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Contains fulltext : 125219.pdf (publisher's version ) (Open Access), BACKGROUND: Growing evidence suggests that alterations of the inflammatory/immune system contribute to the pathogenesis of depression. Indeed, depressed patients exhibit increased levels of inflammatory markers in both the periphery and the brain, and high comorbidity exists between major depression and diseases associated with inflammatory alterations. In order to characterize the link between depression and inflammation, we aimed to investigate whether an altered inflammatory system is present in a genetic model of vulnerability for depression, namely rats with partial or total deletion of the serotonin transporter (SERT) gene. METHODS: Wild-type, heterozygous and homozygous SERT rats were analyzed under basal condition or following a challenge with an acute injection of lipopolysaccharide (LPS) and killed 24 h or 5 days later. RESULTS: We found that SERT mutant rats show altered cytokine expression in the dorsal and ventral hippocampus at basal conditions, and they also display an exacerbated cytokine response to the LPS challenge. Moreover, mutant rats exhibit differences in the expression of markers for microglia activation. CONCLUSION: Based on these data, we suggest that basal or functional alterations of immune/inflammatory systems might contribute to the phenotype of SERT rats and to their heightened susceptibility to depressive-like behavior.

Contains fulltext : 125556.pdf (publisher's version ) (Open Access), Second-generation antipsychotics (SGA) are multi-target agents widely used for the treatment of schizophrenia and bipolar disorder that also hold potential for the treatment of impaired emotional control, thanks to their diverse receptor profiles as well as their potential in modulating neuroadaptive changes in key brain regions. The aim of this study was thus to establish the ability of lurasidone, a novel SGA characterized by a multi-receptor signature, to modulate behavioural and molecular defects associated with a genetic model of impaired emotional control, namely serotonin transporter knockout (SERT KO) rats. At behavioural level, we found that chronic lurasidone treatment significantly increased fear extinction in SERT KO rats, but not in wild-type control animals. Moreover, at molecular level, lurasidone was able to normalize the reduced expression of the neurotrophin brain-derived neurotrophic factor in the prefrontal cortex of SERT KO rats, an effect that occurred through the regulation of specific neurotrophin transcripts (primarily exon VI). Furthermore, chronic lurasidone treatment was also able to restore the reduced expression of different GABAergic markers that is present in these animals. Our results show that lurasidone can improve emotional control in SERT KO rats, with a primary impact on the prefrontal cortex. The adaptive changes set in motion by repeated treatment with lurasidone may in fact contribute to the amelioration of functional capacities, closely associated with neuronal plasticity, which are deteriorated in patients with schizophrenia, bipolar disease and major depression.

Item does not contain fulltext, It is well established that alterations of the serotoninergic system may contribute to the pathophysiology of mood disorders. Accordingly, it has been demonstrated that genetic deletion of the serotonin transporter (SERT) in rodents leads to an anxious and depressive phenotype, which is also associated with reduced neuronal plasticity. Indeed, we have demonstrated that adult SERT(-/-) animals show decreased brain-derived neurotrophic factor (BDNF) expression, as well as reduced levels of transcription factor regulating the neurotrophin transcription. While these changes may represent long-term consequences of impaired function of the transporter during development, no information exists with respect to the developmental profile of such changes. Using SERT(-/-) rats at different ages, we found that the impairment in neuroplasticity originates early in development and worsens during the first 3 weeks of life. Indeed, we observed that BDNF expression was reduced at birth and that the magnitude of these changes became more pronounced starting from PND21, being sustained by epigenetic mechanisms as well as alterations in the expression of specific transcription factors, including Npas4 and CaRF. These results suggest that an impairment of SERT may affect BDNF expression throughout postnatal development. These early changes may increase stress susceptibility during critical windows of brain maturation, which may eventually lead to the heightened predisposition to mood disorders found in individual carrying genetic variants of the serotonin transporter.

Background: Growing compelling evidence from clinical and preclinical studies has demonstrated the primary role of alterations of glutamatergic transmission in cortical and limbic areas in the pathophysiology of mood disorders. Chronic antidepressants have been shown to dampen endogenous glutamate release from rat hippocampal synaptic terminals and to prevent the marked increase of glutamate overflow induced by acute behavioral stress in frontal/prefrontal cortex. Agomelatine, a new antidepressant endowed with MT1/MT2 agonist and 5-HT2C serotonergic antagonist properties, has shown efficacy at both preclinical and clinical levels.Results: Chronic treatment with agomelatine, or with the reference drug venlafaxine, induced a marked decrease of depolarization-evoked endogenous glutamate release from purified hippocampal synaptic terminals in superfusion. No changes were observed in GABA release. This effect was accompanied by reduced accumulation of SNARE protein complexes, the key molecular effector of vesicle docking, priming and fusion at presynaptic membranes.Conclusions: Our data suggest that the novel antidepressant agomelatine share with other classes of antidepressants the ability to modulate glutamatergic transmission in hippocampus. Its action seems to be mediated by molecular mechanisms located on the presynaptic membrane and related with the size of the vesicle pool ready for release.

Item does not contain fulltext, Alterations of the serotonergic system are involved in the pathophysiology of mood disorders and represent an important target for its pharmacological treatment. Genetic deletion of the serotonin transporter (SERT) in rodents leads to an anxious and depressive phenotype, and is associated with reduced neuronal plasticity as indicated by decreased brain-derived neurotrophic factor (Bdnf) expression levels. One of the transcription factors regulating Bdnf is the neuronal PAS domain protein 4 (Npas4), which regulates activity-dependent genes and neuroprotection, and has a critical role in the development of GABA synapses. On the basis of these premises, we investigated the expression of Npas4 and GABAergic markers in the hippocampus and prefrontal cortex of homozygous (SERT(-/-)) and heterozygous (SERT(+/-)) knockout rats, and analyzed the effect of long-term duloxetine treatment on the expression of these targets. We found that Npas4 expression was reduced in both the brain structures of adult SERT(+/-) and SERT(-/-) animals. This effect was already present in adolescent SERT(-/-), and could be mimicked by prenatal exposure to the antidepressant fluoxetine. Moreover, SERT(-/-) rats showed a strong impairment of the GABAergic system, as indicated by the reduction of several markers, including the vesicular transporter (Vgat), glutamic acid decarboxylase-67 (Gad67), the receptor subunit GABA A receptor, gamma 2 (GABA(A)-gamma2), and calcium-binding proteins that label subgroups of the GABAergic neurons. Interestingly, chronic treatment with the antidepressant duloxetine was able to restore the physiological levels of Npas4 and GABAergic markers in SERT(-/-) rats, although some differences in the modulation of GABAergic genes exist between hippocampus and prefrontal cortex. Our results demonstrate that SERT knockout rats, an animal model of mood disorders, have reduced Npas4 expression that correlates with decreased expression of Bdnf exon I and IV. These changes lead to

Stressful life events impact on memory, cognition and emotional responses, and are known to precipitate mood/anxiety disorders. It is increasingly recognized that stress and its neurochemical and endocrine mediators induce changes in glutamate synapses and circuitry, and this in turn modify mental states. Half a century after the monoamine hypothesis, it is widely accepted that maladaptive changes in excitatory/inhibitory circuitry have a primary role in the pathophysiology of mood/anxiety disorders. The neuroplasticity hypothesis posits that volumetric changes consistently found in limbic and cortical areas of depressed subjects are in good part due to remodeling of neuronal dendritic arbors and loss of synaptic spines. A considerable body of work, carried out with in vivo microdialysis as well as alternative methodologies, has shown that both stress and corticosterone treatment induce enhancement of activity-dependent glutamate release. Accordingly, results from preclinical studies suggest that stress- and glucocorticoid-induced enhancement of glutamate release and transmission plays a main role in the induction of maladaptive cellular effects, in turn responsible for dendritic remodeling. Additional recent work has showed that drugs employed for therapy of mood/anxiety disorders (antidepressants) prevent the enhancement of glutamate release induced by stress. Understanding the action of traditional drugs on glutamate transmission could be of great help in developing drugs that may work directly at this level.

Objectives. The association between depression and circadian rhythm disturbances is well established and successful treatment of depressed patients is accompanied by restoration of circadian rhythms. The new antidepressant agomelatine is an agonist of melatonergic MT(1)/MT(2) receptors as well as an antagonist of serotonergic 5-HT(2C) receptors. Animal studies showed that agomelatine resynchronizes disturbed circadian rhythms and reduces depression-like behaviour. Methods. This review analyzes results from different experimental studies. Results. Recent data on the effects of agomelatine on cellular processes involved in antidepressant mechanisms have shown that the drug is able to increase the expression of brain-derived neurotrophic factor in prefrontal cortex and hippocampus, as well as the expression of activity-regulated cytoskeleton associated protein (Arc) in the prefrontal cortex. In line with this, prolonged treatment with agomelatine increases neurogenesis within the hippocampus, particularly via enhancement of neuronal cell survival. Agomelatine attenuates stress-induced glutamate release in the prefrontal/frontal cortex. Treatment with 5-HT(2C) antagonists or melatonin alone failed to reproduce these effects. Conclusions. The unique mode of action of agomelatine may improve the management of major depression by counteracting the pathogenesis of depression at cellular level, thereby relieving the symptoms of depression. These effects are suggested to be due to a synergistic action on MT(1)/MT(2) and 5-HT(2C) receptors.

Background/Aims: Compelling evidence would suggest the involvement of the serotonin 2C receptor in the pathophysiology of affective disorders and in the action of antidepressants. We analyzed the time course of 5-HT2C receptor (5-HTR2C) mRNA expression during antidepressant treatment in the prefrontal/frontal cortex (P/FC) and in the hippocampus (HC) of rats chronically treated with fluoxetine (a selective serotonin reuptake inhibitor) and reboxetine (a selective noradrenaline reuptake inhibitor). We also analyzed the 5-HTR2C RNA-editing levels at the sites called A, B, C, C' and D, which are known to modulate 5-HTR2C receptor function. Results: The expression profile of 5-HTR2C mRNA was modified during treatment with both antidepressants. In particular, we found a general down-regulation of 5-HTR2C mRNA expression in P/FC, which became significant after 3 weeks of treatment with both antidepressants and persisted after a fourth week of drug withdrawal (-46% with fluoxetine, -41% with reboxetine, p < 0.05). In HC, however, reboxetine induced significant down-regulation (-56%, p < 0.05) of 5-HTR2C mRNA after 3 weeks, while fluoxetine induced threefold up-regulation (p < 0.01) by the 2nd and 3rd week, returning to the base level after drug withdrawal of both antidepressants. Moreover, the frequency of 5-HTR2C-edited isoforms showed no significant alterations, although analysis of the RNA-editing level at the single editing sites showed small decreases in the C' and D sites induced by reboxetine in P/FC. Conclusion: Our results suggest that chronic administration of antidepressants in rats slightly modifies the editing levels of 5-HT2C receptor but has considerable influence on its mRNA expression patterns in a way that is area- and time-specific.

1 maart 2010, Contains fulltext : 89453.pdf (Publisher’s version ) (Closed access), In order to identify the molecular mechanisms that may contribute to the enhanced susceptibility to depression under serotonin transporter (SERT) dysfunction, we analyzed the expression of brain-derived neurotrophic factor (BDNF), a key player in neuronal plasticity, which is implicated in the etiology and treatment of depression. We found that BDNF levels were significantly reduced in the hippocampus and prefrontal cortex of SERT knockout rats, through transcriptional changes that affect different neurotrophin isoforms. The reduction of BDNF gene expression observed in prefrontal cortex is due, at least in part, to epigenetic changes that affect the promoter regions of exons IV and VI. Moreover, BDNF gene expression is also significantly reduced in leukocytes from healthy subjects carrying the S allele of the 5-HTTLPR, suggesting that the changes observed in SERT mutant rats may also be present in humans and may confer enhanced vulnerability to mood disorders.

Background. Agomelatine is a melatonergic receptor agonist and a 5HT2C receptor antagonist that has shown ntidepressant efficacy. In order to analyze separately the effect of the two receptorial components, rats were chronically treated with agomelatine, melatonin (endogenous melatonergic agonist), or S32006 (5-HT2C antagonist), and then subjected to acute footshock-stress. Results. Only chronic agomelatine, but not melatonin or S32006, completely prevented the stress-induced increase of glutamate release in the rat prefrontal/frontal cortex. Conclusions. These results suggest a potential synergy between melatonergic and serotonergic pathways in the action of agomelatine.

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.

BACKGROUND: Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release. METHODOLOGY/FINDINGS: Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability. CONCLUSIONS/SIGNIFICANCE: Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of

Stress has been shown to interact with genetic vulnerability in pathogenesis of psychiatric disorders. Here we investigated the outcome of interaction between genetic vulnerability and early-life stress, by employing a rodent model that combines an inherited trait of vulnerability in Flinders Sensitive Line (FSL) rats, with early-life stress (maternal separation). Basal differences in synaptic signaling between FSL rats and their controls were studied, as well as the consequences of early-life stress in adulthood, and their response to chronic antidepressant treatment (escitalopram). FSL rats showed basal differences in the activation of synapsin I and Erk1/2, as well as in alphaCaM kinase II/syntaxin-1 and alphaCaM kinase II/NMDA-receptor interactions in purified hippocampal synaptosomes. In addition, FSL rats displayed a blunted response of Erk-MAP kinases and other differences in the outcome of early-life stress in adulthood. Escitalopram treatment restored some but not all alterations observed in FSL rats after early-life stress. The marked alterations found in key regulators of presynaptic release/neurotransmission in the basal FSL rats, and as a result of early-life stress, suggest synaptic dysfunction. These results show that early gene-environment interaction may cause life-long synaptic changes affecting the course of depressive-like behavior and response to drugs.

Contains fulltext : 79962.pdf (publisher's version ) (Closed access), A gene variant in the human serotonin transporter (SERT) can increase the vulnerability to mood disorders. SERT knockout animals show similarities to the human condition and represent an important tool to investigate the mechanisms underlying the pathologic condition in humans. Along this line of thinking, we used SERT KO rats (SERT(+/-) and SERT(-/-)) to investigate abnormalities in the expression and function of the activity-regulated gene Arc (Activity-regulated cytoskeletal associated protein) and the early inducible gene Zif-268, (zinc finger binding protein clone 268), which are important players in neuronal plasticity. We found lower basal Arc mRNA levels in hippocampus and prefrontal cortex of mutant rats in comparison with wild-type animals. Moreover SERT mutant rats show altered stress responsiveness. Indeed an acute swim stress significantly up-regulated the levels of Arc mRNA in hippocampus and prefrontal cortex, as well as of Zif-268 in frontal cortex, only in SERT(+/-) and SERT(-/-) rats. These alterations may be associated to behavioral traits linked to SERT and may contribute to the neuroplastic and morphological changes observed in depression.

Background: The neurotrophin BDNF has been implicated in the regulation of neuroplasticity, gene expression, and synaptic function in the adult brain, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants. Antidepressant treatments have been shown to increase the expression of BDNF mRNA, although the changes measured were found to be different depending on various factors. A few studies only have measured levels of BDNF protein after antidepressant treatments, and poor correlation was found between mRNA and protein changes. We studied the time course of expression of BDNF mRNA and protein during drug treatments, in order to elucidate the temporal profile of regulation of this effector and whether mRNA and protein levels correlate. Rat groups were treated for 1, 2 or 3 weeks with fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Total BDNF mRNA was measured by Real Time PCR, pro- and mature BDNF proteins were measured by Western blot. Results: We found that mature BDNF protein is induced more rapidly than mRNA, by both drugs in hippocampus (weeks 1-2) and by reboxetine in prefrontal/frontal cortex (week 1). The temporal profile of BDNF protein expression was largely inconsistent with that of mRNA, which followed the protein induction and reached a peak at week 3. Conclusion: These results suggest that BDNF protein is rapidly elevated by antidepressant treatments by posttranscriptional mechanisms, and that induction of BDNF mRNA is a slower process.

Depression is a severe and life-threatening psychiatric illness whose pathogenesis is still essentially unknown. Proteomic analysis of synaptic terminals (synaptoproteomics) in animal models of depression is a powerful approach to gain insight into the molecular mechanisms underlying vulnerability to mood disorders and the long-term action of drug treatments. Here, we employed two different animal models of depression, the Learned Helplessness rats (a classical behavioral model of depression) and a new model of depression with gene - environment interaction (Flinders Sensitive Line rats subjected to early life stress). Both animal models were treated with the antidepressant escitalopram. Analysis of their synaptoproteomic profile revealed a number of protein spots differently regulated by basic vulnerability and/or early life stress. Using this approach, we obtained information regarding biomarkers that may represent predictors of pathology or response/resistance to drug treatment, as well as potential targets for novel pharmacological and therapeutic strategies.

An animal model of depression combining genetic vulnerability and early-life stress (ELS) was prepared by submitting the Flinders Sensitive Line (FSL) rats to a standard paradigm of maternal separation. We analysed hippocampal synaptic transmission and plasticity in vivo and ionotropic receptors for glutamate in FSL rats, in their controls Flinders Resistant Line (FRL) rats, and in both lines subjected to ELS. A strong inhibition of long-term potentiation (LTP) and lower synaptic expression of NR1 subunit of the NMDA receptor were found in FSL rats. Remarkably, ELS induced a remodelling of synaptic plasticity only in FSL rats, reducing inhibition of LTP; this was accompanied by marked increase of synaptic NR1 subunit and GluR2/3 subunits of AMPA receptors. Chronic treatment with escitalopram inhibited LTP in FRL rats, but this effect was attenuated by prior ELS. The present results suggest that early gene-environment interactions cause lifelong synaptic changes affecting functional and molecular aspects of plasticity, partly reversed by antidepressant treatments.

Converging evidence points to adaptive changes in neuroplasticity and gene expression as mediators of therapeutic action of antidepressants. Activation of cAMP response-element binding protein (CREB) and CREB-regulating signalling are considered main effectors in these mechanisms. We analysed the temporal profile of intracellular changes induced by antidepressants, by measuring activation of major CREB-regulating signalling cascades and activation (Ser133 phosphorylation) of CREB. The main aims of the study were to investigate how these different variables are modulated with time, whether stronger activation of signalling cascades corresponds to stronger activation of CREB, and whether these changes are different in distinct brain areas. Rat groups were treated for 1, 2 or 3 wk with the antidepressants fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Activation of CREB and major effectors in signalling cascades were analysed by Western blot analysis with phospho-antibodies, in nuclear and cytosolic fractions from hippocampus and prefrontal/frontal cortex (P/FC). Surprisingly, CREB activation was already maximal after 1-wk treatment. In hippocampus early and stronger CREB activation was consistent with early and stronger activation of signalling. For both drugs, the profile of activation in P/FC was different from that observed in hippocampus. The results also showed that, contrary to the activatory role of MAP-ERKs and CaM kinase IV, nuclear alphaCaM kinase II was inactivated in parallel with activation of CREB.

BACKGROUND: Recent rodent studies reported that antidepressant treatments affect the expression of brain-derived neurotrophic factor (BDNF) mRNA in a way that is dependent on treatment duration, by selective modulation of different BDNF transcripts. However, no data are available for the human BDNF gene. We studied the effect of different antidepressants on BDNF mRNA expression in human neuroblastoma SH-SY5Y cells. RESULTS: Cultured cells were treated with the antidepressants fluoxetine, reboxetine and desipramine for different time lengths (6, 24, 48 hours). Expression of total BDNF mRNA was analyzed by reverse transcription PCR and levels of different BDNF transcripts were detected by hemi-nested PCR with specific primers. Short-term treatment (6 hours) with reboxetine or desipramine reduced total BDNF, whereas long-term treatment (48 hours) significantly increased total BDNF mRNA levels. These changes were accounted for by differential regulation of BDNF IV and VIa/b transcripts. Fluoxetine showed no significant effects. CONCLUSION: This is the first study showing biphasic changes in the expression of total and specific BDNF transcripts in human cells following antidepressant treatments. These findings suggest that biphasic induction of BDNF by antidepressants could be a feature common to rodents and humans and encourage the use of SH-SY5Y cells as a tool for investigation of drug effects on human genes.

Changes in synaptic plasticity are involved in pathophysiology of depression and in the mechanism of antidepressants. Ca2+/calmodulin (CaM) kinase II, a protein kinase involved in synaptic plasticity, has been previously shown to be a target of antidepressants. We previously found that antidepressants activate the kinase in hippocampal neuronal cell bodies by increasing phosphorylation at Thr286, reduce the kinase phosphorylation in synaptic membranes, and in turn its phosphorylation-dependent interaction with syntaxin-1 and the release of glutamate from hippocampal synaptosomes. Here, we investigated the chronic effect of different antidepressants (fluoxetine, desipramine, and reboxetine) on the expression and function of the kinase in distinct subcellular compartments in order to dissect the different kinase pools affected. Acute treatments did not induce any change in the kinase. In total tissue extracts chronic drug treatments induced activation of the kinase; in hippocampus (HC), but not in prefrontal/frontal cortex, this was partially accounted for by increased Thr286 phosphorylation, suggesting the involvement of different mechanisms of activation. In synaptosomes, all drugs reduced the kinase phosphorylation, particularly in HC where, upon fractionation of the synaptosomal particulate into synaptic vesicles and membranes, we found that the drugs induced a redistribution and differential activation of the kinase between membranes and vesicles. Furthermore, a large decrease in the level and phosphorylation of synapsin I located at synaptic membranes was consistent with the observed decrease of CaM kinase II. Overall, antidepressants induce a complex pattern of modifications in distinct subcellular compartments; at presynaptic level, these changes are in line with a dampening of glutamate release