Lucia Carboni

Different susceptibility to social defeat stress of BalbC and C57BL6/J mice

Social stress may precipitate psychopathological disorders in susceptible individuals. The present experiments were focused on the biology beyond the differential susceptibility to social stress. Social defeat, an ethologically relevant stressor known to elicit different coping strategies, was used in two mouse strains differing for baseline emotionality, such as C57BL6/J and BalbC. In separate experiments, in both strains a single social defeat decreased home-cage activity without altering social aversion; it diminished body weight only in defeated BalbC mice. In longitudinal experiments, mice experienced repeated social defeats that induced multiple long-term consequences. Defeated C57BL6/J increased their body weight and food intake; defeated BalbC mice diminished their metabolic efficiency. Only defeated BalbC subjects exhibited increased social avoidance levels; no differences from controls were seen on forced swim test response in defeated mice of either strain. No long-term effects of social defeat were detected in peripheral biomarkers of stress, metabolic, and immune responses, although the analysis of selected internal organs revealed decreases in abdominal fat and gonadal organs in all defeated subjects. These results demonstrated a strain-distinctive profile in the susceptibility to social defeat stress, either acutely or chronically, with metabolic consequences more consistently found in C57BL6/J while social aversion induced predominantly in BalbC subjects.

Proteomic analysis of rat hippocampus after repeated psychosocial stress

Since stress plays a role in the onset and physiopathology of psychiatric diseases, animal models of chronic stress may offer insights into pathways operating in mood disorders. The aim of this study was to identify the molecular changes induced in rat hippocampus by repeated exposure to psychosocial stress with a proteomic technique. In the social defeat model, the experimental animal was defeated by a dominant male eight times. Additional groups of rats were submitted to a single defeat or placed in an empty cage (controls). The open field test was carried out on parallel animal groups. The day after the last exposure, levels of hippocampal proteins were compared between groups after separation by 2-D gel electrophoresis and image analysis. Spots showing significantly altered levels were submitted to peptide fingerprinting mass spectrometry for protein identification. The intensity of 69 spots was significantly modified by repeated stress and 21 proteins were unambiguously identified, belonging to different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism. This work identified molecular changes in protein levels caused by exposure to repeated psychosocial stress. The pattern of changes induced by repeated stress was quantitatively and qualitatively different from that observed after a single exposure. Several changed proteins have already been associated with stress-related responses; some of them are here described for the first time in relation to stress.

Early-life stress and antidepressants modulate peripheral biomarkers in a gene-environment rat model of depression.

BACKGROUND Availability of peripheral biomarkers for depression could aid diagnosis and help to predict treatment response. The objective of this work was to analyse the peripheral biomarker response in a gene-environment interaction model of depression. Genetically selected Flinders Sensitive Line (FSL) rats were subjected to maternal separation (MS), since early-life trauma is an important antecedent of depression. An open-ended approach based on a proteomic analysis of serum was combined with the evaluation of depression-associated proteins. METHODS Rats experienced MS and chronically received escitalopram (ESC) or nortryptiline (NOR). Serum proteins were compared by two-dimensional gel electrophoresis. Corticosterone, cytokines, BDNF and C-reactive protein (CRP) were measured by immunoassays. RESULTS Comparing FSL with the control Flinders Resistant Line (FRL), Apo-AI and Apo-AIV, alpha1-macroglobulin, glutathione peroxidase and complement-C3 were significantly modulated. Significant increases were detected in leptin, interleukin (IL) 1alpha and BDNF. CRP levels were significantly reduced. The impact of early-life stress was assessed by comparing FSL+MS versus FSL. Apo-E, alpha1-macroglobulin, complement-C3, transferrin and hemopexin were significantly modulated. The effect of stress in antidepressant response was then evaluated. In the comparison FSL+ESC+MS versus FSL+ESC, albumin, alpha1-macroglobulin, glutathione peroxidase and complement-C3 were modulated and significant reductions were detected in IL4, IL6, IL10, CRP and BDNF. By comparing FSL+NOR+MS versus FSL+NOR proteins like Apo-AIV, pyruvate dehydrogenase, alpha1-macroglobulin, transferrin and complement-C3 showed different levels. CONCLUSIONS Lipid metabolism and immunity proteins were differently expressed in FSL in comparison with FRL. Exposure to MS induced changes in inflammation and transport proteins which became apparent in response to antidepressant treatments. Modulated proteins could suggest biomarker studies in humans.

Proteomic analysis of rat hippocampus and frontal cortex after chronic treatment with fluoxetine or putative novel antidepressants: CRF1 and NK1 receptor antagonists

Chronic administration of antidepressants is required for their efficacy, suggesting the involvement of long-term modifications. As the impact of antidepressant treatment on the brain molecular machinery is not completely understood, we performed a proteomic analysis of rat hippocampus and frontal cortex after chronic treatment with fluoxetine, with an NK1 receptor antagonist, GR205171, and a CRF receptor 1 antagonist, DMP696. After 2D electrophoresis, protein expression levels were compared with both univariate and multivariate statistical analyses and identified by mass spectrometry. All treatments modified levels of actin isoforms, whereas both fluoxetine and GR205171 reduced synapsin II. Fluoxetine treatment increased ERK2 and NP25 and decreased vacuolar ATP synthase. After GR205171 treatment, protein disulphide isomerase A was reduced; dynamin 1 and aldose reductase increased. DMP696 modulated DRP2, pyruvate kinase, LDH and ATP synthase. Although each compound induced a specific pattern of protein modulation, data suggest that antidepressants share the ability of modulating neural plasticity.

Single exposure to social defeat increases corticotropin-releasing factor and glucocorticoid receptor mRNA expression in rat hippocampus

Stressful life events are able to induce long-term modifications in physiological and neuroendocrine parameters that are related to the onset of several psychiatric disorders. To gain information on molecular modifications involved in long-term changes triggered by stress, we evaluated gene expression in the hippocampus of rats exposed to a single social defeat session. In the social defeat model, the experimental animal is defeated by a dominant male. The defeat induced an increase in body temperature, in distress vocalisations, in serum corticosterone levels and in anxiety-related behaviour measured with an open field test applied 6 h after the exposure to the dominant rat. In the open field test, anxiety-related behaviours were not detectable anymore 30 h after the exposure to the dominant rat and mRNA levels were evaluated at this time-point. The mRNA levels of genes modulated by stress (corticotropin-releasing factor; corticotropin-releasing factor receptor 1; corticotropin-releasing factor binding protein; mineralocorticoid and glucocorticoid receptors; Ca2+/calmodulin-dependent protein kinase-like kinase; Krox20; Bcl-2) and control genes (glyceraldehyde-3-phosphate dehydrogenase; beta-actin and cyclophilin A) were measured with real-time reverse transcription polymerase chain reaction. Corticotropin-releasing factor and glucocorticoid receptor mRNA levels were significantly modulated by the stress procedure, both genes showing an increase in rats exposed to a social defeat. No expression level differences were detected for the other genes. In conclusion, we report that 30 h after an acute social stress, a modification in mRNA levels can be detected in rat hippocampus, thus suggesting potential candidate genes involved in mediating long-term responses.

Expression Profiling of a Genetic Animal Model of Depression Reveals Novel Molecular Pathways Underlying Depressive-Like Behaviours

Background The Flinders model is a validated genetic rat model of depression that exhibits a number of behavioural, neurochemical and pharmacological features consistent with those observed in human depression. Principal Findings In this study we have used genome-wide microarray expression profiling of the hippocampus and prefrontal/frontal cortex of Flinders Depression Sensitive (FSL) and control Flinders Depression Resistant (FRL) lines to understand molecular basis for the differences between the two lines. We profiled two independent cohorts of Flinders animals derived from the same colony six months apart, each cohort statistically powered to allow independent as well as combined analysis. Using this approach, we were able to validate using real-time-PCR a core set of gene expression differences that showed statistical significance in each of the temporally distinct cohorts, representing consistently maintained features of the model. Small but statistically significant increases were confirmed for cholinergic (chrm2, chrna7) and serotonergic receptors (Htr1a, Htr2a) in FSL rats consistent with known neurochemical changes in the model. Much larger gene changes were validated in a number of novel genes as exemplified by TMEM176A, which showed 35-fold enrichment in the cortex and 30-fold enrichment in hippocampus of FRL animals relative to FSL. Conclusions These data provide significant insights into the molecular differences underlying the Flinders model, and have potential relevance to broader depression research.

A role for BDNF/TrkB signaling in behavioral and physiological consequences of social defeat stress.

Accumulating evidences underlie the importance of the interplay between environmental and genetic factors in contributing to the risk to develop mental illness. Brain‐derived neurotrophic factor (BDNF) and its Tyrosine receptor kinase B (TrkB) receptor play a fundamental contribution to brain development and plastic adaptations to life events. In the present study, the potential for the BDNF/TrkB contribution in increasing vulnerability to negative social experiences was assessed by subjecting TrkB.T1 overexpressing mice to a chronic social defeat model. TrkB.T1 mice overexpress the dominant‐negative truncated splice variant of TrkB receptor leading to decreased BDNF signaling. After repeated social defeat, mice were assessed in a longitudinal study for behavioral, physiological, endocrine and immune responses potentially related to psychiatric endophenotypes. TrkB.T1 overexpression corresponded to smaller changes in metabolic parameters such as body weight, food intake, feed efficiency and peripheral ghrelin levels compared with wild‐type (wt) littermates following social defeat. Interestingly, 4 weeks after the last defeat, TrkB.T1 overexpressing mice exhibited more consistent social avoidance effects than what observed in wt subjects. Finally, previously unreported effects of TrkB mutations could be observed on lymphoid organ weight and on peripheral immune biomarker levels, such as interleukin‐1α and regulated on activation, normal, T‐cell expressed, and secreted (RANTES), thus suggesting a systemic role of BDNF signaling in immune function. In conclusion, the present data support a contribution of TrkB to stress vulnerability that, given the established role of TrkB in the response to antidepressant treatment, calls for further studies addressing the link between stress susceptibility and variability in drug efficacy.

Conditioning properties of social subordination in rats: behavioral and biochemical correlates of anxiety.

To develop a socially based model of anxiety, the contextual fear conditioning properties of social defeat were examined in rats. Social threat consisted of exposing intruders to aggressive residents in resident home cage, separated by a partition. During 3 daily encounters, intruders were either defeated or threatened by residents, providing the defeated-threatened (DT) and threatened-threatened (TT) groups respectively. On Day 4, both DT and TT animals were subjected to a social threat only. Additional animals received a 4-day exposure to a novel empty cage (EC group). Further DT, TT, and EC rats were confronted to a different context on Day 4. DT rats exhibited a robust and context-specific anxiety-like response, characterized by significant behavioral and biochemical alterations. DT rats showed increased risk assessment and decreased exploration compared to TT and EC rats that in turn were not different towards each other. DT and TT rats exhibited increased ACTH levels, while only DT rats showed enhanced corticosterone and decreased testosterone levels compared to EC. These differences were context-specific since they were absent confronting animals to a different context and since they were not long lasting. Overall, these data demonstrate the induction of an anxiety-like state in rats through a context conditioning process based upon social factors. The social basis of this paradigm offers good face validity with anxiety disorders, which in humans are mainly related to social factors and associated with HPA axis deregulations. The present procedure may provide a useful experimental model to further investigate the neurobiological mechanisms underlying anxiety-related disorders.

Alterations of behavioral and endocrinological reactivity induced by 3 brief social defeats in rats: Relevance to human psychopathology

In the realm of animal models of psychopathology, social stress based procedures rely on robust theoretical prerequisites to meet construct validity criteria for the target syndromes. In order to further assess the relevance for human psychopathology of a social defeat based model in rats, known to elicit consistent behavioral and hormonal changes, we expanded its characterization on the basis of both behavioral parameters and peripheral biomarkers thought to be pertinent for clinical symptoms. Rats were subjected to 3 daily social defeat experiences that shortly thereafter led to the insurgence of defensive behaviors, anhedonia, and body weight loss. HPA axis showed an activated response when rats were sampled as early as after the first social defeat experience, while none of the peripheral immune, metabolic, and neurotrophic factors examined were concurrently affected. With the aim of determining the long-term bio-behavioral sequelae of the social defeat experience, rats were assessed also 3 weeks after the social defeats. At this time, behavioral changes were still observed, including decreased general activity and sociality in a social avoidance test, increased immobility and decreased escape responses in a forced swim test. These alterations were not paralleled by alterations in anhedonia nor HPA axis responses from controls, nor where evident changes in the humoral component of the immune response nor in brain derived neurotrophic factor levels, whereas a substantial increase in leptin levels was observed in previously socially defeated rats compared to control. Overall these data depict a very complex set of alterations induced both acutely and long-term by social stress in endocrinological and behavioral reactivity of rats.

Social defeat-induced contextual conditioning differentially imprints behavioral and adrenal reactivity: a time-course study in the rat.

The present experiments were based on the rat resident-intruder paradigm and aimed at better understanding the long-term conditioning properties of this social stress model. Intruders were exposed to aggressive conspecifics residents. During 3 daily encounters, intruders were either defeated or threatened by residents, providing the defeated-threatened (DT) and threatened-threatened (TT) groups respectively, or exposed to a novel empty cage (EC). The effect of such exposures was assessed in 3 separate experiments 8, 14, or 21 days following the last session on both behavior and hypothalamus-pituitary-adrenal (HPA) axis parameters. A specific and persistent behavioral conditioning due to social defeat but also to the sole social threat experience was observed as defensive behaviors and anxiety-like behaviors were observed respectively in DT and TT rats, highlighting a lack of habituation for the conditioning properties of this social stressor. On the other hand, at the earlier time points examined a less specific activation of the HPA axis parameters was found, starting to show habituation at day 21 in EC but not in DT or TT rats. These data give further support to the lasting effects of this social stress model, bestowing a special emphasis upon the impact of its psychological component and upon the relevance of its development and maintenance over time.