Characterization of the picrotoxin site of GABAA receptors.

This unit describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics, and competition assays (IC50, Ki determination) are included.

The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription.

Background: The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription.

Characterization and pharmacology of the GHB receptor.

Radioligand binding using [(3)H]NCS-382, an antagonist of the GHB receptor, revealed specific binding sites in the rat cerebrocortical and hippocampal membranes. Scatchard analysis of saturation isotherms revealed two different populations of binding sites. NCS-382 was about 10 times more potent than GHB in inhibiting [(3)H]NCS-382 binding.

Effect of 5-azacytidine on the methylation aspects of NMDA receptor NR2B gene in the cultured cortical neurons of mice.

Our previous study revealed that the exposure of the drug 5-Azacytidine and ethanol to the cultured cortical neurons of mice causes demethylation of cytosine residues in the CpG island of the NMDA receptor NR2B gene (Marutha Ravindran and Ticku, Mol Brain Res 121:19-27, 2004). In the present study, we further analyzed methylation in the CpG island with various concentration frame and time frame of exposure of the cultured cortical neurons with 5-azacytidine to identify whether methylation in the NR2B gene is site specific or region specific. Methylation was studied by digesting the genomic DNA with methylation sensitive HpaII, MspI, AciI or HhaI enzyme following the exposure of cultured cortical neurons of mice with 5-azacytidine by performing PCR and Southern hybridization.

Emerging role of epigenetics in the actions of alcohol.

This review deals with the recent developments on the epigenetic effects of ethanol. A large body of data have come from studies in liver and in neuronal systems and involve post-translational modifications in histones and methylations in DNA. Ethanol causes site selective acetylation, methylation, and phosphorylation in histone.

Effect of chronic administration of ethanol on the regulation of the delta-subunit of GABA(A) receptors in the rat brain.

In the present study, we investigated the effect of chronic ethanol (CE) administration on the polypeptide levels of the delta-subunit of GABA(A) receptors and [(3)H]muscimol binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat brain. CE administration resulted a down-regulation of polypeptide levels of the delta-subunit of GABA(A) receptors in the rat cerebellum and hippocampus, whereas there were no changes in the delta-subunit polypeptide levels in the rat cerebral cortex. Further, CE administration caused a down-regulation of native delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum as determined by [(3)H]muscimol binding to the immunoprecipitated receptor assemblies.

Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain.

Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons.

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression.

Effect of chronic administration of ethanol on the regulation of tyrosine kinase phosphorylation of the GABAA receptor subunits in the rat brain.

One of the many pharmacological targets of ethanol is the GABA inhibitory system, and chronic ethanol (CE) is known to alter the polypeptide levels of the GABA(A )receptor subunits in rat brain regions. In the present study, we investigated the regulation of the tyrosine kinase phosphorylation of the GABA(A) receptor alpha(1)-, beta(2)- and gamma(2)-subunits in the rat cerebellum, cerebral cortex and hippocampus following chronic administration of ethanol to the rats. We observed either down-regulation or no change in the tyrosine kinase phosphorylation of the alpha(1) subunit, whereas there was an up-regulation or no change in the case of beta(2)- and gamma(2)-subunits of the GABA(A) receptors depending on the brain region following chronic administration of ethanol to the rats.

Regulation of platelet-derived growth factor signaling pathway by ethanol, nicotine, or both in mouse cortical neurons.

Background: The higher incidence of smoking among alcoholic subjects suggests the presence of common molecular mechanisms underlying nicotine and alcohol use and abuse. However, these mechanisms are largely unknown. By using cultured fetal mouse cortical neurons as a model system, we sought to identify genes and pathways that are modulated in the cells by ethanol, nicotine, or both.

Tyrosine kinase phosphorylation of GABA(A) receptor alpha1, beta2 and gamma2 subunits following chronic intermittent ethanol (CIE) exposure of cultured cortical neurons of mice.

There is evidence that many of the GABA(A) receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol's regulation of GABA(A) receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the beta(2)- and gamma(2)- subunits and no effect on alpha(1)-subunit of the GABA(A) receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA(A) receptor subunits (alpha(1), beta(2), and gamma(2)) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques.

Comparison of chronic ethanol and chronic intermittent ethanol treatments on the expression of GABA(A) and NMDA receptor subunits.

We examined the mRNA and protein levels of GABA(A) and NMDA receptor (NMDAR) subunits in cultured mouse cortical neurons following exposure to chronic ethanol (CE) or chronic intermittent ethanol (CIE), and after 5 days of withdrawal. With respect to GABA(A) receptor mRNA, both treatments decreased the levels of alpha1 and alpha2 subunits, and increased the level of alpha4. However, only CE treatment caused parallel changes in the protein levels; alpha2 and alpha4 protein levels did not change after CIE.

Tyrosine kinase phosphorylation of GABAA receptor subunits following chronic ethanol exposure of cultured cortical neurons of mice.

Previous studies from our laboratory revealed that acute ethanol exposure inhibits phosphorylation of mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK) in mice. In the present study, we have further investigated effect of chronic administration of ethanol on tyrosine kinase phosphorylation of GABA(A) receptor subunits in the mouse cultured cortical neurons. We observed that there was an up-regulation in tyrosine kinase phosphorylation of the GABA(A) receptor beta(2) and gamma(2) subunits following chronic ethanol exposure, whereas there was no effect on alpha(1) subunit of the GABA(A) receptor in the cultured cortical neurons of mice as determined by Western blotting.

Succinate semialdehyde dehydrogenase deficiency does not down-regulate gamma-hydroxybutyric acid binding sites in the mouse brain.

We investigated whether succinate semialdehyde dehydrogenase deficiency alters gamma-hydroxybutyric acid (GHB) receptor characteristics due to elevation of GHB levels in the mouse brain. The membrane homogenate binding and quantitative autoradiography using [3H]NCS-382 revealed no significant changes in the affinity (Kd), receptor density (Bmax), or displacement potency (IC50) in various brain regions of Aldh5a1-/- vs. Aldh5a1+/+ mice.

Role of AP-1 in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene up-regulation in mouse cortical neurons.

Activator protein 1 (AP-1) has been reported to regulate the gene expression in a wide variety of cellular processes in response to stimuli. In this study, we investigated the DNA-protein binding activities and promoter activity in the N-methyl-D-aspartate R2B (NR2B) gene AP-1 site in normal and ethanol-treated cultured neurons. The identity of the AP-1 site as the functional binding factor is suggested by the specific binding of nuclear extract derived from cultured cortical neurons to the labeled probes and the specific antibody-induced supershift.

Ethers of 3-hydroxyphenylacetic acid as selective gamma-hydroxybutyric acid receptor ligands.

Gamma-hydroxybutyric acid (GHB) is a drug of abuse, a therapeutic, and purportedly a neurotransmitter with a complex mechanism of action in vivo due to direct actions at GABA(B) as well as GHB receptors and because of its metabolism to GABA. Herein, we describe 3-ethers of 3-hydroxyphenylacetic acid, which have relatively high affinity at GHB sites, no significant affinity at GABA receptors, and would not be expected to be rapidly metabolized to GABAergic ligands. The selectivity of these compounds (UMB108, UMB109, and UMB119) could prove to be useful for studying the biology of GHB receptors, free from GABAergic effects.

Methylation of NMDA receptor NR2B gene as a function of age in the mouse brain.

We have previously reported that there is an up-regulation of the NR2B gene expression in the adult cortex and cultured fetal cortical neurons of mice following chronic ethanol treatment due to demethylation of cytosine residues in the NR2B gene CpG island. In the present study, we investigated the methylation pattern of the NR2B CpG island as a function of the mouse age by digesting the cortex genomic DNA with HpaII enzyme, amplifying the interested regions by performing PCR and detecting the methylated regions by Southern hybridization so as to determine whether age affects the methylation process. We observed demethylation of various regions of NR2B gene (5227-5567), (5647-6003), (6091-6445), (6424-7024) of adult mouse cortex.

Potential role of cAMP response element-binding protein in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene transcription in fetal mouse cortical cells.

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days.

Novel gamma-hydroxybutyric acid (GHB) analogs share some, but not all, of the behavioral effects of GHB and GABAB receptor agonists.

gamma-Hydroxybutyrate (GHB), a therapeutic for narcolepsy and a drug of abuse, has several mechanisms of action that involve GHB and GABA(B) receptors, metabolism to GABA, and modulation of dopaminergic signaling. The aim of these studies was to examine the role of GHB and GABA(B) receptors in the behavioral effects of GHB. Three approaches were used to synthesize GHB analogs that bind selectively to GHB receptors and are not metabolized to GABA-active compounds.

Comparison of the behavioral effects of gamma-hydroxybutyric acid (GHB) and its 4-methyl-substituted analog, gamma-hydroxyvaleric acid (GHV).

Gamma-hydroxybutyrate (GHB), a metabolite of GABA, is a drug of abuse and a therapeutic. The illicit use of GHB precursors and analogs reportedly has increased worldwide. Gamma-hydroxyvaleric (GHV) is a 4-methyl-substituted analog of GHB that reportedly is abused and is marketed as a dietary supplement and replacement for GHB.

Neuron-restrictive silencer factor regulates the N-methyl-D-aspartate receptor 2B subunit gene in basal and ethanol-induced gene expression in fetal cortical neurons.

Neuron-restrictive silencer factor (NRSF) is a transcriptional repressor of multiple neuronal genes. This study addressed the role of NRSF in N-methyl-D-aspartate (NMDA) receptor NR2B promoter activity and the molecular mechanisms of ethanol-induced NR2B up-regulation in fetal cortical neurons. The 5'-flanking region of the NR2B gene contains five NRSE-like elements.

Role of CpG islands in the up-regulation of NMDA receptor NR2B gene expression following chronic ethanol treatment of cultured cortical neurons of mice.

Our earlier studies have indicated that chronic ethanol treatment produces an increase in the rate of NR2B gene expression but acute ethanol treatment doesn't cause any change in the expression in adult cortex and cultured fetal cortical neurons. To determine the molecular mechanism involved in the up-regulation of NR2B gene expression, we studied methylation in CpG islands in the regions (5843-6276) and (6477-6763) in mouse fetal cortical cultured neurons. However, acute administration of ethanol (3.

Effect of chronic administration of ethanol on GABAA receptor assemblies derived from alpha2-, alpha3-, beta2- and gamma2-subunits in the rat cerebral cortex.

Chronic administration of ethanol decreased the immunoprecipitation of the [(3)H]flunitrazepam binding activity for GABA(A) receptor assemblies derived from alpha(2)-, alpha(3)- and gamma(2)-subunits in the rat cerebral cortex. However, the [(3)H]muscimol binding sites derived from these subunits were not affected. Thus, chronic ethanol causes the down-regulation of the benzodiazepine sites derived from the alpha(2)-, alpha(3)- and gamma(2)-subunits without affecting the GABA binding sites.

Microarray analysis of ethanol-treated cortical neurons reveals disruption of genes related to the ubiquitin-proteasome pathway and protein synthesis.

Background: Chronic ethanol abuse results in deleterious behavioral responses such as tolerance, dependence, reinforcement, sensitization, and craving. The objective of this research was to identify transcripts that are differentially regulated in ethanol-treated cortical neurons compared with controls by using a pathway-focused complementary DNA microarray.

Methods: Cortical neurons were isolated from postconception day 14 C57BL/6 mouse fetuses and cultured according to a standard protocol.

Ethanol does not alter the binding of the gamma-hydroxybutyric acid (GHB) receptor ligand [3H]NCS-382 in the rat brain.

We investigated the effect of ethanol on the binding of the gamma-hydroxybutyric acid (GHB) receptor ligand [3H]NCS-382 in the rat cerebral cortex and hippocampus. Ethanol (50-100 mM) did not alter the binding of [3H]NCS-382. Furthermore, acute (3g/kg, p.