Prefrontal cortex astroglia modulate anhedonia-like behavior.

Reductions of astroglia expressing glial fibrillary acidic protein (GFAP) are consistently found in the prefrontal cortex (PFC) of patients with depression and in rodent chronic stress models. Here, we examine the consequences of PFC GFAP+ cell depletion and cell activity enhancement on depressive-like behaviors in rodents. Using viral expression of diphtheria toxin receptor in PFC GFAP+ cells, which allows experimental depletion of these cells following diphtheria toxin administration, we demonstrated that PFC GFAP+ cell depletion induced anhedonia-like behavior within 2 days and lasting up to 8 days, but no anxiety-like deficits. Conversely, activating PFC GFAP+ cell activity for 3 weeks using designer receptor exclusively activated by designer drugs (DREADDs) reversed chronic restraint stress-induced anhedonia-like deficits, but not anxiety-like deficits. Our results highlight a critical role of cortical astroglia in the development of anhedonia and further support the idea of targeting astroglia for the treatment of depression.

BDNF release and signaling are required for the antidepressant actions of GLYX-13.

Conventional antidepressant medications, which act on monoaminergic systems, display significant limitations, including a time lag of weeks to months and low rates of therapeutic efficacy. GLYX-13 is a novel glutamatergic compound that acts as an N-methyl-D-aspartate (NMDA) modulator with glycine-like partial agonist properties; like the NMDA receptor antagonist ketamine GLYX-13 produces rapid antidepressant actions in depressed patients and in preclinical rodent models. However, the mechanisms underlying the antidepressant actions of GLYX-13 have not been characterized. Here we use a combination of neutralizing antibody (nAb), mutant mouse and pharmacological approaches to test the role of brain-derived neurotrophic factor-tropomyosin-related kinase B (BDNF-TrkB) signaling in the actions of GLYX-13. The results demonstrate that the antidepressant effects of GLYX-13 are blocked by intra-medial prefrontal cortex (intra-mPFC) infusion of an anti-BDNF nAb or in mice with a knock-in of the BDNF Val66Met allele, which blocks the processing and activity-dependent release of BDNF. We also demonstrate that pharmacological inhibitors of BDNF-TrkB signaling or of L-type voltage-dependent Ca2+ channels (VDCCs) block the antidepressant behavioral actions of GLYX-13. Finally, we examined the role of the Rho GTPase proteins by injecting a selective inhibitor into the mPFC and found that activation of Rac1 but not RhoA is involved in the antidepressant effects of GLYX-13. Together, these findings indicate that enhanced release of BDNF through exocytosis caused by activation of VDCCs and subsequent TrkB-Rac1 signaling is required for the rapid and sustained antidepressant effects of GLYX-13.

Transiently increased glutamate cycling in rat PFC is associated with rapid onset of antidepressant-like effects.

Several drugs have recently been reported to induce rapid antidepressant effects in clinical trials and rodent models. Although the cellular mechanisms involved remain unclear, reports suggest that increased glutamate transmission contributes to these effects. Here, we demonstrate that the antidepressant-like efficacy of three unique drugs, with reported rapid onset antidepressant properties, is coupled with a rapid transient rise in glutamate cycling in the medial prefronal cortex (mPFC) of awake rats as measured by ex vivo 1H-[13C]-nuclear magnetic resonance spectroscopy. Rats were acutely pretreated by intraperitoneal injection with a single dose of ketamine (1, 3, 10, 30 and 80 mg kg-1), Ro 25-6981 (1, 3 and 10 mg kg-1), scopolamine (5, 25 and 100 µg kg-1) or vehicle (controls). At fixed times after drug injection, animals received an intravenous infusion of [1,6-13C2]glucose for 8 min to enrich the amino-acid pools of the brain with 13C, followed by rapid euthanasia. The mPFC was dissected, extracted with ethanol and metabolite 13C enrichments were measured. We found a clear dose-dependent effect of ketamine and Ro 25-6981 on behavior and the percentage of 13C enrichment of glutamate, glutamine and GABA (γ-aminobutyric acid). Further, we also found an effect of scopolamine on both cycling and behavior. These studies demonstrate that three pharmacologically distinct classes of drugs, clinically related through their reported rapid antidepressant actions, share the common ability to rapidly stimulate glutamate cycling at doses pertinent for their antidepressant-like efficacy. We conclude that increased cycling precedes the antidepressant action at behaviorally effective doses and suggest that the rapid change in cycling could be used to predict efficacy of novel agents or identify doses with antidepressant activity.

Prefrontal Cortex Astroglia Modulate Anhedonia-like Behavior.

Reductions of astroglia expressing glial fibrillary acidic protein (GFAP) are consistently found in the prefrontal cortex (PFC) of patients with depression and in rodent chronic stress models. Here, we examine the consequences of PFC GFAP+ cell depletion and cell activity enhancement on depressive-like behaviors in rodents. Using viral expression of diphtheria toxin receptor in PFC GFAP+ cells, which allows experimental depletion of these cells following diphtheria toxin administration, we demonstrated that PFC GFAP+ cell depletion induced anhedonia-like behavior within 2 days and lasting up to 8 days, but no anxiety-like deficits. Conversely, activating PFC GFAP+ cell activity for 3 weeks using designer receptor exclusively activated by designer drugs (DREADDs) reversed chronic restraint stress-induced anhedonia-like deficits, but not anxiety-like deficits. Our results highlight a critical role of cortical astroglia in the development of anhedonia and further support the idea of targeting astroglia for the treatment of depression.

Glial pathology in an animal model of depression: reversal of stress-induced cellular, metabolic and behavioral deficits by the glutamate-modulating drug riluzole.

Growing evidence indicates that glia pathology and amino-acid neurotransmitter system abnormalities contribute to the pathophysiology and possibly the pathogenesis of major depressive disorder. This study investigates changes in glial function occurring in the rat prefrontal cortex (PFC) after chronic unpredictable stress (CUS), a rodent model of depression. Furthermore, we analyzed the effects of riluzole, a Food and Drug Administration-approved drug for the treatment of amyotrophic laterosclerosis, known to modulate glutamate release and facilate glutamate uptake, on CUS-induced glial dysfunction and depressive-like behaviors. We provide the first experimental evidence that chronic stress impairs cortical glial function. Animals exposed to CUS and showing behavioral deficits in sucrose preference and active avoidance exhibited significant decreases in 13C-acetate metabolism reflecting glial cell metabolism, and glial fibrillary associated protein (GFAP) mRNA expression in the PFC. The cellular, metabolic and behavioral alterations induced by CUS were reversed and/or blocked by chronic treatment with the glutamate-modulating drug riluzole. The beneficial effects of riluzole on CUS-induced anhedonia and helplessness demonstrate the antidepressant action of riluzole in rodents. Riluzole treatment also reversed CUS-induced reductions in glial metabolism and GFAP mRNA expression. Our results are consistent with recent open-label clinical trials showing the drug's effect in mood and anxiety disorders. This study provides further validation of hypothesis that glial dysfunction and disrupted amino-acid neurotransmission contribute to the pathophysiology of depression and that modulation of glutamate metabolism, uptake and/or release represent viable targets for antidepressant drug development.

Regulation of cocaine reward by CREB.

Cocaine regulates the transcription factor CREB (adenosine 3', 5'-monophosphate response element binding protein) in rat nucleus accumbens, a brain region that is important for addiction. Overexpression of CREB in this region decreases the rewarding effects of cocaine and makes low doses of the drug aversive. Conversely, overexpression of a dominant-negative mutant CREB increases the rewarding effects of cocaine. Altered transcription of dynorphin likely contributes to these effects: Its expression is increased by overexpression of CREB and decreased by overexpression of mutant CREB. Moreover, blockade of kappa opioid receptors (on which dynorphin acts) antagonizes the negative effect of CREB on cocaine reward. These results identify an intracellular cascade-culminating in gene expression-through which exposure to cocaine modifies subsequent responsiveness to the drug.

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments.

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus.

Recent studies suggest that stress-induced atrophy and loss of hippocampal neurons may contribute to the pathophysiology of depression. The aim of this study was to investigate the effect of antidepressants on hippocampal neurogenesis in the adult rat, using the thymidine analog bromodeoxyuridine (BrdU) as a marker for dividing cells. Our studies demonstrate that chronic antidepressant treatment significantly increases the number of BrdU-labeled cells in the dentate gyrus and hilus of the hippocampus. Administration of several different classes of antidepressant, but not non-antidepressant, agents was found to increase BrdU-labeled cell number, indicating that this is a common and selective action of antidepressants. In addition, upregulation of the number of BrdU-labeled cells is observed after chronic, but not acute, treatment, consistent with the time course for the therapeutic action of antidepressants. Additional studies demonstrated that antidepressant treatment increases the proliferation of hippocampal cells and that these new cells mature and become neurons, as determined by triple labeling for BrdU and neuronal- or glial-specific markers. These findings raise the possibility that increased cell proliferation and increased neuronal number may be a mechanism by which antidepressant treatment overcomes the stress-induced atrophy and loss of hippocampal neurons and may contribute to the therapeutic actions of antidepressant treatment.

Localization of phosphorylated cAMP response element-binding protein in immature neurons of adult hippocampus.

Neurogenesis continues to occur in the adult hippocampus, although many of the newborn cells degenerate 1-2 weeks after birth. The number and survival of newborn cells are regulated by a variety of environmental stimuli, but very little is known about the intracellular signal transduction pathways that control adult neurogenesis. In the present study, we examine the expression of the phosphorylated cAMP response element-binding protein (pCREB) in immature neurons in adult hippocampus and the role of the cAMP cascade in the survival of new neurons. The results demonstrate that virtually all immature neurons, identified by triple immunohistochemistry for bromodeoxyuridine (BrdU) and polysialic acid-neural cell adhesion molecule (PSA-NCAM), are also positive for pCREB. In addition, upregulation of cAMP (via pharmacological inhibition of cAMP breakdown or by antidepressant treatment) increases the survival of BrdU-positive cells. A possible role for pCREB in the regulation of PSA-NCAM, a marker of immature neurons involved in neuronal remodeling and neurite outgrowth, is supported by cell culture studies demonstrating that the cAMP-CREB pathway regulates the expression of a rate-limiting enzyme responsible for the synthesis of PSA-NCAM. These findings indicate that the cAMP-CREB pathway regulates the survival, and possibly the differentiation and function, of newborn neurons.

cAMP response element-mediated gene transcription is upregulated by chronic antidepressant treatment.

Regulation of gene transcription via the cAMP-mediated second messenger pathway has been implicated in the actions of antidepressant drugs, but studies to date have not demonstrated such an effect in vivo. To directly study the regulation of cAMP response element (CRE)-mediated gene transcription by antidepressants, transgenic mice with a CRE-LacZ reporter gene construct were administered one of three different classes of antidepressants: a norepinephrine selective reuptake inhibitor (desipramine), a serotonin selective reuptake inhibitor (fluoxetine), or a monoamine oxidase inhibitor (tranylcypromine). Chronic, but not acute, administration of these antidepressants significantly increased CRE-mediated gene transcription, as well as the phosphorylation of CRE binding protein (CREB), in several limbic brain regions thought to mediate the action of antidepressants, including the cerebral cortex, hippocampus, amygdala, and hypothalamus. These results demonstrate that chronic antidepressant treatment induces CRE-mediated gene expression in a neuroanatomically differentiated pattern and further elucidate the molecular mechanisms underlying the actions of these widely used therapeutic agents.

Region-specific regulation of RGS4 (Regulator of G-protein-signaling protein type 4) in brain by stress and glucocorticoids: in vivo and in vitro studies.

The present study demonstrates that the regulator of G-protein-signaling protein type 4 (RGS4) is differentially regulated in the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus by chronic stress and glucocorticoid treatments. Acute or chronic administration of corticosterone to adult rats decreased RGS4 mRNA levels in the PVN but increased these levels in the LC. Similarly, chronic unpredictable stress decreased RGS4 mRNA levels in the PVN but had a strong trend to increase these levels in the LC. Chronic stress also decreased RGS4 mRNA levels in the pituitary. The molecular mechanisms of RGS4 mRNA regulation were further investigated in vitro in the LC-like CATH.a cell line and the neuroendocrine AtT20 cell line using the synthetic corticosterone analog dexamethasone. Consistent with the findings in vivo, dexamethasone treatment caused a dose- and time-dependent decrease in RGS4 mRNA levels in AtT20 cells but a dose- and time-dependent increase in CATH.a cells. RGS4 mRNA regulation seen in these two cell lines seems to be attributable, at least in part, to opposite changes in mRNA stability. The differential regulation of RGS4 expression in the LC and in key relays of the hypothalamic-pituitary-adrenal axis could contribute to the brain's region-specific and long-term adaptations to stress.

A molecular and cellular theory of depression.

Recent studies have begun to characterize the actions of stress and antidepressant treatments beyond the neurotransmitter and receptor level. This work has demonstrated that long-term antidepressant treatments result in the sustained activation of the cyclic adenosine 3',5'-monophosphate system in specific brain regions, including the increased function and expression of the transcription factor cyclic adenosine monophosphate response element-binding protein. The activated cyclic adenosine 3',5'-monophosphate system leads to the regulation of specific target genes, including the increased expression of brain-derived neurotrophic factor in certain populations of neurons in the hippocampus and cerebral cortex. The importance of these changes is highlighted by the discovery that stress can decrease the expression of brain-derived neurotrophic factor and lead to atrophy of these same populations of stress-vulnerable hippocampal neurons. The possibility that the decreased size and impaired function of these neurons may be involved in depression is supported by recent clinical imaging studies, which demonstrate a decreased volume of certain brain structures. These findings constitute the framework for an updated molecular and cellular hypothesis of depression, which posits that stress-induced vulnerability and the therapeutic action of antidepressant treatments occur via intracellular mechanisms that decrease or increase, respectively, neurotrophic factors necessary for the survival and function of particular neurons. This hypothesis also explains how stress and other types of neuronal insult can lead to depression in vulnerable individuals and it outlines novel targets for the rational design of fundamentally new therapeutic agents.

Neurobiology and treatment of anxiety: signal transduction and neural plasticity.

The stress-dependence and chronic nature of anxiety disorders along with the anxiolytic effectiveness of antidepressant drugs suggests that neuronal plasticity may play a role in the pathophysiology of anxiety. Intracellular signaling pathways are known in many systems to be critical links in the cascades from surface signals to the molecular alterations that result in functional plasticity. Chronic antidepressant treatments can regulate intracellular signaling pathways and can induce molecular, cellular, and structural changes over time. These changes may be important to the anxiolytic effectiveness of these drugs. In addition, the signaling proteins implicated in the actions of chronic antidepressant action, such as cAMP response element binding protein (CREB), have also been implicated in conditioned fear and in anxiety. The cellular mechanisms underlying conditioned fear indicate roles for additional signaling pathways; however, less is known about such mechanisms in anxiety. The challenge to identify intracellular signaling pathways and related molecular and structural changes that are critical to the etiology and treatment of anxiety will further establish the importance of mechanisms of neuronal plasticity in functional outcome and improve treatment strategies.

Novel therapeutic approaches beyond the serotonin receptor.

The influence of serotonin (5-HT) on neuronal function is mediated by regulation of receptor-coupled intracellular signal transduction pathways, and the therapeutic action of 5-HT selective reuptake inhibitors (SSRIs), as well as other types of antidepressants, most likely involves regulation of these intracellular pathways. The cyclic adenosine monophosphate (cAMP) second messenger system is one pathway that could be involved in antidepressant action. Chronic administration of antidepressants, including SSRIs, up-regulates the cAMP pathway at several levels, including increased expression of the cAMP response element binding protein (CREB). Among the multiple target genes that could be regulated by CREB and that could be involved in antidepressant actions and the pathophysiology of depression in brain-derived neurotrophic factor (BDNF). Stress decreases the expression of BDNF, and reduce levels of this neurotrophic factor could contribute to the atrophy and decreased function of stress-vulnerable hippocampal neurons. In contrast, antidepressant treatment increases the expression of BDNF in hippocampus, and could thereby reverse the stress-induced atrophy of neurons or protect these neurons from further damage. Up-regulation of the cAMP and BDNF systems has resulted in a novel model for the mechanism of action of antidepressants and new targets for the development of therapeutic agents.

Neural plasticity to stress and antidepressant treatment.

Adaptations at the cellular and molecular levels in response to stress and antidepressant treatment could represent a form of neural plasticity that contributes to the pathophysiology and treatment of depression. At the cellular level, atrophy and death of stress-vulnerable neurons in the hippocampus, as well as decreased neurogenesis of hippocampal neurons, has been reported in preclinical studies. Clinical studies also provide evidence for atrophy and cell death in the hippocampus, as well as the prefrontal cortex. It is possible that antidepressant treatment could oppose these adverse cellular effects, which may be regarded as a loss of neural plasticity, by blocking or reversing the atrophy of hippocampal neurons and by increasing cell survival and function. The molecular mechanisms underlying these effects are discussed, including the role of the cAMP signal transduction cascade and neurotrophic factors.

Neuronal plasticity and survival in mood disorders.

Studies at the basic and clinical levels demonstrate that neuronal atrophy and cell death occur in response to stress and in the brains of depressed patients. Although the mechanisms have yet to be fully elucidated, progress has been made in characterizing the signal transduction cascades that control neuronal atrophy and programmed cell death and that may be involved in the action of antidepressant treatment. These pathways include the cyclic adenosine monophosphate and neurotrophic factor signal transduction cascades. It is notable that these same pathways have been demonstrated to play a pivotal role in cellular models of neural plasticity. This overlap of plasticity and cell survival pathways, together with studies demonstrating that neuronal activity enhances cell survival, suggests that neuronal atrophy and death could result from a disruption of the mechanisms underlying neural plasticity. The role of these pathways and failure of neuronal plasticity in stress-related mood disorders are discussed.

Expression of the cAMP response element binding protein (CREB) in hippocampus produces an antidepressant effect.

BACKGROUND: Recent studies have demonstrated that chronic antidepressant treatment increases the expression of the cyclic amp (cAMP) response element binding protein (CREB) in rat hippocampus. The study presented here was conducted to determine if CREB is a relevant target that produces an antidepressant-like effect. METHODS: We employed the herpes simplex virus (HSV)-mediated gene transfer technique to overexpress CREB and determined its effect on the learned helplessness and forced swim tests, two established models used for pharmacological screening of antidepressant drugs. RESULTS: In the learned helplessness model, rats that received bilateral microinjection of HSV-CREB into the dentate gyrus showed significantly fewer escape failures in the subsequent conditioned avoidance test than those injected with control vector (HSV-LacZ). In contrast, microinjection of HSV-CREB in either the CA1 pyramidal cell layer of hippocampus or the prefrontal cortex did not produce an antidepressant response. In the forced swim test, CREB expression in the dentate gyrus resulted in a significantly shorter immobility time than those injected with HSV-LacZ. CONCLUSIONS: These results demonstrate that over-expression of CREB in hippocampus results in an antidepressant effect and suggest that CREB may serve as a potential molecular target for novel therapeutic agents.

Stress differentially regulates synaptophysin and synaptotagmin expression in hippocampus.

BACKGROUND: In view of the effects of stress on synaptic plasticity, the regulation of synaptophysin and synaptotagmin expression by immobilization was analyzed by in situ hybridization. METHODS: Rats were exposed to immobilization stress, which induced typical behavioral alterations, such as reduced locomotor activity after stress exposure. Determination of mRNA levels of the integral synaptic vesicle proteins was performed immediately after acute or chronic immobilization. RESULTS: The results demonstrate that stress exposure leads to reduced expression of synaptophysin but increased expression of synaptotagmin in the hippocampus. CONCLUSIONS: This rapid and differential regulation of synaptic vesicle proteins could be responsible for some of the morphological, biochemical, and behavioral changes observed after stress exposure. These changes may be relevant to such clinical disorders as psychoses, depression, and posttraumatic stress disorder that are sensitive to stress and involve changes in neural and synaptic plasticity.

Regulation of adult neurogenesis by antidepressant treatment.

Demonstration of neurogenesis in adult brain represents a major advance in our understanding of the cellular mechanisms underlying neuronal remodeling and complex behavior. Recent studies from our laboratory and others demonstrate that chronic administration of an antidepressant, including either a 5-HT or norepinephrine selective reuptake inhibitor, up-regulates neurogenesis in adult rodent hippocampus. Up-regulation of neurogenesis could block or reverse the effects of stress on hippocampal neurons, which include down-regulation of neurogenesis, as well as atrophy. The possibility that the cAMP signal transduction cascade contributes to the regulation of neurogenesis by antidepressants is supported by previous studies and by recent work. Although additional studies must be conducted to determine the significance of adult neurogenesis in humans, these findings will stimulate new avenues of research to identify the cellular and molecular basis of stress-related mood disorders as well as the development of novel therapeutic strategies.

Administration of a cAMP phosphodiesterase 4 inhibitor enhances antidepressant-induction of BDNF mRNA in rat hippocampus.

The influence of two selective phosphodiesterase 4 (PDE4) inhibitors, rolipram and Ro 20-1724, on the induction of BDNF mRNA by antidepressant treatment was examined. Coadministration of rolipram or Ro 20-1724 with an antidepressant (either desipramine or Org 4428) for 21 d resulted in a significant induction of BDNF mRNA in hippocampus relative to administration of vehicle. Coadministration of a PDE4 inhibitor with an antidepressant for 7 or 14 d also increased levels of BDNF mRNA. In contrast, acute coadministration did not influence levels of BDNF mRNA. In situ hybridization analysis demonstrated that the induction of BDNF mRNA in response to the repeated coadministration paradigm occurs in the dentate gyrus granule and CA1 and CA3 pyramidal cell layers of hippocampus. These findings demonstrate that coadministration shortens the time required for the upregulation of BDNF mRNA, supporting the possibility that this treatment may provide an effective therapy for major depression.