ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH
Vol. 30, No. 2 February 2006
Structural and Functional Modifications in Glutamateric Synapses Following Prolonged Ethanol Exposure L. Judson Chandler, Ezekiel Carpenter-Hyland, Adam W. Hendricson, Regina E. Maldve, Richard A. Morrisett, Feng C. Zhou, Youssef Sari, Richard Bell, and Karen K. Szumlinski
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. Key Words: Ethanol, Nucleus Accumbens, NMDA Receptors, Dendritic Spines, Postsynaptic Density, PSD-95, Homer2, Seizure, Explants, Plasticity.
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HILE IT HAS been clearly established that dopaminergic (DA) neurotransmission plays an important role in alcohol reward and addiction, increasing evidence suggests that many aspects of the plasticity of drug addiction involves changes in glutamatergic (GLU) neurotransmission. Many studies now indicate that GLU-based plasticity, which is critically involved in processes such as learning and memory, is inappropriately coopted by drugs of abuse. In particular, the persistence of drug-induced changes in brain function appears to relate to alterations in GLU synapses and associated signaling events. A unique feature of GLU synapses is that they are located almost entirely on the head of dendritic spines. As the name implies, dendritic spines are tiny thorn-like
From the Alcohol Research Center and Departments of Neurosciences and Psychiatry, Medical University of South Carolina, Charleston, South Carolina (LJC, ECH); The College of Pharmacy, University of Texas at Austin, Austin, Texas (AH, RM, RAM); the Department of Psychology, University of Santa Barbara, Santa Barbara, California (KKS); and the Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana (FCZ, YS, RB). Received for publication October 9, 2005; accepted October 20, 2005. This symposium and the work presented were sponsored by AA10983, RR15776, AA13355, AA010761 and an Alcoholic Beverage Medical Research Foundation Grant (LJC and ECH presentation); AA09230 and AA14068 (AH et al presentation); AA014829 and AA07611 (FCZ et al presentation); and AA1076, and AA013517 (KKS presentation). Reprint request: L. Judson Chandler, Department of Neurosciences, Medical University of South Carolina, 67 President Street, Charleston, SC 29425; E-mail:
[email protected] Copyright r 2006 by the Research Society on Alcoholism. DOI: 10.1111/j.1530-0277.2006.00041.x 368
protuberances that represent the postsynaptic contact site for glutamate synapses. In addition to increasing the surface area for synapse formation (individual neurons typically have thousands to tens of thousand of spines), spines also function to compartmentalize the postsynaptic signal and thus play an important role in dendritic integration of synaptic input within various regions and subregions of the dendritic tree. It has also become clear that spines are not rigid structures that only change during protracted pathology, but can form, retract, and undergo shape changes in response to changes in synaptic activity. Together, these features make spines ideal mediators of the moment to moment functional and structural changes that undoubtedly is critical to the plasticity of the addiction neurocircuitry. Another unique feature of GLU synapses on dendritic spines is the presence of a distinctive postsynaptic density (PSD). While virtually all synapses in the brain possess some form of a postsynaptic region containing neurotransmitter receptors, the PSD of dendritic spines appears as a prominent electron-dense region when viewed by electron microscopy. The apparent reason for this is that the PSD on spines contains a highly compact and complex association of receptors, scaffolding proteins, and a surprisingly large number of signal transduction molecules. Importantly, this organization, compartmentalization, and immobilization of glutamate receptors and related signaling pathways appear to play an important role in plasticity of drug abuse. This symposium presented further evidence that ethanol exposure produces neurochemical and structural changes at dendritic spines and their associated scaffolding proteins that likely mediate protracted changes in brain function associated with alcohol addiction and abuse. Alcohol Clin Exp Res, Vol 30, No 2, 2006: pp 368–376.
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CHRONIC ETHANOL EXPOSURE, N-METHYL-DASPARTATE RECEPTOR DYNAMICS, AND WITHDRAWAL HYPEREXCITABILITY
Adam Hendricson, Regina Maldve, and Richard Morrisett Cessation of compulsive intake of central nervous system (CNS) depressants such as ethanol or g-aminobutyric acid (GABA)ergic drugs is strongly associated with aversive effects ranging from simple irritability to outright electrographic seizure activity. The neurological basis of the ethanol withdrawal (WD) syndrome involves concurrent pathological mechanisms in multiple brain regions. In this complex system, the N-methyl-D-aspartate (NMDA) receptor (NMDAR) has emerged as a prime molecular target owing to the marked ethanol sensitivity of NMDAR currents (Hoffman et al., 1989; Lovinger et al., 1989) and NMDAR-mediated processes of synaptic plasticity and neuroadaptation (Hendricson et al., 2002; Maldve et al., 2002; Morrisett and Swartzwelder, 1993). Chronic ethanol (CET) in vitro induces brain region– specific increases in NR1/NR2A expression (Kalluri et al., 1998; Snell et al., 1996; Trevisan et al., 1994) and has been shown to increase NR2B expression in dissociated neuronal culture (Follesa and Ticku, 1996) and in hippocampal tissue culture (Thomas et al., 1998). Numerous in vivo studies report CET-induced increases in the number of binding sites for [3H]MK-801 (Grant et al., 1990; Gulya et al., 1991; Snell et al., 1996). Interestingly, these findings are contradicted by several reports (Rudolph et al., 1997; Tremwel et al., 1994; Ulrichsen et al., 1996), indicating that the precise nature of CET effects on NMDAR expression remain somewhat controversial. Other lines of evidence suggest that in some systems CET-induced increases in NMDAR function may occur in the absence of increased NMDAR subunit expression. Notably, Chandler et al. (1997) reported increased NMDAR-mediated nitric oxide synthesis in the absence of increases in [3H]MK-801 binding following CET in cortical cultures, a finding suggestive of neuroadaptive pathways by which existing receptors are recruited to synaptic zones during CET. In an elegant study, Carpenter-Hyland et al. (2004) recently reported movement of NMDARs from extrasynaptic to synaptic sites post-CET, functionally correlated with changes in the ratio of NMDAR currents arising from synaptic/extrasynaptic loci. Here we extend previous in vitro work by examining the consequences of CET and subsequent WD on NMDAR dynamics in the intact neural network of hippocampal explant cultures. Our lab has previously developed an explant-based platform for monitoring the electrophysiological and biochemical sequelae of ethanol WD (Thomas and Morrisett, 2000; Thomas et al., 1998). At 10 days in vitro, explants cultured under serum-free conditions were placed in sealed plastic containers humidified with ddH2O supplemented with 95% EtOH so that the medium (EtOH)
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equilibrated at 75 mM. Medium was changed every other day with (EtOH) monitored by enzymatic assay. After 5 days in EtOH, explants were harvested for single-cell patchclamp electrophysiology or biochemistry/ICC. Following a 7-day CET regimen, pre-WD NR1 expression levels measured by Western blot were 59.3 33.6% greater than sham-CET controls and remained significantly elevated through the 20-hour post-WD time course [ po0.035 by analysis of variance (ANOVA), F1,16 5 5.35, n 5 12]. NR2B levels measured prior to WD were 71.8 26.9% greater than control and also remained elevated through the post-WD timecourse (po0.0025 by ANOVA, F1,16 5 13.7, n 5 12). To examine the trafficking of newly synthesized NMDAR subunits following CET, we expressed eGPF in explant neurons via sindbis viral vector, treated slices chronically with ethanol, and then fixed and immunola beled the GFP-infected tissue for NR1. Patterns of NR1 localization to GFP-illuminated dendritic spines were compared in CET versus sham explants. The percentage of spines that were labeled with 1 NR1 puncta did not change following CET (43.1 8.6% in sham slices vs 47.2 11.9% in CET slices, p 5 0.8). However, the area of spine-associated NR1 puncta was significantly greater in CET slices relative to sham controls (53.0 6.8 in sham slices vs 109.3 23.8 in CET slices, po0.04) while the mean area of non–spine-associated puncta did not increase significantly (p 5 0.1). These data indicate that in the present neural network, CET induces increased subunit expression and preferential distribution of NMDARs to likely synaptic sites. Single-cell current-clamp recording was employed to examine functional changes in neurotransmission following WD. Wash to ethanol-free ACSF immediately (EGS latency o5 minutes) elicited WD seizures exhibiting classic tonic-phasic discharge characteristics. Mean seizure duration was 15.2 2.6 minutes. Upon seizure initiation, the mean spike frequency (phasic and tonic spikes combined) increased 295-fold, from 0.02 0.02 to 5.9 0.8 Hz (po0.001, n 5 10); mean intraburst frequency was considerably higher (30.6 2.3 Hz). By contrast, in 5 sham CET slices, no hyperexcitability was observed even with prolonged (430 minutes) recording. EGS initiation was NMDAR dependent: When ethanol was withdrawn in the presence of the NMDAR antagonist (DL)-APV (100 mM), WD seizures were totally absent in all cells (mean pre-WD spike frequency 0.001 0.001 Hz, mean post-WD frequency 0.003 0.001 Hz, n 5 13, p 5 0.2). Chronic cotreatment of explants with ethanol in combination with the protein kinase A antagonist H-89 had no effect on the initiation of WD seizures (n 5 3). To monitor WD-associated changes in the strength of synaptic NMDAR signaling in the absence of network hyperexcitability, we recorded Sr21-supported NMDAR miniature excitatory postsynaptic currents (mEPSCs) in the presence of the AMPA/KA antagonist and potent
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antiepileptic agent 6,7-dinitroquinoxaline-2,3-dione (DNQX). We have previously adapted this technique for use in hippocampus (Hendricson et al., 2004) and nucleus accumbens (Zhang et al., 2005). The mean amplitudes of NMDAR mEPSCs synaptically evoked prior to WD in CET slices (6.0 0.7 pA, n 5 9) and in CET-naı¨ ve slices acutely exposed to ethanol for 25 to 30 minutes prior to recording (7.2 1.0 pA, n 5 9) did not differ significantly (p 5 0.3). Following ethanol washout, the mean mEPSC amplitude in CET slices increased by 163.3 36.1% to 15.8 1.6 pA. By comparison, an identical maneuver in sham CET slices elicited a significantly smaller 48.6 25.1% increase to 10.7 2.1 pA (po0.005) corresponding to the removal of the acute inhibitory effect of ethanol on NMDAR currents. APV reduced the mean amplitude of post-WD mEPSCs to less than pre-WD levels (po0.04, n 5 3). The amplitude of AMPAR mEPSCs was unchanged after WD, indicating that WD effects are most directly attributable to CET-induced changes in NMDAR neurotransmission. In the intact neural network of the hippocampal explant, CET increases the expression of NMDAR subunits and induces a selective partitioning of receptors to synaptic sites. Importantly, NMDAR activation is required for WD seizure expression, and NMDAR mEPSC analysis directly demonstrates enhanced receptor function induced by CET. This enhancement is distinct from WD seizure activity as it was observed in the absence of network hyperexcitability because of the presence of the antiepileptic agent DNQX. These findings centrally implicate CET effects on NMDAR signaling in the pathogenesis of WD hyperexcitability and underscore the utility of networkbased model systems in providing an integrative view of CNS pathologies. ETHANOL-INDUCED SYNAPTIC TARGETING OF NMDA RECEPTORS IS ASSOCIATED WITH ENHANCED PSD-95 CLUSTERING AND SPINE SIZE
Judson Chandler and Ezekiel Carpenter-Hyland A homeostatic adaptation to CET identified in our previous work is the selective targeting of NMDA receptors to synapses (Carpenter-Hyland et al., 2004). This modification to the molecular composition of synapses presumably occurs to stabilize neural networks by counteracting the destabilizing effects of prolonged inhibition of NMDA receptors by ethanol. Cellular models of experience-dependent synaptic plasticity have observed that changes in the subcellular localization of glutamate receptors can be accompanied by molecular reorganization of the PSD and alterations in spine morphology or density (Segal, 2005). Drugs of abuse have also been observed to produce changes in dendritic spines that correlate with various behavioral aspects of addiction (Robinson and Kolb, 2004). Ethanol-induced modifications in synaptic efficacy may therefore involve structural
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changes in synapses associated with the development of chronic tolerance. The postsynaptic scaffolding protein PSD-95 is a core component of the PSD that structurally organizes numerous proteins in GLU synapses and physically links NMDA receptors with downstream signaling pathways. This organizational role is at the center of PSD-95’s involvement in the maturation and plasticity of excitatory synapses. Postsynaptic density-95 can also regulate NMDA receptor trafficking as NMDA receptor binding to PSD-95 has been shown to promote NMDA receptor surface expression while simultaneously reducing NMDA receptor internalization (Lin et al., 2004). N-Methyl-D-aspartate receptors and PSD-95 may also play a role in the regulation of dendritic spines. Pyramidal neurons in the hippocampus exhibit a remarkable diversity in spine shape, and these structures undergo a bidirectional activity-dependent remodeling (Harris, 1999). Glutamate receptor activity has been strongly implicated in mediating structural modifications to spines (Fischer et al., 1998), and chronic inhibition of activity results in a homeostatic increase in spine number (Kirov and Harris, 1999). As the number of NMDA receptors in a spine is proportional to its size (Noguchi et al., 2005), enhanced synaptic clustering of NMDA receptors following CET exposure could have implications for spine size. The capacity for spines to undergo morphological change appears to be largely a function of modifying the actin cytoskeleton (Fischer, 1998), and consistent with this NMDA receptor activity has also been shown to regulate equilibrium between F-actin and G-actin (Okamoto et al., 2004). To further explore the potential relationship between synaptic NMDA receptor targeting and structural adaptations in the development of CET tolerance, we examined the effects of prolonged ethanol exposure on subcellular organization of the synaptic scaffolding protein PSD-95 and dendritic spine morphology. Similar to its effects on NMDA receptors, chronic exposure of hippocampal cultures to either the NMDA antagonist APV (100 mM) or ethanol (50 mM) lead to a significant increase in the clustering of PSD-95. While the increase with APV occurred in both the synaptic and the extrasynaptic compartments, ethanol-induced increase in PSD-95 occurred primarily in the synaptic compartment without altering cluster size in the extrasynaptic compartment. This effect of ethanol is similar to our previous observation of a selective increase in synaptic NR1 receptors. The increase in PSD-95 clustering by CET was completely prevented by coincubation with a low concentration of NMDA (2.5 mM). This suggests that, similar to CET-induced increase in synaptic NMDA receptors, the increase in synaptic PSD-95 clustering is an activity-dependent process. The increase in PSD-95 that accompanied the increase in NMDA receptors at the synapse following CET exposure is consistent with a supposed role for PSD-95 in the
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localization and organization of NMDA receptors and associated signaling molecules at the synapse. Furthermore, increases in the number of receptors and scaffolding proteins at the PSD are likely associated with an expansion of the size of the PSD. We therefore reasoned that this expansion might also be associated with an increase in the size of the spine. Dendritic spines contain extremely high levels of F-actin, and imaging of F-actin labeled with fluorescent-conjugated phalloidin is a convenient method to indirectly estimate the size of the spine. Chronic exposure of neuronal cultures to either APV (100 mM) or ethanol (50 mM) resulted in a large increase in the size of F-actin clusters (interactively thresheld to eliminate background dendritic staining). While APV greatly increased spine density, only minor increases in density were observed with ethanol. As with PSD-95, ethanol-induced increase in F-actin cluster size and density was blocked by coincubation with a low concentration of NMDA. In addition to dendritic spines, F-actin clusters could also represent nonspine structures such as regions of dense cortical actin and filopodia, we examined the colocalization of PSD-95 clusters with actin clusters. In these studies, we operationally define a mature spine as an actin cluster that colocalizes with a PSD-95 clusters. In control cultures, 50% of the actin clusters were found to colocalize with PSD-95. Importantly, it was observed the ethanol-induced increases in actin clusters occurred only in those that contained PSD-95, strongly suggesting that the ethanol-induced increases in actin clustering represented enlargement of mature dendritic spines. Furthermore, the ethanol-induced increases in synaptic clustering of NMDA receptors and PSD-95 and the increase in spine size completely returned to control levels 1 week after ethanol was allowed to evaporate from the culture dish. Previous studies have also suggested that the synaptic localization of PSD-95 is dependent lipid modification via palmitoylation (El-Husseini et al., 2002). We thus examine the effect of blocking palmitoylation upon ethanol-induced increase in PSD-95, NMDA receptors, and spine size. These studies revealed that declustering PSD-95 at the PSD by exposure to the palmitoylation inhibitor 2-Br-palmitate blocked ethanolinduced increases in spine size. Furthermore, this was associated with a similar block of NMDA receptors that was specific for synaptically localized receptors. Taken together, these observations suggest that ethanolinduced homeostatic increase in synaptic NMDA receptors previously reported are associated with an expansion of the PSD and an increase in spine size. As large spines are thought to be more stable and less able to undergo activity-dependent changes compared with smaller spines, it is intriguing to speculate that drug-induced spine enlargement and enhanced clustering of PSD-95 and NMDA receptors may functional and structurally ‘‘lock’’ the addiction neurocircuitry in a maladaptive state.
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PRESYNAPTIC AND POSTSYNAPTIC ALTERATIONS IN THE NUCLEUS ACCUMBENS FOLLOWING CHRONIC ALCOHOL EXPOSURE
Feng Zhou, Youssef Sari, and Richard Bell It is known that chronic alcohol alters brain neurochemistry activities in key brain regions that mediate reward. The changes in GLU and DA transmissions in the extended amygdala may in turn mediate alcohol dependence. Chronic alcohol in rats increases the number of GLU and (its antagonist) MK801 binding sites (Chen et al., 1997; Grant et al., 1990; Sanna et al., 1993; Snell et al., 1996). Nucleus accumbens neurons from ethanol-dependent rats also showed enhanced NMDA sensitivity (Siggins et al., 2003). In chronic alcohol studies of brains of ethanoldependent rats, the extracellular concentrations of GLU are transiently increased by about 3-fold hours after WD (Rossetti and Carboni, 1995). As transient neurochemical changes alone cannot interpret the long-lasting alcohol-seeking behaviors, it is hypothesized that persistent changes in synaptic conformation along with structural protein at synaptic level account for persistent drinking behaviors. To date, it is not known whether there is a structural GLU and DA alteration at the presynaptic and postsynaptic level in chronic alcohol-treated animals, and it is unclear whether this up-regulation of GLU release after chronic alcohol exposure is a result of positive feedback through the NMDA or an alteration of GLU/DA synaptic conformation. To address the microcircuit that is related to GLU and DA in the extended amygdala, we examined changes that occur at the presynaptic and postsynaptic level in brain regions that mediate reward. Alcohol-preferring (P) rats, which are genetically prone to alcohol dependency without additional force, were used for our initial study to avoid stress-related complication. The P rats were given concurrent access to multiple concentrations of ethanol (15 and 30%, v/v) and water; the use of 2 concentrations allows for a greater intake of alcohol than a single concentration (Rodd-Henricks et al., 2001). The continuous alcohol (CAlc) group had free-choice access to ethanol for 14 weeks. The Water group was run in parallel without alcohol. The repeated deprivation (RD-Alc) group had an initial 6 weeks of 24-hour free-choice access to ethanol, followed by 2 cycles of 2 weeks of deprivation from and 2 weeks of reexposure to ethanol access. Our experimental end-points include (a) a presynaptic analysis of GLU and DA terminals with immunocytochemical examination of vesicular glutamate transporter 1 and 2 (vGLUT1 and vGLUT2) and the DA synthetic enzyme tyrosine hydroxylase (TH), (b) postsynaptic analysis with 2-photon microscopic analysis of postsynaptic dendritic spines in median spiny neurons (MSN), and (c) a receptor analysis with Western blot and immunocytochemical staining of major NMDA receptor subunit, NR1, in the extended amygdala.
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The vGLUT, depending on its subtype, is clustered in the synaptic terminals of a subset of GLU innervation in specific regions of the brain. The vGLUT1-bearing GLU fibers innervate the shell of nucleus accumbens in the island-like subregions (patches) that predominately originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus (Fremeau et al., 2001; Hartig et al., 2003). The VGLUT2 has been shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus and is represented as a matrix around the vGLUT1 patches in shell of nucleus accumbens. The complementary expression of VGLUT1 and VGLUT2 defines 2 distinct classes of excitatory synapses. Greater vGLUT2-im staining was found in the shell of the nucleus accumbens in the RD-Alc groups as compared with that of Water control group. The vGLUT1-im is under investigation in shell of the nucleus accumbens and amgdala upon chronic alcohol treatment. The DA in the extended amygdala is an important regulator that fine tunes GLU output in the synaptic GLU-GABA microcircuit. Dopaminergic innervations as indicated by TH immunostaining (TH-im) in the shell of the nucleus accumbens of the central amygdala show that the extended amygdala, with a large population of GABA neurons, received a high density of TH-im fibers. The TH-im fibers in the nucleus accumbens and amygdala are largely DA in nature with very sparse norepinephrine fibers. We demonstrated that the number of TH-im representing primarily terminals of DA innervation in either the shell of nucleus accumbens or the central amygdala is not affected by the C-Alc or RD-Alc treatments. As there is an increase in vGLUT2-im but no change of TH-im terminals in the shell of nucleus accumbens, the synapse ratio of vGLUT2-im GLU:TH, therefore, is increased after repeated deprivation of chronic alcohol drinking. The density of vGLUT1 GLU innervation is still under investigation. The GLU innervation in the shell of nucleus accumbens is primarily on the head of the dendritic spines of the MSN, while DA innervation is on the shaft of the dendritic spines (Freund et al., 1984; Smith and Bolam, 1990). This rearrangement of inputs may have consequences to the response of postsynaptic neurons. It would be very interesting to know how this translates into the electrical activities of the GABA MSNs in the shell of nucleus accumbens. Furthermore, this invites strong speculation regarding the less-regulated excitatory input in the shell of nucleus accumbens. To evaluate the postsynaptic element of the dendritic spines, the MSNs were filled with Alexa488 fluorescent dye and examined using 2-photon laser scanning microscopy in the chronic-alcohol-treated P rats of the above paradigm. It revealed that the density of spines in the nucleus accumbens was lower in the C-Alc and RD-ALC groups compared with that of Water group. However, although reduced in number, there was an increase in their spine head size. There were also more large-size spine heads in
RD-Alc compared with those of Water controls. In addition to larger-sized spines the 2-headed spines were increasingly seen in the RD group, and the dendritic swellings were seen in subpopulation of both the C-Alc and the RD-Alc groups. Thus, prolonged ethanol exposure increased presynaptic vGlut2-im GLU terminals may primarily cluster on fewer but larger-sized dendritic spines. As dendritic spines provide the postsynaptic sites for the vast majority of GLU synapses in the brain, chronic changes in the architecture of the presynaptic and postsynaptic components likely reflect persistent plastic changes and rewiring of MSN following chronic alcohol treatments. In light of changes in the synaptic density and size of the dendritic spine, it is speculated that the major NMDA receptors that are closely associated with the spine function are also involved. Western blot analysis for NMDA subunit NR1 shows that protein expression increased in the C-Alc group in the forebrain at the level of nucleus accumbens compared with that of controls. Immunocytochemical staining of NR1 indicated an upregulation in the nucleus accumbens in the C-Alc group compared with Water group. In summary, current study indicates an alteration of innervation ratio of vGLUT21-GLU and DA in the nucleus accumbens upon chronic alcohol treatments. This information is of significant value in understanding whether environmental factors alter the excitatory or regulatory input into nucleus accumbens. Second, chronic alcohol drinking altered dendrites and their spines size in the nucleus accumbens. Furthermore, the major GLU receptors NMDA subunit NR1 is increased in the nucleus accumbens. As the spine density is reduced, the increase of NMDA receptors is likely clustered in larger spine. The dendritic structures and NMDA receptors of the medium spiny neurons in the nucleus accumbens are differentially altered in the C-Alc and RD-Alc treatments, which indicates a heterogenic mechanism of plasticity between the 2 paradigms. These adaptive changes of neuronal reception, particularly to GLU transmission, imply a persistent change or rewiring of microcircuitry in the key reward processing regions of the brain in animals with genetic propensity for high alcohol drinking. AN ACTIVE ROLE FOR ACCUMBENS HOMER2 EXPRESSION IN ALCOHOL-INDUCED NEURAL PLASTICITY
Karen K. Szumlinski Homer proteins are encoded by 3 genes (Homer1–3), providing both constitutive forms of Homer (Homer1b/c, Homer2, and Homer3) and an immediate early gene product, Homer1a (Xiao et al., 2000). Constitutively expressed Homer proteins interact, via an EVH1 binding domain, with the C-terminus of Group 1 metabotropic receptors (mGluRs), the inositol triphosphate and ryanodine
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receptors, as well as the NMDA receptor scaffolding protein Shank (for reviews, see de Bartolomeis and Iasevoli, 2003; Xiao et al., 2000). In vivo microdialysis studies revealed that deletions of the either the Homer1 or the Homer2 genes in mice produces a ‘‘stimulant presensitized’’ behavioral phenotype that is associated with alterations in basal and drug-stimulated changes in extracellular levels of glutamate in the nucleus accumbens that resemble those produced by repeated cocaine administration (Szumlinski et al., 2004, 2005b). More recent studies conducted in rats revealed an important role for constitutively expressed Homer isoforms in the the neural plasticity induced by repeated cocaine administration as Homer1c, Homer2a, and Homer2b overexpression in the accumbens, following repeated cocaine administration, completely prevented the expression of both behavioral and neurochemical sensitization (Szumlinski et al., 2005a). This report summarizes a series of behavioral and in vivo microdialysis studies employing Homer2 knockout (KO) mice and adenoassociated viral vectors carrying Homer2b (AAV-Homer2b) that were designed to investigate the role for constitutive Homer expression in the accumbens in mediating alcohol-induced behavioral and neurochemical plasticity. Homer2 KO mice exhibit an alcohol-avoiding phenotype when assessed in a 2-bottle-choice procedure (0 vs 3, 6, or 12% alcohol, v/v) and an alcohol-induced place-conditioning paradigm (80–3 g/kg). As the magnitude of the alcohol avoidance exhibited by Homer2 KO mice was largest at higher alcohol concentrations, the effects of gene deletion were assessed on the motor responses to higher alcohol doses. KO mice failed to exhibit tolerance to the locomotor inhibitory effects of 3 g/kg alcohol when administered repeatedly (8 injections) and exhibited a longer latency to regain their righting reflex after the acute administration of an anesthetic dose of alcohol (5 g/kg). Thus, the alcohol-avoiding phenotype of Homer2 KO mice appears to be related to both an increased sensitivity to the motor-impairing effect of alcohol and an inability to develop tolerance to this effect with repeated alcohol administration. Consistent with their lack of alcoholinduced behavioral plasticity, repeated alcohol administration (83 g/kg) did not produce dopamine or glutamate sensitization in the accumbens of Homer2 KO mice, indicating that Homer2 deletion also prevents alcoholinduced neurochemical plasticity in a brain region implicated in the rewarding/reinforcing effects of alcohol (Szumlinski et al., 2005c). To confirm that the alcohol phenotype of Homer2 KO mice resulted from a lack of protein expression, rather than developmental compensations because of gene deletion, a second series of experiments assessed the alcohol phenotype of wild-type (WT) and KO mice following an intraaccumbens infusion of AAV-Homer2b. Importantly, identical genotypic differences were observed between WT and KO mice infused with an AAV-GFP control vector, as observed in our original phenotypic characterization of
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the mice. In support of an active role for Homer2 in mediating the alcohol phenotype of Homer2 KO mice, an intraaccumbens infusion of AAV-Homer2b abolished the genotypic differences in: alcohol preference in a 4-bottlechoice procedure (0, 3, 6, and 12% alcohol), alcoholinduced place conditioning (83 g/kg), the development of locomotor tolerance (83 g/kg), and in alcohol-induced dopamine and glutamate sensitization in the accumbens (83 g/kg). Moreover, intraaccumbens AAV-Homer2b infusion also produced a moderate shift to the left in the alcohol dose-preference function in WT mice, suggesting that the overexpression of Homer2 in the accumbens might actually facilitate certain aspects of alcohol reward (Szumlinski et al., 2005c). To address this possibility, the effects of an intraaccumbens infusion of AAV-Homer2b were assessed in the alcohol-preferring B6 mouse. Consistent with the shift in preference observed in WT Homer2 mice, an intraaccumbens infusion of AAV-Homer2b produced a large shift to the left and upward in the alcohol preference function in B6 mice (Szumlinski et al., 2005c). Although in the 4-bottlechoice assay, no effects of Homer2 overexpression were observed on alcohol consumption, when the behavior of AAV-infused mice were assessed in an operant paradigm it was found that: (1) AAV-Homer2b infusion increased the number of reinforced lever presses for delivery of 6 and 12% alcohol under both preprandial and postprandial conditions; (2) increased the intake of 6 and 12% alcohol under both preprandial and postprandial conditions; and (3) increased the number of contacts with the alcohol delivery fountain when under postprandial conditions. Moreover, intraaccumbens AAV-Homer2b infusion facilitated the development of tolerance to the locomotorinhibitory effects produced by repeated alcohol administration (82 g/kg). Neurochemically, intraaccumbens AAV-Homer2b infusion: (1) increased basal extracellular levels of glutamate in alcohol-naı¨ ve mice; (2) enhanced the elevation in basal glutamate levels observed at 24 hours WD in mice with repeated alcohol experience; (3) enhanced the rise in extracellular glutamate produced by an acute injection of 2 g/kg and facilitated the expression of glutamate sensitization in repeated alcohol-treated mice. Collectively, these data demonstrate that Homer2 overexpression in the accumbens enhances alcohol reward and the development of alcohol-induced behavioral and neurochemical plasticity in mice with moderate, as well as high alcohol-preferring phenotypes. Earlier immunoblotting studies conducted on accumbens tissue of Homer2 WT and KO mice revealed an approximately 25% reduction in the total protein content of the mGluR1a subtype of Group 1 metabotropic glutamate receptor, but no difference in the total protein content of the mGluR5 subtype or any of the NMDA receptor subunits examined (Szumlinski et al., 2004). As selective mGluR1a antagonists abolish the rise in extracellular dopamine and glutamate produced in the accumbens by
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an acute injection of alcohol, enhance alcohol-induced motor impairment, and reduce both appetitive and consummatory aspects of alcohol reward in B6 mice (Lominac et al., 2005), the possibility exists that the alcohol phenotype of Homer2 KO mice may be related to a reduction in the function of mGluR1a. In support of this, Homer2 KO mice exhibit a blunted capacity of the Group 1 mGluR agonist DHPG to elevate extracellular glutamate in the accumbens (Szumlinski et al., 2004), an effect mediated by the stimulation of mGluR1a receptors (Swanson et al., 2001). Alternatively, the lack of alcohol-induced neural plasticity produced by Homer2 deletion may be related to alterations in NMDA receptor function as Homers control the trafficking of NMDA receptors during development through interactions with Shank–GKAP–PSD-95 complexes (Shiraishi et al., 2004). Indeed, Homer KO mice exhibit increased behavioral sensitivity to other NMDA antagonists, injected either systemically or directly into the accumbens (Szumlinski et al., 2004, 2005b, 2005c) and electrophysiological studies conducted on embryonic hippocampal cultures (14 days in vitro) revealed a marked reduction in NMDA-mediated whole-cell current in KO mice that was not associated with altered EtOH sensitivity of the NMDA receptor (Smothers et al., 2003). While electrophysiological studies have not yet been performed in the accumbens, Homer2 KO mice exhibit a reduction in the amount of NR2a and NR2b localized to the cell membrane in accumbens tissue, suggesting that the reduction in NMDA receptor function and heightened sensitivity to the behavioral effects of NMDA receptor blockade observed in Homer2 KO mice may result from a reduction in the cell membrane trafficking of NMDA receptors. While a considerable amount of work remains to elucidate the cellular mechanisms underlying the effects of accumbens Homer2 manipulations upon alcohol-induced changes in brain and behavior, the data to date suggest an active and necessary role for accumbens Homer2 expression in the development of alcohol-induced neural plasticity and implicates Homer regulation of mGluR1a and NMDA receptor function as a cellular mediator of vulnerability to alcoholism. CONCLUSIONS
The presentation by Hendricson, Maldve, and Morrisett demonstrated that up-regulation of NMDA receptors at synapses underlie ethanol WD-induced hyperexcitability. These observations extend previous work carried out in reduced systems by showing similar changes using organotypic explant cultures that maintain intact neural networks. These studies future support a critical role for NMDA receptor signaling in the pathogenesis of ethanol WD. In particular, the enhanced synaptic targeting of NMDA receptors in response to prolonged ethanol exposure lead to their hyperfunctioning in the absence of ethanol. Importantly, while the role NMDA receptors in shaping network activity is often underappreciated, these
studies clearly demonstrate their importance in driving network activity in an intact system. These studies have implications for understanding the pathogenesis and treatment of ethanol-related WD symptomology. The presentation by Chandler and Carpenter-Hyland demonstrated using primarily hippocampal cultures that ethanol-induced increases in synaptic NMDA receptors are associated with an increase in the size of the dendritic spine. This presumably is accompanied by an expansion of the PSD and related scaffolding and signaling complexes as evidenced by an increase in the synaptic clustering of PSD-95. Previous studies of the effects of drug exposure on dendritic spines have focused primarily upon determining changes in spine number. However, recent studies have demonstrated that large spines are more stable and less able to undergo subsequent activity-dependent modifications that are critical for maintaining behavioral plasticity. The combination of a reduction in the number of nimble spines via their conversion to large spines may contribute to the persistence and intractable nature of addictionrelated behaviors. Furthermore, these changes may be especially important in light of recent observations that when lost spines reform, they often do so in their original spot on the dendrite and presumably reestablish the same presynaptic connection. Thus, it is intriguing to speculate that when large spines are lost during abstinence from ethanol, they may leave behind a ‘‘tag’’ for future spinesynapse development that could rapidly ‘‘reawaken’’ the aberrant microcircuitry of the addicted brain. The presentation by Zhou, Sari, and Bell demonstrated in the nucleus accumbens that CET exposure led to a decrease in spine number. However, though less in number, the remaining spines were larger in size. Immunoblot analysis also indicated there was an increase in the number of NMDA receptors. Furthermore, studies of vGlut2 expression suggested that CET exposure resulted in an increase in the number of presynaptic glutamate terminals that cluster on fewer but larger spines. As above, these observations are consistent with the suggestion that CET exposure leads to changes in dendritic spines that may underlie persistent change or rewiring on the brain. As the nucleus accumbens is a brain area that is considered to play a critical role in reward-related processes, aberrant plasticity of spines within this brain region may be especially important in addictive processes. The presentation by Szumlinski demonstrated that Homer2 expression in the nucleus accumbens mediates alcohol-induced behavioral and neurochemical plasticity. Homer2 KO mice fail to exhibit an alcohol avoiding phenotype when assessed using the 2-bottle-choice procedure and alcohol-induced place conditioning. These mice also failed to exhibit tolerance to the locomotor inhibitor effects of ethanol and also show a longer latency to regain righting reflex. Furthermore, repeated daily administration of ethanol failed to produce dopamine or glutamate sensitization in the nucleus accumben that is normally
STRUCTURAL AND FUNCTIONAL MODIFICATIONS IN GLUTAMATERIC SYNAPSES
observed in WT mice. Together, these results suggest that the Homer2 protein normally plays an important role in alcoholinduced behavioral plasticity. As further confirmation of this interpretation, it was observed that reintroduction of the Homer2 gene by an intraaccumbens infusion of AAVHomer-2b abolished the behavioral and neurochemical genotypic differences between the WT and Homer2 KO mice in their response to ethanol. Lastly, Western blot analysis revealed that Homer2 mice have reduced levels of total mGluR1a and a reduction in the cell membrane expression of NR2 subunits. As selective mGluR1a antagonist administration to B6 mice has been shown to abolish the rise in extracellular dopamine and glutamate produced in the accumbens by an acute ethanol injection and to alter other ethanol-related behaviors, it is suggested that the phenotype of Homer2 KO mouse may be related to the function of mGluR1a receptors. Alternatively, Homer regulation of NMDA receptor trafficking may also be a critical cellular mediator of alcohol-induced neural plasticity. In summary, the results of this symposium provide further evidence for a GLU basis of alcoholism and alcoholrelated behaviors. In particular, the dynamic localization of glutamate receptors and related signaling molecules at the PSD is critical in adaptive response to prolonged ethanol exposure. The enhanced targeting of NMDA receptors to the PSD is appears to underlie ethanol-WD hyperactivity and related seizure activity. Furthermore, the increases in the expression of NMDA receptor at the PSD is associated with an increase in spine size, which may be a common feature of drugs of abuse that contributes to persistent and aberrant rewiring of the addiction neurocircuitry. Finally, as Homer proteins are important in scaffolding and organizing NMDA receptors and signaling complexes at the PSD density, it will be of interest to determine in future studies whether a lack of Homer2 results in alterations in the organization of the PSD as a reduction in spine size. REFERENCES de Bartolomeis A, Iasevoli F (2003) The Homer family and the signal transduction system at glutamatergic postsynaptic density: potential role in behavior and pharmacotherapy. Psychopharmacol Bull 37: 51–83. Carpenter-Hyland EP, Woodward JJ, Chandler LJ (2004) Chronic ethanol induces synaptic but not extrasynaptic targeting of NMDA receptors. J Neurosci 24:7859. Chandler LJ, Sutton G, Norwood D, Sumners C, Crews FT (1997) Chronic ethanol increases N-methyl-D-aspartate-stimulated nitric oxide formation but not receptor density in cultured cortical neurons. Mol Pharmacol 51:733–740. Chen X, Michaelis ML, Michaelis EK (1997) Effects of chronic ethanol treatment on the expression of calcium transport carriers and NMDA/ glutamate receptor proteins in brain synaptic membranes. J Neurochem 69:1559–1569. El-Husseini AE, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS (2002) Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108:849–863.
375
Fischer M, Kaech S, Knutti D, Matus A (1998) Rapid actin-based plasticity in dendritic spines. Neuron 20:847–854. Follesa P, Ticku MK (1996) Chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptor polypeptide subunits in mouse cortical neurons in culture. J Biol Chem 271:13297–13299. Fremeau RT Jr, Troyer MD, Pahner I, Nygaard GO, Tran CH, Reimer RJ, Bellocchio EE, Fortin D, Storm-Mathisen J, Edwards RH (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31:247–260. Freund TF, Powell JF, Smith AD (1984) Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 13:1189–1215. Grant KA, Valverius P, Hudspith M, Tabakoff B (1990) Ethanol withdrawal seizures and the NMDA receptor complex. Eur J Pharmacol 176:289–296. Gulya K, Grant KA, Valerius P, Hoffman PL, Tabakoff B (1991) Brain regional specificity and time-course of changes in the NMDA receptor-ionophore complex during ethanol withdrawal. Brain Res 547: 129–134. Harris KM (1999) Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol 9:343–348. Hartig W, Riedel A, Grosche J, Edwards RH, Fremeau RT Jr, Harkany T, Brauer K, Arendt T (2003) Complementary distribution of vesicular glutamate transporters 1 and 2 in the nucleus accumbens of rat: relationship to calretinin-containing extrinsic innervation and calbindinimmunoreactive neurons. J Comp Neurol 465:1–10. Hendricson AW, Miao CL, Lippmann MJ, Morrisett RA (2002) Ifenprodil and ethanol enhance NMDA receptor-dependent long-term depression. J Pharmacol Exp Ther 301:938–944. Hendricson AW, Sibbald JR, Morrisett RA (2004) Ethanol alters the frequency, amplitude and decay kinetics of asynchronous NMDAR mEPSCs in rat hippocampal slices. J Neurophys 91:2568–2577. Hoffman PL, Rabe CS, Moses F, Tabakoff B (1989) N-Methyl-D-aspartate receptors and ethanol: inhibition of calcium flux and cyclic GMP production. J Neurochem 52:1937–1940. Kalluri HS, Mehta AK, Ticku MK (1998) Up-regulation of NMDA receptor subunits in rat brain following chronic ethanol treatment. Brain Res Mol Brain Res 58:221–224. Kirov SA, Harris KM (1999) Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated. Nat Neurosci 2: 878–883. Lin Y, Skeberdis VA, Francesconi A, Bennett MV, Zukin RS (2004) Postsynaptic density protein-95 regulates NMDA channel gating and surface expression. J Neurosci 24:10138–10148. Lominac AD, Hannun R, Szumlinski KK (2005) Subtle, but significant, differences in the effects of mGluR1a versus mGluR5 blockade on alcohol reward in C57BL/6 mice. Alcohol Clin Exp Res 29 Suppl: 96A Lovinger DM, White G, Weight FF (1989) Ethanol inhibits NMDAactivated ion current in hippocampal neurons. Science 243:1721–1724. Maldve RE, Zhang TA, Ferrani-Kile K, Schreiber SS, Lippmann MJ, Snyder GL, Fienberg AA, Leslie SW, Gonzales RA, Morrisett RA (2002) DARPP-32 and regulation of the ethanol sensitivity of NMDA receptors in the nucleus accumbens. Nat Neurosci 5:641–648. Morrisett RA, Swartzwelder HS (1993) Attenuation of hippocampal long-term potentiation by ethanol: a patch-clamp analysis of glutamatergic and GABAergic mechanisms. J Neurosci 13:2264–2272. Noguchi J, Matsuzaki M, Ellis-Davies G, Kasai H (2005) Spine-neck geometry determines NMDA receptor-dependent Ca21 signaling in dendrites. Neuron 46:609–622. Okamoto K, Nagai T, Miyawaki A, Hayashi Y (2004) Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 7: 1104–1112. Robinson TE, Kolb B (2004) Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 47(suppl 1): 33–46.
376
Rodd-Henricks ZA, Bell RL, Kuc KA, Murphy JM, McBride WJ, Lumeng L, Li TK (2001) Effects of concurrent access to multiple ethanol concentrations and repeated deprivations on alcohol intake of alcohol-preferring rats. Alcohol Clin Exp Res 25:1140–1150. Rossetti ZL, Carboni S (1995) Ethanol withdrawal is associated with increased extracellular glutamate in the rat striatum. Eur J Pharmacol 283:177–183. Rudolph JG, Walker DW, Iimuro Y, Thurman RG, Crews FT (1997) NMDA receptor binding in adult rat brain after several chronic ethanol treatment protocols. Alcohol Clin Exp Res 21: 1508–1519. Sanna E, Serra M, Cossu A, Colombo G, Follesa P, Cuccheddu T, Concas A, Biggio G (1993) Chronic ethanol intoxication induces differential effects on GABAA and NMDA receptor function in the rat brain. Alcohol Clin Exp Res 17:115–123. Segal M (2005) Dendritic spines and long-term plasticity. Nat Rev Neurosci 6:277. Shiraishi Y, Mizutani A, Yuasa S, Mikoshiba K, Furuichi T (2004) Differential expression of Homer family proteins in the developing mouse brain. J Comp Neurol 473:582–599. Siggins GR, Martin G, Roberto M, Nie Z, Madamba S, De Lecea L (2003) Glutamatergic transmission in opiate and alcohol dependence. Ann NY Acad Sci 1003:196–211. Smith AD, Bolam JP (1990) The neural network of the basal ganglia as revealed by the study of synaptic connections of identified neurones. Trends Neurosci 13:259–265. Smothers CT, Szumlinski KK, Middaugh LD, Worley PF, Kalivas PW, Woodward JJ (2003) Effects of Homer2 deletion on the development of ethanol tolerance and NMDA receptor function in mice. Alcohol Clin Exp Res 27(suppl): 13A. Snell LD, Nunley KR, Lickteig RL, Browning MD, Tabakoff B, Hoffman PL (1996) Regional and subunit specific changes in NMDA receptor mRNA and immunoreactivity in mouse brain following chronic ethanol ingestion. Brain Res Mol Brain Res 40:71–78. Swanson C, Baker D, Carson D, Worley P, Kalivas PW (2001) Repeated cocaine administration attenuates group I metabotropic glutamate receptor-mediated glutamate release and behavioral activation: a potential role for Homer 1b/c. J Neurosci 21:9043–9052.
CHANDLER ET AL.
Szumlinski KK, Abernathy KE, Oleson EB, Klugmann M, Lominac KD, He D-Y, Ron D, During MJ, Kalivas PW (2005a) Homer isoforms differentially regulate cocaine-induced neuroplasticity. Neuropsychopharmacology, Epub ahead of press. Szumlinski KK, Dehoff MH, Kang SH, Frys KA, Lominac KD, Middaugh LD, Toda S, Tu JC, Berry T, Shealy SE, Worley PF, Kalivas PW (2004) Homer proteins regulate vulnerability to cocaine. Neuron 43:401–413. Szumlinski KK, Lominac KD, Kleschen M, Oleson EB, Dehoff M, Schwartz M, Seeburg PH, Worley PF, Kalivas PW (2005b) Behavioural and neurochemical phenotyping of Homer1 mutant mice: possible implications for schizophrenia. Genes Brain Behav 4:273–288. Szumlinski KK, Lominac KD, Oleson EB, Walker JK, Mason A, Dehoff MH, Klugmann M, Cagle S, Welt K, During MJ, Worley PF, Middaugh LD, Kalivas PW (2005c) Homer2 is necessary for ethanolinduced neuroplasticity. J Neurosci 25:7054–7061. Thomas MP, Monaghan DT, Morrisett RA (1998) Evidence for a causative role of N-methyl-D-asparate receptors in an in vitro model of alcohol withdrawal hyperexcitability. JPET 287:87–97. Thomas MP, Morrisett RA (2000) Dynamics of NMDAR-mediated neurotoxicity during chronic ethanol exposure and withdrawal. Neuropharmacology 39:218–226. Tremwel MF, Anderson KJ, Hunter BE (1994) Stability of [3H]MK-801 binding sites following chronic ethanol consumption. Alcohol Clin Exp Res 18:1004–1008. Trevisan L, Fitzgerald LW, Brose N, Gasic GP, Heinemann SF, Duman RS, Nestler EJ (1994) Chronic ingestion of ethanol up-regulates NMDAR1 receptor subunit immunoreactivity in rat hippocampus. J Neurochem 62:1635–1638. Ulrichsen J, Bech B, Ebert B, Diemer NH, Allerup P, Hemmingsen R (1996) Glutamate and benzodiazepine receptor autoradiography in rat brain after repetition of alcohol dependence. Psychopharmacology 126:31–41. Xiao B, Tu JC, Worley PF (2000) Homer: a link between neural activity and glutamate receptor function. Curr Opin Neurobiol 10:370–374. Zhang TA, Hendricson AW, Morrisett RA (2005) Dual synaptic sites of D(1)-dopaminergic regulation of ethanol sensitivity of NMDA receptors in nucleus accumbens. Synapse 30–44.
