (A) and WFA-positive PNNs (B) in the primary somatosensory cortex, the primary visual cortex, the primary motor cortex, the prefrontal cortex (dAC, PL, and IL), the temporal cortex (PRh, DLEnt, and DIEnt), and the hippocampus (CA1, CA3, and DG) of control or socially isolated mice. Data are expressed as the mean ± SEM (n = 5 mice per group). Statistical significance using a two-way ANOVA followed by Bonferroni t-test. (A) somatosensory cortex; group: F(1,590) = 22.4, layer: F(2,590) = 7.0, group × layer: F(2,590) = 0.5. visual cortex; group: F(1,453) = 0.5, layer: F(2,453) = 3.4, group × layer: F(2,453) = 5.9. motor cortex; group: F(1,369) = 34.7, layer: F(1,369) = 16.7, group × layer: F(1,369) = 0.2. prefrontal cortex; group: F(1,461) = 27.7, region: F(2,461) = 72.0, group × region: F(2,461) = 10.0. temporal cortex; group: F(1,335) = 36.0, region: F(2,335) = 7.7, group × region: F(2,335) = 3.8. hippocampus; group: F(1,734) = 32.8, region: F(2,734) = 5.8, group × region: F(2,734) = 0.5. (B) somatosensory cortex; group: F(1,513) = 4.9, layer: F(2,513) = 180.5, group × layer: F(2,513) = 0.8. visual cortex; group: F(1,396) = 0.1, layer: F(2,396) = 86.7, group × layer: F(2,396) = 0.3. motor cortex; group: F(1,333) = 3.2, layer: F(1,333) = 22.1, 39

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group × layer: F(1,333) = 0.6. prefrontal cortex; group: F(1,231) = 4.3, region: F(2,231) = 6.7, group × region: F(2,231) = 0.9. temporal cortex; group: F(1,199) = 18.2, region: F(2,199) = 9.1, group × region: F(2,199) = 1.6. hippocampus; group: F(1,739) = 1.0, region: F(2,739) = 24.1, group × region: F(2,739) = 0.2. Statistical significance is represented by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.0001. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; DG, dentate gyrus; SEM, standard error of the mean.

Figure 7. Changes in the soma area of PV neurons in each brain region of control or socially isolated mice. Region-specific pattern of the mean soma area of PV neurons in the primary somatosensory cortex, the primary visual cortex, the primary motor cortex, the prefrontal cortex (dAC, PL, and IL), the temporal cortex (PRh, DLEnt, and DIEnt), and the hippocampus (CA1, CA3, and DG) of control or socially isolated mice. Data are expressed as the mean ± SEM (n = 5 mice per group). Statistical significance

using

a

two-way

ANOVA

followed

by

Bonferroni

t-test.

somatosensory cortex; group: F(1,590) = 5.6, layer: F(2,590) = 0.3, group × layer: F(2,590) = 0.3. visual cortex; group: F(1,453) = 1.3, layer: F(2,453) = 5.6, group × layer: F(2,453) = 5.1. motor cortex; group: F(1,369) = 9.5, layer: F(1,369) = 34.6, group × layer: F(1,369) = 0.1. prefrontal cortex; group: F(1,461) = 15.9, region: F(2,461) = 3.7, group ×region: F(2,461) = 0.9. temporal cortex; group: F(1,335) = 0.6, region: F(2,335) 40

Hiroshi Ueno et al.

= 14.2, group × region: F(2,335) = 1.8. hippocampus; group: F(1,734) = 28.4, region: F(2,734) = 20.1, group × region: F(2,734) = 1.4. Statistical significance is represented by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.0001. Abbreviations: PV, parvalbumin; WFA, Wisteria floribunda agglutinin; PNN, perineuronal net; dAC, dorsal anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; PRh, perirhinal cortex; DIEnt, dorsintermed entorhinal cortex; DLEnt, dorsolateral entorhinal cortex; DG, dentate gyrus; SEM, standard error of the mean.

41

A M2 M1

dAC PL

S1

IL

WFA

PV

PV

WFA

control

isolation

1mm

B S1Tr

control

S1BF

isolation CA1

PV

DG

CA3

PV

WFA

WFA

1mm

C Ect PRh DLEnt DIEnt

PV

control

WFA

PV

WFA

isolation

1mm

Figure. 1

A

somatosensory cortex control

PV

WFA

B

control

isolation PV

PV

WFA

C

visual cortex WFA

motor cortex

isolation PV

control

WFA

PV

WFA

isolation PV

WFA

2/3

2/3 2/3 4

4

5/6

5/6

5/6

E

prefrontal cortex control PV

WFA

control

isolation PV

PV

WFA

F

temporal cortex WFA

hippocampus

isolation PV

WFA

PV

control

D

2/3 2/3

WFA

5/6

isolation

PV 5/6

100mm

WFA 50mm

Figure. 2

A

B PV neurons / mm2

somatosensory cortex

0

L 2/3

L4

L4

L 5/6

L 5/6 100

L 2/3

L4

L4

L 5/6

L 5/6 60

motor cortex dAC

80

160

0

60

120

0

60

120

0

40

80

L 2/3

L 5/6

L 5/6

PL

0

40

80

L 2/3

L 2/3

L 5/6

L 5/6

L 2/3

L 2/3

L 5/6

L 5/6

L 2/3

L 2/3

L 5/6

L 5/6

*

IL

prefrontal cortex

120

L 2/3

0

0

temporal cortex

200

L 2/3

0

40

80

0

PRh

PRh

DLEnt

DLEnt

DIEnt

DIEnt 0

hippocampus

120

L 2/3

0

visual cortex

60

WFA (+) PNNs / mm2

40

0

80

CA1

CA1

CA3

CA3

DG

DG

**

40

40

80

80

control isolation

Figure. 3

WFA (+) PNNs / PV neurons (%) somatosensory cortex

0

50

100

L 2/3

control isolation

L4 L 5/6 0

50

100

visual cortex

L 2/3

**

L4

L 5/6 50

100

0

40

80

L 2/3

motor cortex

PL

dAC

L 5/6

L 2/3

**

L 5/6 L 2/3 L 5/6 L 2/3

IL

prefrontal cortex

0

L 5/6

temporal cortex

0

80

PRh DLEnt DIEnt 0

hippocampus

40

CA1

40

80

*

CA3 DG

*

Figure. 4

hippocampus (CA1)

temporal cortex (PRh)

prefrontal cortex (dAC L5/6)

somatosensory cortex (L 4)

control

A PV

A’

isolation

WFA

B PV

B’

C C’ D D’

E E’ F F’

G G’ H H’

WFA

20mm

Figure. 5

A

B

visual cortex

somatosensory cortex

PV fluorescence intensity (a.u.) 0

L 2/3

L4

L4

L 5/6

L 5/6 0

motor cortex prefrontal cortex

30

60

L 2/3

L 2/3

L4

L4

L 5/6

L 5/6 30

60

L 2/3

L 2/3

L 5/6

L 5/6 0

15

30

dAC

***

PL

IL

IL 10

20

PRh

** ***

DLEnt DIEnt

20

40

0

20

40

control isolation

0

20

40

0

10

20

* 0

10

PRh

20

* **

DLEnt DIEnt

0

10

0

20

CA1

** ***

CA3 DG

0

dAC

PL

0

temporal cortex

60

L 2/3

0

hippocampus

30

WFA fluorescence intensity (a.u.)

**

10

20

CA1 CA3 DG

Figure. 6

somatosensory cortex

PV soma area (mm2) 0

120

240

L 2/3

control isolation

L4 L 5/6 0

120

240

0

120

240

0

120

240

visual cortex

L 2/3 L4

prefrontal cortex

motor cortex

L 5/6

L 2/3 L 5/6

dAC PL

**

IL

hippocampus

temporal cortex

0

120

240

PRh DLEnt DIEnt 0

120

240

CA1 CA3 DG

** ***

Figure. 7

Hiroshi Ueno et al.

Highlights

・Social isolation stress affects PV neurons in specific brain regions. ・PV intensity in specific brain regions was lower in socially isolated mice. ・The soma area of PV neurons in specific brain regions was smaller in socially isolated mice. ・Social isolation stress did not affect the density of PV neurons. ・The mouse brain exhibits region-selective vulnerability of PV neurons to environmental factors.

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