The central amygdala (CeA) plays a role in the relationship among stress, corticotropin-releasing factor (CRF), and alcohol abuse. In whole-cell recordings, both CRF and ethanol enhanced gamma-aminobutyric acid-mediated (GABAergic) neurotransmission in CeA neurons from wild-type and CRF2 receptor knockout mice, but not CRF1 receptor knockout mice. CRF1 (but not CRF2) receptor antagonists blocked both CRF and ethanol effects in wild-type mice. These data indicate that CRF1 receptors mediate ethanol enhancement of GABAergic synaptic transmission in the CeA, and they suggest a cellular mechanism underlying involvement of CRF in ethanol's behavioral and motivational effects.

BACKGROUND: Conflicting information exists concerning the actions of ethanol on vesicular release at excitatory synapses. Because long-term alterations in synaptic transmission are thought to underlie neuroadaptive responses to ethanol, we have directly measured the actions of ethanol on release dynamics at an intact central synapse. METHODS: Here we investigated the effects of ethanol on release dynamics in hippocampal slices using confocal microscopy with the lipophilic dye, FM1-43, complemented by a patch clamp analysis of AMPA miniature excitatory postsynaptic currents (mEPSCs). After a pretreatment/loading paradigm with sulforhodamine (S-Rhd) and FM1-43, stable, dense punctate FM1-43 staining in the CA1 stratum radiatum was evident. RESULTS: FM1-43 fluorescence destaining was dose-dependently induced by perfusion with elevated K+ (20-60 mM). Cadmium inhibited K+-induced destaining, whereas nifedipine had no significant effect. Ethanol (25-75 mM) inhibited K+-induced destaining with high efficacy and had no effect on basal destaining. Both omega-Conotoxin GVIA and omega-Agatoxin IVA inhibited K+-induced destaining with high efficacy. The combination of omega-Conotoxin GVIA and omega-Agatoxin IVA occluded the inhibitory effect of ethanol, indicating that ethanol inhibition of release was dependent on inhibition of N/P/Q-voltage-gated calcium channels (VGCCs). Patch clamp studies of AMPA mEPSCs revealed similar findings in that vesicular release was enhanced with K+ depolarization in an ethanol-sensitive manner. CONCLUSIONS: These findings indicate that the FM1-43/S-Rhd method is a stable and powerful approach for direct real-time measurement of vesicular release kinetics in intact brain slice preparations and that ethanol inhibits vesicular release induced by depolarization via inhibition of N/P/Q-VGCCs.

BACKGROUND: The primary goal of this study was to investigate the effects of varying doses of ethanol on cellular activation, as measured by Fos immunoreactivity, in brain areas that have been implicated in the reinforcing and anxiolytic effects of substance abuse and dependence, namely, the extended amygdala and hypothalamus. Specific regions examined included the central nucleus of the amygdala, bed nucleus of the stria terminalis, substantia innominata, and nucleus accumbens of the extended amygdala, as well as the paraventricular nucleus of the hypothalamus. The cholinergic interneurons of the nucleus accumbens were of particular interest, because these cells have recently been reported to play a pivotal role in substance abuse. METHODS: Adult Sprague-Dawley rats underwent 10 days of handling and 5 days of habituation. Animals then received an injection of saline or 0.5, 1, or 2 g/kg of ethanol. Rats were perfused 2 hr after the injections, and brain sections were processed for single Fos or dual Fos/choline acetyltransferase immunolabeling procedures. The number of Fos-positive neurons was calculated from a 0.45-mm sample area from each of the brain regions examined. RESULTS: A dose of 2 g/kg of ethanol significantly increased the number of Fos-immunoreactive neurons in the central nucleus of the amygdala by 149%, in the shell nucleus accumbens by 80%, and in the paraventricular nucleus of the hypothalamus by 321%. Additionally, 1 g/kg of ethanol significantly increased the percentage of Fos-immunoreactive cholinergic neurons in the nucleus accumbens by 59%. CONCLUSIONS: The findings reported in this study reveal region-specific and dose-dependent changes in Fos immunoreactivity in the extended amygdala and hypothalamus and, more specifically, an increase in neuronal activation of cholinergic cells in the shell nucleus accumbens. These findings contribute to our current knowledge of the brain areas and cellular microcircuits involved in the underlying basis of substance abuse and dependence.

gamma-Aminobutyric acid A (GABA(A)) receptors are believed to mediate a number of alcohol's behavioral actions. Because the subunit composition of GABA(A) receptors determines receptor pharmacology, behavioral sensitivity to alcohol (ethanol) may depend on which subunits are present (or absent). A number of knock-out and/or transgenic mouse models have been developed (alpha1, alpha2, alpha5, alpha6, beta2, beta3, gamma2S, gamma2L, delta) and tested for behavioral sensitivity to ethanol. Here we review the current GABA(A) receptor subunit knock-out and transgenic literature for ethanol sensitivity, and integrate these results into those obtained using quantitative trait loci (QTL) analysis and gene expression assays. Converging evidence from these three approaches support the notion that different behavioral actions of ethanol are mediated by specific subunits, and suggest that new drugs that target specific GABA(A) subunits may selectively alter some behavioral actions of ethanol, without altering others. Current data sets provide strongest evidence for a role of alpha1-subunits in ethanol-induced loss of righting reflex, and alpha5-subunits in ethanol-stimulated locomotion. However, three-way validation is hampered by the incomplete behavioral characterization of many of the mutant mice, and additional subunits are likely to be linked to alcohol actions as behavioral testing progresses.

Chronic alcohol exposure induces lasting behavioral changes, tolerance, and dependence. This results, at least partially, from neural adaptations at a cellular level. Previous genome-wide gene expression studies using pooled human brain samples showed that alcohol abuse causes widespread changes in the pattern of gene expression in the frontal and motor cortices of human brain. Because these studies used pooled samples, they could not determine variability between different individuals. In the present study, we profiled gene expression levels of 14 postmortem human brains (seven controls and seven alcoholic cases) using cDNA microarrays (46,448 clones per array). Both frontal cortex and motor cortex brain regions were studied. The list of genes differentially expressed confirms and extends previous studies of alcohol responsive genes. Genes identified as differentially expressed in two brain regions fell generally into similar functional groups, including metabolism, immune response, cell survival, cell communication, signal transduction and energy production. Importantly, hierarchical clustering of differentially expressed genes accurately distinguished between control and alcoholic cases, particularly in the frontal cortex.

A clinical 3-T scanner equipped with a custom-made transmit/receive birdcage coil was used to collect 2D J-resolved single-voxel spectroscopy in vivo of rat brain. Four adult Wistar rats were scanned twice each, with a 2-week interval. Voxel size was approximately 5 x 10 x 5 mm(3). Total spectroscopic acquisition time was 14 min for collection of two 4:20 min water-suppressed acquisitions and one 4:20 min acquisition acquired in the absence of water suppression. The unsuppressed water data were used in post-processing to reduce residual water side bands, as well as for metabolite signal normalization to account for variations in coil loading and voxel size. Peak areas were estimated for resonances from N-acetyl aspartate (NAA), creatine, choline, taurine, glutamate, and combined glutamate and glutamine. T(2)-relaxation times were estimated for NAA and creatine. The average deviation from the mean of repeated measures for glutamate, combined glutamate and glutamine, and taurine ranged from 7.6% to 18.3%, while for NAA, creatine, and choline, the deviation was less than 3%. The estimated T(2) values for NAA (mean +/- SD = 330 +/- 57 ms) and creatine (174 +/- 27 ms) were similar to those reported previously for rat brain and for human gray and white matter. These results indicate that reliable, small animal brain MR spectroscopy can be performed on a human clinical 3-T scanner.

Purpose To examine the feasibility of using product acquisition software on a 3-T human MRI system to acquire high-resolution structural brain images in the rat. Materials and Methods Three sets of dual spin-echo, high-resolution (0.234 × 0.234 mm in-plane, 0.5 mm thick) images covering the entire rat brain were collected and averaged in 66 min. The images had sufficient signal-to-noise ratio (SNR) and resolution for visual identification and manual outlining of exemplary structures, including the lateral ventricles and dorsal and ventral portions of the hippocampus. Further, the data were adequate for unsupervised, automated segmentation, permitting quantification of the dorsolateral ventricles. The images compared favorably with those collected on a 7-T system. Results Interrater reliabilities (intraclass correlations) of manual ventricular scoring were greater than 0.97, and manual vs. automated correlations were 0.97. The variability of lateral ventricular size across animals was substantially higher than that of the hippocampus. Conclusion The large variability of some brain structures that can exist across even a highly selected strain of rats can readily be detected with the use of human 3-T systems for the study of small animals. J. Magn. Reson. Imaging 2004;20:779–785. © 2004 Wiley-Liss, Inc.

The central nucleus of amygdala (CeA) is important in regulating alcohol consumption and plays a major role in the anxiogenic response to ethanol withdrawal. We showed previously that acute ethanol augments GABA(A) receptor-mediated IPSPs and IPSCs, possibly by a presynaptic mechanism. Here, we have examined the interaction of acute ethanol with the GABAergic system in chronic ethanol-treated (CET) rats using an in vitro CeA slice preparation and in vivo brain microdialysis. We found that in CeA slices from CET rats, the baseline evoked IPSP and IPSC amplitudes were increased, and paired-pulse facilitation ratios were lower than in naive rats, suggesting an increased GABAergic transmission after chronic ethanol treatment. Interestingly, acute ethanol (5-66 mm) significantly enhanced IPSPs and IPSCs equally in CET and naive rats, indicating a lack of tolerance for this effect of acute ethanol. Analysis of miniature IPSC frequency suggests that the increased GABAergic transmission by both acute and chronic ethanol arises from a presynaptic mechanism involving enhanced vesicular release of GABA. These data are supported by microdialysis studies showing that CET rats presented a fourfold increase in baseline GABA dialysate content compared with naive rats. In vivo administration of ethanol (0.1, 0.3, and 1.0 m) produced a dose-dependent increase in GABA release in the CeA dialysate in both CET and naive rats. These combined findings suggest that acute and chronic ethanol increases GABA release in CeA and support previous reports that the behavioral actions of ethanol are mediated, in part, by increased GABAergic transmission in the CeA.

The neuropeptide Y (NPY) gene in rat chromosome 4 has been shown to play an important role in alcohol-seeking behavior. NPY knockout mice drink more alcohol than wild-type mice, implicating a link between NPY deficiency and high alcohol intake. This is supported by recent studies showing that intracerebroventricular injections of NPY reduce alcohol intake in both alcohol-preferring (P) and high alcohol-drinking rats. However, it is unknown which anatomical NPY systems are involved in alcohol preference. This study was designed to investigate whether there are innate differences in NPY mRNA in cerebral cortical areas, dentate gyrus (DG) of the hippocampus and medial habenular nucleus (MHb) between P and alcohol-nonpreferring (NP) rats, as these discrete brain regions are rich in NPY mRNA. [(33)P]-labeled 28-mer oligodeoxynucleotide probe was applied for the in situ hybridization study to detect the NPY mRNA, measured using quantitative autoradiography. This study revealed an absence of NPY mRNA in the MHb of P rats. We found that NPY mRNA was significantly lower in the DG of P rats than NP rats. This innate difference of NPY mRNA expression in the DG between P and NP rats is region specific. For example, in most of the cerebral cortical areas examined, an innate difference was not seen. Our study suggests that lower NPY gene expression in the DG and MHb of P rats may be factors contributing to some of the phenotypic differences observed between the P and NP lines of rats.

The Edinger-Westphal nucleus (EW) is a brain region that has recently been implicated as an important novel neural target for ethanol. Thus, the EW is the only brain region consistently showing elevated c-Fos expression following both voluntary and involuntary ethanol administration. Ethanol-induced c-Fos expression in the EW has been shown to occur in urocortin I-positive neurons. Moreover, previous reports using several genetic models have demonstrated that differences in the EW urocortin I system are correlated with ethanol-mediated behaviours such as ethanol-induced hypothermia and ethanol consumption. The aim of this study was to confirm these relationships using a more direct strategy. Thus, ethanol responses were measured following electrolytic lesions of the EW in male C57BL/6J mice. Both EW-lesioned and sham-operated animals were tested for several ethanol sensitivity measures and ethanol consumption in a two-bottle choice test. The results show that lesions of the EW significantly disrupted ethanol-induced hypothermia, while having no effect on pupillary dilation, locomotor activity or ethanol-induced sedation. In addition, EW-lesioned animals showed significantly lower ethanol preference and total ethanol dose consumed in the two-bottle choice test. EW-lesioned animals also consumed less sucrose than sham-operated animals, but did not have altered preferences for sucrose or quinine in a two-bottle choice test. These data support previously observed genetic correlations between EW urocortin I expression and both ethanol-induced hypothermia and ethanol consumption. Taken together, the findings suggest that the EW may function as a sensor for ethanol, which can influence ethanol consumption and preference.

Group III metabotropic glutamate receptors (mGluRs), specifically receptors 4, 6, 7, and 8 (i.e., mGluR4, mGluR6, mGluR7, mGluR8), play an important role in the generation of locomotion as well as in the behavioral effects of some psychostimulants. Because the arousing or stimulant effects of ethanol seem to be relevant behavioral traits associated with its rewarding properties and genetic susceptibility to alcoholism, we addressed the role of mGluR4 by studying behavioral actions of ethanol in mutant mice lacking mGluR4. Null mutant mice showed higher motor response to novelty than did wild-type mice. Ethanol (1.0–2.5 g/kg) stimulated motor activity of wild-type mice, but not of null mutant mice. There were no significant differences between wild-type and knockout strains in ethanol consumption or preference in two-bottle paradigm, severity of ethanol-induced acute withdrawal, or duration of loss of righting reflex. These results show that mGluR4 may play a role in locomotor activity in general and also display specificity for mediation of the motor stimulant effect of ethanol. Consistent with findings of other studies, these results confirm the lack of correlation between ethanol-induced motor stimulation and consumption of ethanol measured in a self-administration paradigm in mice.

Mu opioid receptors mediate positive reinforcement following direct (morphine) or indirect (alcohol, cannabinoids, nicotine) activation, and our understanding of mu receptor function is central to the development of addiction therapies. Recent data obtained in native neurons confirm that mu receptor signaling and regulation are strongly agonist-dependent. Current functional mapping reveals morphine-activated neurons in the extended amygdala and early genomic approaches have identified novel mu receptor-associated proteins. A classification of about 30 genes either promoting or counteracting the addictive properties of morphine is proposed from the analysis of knockout mice data. The targeting of effectors or regulatory proteins, beyond the mu receptor itself, might provide valuable strategies to treat addictive disorders.

Identification of genetic and physiological mechanisms underlying a drug's or mutation's effects on motor performance could be aided by the existence of a simple observation-based rating scale of ataxia for mice. Rating scales were developed to assess ataxia after ethanol (2.75, 3.0, and 3.25 g/kg) in nine inbred mouse strains. Each scale independently rates a single behavior. Raters, blinded to dose, scored four behaviors (splay of hind legs, wobbling, nose down, and belly drag) at each of four time points after injection. The severities of hind leg splaying and wobbling were quantifiable, whereas nose down and belly dragging were expressed in all-or-none fashion. Interrater reliabilities were substantial (0.75 \textlessor= r \textlessor= 0.99). Splay scores (rated 0-5) displayed significant effects of strain, dose, and time point. Wobbling (rated 0-4) was dependent on strain and time point. Ethanol affected wobbling (most strains scored \textgreater0 at some time), but all doses were equally effective. Incidence of nose down and belly dragging behaviors increased strain dependently after ethanol, but strains did not differentially respond to dose. Ethanol-induced splaying was modestly, and negatively, genetically correlated with wobbling. Nose down and belly dragging tended to be associated with splaying and wobbling at later times. Four distinct ataxia-related behaviors were sensitive to ethanol. Strains differed in ethanol sensitivity for all measures. Modest strain mean correlations among behaviors indicate that these behaviors are probably under control of largely different genes and that ataxia rating scales should rate separate behaviors on discrete scales.

Our previous work indicated a role for fyn-kinase in mediating several ethanol- and GABAA agonist-mediated behaviors. In the present work we investigate behavioral sensitivity to ethanol and several GABAA compounds in mice that over-express fyn-kinase in forebrain to further characterize the role of this non-receptor tyrosine kinase in the mediation of ethanol sensitivity. Transgenic mice over-expressing fyn-kinase were tested for sensitivity to ethanol-induced loss of righting reflex and ethanol preference drinking using a two-bottle choice drinking paradigm. Loss of righting reflex induced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; GABAA agonist) and etomidate (GABAA positive allosteric modulator) were also assessed. Fyn over-expressing mice exhibited shorter durations of ethanol-induced loss of righting reflex in the absence of differences in the rate of blood ethanol clearance, and exhibited reduced ethanol preference drinking. The genotypes did not differ in initial sensitivity to ethanol-induced loss of righting reflex suggesting development of greater acute tolerance to this ethanol action. Fyn over-expressing and wild-type mice also did not differ in sensitivity to loss of righting reflex induced by THIP and etomidate. The present results suggest regional specificity for fyn-kinase in the modulation of ethanol and GABAergic behavioral sensitivity. Fyn-kinase over-expression in forebrain structures modulates ethanol's hypnotic actions, as well as ethanol preference and consumption. Moreover, fyn over-expression in forebrain does not alter hypnotic sensitivity to THIP or etomidate, supporting data from fyn null mutant mice suggesting that cerebellar structures mediate the hypnotic actions of these GABAergic compounds.

Animal models designed to examine different facets of alcohol-related behaviors have been developed to study genetic and neurobiological factors underlying alcoholism and alcohol abuse. One goal has been to develop valid, congruent, complementary animal models of alcohol craving and relapse, with the ultimate objective of assessing the effectiveness of pharmacological agents with these models. Animal models of alcohol craving include drug-induced responding (drug reinstatement), cue-induced responding, Pavlovian Spontaneous Recovery (PSR), and appetitive/consummatory responding. A primary experimental approach to study alcohol relapse has been through expression of the Alcohol Deprivation Effect (ADE) following a single deprivation or multiple deprivations. To date, five selectively bred lines of rats have been developed to study alcohol-drinking behavior. These are the ALKO/Alcohol (AA), alcohol-preferring (P), high alcohol-drinking (HAD-1 and HAD-2 replicates), and the Sardinian alcohol-preferring (sP) lines of rats. Findings thus far indicate that only the P line of rats meets all the criteria established for a valid animal model of alcoholism, with progress having been made in characterizing the AA, HAD and sP lines of rats. The focus of the current review will be to analyze the various models of alcohol craving, emphasizing the use of the Indiana University selected rat lines (P and HADs). Overall, the findings indicate substantial progress has been made in developing animal models of alcohol abuse, relapse and craving using these selectively bred rat lines, as well as outbred rats.

This article represents the proceedings of a symposium at the 2002 RSA Meeting in San Francisco, California, organized and co-chaired by L. Judson Chandler and Richard A. Morrisett. The presentations were (1) PKA regulates chronic ethanol-induced synaptic targeting of NMDA receptors, by L. Judson Chandler; (2) Long-lasting potentiation of GABAergic synapses in dopamine neurons after a single in vivo ethanol exposure, by Antonello Bonci; (3) The DARPP-32 cascade and regulation of the ethanol sensitivity of NMDA receptors in the nucleus accumbens, by Richard A. Morrisett; (4) and The cAMP/PKA signal transduction pathway modulates ethanol consumption and sedative effects of ethanol, by Gary S. Wand.

Levels of neuropeptide Y (NPY) mRNA expression in discrete brain regions of alcohol preferring (P) rats and alcohol nonpreferring (NP) rats were examined using in situ hybridization. NPY mRNA expression was significantly lower in the central nucleus of amygdala (CeA) of P rats than NP rats, whereas no differences were found in the medial or basolateral amygdaloid nuclei. This study suggests that reduced NPY gene expression in the CeA may contribute to differences in alcohol preference and other behavioral differences observed between P and NP rats.

The pharmacological profile of allopregnanolone, a neuroactive steroid that is a potent positive modulator of gamma-aminobutyric acidA (GABAA) receptors, is similar to that of ethanol. Recent findings indicate that acute injection of ethanol increased endogenous allopregnanolone to pharmacologically relevant concentrations in male rats. However, there are no comparable data in mice, nor has the effect of ethanol drinking on endogenous allopregnanolone levels been investigated. Therefore, the present studies measured the effect of ethanol drinking and injection on allopregnanolone levels in male and female C57BL/6 mice. One group was given 17 days of 2-h limited access to a 10% v/v ethanol solution in a preference-drinking paradigm, while another group had access to water only. The ethanol dose consumed in 2 h exceeded 2 g/kg. Then, separate groups of mice were injected with either 2 g/kg ethanol or saline. Mice were killed 30 min after the 2-h drinking session or injection. Blood ethanol concentration was significantly higher in the ethanol-injected versus ethanol-drinking groups, even though the dose was similar. Consumption of ethanol significantly increased brain allopregnanolone levels in male but not female mice, compared with animals drinking water, but did not alter plasma corticosterone levels. In contrast, injection of ethanol did not significantly alter brain allopregnanolone levels in male or female mice and only significantly increased plasma corticosterone levels in the male mice, when compared with saline-injected animals. The sex differences in the effect of ethanol administration on endogenous allopregnanolone levels suggest that the hormonal milieu may impact ethanol's effect on GABAergic neurosteroids. Importantly, these data are the first to report the effect of ethanol drinking on allopregnanolone levels and indicate that ethanol consumption and ethanol injection can produce physiologically relevant allopregnanolone levels in male mice. These results have important implications for studies investigating the potential role of endogenous allopregnanolone levels in modulating susceptibility to ethanol abuse.