Chronic restraint stress produces sex-specific behavioral and molecular outcomes in the dorsal and ventral rat hippocampus.

Stress-related disorders display differences at multiple levels according to sex. While most studies have been conducted in male rodents, less is known about comparable outcomes in females. In this study, we found that the chronic restraint stress model (2.5 h/day for 14 days) triggers different somatic responses in male and female adult rats. Chronic restraint produced a loss in sucrose preference and novel location preference in male rats. However, chronic restraint failed to produce loss of sucrose preference in females, while it improved spatial performance. We then characterized the molecular responses associated with these behaviors in the hippocampus, comparing the dorsal and ventral poles. Notably, sex- and hippocampal pole-specific transcriptional signatures were observed, along with a significant concordance between the female ventral and male dorsal profiles. Functional enrichment analysis revealed both shared and specific terms associated with each pole and sex. By looking into signaling pathways that were associated with these terms, we found an ample array of sex differences in the dorsal and, to a lesser extent, in the ventral hippocampus. These differences were mainly present in synaptic TrkB signaling, Akt pathway, and glutamatergic receptors. Unexpectedly, the effects of stress on these pathways were rather minimal and mostly dissociated from the sex-specific behavioral outcomes. Our study suggests that female rats are resilient and males susceptible to the restraint stress exposure in the sucrose preference and object location tests, while the activity of canonical signaling pathways is primarily determined by sex rather than stress in the dorsal and ventral hippocampus.

Sex-Dependent Changes of miRNA Levels in the Hippocampus of Adrenalectomized Rats Following Acute Corticosterone Administration.

We explored sex-biased effects of the primary stress glucocorticoid hormone corticosterone on the miRNA expression profile in the rat hippocampus. Adult adrenalectomized (ADX) female and male rats received a single corticosterone (10 mg/kg) or vehicle injection, and after 6 h, hippocampi were collected for miRNA, mRNA, and Western blot analyses. miRNA profiling microarrays showed a basal sex-biased miRNA profile in ADX rat hippocampi. Additionally, acute corticosterone administration triggered a sex-biased differential expression of miRNAs derived from genes located in several chromosomes and clusters on the X and 6 chromosomes. Putative promoter analysis unveiled that most corticosterone-responsive miRNA genes contained motifs for either direct or indirect glucocorticoid actions in both sexes. The evaluation of transcription factors indicated that almost 50% of miRNA genes sensitive to corticosterone in both sexes was under glucocorticoid receptor regulation. Transcription factor-miRNA regulatory network analyses identified several transcription factors that regulate, activate, or repress miRNA expression. Validated target mRNA analysis of corticosterone-responsive miRNAs showed a more complex miRNA-mRNA interaction network in males compared to females. Enrichment analysis revealed that several hippocampal-relevant pathways were affected in both sexes, such as neurogenesis and neurotrophin signaling. The evaluation of selected miRNA targets from these pathways displayed a strong sex difference in the hippocampus of ADX-vehicle rats. Corticosterone treatment did not change the levels of the miRNA targets and their corresponding tested proteins. Our data indicate that corticosterone exerts a sex-biased effect on hippocampal miRNA expression, which may engage in sculpting the basal sex differences observed at higher levels of hippocampal functioning.

Loss of Social/Non-social Context Discrimination by Movement Acceleration in the Valproate Model of Autism.

Autism spectrum disorder (ASD) is a neurodevelopmental alteration characterized by social/communicative deficits, repetitive/stereotyped movements, and restricted/obsessive interests. However, there is not much information about whether movement alterations in ASD comprise modifications at the basic kinematic level, such as trajectory and velocity, which may contribute to the higher level of processing that allows the perception and interpretation of actions performed by others, and hence, impact social interaction. In order to further explore possible motor alterations in ASD, we analyzed movement parameters in the Valproate (VPA) animal model of autism. We found that VPA-treated rats displayed greater movement acceleration, reduced distance between stops, spent more time in the corner of the open-field arena, and executed a number of particular behaviors; for example, supported rearing and circling, with no major changes in distance and velocity. However, in the social interaction test, we found other alterations in the movement parameters. In addition to increased acceleration, VPA-rats displayed reduced velocity, increased stops, reduced distance/stop and lost the social/non-social area discrimination that is characteristic of control rats in acceleration and stops variables. Hence, even if prenatal VPA-treatment could have a minor effect in motor variables in a non-social context, it has a crucial effect in the capacity of the animals to adjust their kinematic variables when social/non-social context alternation is required.

Region-Specific Reduction of BDNF Protein and Transcripts in the Hippocampus of Juvenile Rats Prenatally Treated With Sodium Valproate.

Autism is a neurodevelopmental disorder characterized by a deep deficit in language and social interaction, accompanied by restricted, stereotyped and repetitive behaviors. The use of genetic autism animal models has revealed that the alteration of the mechanisms controlling the formation and maturation of neural circuits are points of convergence for the physiopathological pathways in several types of autism. Brain Derived Neurotrophic Factor (BDNF), a key multifunctional regulator of brain development, has been related to autism in several ways. However, its precise role is still elusive, in part, due to its extremely complex posttranscriptional regulation. In order to contribute to this topic, we treated prenatal rats with Valproate, a well-validated model of autism, to analyze BDNF levels in the hippocampus of juvenile rats. Valproate-treated rats exhibited an autism-like behavioral profile, characterized by a deficit in social interaction, anxiety-like behavior and repetitive behavior. In situ hybridization (ISH) experiments revealed that Valproate reduced BDNF mRNA, especially long-3'UTR-containing transcripts, in specific areas of the dentate gyrus (DG) and CA3 regions. At the same time, Valproate reduced BDNF immunoreactivity in the suprapyramidal and lucidum layers of CA3, but improved hippocampus-dependent spatial learning. The molecular changes reported here may help to explain the cognitive and behavioral signs of autism and reinforce BDNF as a potential molecular target for this neurodevelopmental disorder.

MicroRNA Profiling and Bioinformatics Target Analysis in Dorsal Hippocampus of Chronically Stressed Rats: Relevance to Depression Pathophysiology.

Studies conducted in rodents subjected to chronic stress and some observations in humans after psychosocial stress, have allowed to establish a link between stress and the susceptibility to many complex diseases, including mood disorders. The studies in rodents have revealed that chronic exposure to stress negatively affects synaptic plasticity by triggering changes in the production of trophic factors, subunit levels of glutamate ionotropic receptors, neuron morphology, and neurogenesis in the adult hippocampus. These modifications may account for the impairment in learning and memory processes observed in chronically stressed animals. It is plausible then, that stress modifies the interplay between signal transduction cascades and gene expression regulation in the hippocampus, therefore leading to altered neuroplasticity and functioning of neural circuits. Considering that miRNAs play an important role in post-transcriptional-regulation of gene expression and participate in several hippocampus-dependent functions; we evaluated the consequences of chronic stress on the expression of miRNAs in dorsal (anterior) portion of the hippocampus, which participates in memory formation in rodents. Here, we show that male rats exposed to daily restraint stress (2.5 h/day) during 7 and 14 days display a differential profile of miRNA levels in dorsal hippocampus and remarkably, we found that some of these miRNAs belong to the miR-379-410 cluster. We confirmed a rise in miR-92a and miR-485 levels after 14 days of stress by qPCR, an effect that was not mimicked by chronic administration of corticosterone (14 days). Our in silico study identified the top-10 biological functions influenced by miR-92a, nine of which were shared with miR-485: Nervous system development and function, Tissue development, Behavior, Embryonic development, Organ development, Organismal development, Organismal survival, Tissue morphology, and Organ morphology. Furthermore, our in silico study provided a landscape of potential miRNA-92a and miR-485 targets, along with relevant canonical pathways related to axonal guidance signaling and cAMP signaling, which may influence the functioning of several neuroplastic substrates in dorsal hippocampus. Additionally, the combined effect of miR-92a and miR-485 on transcription factors, along with histone-modifying enzymes, may have a functional relevance by producing changes in gene regulatory networks that modify the neuroplastic capacity of the adult dorsal hippocampus under stress.

Matrix Metalloproteinase 9 Displays a Particular Time Response to Acute Stress: Variation in Its Levels and Activity Distribution in Rat Hippocampus.

A single stress exposure facilitates memory formation through neuroplastic processes that reshape excitatory synapses in the hippocampus, probably requiring changes in extracellular matrix components. We tested the hypothesis that matrix metalloproteinase 9 (MMP-9), an enzyme that degrades components of extracellular matrix and synaptic proteins such as ß-dystroglycan (ß-DG43), changes their activity and distribution in rat hippocampus during the acute stress response. After 2.5 h of restraint stress, we found (i) increased MMP-9 levels and potential activity in whole hippocampal extracts, accompanied by ß-DG43 cleavage, and (ii) a significant enhancement of MMP-9 immunoreactivity in dendritic fields such as stratum radiatum and the molecular layer of hippocampus. After 24 h of stress, we found that (i) MMP-9 net activity rises at somatic field, i.e., stratum pyramidale and granule cell layers, and also at synaptic field, mainly stratum radiatum and the molecular layer of hippocampus, and (ii) hippocampal synaptoneurosome fractions are enriched with MMP-9, without variation of its potential enzymatic activity, in accordance with the constant level of cleaved ß-DG43. These findings indicate that stress triggers a peculiar timing response in the MMP-9 levels, net activity, and subcellular distribution in the hippocampus, suggesting its involvement in the processing of substrates during the stress response.

Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats.

Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in dorsal and ventral hippocampus (VH). Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day) during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH). By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR) subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR) subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.