2006

This article presents the proceedings of a symposium entitled “The Tipsy Terminal: Presynaptic Effects of Ethanol” (held at the annual meeting of the Research Society on Alcoholism, in Santa Barbara, CA, June 27, 2005). The objective of this symposium was to focus on a cellular site of ethanol action underrepresented in the alcohol literature, but quickly becoming a “hot” topic. The chairs of the session were Marisa Roberto and George Robert Siggins. Our speakers were chosen on the basis of the diverse electrophysiological and other methods used to discern the effects of acute and chronic ethanol on presynaptic terminals and on the basis of significant insights that their data provide for understanding ethanol actions on neurons in general, as mechanisms underlying problematic behavioral effects of alcohol. The 5 presenters drew from their recent studies examining the effects of acute and chronic ethanol using a range of sophisticated methods from electrophysiological analysis of paired-pulse facilitation and spontaneous and miniature synaptic currents (Drs. Weiner, Valenzuela, Zhu, and Morrisett), to direct recording of ion channel activity and peptide release from acutely isolated synaptic terminals (Dr. Treistman), to direct microscopic observation of vesicular release (Dr. Morrisett). They showed that ethanol administration could both increase and decrease the probability of release of different transmitters from synaptic terminals. The effects of ethanol on synaptic terminals could often be correlated with important behavioral or developmental actions of alcohol. These and other novel findings suggest that future analyses of synaptic effects of ethanol should attempt to ascertain, in multiple brain regions, the role of presynaptic terminals, relevant presynaptic receptors and signal transduction linkages, exocytotic mechanisms, and their involvement in alcohol’s behavioral actions. Such studies could lead to new treatment strategies for alcohol intoxication, alcohol abuse, and alcoholism.

The alcohol-preferring, P, rat was developed by selective breeding to study ethanol drinking behavior and its consequences. Characterization of this line indicates the P rat meets all of the criteria put forth for a valid animal model of alcoholism, and displays, relative to their alcohol-non-preferring, NP, counterparts, a number of phenotypic traits associated with alcohol abuse and alcoholism. Behaviorally, compared with NP rats, P rats are less sensitive to the sedative and aversive effects of ethanol and more sensitive to the stimulatory effects of ethanol. Neurochemically, research with the P line indicates the endogenous dopaminergic, serotonergic, GABAergic, opiodergic, and peptidergic systems may be involved in a predisposition for alcohol abuse and alcoholism. Paralleling the clinical literature, genetically selected P rats display levels of ethanol intake during adolescence comparable to that seen during adulthood. Binge drinking has been associated with an increased risk for health and other problems associated with ethanol abuse. A model of binge-like drinking during the dark cycle indicates that P rats will consume 6 g/kg/day of ethanol in as little as three 1-hour access periods/day, which approximates the 24-hour intake of P rats with free-choice access to a single concentration of ethanol. The alcohol deprivation effect (ADE) is a transient increase in ethanol intake above baseline values upon re-exposure to ethanol access after an extended period of deprivation. The ADE has been proposed to be an animal model of relapse behavior, with the adult P rat displaying a robust ADE after prolonged abstinence. Overall, these findings indicate that the P rat can be effectively used in models assessing alcohol-preference, a genetic predisposition for alcohol abuse and/or alcoholism, and excessive drinking using protocols of binge-like or relapse-like drinking.

Since 1996, nearly 100 genes have been studied for their effects related to ethanol in mice using genetic modifications including gene deletion, gene overexpression, gene knock-in, and occasionally by studying existing mutants. Nearly all such studies have concentrated on genes expressed in brain, and the targeted genes range widely in their function, including most of the principal neurotransmitter systems, several neurohormones, and a number of signaling molecules. We review 141 published reports of effects (or lack thereof) of 93 genes on responses to ethanol. While most studies have focused on ethanol self-administration and reward, and/or sedative effects, other responses studied include locomotor stimulation, anxiolytic effects, and neuroadaptation (tolerance, sensitization, withdrawal). About 1/4 of the engineered mutations increase self-administration, 1/3 decrease it, and about 40% have no significant effect. In many cases, the effects on self-administration are rather modest and/or depend on the specific experimental procedures. In some cases, genes in the background strains on which the mutant is placed are important for results. Not surprisingly, review of the systems affected further supports roles for serotonin, gamma-aminobutyric acid, opioids and dopamine, all of which have long been foci of alcohol research. Novel modulatory effects of protein kinase C and G protein-activated inwardly rectifying K+ (GIRK) channels are also suggested. Some newer research with cannabinoid systems is promising, and has led to ongoing clinical trials.

This article summarizes the proceedings of a symposium held at the 2005 Research Society on Alcoholism meeting. The initial presentation by Dr. Wallner provided evidence that selected GABA(A) receptors containing the delta subunit display sensitivity to low intoxicating ethanol concentrations and this sensitivity is further increased by a mutation in the cerebellar alpha6 subunit, found in alcohol-hypersensitive rats. Dr. Mameli reported that ethanol affects gamma-aminobutyric acid (GABA) function by affecting neural circuits that influence GABA release. Dr. Parsons presented data from electrophysiological and microdialysis investigations that ethanol is capable of releasing GABA from presynaptic terminals. Dr. Morrow demonstrated that systemic ethanol increases neuroactive steroids in brain, the absence of which alters various functional responses to ethanol. Dr. Criswell presented evidence that the ability of ethanol to increase GABA was apparent in some, but not all, brain regions indicative of regional specificity. Further, Dr. Criswell demonstrated that neurosteroids alone and when synthesized locally by ethanol act postsynaptically to enhance the effect of GABA released by ethanol in a region specific manner. Collectively, this series of reports support the GABAmimetic profile of acutely administered ethanol being dependent on several specific mechanisms distinct from a direct effect on the major synaptic isoforms of GABA(A) receptors.

Blockade of the mGluR5 subtype of Group 1 metabotropic glutamate receptor (mGluRs) reduces the rewarding effects of ethanol (EtOH), while the effects of mGluR1a blockade remain under-investigated. The present study compared the effects of pretreatment with the mGluR5 antagonist MPEP and the mGluR1a antagonist CPCCPOEt upon behavioral and neurochemical variables associated with EtOH reward in alcohol-preferring C57BL/6J mice. Pretreatment with either antagonist (0-10 mg/kg, IP) dose-dependently reduced measures of EtOH reward in an operant self-administration paradigm and the maximally effective antagonist dose (10 mg/kg) also blocked the expression of EtOH-induced place conditioning, as well as EtOH consumption under 24-h free-access conditions. MPEP pretreatment did not significantly alter the EtOH dose-locomotor response function; however, it prevented EtOH-induced changes in extracellular dopamine, glutamate and GABA in the nucleus accumbens (NAC). In contrast, CPCCOEt shifted the EtOH dose-response function downwards, enhanced the capacity of higher EtOH doses to elevate NAC levels of GABA and lowered extracellular dopamine and glutamate below baseline following EtOH injection. It is suggested that the “anti-alcohol” effects of MPEP may involve an attenuation of the neurochemical signals mediating EtOH reward, whereas those of CPCCOEt may involve an increased sensitivity to the inhibitory effects of EtOH upon brain and behavior.

QTL analysis of behavioral traits and mouse brain gene expression studies were combined to identify candidate genes involved in the traits of alcohol preference and acute functional alcohol tolerance. The systematic application of normalization and statistical analysis of differential gene expression, behavioral and expression QTL location, and informatics methodologies resulted in identification of 8 candidate genes for the trait of alcohol preference and 22 candidate genes for acute functional tolerance. Pathway analysis, combined with clustering by ontology, indicated the importance of transcriptional regulation and DNA and protein binding elements in the acute functional tolerance trait, and protein kinases and intracellular signal transduction elements in the alcohol preference trait. A rudimentary search for transcription control elements that could indicate coregulation of the panels of candidate genes produced modest results, implicating SMAD-3 in the regulation of four of the eight candidate genes for alcohol preference. However, the realization of the many caveats related to transcription factor binding site analysis, and attempts to correlate between transcription factor binding and function, forestalled any definitive global analysis of transcriptional control of differentially expressed candidate genes.

Past research has indicated that chronic ethanol exposure enhances dopamine (DA) neurotransmission in several brain regions. The present study examined the effects of chronic ethanol drinking on dopamine transporter (DAT) function in the nucleus accumbens (Acb) of High-Alcohol-Drinking replicate line 1 (HAD-1) rats. HAD rats were given concurrent 24-h access to 15% ethanol and water or water alone for 8 weeks. Subsequently, DA uptake and the Vmax of the DAT were compared between the two groups using homogenates of the nucleus accumbens. DA uptake was measured following a 2 min incubation at 37°C in the presence of 8 nM [3H]DA. For kinetic analyses, DA uptake was assessed in the presence of 5 concentrations of [3H]DA ranging from 8 nM to 500 nM. Analyses of the data revealed a significant increase in DA uptake in the ethanol group compared to water controls. Kinetic analyses revealed the change in DA uptake to be a consequence of an increase in the Vmax of transport. These findings demonstrate that chronic free-choice oral ethanol consumption in HAD-1 female rats increases DA uptake in the Acb by increasing the Vmax of the transporter. However, it is not known whether the ethanol-induced change in Vmax is caused by differences in the actual number of available transporter sites or from a difference in the velocity of operation of a similar number of transporters. Overall, the data indicate that chronic ethanol consumption by HAD-1 rats produces prolonged neuroadaptations within the mesolimbic DA system, which may be important for the understanding of the neurobiological basis of alcoholism.

The medium spiny neurons (MSNs) of the nucleus accumbens function in a critical regard to examine and integrate information in the processing of rewarding behaviors. These neurons are aberrantly affected by drugs of abuse, including alcohol. However, ethanol is unlike any other common drug of abuse, due to its pleiotropic actions on intracellular and intercellular signaling processes. Intracellular biochemical pathways appear to critically contribute to long-term changes in the level of synaptic activation of these neurons, which have been implicated in ethanol dependence. Additionally, these neurons also display a fascinating pattern of up/down activity, which appears to be, at least in part, regulated by convergent activation of dopaminergic and glutamatergic (NMDA) inputs. Thus, dopaminergic and NMDA receptor-mediated synaptic transmission onto these neurons may constitute a critical site of ethanol action in mesolimbic structures. For instance, dopaminergic inputs alter the ability of ethanol to regulate NMDA receptor-mediated synaptic transmission onto accumbal MSNs. Prior activation of D1-signaling cascade through the cAMP-regulated phosphoprotein-32kD (DARPP-32) and protein phosphatase-1 (PP-1) pathway significantly attenuates ethanol inhibition of NMDA receptor function. Therefore, the interaction of D1-signaling and NMDA receptor signaling may alter NMDA receptor-dependent long-term synaptic plasticity, contributing to the development of ethanol-induced neuroadaptation of the reward pathway.

Alcohol abuse among adolescents continues to be a major health problem for our society. Our laboratory has used the peri-adolescent alcohol-preferring, P, rat as an animal model of adolescent alcohol abuse. Even though peri-adolescent P rats consume more alcohol (g/kg/day) than their adult counterparts, it is uncertain whether their drinking is sufficiently aggregated to result in measurable blood ethanol concentrations (BECs). The objectives of this study were to examine daily alcohol drinking patterns of adolescent and adult, male and female P rats, and to determine whether alcohol drinking episodes were sufficiently aggregated to result in meaningful BECs. Male and female P rats were given 30 days of 24 h free-choice access to alcohol (15%, v/v) and water, with ad lib access to food, starting at the beginning of adolescence (PND 30) or adulthood (PND 90). Water and alcohol drinking patterns were monitored 22 h/day with a “lickometer” set-up. The results indicated that (a) peri-adolescent P rats consumed more water and total fluids than adult P rats, (b) female P rats consumed more water and total fluids than male P rats, (c) there were differences in alcohol, and water, licking patterns between peri-adolescent and adult and female and male P rats, (d) individual licking patterns revealed that alcohol was consumed in bouts often exceeding the amount required to self-administer 1 g/kg of alcohol, and (e) BECs at the end of the dark cycle, on the 30th day of alcohol access, averaged 50 mg%, with alcohol intakes during the last 1 to 2 h averaging 1.2 g/kg. Overall, these findings indicate that alcohol drinking patterns differ across the age and sex of P rats. This suggests that the effectiveness of treatments for reducing excessive alcohol intake may vary depending upon the age and/or sex of the subjects being tested.

The pharmacological profile of a gamma-aminobutyric acid A (GABA(A)) receptor depends upon subunit composition. Studies using recombinant expression systems suggest that delta-subunit containing GABA(A) receptors are particularly sensitive to the actions of the GABA(A) partial agonist, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP, gaboxadol). Here we investigated the actions of THIP in mutant mice lacking the GABA(A) receptor delta-subunit gene. Using the chloride flux assay, we determined that the actions of THIP were reduced by 21% in the cortical, but not cerebellar, membranes of knockout mice. Similar results were seen with another GABA(A) agonist, muscimol. Moreover, delta-subunit knockout mice exhibited a 54% reduction in sensitivity to the hypnotic actions of THIP as assessed by the loss of righting reflex test. These data support the notion that delta-containing GABA(A) receptors are at least partially responsible for the actions of THIP, and contribute to the growing literature suggesting that the pharmacological specificity of GABA(A) receptors depends on which subunits are present or absent.

Brain receptor patterns for the corticotropin-releasing factor (CRF) receptors, CRF1 and CRF2, are dramatically different between monogamous and promiscuous vole species, and CRF physiologically regulates pair bonding behavior in the monogamous prairie vole. However, little is known whether species differences also exist in the neuroanatomical distribution of the endogenous ligands for the CRF1 and CRF2 receptors, such as CRF and urocortin-1 (Ucn1). We compared the expression of CRF and Ucn1 in four vole species, monogamous prairie and pine voles, and promiscuous meadow and montane voles, using in situ hybridization of CRF and Ucn1 mRNA. Our results reveal that CRF mRNA expression patterns in all four vole species appear highly conserved throughout the brain, including the olfactory bulb, nucleus accumbens, bed nucleus of the stria terminalis, medial preoptic area, central amygdala, hippocampus, posterior thalamus, and cerebellum. Similarly, Ucn1 mRNA primarily localized to the Edinger-Westphal nucleus in all four vole species. Immunocytochemistry in prairie and meadow voles confirmed localization of CRF and Ucn1 protein to these previously identified brain regions. These data demonstrate a striking dichotomy between the extraordinary species diversity of brain receptor patterns when compared to the highly conserved brain distributions of their respective ligands. Our findings generate novel hypotheses regarding the evolutionary mechanisms underlying the neural circuitry of species-typical social behaviors.

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.

BACKGROUND: Dopaminergic (DA) activity in the extended amygdala (EA) has been known to play a pivotal role in mediating drug and alcohol addiction. Alterations of DA activity within the EA after chronic exposure to alcohol or substances of abuse are considered a major mechanism for the development of alcoholism and addiction. To date, it is not clear how different patterns of chronic alcohol drinking affect DA receptor levels. Therefore, the current studies investigated the effects of chronic ethanol consumption, with or without deprivations, on D1 and D2 receptor densities within the EA. METHODS: Inbred alcohol-preferring (iP) rats were divided into 3 groups with the following treatments: (1) water for 14 weeks; (2) continuous alcohol (C-Alc) for 14 weeks [24-hour concurrent access to 15 and 30% (v/v) ethanol]; or (3) repeatedly deprived of alcohol (RD-Alc) (24-hour concurrent access to 15 and 30% ethanol for 6 weeks, followed by 2 cycles of 2 weeks of deprivation of and 2 weeks of reexposure to ethanol access). At the end of 14 weeks, the rats were killed for autoradiographic labeling of D1 and D2 receptors. RESULTS: Compared with the water control group, both the C-Alc and the RD-Alc groups displayed increases in D1 receptor binding density in the anterior region of the Acb core, whereas the RD-Alc group displayed additional increases in D1 receptor binding density in anterior regions of the lateral and intercalated nuclei of the amygdala. Additionally, both C-Alc and RD-Alc rats displayed increases in D2 receptor binding density in anterior regions of the Acb shell and core, whereas RD-Alc rats displayed additional increases in D2 receptor binding density in the dorsal striatum. CONCLUSION: The results of this study indicate that 14-week extended alcohol drinking with continuous chronic or repeated deprivations increase binding sites of D1 and D2 receptors in specific regions of the EA with greater sensitivity in the anterior regions. The repeated deprivation has greater effect on altering D1 and D2 receptor binding sites in the Acb, dorsal striatum, and subamygdala regions. The current result indicates that the two drinking paradigms may have common as well as differential mechanisms on alteration of dopamine receptor-binding sites in specific regions of the EA.

Mice from eight inbred strains were studied for their acute sensitivity to ethanol as indexed by the degree of hypothermia (HT), indexed as the reduction from pre-injection baseline of their body temperature. Two weeks later, mice were tested for their loss of righting reflex (LRR) after a higher dose of ethanol. The LRR was tested using the “classical” method of watching for recovery in animals placed on their backs in a V-shaped trough and recording duration of LRR. In a separate test, naive animals of the same strains were tested for HT repeatedly to assess the development of rapid (RTOL) and chronic tolerance (CTOL). We have recently developed a new method for testing LRR that leads to a substantial increase in the sensitivity of the test. Strains also have been found to differ in the new LRR test, as well as in the development of acute functional tolerance (AFT) to this response. In addition, our laboratory has periodically published strain difference data on the older versions of the HT and LRR responses. The earlier tests used some of the exact substrains tested currently, while for some strains, different substrains (usually, Nih versus Jax) were tested. We examined correlations of strain means to see whether patterns of strain differences were stable across time and across different test variants assessing the same behavioral construct. HT strain sensitivity scores were generally highly correlated across a 10-23 years period and test variants. The CTOL to HT was well-correlated across studies, and was also genetically similar to RTOL. The AFT, however, was related to neither RTOL nor CTOL, although this may be because different phenotypic end points were compared. The LRR data, which included a variant of the classical test, were not as stable. Measures of LRR onset were reasonably well correlated, as were those taken at recovery (e.g., duration). However, the two types of measures of LRR sensitivity to ethanol appear to be tapping traits that differ genetically. Also, the pattern of genetic correlation between HT and LRR initially reported in 1983 was not seen in current and contemporaneous studies. In certain instances, substrain seems to matter little, while in others, substrains differed a great deal. These data are generally encouraging about the stability of genetic differences.

Cocaine- and amphetamine-regulated transcript (CART) is a peptide neurotransmitter that has been implicated in drug reward and reinforcement. CART mRNA and peptide expression are highly concentrated in several compartments of the mesolimbic reward pathway. Several lines of evidence suggest that CART peptides may contribute to rewarding behaviors and the addiction liability of psychostimulants; however, there are no reports of basic work concerning CART in relation to alcohol and mechanisms of alcohol dependence development. Therefore, in this study we investigated the response of CART transcript and peptide to acute ethanol administration in vivo. Rats were administered ethanol (1 g/kg or 3.5 g/kg, 1 h, ip) and CART expression was measured by RT-PCR in the nucleus accumbens (NAcc). Ethanol (3.5 g/kg) increased CART transcription markedly. The interactions of dopamine on ethanol-induced CART expression were further evaluated pharmacologically using D1 and D2/D3 receptor antagonists. Both SCH 23390 (0.25 mg/kg) or raclopride (0.2 mg/kg) pre-treatment significantly suppressed ethanol-enhancement of CART mRNA transcription. Confocal immunofluorescence microscopy revealed that CART peptide immunoreactivity was also enhanced in both the core and the shell of the NAcc by ethanol administration. These findings demonstrate that CART mRNA and peptide expression are responsive to acute ethanol administrated in vivo and suggests that CART peptides may be important in regulating the rewarding and reinforcing properties of ethanol.

This article summarizes the proceedings of a symposium at the 2005 Research Society on Alcoholism Meeting in Santa Barbara, California, organized and cochaired by John J. Woodward and Dorit Ron. The purpose of the symposium was to discuss recent findings that extend our understanding of the importance of the N-methyl-D-aspartate (NMDA) receptor as a target for ethanol action in the brain. These receptors are ligand-gated ion channels that are activated by the neurotransmitter glutamate and are critically involved in many forms of synaptic plasticity including those associated with learning and memory. In the first presentation, Dorit Ron presented data showing how activation of Fyn or Src tyrosine kinases differentially regulated the cell surface expression and activity of NR2A and NR2B containing NMDA receptors. Danny Winder discussed the effects of ethanol on NMDA receptor-dependent long-term potentiation in the bed nucleus of the stria terminalis (BNST), a brain region associated with the interaction between stress and drug/alcohol use. In the third presentation, Marisa Roberto described adaptations in the expression and function of NMDA receptors in the central nucleus of the amygdala following chronic exposure to ethanol. Finally, John Woodward described the effects of ethanol on the activity of neurons in deep layers of the prefrontal cortex using a novel slice coculture preparation.

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 knockout 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 knockout 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 stronge)st evidence for a role of alpha1 subunits in ethanol-induced loss of righting reflex and alpha5 subunits in ethanol-stimulated locomotion. Nevertheless, 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.

CONTEXT: Alcohol facilitates gamma-aminobutyric acid (GABA) function, and GABA type A (GABA(A)) receptor-facilitating agents suppress alcohol withdrawal symptoms. Advances in molecular neuroscience, genetics, and neuroimaging provide new insights into the role of brain GABA systems in short- and long-term alcohol effects. OBJECTIVE: To review the role of brain GABA systems in alcohol response, alcohol dependence, alcoholism vulnerability, and alcoholism pharmacotherapy. DESIGN: Literature review. RESULTS: Alcohol increases GABA release, raises neurosteroid levels, and may potently enhance the function of a GABA(A) receptor subclass that shows high affinity for GABA and neurosteroids, relative insensitivity to benzodiazepines, low chloride conductance, and an extrasynaptic location. Variation in GABA(A) receptor subunit genes may contribute to the vulnerability to alcoholism, particularly in the context of environmental risk factors. Alcohol dependence is associated with time-dependent changes in brain GABA(A) receptor density and subunit gene expression levels that contribute to a withdrawal-related deficit in GABA(A) receptor function. However, cortical GABA levels are not reduced during acute withdrawal. Benzodiazepine-assisted detoxification enhances a phasic component of GABA function. However, novel treatments target the tonic component of GABA neurotransmission mediated by benzodiazepine-insensitive GABA(A) receptors. Smoking attenuates withdrawal-related disturbances in brain GABA function, perhaps contributing to comorbid nicotine and alcohol dependence. The GABA systems show recovery with long-term sobriety. CONCLUSIONS: Recent research deepens our understanding of the role of GABA systems in alcohol action, alcohol dependence, and the vulnerability to alcoholism. Also, GABA(A) receptor subtype-selective treatments merit exploration for reducing withdrawal symptoms and drinking in alcohol-dependent individuals.

BACKGROUND: Differences in ethanol metabolizing enzymes expressed in brain have been suggested to contribute to the significant differences in ethanol (alcohol) preference between inbred C57BL/6 and DBA/2 mouse strains. METHODS: We have utilized 2 different platforms of oligonucleotide microarray technology (CodeLink UniSet I BioArray from G.E. Healthcare and MG U74A v2.0 from Affymetrix) to simultaneously assess expression of alcohol and acetaldehyde metabolizing enzymes in the whole brain of naïve (no exposure to alcohol) C57BL/6 and DBA/2 mice. RESULTS: There were no significant differences between the 2 strains of mice in gene expression intensity for alcohol dehydrogenases (ADH), catalase, and a number of the cytochrome P450 family of genes, which can be involved in ethanol catabolism. However, significantly higher expression of mRNA for aldehyde dehydrogenase 2 (ALDH2), an isoform mainly responsible for the catabolism of acetaldehyde, was observed in whole brains of DBA/2 mice with both platforms. Aldehyde dehydrogenase 2 protein was also higher in DBA/2 brain. Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA was found to be higher in brains of DBA/2 mice, when measured with the CodeLink platform, but not when measured with Affymetrix arrays or quantitative reverse transcriptase-real-time polymerase chain reaction (qRT-PCR). The ALDH1A1 protein, however, reflected the results obtained with the CodeLink arrays and was higher in DBA/2 brain, compared with brains of C57BL/6 mice. In contrast, the expression intensity for the aldehyde dehydrogenase 7A1 (ALDH7A1) mRNA and protein was significantly higher in C57BL/6 mice than DBA/2 mice. These expression differences are consistent with more rapid metabolism of acetaldehyde in brains of DBA/2 mice. CONCLUSIONS: The use of 2 different microarray platforms provides important cross-validation of many results, and some discrepancies can be resolved with qRT-PCR and immunoblotting. The expression differences that were validated may affect alcohol/aldehyde metabolism in brain and/or alcohol preference in the 2 strains of mice.

High novelty seeking is a complex personality attribute correlated with risk for substance abuse. There are many putative mouse models of some aspects of novelty seeking, but little is known of genetic similarities among these models. To assess the genetic coherence of “novelty seeking,” we compared the performance of 14 inbred strains of mice in five tests: activity in a novel environment, novel environment preference, head dipping on a hole-board, object preference, and a two-trial version of the spontaneous alternation task. Differences among strains were observed for all tasks, but performance in any given task was generally not predictive of performance in any other. To evaluate similarities among these tasks further, we selectively bred lines of mice for high or low head dipping on the hole-board. Similar to results from the inbred strain experiments, head dipping was not correlated with performance in the other measures but was genetically correlated with differences in locomotor activity. Using two approaches to estimating common genetic influences across tasks, we have found little evidence that these partial models of novelty seeking reflect the influence of common genes or measure a single, unified construct called novelty seeking. Based on the substantial influence of genetic factors, ease of implementation, and relative independence from general locomotion, head dipping on a hole-board is a good task to use in the domain of novelty seeking, but multiple tasks, including others not tested here, would be needed to capture the full genetic range of the behavioral domain.

Alcohol has been shown to affect glutamate (GLU) and dopamine (DA) release and their correlated receptors in the key reward center–extended amygdala–which includes the shell of nucleus accumbens (sNAc) and central nucleus of amygdala (cAmg). It is unclear to date whether there is an alteration in the number of presynaptic GLU/DA nerve terminals. In this study, we investigated the number of GLU and DA terminals in the extended amygdala of alcohol-preferring (P) rats that chronically drank ethanol. P rats have a propensity to drink ethanol to intoxication and develop an alcohol dependency. The P rats were divided into (1) Water group given ad libitum chow and water for 14 weeks; (2) Continuous alcohol group (C-Alc) given ad libitum chow and choice of 15 or 30% (v/v) ethanol or water for 14 weeks; and (3) Repeated deprivation (RD-Alc) group given the same choice of ethanol or water for 6 weeks, followed by a twice repeated cycle of 2 weeks without ethanol followed by 2 weeks with ethanol. Two subpopulations of GLU terminals were labeled by immunostaining for the vesicular GLU transporter 1 (vGLUT1) and vesicular GLU transporter 2 (vGLUT2). DA terminals were labeled by immunostaining for tyrosine hydroxylase (TH). The GLU and DA immunostained (im) varicosities were quantified and analyzed using stereological methods. We found that chronic alcohol did not alter the number of TH-im terminals in the extended amygdala in either the C-Alc or RD-Alc drinking paradigms. Thus, the increases in extracellular levels of DA previously reported following chronic alcohol are likely due to a change in the efficiency of DA release rather than a change in the number of DA terminals. The number of vGLUT1-im terminals was also unchanged in the extended amygdala; however, the number of vGLUT2-im terminals, which represent the greater population of GLU terminals, was increased in the sNAc of the RD-Alc group compared to the Water group. Chronic alcohol is known to affect GLU release, and our findings indicate that repeated alcohol deprivation may preferentially increase GLU terminals in the sNAc bearing the vGLUT2, which are primarily afferents from the thalamus. Our results further indicate that repeated deprivation of alcohol can change the ratio of GLU to DA innervation in the sNAc, a key region of the reward circuitry.

Homer proteins regulate signal transduction, synaptogenesis and receptor trafficking, in addition to maintaining and regulating extracellular glutamate levels in limbo–corticostriatal brain regions. Converging preclinical observations indicate a potential role for both immediate early gene Homer isoforms and constitutively expressed Homer isoforms in behavioral pathologies associated with neuropsychiatric disorders, such as addiction and/or alcoholism, depression, anxiety, epilepsy and schizophrenia.

This article represents the proceedings of a symposium at the Research Society on Alcoholism meeting in Santa Barbara, California. The organizers/chairs were Kristine M. Wiren and Deborah A. Finn. Following a brief introduction by Deborah Finn, the presentations were (1) The Importance of Gender in Determining Expression Differences in Mouse Lines Selected for Chronic Ethanol Withdrawal Severity, by Kristine M. Wiren and Joel G. Hashimoto; (2) Sex Differences in Ethanol Withdrawal Involve GABAergic and Stress Systems, by Paul E. Alele and Leslie L. Devaud; (3) The Influence of Sex on Ethanol Consumption and Reward in C57BL/6 Mice, by Kimber L. Price and Lawrence D. Middaugh; and (4) Sex Differences in Alcohol Self-administration in Cynomolgus Monkeys, by Kathleen A. Grant.

Proton magnetic resonance spectroscopy was used at 3T to measure the uptake and clearance of brain ethanol in rats after bolus intraperitoneal (i.p.) or intragastric (i.g.) alcohol injection, and to estimate the effects of acute alcohol on brain metabolites. The observation duration was 1\textbar[ndash]\textbar1.5\textbar[thinsp]\textbarh with temporal resolution of alcohol sampling ranging from 4\textbar[thinsp]\textbars\textbar[ndash]\textbar4\textbar[thinsp]\textbarmin. The observed time course of alcohol brain concentration followed a consistent pattern characterized by a rapid absorption, an intermediate distribution, and a slower clearance that approached a linear decay. In a sample of eight healthy Wistar rats, the intercept of the linear clearance term, extrapolated back to the time of injection, correlated well with the administered dose per unit of lean body mass. Alcohol concentration estimation based on spectroscopically measured clearance was compared with blood alcohol levels from blood samples at the end of observation, and were in good agreement with the administered dose. Serial proton spectroscopy measurements provide a valid in vivo method for quantifying brain alcohol uptake and elimination kinetics in real time.

Our study examined ethanol self-administration and accumbal dopamine concentration during kappa-opioid receptor (KOPr) blockade. Long-Evans rats were trained to respond for 20 min of access to 10% ethanol (with sucrose) over 7 days. Rats were injected s.c. with the long-acting KOPr antagonist, nor-binaltorphimine (NOR-BNI; 0 or 20 mg/kg) 15-20 h prior to testing. Microdialysis revealed a transient elevation in dopamine concentration within 5 min of ethanol access in controls. NOR-BNI-treated rats did not exhibit this response, but showed a latent increase in dopamine concentration at the end of the access period. The rise in dopamine levels correlated positively with dialysate ethanol concentration but not in controls. NOR-BNI did not alter dopamine levels in rats self-administering 10% sucrose. The transient dopamine response during ethanol acquisition in controls is consistent with previous results that were attributed to ethanol stimulus cues. The altered dopamine response to NOR-BNI during ethanol drinking suggests that KOPr blockade temporarily uncovered a pharmacological stimulation of dopamine release by ethanol. Despite these neurochemical changes, NOR-BNI did not alter operant responding or ethanol intake, suggesting that the KOPr is not involved in ethanol-reinforced behavior under the limited conditions we studied.

BACKGROUND: Tracking the dynamic course of human alcoholism brain pathology can be accomplished only through naturalistic study and without opportunity for experimental manipulation. Development of an animal model of alcohol-induced brain damage, in which animals consume large amounts of alcohol following cycles of alcohol access and deprivation and are examined regularly with neuroimaging methods, would enable hypothesis testing focused on the degree, nature, and factors resulting in alcohol-induced brain damage and the prospects for recovery or relapse. METHODS: We report the results of longitudinal magnetic resonance imaging (MRI) studies of the effects of free-choice chronic alcohol intake on the brains of 2 cohorts of selectively bred alcohol-preferring (P) rats. In the companion paper, we described the MRI acquisition and analysis methods, delineation of brain regions, and growth patterns in total brain and selective structures of the control rats in the present study. Both cohorts were studied as adults for about 1 year and consumed high doses of alcohol for most of the study duration. The paradigm involved a 3-bottle choice with 0, 15 (or 20%), and 30% (or 40%) alcohol available in several different exposure schemes: continuous exposure, cycles of 2 weeks on followed by 2 weeks off alcohol, and binge drinking in the dark. RESULTS: Brain structures of the adult P rats in both the alcohol-exposed and the water control conditions showed significant growth, which was attenuated in a few measures in the alcohol-exposed groups. The region with the greatest demonstrable effect was the corpus callosum, measured on midsagittal images. CONCLUSION: The P rats showed an age-alcohol interaction different from humans, in that normal growth in selective brain regions that continues in adult rats was retarded.

Behavioral studies show that the GABAergic system in the central amygdala (CeA) nucleus has a complex role in the reinforcing effects effects of ethanol and the anxiogenic response to ethanol withdrawal. Opioid peptides and nociceptin/orphanin FQ (nociceptin) within the CeA are implicated also in regulating voluntary ethanol consumption and ethanol relapse. Recently, we reported that basal GABAergic transmission was increased in ethanol-dependent rats, and that acute ethanol increases GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in CeA neurons from both naïve and ethanol-dependent rats to the same extent, suggesting lack of tolerance for the acute effect of ethanol. Here, we investigated the effect of nociceptin on IPSCs in CeA neurons and its interaction with ethanol effects on these GABA synapses. We found that nociceptin moderately decreased IPSC amplitudes, acting mostly presynaptically as it increased paired-pulse facilitation ratio of IPSCs and decreased miniature IPSC frequencies (but not amplitudes). Nociceptin also prevented the ethanol-induced augmentation of IPSCs in CeA of naïve rats. Interestingly, in CeA of ethanol-dependent rats, the nociceptin-induced inhibition of IPSCs was increased, indicating an enhanced sensitivity to nociceptin. Nociceptin also blocked the ethanol-induced augmentation of IPSCs in ethanol-dependent rats. Our data suggest that nociceptin has a role in regulating the GABAergic system and opposing the effect elicited by ethanol. Thus, nociceptin may represent a therapeutic target for alleviating alcohol dependence.

BACKGROUND: Serum treatment of quiescent human dermal fibroblasts induces proliferation, coupled with a complex physiological response that is indicative of their normal role in wound-healing. However, it is not known to what extent such complex transcriptional events are specific to a given cell type and signal, and how these global changes are coordinately regulated. We have profiled the global transcriptional program of human fibroblasts from two different tissue sources to distinct growth stimuli, and identified a striking conservation in their gene-expression signatures. RESULTS: We found that the wound-healing program of gene expression was not specific to the response of dermal fibroblasts to serum but was regulated more broadly. However, there were specific differences among different stimuli with regard to signaling pathways that mediate these transcriptional programs. Our data suggest that the PI3-kinase pathway is differentially involved in mediating the responses of cells to serum as compared with individual peptide growth factors. Expression profiling indicated that let7 and other miRNAs with similar expression profiles may be involved in regulating the transcriptional program in response to proliferative signals. CONCLUSION: This study provides insights into how different stimuli use distinct as well as conserved signaling and regulatory mechanisms to mediate genome-wide transcriptional reprogramming during cell proliferation. Our results indicate that conservation of transcriptional programs and their regulation among different cell types may be much broader than previously appreciated.

OBJECTIVE: Individual differences in subjective responses to alcohol are believed to have a genetic basis and have been associated with increased risk of alcohol-related problems. There are, however, conflicting results from past studies, perhaps owing to differences in subjective alcohol effects by limb of the blood alcohol curve and the passage of time. The current pilot study evaluated relations among serotonin transporter (SERT) genotype, subjective alcohol responses, and drinking behavior across both the ascending and descending limbs of the blood alcohol curve. METHOD: Participants (N=222; 68% male) were administered alcohol (target blood alcohol concentration of .06%) with a subsample (n=86) providing genetic data. Following a social stressor, participants were provided the opportunity to engage in ad libitum alcohol consumption. RESULTS: SERT transporter was not significantly associated with ad lib drinking or subjective alcohol effects at individual time points, although a trend toward a SERT by blood alcohol concentration limb interaction was observed for ad lib drinking. In addition, SERT genotype predicted acute tolerance to alcohol effects, with participants homozygous for the long SERT allele developing acute tolerance more rapidly than other genotypes. CONCLUSIONS: Although SERT genotype was not reliably associated with ad lib drinking behavior, the results suggest that individuals with the long-long (LL) genotype may develop acute tolerance to alcohol effects more rapidly than heterozygotes or individuals homozygous for the short SERT allele.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 hours) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest thath this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Major depression represents a complex mental disorder. The identification of biological markers that define subtypes of major depressive disorder would greatly facilitate appropriate medical treatments, as well as provide insight into etiology. Reduced activity of the cAMP signaling system has been implicated in the etiology of major depression. Previous work has shown low adenylyl cyclase activity in platelets and postmortem brain tissue of depressed individuals. Here, we investigate the role of the brain type VII isoform of adenylyl cyclase (AC7) in the manifestation of depressive symptoms in genetically modified animals, using a combination of in vivo behavioral experiments, gene expression profiling, and bioinformatics. We also completed studies with humans on the association of polymorphisms in the AC7 gene with major depressive illness (unipolar depression) based on Diagnostic and Statistical Manual of Mental Disorders IV criteria. Collectively, our results demonstrate a sex-specific influence of the AC7 gene on a heritable form of depressive illness.

If we conduct the same experiment in two laboratories or repeat a classical study many years later, will we obtain the same results? Recent research with mice in neural and behavioral genetics yielded different results in different laboratories for certain phenotypes, and these findings suggested to some researchers that behavior may be too unstable for fine-scale genetic analysis. Here we expand the range of data on this question to additional laboratories and phenotypes, and, for the first time in this field, we formally compare recent data with experiments conducted 30–50 years ago. For ethanol preference and locomotor activity, strain differences have been highly stable over a period of 40–50 years, and most strain correlations are in the range of r = 0.85–0.98, as high as or higher than for brain weight. For anxiety-related behavior on the elevated plus maze, on the other hand, strain means often differ dramatically across laboratories or even when the same laboratory is moved to another site within a university. When a wide range of phenotypes is considered, no inbred strain appears to be exceptionally stable or labile across laboratories in any general sense, and there is no tendency to observe higher correlations among studies done more recently. Phenotypic drift over decades for most of the behaviors examined appears to be minimal.

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California, USA. The organizer and chair was L. Judson Chandler. The presentations were (1) Chronic Ethanol Exposure, N-Methyl-D-Aspartate (NMDA) Receptor Dynamics, and Withdrawal Hyperexcitability, by Adam Hendricson, Regina Maldve, and Richard Morrisett; (2) Ethanol-Induced Synaptic Targeting of NMDA Receptors Is Associated With Enhanced Postsynaptic Density-95 Clustering and Spine Size, by Judson Chandler and Ezekiel Carpenter-Hyland; (3) Presynaptic and Postsynaptic Alterations in the Nucleus Accumbens Following Chronic Alcohol Exposure, by Feng Zhou, Youssef Sari, and Richard Bell; and (4) An Active Role for Accumbens Homer2 Expression in Alcohol-Induced Neural Plasticity, by Karen Szumlinski.

GABAA receptors mediate the majority of inhibitory neurotransmission in the CNS. Genetic deletion of the alpha1 subunit of GABAA receptors results in a loss of alpha1-mediated fast inhibitory currents and a marked reduction in density of GABAA receptors. A grossly normal phenotype of alpha1-deficient mice suggests the presence of neuronal adaptation to these drastic changes at the GABA synapse. We used cDNA microarrays to identify transcriptional fingerprints of cellular plasticity in response to altered GABAergic inhibition in the cerebral cortex and cerebellum of alpha1 mutants. In silico analysis of 982 mutation-regulated transcripts highlighted genes and functional groups involved in regulation of neuronal excitability and synaptic transmission, suggesting an adaptive response of the brain to an altered inhibitory tone. Public gene expression databases permitted identification of subsets of transcripts enriched in excitatory and inhibitory neurons as well as some glial cells, providing evidence for cellular plasticity in individual cell types. Additional analysis linked some transcriptional changes to cellular phenotypes observed in the knock-out mice and suggested several genes, such as the early growth response 1 (Egr1), small GTP binding protein Rac1 (Rac1), neurogranin (Nrgn), sodium channel beta4 subunit (Scn4b), and potassium voltage-gated Kv4.2 channel (Kcnd2) as cell type-specific markers of neuronal plasticity. Furthermore, transcriptional activation of genes enriched in Bergman glia suggests an active role of these astrocytes in synaptic plasticity. Overall, our results suggest that the loss of alpha1-mediated fast inhibition produces diverse transcriptional responses that act to regulate neuronal excitability of individual neurons and stabilize neuronal networks, which may account for the lack of severe abnormalities in alpha1 null mutants.