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Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain
Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain Anita Kumari, Padmanabh Singh, Meghraj Singh Baghel, M.K. Thakur PII: DOI: Reference:
S0031-9384(16)30076-2 doi: 10.1016/j.physbeh.2016.02.032 PHB 11227
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
25 December 2015 21 February 2016 22 February 2016
Please cite this article as: Kumari Anita, Singh Padmanabh, Baghel Meghraj Singh, Thakur MK, Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.02.032
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ACCEPTED MANUSCRIPT Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in female mouse brain #
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Anita Kumari, Padmanabh Singh , Meghraj Singh Baghel , M. K. Thakur*
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Biochemistry and Molecular Biology, Laboratory, Department of Zoology, Banaras Hindu University, Varanasi
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221005, India
* Corresponding author
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E-mail address, telephone and fax numbers of the corresponding author: Department of Zoology, Biochemistry and Molecular Biology, Laboratory, Banaras Hindu University, Varanasi 221005, India. Tel.: +91 542 6702521;
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Contributed equally
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fax: +91 542 2368174; e-mail: [email protected]
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ACCEPTED MANUSCRIPT
Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in female mouse brain
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Abstract
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Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems.
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Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is
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more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8 weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well known
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molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were upregulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF
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expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore,
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BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by
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social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex.
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Keywords Social isolation; Anxiety; Cerebral cortex; BDNF; HDAC-2; MiRNA-132
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Introduction Early life adverse experiences (social isolation or maternal separation, childhood abuse and neglect, extreme
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poverty) occur worldwide and cannot be eliminated [1]. Exposure to adverse experiences in early life is implicated
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in the later vulnerability to development of psychiatric disorders, including anxiety and affective disorders in humans [2]. More than 30% of mental disorders are directly related to early life stress [3, 4]. Psychological
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problems due to adverse early life experiences cause socio-economic burden as it impairs the normal daily activities of the patients as well as extremely affects their families and caregivers. Therefore, it is utmost and urgent necessity to comprehend the molecular mechanisms underlying the process of psychological problems resulting due to adverse early life experiences. On the other hand, a large body of literature suggests that post-
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weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans [5, 6]. The majority of findings suggest that post-weaning social isolation that encompasses preadolescence produces long-lasting alterations in brain leading to anxiety like behavior [7, 8, 9, 10, 11]. Social
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isolation is a stressor that has been used during early development to examine potential long-lasting effects on behavioral and biological responses to stress in rodents [12, 13]. This chronic stress situation has indeed been shown to have long-lasting effects on various behavioral and neural functions [14]. Social isolation precipitates or
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exacerbates the feelings and symptoms of anxiety. One of the consequences of stress exposure in rodents is
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heightened anxiety [15].
Anxiety is an evolutionary trait that provides a coping mechanism in dangerous environmental situations and is associated with emotional processes. Several studies have revealed that brain regions involved in anxiety are
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parahippocampal gyrus, cingulate cortex, amygdala and frontal cortex [16, 17]. The present study was focused on the cerebral cortex. Furthermore, accumulating evidences suggest that the adverse effect of social isolation on brain is mediated by alteration in gene expression which in turn is regulated by epigenetic mechanisms. Thus, epigenetic mechanisms mediate the link between social isolation, gene expression, neurobiological changes, and
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behavioral variation [18].
Interestingly, neurotrophic factors such as nerve growth factor and brain-derived neurotrophic factor (BDNF) have been implicated in the pathophysiology of anxiety disorders [19, 20, 21]. Several studies have demonstrated that BDNF regulates structural, synaptic, and morphological plasticity to modulate the strength or number of synaptic connections and neurotransmission [22, 23]. Also, BDNF participates in cellular proliferation, migration, phenotypic differentiation and maintenance in the developing central nervous system (CNS) [24]. In addition, its presence is required in the adult CNS for maintenance of neuronal functions and neurogenesis [25, 26]. Besides, there are studies which suggest that BDNF transcription is controlled by Ca2+ regulated transcription factor cAMP response element binding protein (CREB-1) [27]. The CREB-1 binding protein (CBP) is a transcriptional coactivator whose function is critical for CREB-1 activity [28]. Structurally, CBP has several protein-binding regions and a histone acetyltransferase (HAT) domain; functionally, CBP acts as a transcriptional coactivator by facilitating the recruitment of required components of the transcriptional machinery and as a HAT by altering the chromatin structure [29]. Like other synaptic plasticity molecules, BDNF expression is regulated by histone deacetylase-2 (HDAC-2) [30].
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ACCEPTED MANUSCRIPT Furthermore, miRNAs are of particular importance in anxiety disorders due to a number of reasons. MiRNAs and their mRNA targets are highly abundant in the CNS, and there is mounting evidence for the role of miRNAs in numerous CNS functions. Besides, evidences from various sources like animal and human studies indicate towards the involvement of miRNAs in the aetiology of anxiety disorders. In addition, there is preliminary
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evidence for the role of miRNAs as therapeutic targets in anxiety and mood disorders. Also, preclinical models
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illustrate that, by changing the levels of miRNAs, a therapeutically desirable change in anxiety-like behavior can be achieved [31]. Amongst miRNAs, miRNA-132 and miRNA-134 are well studied in case of stress condition
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[32, 33]. Remarkably, BDNF acts in concert with Lim domain kinase-1 (Limk-1), miRNA-132 and miRNA-134 in the regulation of structural and morphological plasticity [34, 35]. Furthermore, one of the most widely documented findings in psychiatric epidemiology is that women are significantly more likely than men to develop an anxiety disorder throughout the life span [36].
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On the basis of the above mentioned evidences, the present study was designed to investigate in female mice the effects of post-weaning social isolation on anxiety like behavior and expression of BDNF and molecules associated with BDNF expression and function including CBP, CREB-1, HDAC-2, Limk-1, miRNA-132, and
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miRNA-134. Elevated plus maze and open field test were performed first to assess the anxiety like behavior of female mice followed by molecular studies. We report that social isolation resulted in anxiety like behavior accompanied by enhancement in the expression of BDNF. This was followed by increased expression of CREB-1,
Material and methods
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CBP, Limk-1 and miRNA-132 with concomitant decreased expression of HDAC-2 and miRNA-134.
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Random hexanucleotides, dNTPs, Taq polymerase, RNase inhibitor and reverse transcriptase enzymes were purchased from New England Biolabs (USA), monoclonal anti-β-actin-peroxidase (A3854), TRI reagent from Sigma–Aldrich (USA), BDNF (SC-20981; Santa Cruz Biotechnology, Santa Cruz, CA, USA); HDAC2 (SAB4300412; Sigma-Aldrich), peroxidase conjugated secondary antibodies from Bangalore Genei (India), and
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polyvinyl difluoride (PVDF) membrane from Millipore (Germany). All other chemical reagents were purchased from Merck (Germany). Animals
Swiss albino strain female mice were maintained at 12 h light and dark schedule with ad libitum standard mice feed and drinking water in the animal house of Department of Zoology, Banaras Hindu University, Varanasi at ambient temperature. At the age of 2 months, the mice were either left in group (as control) or assigned to social isolation by individually housing for another 2 months. The procedure for use and handling of animals has been approved by the Institutional Animal Ethical Committee. Experiments were conducted between 9am-6pm in semisound proof condition. Estrous cycle of female mouse was checked according to Korol et al. [ 37]. and mice at same stage, met-estrous were selected for behavioral and molecular studies. After behavioral analysis, the entire cerebral cortex was isolated. One half of the cerebral cortex dissected from individual mouse was pooled together and used for RNA isolation and other pooled half was used for protein isolation [38].
Elevated plus maze test
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ACCEPTED MANUSCRIPT Elevated plus maze test was conducted according to the protocol followed by Mikaelsson et al. [39]. Young female mice after 8 weeks of social isolation were subjected to elevated plus maze test (Fig. 1A, 1B, 1C, 1D, 1E, 1F). The maze, positioned 300 mm above the floor and illuminated evenly at 15 lux, was constructed of white Perspex and consisted of two exposed open arms (175mm x 78mm, length x width) and two equally sized
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enclosed arms, which had 150-mm high walls. Equivalent arms were arranged opposite to one another. Mice were
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placed at the centre of the maze facing one of the open arms and allowed to freely explore for 5min. Elevated plus maze was wiped with 70 % ethanol after each trial to remove odour of previous mice. Data from each pair of arms
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were combined to generate single open- and closed-arm values (time spent in closed arm, time spent in open arm, distance travelled in open arm, distance travelled in closed arm, number of entries into open arm). The data were analysed by ANY-maze software (version 4.95, Stoelting Co., USA).
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The open-field test
The open-field test was conducted according to the protocol followed by Mikaelsson et al. [39]. The apparatus consisted of a square-shaped arena (750x750mm2, length x width), constructed of white plastic and illuminated
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evenly at 15 lux. Subjects were placed facing the centre of one of the walls and allowed to explore the apparatus for 10 min. The open field was subdivided into two virtual concentric squares. The following parameters (Fig. 2A, 2B, 2C, 2D, 2E) were recorded- time spent in central zone, distance travelled in central zone, number of line
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crossings, number of entries into central zone. Open field apparatus was wiped with 70 % ethanol after each trial
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to remove odour of previous mice. The data were analysed by ANY-maze software (version 4.95, Stoelting Co., USA).
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Semi Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) RT-PCR was performed as mentioned by Kumari and Thakur [40]. Briefly, total RNA was isolated from the cerebral cortex of both the groups using TRI reagent kit. RNA was estimated by taking absorbance at 260 nm and purity was checked by A260/A280 ratio. Total RNA from different groups was resolved on 1% agarose
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containing ethidium bromide and the integrity of RNA was checked by staining of 18S and 28S rRNA. cDNA was synthesized from the total RNA extracted from the cerebral cortex of two mice groups. For BDNF, CBP, CREB1, Limk-1, HDAC-2, and β-actin the following primer pairs were used:
BDNF Forward primer 5’ TGCCAGAGCCCCAGGTGTGA3’ Reverse primer 5’ CTGCCCTGGGCCCATTCACG3’ CBP [41] Forward primer 5’TGGAGTGAACCCCCAGTTAG3’ Forward primer 5’TTGCTTGCTCTCGTCTCTGA3’ CREB-1 Forward primer 5'ACA GTG CCA ACC CCC ATT TA3' Reverse primer 5'GTA CCC CAT CCG TAC CAT TGT T3' HDAC-2 [42] Forward primer 5’GGTCGTAGGAATGTTGCTGAT3’ Reverse primer 5’AAGCCAATGTCCTCAAACAGG3’
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ACCEPTED MANUSCRIPT Limk-1 [43] Forward primer 5’ATGAGGTTGACGCTACTTTGTTG3’ Reverse primer 5’CTACACTCGCAGCACCTGAA3’ β-actin
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Forward primer 5’GTCTCCTGCGACT TCAGC3’,
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Reverse primer 5’TCATTGTCATACCAGGAAATGAGC3’
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PCR condition includes 5 min of initial denaturation at 94 °C, 32 cycles (94 °C for 60 s, 63 ºC for 30s, 72 ºC for 45 s), final extension at 72 ºC for 3 min for BDNF; 5 min of initial denaturation at 94 °C, 28 cycles (94 °C for 30 s, 53 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for CBP; 5 min of initial denaturation at 94 °C, 27 cycles (94 °C for 30 s, 60 ºC for 30 s, 72 °C for 30 s), final extension at 72 ºC for 3 min for CREB-1, 5 min of
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initial denaturation at 94 °C, 28 cycles (94 °C for 30 s, 61 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for HDAC-2; 5 min of initial denaturation at 94 °C, 29 cycles (94 °C for 30 s, 60 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for Limk-1; 94 °C, 32 cycles (94 °C for 30 s, 60 ºC for 30s, 72 ºC for 30 s), final
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extension at 72 ºC for 3 min for β-actin. The PCR products were resolved on 2% agarose gel. The signals were analysed by Alpha-EaseFC software (Alpha Innotech Corp, USA).
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Quantitative real-time PCR
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Quantitative real time PCR (qRT-PCR) was performed as mentioned by Singh et al. [44]. Briefly, total RNA was isolated from the cerebral cortex of mice using TRI reagent kit (Sigma Aldrich, USA) and 2 µg of RNA from each group was reverse transcribed for cDNA synthesis. The cDNA was used as a template for qRT-PCR amplification
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using gene specific primers for miRNA-132 and miRNA-134 [43]. The endogenous control β-actin was used to normalize quantification of the mRNA target.
MiRNA-132
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Forward 5’ACCGTGGCTTTCGATTGTTA3’ Reverse 5’-GGCGACCATGGCTGTAGACT3’ MiRNA-134
Forward 5’-GGGTGTGTGACTGGTTGACCA3’ Reverse 5’-GGGTTGGTGACTAGGTGGCC3’ Western Blotting Western blotting was performed as mentioned by Kumari and Thakur [40]. Briefly, total protein was isolated from the cerebral cortex of mice using lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF and 1 mM EDTA). Protein was estimated (Bradford 1976). For western blotting of HDAC-2 and BDNF, protein was denatured and resolved by 10% and 15% SDS-PAGE, respectively, and electroblotted onto PVDF membrane by semi-dry method. Then the blot was stained with ponceau-S for checking the transfer efficiency. For western blotting of HDAC-2, the blot was blocked with 5% skimmed milk in PBS for 2 hr. The membrane was probed with rabbit anti HDAC2 (1:15000) and secondary antibody goat anti rabbit (1:2000) diluted in blocking buffer. For western blotting of BDNF, the blot was blocked with 5% skimmed
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ACCEPTED MANUSCRIPT milk in PBS for 4 hr. The membrane was probed with rabbit anti BDNF (1:1000) and secondary antibody goat anti rabbit (1:2000) diluted in blocking buffer. The same blot was reprobed with β-actin (1:25,000 dilution) for loading control. The signals were scanned by AlphaImager system and analysed by Alpha-EaseFC software (Alpha Innotech Corp, USA).
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Statistical Analysis
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Each experiment was repeated three times (n = 15 mice/group). In order to analyze qRT-PCR data, the ΔΔCt value was used to calculate relative fold change in mRNA expression and plotted as histograms. The intensity of
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RT PCR and western blotting band was measured by spot densitometry tool of AlphaEaseFC software (Alpha Innotech Corp, San Jose, CA, USA), histogram was plotted as relative density value (Integrated Density Value (IDV) gene/IDV β-actin) and the data are presented as a histogram with the mean±SEM of three values calculated from three independent experiments. For RT-PCR, the signal intensity (IDV) of BDNF, CBP, CREB-1, HDAC-2,
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Limk-1 mRNA was normalized against the signal intensity (IDV) of internal control, β-actin. For western blotting analysis, the signal intensity (IDV) of HDAC2 and BDNF was normalized against signal intensity (IDV) of internal control, β-actin. Pearson’s r statistics method was used for correlation analysis. The data of all
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experiments were analyzed by independent t-test using SPSS for Windows (standard version 16.0).
Results
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1) Anxiety like behavior in female mice by social isolation as shown by elevated plus maze test and open field test
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Elevated plus maze analysis and open field test revealed that social isolation caused anxiety like behavior in female mice. In elevated plus maze paradigm, after social isolation mice spent significantly more time in closed arm and less time in open arm as compared to that of group housed mice (Fig. 1A, 1B). Moreover, distance
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travelled by mice in open arm decreased significantly (Fig. 1D), but distance travelled by mice in closed arm was not altered significantly by social isolation (Fig. 1C). In addition, number of entries into the open arm decreased significantly by social isolation in comparison to that of group housed mice (Fig. 1E). Representative track plot of mice in elevated plus maze is shown in Fig. 1F. In open field analysis, after social isolation, mice spent
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significantly less time in central zone as compared to that of group housed mice (Fig. 2A). Further, distance travelled by mice in central zone decreased significantly by social isolation (Fig. 2B). Moreover, number of line crossings decreased significantly by social isolation (Fig. 2C). In addition, number of entries into central zone decreased significantly by social isolation as compared to that of group housed mice (Fig. 2D). Representative track plot of mice in open field test is shown in Fig. 2E.
2) Expression of BDNF mRNA and protein, CBP, CREB-1and Limk-1 mRNA and miRNA-132 was up-regulated by social isolation RT-PCR analysis of BDNF showed that its expression increased significantly in the cerebral cortex by social isolation as compared to that of group housed mice (Fig. 3A). Western blot analysis also showed significant enhancement in the expression of BDNF in cerebral cortex by social isolation (Fig. 3B). Moreover, the pattern of expression of BDNF mRNA and protein was similar after social isolation; we checked the correlation between expression of BDNF mRNA and protein in the cerebral cortex. A positive correlation was noted between BDNF mRNA and protein expression (r = 0.943663, p< 0.05) (Fig. 3C). RT-PCR analysis revealed significant upregulation in the expression level of CBP, CREB-1 and Limk-1 in the cerebral cortex by social isolation in
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ACCEPTED MANUSCRIPT comparison to that of group housed mice (Figs. 4A, 4B, 5). Real time PCR analysis showed significant upregulation of miRNA-132 in the cerebral cortex by social isolation in comparison to that of group housed mice
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(Fig. 7B).
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3) Expression of HDAC-2 mRNA and protein, and miRNA-134 was down-regulated by social isolation RT-PCR analysis of HDAC-2 revealed that its expression was down-regulated significantly in the cerebral cortex
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by social isolation in comparison to that of group housed mice (Fig. 6A). Western blot analysis also showed significant down-regulation of HDAC-2 in cerebral cortex by social isolation (Fig. 6B). As the pattern of expression of HDAC-2 mRNA and protein was similar after social isolation, we checked the correlation between expression of HDAC-2 mRNA and protein in the cerebral cortex. A positive correlation was noted between
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HDAC-2 mRNA and protein expression (r = 0.98737, p< 0.05) (Fig. 6C). Real time PCR analysis of miRNA-134 revealed that its expression was down-regulated significantly in cerebral cortex by social isolation in comparison
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to that of group housed mice (Fig. 7A).
Discussion
The present study showed that social isolation of female mice results in the development of anxiety like behavior
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as was revealed by elevated plus maze analysis and open field test which are extensively used behavioral
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paradigms for measuring anxiety behavior in case of rodents [39, 45, 46]. Our finding is substantiated by the study of Kwak et al. [46] that social isolation selectively causes anxiety in mice. A great deal of evidences from the clinical literature suggest that the prevalence of anxiety disorders is about twice as high in women as compared to
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men [47, 48, 49, 50]. Several studies suggest that social isolation rearing of male rodents during a critical window (pre- to mid-adolescence) results in long-lasting increase in anxiety-like behavior that are not reversed by resocialization. In contrast, a critical period may not apply for female rodents. Instead, social isolation at any age may enhance anxiety states in female rodents [51]. Sex differences in anxiety like behavior originate due to the
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probable factors including sex-chromosomal gene expression [52], sexually dimorphic developmental differentiation of brain regions, stress response systems, female reproductive hormone fluctuations, protective or vulnerability-inducing effects of reproductive hormones in adulthood, or differences in stressor perception and emotional appraisal. [53]
Remarkably, we found enhancement in the expression of BDNF in cerebral cortex of mice after social isolation. Earlier reports have shown that BDNF Met mutation increases anxiety-like behavior in female rodents [54, 55]. Interestingly, genetic manipulations that increase BDNF expression in mice similarly increased anxiety-like behaviors, but decreased immobility in the forced swim test [56]. Similarly, in humans, genetic variations that result in higher BDNF activity dependent release were associated with higher anxiety traits [57], while a genetic polymorphism reducing BDNF activity was protective against anxiety [58]. Altogether, these studies indicate that genetic variations that increased BDNF levels result in increased anxiety-related phenotypes, while those decreasing BDNF activity decreased anxiety-related phenotypes. Moreover, RT-PCR analysis of CREB-1 mRNA showed that its expression increased in cerebral cortex by social isolation. Similar to CREB-1, CBP mRNA showed up-regulation in cerebral cortex by social isolation. Moreover, HDAC-2 negatively regulates the
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ACCEPTED MANUSCRIPT expression of synaptic plasticity molecules including BDNF [30]. Thus, we found that the expression of HDAC-2 mRNA and protein was down-regulated in cerebral cortex in social isolation condition. Such down-regulation of HDAC-2 might mediate enhancement in the expression of BDNF. Furthermore, alteration in the expression of BDNF was accompanied by the up-regulation of Limk-1 which is the
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brain specific member of Lim kinase family of serine/threonine kinases [59]. A number of studies showed that
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BDNF/TrkB and Limk-1/cofilin-regulated actin dynamics are involved in growth cone motility, neurite extension, synaptogenesis, and long term potentiation in neurons [60, 61, 62, 63]. Moreover, studies revealed that BDNF
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increases the expression of Limk-1 by relieving the miRNA-134 inhibition of Limk-1 translation [64]. Interestingly, we found that Limk-1 mRNA was up-regulated by social isolation in cerebral cortex. Also, our data revealed down-regulation of miRNA-134 in the cerebral cortex which in turn might mediate enhancement in the expression of Limk-1 of mice after social isolation. It is further substantiated by the finding that chronic stress
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decreases the level of miRNA-134 [32].
In addition, miRNA-132 is one of the most highly inducible CREB-1 targets, and plays a role in neurite outgrowth and neuronal plasticity. It contributes to the action of BDNF. In fact, the over expression of miRNA-132 causes
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enhanced neurite outgrowth and dendritic morphogenesis [65, 66]. Therefore, we studied the expression level of miRNA-132 and found that it increased in cerebral cortex which might be mediated by BDNF. This was also shown by [67], who reported that BDNF up-regulated the expression of miRNA-132 in cultured neurons.
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Moreover, miRNA-132 expression increases in chronic stress condition [32] as well as after maternal separation
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[68].
The alteration in the expression of BDNF and molecules associated with its expression and function might have important role after social isolation. These molecules are primarily associated with synaptic transmission. Also,
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the findings from different laboratories have shown that excitatory neurotransmission is increased in the brain regions linked with anxiety [16, 17]. Therefore, enhancement in the expression of BDNF, CREB-1, CBP, Limk-1, miRNA-132 and decline in the expression of miRNA-134, HDAC-2 in cerebral cortex after social isolation in
Conclusion
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female mice might lead to increased excitatory neurotransmission which is reported in case of anxiety.
Altogether it can be concluded that anxiety like behavior caused by social isolation might be mediated by upregulation in the expression of BDNF with concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex. Acknowledgments Anita Kumari and Padmanabh Singh are recipients of senior research fellowship from the Council of Scientific and Industrial Research, India. Meghraj Singh Baghel is a recipient of junior research fellowship from the Department of Biotechnology, India. This work was supported by Grants from Department of Science & Technology, India and Council of Scientific & Industrial Research, India. We acknowledge DBT-BHU Interdisciplinary School of Life Sciences, Banaras Hindu University for real-time PCR facility. The funding sources had no involvement in study design; collection, analysis and interpretation of data; writing of the report; and in the decision to submit the article for publication.
Conflict of interest:
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Legends:
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Fig. 1 Elevated plus maze analysis showing A time spent by two groups of mice in closed arm B time spent in open arm C distance travelled in closed arm D distance travelled in open arm E number of entries into open arm F Representative track plot: (a) G (b) I. G- Group, I- Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test), (n=15 mice/group).
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Fig. 2 Open field test showing A time spent by two groups of mice in central zone B distance travelled in central
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zone C number of line crossings D number of entries into open arm E Representative track plot: (a) G (b) I. GGroup, I- Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test),
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(n=15 mice/group).
Fig. 3 A RT-PCR analysis of BDNF mRNA in cerebral cortex of two groups of mice. Signal intensity for BDNF was normalized against signal intensity for β-actin. B Western blot analysis of BDNF protein in cerebral cortex of two groups of mice. Signal intensity for BDNF was normalized against signal intensity for β-actin. The data
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are presented as a histogram with the mean (±SEM) of three values calculated as RDV of BDNF/β-actin. C Correlation analysis between BDNF mRNA and protein expression in two groups of mice.
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Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test).
Fig. 4 RT-PCR analysis of A CBP mRNA and B CREB-1 mRNA in cerebral cortex of two groups of mice. Signal intensity for CBP and CREB-1 was normalized against signal intensity for β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of CBP/β-actin and CREB-1/ β-actin. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, IIsolation.
Fig. 5 RT-PCR analysis of Limk-1 mRNA in cerebral cortex of two groups of mice. Signal intensity for Limk-1 was normalized against signal intensity for β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of Limk-1/β-actin. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, I- Isolation.
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Fig. 6 A RT-PCR analysis of HDAC-2 mRNA in cerebral cortex of two groups of mice. Signal intensity for HDAC-2 was normalized against signal intensity for β-actin. B Western blot analysis of HDAC-2 protein in
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cerebral cortex of two groups of mice. Signal intensity for HDAC-2 was normalized against signal intensity for
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β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of HDAC-2 /β-actin. C Correlation analysis between HDAC-2 mRNA and protein expression in two groups of
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mice. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, IIsolation.
Fig. 7 Quantitative real-time PCR analysis representing fold change in A miRNA-134 expression B miRNA-132
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expression in cerebral cortex in two groups of mice. G- Group, I- Isolation. Data are represented the mean
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Highlights Elevated plus maze and open field test analysis revealed that post-weaning social isolation of
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young female mice caused anxiety like behaviour.
BDNF, CREB-1, Limk-1 and mir-132 mRNA were up-regulated in the cerebral cortex by
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HDAC-2 and Mir-134 were down-regulated in the cerebral cortex by social isolation.
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social isolation.
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S0031-9384(16)30076-2 doi: 10.1016/j.physbeh.2016.02.032 PHB 11227
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
25 December 2015 21 February 2016 22 February 2016
Please cite this article as: Kumari Anita, Singh Padmanabh, Baghel Meghraj Singh, Thakur MK, Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in the female mouse brain, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.02.032
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ACCEPTED MANUSCRIPT Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in female mouse brain #
#
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Anita Kumari, Padmanabh Singh , Meghraj Singh Baghel , M. K. Thakur*
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Biochemistry and Molecular Biology, Laboratory, Department of Zoology, Banaras Hindu University, Varanasi
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221005, India
* Corresponding author
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E-mail address, telephone and fax numbers of the corresponding author: Department of Zoology, Biochemistry and Molecular Biology, Laboratory, Banaras Hindu University, Varanasi 221005, India. Tel.: +91 542 6702521;
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Contributed equally
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#
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fax: +91 542 2368174; e-mail: [email protected]
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Social isolation mediated anxiety like behavior is associated with enhanced expression and regulation of BDNF in female mouse brain
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Abstract
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Adverse early life experience is prominent risk factors for numerous psychiatric illnesses, including mood and anxiety disorders. It imposes serious long-term costs on the individual as well as health and social systems.
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Hence, developing therapies that prevent the long-term consequences of early life stress is of utmost importance, and necessitates a better understanding of the mechanisms by which early life stress triggers long-lasting alterations in gene expression and behavior. Post-weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans and it is reported to cause anxiety like behavior which is
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more common in case of females. Therefore, in the present study, we have studied the impact of social isolation of young female mice for 8 weeks on the anxiety like behavior and the underlying molecular mechanism. Elevated plus maze and open field test revealed that social isolation caused anxiety like behavior. BDNF, a well known
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molecule implicated in the anxiety like behavior, was up-regulated both at the message and protein level in cerebral cortex by social isolation. CREB-1 and CBP, which play a crucial role in BDNF transcription, were upregulated at mRNA level in cerebral cortex by social isolation. HDAC-2, which negatively regulates BDNF
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expression, was down-regulated at mRNA and protein level in cerebral cortex by social isolation. Furthermore,
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BDNF acts in concert with Limk-1, miRNA-132 and miRNA-134 for the regulation of structural and morphological plasticity. Social isolation resulted in up-regulation of Limk-1 mRNA and miRNA-132 expression in the cerebral cortex. MiRNA-134, which inhibits the translation of Limk-1, was decreased in cerebral cortex by
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social isolation. Taken together, our study suggests that social isolation mediated anxiety like behavior is associated with up-regulation of BDNF expression and concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex.
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Keywords Social isolation; Anxiety; Cerebral cortex; BDNF; HDAC-2; MiRNA-132
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Introduction Early life adverse experiences (social isolation or maternal separation, childhood abuse and neglect, extreme
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poverty) occur worldwide and cannot be eliminated [1]. Exposure to adverse experiences in early life is implicated
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in the later vulnerability to development of psychiatric disorders, including anxiety and affective disorders in humans [2]. More than 30% of mental disorders are directly related to early life stress [3, 4]. Psychological
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problems due to adverse early life experiences cause socio-economic burden as it impairs the normal daily activities of the patients as well as extremely affects their families and caregivers. Therefore, it is utmost and urgent necessity to comprehend the molecular mechanisms underlying the process of psychological problems resulting due to adverse early life experiences. On the other hand, a large body of literature suggests that post-
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weaning isolation rearing of rodents models the behavioral consequences of adverse early life experiences in humans [5, 6]. The majority of findings suggest that post-weaning social isolation that encompasses preadolescence produces long-lasting alterations in brain leading to anxiety like behavior [7, 8, 9, 10, 11]. Social
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isolation is a stressor that has been used during early development to examine potential long-lasting effects on behavioral and biological responses to stress in rodents [12, 13]. This chronic stress situation has indeed been shown to have long-lasting effects on various behavioral and neural functions [14]. Social isolation precipitates or
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exacerbates the feelings and symptoms of anxiety. One of the consequences of stress exposure in rodents is
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heightened anxiety [15].
Anxiety is an evolutionary trait that provides a coping mechanism in dangerous environmental situations and is associated with emotional processes. Several studies have revealed that brain regions involved in anxiety are
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parahippocampal gyrus, cingulate cortex, amygdala and frontal cortex [16, 17]. The present study was focused on the cerebral cortex. Furthermore, accumulating evidences suggest that the adverse effect of social isolation on brain is mediated by alteration in gene expression which in turn is regulated by epigenetic mechanisms. Thus, epigenetic mechanisms mediate the link between social isolation, gene expression, neurobiological changes, and
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behavioral variation [18].
Interestingly, neurotrophic factors such as nerve growth factor and brain-derived neurotrophic factor (BDNF) have been implicated in the pathophysiology of anxiety disorders [19, 20, 21]. Several studies have demonstrated that BDNF regulates structural, synaptic, and morphological plasticity to modulate the strength or number of synaptic connections and neurotransmission [22, 23]. Also, BDNF participates in cellular proliferation, migration, phenotypic differentiation and maintenance in the developing central nervous system (CNS) [24]. In addition, its presence is required in the adult CNS for maintenance of neuronal functions and neurogenesis [25, 26]. Besides, there are studies which suggest that BDNF transcription is controlled by Ca2+ regulated transcription factor cAMP response element binding protein (CREB-1) [27]. The CREB-1 binding protein (CBP) is a transcriptional coactivator whose function is critical for CREB-1 activity [28]. Structurally, CBP has several protein-binding regions and a histone acetyltransferase (HAT) domain; functionally, CBP acts as a transcriptional coactivator by facilitating the recruitment of required components of the transcriptional machinery and as a HAT by altering the chromatin structure [29]. Like other synaptic plasticity molecules, BDNF expression is regulated by histone deacetylase-2 (HDAC-2) [30].
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ACCEPTED MANUSCRIPT Furthermore, miRNAs are of particular importance in anxiety disorders due to a number of reasons. MiRNAs and their mRNA targets are highly abundant in the CNS, and there is mounting evidence for the role of miRNAs in numerous CNS functions. Besides, evidences from various sources like animal and human studies indicate towards the involvement of miRNAs in the aetiology of anxiety disorders. In addition, there is preliminary
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evidence for the role of miRNAs as therapeutic targets in anxiety and mood disorders. Also, preclinical models
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illustrate that, by changing the levels of miRNAs, a therapeutically desirable change in anxiety-like behavior can be achieved [31]. Amongst miRNAs, miRNA-132 and miRNA-134 are well studied in case of stress condition
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[32, 33]. Remarkably, BDNF acts in concert with Lim domain kinase-1 (Limk-1), miRNA-132 and miRNA-134 in the regulation of structural and morphological plasticity [34, 35]. Furthermore, one of the most widely documented findings in psychiatric epidemiology is that women are significantly more likely than men to develop an anxiety disorder throughout the life span [36].
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On the basis of the above mentioned evidences, the present study was designed to investigate in female mice the effects of post-weaning social isolation on anxiety like behavior and expression of BDNF and molecules associated with BDNF expression and function including CBP, CREB-1, HDAC-2, Limk-1, miRNA-132, and
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miRNA-134. Elevated plus maze and open field test were performed first to assess the anxiety like behavior of female mice followed by molecular studies. We report that social isolation resulted in anxiety like behavior accompanied by enhancement in the expression of BDNF. This was followed by increased expression of CREB-1,
Material and methods
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CBP, Limk-1 and miRNA-132 with concomitant decreased expression of HDAC-2 and miRNA-134.
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Random hexanucleotides, dNTPs, Taq polymerase, RNase inhibitor and reverse transcriptase enzymes were purchased from New England Biolabs (USA), monoclonal anti-β-actin-peroxidase (A3854), TRI reagent from Sigma–Aldrich (USA), BDNF (SC-20981; Santa Cruz Biotechnology, Santa Cruz, CA, USA); HDAC2 (SAB4300412; Sigma-Aldrich), peroxidase conjugated secondary antibodies from Bangalore Genei (India), and
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polyvinyl difluoride (PVDF) membrane from Millipore (Germany). All other chemical reagents were purchased from Merck (Germany). Animals
Swiss albino strain female mice were maintained at 12 h light and dark schedule with ad libitum standard mice feed and drinking water in the animal house of Department of Zoology, Banaras Hindu University, Varanasi at ambient temperature. At the age of 2 months, the mice were either left in group (as control) or assigned to social isolation by individually housing for another 2 months. The procedure for use and handling of animals has been approved by the Institutional Animal Ethical Committee. Experiments were conducted between 9am-6pm in semisound proof condition. Estrous cycle of female mouse was checked according to Korol et al. [ 37]. and mice at same stage, met-estrous were selected for behavioral and molecular studies. After behavioral analysis, the entire cerebral cortex was isolated. One half of the cerebral cortex dissected from individual mouse was pooled together and used for RNA isolation and other pooled half was used for protein isolation [38].
Elevated plus maze test
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ACCEPTED MANUSCRIPT Elevated plus maze test was conducted according to the protocol followed by Mikaelsson et al. [39]. Young female mice after 8 weeks of social isolation were subjected to elevated plus maze test (Fig. 1A, 1B, 1C, 1D, 1E, 1F). The maze, positioned 300 mm above the floor and illuminated evenly at 15 lux, was constructed of white Perspex and consisted of two exposed open arms (175mm x 78mm, length x width) and two equally sized
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enclosed arms, which had 150-mm high walls. Equivalent arms were arranged opposite to one another. Mice were
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placed at the centre of the maze facing one of the open arms and allowed to freely explore for 5min. Elevated plus maze was wiped with 70 % ethanol after each trial to remove odour of previous mice. Data from each pair of arms
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were combined to generate single open- and closed-arm values (time spent in closed arm, time spent in open arm, distance travelled in open arm, distance travelled in closed arm, number of entries into open arm). The data were analysed by ANY-maze software (version 4.95, Stoelting Co., USA).
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The open-field test
The open-field test was conducted according to the protocol followed by Mikaelsson et al. [39]. The apparatus consisted of a square-shaped arena (750x750mm2, length x width), constructed of white plastic and illuminated
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evenly at 15 lux. Subjects were placed facing the centre of one of the walls and allowed to explore the apparatus for 10 min. The open field was subdivided into two virtual concentric squares. The following parameters (Fig. 2A, 2B, 2C, 2D, 2E) were recorded- time spent in central zone, distance travelled in central zone, number of line
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crossings, number of entries into central zone. Open field apparatus was wiped with 70 % ethanol after each trial
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to remove odour of previous mice. The data were analysed by ANY-maze software (version 4.95, Stoelting Co., USA).
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Semi Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) RT-PCR was performed as mentioned by Kumari and Thakur [40]. Briefly, total RNA was isolated from the cerebral cortex of both the groups using TRI reagent kit. RNA was estimated by taking absorbance at 260 nm and purity was checked by A260/A280 ratio. Total RNA from different groups was resolved on 1% agarose
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containing ethidium bromide and the integrity of RNA was checked by staining of 18S and 28S rRNA. cDNA was synthesized from the total RNA extracted from the cerebral cortex of two mice groups. For BDNF, CBP, CREB1, Limk-1, HDAC-2, and β-actin the following primer pairs were used:
BDNF Forward primer 5’ TGCCAGAGCCCCAGGTGTGA3’ Reverse primer 5’ CTGCCCTGGGCCCATTCACG3’ CBP [41] Forward primer 5’TGGAGTGAACCCCCAGTTAG3’ Forward primer 5’TTGCTTGCTCTCGTCTCTGA3’ CREB-1 Forward primer 5'ACA GTG CCA ACC CCC ATT TA3' Reverse primer 5'GTA CCC CAT CCG TAC CAT TGT T3' HDAC-2 [42] Forward primer 5’GGTCGTAGGAATGTTGCTGAT3’ Reverse primer 5’AAGCCAATGTCCTCAAACAGG3’
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Forward primer 5’GTCTCCTGCGACT TCAGC3’,
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Reverse primer 5’TCATTGTCATACCAGGAAATGAGC3’
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PCR condition includes 5 min of initial denaturation at 94 °C, 32 cycles (94 °C for 60 s, 63 ºC for 30s, 72 ºC for 45 s), final extension at 72 ºC for 3 min for BDNF; 5 min of initial denaturation at 94 °C, 28 cycles (94 °C for 30 s, 53 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for CBP; 5 min of initial denaturation at 94 °C, 27 cycles (94 °C for 30 s, 60 ºC for 30 s, 72 °C for 30 s), final extension at 72 ºC for 3 min for CREB-1, 5 min of
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initial denaturation at 94 °C, 28 cycles (94 °C for 30 s, 61 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for HDAC-2; 5 min of initial denaturation at 94 °C, 29 cycles (94 °C for 30 s, 60 ºC for 30s, 72 ºC for 30 s), final extension at 72 ºC for 3 min for Limk-1; 94 °C, 32 cycles (94 °C for 30 s, 60 ºC for 30s, 72 ºC for 30 s), final
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extension at 72 ºC for 3 min for β-actin. The PCR products were resolved on 2% agarose gel. The signals were analysed by Alpha-EaseFC software (Alpha Innotech Corp, USA).
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Quantitative real-time PCR
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Quantitative real time PCR (qRT-PCR) was performed as mentioned by Singh et al. [44]. Briefly, total RNA was isolated from the cerebral cortex of mice using TRI reagent kit (Sigma Aldrich, USA) and 2 µg of RNA from each group was reverse transcribed for cDNA synthesis. The cDNA was used as a template for qRT-PCR amplification
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using gene specific primers for miRNA-132 and miRNA-134 [43]. The endogenous control β-actin was used to normalize quantification of the mRNA target.
MiRNA-132
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Forward 5’ACCGTGGCTTTCGATTGTTA3’ Reverse 5’-GGCGACCATGGCTGTAGACT3’ MiRNA-134
Forward 5’-GGGTGTGTGACTGGTTGACCA3’ Reverse 5’-GGGTTGGTGACTAGGTGGCC3’ Western Blotting Western blotting was performed as mentioned by Kumari and Thakur [40]. Briefly, total protein was isolated from the cerebral cortex of mice using lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF and 1 mM EDTA). Protein was estimated (Bradford 1976). For western blotting of HDAC-2 and BDNF, protein was denatured and resolved by 10% and 15% SDS-PAGE, respectively, and electroblotted onto PVDF membrane by semi-dry method. Then the blot was stained with ponceau-S for checking the transfer efficiency. For western blotting of HDAC-2, the blot was blocked with 5% skimmed milk in PBS for 2 hr. The membrane was probed with rabbit anti HDAC2 (1:15000) and secondary antibody goat anti rabbit (1:2000) diluted in blocking buffer. For western blotting of BDNF, the blot was blocked with 5% skimmed
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ACCEPTED MANUSCRIPT milk in PBS for 4 hr. The membrane was probed with rabbit anti BDNF (1:1000) and secondary antibody goat anti rabbit (1:2000) diluted in blocking buffer. The same blot was reprobed with β-actin (1:25,000 dilution) for loading control. The signals were scanned by AlphaImager system and analysed by Alpha-EaseFC software (Alpha Innotech Corp, USA).
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Statistical Analysis
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Each experiment was repeated three times (n = 15 mice/group). In order to analyze qRT-PCR data, the ΔΔCt value was used to calculate relative fold change in mRNA expression and plotted as histograms. The intensity of
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RT PCR and western blotting band was measured by spot densitometry tool of AlphaEaseFC software (Alpha Innotech Corp, San Jose, CA, USA), histogram was plotted as relative density value (Integrated Density Value (IDV) gene/IDV β-actin) and the data are presented as a histogram with the mean±SEM of three values calculated from three independent experiments. For RT-PCR, the signal intensity (IDV) of BDNF, CBP, CREB-1, HDAC-2,
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Limk-1 mRNA was normalized against the signal intensity (IDV) of internal control, β-actin. For western blotting analysis, the signal intensity (IDV) of HDAC2 and BDNF was normalized against signal intensity (IDV) of internal control, β-actin. Pearson’s r statistics method was used for correlation analysis. The data of all
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experiments were analyzed by independent t-test using SPSS for Windows (standard version 16.0).
Results
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1) Anxiety like behavior in female mice by social isolation as shown by elevated plus maze test and open field test
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Elevated plus maze analysis and open field test revealed that social isolation caused anxiety like behavior in female mice. In elevated plus maze paradigm, after social isolation mice spent significantly more time in closed arm and less time in open arm as compared to that of group housed mice (Fig. 1A, 1B). Moreover, distance
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travelled by mice in open arm decreased significantly (Fig. 1D), but distance travelled by mice in closed arm was not altered significantly by social isolation (Fig. 1C). In addition, number of entries into the open arm decreased significantly by social isolation in comparison to that of group housed mice (Fig. 1E). Representative track plot of mice in elevated plus maze is shown in Fig. 1F. In open field analysis, after social isolation, mice spent
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significantly less time in central zone as compared to that of group housed mice (Fig. 2A). Further, distance travelled by mice in central zone decreased significantly by social isolation (Fig. 2B). Moreover, number of line crossings decreased significantly by social isolation (Fig. 2C). In addition, number of entries into central zone decreased significantly by social isolation as compared to that of group housed mice (Fig. 2D). Representative track plot of mice in open field test is shown in Fig. 2E.
2) Expression of BDNF mRNA and protein, CBP, CREB-1and Limk-1 mRNA and miRNA-132 was up-regulated by social isolation RT-PCR analysis of BDNF showed that its expression increased significantly in the cerebral cortex by social isolation as compared to that of group housed mice (Fig. 3A). Western blot analysis also showed significant enhancement in the expression of BDNF in cerebral cortex by social isolation (Fig. 3B). Moreover, the pattern of expression of BDNF mRNA and protein was similar after social isolation; we checked the correlation between expression of BDNF mRNA and protein in the cerebral cortex. A positive correlation was noted between BDNF mRNA and protein expression (r = 0.943663, p< 0.05) (Fig. 3C). RT-PCR analysis revealed significant upregulation in the expression level of CBP, CREB-1 and Limk-1 in the cerebral cortex by social isolation in
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ACCEPTED MANUSCRIPT comparison to that of group housed mice (Figs. 4A, 4B, 5). Real time PCR analysis showed significant upregulation of miRNA-132 in the cerebral cortex by social isolation in comparison to that of group housed mice
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(Fig. 7B).
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3) Expression of HDAC-2 mRNA and protein, and miRNA-134 was down-regulated by social isolation RT-PCR analysis of HDAC-2 revealed that its expression was down-regulated significantly in the cerebral cortex
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by social isolation in comparison to that of group housed mice (Fig. 6A). Western blot analysis also showed significant down-regulation of HDAC-2 in cerebral cortex by social isolation (Fig. 6B). As the pattern of expression of HDAC-2 mRNA and protein was similar after social isolation, we checked the correlation between expression of HDAC-2 mRNA and protein in the cerebral cortex. A positive correlation was noted between
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HDAC-2 mRNA and protein expression (r = 0.98737, p< 0.05) (Fig. 6C). Real time PCR analysis of miRNA-134 revealed that its expression was down-regulated significantly in cerebral cortex by social isolation in comparison
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to that of group housed mice (Fig. 7A).
Discussion
The present study showed that social isolation of female mice results in the development of anxiety like behavior
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as was revealed by elevated plus maze analysis and open field test which are extensively used behavioral
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paradigms for measuring anxiety behavior in case of rodents [39, 45, 46]. Our finding is substantiated by the study of Kwak et al. [46] that social isolation selectively causes anxiety in mice. A great deal of evidences from the clinical literature suggest that the prevalence of anxiety disorders is about twice as high in women as compared to
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men [47, 48, 49, 50]. Several studies suggest that social isolation rearing of male rodents during a critical window (pre- to mid-adolescence) results in long-lasting increase in anxiety-like behavior that are not reversed by resocialization. In contrast, a critical period may not apply for female rodents. Instead, social isolation at any age may enhance anxiety states in female rodents [51]. Sex differences in anxiety like behavior originate due to the
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probable factors including sex-chromosomal gene expression [52], sexually dimorphic developmental differentiation of brain regions, stress response systems, female reproductive hormone fluctuations, protective or vulnerability-inducing effects of reproductive hormones in adulthood, or differences in stressor perception and emotional appraisal. [53]
Remarkably, we found enhancement in the expression of BDNF in cerebral cortex of mice after social isolation. Earlier reports have shown that BDNF Met mutation increases anxiety-like behavior in female rodents [54, 55]. Interestingly, genetic manipulations that increase BDNF expression in mice similarly increased anxiety-like behaviors, but decreased immobility in the forced swim test [56]. Similarly, in humans, genetic variations that result in higher BDNF activity dependent release were associated with higher anxiety traits [57], while a genetic polymorphism reducing BDNF activity was protective against anxiety [58]. Altogether, these studies indicate that genetic variations that increased BDNF levels result in increased anxiety-related phenotypes, while those decreasing BDNF activity decreased anxiety-related phenotypes. Moreover, RT-PCR analysis of CREB-1 mRNA showed that its expression increased in cerebral cortex by social isolation. Similar to CREB-1, CBP mRNA showed up-regulation in cerebral cortex by social isolation. Moreover, HDAC-2 negatively regulates the
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ACCEPTED MANUSCRIPT expression of synaptic plasticity molecules including BDNF [30]. Thus, we found that the expression of HDAC-2 mRNA and protein was down-regulated in cerebral cortex in social isolation condition. Such down-regulation of HDAC-2 might mediate enhancement in the expression of BDNF. Furthermore, alteration in the expression of BDNF was accompanied by the up-regulation of Limk-1 which is the
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brain specific member of Lim kinase family of serine/threonine kinases [59]. A number of studies showed that
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BDNF/TrkB and Limk-1/cofilin-regulated actin dynamics are involved in growth cone motility, neurite extension, synaptogenesis, and long term potentiation in neurons [60, 61, 62, 63]. Moreover, studies revealed that BDNF
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increases the expression of Limk-1 by relieving the miRNA-134 inhibition of Limk-1 translation [64]. Interestingly, we found that Limk-1 mRNA was up-regulated by social isolation in cerebral cortex. Also, our data revealed down-regulation of miRNA-134 in the cerebral cortex which in turn might mediate enhancement in the expression of Limk-1 of mice after social isolation. It is further substantiated by the finding that chronic stress
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decreases the level of miRNA-134 [32].
In addition, miRNA-132 is one of the most highly inducible CREB-1 targets, and plays a role in neurite outgrowth and neuronal plasticity. It contributes to the action of BDNF. In fact, the over expression of miRNA-132 causes
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enhanced neurite outgrowth and dendritic morphogenesis [65, 66]. Therefore, we studied the expression level of miRNA-132 and found that it increased in cerebral cortex which might be mediated by BDNF. This was also shown by [67], who reported that BDNF up-regulated the expression of miRNA-132 in cultured neurons.
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Moreover, miRNA-132 expression increases in chronic stress condition [32] as well as after maternal separation
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[68].
The alteration in the expression of BDNF and molecules associated with its expression and function might have important role after social isolation. These molecules are primarily associated with synaptic transmission. Also,
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the findings from different laboratories have shown that excitatory neurotransmission is increased in the brain regions linked with anxiety [16, 17]. Therefore, enhancement in the expression of BDNF, CREB-1, CBP, Limk-1, miRNA-132 and decline in the expression of miRNA-134, HDAC-2 in cerebral cortex after social isolation in
Conclusion
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female mice might lead to increased excitatory neurotransmission which is reported in case of anxiety.
Altogether it can be concluded that anxiety like behavior caused by social isolation might be mediated by upregulation in the expression of BDNF with concomitant increase in the expression of CBP, CREB-1, Limk-1 and miRNA-132, and decrease in the expression of HDAC-2 and miRNA-134 in the cerebral cortex. Acknowledgments Anita Kumari and Padmanabh Singh are recipients of senior research fellowship from the Council of Scientific and Industrial Research, India. Meghraj Singh Baghel is a recipient of junior research fellowship from the Department of Biotechnology, India. This work was supported by Grants from Department of Science & Technology, India and Council of Scientific & Industrial Research, India. We acknowledge DBT-BHU Interdisciplinary School of Life Sciences, Banaras Hindu University for real-time PCR facility. The funding sources had no involvement in study design; collection, analysis and interpretation of data; writing of the report; and in the decision to submit the article for publication.
Conflict of interest:
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ACCEPTED MANUSCRIPT The authors declare that they do not have any actual or potential conflict of interest. References:
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Legends:
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Fig. 1 Elevated plus maze analysis showing A time spent by two groups of mice in closed arm B time spent in open arm C distance travelled in closed arm D distance travelled in open arm E number of entries into open arm F Representative track plot: (a) G (b) I. G- Group, I- Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test), (n=15 mice/group).
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Fig. 2 Open field test showing A time spent by two groups of mice in central zone B distance travelled in central
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zone C number of line crossings D number of entries into open arm E Representative track plot: (a) G (b) I. GGroup, I- Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test),
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(n=15 mice/group).
Fig. 3 A RT-PCR analysis of BDNF mRNA in cerebral cortex of two groups of mice. Signal intensity for BDNF was normalized against signal intensity for β-actin. B Western blot analysis of BDNF protein in cerebral cortex of two groups of mice. Signal intensity for BDNF was normalized against signal intensity for β-actin. The data
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are presented as a histogram with the mean (±SEM) of three values calculated as RDV of BDNF/β-actin. C Correlation analysis between BDNF mRNA and protein expression in two groups of mice.
G- Group, I-
Isolation. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test).
Fig. 4 RT-PCR analysis of A CBP mRNA and B CREB-1 mRNA in cerebral cortex of two groups of mice. Signal intensity for CBP and CREB-1 was normalized against signal intensity for β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of CBP/β-actin and CREB-1/ β-actin. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, IIsolation.
Fig. 5 RT-PCR analysis of Limk-1 mRNA in cerebral cortex of two groups of mice. Signal intensity for Limk-1 was normalized against signal intensity for β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of Limk-1/β-actin. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, I- Isolation.
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Fig. 6 A RT-PCR analysis of HDAC-2 mRNA in cerebral cortex of two groups of mice. Signal intensity for HDAC-2 was normalized against signal intensity for β-actin. B Western blot analysis of HDAC-2 protein in
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cerebral cortex of two groups of mice. Signal intensity for HDAC-2 was normalized against signal intensity for
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β-actin. The data are presented as a histogram with the mean (±SEM) of three values calculated as RDV of HDAC-2 /β-actin. C Correlation analysis between HDAC-2 mRNA and protein expression in two groups of
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mice. ‘*’ denotes significant differences (p < 0.05) as compared to G (Independent-samples t-test). G- Group, IIsolation.
Fig. 7 Quantitative real-time PCR analysis representing fold change in A miRNA-134 expression B miRNA-132
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expression in cerebral cortex in two groups of mice. G- Group, I- Isolation. Data are represented the mean
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Fig. 7AB
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Highlights Elevated plus maze and open field test analysis revealed that post-weaning social isolation of
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young female mice caused anxiety like behaviour.
BDNF, CREB-1, Limk-1 and mir-132 mRNA were up-regulated in the cerebral cortex by
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HDAC-2 and Mir-134 were down-regulated in the cerebral cortex by social isolation.
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social isolation.
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