Rajesh R. Ugale

Brain Chromatin Remodeling: A Novel Mechanism of Alcoholism

The treatment of alcoholism requires the proper management of ethanol withdrawal symptoms, such as anxiety, to prevent further alcohol use and abuse. In this study, we investigated the potential role of brain chromatin remodeling, caused by histone modifications, in alcoholism. We found that the anxiolytic effects produced by acute alcohol were associated with a decrease in histone deacetylase (HDAC) activity and increases in acetylation of histones (H3 and H4), levels of CREB (cAMP-responsive element binding) binding protein (CBP), and neuropeptide Y (NPY) expression in the amygdaloid brain regions of rats. However, the anxiety-like behaviors during withdrawal after chronic alcohol exposure were associated with an increase in HDAC activity and decreases in acetylation of H3 and H4, and levels of both CBP and NPY in the amygdala. Blocking the observed increase in HDAC activity during alcohol withdrawal with the HDAC inhibitor, trichostatin A, rescued the deficits in H3 and H4 acetylation and NPY expression (mRNA and protein levels) in the amygdala (central and medial nucleus of amygdala) and prevented the development of alcohol withdrawal-related anxiety in rats as measured by the elevated plus maze and light/dark box exploration tests. These results reveal a novel role for amygdaloid chromatin remodeling in the process of alcohol addiction and further suggest that HDAC inhibitors may be potential therapeutic agents in treating alcohol withdrawal symptoms.

Effector Immediate-Early Gene Arc in the Amygdala Plays a Critical Role in Alcoholism

The immediate early gene, activity-regulated cytoskeleton-associated protein (Arc), has been implicated in synaptic plasticity. However, the role of Arc in alcoholism is unknown. Here, we report that the anxiolytic effects of acute ethanol were associated with increased brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (trkB) expression, increased phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), Elk-1, and cAMP responsive element-binding protein (CREB), increased Arc expression, and increased dendritic spine density (DSD) in both the central amygdala (CeA) and medial amygdala (MeA) but not in the basolateral amygdala (BLA) of rats. Conversely, the anxiogenic effects of withdrawal after long-term ethanol exposure were associated with decreased BDNF and trkB expression, decreased phosphorylation of Erk1/2, Elk-1, and CREB, decreased Arc expression, and decreased DSD in both the CeA and MeA but not in the BLA of rats. We also showed that BDNF infusion into the CeA normalized phosphorylation of Erk1/2, Elk-1, and CREB, and normalized Arc expression, thereby protecting against the onset of ethanol withdrawal-related anxiety. We further demonstrated that arresting Arc expression in the CeA decreased DSD, thereby increasing anxiety-like and alcohol-drinking behaviors in control rats. These results revealed that BDNF–Arc signaling and the associated DSD in the CeA, and possibly in the MeA, may be involved in the molecular processes of alcohol dependence and comorbidity of anxiety and alcohol-drinking behaviors.

Antidepressant like effect of selective serotonin reuptake inhibitors involve modulation of imidazoline receptors by agmatine.

Recent findings demonstrated the dysregulation of imidazoline receptor binding sites in major depression and their normalization by chronic treatment with antidepressants including selective serotonin reuptake inhibitors (SSRIs). Present study investigated the role of agmatine and imidazoline receptors in antidepressant like effect of SSRIs and imipramine in mouse forced swimming test (FST) paradigm. The antidepressant like effect of fluoxetine or paroxetine was potentiated by imidazoline I(1)/I(2) receptor agonist agmatine (5-10 mg/kg, ip), imidazoline I(1) receptor agonists, moxonidine (0.25-0.5 mg/kg, ip) and clonidine (0.015-0.03 mg/kg, ip), imidazoline I(2) receptor agonist, 2-(2-benzofuranyl)-2-imidazoline (5-10 mg/kg, ip) as well as by the drugs known to increase endogenous agmatine levels in brain viz., L-arginine, an agmatine biosynthetic precursor (40 microg/mouse, icv), ornithine decarboxylase inhibitor, difluoromethyl ornithine (12.5 microg/mouse, icv), diamine oxidase inhibitor, aminoguanidine (6.5 microg/mouse, icv) and agmatinase inhibitor, arcaine (50 microg/mouse, icv). Conversely, prior administration of I(1) receptor antagonist, efaroxan (1 mg/kg, ip), I(2) receptor antagonist, idazoxan (0.25 mg/kg, ip) and arginine decarboxylase inhibitor, d-arginine (100 mg/kg, ip) blocked the antidepressant like effect of paroxetine (10 mg/kg, ip) and fluoxetine (20 mg/kg, ip). On the other hand, antidepressant like effect of imipramine was neither augmented nor attenuated by any of the above drugs. Mice pretreated with SSRIs but not imipramine and exposed to FST showed higher concentration of agmatine in brain as compared to saline control. This effect of SSRIs on agmatine levels was completely blocked by arginine decarboxylase inhibitor d-arginine but not by imidazoline receptor antagonists, efaroxan or idazoxan. These results demonstrate that modulation of imidazoline receptors by agmatine are implicated in the antidepressant like effect of SSRIs and may be projected as a potential therapeutic target for the treatment of depressive disorders.

Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats.

Present study investigated the role of agmatine in ethanol-induced anxiolysis and withdrawal anxiety using elevated plus maze (EPM) test in rats. The anxiolytic-like effect of ethanol was potentiated by pretreatment with imidazoline I(1)/I(2) receptor agonist agmatine (10-20 mg/kg, i.p.), imidazoline I(1) receptor agonists, moxonidine (0.25 mg/kg, i.p.) and clonidine (0.015 mg/kg, i.p.), imidazoline I(2) receptor agonist, 2-BFI (5 mg/kg, i.p.) as well as by the drugs known to increase endogenous agmatine levels in brain viz., L-arginine, an agmatine biosynthetic precursor (100 microg/rat, i.c.v.), ornithine decarboxylase inhibitor, DFMO (125 microg/rat, i.c.v.), diamine oxidase inhibitor, aminoguanidine (65 microg/rat, i.c.v.) and agmatinase inhibitor, arcaine (50 microg/rat, i.c.v.). Conversely, prior administration of I(1) receptor antagonist, efaroxan (1 mg/kg, i.p.), I(2) receptor antagonist, idazoxan (0.25mg/kg, i.p.) and arginine decarboxylase inhibitor, D-arginine (100 microg/rat, i.c.v.) blocked the anxiolytic-like effect of ethanol. Moreover, ethanol withdrawal anxiety was markedly attenuated by agmatine (10-20 mg/kg, i.p.), moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.), 2-BFI (5 mg/kg, i.p.), L-arginine (100 microg/rat, i.c.v.), DFMO (125 microg/rat, i.c.v.), aminoguanidine (65 microg/rat, i.c.v.) and arcaine (50 microg/rat, i.c.v.). The anti-anxiety effect of agmatine in ethanol-withdrawn rats was completely blocked by efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.). These results suggest that agmatine and imidazoline receptor system may be implicated in ethanol-induced anxiolysis and withdrawal anxiety and strongly support further investigation of agmatine in ethanol dependence mechanism. The data also project agmatine as a potential therapeutic target in overcoming alcohol withdrawal symptoms such as anxiety.

Neuropeptide Y Signaling in the Central Nucleus of Amygdala Regulates Alcohol-Drinking and Anxiety-Like Behaviors of Alcohol-Preferring Rats

BACKGROUND The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol-preferring (P) rats as an animal model. METHODS Alcohol-preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety-like behaviors were assessed by the 2-bottle free-choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT-PCR. Double-immunofluorescence labeling was performed to observe the colocalization of NPY and Ca(2+)/calmodulin-dependent protein kinase IV (CaMK IV). RESULTS We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element-binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic alpha-subunit of protein kinase A (PKA-Calpha) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA-Calpha without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV-positive cells were co-localized with NPY in amygdaloid structures of P rats. CONCLUSIONS These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV-dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.

Role of Neuroactive Steroid Allopregnanolone in Antipsychotic-like Action of Olanzapine in Rodents

Olanzapine increases brain allopregnanolone (ALLO) levels sufficiently to modulate neuronal activity by allosterically regulating GABAA receptors. Recently, we reported the antipsychotic-like profile of ALLO in rodents. The present study examined the hypothesis that olanzapine-induced elevation of endogenous neurosteroid ALLO is vital for its neuroleptic-like action. The conditioned avoidance response (CAR) and apomorphine-induced climbing behavioral paradigms were used in rodents. Administration of ALLO (1 μg, intracerebroventricular (i.c.v.)) or neurosteroidogenic agents such as the mitochondrial diazepam binding inhibitor receptor agonist, FGIN 1-27 (0.5 μg, i.c.v.) or the ALLO precursor, progesterone (10 mg/kg, i.p.) significantly potentiated olanzapine-induced blockade of CAR and apomorphine-induced climbing. In contrast, these agents failed to alter the antipsychotic-like effect of risperidone and haloperidol. On the other hand, inhibition of the endogenous biosynthesis of neurosteroids by the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane (30 mg/kg, i.p.), the 3α-hydroxysteroid oxidoreductase inhibitor, indomethacin (5 mg/kg, i.p.), or the GABAA receptor antagonist bicuculline (1 mg/kg, i.p.) and dehydroepiandrosterone sulfate (DHEAS) (1 mg/kg, i.p.) blocked the effect of olanzapine, but not of risperidone and haloperidol. Socially isolated animals, known to exhibit decreased brain ALLO and GABAA receptor functions, displayed a shortening in the muscimol-induced loss of righting reflex and an increased susceptibility to apomorphine-induced climbing. Administration of olanzapine, but not of haloperidol and risperidone, normalized the duration of muscimol-elicited loss of righting reflex. Although all three antipsychotics proved capable of antagonizing the apomorphine-induced climbing, a dose almost five times higher of olanzapine was required in socially isolated animals. The data obtained suggest that enhancement of the GABAergic tone plays a key role in the antipsychotic-like effect exerted by olanzapine in rodents, likely as a consequence of augmented levels of neuroactive steroids, in particular ALLO, in the brain. The present findings provide the first specific behavioral evidence in support of the hypothesis that neuroactive steroid ALLO- mediated GABAergic modulation is essential for the antipsychotic-like action of olanzapine.

Essentiality of central GABAergic neuroactive steroid allopregnanolone for anticonvulsant action of fluoxetine against pentylenetetrazole-induced seizures in mice.

Fluoxetine, a selective serotonin reuptake inhibitor, is known to increase the cortical content of allopregnanolone (ALLO) without altering the level of other neurosteroids. In contrast to the proconvulsant effect of many antidepressants, fluoxetine exhibits anticonvulsant effects. The present study was undertaken to examine the role of ALLO in the anticonvulsant action of fluoxetine against pentylenetetrazole (PTZ)-induced seizures in mice. Prior administration of GABA(A) receptor agonist muscimol or neurosteroid ALLO or progesterone, a precursor of ALLO or neurosteroidogenic drugs like FGIN 1-27, an agonist at the mitochondrial diazepam binding inhibitor receptor (MDR) or metyrapone, an 11beta-hydroxylase inhibitor, significantly potentiated the anticonvulsant effect of fluoxetine. In contrast, the effect of fluoxetine was counteracted by inhibition of the neurosteroid biosynthesis using drugs like 5alpha-reductase inhibitor, finasteride; 3beta-hydroxysteroid dehydrogenase inhibitor, trilostane; 3alpha-hydroxysteroid dehydrogenase inhibitor, indomethacin; MDR antagonist, PK 11195; or the GABA(A) receptor antagonist, bicuculline. Further, bilateral adrenalectomy had no significant effect on the anticonvulsant action of fluoxetine, suggesting negligible contribution from peripheral steroidogenesis. The anticonvulsant effect of fluoxetine was partially abolished in 5,7-DHT treated mice, indicating that the effect may also, in part, be dependent on serotonergic transmission. Thus, our data indicate that increased synthesis of ALLO in CNS is a major factor that ultimately leads to anticonvulsant effects of fluoxetine against PTZ-induced seizures.

Neurosteroid allopregnanolone mediates anxiolytic effect of etifoxine in rats

Etifoxine (6-chloro-2-ethylamino-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride), a nonbenzodiazepine anxiolytic drug, potentiates GABA(A) receptor function perhaps through stimulation of neurosteroid biosynthesis. However, the exact mechanism of etifoxine action is not fully understood. In this study, we have assessed the possible role of GABAergic neurosteroid like allopregnanolone (ALLO) in the anxiolytic-like effect of etifoxine in rats using elevated plus maze test. Selective GABA(A) receptor agonist, muscimol, ALLO or neurosteroidogenic agents like progesterone, metyrapone or mitochondrial diazepam binding inhibitor receptor (MDR) agonist, FGIN 1-27 significantly heightened the etifoxine-induced anxiolysis. On the other hand, GABA(A) receptor antagonist, bicuculline or neurosteroid biosynthesis inhibitors like finasteride, indomethacin, trilostane or PBR antagonist, PK11195 significantly blocked the effect of etifoxine. Bilateral adrenalectomy did not influence anti-anxiety effect of etifoxine thereby ruling out contribution of adrenal steroids. Thus, our results provide behavioral evidence for the role of neurosteroids like ALLO in the anti-anxiety effect of etifoxine.

Agmatine in the hypothalamic paraventricular nucleus stimulates feeding in rats: involvement of neuropeptide Y

BACKGROUND AND PURPOSE Agmatine, a multifaceted neurotransmitter, is abundantly expressed in the hypothalamic paraventricular nucleus (PVN). Our aim was to assess (i) the effect of agmatine on feeding behaviour and (ii) its association, if any, with neuropeptide Y (NPY).

GluN2C/GluN2D subunit-selective NMDA receptor potentiator CIQ reverses MK-801-induced impairment in prepulse inhibition and working memory in Y-maze test in mice.

Despite ample evidence supporting the N‐methyl‐d‐aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, progress in the development of effective therapeutics based on this hypothesis has been limited. Facilitation of NMDA receptor function by co‐agonists (d‐serine or glycine) only partially alleviates the symptoms in schizophrenia; other means to facilitate NMDA receptors are required. NMDA receptor sub‐types differ in their subunit composition, with varied GluN2 subunits (GluN2A‐GluN2D) imparting different physiological, biochemical and pharmacological properties. CIQ is a positive allosteric modulator that is selective for GluN2C/GluN2D‐containing NMDA receptors (Mullasseril et al.).