Neurite Outgrowth and Gene Expression Profile Correlate with Efficacy of Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts.

Transplantation of human induced pluripotent stem cell-derived dopaminergic (iPSC-DA) neurons is a promising therapeutic strategy for Parkinson's disease (PD). To assess optimal cell characteristics and reproducibility, we evaluated the efficacy of iPSC-DA neuron precursors from two individuals with sporadic PD by transplantation into a hemiparkinsonian rat model after differentiation for either 18 (d18) or 25 days (d25). We found similar graft size and dopamine (DA) neuron content in both groups, but only the d18 cells resulted in recovery of motor impairments. In contrast, we report that d25 grafts survived equally as well and produced grafts rich in tyrosine hydroxylase-positive neurons, but were incapable of alleviating any motor deficits. We identified the mechanism of action as the extent of neurite outgrowth into the host brain, with d18 grafts supporting significantly more neurite outgrowth than nonfunctional d25 grafts. RNAseq analysis of the cell preparation suggests that graft efficacy may be enhanced by repression of differentiation-associated genes by REST, defining the optimal predifferentiation state for transplantation. This study demonstrates for the first time that DA neuron grafts can survive well in vivo while completely lacking the capacity to induce recovery from motor dysfunction. In contrast to other recent studies, we demonstrate that neurite outgrowth is the key factor determining graft efficacy and our gene expression profiling revealed characteristics of the cells that may predict their efficacy. These data have implication for the generation of DA neuron grafts for clinical application.

Selective inhibition of monoacylglycerol lipase is associated with passive coping behavior and attenuation of stress-induced dopamine release in the medial prefrontal cortex.

The endocannabinoid system is involved in the regulation of the stress response, but the relative contribution of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) and their mechanisms have to be elucidated. In this study, we compared the effects of the pharmacological inhibition of the two major endocannabinoid-degrading enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) for AEA and 2-AG, respectively] on stress-coping [forced swim test (FST) and tail suspension test (TST)] and anxiety-like [elevated-plus maze (EPM) and light-dark test (LDT)] behaviors in wild-type and FAAH knockout mice. In vivo microdialysis estimated the effects of FAAH and MAGL inhibition on dopamine (DA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) during an FST. Mice were treated with PF-3845 (FAAH inhibitor), JZL184 (MAGL inhibitor), JZL195 (dual FAAH/MAGL inhibitor) or vehicle. Our data showed that PF-3845 increased latency to immobility and decreased total immobility time in FST, but no effects were observed in TST compared with vehicle-treated wild-type mice. By contrast, JZL184 decreased latency and increased immobility in TST and FST. JZL195 in wild-type mice and JZL184 in FAAH knockout mice reproduced the same passive coping behaviors as JZL184 in wild-type mice in TST and FST. In the microdialysis experiment, FST was associated with increased DA and 5-HT levels in the mPFC. However, JZL184-treated wild-type mice displayed a significant attenuation of forced swim stress-induced DA release compared with vehicle-treated wild-type mice and PF-3845-treated wild-type mice. Finally, FAAH and/or MAGL inhibitors induced robust and consistent anxiolytic-like effects in EPM and LDT. These results suggested differences between FAAH and MAGL inhibition in stress-coping behaviors. Notably, MAGL inhibition induced a consistent avoidant coping behavior and attenuated the stress-induced mPFC DA response in FST. However, more investigation is needed to elucidate the functional association between DA and 2-AG signaling pathways, and the molecular mechanism in the regulation of passive coping strategies during inescapable stress.

COX-2 Inhibition Antagonizes Intra-Accumbens 2-Arachidonoylglycerol-Mediated Reduction in Ethanol Self-Administration in Rats.

BACKGROUND: Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake. METHODS: In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide. RESULTS: Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration. CONCLUSIONS: We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non-CB1 receptor mechanism involving the COX-2 pathway.

Ethanol-induced alterations in endocannabinoids and relevant neurotransmitters in the nucleus accumbens of fatty acid amide hydrolase knockout mice.

Deletion of fatty acid amide hydrolase (FAAH), enzyme responsible for degrading endocannabinoids, increases alcohol consumption and preference. However, there is a lack of data on neurochemical events in mice exposed to alcohol in the absence of FAAH. Extracellular levels of endocannabinoids and relevant neurotransmitters were measured by in vivo microdialysis in the nucleus accumbens (NAc) of FAAH knockout (KO) and wild-type (WT) mice during an ethanol (EtOH; 2 g/kg, ip) challenge in EtOH-naive and repeated (r) EtOH-treated mice. In both genotypes, EtOH treatment caused no changes in baseline endocannabinoid levels, although FAAH KO mice displayed higher baseline N-arachidonoylethanolamine levels than WT mice. EtOH challenge caused a sustained increase in 2-arachidonoylglycerol (2-AG) levels in EtOH-naive WT mice but not in FAAH KO mice. In contrast, 2-AG levels were decreased following EtOH challenge in (r)EtOH-treated mice in both genotypes. Whereas (r)EtOH-treated mice showed higher baseline dopamine and serotonin levels than EtOH-naive mice in WT mice, these differences were attenuated in FAAH KO mice. Significant differences in baseline γ-aminobutyric acid (GABA) and glutamate levels by EtOH history were observed in WT mice but not in FAAH KO mice. Moreover, opposed effects on glutamate response were observed after EtOH challenge in EtOH-naive and (r)EtOH-treated FAAH KO mice. Finally, FAAH deletion failed to show EtOH-induced locomotion sensitivity. These data provide evidence of a potential influence of 2-AG in the neurochemical response to EtOH exposure in the NAc.

Cannabinoid dependence induces sustained changes in GABA release in the globus pallidus without affecting dopamine release in the dorsal striatum: A dual microdialysis probe study.

A dual probe microdialysis study was designed to characterize GABA and dopamine (DA) release in the basal ganglia of cannabinoid-dependent Wistar rats. Whereas chronic administration of the cannabinoid receptor agonist WIN55,212 (WIN) resulted in increased basal GABA release, the D2 agonist receptor-mediated control of GABA and DA release elicited by quinpirole was similar in both cannabinoid-dependent and non dependent animals. However, quinpirole did induce a greater number of more stereotypies in cannabinoid-dependent animals, indicating a dysregulated behavioral response.

Deficient endocannabinoid signaling in the central amygdala contributes to alcohol dependence-related anxiety-like behavior and excessive alcohol intake.

Negative emotional states that are associated with excessive alcohol intake, particularly anxiety-like states, have been linked to opponent processes in the central nucleus of the amygdala (CeA), affecting stress-related transmitters and monoamines. This study extends these observations to include endocannabinoid signaling in alcohol-dependent animals. Rats and mice were exposed to chronic intermittent alcohol with vapor inhalation or liquid diet to induce dependence. In vivo microdialysis was used to estimate interstitial concentrations of endocannabinoids [N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG)] and amino acids (glutamate and GABA) in rat CeA. Additionally, we evaluated the inhibition of endocannabinoids clearance enzymes [monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase] on anxiety-like behavior and alcohol consumption in alcohol-dependent rats and mice. Results revealed that alcohol dependence produced decreases in baseline 2-AG dialysate levels and increases in baseline levels of glutamate and GABA. Acute alcohol abstinence induced an enhancement of these dependence-induced effects and the levels of 2-AG and GABA were restored upon alcohol re-exposure. Additional studies showed that the increased CeA 2-AG levels induced by restraint stress and alcohol self-administration were blunted in alcohol-dependent rats. Pharmacological studies in rats and mice showed that anxiety-like behavior and alcohol consumption were increased in alcohol-dependent animals, and these behavioral effects were attenuated mainly by MAGL inhibitors [MJN110 (10 and 20 mg/kg) in rats and JZL184 (1 and 3 mg/kg) in mice]. The present results suggest a key role for endocannabinoid signaling in motivational neuroadaptations during alcohol dependence, in which a deficiency in CeA 2-AG signaling in alcohol-dependent animals is linked to stress and excessive alcohol consumption.

Unique treatment potential of cannabidiol for the prevention of relapse to drug use: preclinical proof of principle.

Cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, has received attention for therapeutic potential in treating neurologic and psychiatric disorders. Recently, CBD has also been explored for potential in treating drug addiction. Substance use disorders are chronically relapsing conditions and relapse risk persists for multiple reasons including craving induced by drug contexts, susceptibility to stress, elevated anxiety, and impaired impulse control. Here, we evaluated the "anti-relapse" potential of a transdermal CBD preparation in animal models of drug seeking, anxiety and impulsivity. Rats with alcohol or cocaine self-administration histories received transdermal CBD at 24 h intervals for 7 days and were tested for context and stress-induced reinstatement, as well as experimental anxiety on the elevated plus maze. Effects on impulsive behavior were established using a delay-discounting task following recovery from a 7-day dependence-inducing alcohol intoxication regimen. CBD attenuated context-induced and stress-induced drug seeking without tolerance, sedative effects, or interference with normal motivated behavior. Following treatment termination, reinstatement remained attenuated up to ≈5 months although plasma and brain CBD levels remained detectable only for 3 days. CBD also reduced experimental anxiety and prevented the development of high impulsivity in rats with an alcohol dependence history. The results provide proof of principle supporting potential of CBD in relapse prevention along two dimensions: beneficial actions across several vulnerability states and long-lasting effects with only brief treatment. The findings also inform the ongoing medical marijuana debate concerning medical benefits of non-psychoactive cannabinoids and their promise for development and use as therapeutics.

Inhaled delivery of Δ(9)-tetrahydrocannabinol (THC) to rats by e-cigarette vapor technology.

Most human Δ(9)-tetrahydrocannabinol (THC) use is via inhalation, and yet few animal studies of inhalation exposure are available. Popularization of non-combusted methods for the inhalation of psychoactive drugs (Volcano(®), e-cigarettes) further stimulates a need for rodent models of this route of administration. This study was designed to develop and validate a rodent chamber suitable for controlled exposure to vaporized THC in a propylene glycol vehicle, using an e-cigarette delivery system adapted to standard size, sealed rat housing chambers. The in vivo efficacy of inhaled THC was validated using radiotelemetry to assess body temperature and locomotor responses, a tail-flick assay for nociception and plasma analysis to verify exposure levels. Hypothermic responses to inhaled THC in male rats depended on the duration of exposure and the concentration of THC in the vehicle. The temperature nadir was reached after ∼40 min of exposure, was of comparable magnitude (∼3 °Celsius) to that produced by 20 mg/kg THC, i.p. and resolved within 3 h (compared with a 6 h time course following i.p. THC). Female rats were more sensitive to hypothermic effects of 30 min of lower-dose THC inhalation. Male rat tail-flick latency was increased by THC vapor inhalation; this effect was blocked by SR141716 pretreatment. The plasma THC concentration after 30 min of inhalation was similar to that produced by 10 mg/kg THC i.p. This approach is flexible, robust and effective for use in laboratory rats and will be of increasing utility as users continue to adopt "vaping" for the administration of cannabis.

GIRK3 gates activation of the mesolimbic dopaminergic pathway by ethanol.

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.

Aß induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss.

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer's disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-ß peptide (Aß) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aß induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aß-induced damage both in vitro and in vivo.

Corticotropin releasing factor-induced amygdala gamma-aminobutyric Acid release plays a key role in alcohol dependence.

BACKGROUND: Corticotropin-releasing factor (CRF) and gamma-aminobutyric acid (GABA)ergic systems in the central amygdala (CeA) are implicated in the high-anxiety, high-drinking profile associated with ethanol dependence. Ethanol augments CeA GABA release in ethanol-naive rats and mice. METHODS: Using naive and ethanol-dependent rats, we compared electrophysiologic effects and interactions of CRF and ethanol on CeA GABAergic transmission, and we measured GABA dialyzate in CeA after injection of CRF(1) antagonists and ethanol. We also compared mRNA expression in CeA for CRF and CRF(1) using real-time polymerase chain reaction. We assessed effects of chronic treatment with a CRF(1) antagonist on withdrawal-induced increases in alcohol consumption in dependent rats. RESULTS: CRF and ethanol augmented CeA GABAergic transmission in naive rats via increased GABA release. Three CRF1 receptor (CRF(1)) antagonists decreased basal GABAergic responses and abolished ethanol effects. Ethanol-dependent rats exhibited heightened sensitivity to CRF and CRF(1) antagonists on CeA GABA release. Intra-CeA CRF(1) antagonist administration reversed dependence-related elevations in GABA dialysate and blocked ethanol-induced increases in GABA dialyzate in both dependent and naive rats. Polymerase chain reaction studies indicate increased expression of CRF and CRF(1) in CeA of dependent rats. Chronic CRF(1) antagonist treatment blocked withdrawal-induced increases in alcohol drinking by dependent rats and tempered moderate increases in alcohol consumption by nondependent rats in intermittent testing. CONCLUSIONS: These combined findings suggest a key role for specific presynaptic CRF-GABA interactions in CeA in the development and maintenance of ethanol dependence.

Chronic ethanol treatment potentiates ethanol-induced increases in interstitial nucleus accumbens endocannabinoid levels in rats.

We employed in vivo microdialysis to characterize the effect of an ethanol challenge injection on endocannabinoid levels in the nucleus accumbens of ethanol-naïve and chronic ethanol-treated rats. Ethanol (0.75 and 2 g/kg, i.p.) dose-dependently increased dialysate 2-arachidonoylglycerol (to a maximum 157 +/- 20% of baseline) and decreased anandamide (to a minimum 52 +/- 9% of baseline) in ethanol-naïve rats. The endocannabinoid clearance inhibitor N-(4-hydrophenyl) arachidonoylamide (AM404; 3 mg/kg) potentiated ethanol effects on 2-arachidonoylglycerol levels but did not alter ethanol-induced decreases in anandamide. AM404 alone did not alter dialysate levels of either endocannabinoid. Then, we characterized the effect of ethanol challenge on nucleus accumbens endocannabinoid levels in rats previously maintained on an ethanol-containing liquid diet. Ethanol challenge produced a greater and more prolonged increase in 2-arachidonoylglycerol (to a maximum 394 +/- 135% of baseline) in ethanol-experienced than in ethanol-naïve rats. The profile in ethanol-experienced rats was similar to that produced by AM404 pre-treatment in ethanol-naïve rats. AM404 in ethanol-experienced rats led to a further enhancement in the 2-arachidonoylglycerol response to ethanol challenge (to a maximum 704 +/- 174% of baseline). Our findings demonstrate that ethanol-induced increases in nucleus accumbens 2-arachidonoylglycerol are potentiated in animals with a history of ethanol consumption.

Central cannabinoid 1 receptor antagonist administration prevents endotoxic hypotension affecting norepinephrine release in the preoptic anterior hypothalamic area.

It is widely assumed that LPS lowers arterial pressure during sepsis by stimulating release of TNF-alpha and other vasoactive mediators from macrophages. However, recent data from this and other laboratories have shown that LPS hypotension can be prevented by inhibiting afferent impulse flow in the vagus nerve, by blocking neuronal activity in the nucleus of the solitary tract, or by blocking alpha-adrenergic receptors in the preoptic area/anterior hypothalamic area (POA). These findings suggest that the inflammatory signal is conveyed from the periphery to the brain via the vagus nerve, and that endotoxic shock is mediated through a central mechanism that requires activation of POA neurons. In the present study, we tested whether central cannabinoid 1 (CB1) receptors participate in the control of arterial pressure during endotoxemia based on evidence that hypothalamic neurons express CB1 receptors and synthesize the endogenous CB anandamide. We found that intracerebroventricular administration of rimonabant, a CB1 receptor antagonist, inhibited the fall in arterial pressure evoked by LPS significantly in both conscious and anesthetized rats. Rimonabant attenuated both the immediate fall in arterial pressure evoked by LPS and the second, delayed hypotensive phase that leads to tissue ischemia and death. Rimonabant also prevented the associated LPS-induced rise in extracellular fluid norepinephrine concentrations in the POA. Furthermore, rimonabant attenuated the associated increase in plasma TNF-alpha concentrations characteristic of the late phase of endotoxic hypotension. These data indicate that central CB1 receptors may play an important role in the initiation of endotoxic hypotension.

Specific alterations of extracellular endocannabinoid levels in the nucleus accumbens by ethanol, heroin, and cocaine self-administration.

Ethanol and opiate self-administration are sensitive to manipulations of cannabinoid CB1 receptor function and, from this, a role for the endogenous cannabinoid system in the modulation of drug reward has been hypothesized. However, direct in vivo evidence of drug-induced alterations in brain endocannabinoid (eCB) formation has been lacking. To address this issue, we explored the effect of drug self-administration on interstitial eCB levels in the nucleus accumbens (NAc) shell using in vivo microdialysis. Ethanol, heroin, and cocaine were compared because the rewarding properties of ethanol and heroin are reduced by CB1 receptor inactivation, whereas cocaine reward is less sensitive to these manipulations. Ethanol self-administration significantly increased dialysate 2-arachidonoylglycerol (2-AG) levels with no concomitant change in dialysate anandamide (AEA) concentrations. Conversely, heroin self-administration significantly increased dialysate AEA levels, and induced a subtle but significant decrease in dialysate 2-AG levels. In each case, the relative change in dialysate eCB content was significantly correlated with the amount of drug consumed. In contrast, cocaine self-administration did not alter dialysate levels of either AEA or 2-AG. Local infusion of the CB1 antagonist SR 141716A into the NAc significantly reduced ethanol, but not cocaine, self-administration. Together with our previous observation that intra-NAc SR 141716A reduces heroin self-administration, these data provide novel in vivo support for an eCB involvement in the motivational properties of ethanol and heroin but not cocaine. Furthermore, the selective effects of ethanol and heroin on interstitial 2-AG and AEA provide new insight into the distinct neurochemical profiles produced by these two abused substances.

Biphasic alterations in serotonin-1B (5-HT1B) receptor function during abstinence from extended cocaine self-administration.

Alterations in 5-HT1B receptor function during cocaine abstinence were evaluated in rats given either limited- or extended access (LA and EA, respectively) to cocaine self-administration. The locomotor response to the 5-HT1B/1A agonist RU24969 was significantly reduced in cocaine-experienced animals relative to cocaine-naïve controls following 6 h of abstinence but became sensitized over the subsequent 14 days of abstinence. Both the early phase subsensitivity and later phase supersensivity to RU 24969-induced activity were greater in EA versus LA animals. Intra-nucleus accumbens administration of the 5-HT1B agonist CP 93, 129 produced significantly greater increases in dialysate dopamine levels in EA versus control animals following 14 days of abstinence. However, there was no difference between EA and cocaine-naïve control animals in the augmentation of cocaine-induced increases in nucleus accumbens DA produced by intra-VTA CP 93, 129. Collectively these findings demonstrate that 5-HT1B receptor function is persistently altered by cocaine self-administration.

Increased GABA release in the central amygdala of ethanol-dependent rats.

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

Acute and chronic ethanol alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis.

The modulation of glutamatergic transmission by ethanol may contribute to ethanol intoxication, reinforcement, tolerance, and dependence. Therefore, we used in vitro electrophysiological and in vivo microdialysis techniques to investigate the effects of acute and chronic ethanol on glutamatergic transmission in the central nucleus of amygdala (CeA). Superfusion of 5-66 mM ethanol decreased compound glutamatergic EPSPs and EPSCs in CeA neurons, with half-maximal inhibition elicited by 14 mM ethanol. Ethanol (44 mM) decreased both non-NMDAR- and NMDAR-mediated EPSPs and EPSCs by 21%. Both the ethanol- and ifenprodil-induced depression of NMDAR-mediated EPSPs and EPSCs was enhanced in rats that received chronic ethanol treatment (CET). Ifenprodil also occluded the ethanol effect, suggesting that NR2B subunit-containing receptors may be involved. With local applications of NMDA, acute ethanol elicited a greater inhibition of NMDA currents in slices taken from CET (47%) compared with naive (30%) animals, suggesting that CET sensitizes NMDA receptors to ethanol. Acute ethanol also reduced paired pulse facilitation of EPSPs and EPSCs only in CET animals, suggesting acute ethanol-induced increase of glutamate release. This finding was supported by in vivo experiments showing that infusion of ethanol (0.1-1 M) via reverse microdialysis significantly increased glutamate release into the CeA dialysate but only after CET. Moreover, baseline CeA glutamate content was significantly higher in CET compared with naive animals. These combined findings suggest that CET and withdrawal lead to neuroadaptations of glutamatergic transmission at both presynaptic and postsynaptic sites in CeA, and glutamatergic synapses in CeA may play an important role in ethanol dependence.