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.