Alcoholism in the United States is a serious health concern. Our long-term goal is to elucidate the mechanisms underlying alcohol addiction, a necessary prerequisite to the development of effective therapy. The specific hypothesis is that acute ethanol increases glutamate release via activation of dopamine D1 receptors (D1R). The increased glutamatergic transmission in turn modulates dopaminergic cell activity in the reward pathway and thus plays a significant role in the processes involved in alcohol addiction. We base this hypothesis on the following observations: a) clinically relevant concentrations of ethanol (10-80 mM) increase the amplitude of evoked excitatory postsynaptic currents (EPSCs) mediated by AMPA receptors. In addition, ethanol reduces paired-pulse facilitation of evoked EPSCs and increased the frequency but not the amplitude of spontaneous EPSCs. Furthermore, ethanol increases extracellular glutamate levels in the ventral tegmental area (VTA) in midbrain slices and in vivo in rats; and b) the effects of ethanol are mimicked by a D1R agonist or a dopamine reuptake inhibitor, and they are blocked by a D1R antagonist or by depleting dopamine stores with reserpine. This hypothesis will be tested in the VTA of rats or mice by a combination of electrophysiological and pharmacological techniques. This includes measurements of extracellular glutamate and dopamine levels in both the VTA and the nucleus accumbens, in brain slices and/or in vivo.
The Specific Aims are to determine: 1) The effects of ethanol on glutamatergic transmission to VTA dopamine neurons. We will compare AMPA receptor-mediated EPSCs and extracellular glutamate and dopamine levels in the absence and presence of ethanol. 2) The role of presynaptic D1Rs in the ethanol-induced increase in glutamatergic transmission to VTA dopamine neurons. We will compare the effects of ethanol on EPSCs and on extracellular glutamate levels in the absence and presence of a D1R agonist or antagonist. 3) The functional consequences of ethanol-mediated facilitation of glutamate release on the output of VTA dopamine neurons. We will determine whether glutamate antagonists: 1) attenuate the effects of ethanol on the excitability of VTA DA neurons, and 2) block the ability of systemic ethanol to increase dopamine release in the nucleus accumbens when the antagonists are infused into the VTA.
The results of these studies will clarify a novel mechanism that is a significant component of the action of ethanol on the brain`s reward pathways. A better understanding of cellular mechanisms of alcohol addiction will improve the treatment and prevention of alcoholism. __SpecificAimsTextDelimiter__ Principal Investigator/Program Director (Last; First; Middle):A. SPECIFIC AIMSThe mechanisms underlying alcohol addiction are not well understood. Despite much evidence linking thedopaminergic (DA) neurons of the ventral tegmental area (VTA) to ethanol (EtOH); the specific mechanismsthrough which EtOH enhances VTA DA cell excitability are not totally clear. One of our long-term goals is toelucidate the molecular; cellular and synaptic mechanisms underlying alcohol addiction. Glutamatergic(GLUergic) inputs to VTA DA neurons provide the major excitatory control of VTA activity and ultimately ofdopamine release in the reward pathway. Dopamine receptors; in particular the D1 receptors (D1Rs) situated onGLUergic axons afferent to DA neurons in the VTA; could be a locus for facilitating GLUergic synaptictransmission. Since glutamate is important in the mechanism of addiction; this facilitation may further thedevelopment of alcohol addiction. The objective of this proposal is to study the acute effects of EtOH onGLUergic transmission and their impact on the excitability of VTA DA neurons; a necessary first step towardsour long-term goal. In several areas of the brain; acute EtOH depresses glutamate-mediated excitatorytransmission; but the effect of EtOH on excitatory synapses on DA neurons of the VTA remains unknown. Our preliminary data show a) that clinically relevant concentrations of EtOH (10-80 mM) increase theamplitude of evoked excitatory postsynaptic currents (EPSCs) mediated by AMPA receptors. In addition; EtOHreduces paired-pulse facilitation of evoked EPSCs and increases the frequency but not the amplitude ofspontaneous EPSCs. Furthermore; EtOH increases extracellular glutamate levels in the VTA in midbrain slicesand in vivo in rats; and b) that the effects of EtOH are mimicked by a dopamine D1R agonist or a dopaminereuptake inhibitor; and they are blocked by a D1R antagonist or by depleting dopamine stores with reserpine(see preliminary data section for details). Based on these unexpected findings and the literature; we hypothesizethat acute EtOH increases glutamate release via activation of D1Rs. The increased GLUergictransmission in turn modulates DA cell activity and output; and this could play a significant role in theprocesses involved in alcohol addiction. This hypothesis will be tested in the VTA of rats or mice by acombination of electrophysiological and pharmacological techniques. These include the measurements ofextracellular glutamate and dopamine levels in both the VTA and the nucleus accumbens (NAcc); in brain slicesand/or in vivo (in collaboration with Dr. Shao at UCLA and Dr. Olive at MUSC).We therefore propose studies with the following Specific Aims:1) To determine EtOH's effects on GLUergic transmission to VTA DA neurons. We will compare AMPAreceptor-mediated EPSCs and extracellular glutamate and dopamine levels in the absence and presence ofEtOH. We expect that EtOH will enhance EPSCs and extracellular levels of glutamate and dopamine.2) To determine the role of presynaptic D1Rs in EtOH-induced increase in GLUergic transmission toVTA DA neurons. We will compare the effects of EtOH on EPSCs and on extracellular glutamate levels in theabsence and presence of a D1R agonist or antagonist. We expect that the D1R agonist and EtOH will enhanceGLUergic transmission and extracellular glutamate levels. Furthermore; the EtOH-induced enhancement willbe prevented by a D1R antagonist.3) To assess the functional consequences of EtOH-mediated facilitation of glutamate release on the outputof VTA DA neurons. We will determine whether glutamate antagonists: 1) attenuate the effects of EtOH on theexcitability of VTA DA neurons; and 2) block the ability of systemic EtOH to increase dopamine release inNAcc when the antagonists are infused into VTA.The proposed work is innovative as a multidisciplinary approach that includes electrophysiology;pharmacology; neurochemistry; immunocytochemistry and molecular genetics on three different preparationsto investigate a previously unrecognized effect of EtOH on GLUergic transmission in the VTA. The results ofthese studies will clarify a novel mechanism that is a significant component of the action of EtOH on the brain`sreward pathways. A better understanding of cellular mechanisms of alcohol addiction will improve thetreatment and prevention of alcoholism.To help the reviewers put this hypothesis into perspective; the following scheme illustrates how presynapticD1Rs and EtOH are proposed to influence GLUergic transmission; extracellular glutamate levels; and cellexcitability.PHS 398/2590 (Rev. 09/04) Page Continuation Format PagePrincipal Investigator/Program Director (Last; First; Middle):Fig. 1. Simplified diagram shows the major targets in this project. Aim 1will study EtOH effects on GLUergic transmission to VTA DA neurons.Aim 2 will study the role of presynaptic D1Rs on EtOH-inducedenhancement of GLUergic transmission. Aim 3 will study the functionalconsequences of EtOH-induced increase of glutamate release in VTA.This project focuses on the somatodendritic release of dopamine andthe D1Rs on GLUergic axons forming synapses on VTA DA cells. Notethat the extrinsic DA projection to VTA is negligible [1]. Thus; the onlyimportant source of dopamine in VTA is somatodendritic release. D1Rsare located on the glutamate-releasing afferent to the VTA DA neuron;but not on the DA neurons.CONCEPTUAL FRAMEWORKEtOH could act by increasing a dopamine-mediated facilitation of GLUergic inputs onto DA cells by one (or some combination) of thefollowing four mechanisms: 1) directly potentiates somatodendritic dopamine release in the VTA if EtOH directly excites DA neurons [19]; 2)raises extracellular dopamine levels by blocking dopamine reuptake (but see [168]; 3) sensitizes D1Rs on GLUergic axons to dopamine; and 4)directly modulates D1R signaling [122;123]. In addition; EtOH could disinhibit the VTA DA neurons by reducing glutamate release onto GABAergic interneurons [142]. The EtOH-induced facilitation of DA cell firing may be partly counteracted by EtOH-induced inhibition of postsynaptic NMDA receptors (situated on DAcells) and by the activation of D2Rs on the GLUergic terminals and on the DA cells. Note that ambient dopamine levels are much lower inVTA than in the NAcc; and that in the prefrontal cortex; D2Rs are relatively insensitive to dopamine; compared to D1Rs [3;149]. Furthermore;D2Rs in the VTA are less sensitive to dopamine; as implied by a study showing that somatodendritic release in VTA is not under significantD2R control [34]. Thus; presynaptic D1Rs are likely to be the predominant target for dopamine released by EtOH in the VTA.PHS 398/2590 (Rev. 09/04) Page Continuation Format Page
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