11ß-hydroxysteroid dehydrogenase inhibition as a new potential therapeutic target for alcohol abuse.

The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11ß-hydroxysteroid dehydrogenases (11ß-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11ß-HSD inhibitor carbenoxolone (CBX, 18ß-glycyrrhetinic acid 3ß-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11ß-HSD1 and 11ß-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3ß-O-hemisuccinate (αCBX), which we found to be selective for 11ß-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11ß-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11ß-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment.

Protein degradation by the proteasome is required for synaptic tagging and the heterosynaptic stabilization of hippocampal late-phase long-term potentiation.

Activity-dependent regulation of synaptic efficacy is believed to underlie learning and memory formation. Here we show that protein degradation by the proteasome is required for the induction of the protein synthesis-dependent late-phase of long-term potentiation (late-LTP) but not for its maintenance. Proteasome activity was also key to the polarity of heterosynaptic interactions between synapses expressing synaptic plasticity and newly activated synapses. In fact, proteasome activity was required for the consolidation of an otherwise transient potentiation (early-LTP) into late-LTP by strong tetanization of a separate afferent pathway both in the "weak-before-strong" and in the "strong-before-weak" two-pathway paradigms [Frey and Morris (1997) Nature 385:533-536; Frey and Morris (1998) Neuropharmacology 37:545-552], suggesting that proteasome activity plays a role in the synaptic tagging and capture of plasticity-related proteins at stimulated synapses. Additionally, proteasome inhibition abrogated immunity against heterosynaptic depotentiation of an established late-LTP when applied during weak tetanic stimulation in the "strong-before-weak" two-pathway paradigm. Such a heterosynaptic destabilizing effect of proteasome inhibition was abolished by concomitant inhibition of N-methyl-d-aspartate (NMDA) receptors, suggesting that it is an active process. Together, these results indicate that the proteasome plays important roles in the establishment of late-LTP and in the preservation of potentiated synapses when a subsequent synaptic plasticity is induced within the same neuronal population.

Region-specific transcriptional changes following the three antidepressant treatments electro convulsive therapy, sleep deprivation and fluoxetine.

The significant proportion of depressed patients that are resistant to monoaminergic drug therapy and the slow onset of therapeutic effects of the selective serotonin reuptake inhibitors (SSRIs)/serotonin/noradrenaline reuptake inhibitors (SNRIs) are two major reasons for the sustained search for new antidepressants. In an attempt to identify common underlying mechanisms for fast- and slow-acting antidepressant modalities, we have examined the transcriptional changes in seven different brain regions of the rat brain induced by three clinically effective antidepressant treatments: electro convulsive therapy (ECT), sleep deprivation (SD), and fluoxetine (FLX), the most commonly used slow-onset antidepressant. Each of these antidepressant treatments was applied with the same regimen known to have clinical efficacy: 2 days of ECT (four sessions per day), 24 h of SD, and 14 days of daily treatment of FLX, respectively. Transcriptional changes were evaluated on RNA extracted from seven different brain regions using the Affymetrix rat genome microarray 230 2.0. The gene chip data were validated using in situ hybridization or autoradiography for selected genes. The major findings of the study are: 1. The transcriptional changes induced by SD, ECT and SSRI display a regionally specific distribution distinct to each treatment. 2. The fast-onset, short-lived antidepressant treatments ECT and SD evoked transcriptional changes primarily in the catecholaminergic system, whereas the slow-onset antidepressant FLX treatment evoked transcriptional changes in the serotonergic system. 3. ECT and SD affect in a similar manner the same brain regions, primarily the locus coeruleus, whereas the effects of FLX were primarily in the dorsal raphe and hypothalamus, suggesting that both different regions and pathways account for fast onset but short lasting effects as compared to slow-onset but long-lasting effects. However, the similarity between effects of ECT and SD is somewhat confounded by the fact that the two treatments appear to regulate a number of transcripts in an opposite manner. 4. Multiple transcripts (e.g. brain-derived neurotrophic factor (BDNF), serum/glucocorticoid-regulated kinase (Sgk1)), whose level was reported to be affected by antidepressants or behavioral manipulations, were also found to be regulated by the treatments used in the present study. Several novel findings of transcriptional regulation upon one, two or all three treatments were made, for the latter we highlight homer, erg2, HSP27, the proto oncogene ret, sulfotransferase family 1A (Sult1a1), glycerol 3-phosphate dehydrogenase (GPD3), the orphan receptor G protein-coupled receptor 88 (GPR88) and a large number of expressed sequence tags (ESTs). 5. Transcripts encoding proteins involved in synaptic plasticity in the hippocampus were strongly affected by ECT and SD, but not by FLX. The novel transcripts, concomitantly regulated by several antidepressant treatments, may represent novel targets for fast onset, long-duration antidepressants.

Involvement of multiple phosphatidylinositol 3-kinase-dependent pathways in the persistence of late-phase long term potentiation expression.

The mechanisms responsible for the stabilization and persistence of synaptic plasticity remain largely unknown. In this study, we investigated the time course of the dependence of late-phase long term potentiation of field excitatory post-synaptic potential on phosphatidylinositol 3-kinase and its downstream effectors mTOR and AKT. In agreement with our previous results obtained on an early-phase long-term potentiation paradigm we observed that application of a nanomolar concentration of wortmannin (100 nM) 1 h after late-phase long term potentiation induction reversed potentiation completely. However, application of wortmannin 4 h after late-phase long term potentiation induction resulted in a more limited reduction of field excitatory post-synaptic potential suggesting that the dependence of late-phase long term potentiation expression on phosphatidylinositol 3-kinase decreases over time. Application of a nanomolar concentration of rapamycin (200 nM) during the tetanization paradigm prevented the induction of late-phase long term potentiation consistent with our earlier results. Application of rapamycin 1 h after late-phase long term potentiation induction resulted in a less pronounced though significant decline of field excitatory post-synaptic potential. Immunohistological analysis demonstrated that the concentration of rapamycin used was effective in inhibiting the phosphorylation of p70S6K at Thr389, the main determinant of its pro-translational activity, and that Thr389 phosphorylation recovered after washout. Lastly, a transient application of Akt inhibitor I (10 microM) one hour after late-phase long term potentiation induction also induced a partial although significant reduction of potentiated field excitatory post-synaptic potential that stabilized at a level of approximately 114% of baseline three hours after application, suggesting that AKT also contributes to the stabilization of late-phase long term potentiation expression. These results confirm and extend previous observations that the expression of long term potentiation in the CA1 of rat hippocampus involves several elements of the phosphatidylinositol 3-kinase signaling pathway.

The transient depression of hippocampal CA1 LTP induced by chronic intermittent ethanol exposure is associated with an inhibition of the MAP kinase pathway.

Using electrophysiological and biochemical approaches, we investigated the effects of chronic, intermittent ethanol (CIE) treatment on activation of the mitogen activated protein kinase (MAPK), also known as extracellular signal regulated protein kinase 1 and 2. In hippocampal slices taken from control rats, brief high-frequency stimulation to Schaffer collateral fibers induced a large post-tetanic potentiation (PTP) in the CA1 region that decayed to stable long-term potentiation (LTP) of field extracellular postsynaptic potentials. Western blot analyses showed that phosphorylation of MAPK was increased during PTP and returned to baseline levels during LTP. In slices from the rats removed immediately from CIE treatment, PTP and MAPK activation during the PTP was significantly less than that observed in control slices and LTP was absent. In slices from rats subjected to 1 day withdrawal from CIE treatment, both the reduction in MAPK phosphorylation during PTP and the impairment of PTP and LTP were still evident. Recovery of PTP and partial recovery of LTP was observed in slices obtained from 5-day withdrawn rats. However, MAPK activation during PTP was still attenuated significantly. Interestingly, MAPK activation was enhanced significantly during LTP in 5-day withdrawn rats as well as the sensitivity to MAPK inhibitor PD 098059. In addition to these changes in HFS-induced MAPK activation, we also observed a significant reduction in the basal phosphorylation of MAPK in slices removed from rats immediately after CIE treatment. These results implicate the MAPK signal transduction pathway as a potential cellular target of ethanol. Alterations in MAPKs could play an important role in the alcohol-induced changes in synaptic plasticity associated with the effects of alcohol abuse on learning and memory processes.

Inhibition of the ERK pathway prevents HIVgp120-induced REM sleep increase.

Approximately 35% of HIV-infected subjects, both children and adults, exhibit alterations in the sleep-waking cycle. HIV surface glycoprotein gp120 has been postulated to contribute to this abnormality. For example, it has been reported that HIVgp120 modifies sleep in freely-moving rats and that it also activates the ERK pathway in brain slices. The goal of this work was to determine if sleep changes induced by HIVgp120 in normal rats are mediated by the MAPK pathway. Our results show that a single intraventricular administration of HIVgp120 selectively increases REMS and that such an increase can be prevented by U0126, an inhibitor of ERK activating enzyme, MEK. In contrast, SB202190, a MAPK-p38 inhibitor, had no effect on HIVgp120-induced increase in REMS. These results suggest that HIVgp120 increases REMS in the rat by specifically affecting the ERK signal transduction pathway.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: reversal by D(1) antagonists.

The conditioning of cocaine's subjective actions with environmental stimuli may be a critical factor in long-lasting relapse risk associated with cocaine addiction. To study the significance of learning factors in persistent addictive behavior as well as the neurobiological basis of this phenomenon, rats were trained to associate discriminative stimuli (S(D)) with the availability of i.v. cocaine vs. nonrewarding saline solution, and then placed on extinction conditions during which the i.v. solutions and S(D)s were withheld. The effects of reexposure to the S(D) on the recovery of responding at the previously cocaine-paired lever and on Fos protein expression then were determined in two groups. One group was tested immediately after extinction, whereas rats in the second group were confined to their home cages for an additional 4 months before testing. In both groups, the cocaine S(D), but not the non-reward S(D), elicited strong recovery of responding and increased Fos immunoreactivity in the basolateral amygdala and medial prefrontal cortex (areas Cg1/Cg3). The response reinstatement and Fos expression induced by the cocaine S(D) were both reversed by selective dopamine D(1) receptor antagonists. The undiminished efficacy of the cocaine S(D) to elicit drug-seeking behavior after 4 months of abstinence parallels the long-lasting nature of conditioned cue reactivity and cue-induced cocaine craving in humans, and confirms a significant role of learning factors in the long-lasting addictive potential of cocaine. Moreover, the results implicate D(1)-dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala as substrates for cocaine-seeking behavior elicited by cocaine-predictive environmental stimuli.

Effect of nociceptin/orphanin FQ on the rewarding properties of morphine.

The present study investigated the effect of nociceptin/orphanin FQ, the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, on the rewarding properties of morphine in the place conditioning paradigm. Intracerebroventricular (i.c.v.) injections of nociceptin/orphanin FQ, 500 or 1000 (but not 250) ng/rat, abolished conditioned place preference induced by subcutaneous (s.c.) injections of morphine (3 mg/kg). These doses of nociceptin/orphanin FQ induced neither place aversion nor preference per se. The same doses did not modify the rat performance in the Morris water test, suggesting that they do not disrupt spatial learning and memory. Moreover, these doses of nociceptin/orphanin FQ did not modify the development of morphine-induced locomotor sensitization, suggesting that they do not interfere with sensitization processes to morphine. The present results confirm and extend previous reports that nociceptin/orphanin FQ is able to abolish morphine-induced conditioned place preference, and raise interest for the possible role of nociceptin/orphanin FQ and ORL1 receptors in the control of opiate abuse.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus.

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg(2+)-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg(2+)-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Synergistic interactions of antibodies in rate of virus neutralization.

Antibodies and antibody combinations are often evaluated only by their potency in inactivating a known quantity of virus in dose-effect assays. However, a crucial additional parameter is the rate at which neutralization takes place, or kinetics. Synergism of certain antibody combinations in dose-effect assays has been previously demonstrated. In the present report, using a battery of murine monoclonal antibodies to herpes simplex virus (HSV), we investigated whether antiviral antibodies can also synergize in neutralization kinetics. To determine whether synergism in dose-effect assays can predict synergism in neutralization rate, the ability of neutralizing antibodies to synergize in neutralization rate (kinetics) was compared to their ability to synergize in dose-effect assays (potency) in cell-free assays. Although certain antibody combinations synergized in both neutralization rate and potency, combinations that did not clearly synergize in potency could still significantly synergize in neutralization rate. Weak neutralizing antibodies could also greatly increase the neutralization rate of more potent antibodies. These results suggest that evaluating antibody combinations in dose-effect assays but not in neutralization kinetics provides a partial picture of neutralizing antibody dynamic interactions and may prevent the identification of certain favorable antibody combinations. These findings also support the importance of establishing defined antibody cocktails for prophylactic and therapeutic purposes. A simple strategy to evaluate antibody interactions in neutralization kinetics is proposed in which a quantitative prediction of additivity is made on the basis of the neutralization rate constants of the individual antibodies in the combination.

Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA.

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as 'target-derived factors'. During differentiation, muscle cells also express some neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform cells of the TE-671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased the fusion rate of both primary and L6 myoblasts, as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low microM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing L6 cells do not fuse and present changes in their morphology similar to the TrKA-inhibitors treated L6 cells. These data support the notion that NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

Localization of a passively transferred human recombinant monoclonal antibody to herpes simplex virus glycoprotein D to infected nerve fibers and sensory neurons in vivo.

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea--identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase--and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.

Neutralizing antibodies inhibit axonal spread of herpes simplex virus type 1 to epidermal cells in vitro.

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.

pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies.

We have constructed a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies and their expression in eukaryotic cells. This vector, named pFab-CMV, utilizes the same unique cloning sites present on the pComb3 phagemid thus allowing for the direct subcloning of light chains and heavy chain Fd regions. pFab-CMV also allows for the expression of recombinant Fabs in eukaryotic cells by removal of a cassette containing part of the hinge, CH2 and CH3 sequences. Stable cell lines are rapidly obtained with pFab-CMV by NEO selection without the need for co-transfection of heavy and light chain expressing vectors.

Modulation of serotonin 5-HT3 receptor expression in injured adult rat spinal cord motoneurons.

The effects of sciatic nerve lesions on the expression of serotonin 5-HT3 receptor (5-HT3R) alpha subunit in motoneurons of the spinal cord was investigated by semi-quantitative immunohistochemistry. Following sciatic nerve crush, a significant reduction in density of staining in motoneurons was observed in longitudinal sections of the ventral horn at 3 and 15 days on the lesioned side when compared to the contralateral side (p<0.01). At 30 days after crush, after completion of sciatic nerve regeneration and reinnervation of peripheral targets, intensity of staining had returned to normal. Conversely, after sciatic nerve cut, a lesion that does not allow for target reinnervation, highly significant reductions were observed at 3, 15, 30 and 45 days. These results suggest a role for functional contacts with muscular targets in the maintenance of 5-HT3R expression in spinal motoneurons.

Characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential.

We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.

Acute ethanol induces c-fos immunoreactivity in GABAergic neurons of the central nucleus of the amygdala.

The central nucleus of the amygdala (CNA) is a component of the brain reward pathway which is believed to represent an anatomical substrate for drugs of abuse. Previous studies have shown that acute ethanol administration induces the expression of c-fos in the CNA of rat brains. We report here, that over 70% of these c-fos immunoreactive neurons are GABAergic. This observation provides the first anatomical evidence that GABAergic neurons of the CNA are responsive to acute ethanol exposure and suggest that the GABAergic system of the CNA is a key neuronal substrate for ethanol actions on the central nervous system.