Husseini K. Manji - US grants

Bipolar affective disorder (manic-depressive illness) and unipolar depression are common, severe, chronic and often life-threatening illnesses. Impairment in physical and social functioning resulting from depression can be just as severe as other chronic medical illnesses. Recent preclinical and clinical studies suggest that the glutamatergic system is involved in the mechanism of action of antidepressants. In two separate trials, we tested riluzole (an inhibitor of glutamate release) and found it to have antidepressant properties in patients with unipolar and bipolar depression (Zarate et al. 2004, 2005). In another study, we found that the non-competitive NMDA antagonist (ketamine) was effective in treatment-resistant major depression. Ketamine resulted in rapid, robust and relatively sustained antidepressant effects. Response with ketamine occurred within 2 hours and last approximately 1 week (Zarate et al in press). The current protocol consists of 3 studies designed to address 3 major questions: [unreadable] [unreadable] Study 1 (Rapid improvement research in unipolar depression)[unreadable] OBJECTIVE: To determine whether a rapid antidepressant effect can be achieved with an antagonist at the N-methyl-D-aspartate receptor in subjects with major depression. DESIGN: A randomized, placebo-controlled, double-blind crossover study from November 2004 to September 2005. SETTING: Mood Disorders Research Unit at the National Institute of Mental Health.Patients Eighteen subjects with DSM-IV major depression (treatment resistant). INTERVENTIONS: After a 2-week drug-free period, subjects were given an intravenous infusion of either ketamine hydrochloride (0.5 mg/kg) or placebo on 2 test days, a week apart. Subjects were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days postinfusion.Main Outcome Measure Changes in scores on the primary efficacy measure, the 21-item Hamilton Depression Rating Scale. RESULTS: Subjects receiving ketamine showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week. The effect size for the drug difference was very large (d = 1.46 95% confidence interval, 0.91-2.01) after 24 hours and moderate to large (d = 0.68 95% confidence interval, 0.13-1.23) after 1 week. Of the 17 subjects treated with ketamine, 71% met response and 29% met remission criteria the day following ketamine infusion. Thirty-five percent of subjects maintained response for at least 1 week. CONCLUSIONS: Robust and rapid antidepressant effects resulted from a single intravenous dose of an N-methyl-D-aspartate antagonist; onset occurred within 2 hours postinfusion and continued to remain significant for 1 week.[unreadable] [unreadable] [unreadable] Study 2 (Rapid improvement research in bipolar depression)[unreadable] Does the NMDA antagonist ketamine produce rapid antidepressant effects in patients with treatment-resistant bipolar depression? Patients, ages 18 to 65 years with treatment-resistant bipolar depression will in a double-blind crossover study receive either intravenous ketamine or saline solution added to a mood stabilizer (lithium or valproate). [unreadable] [unreadable] Study 3 (Rapid and sustained improvement research in unipolar depression)[unreadable] Does riluzole (an inhibitor of glutamate release) prevent relapse in patients with treatment-resistant major depression who have rapidly responded to a single intravenous dose of ketamine? Patients, ages 18 to 65 years, with treatment-resistant major (unipolar) depression who have rapidly responded to a single intravenous dose of ketamine will in a double-blind study receive either riluzole or placebo to determine if the rapid response obtained can be sustained.[unreadable] [unreadable] Our primary hypotheses for these studies are: 1) rapid response (same or next day) can be achieved in patients with treatment-resistant major (unipolar) depression, 2) rapid response (same or next day) can be achieved in patients with treatment-resistant bipolar depression, and 3) rapid response (same or next day) can be sustained in patients with treatment-resistant unipolar depression.

The mood stabilizer lithium inhibits a select group of enzymes, including glycogen synthase kinase-3 (GSK-3). However, it is unclear if lithiums inhibition of GSK-3 is relevant for its antimanic and antidepressant effectiveness. We are utilizing biochemical, cellular, histochemical, genomic, and behavioral validation approaches to investigate whether the inhibition of GSK-3 is an integral part of the mechanism of lithiums clinical effects.[unreadable] [unreadable] We are utilizing rodent behavioral models and two distinct but complementary approaches (pharmacologic inhibition and transgenic gene expression) in an attempt to further validate GSK-3 as a possible mediator of lithiums therapeutic effects. Specifically, one of the primary targets of GSK-3 is the transcription factor beta-catenin. We have shown that lithium administration to rats, in a clinically relevant paradigm, results in an increase in beta- catenin levels. We are studying the effects of both over-expression and under-expression of beta-catenin in the mouse brain as well as pharmacological mechanisms to increase beta-catenin. Using both approaches, we have found the rodents exhibit both antidepressant-like and antimanic-like behavior. Combined, these data support the hypothesis that lithium may exert its antidepressant and [unreadable] antimanic effects through inhibition of GSK-3, and that novel small-molecule GSK-3 inhibitors may represent a truly novel class of medications useful for the treatment of bipolar disorder and depression. Validation of lithiums therapeutic target will require [unreadable] clinical trials with novel inhibitors, the development of which is in progress.[unreadable] It is now clear that severe mood disorders arise from abnormalities in cellular plasticity cascades that lead to aberrant information processing in critical circuits regulating mood, cognition, motor function, and neurovegetative symptomatology. In this context, it is noteworthy that GSK-3 is at the nexus of convergent neurotransmitter/ neurotrophic pathways, and demonstrates both anti-manic and antidepressant effects in mood-associated behaviors. In addition, AMPA receptor synaptic localization and phosphorylation are increased after antidepressant treatment, and decreased after treatment with anti-manic agents in vivo, therefore becoming a convergent point for therapeutic agents for mood disorders. We therefore sought to determine how GSK-3 regulates AMPA receptor trafficking and whether this mechanism is involved in mood-associated behaviors. We found that two distinct, small-molecule GSK-3 inhibitors decreased AMPA- and insulin-induced GluR1 and GluR2 internalization in cultured hippocampal neurons, suggesting that GSK-3 is involved in the regulation of AMPA receptor internalization. Treatment with AR-A014418 in mice significantly enhanced surface AMPA receptor levels in vivo in the hippocampus, but not in the frontal cortex, suggesting a regional-specific effect. The behavioral consequence of this stabilizing mechanism of GSK-3 inhibition to AMPA trafficking is under investigation. The novel mechanisms of GSK-3 regulation of AMPA receptor trafficking are anticipated to provide new mechanism for the treatment of these devastating disorders.

Kainate receptors (KARs) regulate the release of glutamate and GABA and synaptic plasticity in different brain regions involved in mood regulation, including the anterior-cingulate cortex, hippocampus, and amygdala. The KAR family includes five subunits: GluR5-7 and KA-1-2 (also called glutamate receptor ionotropic kainate (GRIK) 1-5 respectively). The receptors form hetereotrimers or homotrimers (GluR5-7 only). GluR5 and 6 can undergo RNA editing, resulting in altered channel permeability to different ions. The cell surface expression of the different KAR subunits is regulated by alternative splicing of receptor transcripts and trafficking properties of the receptor subtypes. The GluR6 gene is on chromosome 6q16.3-q21, a region of which has been implied in several bipolar disorder linkage studies. Recent genetic association studies directly implicates GluR6 as a contributing factor to increased risk of mood disorders. However, the roles of kainate receptors in mood regulation are largely unknown. Therefore we conducted studies in GluR5 and GluR6 knockout (KO) and wild-type control mice using a behavioral test battery for mood disorders and neurochemical experiments.
GluR5 and 6 KO mice appeared to attain normal growth, and lacked neurological abnormalities. Compared to WT and GluR5 KO mice, GluR6 KO consumed more saccharin sweeten solution, an indicator of hedonic activity. GluR6 KO traveled longer distances, entered and spent more time in the center of the open field (which can serve as indices for general activity as well as explorative-risk-taking activities). The hyperactivity of GluR6 KO persisted throughout the entire experimental period and they showed no habituation to the open-field arena. GluR6 KO exhibited aggressiveness in both the social interaction and resident-intruder tests. GluR6 KO had more activity in the open-arm of an elevated plus maze and less immobility in the forced swim test. GluR6 KO also exhibited higher responses in amphetamine challenge test. Thus, GluR6, but not GluR5, KO mice display behavioral excitement throughout experiments, greater aggressiveness, and hyperhedonia; these appear to phenocopy of the clinical manic state. Furthermore, chronic lithium treatment relieved key behavioral alterations of GluR6 KO mice including heightened locomotor activity, aggravated aggression and supersensitivity to amphetamine. Neurochemical experiments did not reveal any significant alterations of AMPA and NMDA receptors, however, hippocampal and prefrontal cortical membrane levels of GluR5 and KA-2 receptors were decreased in GluR6 KO mice, and chronic lithium treatment did not affect these decreases. Taken together, the data strongly support the notion that GluR6 neurotransmission is a critical modulator of mood related behavior and that genetic dysfunction of GluR6 neurotransmission can result in behavioral display of mania-like behavior. Future studies are needed to elucidate the effects of human GluR6 SNPs on the function of GluR6 and related neurotransmission, brain regions involved in GluR6 medicated mood-like behavioral plasticity, and GluR6 as novel targets for developing mood affecting agents.

There is now compelling evidence that radial glial cells have the potential, not only to guide newly born neurons, but also to self-renew and to generate both neurons and astrocytes. Recent data has also shown that astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold, demonstrating that CNS synapse numbers can be profoundly regulated by glia. Glial cells also play critical roles in regulating synaptic glutamate levels, CNS energy homeostasis, liberation of trophic factors, and form dynamic, complex synaptic networks with neurons. Nevertheless, the possibility of glial dysfunction in major psychiatric disorders has only recently received serious consideration due to the converging neuroimaging, postmortem morphometric and microarray studies, which have clearly revealed glial abnormalities in schizophrenia and mood disorders. To examine the effect of lithium (Li) on glia and neuron growth, we have established an astrocyte and neuronal primary culture system.[unreadable] [unreadable] We found that astrocytes, whose proliferation is increased by lithium, may indirectly (via liberation of factors from glial cells) regulate neuronal differentiation. Astrocytes may induce the pluripotent immature neuron to express an astrocytic phenotype. Next, we examined the alteration of cell signaling in astrocyte proliferation and neuronal differentiation to study the possible molecular mechanism of Li-induced action In addition, we examined whether Li affected growth of oligodendricytes, another type of glial cell. To investigate this more definitively, we began a series of in vitro and in vivo studies examining lithium's effects on oligodendrocytes. Chronic lithium treatment significantly increased the total number of oligodendrocytes in a dose-dependent manner, with a maximal effect observed with 1.0 mM lithium. To determine whether lithium affects BrdU incorporation, oligodendrocytes were treated with BrdU for 6 h in the absence or presence of lithium (1.0 mM). BrdU incorporation was determined by immunocytochemistry. BrdU-labeled cells were markedly increased by lithium treatment. O4 expression was examined by immunocytochemistry to further determine the cell phenotype of these BrdU-labeled cells. BrdU-positive cells were also O4+. Quantitatively, the percentage of BrdU-positive oligodendrocytes, as well as that of BrdU+O4+ cells, were significantly increased by the lithium treatment. Our data demonstrate for the first time that chronic lithium exerts a major effect on oligodendrocytes, increasing their proliferation. These observations raise the possibility that lithium may serve to correct abnormalities in white matter tracts, thereby restoring the functioning of critical circuits mediating affective, cognitive and motor symptom. These mechanisms may provide a potential target for improved long-term therapeutics for severe neuropsychiatric disorders.[unreadable] [unreadable] Recent evidence suggests that ATP acting via ionotropic (P2X) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. The P2X7 receptor, known as the cytolytic P2Z receptor, has been implicated in signaling between neuron and astrocytes, and has recently been postulated to represent a candidate gene for recurrent mood disorders. P2X7 receptors have been proposed as mediators of inflammation, and a potential role in neurodegeneration has been suggested. The P2X7 receptor shares 35-40% homology with other P2X receptors. It has two hydrophobic membrane-spanning domains and an extracellular loop, and forms transmembrane ion channels. Under normal conditions, extracellular nucleotides are present in only low concentrations. However, activated immune cells, such as lymphocytes, macrophages, microgli, and platelets, and dying cells may release high concentrations of different nucleotide di- and tri-phosphates into the extracellular space. Under inflammatory conditions, P2X7 receptor activation stimulates the induction of multiple cytokine pathways that may co-ordinate inflammatory responses, and triggers massive transmembrane ion fluxes (particularly influx of Ca2+ and Na+, and efflux of K+) and the formation of non-selective plasma membrane pores that result in cell death. In contrast to their neuronal counterpart, the function of P2X receptors in CNS glial cells is largely unknown. By Westen blot protein analysis, immunocytochemistry and immunohistochemistry, we examined expression of P2X7 receptors in astrocytes in vivo and in vitro and examined effect of lithium (Li) on the expression of P2X7 in astrocytes. We found that P2X7 receptor is expressed in astrocytes. P2X7 positive cells can be GFAP- and S100beta- positive, suggesting a colocalization of astrocyte proteins and P2X7 receptor in CNS. Moreover, we also found that Li (0.5-1.0 mM) resulted in significantly decreased expression of P2X7 receptor in cultured astrocytes. Finally, we also found chronic (5-day) Li-treatment significantly blocked ATP-induced influx of Ca2+ in astrocytes. Our data demonstrated that P2X7 is expressed in astrocytes, and its expression levels can be regulated by chronic treatment of lithium at therapeutically relevant concentrations. Considering P2X7 receptor.s role in the regulation of Ca2+ by ATP, this may be the molecular mechanism by which ATP-induced influx of Ca2+ can be blocked by lithium in astrocytes. Our data provide evidence showing that the P2X7 receptor in astrocytes may be a therapeutic target for mood disorder treatments[unreadable] [unreadable] Lithium has demonstrated to exert positive regulatory effects on cellular neurotrophic signaling, protecting neurons and astrocytes against a variety of insults. Lithium also has shown to robustly up-regulate the anti-apoptotic protein Bcl-2 expression in vitro and in vivo. Bcl-2 is localized to the ER and mitochondria and regulates calcium dynamics in these intracellular compartments. The ATP-gated calcium channel P2X7 is known to play a role in regulating calcium dynamics, and in glial and neuronal cytotoxicity. Therefore we sought to determine the role of Bcl-2 in lithium-mediated regulation on mitochondrial calcium uptake, utilizing Lenti-virus containing Bcl-2siRNA. Lithium dose-dependently increased Bcl-2 levels in astrocytes. BzATP (3005M) activated P2X7 ATP receptor and enhanced mitochondrial Ca2+ in cultured primary astrocytes. This effect was completely blocked by P2X7 antagonist ox-ATP, thus suggesting a P2X7- specific effect. We found that the mitochondrial calcium-uptake significantly increased during thapsigargin- (2uM) or P2X7-evoked intracellular calcium increase in lithium-treated astrocytes. This effect was abolished in Bcl-2 SiRNA lenti-virus infected cells compared to scrambled sequence infected cells (p< 0.001). These results suggest an essential role for Bcl-2 in lithium-mediated increase in mitochondrial-Ca++ uptake in astrocytes. These findings might shed light on new mechanisms for the neuroprotective effect of lithium and lead to the development of novel, improved therapeutics.

The mood stabilizers lithium and valproate are both effective in the treatment of bipolar disorder; however, their therapeutic mechanisms remain unclear. Because of the delayed onset of clinical efficacy (days to weeks), it has been proposed that adaptive changes in gene expression, rather than initial pharmacological actions, may be directly responsible. Using cDNA microarray as the initial screening method, we discovered that chronic administration of both agents at therapeutic doses increased the expression of BAG-1 (bcl-2 associated athanogene) in rat hippocampus. Furthermore, these findings were validated in the hippocampus at the protein level, the effects were seen in a time frame consistent with therapeutic effects, and were specific for mood stabilizers. BAG -1 is an important chaperone of bcl-2 (B-cell CLL/lymphoma 2), and enhances bcl-2_s anti-apoptotic functions; furthermore, through interaction with raf (v-raf-1 murine leukemia viral oncogene homolog 1), BAG-1 is able to activate ERK (extracellular signal-regulated protein kinase) MAP (mitogen-activated protein) kinases. Consistent with this, we previously found that lithium and valproate activate ERK MAP kinases. Bag-1 also inhibits GR (glucocorticoid receptor) activation, which may counteract the deleterious effects of hypercortisolemia seen in bipolar disorder. Anti-GR antibody immunostaining plus double staining with DAPI (4',6-Diamidino-2-phenylindole) showed either lithium or VPA, at therapeutically relevant levels, inhibited dexamethasone induced GR nuclear translocation. In addition, glucocorticoid response element (GRE) transfection assay showed lithium, at therapeutically relevant levels, inhibited GR activity in cultured human cells. Evaluated through siRNA (short interference RNA) silencing of BAG-1, the inhibition of mood stabilizers to GR nuclear translocation and to GR activity is mediated, at least in part, by BAG-1. The effect that BAG-1 inhibits glucocorticoid activation suggests mood stabilizers may counteract the deleterious effects of hypercortisolemia seen in bipolar disorder by up-regulating BAG-1. The role of BAG-1 in behavioral plasticity relevant to mood disorders was further investigated in wild-type and neuron-selective BAG-1 transgenic mice using a battery of behavioral tests. The BAG1 mice appeared normal in growth and in neurological and sensory tests. Mice were then subjected to behavioral tests related to anxiety, depression, and mania. BAG1 transgenic mice showed less anxious-like behavior on the elevated plus maze test. The mice did not differ from wild-type mice in the forced swim test, and exhibited comparable rates of developing helplessness behavior in the learned helplessness paradigm. However, the BAG1 transgenic mice showed higher spontaneous recovery rates from the helplessness behavior. On mania-related tests, BAG1 transgenic mice recovered much faster in the amphetamine-induced hyperlocomotion test, and displayed a clear resistance to cocaine-induced behavioral sensitization. BAG1 transgenic mice exhibited specific hippocampal neurochemical alterations including increased Hsp70 and decreased FKBP51 levels. Together, the data support that BAG-1 represent a novel, highly therapeutically relevant target in the long-term treatment of bipolar disorder and play role in mood stability. This role of BAG-1 will be further investigated in the BAG-1 knockout mice.

Our recent double blind clinical trials demonstrated that ketamine (an NMDA antagonist) produced rapid onset, robust, and long-pasting antidepressive actions in the patients who are resistant to typical antidepressant treatment. We have hypothesized that targeting AMPA/NMDA receptor throughput is an effective strategy for rapid relief of depression symptoms. To further test the AMPA/NMDA throughput hypothesis and to develop new medication based on this ketamine phenomenon, we conducted a series of animal experiments. We found that subanesthetic doses of ketamine treatment produced rapid onset antidepressant-like effects in two behavioral paradigms, the learned helpless test and the forced swim test. The effects lasted at least more than a week after a single drug administration. MK-801 (dizocilpine) and Ro25-6981, a NR2B selective antagonist, also exerted antidepressant-like effects; these effects, however, were not sustained as long as those of ketamine. Animals treated with ketamine in a similar regiment to the learned helpless test performed well on passive avoidance tests, thus the antidepressant-like effects of ketamine in animal is unlikely due to learning and memory deterioration associated with ketamine use. Pre-treatment with NBQX, an AMPA receptor antagonist, blocked ketamine-, MK-801- and Ro25-6981-induced immobility reductions in the forced swim test, indicating the antidepressant-like effects of these agents require AMPA throughput. Ketamine altered GluR1 phosphorylation in hippocampal tissues from ketamine treated animals, the effects was also blocked by NBQX pretreatment. Taken together, our animal findings strongly support the involvement of AMPA receptors in ketamine_s antidepressant-like action and the AMPA/NMDA throughput strategy to develop novel antidepressive agents. Future studies are required to further elucidate the receptor subtype(s) and the brain region(s) involved in this ketamine antidepressant-like action.

Mood disorders have traditionally been considered to be neurochemical disorders, but new evidence demonstrates impairments of structural plasticity and cellular resilience in the central nervous system. Recent preclinical and clinical studies have shown that signaling pathways involved in regulating cell survival and death are long-term targets for the actions of mood stabilizers. Lithium and valproate are common mood stabilizers for bipolar disorder and are shown to indirectly regulate cell pathways, including CREB, BDNF, Bcl-2, and MAP kinases, which may stimulate some of their delayed long-term beneficial effects. Specifically, lithium and valproate upregulate Bcl-2 in vivo and in vitro, preventing neuronal cell death through blocking mitochondrial program for apoptosis. In this study, rat primary cortical neurons and human neuroblastoma SH-SY5Y cells were treated chronically with valproate and lithium at therapeutically relevant concentrations. Mitochondrial protein levels were determined with western blot analysis; respiratory activity was measured using the Oxygraph system. Western blot analysis showed upregulation of porin (a voltage dependent anion selective channel) and anti-apoptotic proteins Bcl-2 and Bcl-x/L in the mitochondrial fraction of cells treated with lithium and valproate. Oxygraph measurements demonstrated that lithium and valproate increased respiratory rate both time- and dose-dependently. It is postulated that Bcl-xL and Bcl-2 may act to influence the properties of other outer membrane proteins to maintain their ability to pass complex anions. Recently it was reported that Bcl-xL can interact with porin and regulate its gating properties in vitro. It has also been shown that Bcl-2 over-expressing cells result in an increased mitochondrial volume and structural complexity. [unreadable] Since Bcl-2 is a key regulator of cell survival, we have used SiRNAs (small interfering RNAs) to knock down bcl-2 levels and determine the consequences of this manipulation on the biochemical effects of mood stabiliziers. In this study, we investigated the role of Bcl-2 as a regulator of mitochondrial function, through the use of siRNA knockdown techniques. Bcl-2 siRNA/pSilencer significantly knocked down Bcl-2 gene expression, resulting in 40% reductions in Bcl-2 protein levels in human neuroblastoma SH-SY5Y cells. 3 different dyes were used to study the effects of lithium and valproate (VPA) on quantity of mitochondria, mitochondrial membrane potential, mitochondrial oxidation and generation of free radicals (ROS) in neuronal cells. Mitotracker green becomes fluorescent in the lipid environment of mitochondria regardless of the membrane potential; JC-1 accumulates in proportion to the mitochondrial membrane potential; Mitotracker red oxidizes to a fluorescent product once inside the mitochondria and 2',7'-dichlorofluorescin diacetate (H2DCFDA) for assessing oxidative stress by staining ROS. Confocal microscope study showed that chronic lithium treatment increased mitochondrial membrane potential of the SH-SY5Y cells. The knockdown of Bcl-2 gene in SH-SY5Y cells was accompanied by clear reductions in mitochondrial oxidation and significantly reduced Bcl-2-protein levels. Previous studies from our lab showed that lithium and VPA enhanced Bcl-2 protein expression in vivo and in vitro. There is some evidence that mood stabilizers might stabilize mitochondrial function by enhancing their bioenergetic capacity and avoiding apoptosis through Bcl-2 dependent mechanisms. In order to elucidate how mood-stabilizers exert its neuroprotective effects, we investigated mitochondrial function after lithium and valproate treatment in vitro. Both, lithium- and VPA- treatments enhanced membrane potential and mitochondrial oxidation in SH-SY5Y human neuroblastoma cells in a time dependent manner. In addition, Bcl-2 siRNA significantly knocked down Bcl-2 gene expression and caused clear reductions in mitochondrial membrane potential and mitochondrial oxidation in lithium- or valproate- treated SH-SY5Y neuroblastoma cells. These findings indicate that mood stabilizers regulated mitochondrial function partially through the enhanced Bcl-2-gene expression in mitochondria. Bcl-2 mediated increase of mitochondrial function after lithium- or valproate- treatment may have utility in the long-term treatment of a variety of disorders where mitochondrial dysfunction contributes to the diseases pathophysiology/progression, it might be a novel therapeutics for the treatment of mitochondrial disorders. [unreadable] To further investigate the chronic effects of mood-stabilizers on mitochondrial function in vivo, adult Wistar rats were treated with lithium or VPA chow for 4 weeks, then challenged with neurotoxin methamphetamine (5mg/kg, i.p, 4 times in 8h). We found that chronic lithium and VPA treatment attenuated methamphetamine-induced decrease of Bcl-2 and increase of Bax in rat frontal cortex mitochondrial fraction. VPA treatment preserved rat frontal cortex mitochondrial electron transport complex IV-- cytochrome C oxidase activity, while lithium pretreatment significantly prevented methamphetamine-induced hyperthermia and high-dose toxic mortality. These novel results suggest that lithium and valproate may exert some of their long-term effects on neuroplasticity and cellular resilience via hitherto underappreciated effects on mitochondrial proteins and mitochondrial function.[unreadable] In addition, calcium dysfunction in BPD is considered the most reproducible biological abnormality described in BPD, and recent data support a role for calcium dysregulation in the mitochondrial and endoplasmic reticulum (ER) of individuals with BPD. Mitochondria and the ER critically regulate intracellular calcium signaling through several different mechanisms. Furthermore, the anti-apoptotic protein Bcl-2 has been shown to play an important role in regulating calcium kinetics within the ER and mitochondria. This role of Bcl-2 in intracellular calcium kinetics contributes to the modulation of many cellular functions, including gene expression, synaptic plasticity and cellular resilience. In addition, recent human genetic data suggest that single nucleotide polymorphism (SNPs) in the Bcl-2 gene may be associated with BPD. We thus sought to evaluate the functional role of Bcl-2 in intracellular calcium regulation in lymphoblasts from eighteen BPD patients with the SNP rs956572 (variants AA, AG, or GG, six subjects per group). We found that the Bcl-2 gene variant AA (associated with increased risk for BPD) decreased Bcl-2 levels, and significantly increased cell apoptosis. Using different calcium ratiometric dyes (Fura-2 and Fura-2FF), we observed increased baseline cytosolic calcium levels Reinforcing the role of Bcl-2 in calcium dynamics, baseline cytosolic calcium levels showed a significant inverse correlation with Bcl-2 levels in the total sample (r=-0.59). Furthermore, electron microscopy showed mitochondrial morphological alterations in some BPD samples. The results demonstrate for the first time that the BPD-associated SNP variations of Bcl-2 play a critical role in cytosolic calcium dynamics. Overall, these findings may provide a better understanding of the genetic association between BPD SNPs and the pathophysiology of this devastating illness.

Sleep deprivation is one of the only interventions that have consistently been demonstrated to produce rapid antidepressant effects. The mechanisms by which sleep deprivation brings about rapid antidepressant effects have not been elucidated. It is noteworthy; however, that recent genomic and proteomic studies have shown that acute sleep deprivation rapidly brings about an upregulation of several mediators of neuronal plasticity, most notably CREB and BDNF. Intriguingly, these very same molecules are upregulated by chronic antidepressants, and may underlie the delayed therapeutic effects of most antidepressants. Additional investigation of the regulation of CREB and BDNF by sleep deprivation has revealed that these changes are critically dependent upon the activation of the noradrenergic system. This is particularly noteworthy, since the locus coeruleus noradrenergic projection is quiescent only during rapid eye movement sleep (REM), when the target tissues display their greatest sensitivity; indeed, the temporal dissociation between the firing of the locus coeruleus noradrenergic neurons, and the sensitivity of its postsynaptic targets in the cortex may have considerable relevance for the antidepressant effects of sleep deprivation. In this context, biological rhythms have the capacity to temporally dissociate biochemical processes, and imposing a temporal coincidence on normally dissociated events can have striking and unexpected effects. Thus, it is our hypothesis that activating the normally quiescent noradrenergic system during REM sleep (i.e. when its postsynaptic target system displays its greatest sensitivity) will robustly upregulate CREB and BDNF, thereby bringing about a rapid antidepressant effect. We propose to activate the noradrenergic system during REM sleep by infusing an alpha-2 antagonist, yohimbine. Since it is our hypothesis that activating the noradrenergic system during REM sleep will bring about an antidepressant effect by a similar mechanism as sleep deprivation, we will enrich our sample with sleep deprivation responders in this pilot study.[unreadable] Patients, ages 18 to 60 with a diagnosis of major depressive disorder, currently depressed without psychotic features will be recruited into this study. This experimental proof-of-concept study has two Study Phases. Study Phase I consists of total sleep deprivation. Responders to total sleep deprivation who subsequently relapse will enter Study Phase II. Study Phase II is a double-blind crossover administration of either intravenous yohimbine or saline solution during REM sleep. [unreadable] The specific aim of this study is to assess the efficacy of a single dose of intravenous yohimbine hydrochloride (0.125 mg/kg given over 3 minutes) compared with placebo in improving overall depressive symptomatology when administered during REM sleep. [unreadable] Our primary hypothesis is that the intravenous use of an antagonist in patients with major depression during REM sleep will activate the LC and thus increase noradrenergic activity during a time when the locus coeruleus is normally quiescent- namely REM sleep. If the hypothesis that the timing of the activation of the noradrenergic system is crucial in the antidepressant effect of sleep deprivation is correct then an acute antidepressant effect should be observed in patients despite minimal to no disruption of sleep.

Chronic stress has been shown to be associated clinically with formation of depression in patients and hormones are known to mediate certain clinical manifestations of mood disorders. Chronic restraint stress induces a morphological reorganization in the areas of rodent brain, effects which are also accompanied by behavioral changes. Although the precise mechanisms underlying these effects remain to be elucidated, increasing data suggests that an alteration in neuroprotection and mitochondrial functions may play an important role in regulating various forms of synaptic and neural plasticity; we have sought to investigate the mitochondrial functions regulated by hormones during chronic stress. [unreadable] [unreadable] Cortical neuronal cultures were established in order to determine the localization and function of glucocorticoid receptors in the mitochondria. Glucocorticoid receptors translocated into mitochondria after 1.5 hour treatment with low concentration (100 nM) and high concentration (1,000 nM) of corticosterone in cultured cortical neurons. Consistent with the enhancement of mitochondrial function, mitochondrially encoded gene cytochrome oxidase I (COXI) (has GRE in its promoter region) expression was also increased in mitochondria fraction after 24 hours treatment. However, after three days of treatment, 1uM corticosterone resulted in a decrease in GR levels in mitochondria and 100nM corticosterone treatment did not. Similarly, mitochondrial membrane potential were enhanced after one day treatment with high (1uM) and low (100nM) concentration of corticosterone in a similar extend, and only high concentration (1uM) significantly decreased in mitochondrial membrane potential after 3 day treatment in comparison to the 100nM corticosterone. In addition, mitochondrial oxidation were enhanced after one day treatment with high (1uM) and low (100nM) concentration of corticosterone in a similar extend, and only high concentration (1uM) significantly decreased in mitochondrial membrane potential after 3 day treatment in comparison to the 100nM corticosterone and untreated control. To determine the situation under chronic stress, we found that glucocorticoid receptor levels in mitochondria were significantly decreased in the mitochondrial fraction from prefrontal cortex tissue after chronic stress, suggesting a similar change after high concentration and long-term corticosterone treatment in vitro. These studies may provide additional insights into the mechanisms by which glucocorticoid regulate mitochondrial function and neuronal signaling. Furthermore, this research also has the potential to contribute to a more complete understanding of the mechanisms by which chronic stress and hormones regulate cellular plasticity and resilience and to the future development of improved therapeutics.

Bipolar Disorder (BD) is a common, severe, chronic and often life-threatening illness. The discovery of lithium's efficacy as a mood-stabilizing agent has since revolutionized the treatment of patients with BD. Elucidation of the mechanism(s) by which lithium stabilizes an underlying dysregulation of limbic and limbic-associated function also offers the potential to delineate the underlying pathophysiology of BD; however, a major problem inherent in neuropharmacologic research is the difficulty in attributing therapeutic relevance to any observed biochemical finding. One powerful approach is to identify common biochemical targets, which are modified by drugs belonging to the same therapeutic class (e.g. mood-stabilizing agents) but possessing distinct chemical structures when administered in a "therapeutically relevant" paradigm (i.e., effects which are observed upon chronic drug administration, and yet persist beyond abrupt drug discontinuation). In this context, it is noteworthy that both valproic acid (VPA) and lithium, with different chemical structures, belong to the same therapeutic class and cause considerable inhibition of protein kinase C (PKC). The PKC signaling pathway is clearly a target for the actions of two structurally highly dissimilar antimanic agents -- lithium and VPA. Do these effects of lithium and VPA on PKC signaling have any clinical relevance? There is thus a clear need to investigate the potential efficacy of a direct PKC inhibitor in the treatment of acute mania. There is currently only one relatively selective PKC inhibitor available for human use- Tamoxifen. Tamoxifen (TAM), a synthetic nonsteroidal antiestrogen, has been widely used in the treatment of breast cancer. TAMs potent inhibitory effects on PKC are striking. Recently, our group conducted the first open-label study with TAM in acute mania. In this study, TAM resulted in a significant decrease in manic symptoms within a short period of time (3-7 days). The overarching goal of this proposal is to test the hypothesis that PKC inhibition is part of the mechanism of the therapeutic effect of mood stabilizing drugs. The proposal derives from and builds on our published open-label study of TAM in acute mania (Bebchuk et al., 2000). The study has been completed. In this double-blind, placebo-controlled study, 16 subjects with bipolar disorder, manic or mixed, with or without psychotic features were randomly assigned to receive tamoxifen (20-140 mg/day; n=8) or placebo (n=8) for 3 weeks. Subjects on tamoxifen showed significant improvement in mania compared to placebo as early as 5 days, an effect that remained significant throughout the 3 week trial. The effect size for the drug difference was very large (d=1.08, 95% C.I. = 0.45 to 1.71) after 3 weeks (p=.001). At study endpoint, response rates were 63% for tamoxifen and 13% for placebo.[unreadable] [unreadable] The study is now completed. We will now pursue to conduct clinical trials in acute mania with more selective PKC inhibitors.

Despite the availability of a wide range of antidepressant drugs, 30% to 40% of patients with major depression fail to respond to first-line antidepressant (e.g., selective serotonin reuptake inhibitors SSRIs) treatment, despite adequate dosage, duration, and compliance. Furthermore, these medications may take weeks to months to achieve their full effects, and in the meantime patients continue to suffer from their symptoms and continue to be at risk of self-harm as well as harm to their personal and professional lives. Thus, there is a clear need to develop novel and improved therapeutics for treatment-resistant major depression that are more effective and have a rapid onset of action. Preclinical and clinical studies suggest that antidepressants with a combined mechanism of action (e.g., combination of a selective serotonin-reuptake inhibitor (SSRI) and a norepinephrine reuptake inhibitor) may be more effective than either agent alone in achieving remission (Nelson et al 2004). Thus, it stands to reason that other combinations of antidepressants with other mechanisms of action when combined may have a synergistic effect that is superior to an antidepressant with a single mechanism of action. Preclinical and clinical studies suggest that the dopaminergic system may play a major role in the pathophysiology of depression. Preclinical studies suggest synergistic antidepressant effects with the combination of a SSRI and a selective D3 receptor agonist in animal models of depression. Similarly, preliminary clinical studies suggest synergism with combination treatment that affects the serotonin and dopamine systems. Together, these data suggest that treatments which affect the serotonin and dopamine systems will be more effective than agents which use a single mechanism. We propose to compare the combination of a selective dopaminergic agonist and a SSRI in patients with treatment-resistant major depression. To our knowledge, this will be the only controlled double-blind study to date that will examine the efficacy of a serotonin and dopamine combination given from the start of treatment.[unreadable] Patients, ages 18 years or older, with a diagnosis of major depression (without psychotic features), will be randomized to the combination of a selective dopaminergic receptor agonist and a SSRI or either drug alone for a period of 6 weeks. Acute efficacy will be determined by demonstrating a greater remission rate using specified criteria. Approximately 85 patients with acute major depression will be enrolled in the study.

Patients with bipolar depression often have indices of hypercortisolism, which potentially contribute to central nervous system dysfunction responsible for the cognitive and affective abnormalities in bipolar disorder. Recent data reveal that mifepristone is capable of ameliorating the mood and thought disturbances of psychotic depression. Clinical responsiveness to mifepristone in bipolar depression would implicate cortisol and an abnormal hypothalamic-pituitary axis (HPA) axis directly in mood abnormalities and specific cognitive deficits in bipolar depression. A detailed dissection of the HPA axis in bipolar patients should identify a distinctive mifepristone -responsive pathophysiological subtype based on neuroendocrine profile. Patients, in addition to careful clinical evaluation, will be carefully evaluated by physiologic stimuli that probe discreet aspects of HPA axis organization including diurnal rhythm, pulsatile secretion, feedback responsiveness to cortisol, and pituitary responsiveness to corticotrophin releasing hormone challenge. Patients will also receive formal neuropsychological and psychophysiological testing of various components of information processing, including those that are cortisol-dependent and associated with bipolar illness.

The treatments for acute unipolar depression have been extensively researched. However, despite the availability of a wide range of antidepressant drugs, clinical trials indicate that 30% to 40% of depressed patients fail to respond to first-line antidepressant treatment, despite adequate dosage, duration, and compliance. Very few studies have examined the efficacy of somatic treatments for the acute phase of bipolar depression. Thus, there is a clear need to develop novel and improved therapeutics for bipolar depression. Recent preclinical studies suggest that antidepressants may exert delayed indirect effects on the glutamatergic system. Clinical data suggests that lamotrigine an inhibitor of glutamate release and the NMDA antagonist ketamine may have antidepressant effects. Finally, our group recently found in two separate studies that the glutamate modulating agent riluzole was effective in treatment-resistant unipolar and bipolar depression (Zarate et al 2004; Zarate et al. 2005). Together, these data suggest that the glutamatergic system may play a role in the pathophysiology and treatment of depression, and that agents which more directly reduce glutamatergic neurotransmission, may represent a novel class of antidepressants. [unreadable] In this study, we propose to extend our findings from open-label studies with riluzole in treatment-resistant depression by investigating its efficacy in a double-blind placebo-controlled study in bipolar depression. [unreadable] Patients, ages 18 to 70 years with a diagnosis of bipolar disorder I or II current episode depressed (without psychotic features), will be randomized to double-blind treated to receive either riluzole (50-200 mg/day) or placebo for a period of 8 weeks. Acute efficacy will be determined by demonstrating a greater response rate using specified criteria. [unreadable] Approximately 78 patients with acute bipolar depression will be enrolled in this study.

Depression is a devastating illness that affects approximately 12 to 17% of the population at some point during the lifetime of an individual (Kessler et al 1994). Despite the availability of a wide range of antidepressant drugs, 30% to 40% of patients with major depression fail to respond to first-line antidepressant (e.g., selective serotonin reuptake inhibitors SSRIs) treatment, despite adequate dosage, duration, and compliance. Thus there is a clear need to develop novel and improved therapeutics for major depression. Current pathophysiological theories regarding the neurobiology of depression include alterations in intracellular signaling cascades, and impairments of cellular plasticity and resilience. There is recent evidence suggesting that promoting growth factors such as brain derived neurotrophic factor (BDNF) may provide a mechanism for the treatment of depression. New information indicating modulation of glutamate receptors in the actions of antidepressant treatments suggests a novel approach to develop a new class of antidepressants. Studies have shown that the biarylpropylsulfonamide AMPA receptor potentiators (LY392098 and LY451616) have antidepressant effects in animal models of depression. Several studies have demonstrated that AMPA receptor activation can increase expression of BDNF both in vitro and in vivo. Thus, one possible new approach for the treatment of depression is to use an AMPA receptor potentiator. [unreadable] In this study we propose to compare the ampakine receptor potentiator Org 24448 to placebo for the treatment of major depression. Inpatients and outpatients (primarily outpatients), ages 21-55, with a diagnosis of major depression (without psychotic features), will be randomized to double-blind treatment to either Org 24448 or placebo for a period of 8 weeks. Acute efficacy will be determined by demonstrating a greater response rate using specified criteria. Approximately 90 patients with acute major depression will be enrolled in the study in order to reach the goal of randomizing 70 patients in the controlled trial.

A growing body of data shows that the AMPA subtype of glutamate receptors play a major role in regulating short and long term forms of synaptic plasticity. Furthermore, it is now clear that regulation of plasticity occurs predominantly by regulating the trafficking of AMPA receptor subunits, and their insertion and removal from the synapse. Notably, this trafficking is primarily dependent upon AMPA receptor subunit phosphorylation by 3 major signaling pathways known to be targets for mood stabilizers: the PKC, PKA and MAPK cascades. In view of the growing body of data suggesting that severe mood disorders may be associated with impairments of cellular plasticity, we undertook the present series of studies to determine if two clinically effective, but structurally highly dissimilar antimanic agents, lithium and VPA, regulate synaptic expression of AMPA receptor subunit GluR1. Administration of chronic lithium or valproate (at therapeutically relevant concentrations) reduced rat hippocampal synaptosomal levels of GluR1 after by 40% and 20%, respectively. In cultured hippocampal neurons, both lithium and VPA significantly down regulated the surface expression of GluR1 40% in a dose and time-dependent manner. Surface staining with an anti-N terminal GluR1 antibody confirmed the result. Double-immunostaining of GluR1 and synaptotagmin showed that chronic treatment attenuated the numbers of GluR1 positive synapses of lithium and valproate-treated neurons. However, total protein levels of GluR1, and synaptotagmin remained unchanged after lithium and valproate treatment in vitro and in vivo. Lithium and valproate treatment also attenuated the phosphorylation of a specific PKA site (GluRp845) by 52 and 33% respectively. Sp-cAMP treatment reversed the attenuation of phosphorylation by lithium and valproate and also brought GluR1s back to the surface, suggesting that phosphorylation of GluRp845 is involved in the mechanism of GluR1 surface attenuation. In striking contrast, drugs, such as imipramine, which induce mania, increase the synaptic expression of GluR1 in vivo in hippocampus. These studies suggest that regulation of glutamatergically mediated synaptic plasticity may play a role in the treatment of mood disorders, and raises the possibility that agents more directly affecting synaptic GluR1 may represent novel therapies for this devastating illness.[unreadable] [unreadable] In order to develop a new potential drug which mimics the effect of mood stabilizers on GluR1 phosphorylation, TAT-peptides (TAT-p845 and TAT-SRC) were designed and synthesized. Tat peptide (YGRKKRRQRRR) is a leading peptide, which enables the functional peptide to pass through the blood brain barrier and cell membrane, allowing it to get into cytosol or synapses of the neurons. A previous study has successfully utilized peptide injection into animals to disrupt the interaction of PSD-95 with NMDA receptors in the brain and to provide a neuroprotective effect on a stroke animal model. TAT-p845 was able to inhibit the phosphorylation of AMPA receptors at its PKA site and down-regulate the surface expression of GluR1 in cultured hippocampal neurons, which is the same effect produced by lithium and valproate. Moreover, this TAT-p845 was able to pass the blood brain barrier and inhibit the phosphorylation of GluR1 in the hippocampus in vivo, which again demonstrated its ability to induce the same effects as lithium and valproate. In addition, reduction of GluR1 phosphorylation at its PKA site by Tat-p845, was sufficient to attenuate synaptic GluR1/2 in hippocampal neurons in vivo. Intra-hippocampal infusion of AMPA-specific inhibitor GYKI54226, GluR1-specific TAT-p845 peptide and GluR1-PDZ-specific TAT-TGL peptide were able to attenuate amphetamine-induced hyperactivity and/or amphetamine-induced conditioned-place preference in the mania animal model. These studies provide novel mechanisms for anti-manic effect through attenuationof AMPA receptor activity and avenues for new drug development for mood disorders. TAT-p845, which attenuates AMPA receptor levels at synapses, may offer exciting possibilities as a new class of medicine with the potential for treatment of: bipolar disorder. [unreadable] [unreadable] Chronic treatment with the antimanic agents, lithium and valproate resulted in reduced synaptic expression of the AMPA receptor subunit GluR1 in the hippocampus while treatment with an antidepressant (imipramine) enhanced the synaptic expression of GluR1. The anticonvulsants, lamotrigine and riluzole have been demonstrated to have efficacy in the depressive phase of bipolar disorder. We therefore sought to determine the role of these anticonvulsants, compared to that of the predominantly antimanic anticonvulsant valproate, on AMPA receptor localization. We found that the agents with a predominantly antidepressant profile, namely lamotrigine and riluzole, significantly enhanced the surface expression of GluR1 and GluR2 in a time- and dose-dependent-manner in cultured hippocampal neurons. By contrast, the predominantly antimanic agent, valproate, significantly reduced surface expression of GluR1 and GluR2. Concomitant with the GluR1 and GluR2 changes, the peak value of depolarized membrane potential evoked by AMPA was significantly higher in lamotrigine and riluzole treated neurons, supporting the surface receptor changes. Phosphorylation of GluR1 at the PKA site (S845) was enhanced in both lamotrigine- and riluzole- treated hippocampal neurons, but reduced in valproate treated neurons. In addition, lamotrigine and riluzole, as well as the traditional antidepressant imipramine, also increased GluR1 phosphorylation at GluR1 (S845) in the hippocampus after chronic in vivo treatment. Our findings suggest that regulation of GluR1/2 surface levels and function may be responsible for the different clinical profile of anticonvulsants (antimanic or antidepressant), and may suggest avenues for the development of novel therapeutics for these illnesses.

Mood and anxiety disorders are common psychiatric disorders that affect patients chronically and can adversely complicate the prognosis of other medical conditions. As stressful life events are often associated with the development of depressive- and anxiety-like behaviors, both disorders have been described as stress-related. Stress, particularly when chronic, can aggravate and increase susceptibility for developing depression, but does not seem to confer the disorder per se. In contrast, acute stress has been shown to aid in the development of anxiety-like symptoms as well as lead to specific anxiety disorders such as post-traumatic stress disorder (PTSD). The cellular and molecular mechanisms underlying depressive and anxiety disorders remain largely unknown. Clinically, these disorders show high rates of co-morbidity and both respond to antidepressant therapy, but the extent to which they share underlying mechanisms is unclear. [unreadable] One approach to studying the pathogenesis of these disorders is to examine how the brain controls the physiological and behavioral response to acute and chronic stressors. Upon exposure to an acutely stressful situation, the hypothalamic-pituitary-adrenal (HPA) axis is activated, resulting in a rapid increase in plasma glucocorticoid (GC) levels. The acute stress system also encompasses the sympathomedullary component, which induces the secretion of adrenalin from the adrenal medulla, leading to physiological changes including an increase in body temperature and heart rate as well as the classical fright, fight or flight responses. A critical brain structure that exerts powerful negative regulation on the stress-response system by inhibiting the HPA axis is the hippocampus, which receives and processes information to determine what is stressful and whether or not the body needs to mount an appropriate, adaptive response. While critical for the animal's short-term survival, these stress responses can become harmful if uncontrolled, leading to maladaptation that results in an elevated HPA response. This can ultimately lead to damage and atrophy of neurons in the hippocampus as well as development of depressive- and anxiety-like phenotypes. [unreadable] Interestingly, acute stress also enables long-term depression (LTD) in the adult hippocampus, which, unlike the juvenile hippocampus, does not express LTD under normal conditions. In contrast to the well-studied role of long-term potentiation (LTP) in learning and memory, the biological functions of hippocampal LTD, especially in the adult, remain obscure. Recent studies have begun to provide some clues. Blockade of LTD in the nucleus accumbens (NAc) dampens amphetamine-induced behavioral sensitization. Also, in the hippocampus, genetic manipulation that impairs LTD is correlated with behavioral inflexibility. These findings suggest that LTD may be a cellular mechanism to ensure adequate or proper behavioral responses to environmental changes. In the present study, we hypothesized that acute, stress-enabled hippocampal LTD might be a necessary coping mechanism to aid the organism in its recovery effort. We attempted to develop strategies that selectively block stress-enabled hippocampal LTD. One such strategy was to take advantage of p75NTR knockout (KO) mice, which have recently been shown to exhibit no LTD in the adolescent hippocampus. Surprisingly, the p75NTR mutants behave anxiously under stressful conditions, without any signs of depression. In the adult brain, p75NTR is primarily expressed in basal forebrain cholinergic neurons (BFCN), which have a major cholinergic projection to the hippocampus via the medial septum. Inhibition of the cholinergic transmission by the muscarinic antagonist scopolamine also blocked stress-enabled LTD, leading to anxiety. A final strategy is to block LTD directly using a specific peptide that prevents AMPA receptor endocytosis. Systemic injection of the peptide inhibitor exacerbated anxiety-like behavior after the animal was exposed to an acute stressor. Thus, anxiety- and depressive-like behaviors can be separated mechanistically, and p75NTR-cholinergic transmission-LTD might be a major pathway that the brain uses to manage stress-induced anxiety.

A growing body of data shows that the AMPA subtype of glutamate receptors play a major role in regulating short and long term forms of synaptic plasticity. Furthermore, it is now clear that regulation of plasticity occurs predominantly by regulating the trafficking of AMPA receptor subunits, and their insertion and removal from the synapse. Notably, this trafficking is primarily dependent upon AMPA receptor subunit phosphorylation by 3 major signaling pathways known to be targets for mood stabilizers: the PKC, PKA and MAPK cascades. In view of the growing body of data suggesting that severe mood disorders may be associated with impairments of cellular plasticity, we undertook the present series of studies to determine if two clinically effective, but structurally highly dissimilar antimanic agents, lithium and VPA, regulate synaptic expression of AMPA receptor subunit GluR1. Administration of chronic lithium or valproate (at therapeutically relevant concentrations) reduced rat hippocampal synaptosomal levels of GluR1 after by 40% and 20%, respectively. In cultured hippocampal neurons, both lithium and VPA significantly down regulated the surface expression of GluR1 40% in a dose and time-dependent manner. Surface staining with an anti-N terminal GluR1 antibody confirmed the result. Double-immunostaining of GluR1 and synaptotagmin showed that chronic treatment attenuated the numbers of GluR1 positive synapses of lithium and valproate-treated neurons. However, total protein levels of GluR1, and synaptotagmin remained unchanged after lithium and valproate treatment in vitro and in vivo. Lithium and valproate treatment also attenuated the phosphorylation of a specific PKA site (GluRp845) by 52 and 33% respectively. Sp-cAMP treatment reversed the attenuation of phosphorylation by lithium and valproate and also brought GluR1s back to the surface, suggesting that phosphorylation of GluRp845 is involved in the mechanism of GluR1 surface attenuation. In striking contrast, drugs, such as imipramine, which induce mania, increase the synaptic expression of GluR1 in vivo in hippocampus. [unreadable] In order to develop a new potential drug which mimics the effect of mood stabilizers on GluR1 phosphorylation, TAT-peptides (TAT-p845 and TAT-SRC) were designed and synthesized. Tat peptide (YGRKKRRQRRR) is a leading peptide, which enables the functional peptide to pass through the blood brain barrier and cell membrane, allowing it to get into cytosol or synapses of the neurons. A previous study has successfully utilized peptide injection into animals to disrupt the interaction of PSD-95 with NMDA receptors in the brain and to provide a neuroprotective effect on a stroke animal model. TAT-p845 was able to inhibit the phosphorylation of AMPA receptors at its PKA site and down-regulate the surface expression of GluR1 in cultured hippocampal neurons, which is the same effect produced by lithium and valproate. Moreover, this TAT-p845 was able to pass the blood brain barrier and inhibit the phosphorylation of GluR1 in the hippocampus in vivo, which again demonstrated its ability to induce the same effects as lithium and valproate. In addition, reduction of GluR1 phosphorylation at its PKA site by Tat-p845, was sufficient to attenuate synaptic GluR1/2 in hippocampal neurons in vivo. Intra-hippocampal infusion of AMPA-specific inhibitor GYKI54226, GluR1-specific TAT-p845 peptide and GluR1-PDZ-specific TAT-TGL peptide were able to attenuate amphetamine-induced hyperactivity and/or amphetamine-induced conditioned-place preference in the mania animal model. These studies provide novel mechanisms for anti-manic effect through attenuationof AMPA receptor activity and avenues for new drug development for mood disorders. TAT-p845, which attenuates AMPA receptor levels at synapses, may offer exciting possibilities as a new class of medicine with the potential for treatment of: bipolar disorder. [unreadable] [unreadable] Chronic treatment with the antimanic agents, lithium and valproate resulted in reduced synaptic expression of the AMPA receptor subunit GluR1 in the hippocampus while treatment with an antidepressant (imipramine) enhanced the synaptic expression of GluR1. The anticonvulsants, lamotrigine and riluzole have been demonstrated to have efficacy in the depressive phase of bipolar disorder. We therefore sought to determine the role of these anticonvulsants, compared to that of the predominantly antimanic anticonvulsant valproate, on AMPA receptor localization. We found that the agents with a predominantly antidepressant profile, namely lamotrigine and riluzole, significantly enhanced the surface expression of GluR1 and GluR2 in a time- and dose-dependent-manner in cultured hippocampal neurons. By contrast, the predominantly antimanic agent, valproate, significantly reduced surface expression of GluR1 and GluR2. Concomitant with the GluR1 and GluR2 changes, the peak value of depolarized membrane potential evoked by AMPA was significantly higher in lamotrigine and riluzole treated neurons, supporting the surface receptor changes. Phosphorylation of GluR1 at the PKA site (S845) was enhanced in both lamotrigine- and riluzole- treated hippocampal neurons, but reduced in valproate treated neurons. In addition, lamotrigine and riluzole, as well as the traditional antidepressant imipramine, also increased GluR1 phosphorylation at GluR1 (S845) in the hippocampus after chronic in vivo treatment. Our findings suggest that regulation of GluR1/2 surface levels and function may be responsible for the different clinical profile of anticonvulsants (antimanic or antidepressant), and may suggest avenues for the development of novel therapeutics for these illnesses.In addition, we found that glutamate receptors are targets of Protein Kinase C (PKC), which plays an important role in the pathophysiology and treatment of mania. Considerable biochemical evidence suggests that the protein kinase C (PKC) signaling cascade may be a convergent point for the actions of anti-manic agents, and that excessive PKC activation can disrupt prefrontal cortical regulation of thinking and behavior. Currently, however, brain protein targets of PKCs anti-manic effects remains unclear. Here we showed that PKC activity was enhanced in the prefrontal cortex brain region of animals treated with the psychostimulant amphetamine and the antidepressant imipramine. Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a marker of PKC activity, was increased in the prefrontal cortex of psychstimulant amphetamine treated animals, as well as in sleep-deprived animals (another animal model of mania), but decreased in lithium-treated animals. Antidepressant imipramine, which shows promanic property on bipolar patients, also enhanced pMARCKS in prefrontal cortex in vivo. We further explored the functional targets of PKC in mania-associated behaviors. Neurogranin is a brain-specific, postsynaptically located PKC substrate. PKC phosphorylation of neurogranin was robustly increased by pro-manic manipulations and decreased by anti-manic agent. PKC phosphorylation of the NMDA receptor site NR1S896 and the AMPA receptor site GluR1T840 was also enhanced in the prefrontal cortex of animals treated with antidepressant imipramine, as well as behaviorally sleep-deprived, in striking contrast to the reduced activity seen in lithium-treated animals. These results suggest that PKC may play an important role in regulating NMDA and AMPA receptor functions. The biochemical profile of the PKC pathway thus encompasses both pro- and anti-manic effects on behavior. These results suggest that PKC modulators or their intracellular targets may ultimately represent novel avenues for the development of new therapeutics for mood disorders.