. Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABA A receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 M) and decreased by Zn 2ϩ (50 M) but was unaffected by zolpidem (0.3 M) or midazolam (0.2 M). The pharmacological profile of the tonic current is consistent with its generation by activation of GABA A receptors that do not contain the ␣ 1 or ␥ 2 subunits. GABA A receptors expressed in HEK 293 cells that contained ␣ 4  2 ␦ subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from ␣ 1  2 ␦, ␣ 4  2 ␥ 2s, or ␣ 1  2 ␥ 2s subunits. Western blot analysis revealed that there is little, if any, ␣ 3 or ␣ 5 subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the ␦ subunit could precipitate ␣ 4 , but not ␣ 1 subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that ␣ 4 and ␦ subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABA A receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of ␣ 4  2 ␦ subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol. I N T R O D U C T I O NThe activation of GABA A receptors inhibits neurons in two ways-via a fast or transient inhibition after GABA binding to synaptically localized receptors and by a sustained inhibition due to GABA binding to extrasynaptic receptors (Brickley et al. 1996; Farrant and Nusser 2005;Kaneda et al. 1995;Mody 2001). Extrasynaptic receptors are excellent sensors for extracellular GABA due to their high affinity for GABA and slow rates of desensitization (Bai et al. 2001; Brickley et al. 1999;Yeung et al. 2003). The activation of these receptors regulates neuronal input resistance and, hence, excitability (Brickley et al. 2001;Semyanov et al. 2003). The ␦ subunit appears to be present in many extrasynaptic GABA A receptors. In cerebellar granule cells, these receptors likely contain ␣ 6 , , and ␦ subunits (Brickley et al. 2001;Nusser et al. 1998;Pirker et al. 2000), whereas granule cells in the dentate gyrus likely contain ␣ 4 , , and ␦ subunits (Nusser and Mody 2002;Sperk et al. 1997;Sun et al. 2004;Wei et al. 2003). Extrasynaptic GABA A receptors in CA1 pyramidal neurons in the hippocampus, however, likely contain ␣ 5 ,  2/3 , and ␥ 2 subunits (Caraiscos et al. 2004).Several GABA A receptor subunits are expressed in the thalamus. ␣ 1 , ␣ 4 ,  2 , ␥ 2 , and ␦ subunits are found in thalamocortical neurons in the ventrobasal (VB) complex, whereas ␣ 3 ,  3 , and ␥ 2 subunits...
Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10 -60 mM), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.
Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/ aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Mü ller cells in GLAST Ϫ/Ϫ mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the K m value for glutamate uptake was similar in wild-type and GLAST Ϫ/Ϫ mouse retinas, but the V max was ϳ50% lower in the mutant. In Na ϩ -free medium, the V max was further reduced by 40%. In patch-clamp recordings of dissociated Mü ller cells from GLAST Ϫ/Ϫ mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Mü ller cells. [ 3 H]D-Aspartate uptake autoradiography, however, showed that Na ϩ -dependent, high-affinity transporters account for most of the glutamate uptake by Mü ller cells, and that Na ϩ -independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Mü ller cells in the GLAST Ϫ/Ϫ mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Mü ller cells, new Na ϩ -dependent, high-affinity glutamate transporters remain to be identified.
In recent years there has been an explosion of interest in how genes regulate alcohol drinking and contribute to alcoholism. This work has been stimulated by the completion of the human and mouse genome projects and the resulting availability of gene microarrays. Most of this work has been performed in drinking animals, and has utilized the extensive genetic variation among different mouse strains. At the same time, a much smaller amount of effort has gone into the in vitro study of the mechanisms underlying the regulation of individual genes by alcohol. These studies at the cellular and sub-cellular level are beginning to reveal the ways in which alcohol can interact with the transcriptional, translational and post-translational events inside the cell. Detailed studies of the promoter regions within several individual alcohol-responsive genes (ARGs) have been performed and this work has uncovered intricate signaling pathways that may be generalized to larger groups of ARGs. In the last few years several distinct ARGs have been identified from 35,000 mouse genes, by both the “top-down” approach (ex vivo gene arrays) and the “bottom-up” methods (in vitro promoter analysis). These divergent methodologies have converged on a surprisingly small number of genes encoding ion channels, receptors, transcription factors and proteins involved in synaptic function and remodeling. In this review we will describe some of the most interesting cellular and microarray work in the field, and will outline specific examples of genes for which the mechanisms of regulation by alcohol are now somewhat understood.
Many synapses within the central nervous system are sensitive to ethanol. Although alcohol is known to affect the probability of neurotransmitter release in specific brain regions, the effects of alcohol on the underlying synaptic vesicle fusion machinery have been little studied. To identify a potential pathway by which ethanol can regulate neurotransmitter release, we investigated the effects of acute alcohol exposure (1–24 hours) on the expression of the gene encoding Synaptotagmin 1 (Syt1), a synaptic protein that binds calcium to directly trigger vesicle fusion. Syt1 was identified in a microarray screen as a gene that may be sensitive to alcohol and heat shock. We found that Syt1 mRNA and protein expression are rapidly and robustly up-regulated by ethanol in mouse cortical neurons, and that the distribution of Syt1 protein along neuronal processes is also altered. Syt1 mRNA up-regulation is dependent on the activation of the transcription factor heat shock factor 1 (HSF1). The transfection of a constitutively active Hsf1 construct into neurons stimulates Syt1 transcription, while transfection of Hsf1 siRNA or a constitutively inactive Hsf1 construct into neurons attenuates the induction of Syt1 by ethanol. This suggests that the activation of HSF1 can induce Syt1 expression and that this may be a mechanism by which alcohol regulates neurotransmitter release during brief exposures. Further analysis revealed that a subset of the genes encoding the core synaptic vesicle fusion (SNARE) proteins share this property of induction by ethanol, suggesting that alcohol may trigger a specific coordinated adaptation in synaptic function. This molecular mechanism could explain some of the changes in synaptic function that occur following alcohol administration, and may be an important step in the process of neuronal adaptation to alcohol.
Astrocytes are critical for maintaining homeostasis in the central nervous system (CNS), and also participate in the genomic response of the brain to drugs of abuse, including alcohol. In this study, we investigated ethanol regulation of gene expression in astrocytes. A microarray screen revealed that a brief exposure of cortical astrocytes to ethanol increased the expression of a large number of genes. Among the alcohol-responsive genes (ARGs) are glial-specific immune response genes, as well as genes involved in the regulation of transcription, cell proliferation, and differentiation, and genes of the cytoskeleton and extracellular matrix. Genes involved in metabolism were also upregulated by alcohol exposure, including genes associated with oxidoreductase activity, insulin-like growth factor signaling, acetyl-CoA, and lipid metabolism. Previous microarray studies performed on ethanol-treated hepatocyte cultures and mouse liver tissue revealed the induction of almost identical classes of genes to those identified in our microarray experiments, suggesting that alcohol induces similar signaling mechanisms in the brain and liver. We found that acute ethanol exposure activated heat shock factor 1 (HSF1) in astrocytes, as demonstrated by the translocation of this transcription factor to the nucleus and the induction of a family of known HSF1-dependent genes, the heat shock proteins (Hsps). Transfection of a constitutively transcriptionally active Hsf1 construct into astrocytes induced many of the ARGs identified in our microarray study supporting the hypothesis that HSF1 transcriptional activity, as part of the heat shock cascade, may mediate the ethanol induction of these genes. These data indicate that acute ethanol exposure alters gene expression in astrocytes, in part via the activation of HSF1 and the heat shock cascade.
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