Genes Involved by Dexamethasone in Prevention of Long-Term Memory Impairment Caused by Lipopolysaccharide-Induced Neuroinflammation.

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.

Comparative Investigation of Expression of Glutamatergic and GABAergic Genes in the Rat Hippocampus after Focal Brain Ischemia and Central LPS Administration.

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.

LPS Administration Impacts Glial Immune Programs by Alternative Splicing.

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.

Identifying the Involvement of Pro-Inflammatory Signal in Hippocampal Gene Expression Changes after Experimental Ischemia: Transcriptome-Wide Analysis.

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.

Changes in Gene Expression and Neuroinflammation in the Hippocampus after Focal Brain Ischemia: Involvement in the Long-Term Cognitive and Mental Disorders.

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.

Anxiogenic-like effect of chronic lipopolysaccharide is associated with increased expression of matrix metalloproteinase 9 in the rat amygdala.

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.

Stress-induced expression pattern of glutamate signaling genes associated with anhedonia.

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.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

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.

Behavioral effects of glucocorticoids during the first exposures to the forced swim stress.

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.

Rodent models of depression: neurotrophic and neuroinflammatory biomarkers.

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.

A critical point of male gonad development: neuroendocrine correlates of accelerated testicular growth in rats during early life.

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.

Increased expression of the anti-apoptotic protein Bcl-xL in the brain is associated with resilience to stress-induced depression-like behavior.

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.

Induction of tyrosine hydroxylase gene expression by glucocorticoids in the perinatal rat brain is age-dependent.

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.

Stress-induced activation of the brainstem Bcl-xL gene expression in rats treated with fluoxetine: correlations with serotonin metabolism and depressive-like behavior.

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'.

Resistance to the development of stress-induced behavioral despair in the forced swim test associated with elevated hippocampal Bcl-xl expression.

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.

Serotonergic changes produced by repeated exposure to forced swimming: correlation with 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.

Neonatal programming of rat behavior by downregulation of alpha2A-adrenoreceptor gene expression in the brain.

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.

Influence of neonatal short-term reduction in brainstem alpha2A-adrenergic receptors on receptor ontogenesis, acoustic startle reflex, and prepulse inhibition in rats.

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.

Clonidine increases caspase-3 mRNA level and DNA fragmentation in the developing rat brainstem.

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.