Prefrontal Cortex Cytosolic Proteome and Machine Learning-Based Predictors of Resilience toward Chronic Social Isolation in Rats.

Chronic social isolation (CSIS) generates two stress-related phenotypes: resilience and susceptibility. However, the molecular mechanisms underlying CSIS resilience remain unclear. We identified altered proteome components and biochemical pathways and processes in the prefrontal cortex cytosolic fraction in CSIS-resilient rats compared to CSIS-susceptible and control rats using liquid chromatography coupled with tandem mass spectrometry followed by label-free quantification and STRING bioinformatics. A sucrose preference test was performed to distinguish rat phenotypes. Potential predictive proteins discriminating between the CSIS-resilient and CSIS-susceptible groups were identified using machine learning (ML) algorithms: support vector machine-based sequential feature selection and random forest-based feature importance scores. Predominantly, decreased levels of some glycolytic enzymes, G protein-coupled receptor proteins, the Ras subfamily of GTPases proteins, and antioxidant proteins were found in the CSIS-resilient vs. CSIS-susceptible groups. Altered levels of Gapdh, microtubular, cytoskeletal, and calcium-binding proteins were identified between the two phenotypes. Increased levels of proteins involved in GABA synthesis, the proteasome system, nitrogen metabolism, and chaperone-mediated protein folding were identified. Predictive proteins make CSIS-resilient vs. CSIS-susceptible groups linearly separable, whereby a 100% validation accuracy was achieved by ML models. The overall ratio of significantly up- and downregulated cytosolic proteins suggests adaptive cellular alterations as part of the stress-coping process specific for the CSIS-resilient phenotype.

Prefrontal cortical synaptoproteome profile combined with machine learning predicts resilience towards chronic social isolation in rats.

Chronic social isolation (CSIS) of rats serves as an animal model of depression and generates CSIS-resilient and CSIS-susceptible phenotypes. We aimed to investigate the prefrontal cortical synaptoproteome profile of CSIS-resilient, CSIS-susceptible, and control rats to delineate biochemical pathways and predictive biomarker proteins characteristic for the resilient phenotype. A sucrose preference test was performed to distinguish rat phenotypes. Class separation and machine learning (ML) algorithms support vector machine with greedy forward search and random forest were then used for discriminating CSIS-resilient from CSIS-susceptible and control rats. CSIS-resilient compared to CSIS-susceptible rat proteome analysis revealed, among other proteins, downregulated glycolysis intermediate fructose-bisphosphate aldolase C (Aldoc), and upregulated clathrin heavy chain 1 (Cltc), calcium/calmodulin-dependent protein kinase type II (Cam2a), synaptophysin (Syp) and fatty acid synthase (Fasn) that are involved in neuronal transmission, synaptic vesicular trafficking, and fatty acid synthesis. Comparison of CSIS-resilient and control rats identified downregulated mitochondrial proteins ATP synthase subunit beta (Atp5f1b) and citrate synthase (Cs), and upregulated protein kinase C gamma type (Prkcg), vesicular glutamate transporter 1 (Slc17a7), and synaptic vesicle glycoprotein 2 A (Sv2a) involved in signal transduction and synaptic trafficking. The combined protein differences make the rat groups linearly separable, and 100% validation accuracy is achieved by standard ML models. ML algorithms resulted in four panels of discriminative proteins. Proteomics-data-driven class separation and ML algorithms can provide a platform for accessing predictive features and insight into the molecular mechanisms underlying synaptic neurotransmission involved in stress resilience.

Transformation of cereal grains: Botanical and chemical analysis of food residues encrusted on pottery from the Funnel Beaker settlement of Oldenburg LA 77, northern Germany.

An integrated botanical and chemical approach is used to study surface residues on Funnel Beaker ceramics from the site of Oldenburg LA 77, in northern Germany. Organic residues were discovered adhering to fragments of thick-walled, undecorated ceramic vessels (n = 19) and ceramic discs (n = 2). The surface residues were studied using scanning electron microscopy (SEM), to examine remains of cereals and other plant tissues that survived food preparation and cooking, and using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and direct time-resolved mass spectrometry (DTMS), to chemically identify specific food components. The SEM results show a reoccurring presence of cereal grain (emmer and barley) and one case of co-occurrence of emmer and fat-hen seeds. The SEM evidence for the use of sprouted emmer grain and milk-ripe barley from the Oldenburg residues greatly enhances our understanding of Neolithic foodways in northwestern Europe. The ATR-FTIR results showed that roughly a third of the surface residues contain traces of the original foods prepared or processed and DTMS results confirm that most of the residues primarily contain polysaccharides and a minimal amount of plant protein and that they lack lipids. Only one residue presents minor indications for a (partly) animal origin. The ceramic vessels were thus used almost exclusively for the processing or cooking of cereal grains. This study offers an intimate view of the cuisine and cooking practices (and in some cases their seasonal timing) in an early agricultural village located in a marginal farming region on the south coast of the Baltic Sea.

Olanzapine Effects on Parvalbumin/GAD67 Cell Numbers in Layers/Subregions of Dorsal Hippocampus of Chronically Socially Isolated Rats.

Depression is linked to changes in GABAergic inhibitory neurons, especially parvalbumin (PV) interneurons, which are susceptible to redox dysregulation. Olanzapine (Olz) is an atypical antipsychotic whose mode of action remains unclear. We determined the effect of Olz on PV-positive (+) and glutamate decarboxylase 67 (GAD67) + cell numbers in the layers of dorsal hippocampus (dHIPP) cornu ammonis (CA1-CA3) and dentate gyrus (DG) subregions in rats exposed to chronic social isolation (CSIS), which is an animal model of depression. Antioxidative enzymes and proinflammatory cytokine levels were also examined. CSIS decreased the PV+ cell numbers in the Stratum Oriens (SO) and Stratum Pyramidale (SP) of dCA1 and dDG. It increased interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and copper-zinc superoxide dismutase (CuZnSOD) levels, and it decreased catalase (CAT) protein levels. Olz in CSIS increased the number of GAD67+ cells in the SO and SP layers of dCA1 with no effect on PV+ cells. It reduced the PV+ and GAD67+ cell numbers in the Stratum Radiatum of dCA3 in CSIS. Olz antagonizes the CSIS-induced increase in CuZnSOD, CAT and SOCS3 protein levels with no effect on IL-6. Data suggest that the protective Olz effects in CSIS may be mediated by altering the number of PV+ and GAD67+ cells in dHIPP subregional layers.

Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.

Chronic fluoxetine treatment in socially-isolated rats modulates the prefrontal cortex synaptoproteome.

Exposure to chronic social isolation (CSIS) and synapse dysfunction have been implicated in the etiology of major depressive disorder (MDD). Fluoxetine (Flx) has been widely used to treat MDD, but its mechanisms of action remain elusive. We employed comparative synaptoproteomics to investigate the changes in the levels of proteins and molecular signaling pathways in prefrontal cortical samples of adult male Wistar rats exposed to CSIS, a rat model of depression, and CSIS rats treated with chronic Flx and controls, using liquid chromatography coupled to tandem mass spectrometry. Flx-treated control rats showed a decreased level of proteins involved in vesicle-mediated transport, and a predominantly increased level of exocytosis-associated proteins. CSIS significantly reduced the level of proteins involved in the ATP metabolic process, clathrin-dependent endocytosis, and proteolysis. Flx treatment in CSIS rats stimulated synaptic vesicle trafficking by increasing the regulation of exo/endocytosis-associated proteins, proteins involved in synaptic plasticity including neurogenesis, Cox5a, mitochondria-associated proteins involved in oxidative phosphorylation, and ion transport proteins (Slc8a2, Atp1b2). Flx treatment resulted in an increased synaptic vesicle dynamic, plasticity and mitochondrial functionality, and a suppression of CSIS-induced impairment of these processes. BIOLOGICAL SIGNIFICANCE: Identifying biomarkers of MDD and treatment response is the goal of many studies. Contemporary studies have shown that many molecular alterations associated with the pathophysiology of MDD reside within the synapse. As part of this research, a growing importance is the use of proteomics, as monitoring the changes in protein levels enables the identification of (possible) biochemical pathways and processes of importance for the development of depressive-like behavior and the efficacy of antidepressant treatments. We profiled proteomic changes representative of the development of CSIS-induced depressive-like behavior and the antidepressant effects of Flx. Our study has identified synaptosomal proteins and altered molecular pathways that may be potential markers of prefrontal cortical synaptic dysfunction associated with depressive-like behavior, and further clarified the mechanisms of depressive-like behavior and mode of action of Flx. Our findings indicate potential PFC synaptic targets for antidepressant treatment.

Fluoxetine Enhances Synaptic Vesicle Trafficking and Energy Metabolism in the Hippocampus of Socially Isolated Rats.

Chronic social isolation (CSIS)-induced alternation in synaptic and mitochondrial function of specific brain regions is associated with major depressive disorder (MDD). Despite the wide number of available medications, treating MDD remains an important challenge. Although fluoxetine (Flx) is the most frequently prescribed antidepressant, its mode of action is still unknown. To delineate affected molecular pathways of depressive-like behavior and identify potential targets upon Flx treatment, we performed a comparative proteomic analysis of hippocampal purified synaptic terminals (synaptosomes) of rats exposed to six weeks of CSIS, an animal model of depression, and/or followed by Flx treatment (lasting three weeks of six-week CSIS) to explore synaptic protein profile changes. Results showed that Flx in controls mainly induced decreased expression of proteins involved in energy metabolism and the redox system. CSIS led to increased expression of proteins that mainly participate in Ca2+/calmodulin-dependent protein kinase II (Camk2)-related neurotransmission, vesicle transport, and ubiquitination. Flx treatment of CSIS rats predominantly increased expression of proteins involved in synaptic vesicle trafficking (exocytosis and endocytosis), and energy metabolism (glycolytic and mitochondrial respiration). Overall, these Flx-regulated changes in synaptic and mitochondrial proteins of CSIS rats might be critical targets for new therapeutic development for the treatment of MDD.

Chronic Fluoxetine Treatment of Socially Isolated Rats Modulates Prefrontal Cortex Proteome.

Fluoxetine (Flx) is the most commonly used antidepressant to treat major depressive disorder. However, its molecular mechanisms of action are not defined as yet. A comparative proteomic approach was used to identify proteome changes in the prefrontal cortex (PFC) cytosolic and non-synaptic mitochondria (NSM)-enriched fractions of adult male Wistar rats following chronic social isolation (CSIS), a rat model of depression, and Flx treatment in CSIS and control rats, using liquid chromatography online tandem mass spectrometry. Flx reversed CSIS-induced depressive - like behavior according to preference for sucrose and immobility in the forced swim test, indicating its antidepressant effect. Flx treatment in controls led to an increase of the expression of cytosolic proteins involved in the microtubule cytoskeleton and intracellular calcium homeostasis and of enzymes involved in bioenergetic and transmembrane transport in NSM. CSIS downregulated the cytosolic proteins involved in proteasome pathway, and glutathione antioxidative system, and upregulated the expression of enzymes participating in mitochondrial-energy metabolism and transport. The presence of cytochrome c in the cytosol may suggest compromised mitochondrial membrane integrity. Flx treatment in CSIS rats downregulated protein involved in oxidative phosphorylation, such as complex III and manganese superoxide dismutase, and upregulated vesicle-mediated transport and synaptic signaling proteins in the cytosol, and neuronal calcium-binding protein 1 in NSM. Our study identified PFC modulated proteins and affected biochemical pathways that may represent potential markers/targets underlying CSIS-induced depression and effective Flx treatment, and highlights the role of protein systems involved in NSM and various metabolic pathways potentially involved in neuronal plasticity.

Metabolomic profiling relates tianeptine effectiveness with hippocampal GABA, myo-inositol, cholesterol, and fatty acid metabolism restoration in socially isolated rats.

RATIONALE: Discovering biomarkers of major depressive disorder (MDD) can give a deeper understanding of this mood disorder and improve the ability to screen for, diagnose, and treat MDD. OBJECTIVES: In this study, metabolomics was used in unraveling metabolite fluctuations of MDD and drug outcome by creating specific metabolomic fingerprints. We report metabolomic patterns of change of the hippocampus of adult male Wistar rats following chronic social isolation (CSIS) (6 weeks), an animal model of depression, and/or chronic tianeptine (Tian) treatment (10 mg kg-1 per day) (lasting 3 weeks of 6-week CSIS), monitored by using comprehensive GC × GC-MS. RESULTS: The comparative metabolomic analysis highlighted the role of gamma aminobutyric acid (GABA), iso-allocholate, and unsaturated fatty acid metabolism alterations following the CSIS, which was corroborated with moderate to strong negative Pearson's correlation of GABA, docosahexaenoic, 9-hexadecenoic acid, 5,8,11,14-eicosatetraynoic, and arachidonic acids with immobility behavior in the forced swim test. The antidepressant effect of Tian restored GABA levels, which was absent in Tian resilient rats. Tian decreased myo-inositol and increased TCA cycle intermediates, amino acids, and cholesterol and its metabolite. As key molecules of divergence between Tian effectiveness and resilience, metabolomics revealed myo-inositol, GABA, cholesterol, and its metabolite. A significant moderate positive correlation between myo-inositol and immobility was revealed. Tian probably acted by upregulating NMDAR's and α2 adrenergic receptors (AR) or norepinephrine transporter in both control and stressed animals. CONCLUSION: Metabolomics revealed several dysregulations underlying CSIS-induced depressive-like behavior and responsiveness to Tian, predominantly converging into NMDAR-mediated glutamate and myo-inositol signalization and GABA inhibitory pathways.

Tianeptine modulates synaptic vesicle dynamics and favors synaptic mitochondria processes in socially isolated rats.

Deregulation of synaptic function and neurotransmission has been linked with the development of major depression disorder (MDD). Tianeptine (Tian) has been used as antidepressant with anxiolytic properties and recently as a nootropic to improve cognitive performance, but its mechanism of action is unknown. We conducted a proteomic study on the hippocampal synaptosomal fractions of adult male Wistar rats exposed to chronic social isolation (CSIS, 6 weeks), an animal model of depression and after chronic Tian treatment in controls (nootropic effect) and CSIS-exposed rats (lasting 3 weeks of 6-week CSIS) (therapeutic effect). Increased expression of Syn1 and Camk2-related neurotransmission, vesicle transport and energy processes in Tian-treated controls were found. CSIS led to upregulation of proteins associated with actin cytoskeleton, signaling transduction and glucose metabolism. In CSIS rats, Tian up-regulated proteins involved in mitochondrial energy production, mitochondrial transport and dynamics, antioxidative defense and glutamate clearance, while attenuating the CSIS-increased glycolytic pathway and cytoskeleton organization proteins expression and decreased the expression of proteins involved in V-ATPase and vesicle endocytosis. Our overall findings revealed that synaptic vesicle dynamics, specifically exocytosis, and mitochondria-related energy processes might be key biological pathways modulated by the effective nootropic and antidepressant treatment with Tian and be a potential target for therapeutic efficacy of the stress-related mood disorders.

Fluoxetine exerts subregion/layer specific effects on parvalbumin/GAD67 protein expression in the dorsal hippocampus of male rats showing social isolation-induced depressive-like behaviour.

The molecular background of depression is intensively studied in terms of alterations of inhibitory circuits, mediated by gamma aminobutyric acid (GABA) signalization. We investigated the effects of chronic social isolation (CSIS) and chronic fluoxetine (Flx) treatment (15 mg/kg/day) (3 weeks), on Parvalbumin (PV) and GAD67 expression in a layer-specific manner in rat dorsal hippocampal subregions. CSIS-induced depressive- and anxiety-like behaviours were confirmed with decrease in sucrose preference and increase in marble burying during behavioural testing, while Flx antagonized these effects. CSIS altered PV expression in stratum pyramidale (SP) of dorsal cornu ammonis 1 (dCA1) and stratum radiatum (SR) of dCA3. Flx antagonized this effect, and boosted PV expression in SP of the entire dCA and the dorsal dentate gyrus (dDG), as well as in the SR of dCA1/CA3. CSIS showed no significant effects on GAD67 expression, while Flx boosted its expression within the SR of the entire CA and SO of the dCA3. A correlation between SP of dCA1 and SR of dCA3 with regard to PV changes, implicates their possible role in the inhibitory circuit alterations. Flx-induced increase in GAD67 expression, specifically in SR of the entire dHIPP, may impose its involvement in the cell metabolic processes. Strong negative correlation between GAD67 and sucrose preference following Flx-treatment of CSIS rats was revealed. PV + cells of the SP layer of dCA1 and CA2 could be a potential target for the antidepressant action of Flx, while strong effect of Flx on GAD67 expression in the SR should be more extensively studied.

Mapping past human land use using archaeological data: A new classification for global land use synthesis and data harmonization.

In the 12,000 years preceding the Industrial Revolution, human activities led to significant changes in land cover, plant and animal distributions, surface hydrology, and biochemical cycles. Earth system models suggest that this anthropogenic land cover change influenced regional and global climate. However, the representation of past land use in earth system models is currently oversimplified. As a result, there are large uncertainties in the current understanding of the past and current state of the earth system. In order to improve representation of the variety and scale of impacts that past land use had on the earth system, a global effort is underway to aggregate and synthesize archaeological and historical evidence of land use systems. Here we present a simple, hierarchical classification of land use systems designed to be used with archaeological and historical data at a global scale and a schema of codes that identify land use practices common to a range of systems, both implemented in a geospatial database. The classification scheme and database resulted from an extensive process of consultation with researchers worldwide. Our scheme is designed to deliver consistent, empirically robust data for the improvement of land use models, while simultaneously allowing for a comparative, detailed mapping of land use relevant to the needs of historical scholars. To illustrate the benefits of the classification scheme and methods for mapping historical land use, we apply it to Mesopotamia and Arabia at 6 kya (c. 4000 BCE). The scheme will be used to describe land use by the Past Global Changes (PAGES) LandCover6k working group, an international project comprised of archaeologists, historians, geographers, paleoecologists, and modelers. Beyond this, the scheme has a wide utility for creating a common language between research and policy communities, linking archaeologists with climate modelers, biodiversity conservation workers and initiatives.

Hippocampal synaptoproteomic changes of susceptibility and resilience of male rats to chronic social isolation.

The susceptibility of an individual to chronic social isolation (CSIS) stress may cause major depression (MD) whereby some individuals are resistant to the stress. Recent studies relate MD with altered expression of synaptic proteins in specific brain regions. To explore the neurobiological underpinnings and identify candidate biomarkers of susceptibility or resilience to CSIS, a comparative proteomic approach was used to map hippocampal synaptic protein alterations of rats exposed to 6 weeks of CSIS, an animal model of depression. This model generates two stress-response phenotypes: CSIS-sensitive (depressive-like behaviour) and CSIS-resilience assessed by means of sucrose preference and forced swim tests. Our aim was to characterize the synaptoproteome changes representative of potential long-term changes in protein expression underlying susceptibility or resilience to stress. Proteomic data showed increased expression of glycolytic enzymes, the energy-related mitochondrial proteins, actin cytoskeleton, signalling transduction and synaptic transmission proteins in CSIS-sensitive rats. Protein levels of glutamate-related enzymes such as glutamate dehydrogenase and glutamine synthetase were also increased. CSIS-resilient rats showed similar proteome changes, however with a weaker increase compared to CSIS-sensitive rats. The main difference was observed in the level of protein expression of vesicle-mediated transport proteins. Nonetheless, only few proteins were uniquely up-regulated in the CSIS-resilient rats, whereby Cytochrome b-c1 complex subunit 2, mitochondrial (Uqcrc2) and Voltage-dependent anion-selective channel protein 1 (Vdac1) were uniquely down-regulated. Identified altered activated pathways and potential protein biomarkers may help us better understand the molecular mechanisms underlying synaptic neurotransmission in MD or resilience, crucial for development of new therapeutics.

Tianeptine Enhances Energy-related Processes in the Hippocampal Non-synaptic Mitochondria in a Rat Model of Depression.

Tianeptine (Tian) has been widely used in treating mood and anxiety disorders, and recently as a nootropic to improve cognitive performance. However, its mechanisms of action are insufficiently clear. We used a comparative proteomic approach to identify sub-proteome changes in hippocampal cytosol and non-synaptic mitochondria (NSM) following chronic Tian treatment (3 weeks, 10 mg/kg/day) of adult male Wistar rats and rats exposed to chronic social isolation stress (CSIS) (6 weeks), an animal model of depression. Behavioural assessment of depressive and anxiety-like behaviours was based on sucrose preference, forced swim test and marble burying. Selected differently expressed proteins were validated by Western blot and/or immunohistochemical analysis. Tian normalized the behavioural alternations induced by CSIS, indicating its antidepressant and anxiolytic efficacy. Proteomic data showed that Tian increased the expression of proteasome system elements and redox system enzymes, enhanced energy metabolism and increased glyceraldehyde-3-phosphate dehydrogenase expression bound to NSM in control rats. Tian-treatment of CSIS-stressed rats resulted in a minor suppression of the increase in proteasome elements and antioxidative enzymes, except for an increase in Cu-Zn superoxide dismutase, and increased the level of Lactate dehydrogenase. Our results indicate on an increased NSM functionality in controls and suppression of the CSIS-induced impairment of NSM functionality by Tian treatment as well as on the CSIS-caused discrepancy in Tian effects relative to controls.

New AMS 14C dates track the arrival and spread of broomcorn millet cultivation and agricultural change in prehistoric Europe.

Broomcorn millet (Panicum miliaceum L.) is not one of the founder crops domesticated in Southwest Asia in the early Holocene, but was domesticated in northeast China by 6000 BC. In Europe, millet was reported in Early Neolithic contexts formed by 6000 BC, but recent radiocarbon dating of a dozen 'early' grains cast doubt on these claims. Archaeobotanical evidence reveals that millet was common in Europe from the 2nd millennium BC, when major societal and economic transformations took place in the Bronze Age. We conducted an extensive programme of AMS-dating of charred broomcorn millet grains from 75 prehistoric sites in Europe. Our Bayesian model reveals that millet cultivation began in Europe at the earliest during the sixteenth century BC, and spread rapidly during the fifteenth/fourteenth centuries BC. Broomcorn millet succeeds in exceptionally wide range of growing conditions and completes its lifecycle in less than three summer months. Offering an additional harvest and thus surplus food/fodder, it likely was a transformative innovation in European prehistoric agriculture previously based mainly on (winter) cropping of wheat and barley. We provide a new, high-resolution chronological framework for this key agricultural development that likely contributed to far-reaching changes in lifestyle in late 2nd millennium BC Europe.

Fluoxetine modulates neuronal activity in stress-related limbic areas of adult rats subjected to the chronic social isolation.

Antidepressant fluoxetine (Flx) is the first therapeutic choice for the treatment of major depression (MD), however neuroanatomical spots of its action remain unclear. Immunohistochemical detection of c-Fos protein expression has been used for mapping activated neuronal circuits upon various stressors and drugs. We investigated the effect of 3 weeks of Flx treatment (15 mg/kg/day) on changes in neuronal activity, by mapping the number of c-Fos+ cells, in several brain subregions in adult male rats of control and following 3 weeks of chronic social isolation (CSIS), an animal model of depression. The aim was to identify brain subregions activated by vehicle or Flx treatment in both controls or simultaneously applied with CSIS. Flx prevented depressive- and anxiety-like behaviors in CSIS rats. In controls, Flx increased the number of c-Fos+ cells in the anterior/posterior piriform cortex (aPirCx, pPirCx), retrosplenial cortex dysgranular (RSD) and granular, c region (RSGc), dorsal hippocampal subregions (CA1d, CA2, CA3d, DGd), lateral habenula (LHB), paraventricular thalamic nucleus, posterior part (PVP) and lateral/basolateral complex of amygdala (LA/BL). CSIS-induced neuronal activation was observed in brain subregions implicated in mood and other mental disorders such as aPirCx, pPirCx, caudate putamen (CPu), acumbens nucleus shell (AcbSh), RSD, RSGc, DGd, PVP and LA/BL. Flx increased neuronal activation in both controls and CSIS rats in the CA1d, CA2, CA3d, PVP, LA/BL, while in striatum increased neuronal activation was observed only in CSIS. Our data identify activated CSIS-related brain subregions and/or Flx treatment, in which Flx increased c-Fos protein expression in CSIS rats.

Social isolation stress-resilient rats reveal energy shift from glycolysis to oxidative phosphorylation in hippocampal nonsynaptic mitochondria.

AIMS: To examine the differences in the hippocampal proteome profiles of resilience or susceptibility to chronic social isolation (CSIS), animal model of depression, and to identify biomarkers that can distinguish the two. MAIN METHODS: Comparative subproteomic approach was used to identify changes in hippocampal cytosol and nonsynaptic mitochondria (NSM) of CSIS-resilient compared to CSIS-sensitive or control rats. The resilient and sensitive phenotypes of CSIS rats were distinguished based on their sucrose preference values. Selected proteins were validated by Western blot or immunofluorescence. KEY FINDINGS: Predominantly down-regulated processes such as cytosolic cytoskeleton organization, the calcium signaling pathway, ubiquitin proteasome degradation, redox system, malate/aspartate shuttling and glutamate metabolism in CSIS-resilient compared to CSIS-sensitive rats were found. Decreased protein expression of glycolytic enzymes with simultaneous increased expression of Aco2 involved in tricarboxylic acid cycle and expression of several subunits composing oxidative phosphorylation involved enzymes (Uqcrc2, Atp5f1a, Atp5f1b) were found, indicating shift in energy production from glycolysis to oxidative phosphorylation in NSM. The four-fold higher level of mitochondrial glyceraldehyde-3-phosphate dehydrogenase of resilient rats indicated its transfer from the cytosol to the NSM. An increased level of transketolase along with the reduced pyruvate kinase level suggested an activated pentose phosphate pathway in CSIS-resilient relative to control rats. Cytosolic up-regulated CSIS proteins were implicated in antioxidative and proteasomal systems, while down-regulated NSM protein was involved in oxidative phosphorylation. SIGNIFICANCE: The identified altered activated pathways and potential biomarkers enhance understanding of molecular mechanisms underlying resilience or susceptibility to CSIS, crucial in developing new therapeutic strategies.

Clozapine increased c-Fos protein expression in several brain subregions of socially isolated rats.

Chronic social stress and/or pharmacological treatments differentially modulate the expression of c-Fos, a marker of neuronal activity, in subregions of the rat brain. Here, we examined the effect of the atypical antipsychotic Clozapine (Clz) (20 mg/kg/day for 3 weeks) on the neuronal activation pattern of c-Fos protein expression in stress-relevant brain subregions of adult male Wistar rats exposed to chronic social isolation (CSIS: 3 weeks), an animal model of depression and schizophrenia, and controls. The protein expression of c-Fos was also used to map neuronal populations in brain subregions activated by CSIS alone. Subregions which showed significantly increased c-Fos protein expression following CSIS included the retrosplenial cortex (RSC), (subregions:RSC granular cortex, c region (RSGc) and dysgranular (RSD)), dentate gyrus, dorsal (DGd), paraventricular thalamic nucleus, posterior part (PVP), lateral (LA)/basolateral (BL) complex of amygdala, caudate putamen (CPu) and accumbens nucleus, shell (AcbSh). Increases in c-Fos protein expression in the RSGc, RSD, DGd, PVP, LA/BL complex of amygdala and striatum (CPu, Acb Core (AcbC) and AcbSh) following Clz treatment in controls were found. Clz applied simultaneously with CSIS modulated neuronal activity in CPu, AcbC and AcbSh subregions compared to CSIS alone, increasing c-Fos protein expression. Furthermore, Clz revealed synergistic effects with CSIS in the CA1d and PVP. These identified neural circuits reflect brain subregions activated following CSIS and/or Clz administration. These data further contribute to the understanding of the effectiveness of Clz in the modulation of brain subregion activation in response to CSIS.

Brain Sub/Region-Specific Effects of Olanzapine on c-Fos Expression of Chronically Socially Isolated Rats.

Olanzapine (Olz) is an atypical antipsychotic used to treat depression, anxiety and schizophrenia, which can be caused by chronic psychosocial stress. c-Fos protein expression has been used as an indirect marker of neuronal activity in response to various forms of stress or pharmacological treatments. We examined the effects of a 3-week treatment of Olz (7.5 mg/kg/day) on c-Fos protein expression in stress-relevant brain sub/regions, its relationship with isolation-induced behavioral changes, and potential sites of Olz action on control and male rats exposed to 6 weeks of chronic social isolation (CSIS), an animal model of depression. Olz treatment reversed depression- and anxiety-like behaviors induced by CSIS and suppressed a CSIS-induced increase in the number of c-Fos-positive cells in subregions of the dorsal hippocampus, ventral (v) DG, retrosplenial cortex, and medial prefrontal cortex. In contrast, no change in c-Fos expression was seen in the CA3v, amygdala and thalamic, hypothalamic or striatal subregions in Olz-treated CSIS rats, suggesting different brain sub/regions' susceptibility to Olz. An increased number of c-Fos-positive cells in the CA1v, amygdala and thalamic, hypothalamic and striatal subregions in controls as well as in the CA1v and subregion of the hypothalamus and nucleus accumbens in Olz-treated CSIS rats was found. Results suggest the activation of brain sub/regions following CSIS that may be involved in depressive and anxiety-like behaviors. Olz treatment showed region-specific effects on neuronal activation. Our data contribute to a better understanding of the mechanisms underlying the CSIS response and potential brain targets of Olz in socially isolated rats.

Tianeptine antagonizes the reduction of PV+ and GAD67 cells number in dorsal hippocampus of socially isolated rats.

Adult male rats exposed to chronic social isolation (CSIS) show depressive- and anxiety-like behaviors and reduce the numbers of parvalbumin-positive (PV+) interneurons in the dorsal hippocampus. We aimed to determine whether tianeptine (Tian), administered during the last three weeks of a six-week-social isolation (10 mg/kg/day), may reverse CSIS-induced behavioral changes and antagonize the CSIS-induced reduction in the number of PV+ interneurons. We also studied whether Tian affects the GABA-producing enzyme GAD67+ cells, in Stratum Oriens (SO), Stratum Pyramidale (SP), Stratum Radiatum (SR) and Stratum Lacunosum Moleculare (LM) of CA1-3, as well as in molecular layer-granule cell layer (ML-GCL) and Hilus (H) of the dentate gyrus (DG). CSIS-induced reduction in the number of PV+ cells was layer/subregion-specific with the greatest decrease in SO of CA2. Reduction in the number of PV+ cells was significantly higher than GAD67+ cells, indicating that PV+ cells are the main target following CSIS. Tian reversed CSIS-induced behavior phenotype and antagonized the reduction in the number of PV+ and GAD67+ cells in all subregions. In controls, Tian led to an increase in the number of PV+ and GAD67+ cells in SP of all subregions and PV+ interneurons in ML-GCL of DG, while treatment during CSIS, compared to CSIS alone, resulted with an increase of PV+ interneurons in SO and SP CA1, SP CA2/CA3 and ML-GCL DG with simultaneous increase in GAD67+ cells in all CA1, LM CA2, SO/SR/LM CA3. Data show that Tian offers protection from CSIS via modulation of the dorsal hippocampal GABAergic system.