Marisa Roberto

Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons

We examined the interaction of ethanol with the γ-aminobutyric acid (GABA)ergic system in neurons of slices of the rat central amygdala nucleus (CeA), a brain region thought to be critical for the reinforcing effects of ethanol. Brief superfusion of 11–66 mM ethanol significantly increased GABA type A (GABAA) receptor-mediated inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in most CeA neurons, with a low apparent EC50 of 20 mM. Acute superfusion of 44 mM ethanol increased the amplitude of evoked GABAA IPSPs and IPSCs in 70% of CeA neurons. The ethanol enhancement of IPSPs and IPSCs occurred to a similar extent in the presence of the GABA type B (GABAB) receptor antagonist CGP 55845A, suggesting that this receptor is not involved in the ethanol effect on CeA neurons. Ethanol superfusion also decreased paired-pulse facilitation of evoked GABAA IPSPs and IPSCs and always increased the frequency and sometimes the amplitude of spontaneous miniature GABAA IPSCs as well as responses to local GABA application, indicating both presynaptic and postsynaptic sites of action for ethanol. Thus, the CeA is the first brain region to reveal, without conditional treatments such as GABAB antagonists, consistent, low-dose ethanol enhancement of GABAergic transmission at both pre- and postsynaptic sites. These findings add further support to the contention that the ethanol–GABA interaction in CeA plays an important role in the reinforcing effects of ethanol.

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.

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.

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 GABAA 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.

CRF system recruitment mediates dark side of compulsive eating

Dieting to control body weight involves cycles of deprivation from palatable food that can promote compulsive eating. The present study shows that rats withdrawn from intermittent access to palatable food exhibit overeating of palatable food upon renewed access and an affective withdrawal-like state characterized by corticotropin-releasing factor-1 (CRF1) receptor antagonist-reversible behaviors, including hypophagia, motivational deficits to obtain less palatable food, and anxiogenic-like behavior. Withdrawal was accompanied by increased CRF expression and CRF1 electrophysiological responsiveness in the central nucleus of the amygdala. We propose that recruitment of anti-reward extrahypothalamic CRF-CRF1 systems during withdrawal from palatable food, analogous to abstinence from abused drugs, may promote compulsive selection of palatable food, undereating of healthier alternatives, and a negative emotional state when intake of palatable food is prevented.

NMDA receptors regulate nicotine-enhanced brain reward function and intravenous nicotine self-administration: role of the ventral tegmental area and central nucleus of the amygdala.

Nicotine is considered an important component of tobacco responsible for the smoking habit in humans. Nicotine increases glutamate-mediated transmission throughout brain reward circuitries. This action of nicotine could potentially contribute to its intrinsic rewarding and reward-enhancing properties, which motivate consumption of the drug. Here we show that the competitive N-methyl-D-aspartate (NMDA) receptor antagonist LY235959 (0.5–2.5 mg per kg) abolished nicotine-enhanced brain reward function, reflected in blockade of the lowering of intracranial self-stimulation (ICSS) thresholds usually observed after experimenter-administered (0.25 mg per kg) or intravenously self-administered (0.03 mg per kg per infusion) nicotine injections. The highest LY235959 dose (5 mg per kg) tested reversed the hedonic valence of nicotine from positive to negative, reflected in nicotine-induced elevations of ICSS thresholds. LY235959 doses that reversed nicotine-induced lowering of ICSS thresholds also markedly decreased nicotine self-administration without altering responding for food reinforcement, whereas the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist NBQX had no effects on nicotine intake. In addition, nicotine self-administration upregulated NMDA receptor subunit expression in the central nucleus of the amygdala (CeA) and ventral tegmental area (VTA), suggesting important interactions between nicotine and the NMDA receptor. Furthermore, nicotine (1 μM) increased NMDA receptor-mediated excitatory postsynaptic currents in rat CeA slices, similar to its previously described effects in the VTA. Finally, infusion of LY235959 (0.1–10 ng per side) into the CeA or VTA decreased nicotine self-administration. Taken together, these data suggest that NMDA receptors, including those in the CeA and VTA, gate the magnitude and valence of the effects of nicotine on brain reward systems, thereby regulating motivation to consume the drug.

Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala

We recently reported that chronic ethanol treatment (CET) and early withdrawal (2–8 h) altered glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of the amygdala (CeA). Acute ethanol (44 mM) inhibited the NMDA receptor (NMDAR)-mediated EPSCs (NMDA-EPSCs) more in CeA neurons from CET rats than from naïve rats and also decreased paired-pulse facilitation (PPF) of NMDA-EPSCs only in CET rats. To determine whether these CET effects persisted after prolonged withdrawal, we recorded intracellularly in rat CeA slices and measured mRNA and protein expression of CeA NMDAR subunits from CET rats and those withdrawn from ethanol for 1 or 2 weeks. At 1 week withdrawal, acute ethanol decreased evoked NMDA-EPSC amplitudes and NMDA currents induced by exogenous NMDA (∼20%) equally to that in naïve rats, indicating that CET effects on postsynaptic mechanisms reversed 1 week after CET cessation. However, acute ethanol still decreased PPF of NMDA-EPSCs, indicating that the acute ethanol-induced increase in glutamate release in CeA seen in CET rats was still present at this time. CET also significantly increased mRNA levels of NR1 and NR2B NMDAR subunits compared to control rats. At 1 week withdrawal, mRNA levels for NR1 and NR2B subunits were significantly decreased. These changes reversed at 2 weeks withdrawal. In Western blots, a significant increase in protein for all three subunits occurred in CeA from CET rats, but not after 1 and 2 weeks of withdrawal. These data indicate that CET induces reversible neuroadaptations in synaptic function, gene expression, and protein composition of NMDAR at CeA synapses.

Protein kinase C epsilon mediation of CRF- and ethanol-induced GABA release in central amygdala

In the central amygdala (CeA), ethanol acts via corticotrophin-releasing factor (CRF) type 1 receptors to enhance GABA release. Amygdala CRF mediates anxiety associated with stress and drug dependence, and it regulates ethanol intake. Because mutant mice that lack PKCε exhibit reduced anxiety-like behavior and alcohol consumption, we investigated whether PKCε lies downstream of CRF1 receptors in the CeA. Compared with PKCε+/+ CeA neurons, PKCε−/− neurons showed increased GABAergic tone due to enhanced GABA release. CRF and ethanol stimulated GABA release in the PKCε+/+ CeA, but not in the PKCε−/− CeA. A PKCε-specific inhibitor blocked both CRF- and ethanol-induced GABA release in the PKCε+/+ CeA, confirming findings in the PKCε−/− CeA. These results identify a PKCε signaling pathway in the CeA that is activated by CRF1 receptor stimulation, mediates GABA release at nerve terminals, and regulates anxiety and alcohol consumption.

Synaptic Effects Induced by Alcohol

Ethanol (EtOH) has effects on numerous cellular molecular targets, and alterations in synaptic function are prominent among these effects. Acute exposure to EtOH activates or inhibits the function of proteins involved in synaptic transmission, while chronic exposure often produces opposing and/or compensatory/homeostatic effects on the expression, localization, and function of these proteins. Interactions between different neurotransmitters (e.g., neuropeptide effects on release of small molecule transmitters) can also influence both acute and chronic EtOH actions. Studies in intact animals indicate that the proteins affected by EtOH also play roles in the neural actions of the drug, including acute intoxication, tolerance, dependence, and the seeking and drinking of EtOH. This chapter reviews the literature describing these acute and chronic synaptic effects of EtOH and their relevance for synaptic transmission, plasticity, and behavior.

Long-lasting hippocampal potentiation and contextual memory consolidation

In order to ascertain whether there are hippocampal electrophysiological modifications specifically related to memory, exploratory activity and emotional stress, extracellular electrical activity was recorded in hippocampal slices prepared from the brains of male adult rats. Several groups of animals were employed: (i) rats which had freely explored the experimental apparatus (8 min exposure); (ii) rats which had been subjected, in the same apparatus, to a fear conditioning paradigm training entailing the administration of aversive electrical footshocks (8 min exposure); (iii) rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make difficult the association between painful stimuli and the apparatus (30 s exposure); (iv) naïve rats never placed in the apparatus. Half of the rats from each treatment group were used for retrieval testing and the other half for hippocampal excitability testing. The conditioned freezing response was exhibited for no less than 4 weeks. Hippocampal excitability was measured by means of input–output curves (IOC) and paired‐pulse facilitation curves (PPF). Retrieval testing or brain slices preparation were performed at increasing delays after the training sessions: immediately afterwards or after 1, 7 or 28 days. Only the rats subjected to the fear conditioning training exhibited freezing when placed again in the apparatus (retrieval testing). It was found that IOCs, with respect to naïve rats, increased in the conditioned animals up to the 7‐day delay. In free exploration animals the IOCs increased only immediately after the training session. In all other rats no modification of the curves was observed. IOC increases do not appear to imply presynaptic transmitter release modifications, because they were not accompanied by PPF modifications. In conclusion, a clear‐cut correlation was found between the increase in excitability of the Schaffer collateral–CA1 dendrite synapses and freezing response consolidation.