Chronic antidepressant treatment selectively increases expression of plasticity-related proteins in the hippocampus and medial prefrontal cortex of the rat

Antidepressants protect against hippocampal volume loss in humans and reverse stress-induced atrophic changes in animals thus supporting the hypothesis that the pathophysiology of stress-related disorders such as depression involves reductions in neuronal connectivity and this effect is reversible by antidepressant treatment. However, it is unclear which brain areas demonstrate such alterations in plasticity in response to antidepressant treatment. The aim of the present study was to examine the effect of antidepressant treatment on the expression of three plasticity-associated marker proteins, the polysialylated form of nerve cell adhesion molecule (PSA-NCAM), phosphorylated cyclic-AMP response element binding protein (pCREB) and growth-associated protein 43 (GAP-43), in the rat brain. To this end, rats were treated either acutely (60 min) or chronically (21 days) with imipramine (30 and 15 mg/kg, respectively) and the expression of PSA-NCAM, pCREB, and GAP-43 was assessed using immunohistochemistry. Initial mapping revealed that chronic imipramine treatment increased expression of these plasticity-associated proteins in the hippocampus, medial prefrontal cortex and piriform cortex but not in the other brain regions examined. Since PSA-NCAM and pCREB are expressed in recently-generated neurons in the dentate gyrus, it is likely that chronic imipramine treatment increased their expression in the hippocampus at least partially by increasing neurogenesis. In contrast, since chronic imipramine treatment is not associated with neurogenesis in the medial prefrontal cortex, increased expression of PSA-NCAM and pCREB in the prelimbic cortex implicates changes in synaptic connectivity in this brain region. Acute treatment with imipramine increased the number of pCREB positive nuclei in the hippocampus and the prefrontal cortex but did not alter expression of GAP-43 or PSA-NCAM in any of the brain regions examined. Taken together, the results of the present study suggest that antidepressant treatment increases synaptic plasticity and connectivity in brain regions associated with mood disorders.     

Chronic restraint stress and chronic corticosterone treatment modulate differentially the expression of molecules related to structural plasticity in the adult rat piriform cortex

Stress and stress-related hormones induce structural changes in neurons of the adult CNS. Neurons in the hippocampus, the amygdala and the prefrontal cortex undergo neurite remodeling after chronic stress. In the hippocampus some of these effects can be mimicked with chronic administration of adrenal steroids. These changes in neuronal structure may be mediated by certain molecules related to plastic events such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). The expression of PSA-NCAM persists in the adult hippocampus and it is up-regulated after chronic stress. The piriform cortex also displays considerable levels of PSA-NCAM during adulthood and indirect evidence suggests that it may also be the target of stress and stress related-hormones. Using immunohistochemistry we have studied the expression of PSA-NCAM and doublecortin (DCX; another protein implicated in neuronal structural plasticity) in the piriform cortex of adult rats subjected either to 21 days of chronic restraint stress or to oral corticosterone administration during the same period. Our results indicate that chronic stress and chronic corticosterone administration have differential effects on the expression of PSA-NCAM and DCX. While chronic stress increases the number of PSA-NCAM- and DCX-immunoreactive cells in the piriform cortex layer II, chronic corticosterone administration decreases these numbers. These findings indicate that stress and adrenal steroids affect the piriform cortex and suggest that in this region, as in the hippocampus, they may induce structural changes. This is a potential mechanism by which stress and corticosterone modulate functions of this limbic region, such as its participation in olfactory memory.     

PSA-NCAM expression in the human prefrontal cortex

The prefrontal cortex (PFC) of adult rodents is capable of undergoing neuronal remodeling and neuroimaging studies in humans have revealed that the structure of this region also appears affected in different psychiatric disorders. However, the cellular mechanisms underlying this plasticity are still unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) may mediate these structural changes through its anti-adhesive properties. PSA-NCAM participates in neurite outgrowth and synaptogenesis and changes in its expression occur parallel to neuronal remodeling in certain regions of the adult brain. PSA-NCAM is expressed in the hippocampus and temporal cortex of adult humans, but it has not been studied in the PFC. Employing immunohistochemistry on sections from the rostromedial superior frontal gyrus we have found that PSA-NCAM is expressed in the human PFC neuropil following a laminated pattern and in a subpopulation of mature neurons, which lack doublecortin expression. Most of these cells have been identified as interneurons expressing calbindin. The expression of PSA-NCAM in the human PFC is similar to that of rodents. Since this molecule has been linked to the neuronal remodeling found in experimental models of depression, it may also participate in the structural plasticity described in the PFC of depressed patients.     

Chronic restraint stress down-regulates amygdaloid expression of polysialylated neural cell adhesion molecule

The amygdala is a brain area which plays a decisive role in fear and anxiety. Since exposure to chronic stress can induce profound effects in emotion and cognition, plasticity in specific amygdaloid nuclei in response to prior stress has been hypothesized to account for stress-induced emotional alterations. In order to identify amygdala nuclei which may be affected under chronic stress conditions we evaluated the effects of 21-days chronic restraint stress on the expression of a molecule implicated crucially in alterations in structural plasticity: the polysialylated neural cell adhesion molecule. We found that polysialylated neural cell adhesion molecule-immunoreactivity within the amygdala, present in somata and neuronal processes, has a regional gradient with the central medial and medial amygdaloid nuclei showing the highest levels. Our results demonstrate that chronic restraint stress induced an overall reduction in polysialylated neural cell adhesion molecule-immunoreactivity in the amygdaloid complex, mainly due to a significant decrease in the central medial amygdaloid and medial amygdaloid nuclei. Our data suggest that polysialylated neural cell adhesion molecule in these nuclei may play a prominent role in functional and structural remodeling induced by stress, being a potential mechanism for cognitive and emotional modulation. Furthermore, these finding provide the first clear evidence that life experiences can regulate the expression of polysialylated neural cell adhesion molecule in the amygdaloid complex.     

Differential evolution of PSA-NCAM expression during aging of the rat telencephalon

Changes in the ability of neuronal networks to undergo structural remodeling may be involved in the age-associated cognitive decline. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) declines dramatically during postnatal development, but persists in several regions of the young-adult rat telencephalon, where it participates, through its anti-adhesive properties, in neuronal structural plasticity. However, PSA-NCAM expression during aging has only been studied in the dentate gyrus and the piriform cortex layer II, where it is strongly downregulated in adult (middle-aged) individuals. Using immunohistochemistry, we have observed that in most of the telencephalic areas studied the number of PSA-NCAM expressing cells and the intensity of PSA-NCAM expression in the neuropil remains stable during aging. Old rats only show decreases in the number of PSA-NCAM expressing cells in the lateral amygdala and retrosplenial cortex, and in neuropil expression of stratum lucidum. Given the role of PSA-NCAM in neuronal plasticity, the present results indicate that, even during aging, many regions of the CNS may display neurite, spine or synaptic remodeling.     

Chronic fluoxetine treatment induces structural plasticity and selective changes in glutamate receptor subunits in the rat cerebral cortex

It has been postulated that chronic administration of antidepressant drugs induces delayed structural and molecular adaptations at glutamatergic forebrain synapses that might underlie mood improvement. To gain further insight into these changes in the cerebral cortex, rats were treated with fluoxetine (flx) for 4 weeks. These animals showed decreased anxiety and learned helplessness. N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit levels (NR1, NR2A, NR2B, GluR1 and GluR2) were analysed in the forebrain by both western blot of homogenates and immunohistochemistry. Both methods demonstrated an upregulation of NR2A, GluR1 and GluR2 that was especially significant in the retrosplenial granular b cortex (RSGb). However, when analysing subunit content in postsynaptic densities and synaptic membranes, we found increases of NR2A and GluR2 but not GluR1. Instead, GluR1 was augmented in a microsomal fraction containing intracellular membranes. NR1 and GluR2 were co-immunoprecipitated from postsynaptic densities and synaptic membranes. In the immunoprecipitates, NR2A was increased while GluR1 was decreased supporting a change in receptor stoichiometry. The changes of subunit levels were associated with an upregulation of dendritic spine density and of large, mushroom-type spines. These molecular and structural adaptations might be involved in neuronal network stabilization following long-term flx treatment.     

Calcineurin (protein phosphatase 2B) is involved in the mechanisms of action of antidepressants

Calcineurin (PP2B) is a Ca(2+)-dependent protein phosphatase enriched in the brain that takes part in intracellular signaling pathways regulating synaptic plasticity and neuronal functions. Calcineurin-dependent pathways are important for complex brain functions such as learning and memory. More recently, they have been suggested to play a role in the processing of emotional information. The aim of this study was to investigate whether calcineurin may be involved in the effect of antidepressants. We first found that chronic antidepressant treatment in mice leads to an increase of calcineurin levels in the hippocampus. We then studied the behavioral and molecular responses to fluoxetine of mice with a genetic overactivation of calcineurin in the hippocampus (constitutively-activated calcineurin transgenic mouse line #98, CN98 mice). We observed that CN98 mice are more sensitive to the behavioral effect of fluoxetine and desipramine tested in the tail suspension test. Moreover, the basal expression of growth factor brain-derived neurotrophic factor and subunit 1 of AMPA glutamate receptor, GluR1, both of which are modified after chronic antidepressant administration, are altered in the hippocampus of CN98 mice. These results suggest that calcineurin-dependent dephosphorylation plays an important role in the mechanisms of action of antidepressants, providing a new starting point for developing improved therapeutic treatments for depression.     

Glucocorticoid receptor-dependent desensitization of 5-HT1A autoreceptors by sleep deprivation: studies in GR-i transgenic mice

Sleep deprivation for one night induces mood improvement in depressed patients, an action that probably involves the serotonergic (5-HT) system. In animals, sleep deprivation and pharmacologic treatment with antidepressants exert similar effects on 5-HT neurotransmission, notably functional desensitization of 5-HT1A autoreceptors located on 5-HT neurons in the dorsal raphe nucleus (DRN). However, in stressful conditions, corticosterone can also induce a desensitization of these autoreceptors. STUDY OBJECTIVES: To investigate the mechanisms of this adaptation during sleep deprivation and the possible involvement of corticosterone, we studied the effects of an 18-hour sleep deprivation, by forced locomotion, on 5-HT1A receptor-mediated firing response of DRN 5-HT neurons in transgenic mice with impaired glucocorticoid-receptor expression (GR-i) and in wild-type animals. We also examined the effects of chronic treatment with the antidepressant drug fluoxetine in the same paradigm. MEASUREMENTS AND RESULTS: In both wild-type and GR-i mice, the 18-hour sleep deprivation or fluoxetine treatment had no effect on the spontaneous firing of 5-HT neurons recorded under anesthesia. However, sleep deprivation decreased the potency of the 5-HT1A agonist 8-OH-DPAT to inhibit 5-HT neuronal firing in wild-type mice, whereas it had no effect in GR-i animals. Conversely, after chronic fluoxetine treatment, the induced reduction of this 5-HT1A autoreceptor-driven response was of larger amplitude in GR-i than in wild-type mice. CONCLUSIONS: These data suggest that glucocorticoid-receptor activation by corticosterone participates in the antidepressant-like adaptive changes in 5-HT1A autoreceptors in sleep-deprived mice. On the other hand, GR-i animals exhibited enhanced 5-HT1A autoreceptor desensitization induced by fluoxetine, in line with data in other animal models of depression.     

Alterations in the expression of PSA-NCAM and synaptic proteins in the dorsolateral prefrontal cortex of psychiatric disorder patients

Alterations in the structure and physiology of the prefrontal cortex (PFC) have been found in different psychiatric disorders and some of them involve inhibitory networks, especially in schizophrenia and major depression. Changes in the structure of these networks may be mediated by the polysialylated neural cell adhesion molecule (PSA-NCAM), a molecule related to neuronal structural plasticity, expressed in the PFC exclusively by interneurons. Different studies have found that PSA-NCAM expression in the hippocampus and the amygdala is altered in schizophrenia, major depression and animal models of these disorders, in parallel to changes in the expression of molecules related to inhibitory neurotransmission and synaptic plasticity. We have analyzed post-mortem sections of the dorsolateral PFC from the Stanley Neuropathology Consortium, which includes controls, schizophrenia, bipolar and major depression patients, to check whether similar alterations occur. PSA-NCAM was found in neuronal somata and neuropil puncta, many of which corresponded to interneurons. PSA-NCAM expression was only reduced significantly in schizophrenic patients, in parallel to a decrease in glutamic acid-decarboxylase-67 (GAD67) and to an increased expression of vesicular glutamate transporter 1 (VGLUT1) in the white matter. Depressed patients showed significant decreases in synaptophysin (SYN) and VGLUT1 expression. Whereas in bipolar patients, decreases in VGLUT1 expression have also been found, together with a reduction of GAD67. These results indicate that the expression of synaptic proteins is altered in the PFC of patients suffering from these disorders and that, particularly in schizophrenia, abnormal PSA-NCAM and GAD67 expression may underlie the alterations observed in inhibitory neurotransmission.     

Distribution of PSA-NCAM expression in the amygdala of the adult rat

Synaptic plasticity in the amygdala appears to be necessary for the generation of emotional memories. However, the molecular bases of this plasticity are not fully understood. Because the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) has been implicated in memory consolidation in the hippocampus and temporal cortex, we have studied in detail the expression of this molecule in the adult rat amygdala with an antibody against PSA-NCAM. Our results demonstrate for the first time the presence of PSA-NCAM in the adult rat amygdala. Immunoreactive somata and processes are abundant in the amygdalo-hippocampal transition area, central nucleus, intra-amygdaloid bed nucleus of the stria terminalis, anterior and posterior cortical nuclei, periamygdaloid cortex and medial nucleus of the amygdala. In addition PSA-NCAM immunoreactive neuronal somata and processes exist in the lateral, basal and accessory basal nuclei, anterior amygdaloid area and amygdalo-striatal area. The presence of this molecule in areas that receive olfactory or vomeronasal input could reflect the intrinsic plasticity of these chemosensory systems. PSA-NCAM expression in the lateral amygdala could indicate its participation in the plastic events that lead to the generation of emotional memories such as those related to fear conditioning.     

Chronic antidepressant treatment alters serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons

The serotonin system is highly involved in the pathophysiology of mood disorders such as depression and anxiety. Currently, the most widely used treatment for these illnesses is selective serotonin (5-HT)reuptake inhibitors, such as fluoxetine. Because of the multiplicity of 5-HT receptors and their different adaptive properties, the chronic effects of fluoxetine have remained unclear. In this study, we investigated the alteration of 5-HT functions by long-term antidepressant treatment in pyramidal neurons of prefrontal cortex (PFC), a brain region crucial for the control of emotion and cognition. One prominent function of serotonin in PFC is to regulate GABAergic inhibitory transmission. Application of 5-HT induced a large, desensitizing enhancement of the amplitude and frequency of spontaneous inhibitory postsynaptic currents (sIPSC), as well as a potent reduction of electrically evoked IPSC (eIPSC). Chronic fluoxetine treatment did not alter basal sIPSC, but reduced eIPSC in response to different stimulus strengths. Moreover, chronic (but not acute) fluoxetine treatment caused a much faster desensitization of the 5-HT effect on sIPSC, and significantly attenuated the 5-HT effect on eIPSC. Application of a 5-HT(2) receptor agonist produced similar effects as 5-HT on sIPSC and eIPSC, and these effects were similarly altered by long-term fluoxetine treatment. These electrophysiological results suggest that chronic antidepressant treatment resulted in a down-regulation of the synaptic function of forebrain 5-HT(2) receptors. Given the key role of GABAergic inhibitory transmission in controlling PFC functions, its altered regulation by serotonin after chronic fluoxetine treatment may provide a mechanism underlying the therapeutic action of antidepressants.     

Cerebellar granular cell cultures as an in vitro model for antidepressant drug-induced neurogenesis

Both preclinical and clinical evidence suggested that antidepressant drugs upregulate hippocampal cell proliferation and neurogenesis. In addition, direct evidence was recently published that hippocampal de novo cell proliferation is necessary for antidepressant action. Within this frame, we used primary cultures of rat cerebellar granule cells (CGC) as an in vitro model of central nervous system (CNS) to investigate whether a neurogenic response could be elicited also in the cerebellum, upon chronic treatment with selective serotonin reuptake inhibitors (SSRIs). Furthermore, we assayed the presence of neural precursor cells in CGC, possibly responsive to proliferation and differentiation stimuli. We found that 1 microM fluoxetine increased cell proliferation, as assayed by [3H]-thymidine incorporation. CGC immunocytochemical analysis with neural cell-specific markers revealed the presence of granule neurons, glial cells, and a cell component that we named "round cells." Because only round cells displayed proliferation ability, as revealed by 5-bromo-2'-deoxyuridine (BrdU) labeling, they were further characterized. For this purpose, round cells were isolated and expanded by culturing in a serum-free medium, containing basic fibroblast growth factor (bFGF), before immunocytochemical analysis. We found that round cells were not immunoreactive for glial, neuronal, and oligodendrocyte markers, whereas they were immunoreactive for several immature neuronal markers. Accordingly, round cells could be induced to differentiate into astrocytes, neurons, and oligodendrocytes, either by withdrawing the mitogen bFGF or by exposing them to fluoxetine. These findings suggest that round cells in CGC possess the features and potentials of neural precursors, able to differentiate in mature neural cells upon a pharmacological simulum.     

G protein-activated inwardly rectifying K+ channel inhibition and rescue of weaver mouse motor functions by antidepressants

Antidepressants, including tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), have been widely used for the treatment of not only depression but also other psychiatric disorders, although the molecular mechanisms of the drug effects have not yet been sufficiently revealed. Here, we investigated the in vivo effects of these antidepressants on G protein-activated inwardly rectifying K+ (GIRK) channels, which are important for regulating the excitability of various cells, by using weaver (wv) mice, which have mutant GIRK channels and show abnormal neuronal cell death and motor disturbances. First, we found that a widely used SSRI fluoxetine (also known as Prozac) effectively inhibited wv GIRK2 channels like wild-type GIRK channels, expressed in Xenopus oocytes. Next, we found that weaver motor disturbances were remarkably alleviated by chronic treatment with fluoxetine or desipramine. Furthermore, the chronic fluoxetine treatment substantially suppressed the abnormal neuronal cell death in the weaver mouse cerebellum and pontine nuclei. These results suggest that continuous inhibition of wv GIRK2 channels by a group of antidepressants caused substantial suppression of the neuronal cell death and resulted in improvement of motor abilities in weaver mice. These results provide evidence for in vivo GIRK channel inhibition by a group of antidepressants.     

Antidepressants reverse corticosterone-mediated decrease in brain-derived neurotrophic factor expression: differential regulation of specific exons by antidepressants and corticosterone

Earlier studies have implicated brain-derived neurotrophic factor in stress and in the mechanism of action of antidepressants. It has been shown that antidepressants upregulate, whereas corticosterone downregulates, brain-derived neurotrophic factor expression in rat brain. Whether various classes of antidepressants reverse corticosterone-mediated downregulation of brain-derived neurotrophic factor is unclear. Also not known is how antidepressants or corticosterone regulates brain-derived neurotrophic factor expression. To clarify this, we examined the effects of various classes of antidepressants and corticosterone, alone and in combination, on the mRNA expression of total brain-derived neurotrophic factor and of individual brain-derived neurotrophic factor exons, in rat brain. Normal or corticosterone pellet-implanted (100 mg, 21 days) rats were injected with different classes of antidepressants, fluoxetine, desipramine, or phenelzine, intraperitoneally for 21 days and killed 2 h after the last injection. mRNA expression of total brain-derived neurotrophic factor and of exons I-IV was measured in frontal cortex and hippocampus. Given to normal rats, fluoxetine increased total brain-derived neurotrophic factor mRNA only in hippocampus, whereas desipramine or phenelzine increased brain-derived neurotrophic factor mRNA in both frontal cortex and hippocampus. When specific exons were examined, desipramine increased expression of exons I and III in both brain areas, whereas phenelzine increased exon I in both frontal cortex and hippocampus but exon IV only in hippocampus. On the other hand, fluoxetine increased only exon II in hippocampus. Corticosterone treatment of normal rats decreased expression of total brain-derived neurotrophic factor mRNA in both brain areas, specifically decreasing exons II and IV. Treatment with desipramine or phenelzine of corticosterone pellet-implanted rats reversed the corticosterone-induced decrease in total brain-derived neurotrophic factor expression in both brain areas; however, fluoxetine reversed the decrease only partially in hippocampus. Interestingly, antidepressant treatment of corticosterone pellet-implanted rats increased only those specific exons that are increased during treatment of normal rats with each particular antidepressant. We found that although corticosterone and antidepressants both modulate brain-derived neurotrophic factor expression, and antidepressants reverse the corticosterone-induced brain-derived neurotrophic factor decrease, antidepressants and corticosterone differ in how they regulate the expression of brain-derived neurotrophic factor exon(s).     

GABA, depression and the mechanism of action of antidepressant drugs: a neuroendocrine approach

Recent evidence has suggested the involvement of the GABAergic system in depression and in the mechanism of action of somatic antidepressant treatments. In particular, GABAB receptors have been found to be increased in the rat frontal cortex following chronic antidepressant therapies. In the present study, the sensitivity of GABAB binding sites was assessed in nine healthy men and 10 depressed patients via the plasma growth hormone (GH) response to acute baclofen administration (20 mg p.o.). Depressed subjects were tested before and after 15 and 35 days of treatment with amitriptyline (100 mg/day), imipramine (100 mg/day) and fluoxetine (20 mg/day). GH response to acute GABAB receptor activation did not differ between depressed subjects and healthy controls. Moreover, chronic antidepressant treatment did not significantly modify this response, even when a clear therapeutic effect was obtained. These results do not support the idea that GABAergic mechanisms are involved in the pathophysiology of depression and in the mechanism of action of antidepressant drugs.     

Structural basis for the role of inhibition in facilitating adult brain plasticity

Although inhibition has been implicated in mediating plasticity in the adult brain, the underlying mechanism remains unclear. Here we present a structural mechanism for the role of inhibition in experience-dependent plasticity. Using chronic in vivo two-photon microscopy in the mouse neocortex, we show that experience drives structural remodeling of superficial layer 2/3 interneurons in an input- and circuit-specific manner, with up to 16% of branch tips undergoing remodeling. Visual deprivation initially induces dendritic branch retractions, and this is accompanied by a loss of inhibitory inputs onto neighboring pyramidal cells. The resulting decrease in inhibitory tone, also achievable pharmacologically using the antidepressant fluoxetine, provides a permissive environment for further structural adaptation, including addition of new synapse-bearing branch tips. Our findings suggest that therapeutic approaches that reduce inhibition, when combined with an instructive stimulus, could facilitate restructuring of mature circuits impaired by damage or disease, improving function and perhaps enhancing cognitive abilities.     

Abnormal 5-HT modulation of stress behaviors in the Kv4.2 knockout mouse

The Kv4.2 gene codes for an essential subunit of voltage-gated A-type potassium channels that are involved in dendritic signal integration and synaptic plasticity. Detailed cellular characterization in CA1 pyramidal neurons of the hippocampus has shown that knocking out the Kv4.2 gene increases neuronal excitability and promotes long-term potentiation. However, the overall behavioral consequences of these modifications have not been fully explored. Given the growing connection between neuronal plasticity and affect processing in the hippocampus and other Kv4.2 expressing regions, we proposed to investigate whether the absence of this gene would alter the stress response of mice to the forced swimming and tail suspension tests (TSTs) for depression-like behavior. Kv4.2 knockout (KO) mice, generated in the 129SvEv background, demonstrated elevated immobility and a loss of swimming, as well as antidepressant resistance to the selective 5-HT reuptake inhibitor fluoxetine (FLX). Characterization of a relatively new head movement behavior category, responsive to serotonergic treatment in wildtype (WT) mice, supported conclusions of abnormal 5-HT modulation. Electrophysiology recordings in the prefrontal cortex showed a blunting of postsynaptic response to direct 5-HT application following a single period of swim stress only in the animals without the Kv4.2 subunit. Based on our findings, we hypothesize that Kv4.2 KO mice may have an exaggerated 5-HT response to stress leading to a premature desensitization of postsynaptic receptors and a loss of continued behavior modulation. These results may shed some light on the involvement of A-type potassium channels in the effective action of selective serotonin reuptake inhibitor (SSRI) antidepressants