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Inflammatory activation within the brain is linked to a decrease in cognitive abilities; however, the molecular mechanisms implicated in the development of inflammatory-related cognitive dysfunction and its prevention are poorly understood. This study compared the responses of hippocampal transcriptomes 3 months after the striatal infusion of lipopolysaccharide (LPS; 30 µg), resulting in memory loss, or with dexamethasone (DEX; 5 mg/kg intraperitoneal) pretreatment, which abolished the long-term LPS-induced memory impairment. After LPS treatment, a significant elevation in the expression of immunity/inflammatory-linked genes, including chemokines ( Cxcl13 ), cytokines ( Il1b and Tnfsf13b ), and major histocompatibility complex (MHC) class II members ( Cd74 , RT1-Ba , RT1-Bb , RT1-Da , and RT1-Db1 ) was observed. DEX pretreatment did not change the expression of these genes, but significantly affected the expression of genes encoding ion channels, primarily calcium and potassium channels, regulators of glutamate ( Slc1a2 , Grm5 , Grin2a ), and GABA ( Gabrr2 , Gabrb2 ) neurotransmission, which enriched in such GO biological processes as "Regulation of transmembrane transport", "Cognition", "Learning", "Neurogenesis", and "Nervous system development". Taken together, these data suggest that (1) pretreatment with DEX did not markedly affect LPS-induced prolonged inflammatory response; (2) DEX pretreatment can affect processes associated with glutamatergic signaling and nervous system development, possibly involved in the recovery of memory impairment induced by LPS.

Among the responses in the early stages of stroke, activation of neurodegenerative and proinflammatory processes in the hippocampus is of key importance for the development of negative post-ischemic functional consequences. However, it remains unclear, what genes are involved in these processes. The aim of this work was a comparative study of the expression of genes encoding glutamate and GABA transporters and receptors, as well as inflammation markers in the hippocampus one day after two types of middle cerebral artery occlusion (according to Koizumi et al. method, MCAO-MK, and Longa et al. method, MCAO-ML), and direct pro-inflammatory activation by central administration of bacterial lipopolysaccharide (LPS). Differences and similarities in the effects of these challenges on gene expression were observed. Expression of a larger number of genes associated with activation of apoptosis and neuroinflammation, glutamate reception, and markers of the GABAergic system changed after the MCAO-ML and LPS administration than after the MCAO-MK. Compared with the MCAO-ML, the MCAO-MK and LPS challenges caused changes in the expression of more genes involved in glutamate transport. The most pronounced difference between the responses to different challenges was the changes in expression of calmodulin and calmodulin-dependent kinases genes observed after MCAO, especially MCAO-ML, but not after LPS. The revealed specific features of the hippocampal gene responses to the two types of ischemia and a pro-inflammatory stimulus could contribute to further understanding of the molecular mechanisms underlying diversity of the post-stroke consequences both in the model studies and in the clinic.

We performed transcriptome analysis in the hippocampus 24 h after lipopolysaccharide (LPS) administration. We observed glial-specific genes, comprised of two-thirds of all differentially expressed genes (DEGs). We found microglial DEGs that were the most numerous in LPS group. On the contrary, differential alternative splicing (DAS) analysis revealed the most numerous DAS events in astrocytes. Besides, we observed distinct major isoform switching in the Ptbp1 gene, with skipping of exon 8 in LPS group. Ptbp1 usually considered a pluripotency sustaining agent in brain embryonic development, according to the previous studies. Analyzing the splicing tune-up upon LPS exposure, we came to a supposition that the short Ptbp1 isoform de-represses immune-specific response by Ptbp1 adjusted splicing architecture. Additionally, the Ptbp3 ( NOD1 ) immune-specific splicing factor has apparently been de-repressed by the Ptbp1 short isoform in glial cells. Notably, both the Ptbp1 and Ptbp3 genes express primarily in microglial/endothelial brain cells. We also report immune-related genes, altering their major isoforms upon LPS exposure. The results revealed immune modulating role of alternative splicing in brain.

Acute cerebral ischemia induces distant inflammation in the hippocampus; however, molecular mechanisms of this phenomenon remain obscure. Here, hippocampal gene expression profiles were compared in two experimental paradigms in rats: middle cerebral artery occlusion (MCAO) and intracerebral administration of lipopolysaccharide (LPS). The main finding is that 10 genes ( Clec5a, CD14, Fgr, Hck, Anxa1, Lgals3, Irf1, Lbp, Ptx3, Serping1 ) may represent key molecular links underlying acute activation of immune cells in the hippocampus in response to experimental ischemia. Functional annotation clustering revealed that these genes built the same clusters related to innate immunity/immunity/innate immune response in all MCAO differentially expressed genes and responded to the direct pro-inflammatory stimulus group. The gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses also indicate that LPS-responding genes were the most abundant among the genes related to "positive regulation of tumor necrosis factor biosynthetic process", "cell adhesion", "TNF signaling pathway", and "phagosome" as compared with non-responding ones. In contrast, positive and negative "regulation of cell proliferation" and "HIF-1 signaling pathway" mostly enriched with genes that did not respond to LPS. These results contribute to understanding genomic mechanisms of the impact of immune/inflammatory activation on expression of hippocampal genes after focal brain ischemia.

Ischemic brain injuries are accompanied by the long-term changes in gene expression in the hippocampus, the limbic system structure, involved in the regulation of key aspects of the higher nervous activity, such as cognitive functions and emotions. The altered expression of genes and proteins encoded by them may be related to the development of post-ischemic psycho-emotional and cognitive disturbances. Activation of neuroinflammation following stroke in the hippocampus has been suggested to play an essential role in induction of long-lasting consequences. Identification of changes in the gene expression patterns after ischemia and investigation of the dynamics of these changes in the hippocampus are the necessary first steps toward understanding molecular pathways responsible for the development of post-stroke cognitive impairments and mental pathologies.

The review briefly summarizes experimental and preclinical data of the role of pro-inflammatory cytokines in triggering pathophysiological changes associated with depression, primarily major depressive disorder (MDD), as well as the possibility of using anti-inflammatory drugs as antidepressants.

Pathways by which inflammatory stimuli influence behaviors can involve changes in neuronal plasticity, however, the evidence for this is still insufficient. This study aimed to evaluate the effects of chronic lipopolysaccharide (LPS) injected alone or together with tetracycline antibiotic doxycycline (Dox) on the levels of Iba-1, BDNF, Bcl-xL and MMP-9 in brain regions in relation to stress-induced behaviors in the elevated plus-maze (EPM). LPS injected to adult rats every 2 days for a total of 7 injections reduced body weight gain, increased spleen and adrenal weights, decreased locomotor activity, and increased anxiety-like behavior. These effects were associated with increased expression of Iba-1, a well-known marker for activated microglia, in most brain regions investigated. Co-treatment of LPS with Dox attenuated LPS-induced microglial activation and behavioral changes, supporting their relation to the neuroinflammation. LPS administration also produced pro-apoptotic changes in the brain. In the hypothalamus and striatum, the levels of anti-apoptotic protein Bcl-xL were decreased, whereas in the amygdala, a significant increase in MMP-9 protein levels was observed. The levels of Iba-1 as well as MMP-9 in the amygdala positively correlated with the numbers of defecation. The data suggest that mechanisms of anxiety associated with neuroinflammation may involve the increase in MMP-9 levels in the amygdala.

Chronic stress can predispose vulnerable individuals to mood disorders, including depression. Glutamate, one of the key participants in this process, may exert both pathological and therapeutic psycho-emotional effects. However, the role of expression of genes encoding proteins that provide glutamatergic signal is still unclear. In this study, we attempted to distinguish changes in expression of glutamatergic genes associated with stress-induced anhedonia, a core symptom of depression, from those related to other stress-related effects. For this, expression of genes was compared between rats after a short-term stress, which did not yet cause depressive-like symptoms, and animals exposed chronically to different stressors that produce anhedonia-like responses. The changes in gene expression induced by chronic restraint or forced swimming concomitantly with anhedonia development demonstrated similar for both stressors patterns. Main features of the expression patterns include the decrease in mRNA levels for AMPA and NMDA subunits in the midbrain and hippocampus that is consistent with the hypothesis that "monoamine (serotonin)-Glutamate/GABA long neural circuit" involved in mood regulation. The decrease in expression of these subunits in the midbrain may attenuate glutamatergic drive on the serotonergic neurons promoting a shift of excitation/inhibition balance between glutamate and GABA in the forebrain regions resulting in anhedonia. In general, changes in expression of multiple genes involved in glutamatergic neurotransmission in the forebrain and brainstem regions suggest that stress-induced anhedonia may result from the network dysfunction of this neurotransmitter system.

Abstract-Chemogenetic activation of glutamatergic neurons of the prefrontal cortex reduces the manifestations of psychoemotional anxiety during the juvenile period of ontogenesis. This result is the first evidence of feasibility of targeted chemogenetic control of neuronal activity during the early stages of brain development.

Doxycycline (Dox)-inducible transgenic approach is used to examine the neural mechanisms of anxiety and depression; however, its own effects on related behaviors are not clear. To address this, in the present study, we tested the anxiety- and depression-like behaviors in rats treated with Dox in drinking water (2 mg/ml) in the elevated plus-maze (EPM; on day 5) and forced swim (FST; on day 8) tests, respectively. In addition, the levels of mRNAs and proteins of brain-derived neurotrophic factor (BDNF) and anti-apoptotic protein Bcl-xL in the hippocampus (HIPP) and frontal cortex (FC) were also analyzed. Consumption of Dox for 4 days induced an anxiogenic-like phenotype that was manifested by the decreased percentages of open arm entries and time spent on the open arms of the EPM. After Dox for 7 days, animals demonstrated more active behavior in the FST than control rats as evidenced by the increase in climbing time. When assessed after the FST, expression of Bcl-xL was increased in the hippocampus of Dox-treated animals. Furthermore, hippocampal Bcl-xL content correlated positively with the duration of climbing in the test. This study is the first to find that Dox in treatment regime used to control transgene expression can affect anxiety- and depression-like behaviors in rats. Dox-induced increase in Bcl-xL expression in the hippocampus may be involved in the moderate activation of FST behavior.

The antiapoptotic protein Bcl-xL is involved in development of neurobiological resilience to stress; hence, the possibility of use of psychotropic drugs to increase its expression in brain in response to stress is of considerable interest. Lithium is a neurotropic drug widely used in psychiatry. In work, we studied effects of lithium administration (for 2 or 7 days) on the expression of Bcl-xL mRNA and protein in the hippocampi and cortices of rats subjected to stress that induced depression-like behavior in the animals. In contrast to the brain-derived neurotrophic factor (BDNF), whose expression decreased in the hippocampus in response to acute stress, stress increased the level of Bcl-xL mRNA in the hippocampus, but decreased it in the frontal cortex. Treatment of stressed animals with lithium for 2 or 7 days increased Bcl-xL protein levels 1.5-fold in the hippocampus, but it decreased them in the cortex. Therefore, Bcl-xL expression in the brain can be modulated by both stress and psychotropic drugs, and the effects of these factors are brain region-specific: both stress exposure and lithium administration activated Bcl-xL expression in the hippocampus and suppressed it in the frontal cortex. The activation of Bcl-xL expression in the hippocampus by lithium, demonstrated for the first time in this study, suggests an important role of this protein in the therapeutic effects of lithium in the treatment of stress-induced psychoemotional disorders.

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

Rationale: Glucocorticoids facilitate coping with stress, but their high levels have been also implicated in mood disorders. Due to this duality, the role of glucocorticoid signaling in the development of the first episodes of stress-induced depression remains unclear., Objectives: To address this issue, effects of the glucocorticoid signal modulation on depressive-like behavior during pretest and test Porsolt swim sessions were examined., Methods: Metyrapone (MET; 150 mg/kg, i.p.) was injected 3 h before pretest to block stress-induced increase in corticosterone levels. Dexamethasone (DEX; 0.2 mg/kg, s.c.) was applied to MET-treated rats 1 h before both pretest and test sessions. In addition to behavior during these sessions, glucocorticoid receptor (GR) expression was analyzed by immunohistochemistry 2 h after the second swim., Results: In pretest, MET-treated rats exhibited increased latency to immobility and shortened immobility. DEX reversed the behavioral effects of MET in the pretest. In the test, animals from MET + DEX group unexpectedly exhibited an antidepressant-like behavior. Swim stress increased GR expression in the frontal cortex irrespective of the pharmacological treatment. A significant elevation in GR expression was found in the prefrontal cortex (PFC) of stressed MET + DEX-treated rats and in the PFC of unstressed rats 6 h after injection of DEX alone., Conclusion: The data suggest that the increase in glucocorticoid levels under swim stress during pretest directly contributes to the development of the immobility response. Transition of DEX effect from prodepressant in the pretest to an antidepressant in the test was associated with the elevation in the PFC GR expression.

Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during "proportional" period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during "proportional" period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during "proportional" period but the same doses of this GnRH antagonist significantly inhibited "accelerated" testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during "accelerated" period of testicular growth than during "proportional" period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling.

Triptorelin, a synthetic analogue of neurohormone gonadoliberin (gonadotropin-releasing hormone, GnRH) administered daily to rats on postnatal days 5-7 suppressed the expression of GnRH receptor in the pituitary gland, but did not change functioning of the pituitary-testicular complex. Administration of triptorelin on postnatal days 12-14 (i.e. during the formation of pulsatile pattern of GnRH secretion and increasing levels of its mRNA receptor in the pituitary gland) had no effect on receptor expression, but increased the levels of luteinizing hormone mRNA in the pituitary gland and the weight of testes. At that time, blood levels of testosterone were lowered, which indicated disturbed pulsatile pattern of GnRH secretion.

Rodent models are an indispensable tool for studying etiology and progress of depression. Since interrelated systems of neurotrophic factors and cytokines comprise major regulatory mechanisms controlling normal brain plasticity, impairments of these systems form the basis for development of cerebral pathologies, including mental diseases. The present review focuses on the numerous experimental rodent models of depression induced by different stress factors (exteroceptive and interoceptive) during early life (including prenatal period) or adulthood, giving emphasis to the data on the changes of neurotrophic factors and neuroinflammatory indices in the brain. These parameters are closely related to behavioral depression-like symptoms and impairments of neuronal plasticity and are both gender- and genotype-dependent. Stress-related changes in expression of neurotrophins and cytokines in rodent brain are region-specific. Some contradictory data reported by different groups may be a consequence of differences of stress paradigms or their realization in different laboratories. Like all experimental models, stress-induced depression-like conditions are experimental simplification of clinical depression states; however, they are suitable for understanding the involvement of neurotrophic factors and cytokines in the pathogenesis of the disease-a goal unachievable in the clinical reality. These major regulatory systems may be important targets for therapeutic measures as well as for development of drugs for treatment of depression states.

Stress predisposes to depression by enhancing apoptosis and reducing neurogenesis in the brain. There are significant individual differences in resilience to the effects of stress on mood. These differences, at least in part, may be related to the expression of the anti-apoptotic protein Bcl-xL in the brain. Increased expression of this protein in the hippocampus may be an important factor of resilience to stress-induced depression. Expression of the Bcl-xL in the brainstem acquires the ability to respond to stress induction during the course of treatment with prozac concomitantly with the emergence of the therapeutic effect of this antidepressant on behavior. Processes linking stress and behavior in which the protein Bcl-xL is involved, are considered in this review.

ACTH1-24(1 mg/kg) administered to 5-7-day-old rats reduced their locomotor activity and did not alter blood levels of corticosterone. ACTH administration on postnatal days 12-14 increased corticosterone levels, but had no effect on locomotor activity. Thus, the behavioral effect of ACTH is not due to corticotropic action of the hormone and is not associated with blood levels of glucocorticoids.

Clinical observations and the results of animal studies have implicated changes in neuronal survival and plasticity in both the etiology of mood disorders, especially stress-induced depression, and anti-depressant drug action. Stress may predispose individuals toward depression through down-regulation of neurogenesis and an increase in apoptosis in the brain. Substantial individual differences in vulnerability to stress are evident in humans and were found in experimental animals. Recent studies revealed an association between the brain anti-apoptotic protein B cell lymphoma like X, long variant (Bcl-xL) expression and individual differences in behavioral vulnerability to stress. The ability to increase Bcl-xL gene expression in the hippocampus in response to stress may be an important factor for determining the resistance to the development of stress-induced depression. Treatment with anti-depressant drugs may change Bcl-xL response properties. In the rat brainstem, expression of this anti-apoptotic gene becomes sensitive to swim stress during the long-term fluoxetine treatment, an effect that appeared concomitantly with the anti-depressant-like action of the drug in the forced swim test, suggesting that Bcl-xL may be a new target for depression therapy. The processes and pathways linking stress stimuli to behavior via intracellular anti-apoptotic protein are discussed here in the context of Bcl-xL functions in the mechanisms of individual differences in behavioral resilience to stress and anti-depressant-induced effects on the behavioral despair.

Changes in gene expression of the brain serotonin (5-HT) 1A receptors may be important for the development and ameliorating depression, however identification of specific stimuli that activate or reduce the receptor transcriptional activity is far from complete. In the present study, the forced swim test (FST) exposure, the first stress session of which is already sufficient to induce behavioral despair in rats, significantly increased 5-HT1A receptor mRNA levels in the brainstem, frontal cortex, and hippocampus at 24 h. In the brainstem and frontal cortex, the elevation in the receptor gene expression after the second forced swim session was not affected following chronic administration of fluoxetine, while in the cortex, both control and FST values were significantly reduced in fluoxetine-treated rats. In contrast to untreated rats, no increase in hippocampal 5-HT1A receptor mRNA was observed in response to FST in rats chronically treated with fluoxetine. Metabolism of 5-HT (5-HIAA/5-HT) in the brainstem was significantly decreased by fluoxetine and further reduced by swim stress, showing a certain degree of independence of these changes on 5-HT1A receptor gene expression that was increased in this brain region only after the FST, but not after fluoxetine. FST exposure also decreased the brainstem dopamine metabolism, which was unexpectedly positively correlated with 5-HT1A receptor mRNA levels in the frontal cortex. Together, these data suggest that the effects of the forced swim stress as well as fluoxetine involve brain region-dependent alterations in 5-HT1A receptor gene transcription, some of which may be interrelated with concomitant changes in catecholamine metabolism.

Brain noradrenergic system has been implicated in early-life stress effects on adult physiology and behavior; however, the mechanisms for this relationship are not clear. Here we tested the hypothesis that stress hormones, glucocorticoids, may affect noradrenergic system activity by modulating gene expression and function of tyrosine hydroxylase (TH), the key enzyme for catecholamine synthesis, in the rat brain during perinatal life. We have shown that TH mRNA levels and enzyme activity increase in the fetal rat brainstem during the last days of pregnancy. Administration of hydrocortisone or dexamethasone to female rats on day 20 of pregnancy significantly increased TH mRNA levels (real-time PCR) and enzyme activity (DOPA accumulation after inhibition of aromatic L: -amino acid decarboxylase with NSD-1015) as well as noradrenaline concentrations in the brainstem of fetuses 6 h after the treatment. Similar glucocorticoid effects on fetal TH and noradrenaline were observed 72 h after the treatment with hydrocortisone on days 16 and 18 of pregnancy. In contrast to fetuses, no effects on the TH were revealed in the brainstem of neonatal pups after single or repeated injections of hydrocortisone or dexamethasone. TH gene expression remains at a relatively constant level in the early neonatal rat brain. The results suggest that glucocorticoids are capable of inducing TH at both transcriptional and enzyme activity levels in the brainstem of near-term fetuses.

Mechanisms underlying stress-induced depression and antidepressant drug action were shown to involve alterations in serotonergic (5-HT) neurotransmission and expression of genes coding for proteins associated with neurotrophic signaling pathways and cell-survival in the hippocampus and cortex. Expression of these genes in the brainstem containing 5-HT neurons may also be related to vulnerability or resilience to stress-related psychopathology. Here we investigated 5-HT markers and expression of genes for Brain-Derived Neurotrophic Factor (BDNF) and apoptotic proteins in the brainstem in relation to swim stress-induced behavioral despair. We found that anti-apoptotic Bcl-xL gene is sensitive to stress during the course of fluoxetine administration. Responsiveness of this gene to stress appeared concomitantly with an antidepressant-like effect of fluoxetine in the forced swim test. Bcl-xL transcript levels showed negative correlations with duration of immobility in the test and 5-HT turnover in the brainstem. In contrast, BDNF and pro-apoptotic protein Bax mRNA levels were unchanged by either fluoxetine or stress, suggesting specificity of Bcl-xL gene responses to these treatments. We also found that the levels of mRNAs for tryptophan hydroxylase-2 (TPH2) and 5-HT transporter (5-HTT) were significantly down-regulated following prolonged treatment with fluoxetine, but were not affected by stress. Unlike TPH2 and 5-HTT, 5-HT1A receptor mRNA levels were not altered by fluoxetine but significantly increased in response to swim stress. These data show that long-term fluoxetine treatment leads to changes in 5-HT and Bcl-xL responses to stress associated with antidepressant-like effects of the drug. This article is part of a Special Issue entitled 'Anxiety and Depression'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A single exposure of rats to the forced-swimming stress decreased BDNF mRNA levels in the cortex and increased Bcl-xl gene expression in the hippocampus and amygdala 24 h after the stress. The animals demonstrated a depressive-like behavior and elevated blood corticosterone level. There was a significant negative correlation between BDNF mRNA level in the cortex and immobility time during swimming. Repeated exposure to swimming stress caused the elevation of the hippocampal BDNF mRNA level assessed 24 h after the second swimming session. The data suggest that stress-induced down-regulation of cortical BDNF gene expression and behavioral despair in the forced-swimming test may be interrelated. The increase in the BDNF and Bcl-xl mRNA levels may contribute to the mechanisms protecting the brain against negative effects of stress.

Stress may predispose individuals toward depression through down-regulation of neurogenesis and increase in apoptosis in the brain. However, many subjects show high resistance to stress in relation to psychopathology. In the present study, we assessed the possibility that individual-specific patterns of gene expression associated with cell survival and proliferation may be among the molecular factors underlying stress resilience. Brain-derived neurotrophic factor (BDNF), anti-apoptotic B cell lymphoma like X (Bcl-xl) and pro-apoptotic bcl2-associated X protein (Bax) expression were determined in the hippocampus and frontal cortex of rats naturally differed in despair-like behavior in the forced swim test. In the hippocampus, BDNF messenger RNA (mRNA) level was significantly down-regulated 2h after the forced swim test exposure, and at this time point, Bcl-xl mRNA and protein levels were significantly higher in stressed than in untested animals. The ratios of hippocampal Bcl-xl to Bax mRNA negatively correlated with the total time spent immobile in the test. When animals were divided in two groups according to immobility responses in two consecutive swim sessions and designated as stress resilient if their immobility time did not increase in the second session as it did in stress sensitive rats, it was found that resilient rats had significantly higher Bcl-xl/Bax ratios in the hippocampus than stress sensitive animals. The data suggest that naturally occurring variations in the Bcl-xl/Bax ratio in the hippocampus may contribute to individual differences in vulnerability to stress-induced depression-like behaviors., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Depression represents a complex mental disorder which results from the contribution of multiple genetic and environmental factors. A depressive state can be characterized by abnormalities in the functions of monoaminergic neurotransmission, hypothalamic-pituitary-adrenocortical system, neurotrophins and cytokines in genetically predisposed individuals that endured the impact of stress or infection. Genetic, epigenetic and ontogenetic factors underlie these abnormalities. Manifestations of the pathology demonstrate associations with specific alleles of candidate genes. Epigenetic mechanisms of DNA methylation, acetylation and methylation of histones and processes of transcription and translation are also involved in the development of depressive symptoms and their amelioration under prolonged treatment with antidepressants. These processes are regulated by transcription factors and specific microRNAs. The search for effective means to affect activity of these intracellular targets with traditional pharmaceutical agents altering intercellular communication performed by neurotransmitters, hormones and tissue factors that provide long-lasting neuronal plasticity would be useful for unraveling the pathophysiological mechanisms of depression and for development of new approaches to its therapy.

Short-term knockdown of alpha2A-adrenergic receptor gene expression in the rat brain by siRNA or antisense oligodeoxynucleotide during the first days of life induced acute and long-lasting neurochemical and behavioral alterations. The acute effects in the neonatal rats were consistent with the known functions of the alpha2A-adrenergic receptors in the mature animals. The long-lasting alterations suggested involvement of receptor-specific gene expression during the critical period of brain development in early-life programming of anxiety-related behavior.

Repeated forced swim resulted in a decrease in the concentrations of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid in the hypothalamus and amygdala 24 h after the second swim session. This stressor also increased the mRNA levels for tryptophan hydroxylase-2, the rate-limiting enzyme in neuronal 5-HT synthesis, and 5-HT transporter in the midbrain as well as 5-HT1A receptor in the frontal cortex. Some of these serotonergic changes may be involved in the mechanisms of a depressive-like behavior induced by a stress of repeated swim in these animals.

Tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme in 5-HT synthesis in the brain, is a candidate for participation in a mechanism mediating the antidepressant effect of selective 5-HT reuptake inhibitors such as fluoxetine. Using real-time reverse transcription-polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR techniques, we have examined the effects of fluoxetine administration with drinking water (7.5 mg/kg/day) for 2, 4 and 8 weeks on TPH2 mRNA expression in the midbrain part of the dorsal raphe nucleus (DRN) and in the brainstem containing the rest of the raphe complex. Fluoxetine treatment for 4 and 8 weeks significantly increased basal TPH2 mRNA levels in the midbrain, an effect that was correlated with the appearance of antidepressant-like effects in the forced swim test. A significant induction of TPH2 and 5-HT transporter (5-HTT) mRNAs was detected in the midbrain of untreated rats 24 h after the swim test. In these animals, the swim test also produced a marked decrease in 5-HT metabolite (5-hydroxyindoleacetic acid (5-HIAA)) content in the amygdala. Fluoxetine treatment for 4 and 8, but not for 2 weeks, abolished these swim-induced changes in TPH2 and 5-HTT mRNAs levels in the midbrain and 5-HIAA content in the amygdala. The results of the present study suggest that TPH2 gene expression in the midbrain part of the DRN is implicated in depression and stress response, as well as in the antidepressant fluoxetine action.

Selective serotonin reuptake inhibitors such as fluoxetine that are widely used for the treatment of depression and anxiety disorders produce neuroadaptive change not only in the serotoninergic system but also in other neuromediator systems. These changes may be involved in the therapeutic as well as in side effects of the drugs.

Treatment of the adult rats with selective serotonin (5-HT) reuptake inhibitor: fluoxetine and its complexes with glycyrrizhinic acid during 2 weeks (25 mg/kg/day) significantly increased plasma corticosterone levels that were measured after 5-min plus-maze. All the drugs decreased the content of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the striatum as well as 5-HT in the hippocampus. There was a significant negative correlation between 5-HT in the striatum and corticosterone levels. These data suggest that fluoxetine induces serotoninergic changes in the striatum that might be related to neuroendocrine and behavioural effects of the drug.

Selective serotonin (5-HT) reuptake inhibitors such as fluoxetine are widely used in the treatment of depression and anxiety; however, the mechanisms underlying their action and particularly the delay in therapeutic onset remain unclear. It is proposed that 5-HT reuptake inhibitors exert their therapeutic activity by increasing serotonergic neurotransmission; therefore, the aim of the present study was to investigate the effects of repeated treatment with fluoxetine (25 mg/kg/day p.o., 14 days) on expression of genes coding for proteins that involved in the synthesis and reuptake of 5-HT. Exposure of animals to plus-maze conditions on the first day of drug administration produced an increase in baseline anxiety on subsequent trial 2 weeks later. Fluoxetine strengthened the anxiogenic effects of maze experience. Two-week fluoxetine treatment also significantly reduced expression of tryptophan hydroxylase-2 (TPH2) and 5-HT transporter mRNAs as determined by RT-PCR in the brainstem. These changes were consistent with the decreased 5-HT levels and 5-HT turnover in the brain, and might contribute to the anxiogenic effects of the drug. The results also suggest that recently found association between treatment responses to fluoxetine and polymorphic variants of human TPH2 gene [Peters EJ, Slager SL, McGrath PJ, Knowles JA, Hamilton SP. Investigation of serotonin-related genes in antidepressant response. Mol Psychiatry 2004; 9:879-889] may be related to the drug effect on the TPH2 gene expression.

Plus-maze benavior of adult mail rats was assessed and contents of 5-hydroxyindoleacetic acid (5-HIAA) and dopamine (DA) in different brain regions were analyzed after two-week fluoxetine treatment (25 mg/kg/day, per os). Chronic fluoxetine treatment produced a general decrease in the brain 5-HIAA content and a decrease in DA content in the frontal cortex and striatum. Behaviorally, fluoxetine-treated animals displayed enhanced anxiety and decreased locomotor activity. The DA depletion os supposed to be responsible for fluoxetine-produced hypolocomotion. This is supported by a significant correlation between the dopamine content in the frontal cortex and the number of entries into the closed arms of the plus-maze. The results suggest that the hypolocomotor effect of fluoxetine may involve the changes in the dopaminergic system.

The effects of the serotonin reuptake inhibitor fluoxetine (FL) and its complexes with glycyrrhizic acid (GA) in molar ratios of 1:1 (FLG-1) and 4:1 (FLG-4) on the behavior of adult rats were studied in an elevated cross maze, with measurement of brain monoamine and monamine metabolite levels. Agents were given via the intragastric route using a cannula at a dose of 25 mg/kg 1 h before testing. FL increased anxiety in the rats and decreased their movement activity; FLG-1 and FLG-4 had no effect on behavior. None of the agents affected brain serotonin content, though all decreased the levels of its metabolite 5-hydroxyindoleacetic acid in the hypothalamus, FLG-4 also decreasing this in the cortex. Noradrenaline levels in the hypothalamus were increased after FLG-1 and FLG-4. In the striatum, FL increased the levels of dopamine and its metabolite dihydroxyphenylacetic acid but had no effect on the level of transmitter catabolism. Unlike FL, FLG-1 activated dopamine metabolism in the striatum. Overall, use of FL complexed with GA significantly modified its behavioral effects, which appears to be associated with the effects of FL and its complexes on the function of the monoaminergic systems involved in controlling behavior.

Effects of serotonin uptake inhibitor fluoxetine (F) and it's complexes with glycyrrizhinic acid (GA) in molar proportions 1GA : 1F (FGA-1) and 4GA : 1F (FGA-4) on rat behavior in elevated plus-maze and brain monoamine concentrations were studied. Drugs (25 mg/kg) were administered per os 1 h before investigations. F-treated rats showed increased anxiety and reduced locomotor activity, whereas FGA-1 and FGA-4 had no effects on the behaviors. None of the compounds modified brain tissue serotonin content, but all of them decreased the level of its metabolite 5-hydroxyindole-3-acetic acid level in the hypothalamus, and FGA-4 also decreased it in the cortex. Noradrenaline levels were increased in the hypothalamus of rats treated with F in both combinations with GA. In the striatum, F increased dopamine and its metabolite DOPAC levels, but their ratio (an indicator of the neurotransmitter turnover) was not altered by this drug. Unlike F, FGA-1 significantly activated dopamine turnover in the striatum. The data obtained suggested that application of F in complexes with GA significantly modified the drug behavioral effects and these alterations may be related to specific effects of the pure compound and its complexes on the functions of the brain monoaminergic systems that regulate investigated behavior.

Neonatal treatments can disrupt prepulse inhibition (PPI) of startle response later in life. Alpha2A-adrenergic receptors (alpha2A-ARs) regulate the release of brain neurotransmitters that may influence PPI. The authors examined the effects of short-term reduction in the neonatal brainstem alpha2A-ARs on subsequent development of this receptor system and acoustic startle reflex in rats. Administration of antisense oligodeoxynucleotide complementary to the alpha2A-ARs on Days 2-4 of life reduced receptor expression in the brainstem by Day 5. The treatment increased alpha2-AR numbers in the cortex, hippocampus, and amygdala at 40 days of age, and in cortex and hypothalamus at 90 days of age. Transient increases in hippocampal and amygdalar alpha2-ARs were accompanied by attenuation of acoustic startle response and impairment of PPI.

The densities of alpha2-adrenergic receptors, labeled by 3H-clonidine or 3H-RX821002, reach a peak in the rat brainstem during the first week of its life. This enables the agonist of alpha2-adrenergic receptor clonidine, which is used as a component of anaesthetic solution in infants and children, to have specific effects in this structure of the developing brain. Clonidine was injected into the fetal brain (5 microg in 5 microl of saline) or subcutaneously to the pups (1, 10 microg in 50 microl of saline) 3 days before investigation. Clonidine increased the level of apoptotic enzyme caspase-3 mRNA expression, as measured by RT-PCR and enhanced the DNA fragmentation, as determined by gel electrophoresis, in the brainstem of the 21-day-old fetuses and 8-day-old rats. In the cortex of 8-day-old rat, the alpha2-adrenergic receptors are at a much lower level than the brainstem. Clonidine treatment had no evident effects on caspase-3 mRNA level and DNA fragmentation in the cortex of an 8-day-old rat. The data suggest that clonidine facilitates cell death in the developing brainstem. This drug effect provides a potential mechanism whereby clonidine during early life could induce long-lasting alterations in brain neurochemistry, autonomic functions and behavior.

Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain development may be involved in early-life programming of anxiety-related behavior.

Involvement of alpha 2A-adrenoceptors in control of motor activity in neonatal rats was investigated using intrabrain injections of antisense deoxyoligonucleotide targeting mRNA of these receptors. Antisense-induced decrease in the receptor expression in the brain accompanied by an augmentation of animal's motor activity, which was evaluated as a number of paddling movements with the right forepaw. This antisense effect was stimulatory and was not related to somatic growth and maturation of reflexes in the animals. The antisensetreated pups differed from controls neither in body weight nor the latency of righting reflex. The data suggest that alpha 2A-adrenoceptors are involved in inhibitory control of motor activity of rat pups from the first day of life.

Androgens are involved in regulation of behaviour through intracellular mechanisms owing to their receptors. Involvement of intercellular messengers such as brain norepinephrine and adrenergic receptors (ARs) is seemed to be necessary to realise hormone-dependent behavioural effects. Castration of adult male rats, which decreases copulatory activity in the animals, was accompanied by a significant increase in 3H-clonidine (alpha2-AR agonist) binding site density in the frontal cortex. The levels of mRNA for the alpha2A-ARs (measured by RT-PCR) were increased in the brainstem of castrated males in parallel to the changes in cortical ARs densities. Testosterone treatment, that activates copulatory behaviour in castrates, down regulated alpha2A-AR mRNA levels in the brainstem and 3H-clonidine binding sites densities in the cortex, where terminals of the brain stem neurones are situated. Unlike in the brainstem, castration caused a decrease in alpha2A-AR mRNA in the cortex and testosterone up-regulated this mRNA in the cortical region. The data suggested that down-regulation of alpha2-ARs densities in the cortex that is induced by testosterone can be preferentially related to alpha2-ARs subpopulation which is expressed by the brainstem neurones and imported into the cortex by axons of these neurones.

Antisense strategy was used to investigate the role of alpha2A-adrenoceptor (alpha2A-AR) subtype in anxiety-related behavior. A 18-mer phosphorothioate oligodeoxynucleotide (AS-ODN) complementary to the alpha2A-AR mRNA was administered to the adult male rats for 3 days (1 nmol/5 microl/day) into the region of locus coeruleus (LC). Control groups received infusions of either oligodeoxynucleotide of a random sequence (RS-ODN) or saline. Treatment with AS-ODN significantly reduced the levels of alpha2A-AR mRNA in the brain stem. At the same time, AS-ODN treatment caused only a small reduction in [(3)H]clonidine binding (by 26-32%) in the brain stem which was not significant. Compared to both RS-ODN and saline controls, treatment with AS-ODN significantly increased the percentage of open arm entries in the elevated plus-maze while the total number of arm entries was unaltered. Also, AS-ODN treatment elevated basal levels of plasma corticosterone by 217% and 96% compared to both RS-ODN and saline controls. These changes in the hormone concentrations were at a level of marginal significance (p<0.1 versus random group). Taken together, the data indicate that administration of AS-ODN against alpha2A-ARs in the LC significantly reduced expression of alpha2A-AR mRNA in brain stem, moderately increased plasma corticosterone and had anxiolytic-like effect in the elevated plus-maze.

A- B- and C-subtypes of alpha 2-adrenoreceptors present in all mammals are involved in responses to currently existing subtype-nonselective ligands of these receptors widely used in medicine. Each of the subtypes has its own specific distribution in tissue and cells, onthogenetic pattern, specific regulation of activity and expression, and, as result, specific physiological functions. The latter suggests opportunities of using the subtype-specific for correction of the functions depending on this receptor. The article reviews the role of individual subtypes of alpha 2-adrenoreceptors in regulation of neurochemical transmission of cardiovascular system, psychoemotional state and development of psychic disorders, and also male sexual behaviour.

Clinical and experimental findings have implicated brain alpha2-adrenoceptors in the regulation of many physiological functions, including sexual activity and stress-related behavior. However, which subtypes of the three alpha2-adrenoceptors that have now been cloned (alpha2A, alpha2B, and alpha2C) are involved in these controls have yet to be established. Here, we investigated the contribution of alpha2A-adrenoceptors of the locus ceruleus, the principal source of brain noradrenaline, to exploratory and sexual behaviors. Using administration of antisense oligodeoxynucleotide to inhibit the receptor expression, we found that reductions in brainstem alpha2A-adrenoceptor mRNA levels and alpha2-adrenoceptor densities induced by antisense treatment were not accompanied by any changes in the major characteristics of male sexual activity, such as mount latencies and numbers of mounts. However, in sexual behavior tests, antisense-treated male rats had decreased numbers of rearings and thus have higher percentages of behaviors positively correlated with sexual activity. Besides, antisense-treated animals had decreased anxiety in plus-maze tests. The data demonstrate that inhibition of alpha2A-adrenoceptor expression in the region of the locus ceruleus has an anxiolytic-like effect and facilitates male's attention to female in sexual behavior test.

Now available nucleotide sequences of neurotransmitter receptor genes enable to apply oligonucleotides targeted to mRNAs of these genes for highly selective inactivation of their expression (antisense-knockdown) and for function determination of single receptor subtype by this experimental approach. The antisense-knockdown may be of special importance in case of receptor families members of which are pharmacologically similar. Advantages of the antisense technology for investigation into the brain neurotransmitter receptor function in regulation of behaviour, are discussed.

The activity of tyrosine hydroxylase, the key enzyme in catecholamine biosynthesis, was studied along with adrenoceptor density in the brains of male CBA/Lac, BALB/cLac, and C57BL/6J mice, which show different responses to novel environments. C57BL mice showed the highest level of movement activity and the lowest level of emotionality in a novel environment. Mice of this line also showed the highest brainstem tyrosine hydroxylase activity. At the same time, the density of beta-adrenoceptors in the cortex and hypothalamus of C57BL mice was lower than in the other two lines of mice, while the density of alpha2-adrenoceptors in these parts of the brain was lower than in CBA mice. In BALB mice, movement activity was twice as high as in CBA mice, while levels of emotionality were similar in these two lines. Tyrosine hydroxylase activity was higher in the cerebral cortex of BALB mice, while the density of alpha2-adrenoceptors was lower than in CBA mice. These results show that increased investigative activity and decreased emotionality were seen in animals with higher levels of noradrenaline synthesis and decreased density of adrenergic receptors in the brain.

The role of the serotoninergic system in acceleration of the sexual development of domesticated rats (Rattus norvegicus) was assessed. The onset of age-related changes in hypothalamic serotonin during prepubertal period occurred earlier in domesticated than in aggressive male rats. Blockade of the serotoninergic system after p-chlorophenylalanine (PCPA) administration on days 40 and 44 delayed the development of the reproductive system in both aggressive and domesticated males. In 60-day-old rats treated with PCPA, levels of testosterone in plasma and the number of mature spermatozoa in epididymis were decreased compared to controls. At the same time, the administration of PCPA on days 30 and 34 did not modify basal testosterone secretion and other parameters in 60-day-old aggressive rats and produced a decrease similar to PCPA injections on days 40 and 44, although less pronounced, in the weights of testes in domesticated animals. Administration of 5-hydroxytryptophan (5-HTP), a precursor of serotonin synthesis, on days 30, 32, 34, 36 and 38 increased plasma testosterone levels and weights of the sex organs in 60-day-old domesticated males, but did not significantly affect the development of reproductive system in aggressive animals. These data indicate that serotonin stimulates sexual development of males during prepubertal period and this activating effect of serotonin occurs earlier in domesticated than in aggressive males. They also suggest that the acceleration in sexual maturation of domesticated rats could result from changes in the ontogenetic dynamic of hypothalamic serotonin induced by a selection for low aggressiveness towards man.

We studied the level of noradrenaline and the density of alpha 2- and beta-adrenoreceptors in the brain stem and cerebral cortex of 12-day- and 21-day-old rat fetuses, as well as of rats at the ages of 1, 2, 5, 7, 9, 16, 21, 35, and 70 days. We found a positive correlation between the level of noradrenaline in the brain stem and the density of beta-receptors in the cerebral cortex, and between the amount of alpha 2- and beta receptors in the cerebral cortex, as well as between the values of each of these indices of the neurochemical system and body weight. Significant negative correlations (r = -0.72 and r = -0.88, respectively) were found between the amount of alpha 2-adreno-receptors in the brain stem and the content of noradrenaline in this brain region, as well as in the cerebral cortex. Explanations of these positive and negative correlations between the level of noradrenaline and the amount of adrenergic receptors in the rat brain during ontogenesis are discussed.