Acupuncture Inhibits GABA Neuron Activity in the Ventral Tegmental Area and Reduces Ethanol Self‐Administration

Background: Withdrawal from chronic ethanol enhances ventral tegmental area (VTA) GABA neuron excitability and reduces mesolimbic dopamine (DA) neurotransmission, which is suppressed by acupuncture at Shenmen (HT7) points (Zhao et al., 2006). The aim of this study was to evaluate the effects of HT7 acupuncture on VTA GABA neuron excitability, ethanol inhibition of VTA GABA neuron firing rate, and ethanol self‐administration. A role for opioid receptors (ORs) in ethanol and acupuncture effects is also explored. Methods: Using electrophysiological methods in mature rats, we evaluated the effects of HT7 stimulation and opioid antagonists on VTA GABA neuron firing rate. Using behavioral paradigms in rats, we evaluated the effects of HT7 stimulation and opioid antagonists on ethanol self‐administration using a modification of the sucrose‐fading procedure. Results: HT7 stimulation produced a biphasic modulation of VTA GABA neuron firing rate characterized by transient enhancement followed by inhibition and subsequent recovery in 5 minutes. HT7 inhibition of VTA GABA neuron firing rate was blocked by systemic administration of the nonselective μ‐opioid receptor antagonist naloxone. HT7 stimulation significantly reduced ethanol suppression of VTA GABA neuron firing rate, which was also blocked by naloxone. HT7 acupuncture reduced ethanol self‐administration without affecting sucrose consumption. Systemic administration of the δ‐opioid receptor (DOR) antagonist naltrindole blocked ethanol suppression of VTA GABA neuron firing rate and significantly reduced ethanol self‐administration without affecting sucrose consumption. Conclusions: These findings suggest that DOR‐mediated opioid modulation of VTA GABA neurons may mediate acupuncture’s role in modulating mesolimbic DA release and suppressing the reinforcing effects of ethanol.     

Ethanol Acutely Inhibits Ionotropic Glutamate Receptor‐Mediated Responses and Long‐Term Potentiation in the Developing CA1 Hippocampus

Background: Developmental ethanol (EtOH) exposure damages the hippocampus, causing long‐lasting alterations in learning and memory. Alterations in glutamatergic synaptic transmission and plasticity may play a role in the mechanism of action of EtOH. This signaling is fundamental for synaptogenesis, which occurs during the third trimester of human pregnancy (first 12 days of life in rats). Methods: Acute coronal brain slices were prepared from 7‐ to 9‐day‐old rats. Extracellular and patch‐clamp electrophysiological recording techniques were used to characterize the acute effects of EtOH on α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate receptor (AMPAR)‐ and N‐methyl‐d ‐aspartate receptor (NMDAR)‐mediated responses and long‐term potentiation (LTP) in the CA1 hippocampal region. Results: Ethanol (40 and 80 mM) inhibited AMPAR‐ and NMDAR‐mediated field excitatory postsynaptic potentials (fEPSPs). EtOH (80 mM) also reduced AMPAR‐mediated fEPSPs in the presence of an inhibitor of Ca²⁺ permeable AMPARs. The effect of 80 mM EtOH on NMDAR‐mediated fEPSPs was significantly greater in the presence of Mg²⁺. EtOH (80 mM) neither affected the paired‐pulse ratio of AMPAR‐mediated fEPSPs nor the presynaptic volley. The paired‐pulse ratio of AMPAR‐mediated excitatory postsynaptic currents was not affected either, and the amplitude of these currents was inhibited to a lesser extent than that of fEPSPs. EtOH (80 mM) inhibited LTP of AMPAR‐mediated fEPSPs. Conclusions: Acute EtOH exposure during the third‐trimester equivalent of human pregnancy inhibits hippocampal glutamatergic transmission and LTP induction, which could alter synapse refinement and ultimately contribute to the pathophysiology of fetal alcohol spectrum disorder.     

Ethanol self-administration is regulated by CB1 receptors in the nucleus accumbens and ventral tegmental area in alcohol-preferring AA rats

Background: Endogenous cannabinoids and their receptors, CB1 receptors in particular, have been implicated in mediation of ethanol reinforcement. Previously, suppression of ethanol drinking by CB1 antagonists has been demonstrated in many experimental paradigms. However, the exact mechanism by which CB1 antagonists modulate ethanol drinking remains elusive. In the present study, we assessed the role of CB1 receptors within the key regions of the mesolimbic dopamine pathway, the nucleus accumbens (NAcc) and ventral tegmental area (VTA), in regulation of ethanol self‐administration. Methods: Adult male alcohol‐prefer AA rats were trained to self‐administer either 10% (w/v) ethanol or 0.1% (w/v) saccharin under an FR1 schedule during daily 30‐minute sessions. Following stable baseline responding, rats were tested after systemic administration of the CB1 antagonist SR141716A (0 to 10 mg/kg) and the agonist WIN55,212‐2 (0 to 2 mg/kg). Separate groups of rats were implanted with bilateral cannulas aimed at the NAcc or VTA, and tested after microinjections of SR141716A (0 to 3 μg) and WIN55,212‐2 (0 to 5 μg) into the NAcc or VTA. The highest intracerebral doses were tested also in rats responding for a 0.1% saccharin solution. Results: SR141617A dose‐dependently suppressed ethanol responding after systemic administration. Microinjections of SR141617A both into NAcc and VTA attenuated ethanol responding. In addition, intra‐NAcc injections of SR141617A suppressed saccharin intake. Although low doses of systemically given WIN55,212‐2 increased ethanol responding, no effects were seen after WIN55,212‐2 microinjections into NAcc or VTA. Conclusions: Bidirectional changes in ethanol self‐administration by the systematically administered CB1 agonist and antagonist show that ethanol reinforcement is controlled by CB1 receptors in alcohol‐preferring AA rats. Replication of the suppressive effects by CB1 antagonism in the NAcc and VTA suggests that endocannabinoids and their receptors mediate ethanol reinforcement through interaction with the mesolimbic dopamine pathway.     

Ethanol enhances GABAergic transmission onto dopamine neurons in the ventral tegmental area of the rat

Background:  Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. At most central synapses, ethanol generally increases inhibitory synaptic transmission; however, no studies have explored the effect of acute ethanol on GABAergic transmission in the VTA.
Methods:  Whole-cell patch clamp recordings of inhibitory postsynaptic currents (IPSCs) from VTA-DA neurons in midbrain slices from young rats.
Results  Acute exposure of VTA-DA neurons to ethanol (25 to 50 mM) robustly enhanced GABAergic spontaneous and miniature IPSC frequency while inducing a slight enhancement of spontaneous IPSC (sIPSC) amplitude. Ethanol (50 mM) enhanced paired-pulse depression of evoked IPSCs, further suggesting enhanced GABA release onto VTA-DA neurons. The frequency of sIPSCs was suppressed by the GABA_B agonist, baclofen (1.25 μM) and enhanced by the antagonist, SCH50911 (20 μM); however, neither appeared to modulate or occlude the effects of ethanol on sIPSC frequency.
Conclusions:  The present results indicate that ethanol increases postsynaptic GABA_A receptor sensitivity, enhances action potential-independent GABA release onto VTA-DA neurons, and that this latter effect is independent of GABA_B auto-receptor inhibition of GABA release.     

Axonal α7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons

In association with NMDA receptors (NMDARs), neuronal α7 nicotinic ACh receptors (nAChRs) have been implicated in neuronal plasticity as well as neurodevelopmental, neurological, and psychiatric disorders. However, the role of presynaptic NMDARs and their interaction with α7 nAChRs in these physiological and pathophysiological events remains unknown. Here we report that axonal α7 nAChRs modulate presynaptic NMDAR expression and structural plasticity of glutamatergic presynaptic boutons during early synaptic development. Chronic inactivation of α7 nAChRs markedly increased cell surface NMDAR expression as well as the number and size of glutamatergic axonal varicosities in cortical cultures. These boutons contained presynaptic NMDARs and α7 nAChRs, and recordings from outside-out pulled patches of enlarged presynaptic boutons identified functional NMDAR-mediated currents. Multiphoton imaging of presynaptic NMDAR-mediated calcium transients demonstrated significantly larger responses in these enlarged boutons, suggesting enhanced presynaptic NMDAR function that could lead to increased glutamate release. Moreover, whole-cell patch clamp showed a significant increase in synaptic charge mediated by NMDAR miniature EPSCs but no alteration in the frequency of AMPAR miniature EPSCs, suggesting the selective enhancement of postsynaptically silent synapses upon inactivation of α7 nAChRs. Taken together, these findings indicate that axonal α7 nAChRs modulate presynaptic NMDAR expression and presynaptic and postsynaptic maturation of glutamatergic synapses, and implicate presynaptic α7 nAChR/NMDAR interactions in synaptic development and plasticity.     

Competing presynaptic and postsynaptic effects of ethanol on cerebellar purkinje neurons

BACKGROUND: Ethanol has actions on cerebellar Purkinje neurons that can result either in a net excitation or in inhibition of neuronal activity. The present study examines the interplay of presynaptic and postsynaptic mechanisms to determine the net effect of ethanol on the neuronal firing rate of cerebellar Purkinje neurons. METHODS: Whole-cell voltage-clamp recording of miniature inhibitory postsynaptic currents (mIPSCs) from Purkinje neurons in cerebellar slices was used to examine the effect of ethanol on presynapticsynaptic release of gamma-aminobutyric acid (GABA) and glutamate. Extracellular recording was used to examine the net action of both presynaptic and postsynaptic effects of ethanol on the firing rate of Purkinje neurons. RESULTS: Under whole-cell voltage clamp, the frequency of bicuculline-sensitive miniature postsynaptic currents (mIPSCs) was increased dose-dependently by 25, 50, and 100 mM ethanol without any change in amplitude or decay time. Despite this evidence of increased release of GABA by ethanol, application of 50 mM ethanol caused an increase in firing in some neurons and a decrease in firing in others with a nonrandom distribution. When both glutamatergic and GABAergic influences were removed by simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione and picrotoxin, respectively, ethanol caused only an increase in firing rate. CONCLUSIONS: These data are consistent with a dual action of ethanol on cerebellar Purkinje neuron activity. Specifically, ethanol acts presynaptically to increase inhibition by release of GABA, while simultaneously acting postsynaptically to increase intrinsic excitatory drive.     

Acute and Chronic Ethanol Modulate Dopamine D2-Subtype Receptor Responses in Ventral Tegmental Area GABA Neurons

Background: Ventral tegmental area (VTA) γ‐aminobutyric acid (GABA) neurons appear to be critical substrates underlying the acute and chronic effects of ethanol on dopamine (DA) neurotransmission in the mesocorticolimbic system implicated in drug reward. VTA GABA neuron firing rate is reduced by acute ethanol and enhanced by DA via D2 receptor activation. The objective of this study was to evaluate the role of D2 receptors in acute ethanol inhibition of VTA GABA neuron activity, as well as the adaptation of D2 receptors by chronic ethanol consumption. Methods: Using electrophysiological methods, we evaluated the effects of intraperitoneal ethanol on DA activation of VTA GABA neurons, the effects of DA antagonists on ethanol inhibition of their firing rate, as well as adaptations in firing rate following chronic ethanol consumption. Using single cell quantitative RT‐polymerase chain reaction (PCR), we evaluated the expression of VTA GABA neuron D2 receptors in rats consuming ethanol versus pair‐fed controls. Results: In acute ethanol studies, microelectrophoretic activation of VTA GABA neurons by DA was inhibited by acute intraperitoneal ethanol, and intravenous administration of the D2 antagonist eticlopride blocked ethanol suppression of VTA GABA neuron firing rate. In chronic ethanol studies, while there were no signs of withdrawal at 24 hours, or significant adaptation in firing rate or response to acute ethanol, there was a significant down‐regulation in the expression of D2 receptors in ethanol‐consuming rats versus pair‐fed controls. Conclusions: Inhibition of DA activation of VTA GABA neuron firing rate by ethanol, as well as eticlopride block of ethanol inhibition of VTA GABA neuron firing rate, suggests an interaction between ethanol and DA neurotransmission via D2 receptors, perhaps via enhanced DA release in the VTA subsequent to ethanol inhibition of GABA neurons. Down‐regulation of VTA GABA neuron D2 receptors by chronic ethanol might result from persistent DA release onto GABA neurons.     

Effects of ethanol on excitatory and inhibitory synaptic transmission in rat cortical neurons

BACKGROUND: The gamma-aminobutyric acid-A (GABA(A)) receptor and glutamate receptors are among the most important target sites for the behavioral effects of ethanol. However, data in the literature concerning the ethanol modulation of the GABA(A) and glutamate receptors have been controversial. The activity of the neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) has recently been reported to be potently augmented by ethanol. The activation of nAChRs is also known to cause the release of various neurotransmitters including GABA and glutamate. Thus, ethanol potentiation of nAChRs is expected to stimulate the GABAergic and glutamatergic systems. METHODS: Whole-cell patch clamp experiments were performed using rat cortical neurons in primary culture to record spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Two types of neurons were distinguished: bipolar neurons possessed alpha4beta2 nAChRs generating a steady current in response to 30 nM ACh, and multipolar neurons that did not generate a current by ACh application. Acetylcholine greatly increased the frequency of mEPSCs and mIPSCs in bipolar neurons but not in multipolar neurons. The amplitude of neither type of neuron was affected by ACh. Ethanol at 10 to 100 mM suppressed the amplitude of mEPSCs while augmenting the amplitude of mIPSCs in both bipolar and multipolar neurons, indicating the direct action on the respective receptors. In bipolar neurons, ACh plus 100 mM ethanol greatly increased the frequency of mIPSCs beyond the levels achieved by ACh alone, while no such increases were observed in multipolar neurons. CONCLUSIONS: It is concluded that ethanol stimulation of nAChRs modulates the activity of both glutamate and GABA receptors in rat cortical bipolar neurons.     

Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter

Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and beta-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that beta-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to beta-neurexin and was not observed with other synaptic cell adhesion molecules such as N-cadherin or SynCAM. NMDA receptors, but not alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this beta-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic beta-neurexin and that glutamate release also is required for maturation of synapses.     

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.     

Reward and relapse: complete gene-induced dissociation in an animal model of alcohol dependence

Background: In animal models of continuous alcohol self‐administration, in which physical dependence does not constitute the major factor of ethanol intake, 2 factors likely contribute to the perpetuation of alcohol self‐administration: (i) the rewarding effects of ethanol and (ii) the contextual conditioning cues that exist along with the process of self‐administration. Present studies are aimed at understanding the relative contribution of these factors on the perpetuation of heavy alcohol self‐administration, as an indication of relapse. Methods: Wistar‐derived UChB high ethanol drinker rats were allowed access to 10% ethanol and water on a 24‐hour basis. In initial studies, an anticatalase shRNA gene‐coding lentiviral vector aimed at inhibiting acetaldehyde generation was administered into the ventral tegmental area (VTA) of the animals prior to ethanol access. In subsequent studies, the lentiviral vector was administered to animals, which had consumed ethanol on a 24‐hour basis, or a 1‐hour basis, after the animals had reached high levels of ethanol intake for 60 to 80 days. In final studies, quinine (0.01%) was added to the ethanol solution to alter the conditioning taste/smell cues of alcohol that animals had chronically ingested. Results: Data indicate that the administration of an anticatalase vector into the VTA of naïve animals blocked reward and alcohol self‐administration, while it was, nevertheless, inactive in inhibiting alcohol self‐administration in rats that had been conditioned to ingest ethanol for over 2 months. The lack of inhibitory effect of the anticatalase vector on ethanol intake in animals that had chronically self‐administered ethanol was fully reversed when the contextual conditioning cues of the alcohol solution were changed. Conclusions: Data highlight the importance of conditioning factors in relapse and suggest that only abolishing or blunting it, along with long‐lasting pharmacological treatment to reduce ethanol reward, may have protracted effects in reducing alcohol self‐administration.     

Dehydration-induced synaptic plasticity in magnocellular neurons of the hypothalamic supraoptic nucleus

Norepinephrine plays a critical role in the regulation of hypothalamic neuroendocrine function, in large part through modulation of synaptic glutamate and gamma-aminobutyric acid (GABA) release. Hypothalamic magnocellular neuroendocrine cells undergo dramatic changes in synaptic organization under conditions of increased hormone release, including increased numbers of glutamatergic, GABAergic and noradrenergic synapses. We studied the functional plasticity of magnocellular neurons of the rat supraoptic nucleus induced by chronic dehydration using whole-cell recordings in hypothalamic slices. Dehydrated rats showed increases in glutamate and GABA release onto magnocellular neurons, as evidenced by an increase in the frequency of spontaneous excitatory (29%) and inhibitory (33%) postsynaptic currents. The change in glutamate release was likely due to increased numbers of release sites because paired-pulse facilitation analysis did not reveal a change in the probability of transmitter release. In untreated rats, norepinephrine facilitates glutamate release and attenuates GABA release onto magnocellular neurons. Dehydration resulted in a marked enhancement of norepinephrine's actions, doubling both the norepinephrine-induced increase in glutamate release and decrease in GABA release. The norepinephrine dose-response curve was shifted to the left with dehydration, revealing an increase in norepinephrine sensitivity. Thus, dehydration leads to an increase in glutamate and GABA release onto supraoptic magnocellular neurons as well as a marked enhancement of the facilitatory effect of norepinephrine on glutamate release and inhibitory effect on GABA release. This synaptic plasticity would be expected to increase the excitability of the magnocellular neurons and support the enhanced bursting capacity and facilitated hormone secretion observed in vivo with chronic dehydration.     

Early Social Isolation in Male Long-Evans Rats Alters Both Appetitive and Consummatory Behaviors Expressed During Operant Ethanol Self-Administration

Background: Postweaning social isolation in rats produces profound and long‐lasting cognitive and behavioral deficits in adult animals. Importantly, this housing manipulation alters sensitivity to a number of drugs of abuse including ethanol. However, most studies with ethanol have utilized continuous or limited home‐cage access to examine interactions between juvenile social experience and drinking. More recently, social isolation was shown to increased ethanol responding in a “dipper” model of self‐administration ( Deehan et al., 2007 ). In the current study, we utilize a “sipper” operant self‐administration model to distinguish the effects of isolation rearing on ethanol seeking‐ and drinking‐related behaviors. Methods: Postweaning juvenile male Long‐Evans rats were placed into 2 housing groups for 6 weeks: one group consisted of individually housed animals; the second group was housed 4 animals per cage. Following the isolation period, anxiety‐like behavior was assessed to confirm the efficacy of the isolation procedure. In some animals, ethanol drinking in the home cage was assessed using a continuous access, 2‐bottle choice paradigm. All animals were then individually housed and trained to lever‐press for a sipper tube containing either an ethanol solution or a sucrose solution. Results: Postweaning social isolation increased the expression of anxiety‐like behavior in the elevated plus maze but not the light‐dark box. Ethanol consumption was also increased during continuous home‐cage access with the 2‐bottle choice paradigm. During operant self‐administration, isolation housing increased the response rate and increased ethanol consumption but did not alter responding for or consumption of sucrose. The housing manipulation did not change the total number of lever responses during extinction sessions. Paired‐pulse inhibition deficits that are characteristic of juvenile isolation remained intact after prolonged experience with sucrose self‐administration. Discussion: The effects of postweaning social isolation on ethanol drinking in the home cage are also manifest during operant self‐administration. Importantly, these alterations in adult operant self‐administration are ethanol‐specific.     

Ghrelin Receptor Antagonism Decreases Alcohol Consumption and Activation of Perioculomotor Urocortin‐Containing Neurons

Background: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut‐derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6. Methods: In Experiment 1 , male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2‐bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D‐Lys3‐GHRP‐6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2 , the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c‐Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D‐Lys3‐GHRP‐6 affects alcohol intake, in Experiment 3 , the effect of D‐Lys3‐GHRP‐6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c‐Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4 , to test if blood ethanol concentrations were affected by D‐Lys3‐GHRP‐6, blood samples were taken at 2 time‐points after D‐Lys3‐GHRP‐6 pretreatment and systemic ethanol administration. Results: In Experiment 1 , D‐Lys3‐GHRP‐6 reduced preference to alcohol and in a follow‐up experiment ( Experiment 2 ) also dramatically reduced alcohol intake when compared to saline‐treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c‐Fos showed fewer immunopositive cells in the pIIIu of the antagonist‐treated mice but no difference was seen in the VTA or Arc. In Experiment 3 , D‐Lys3‐GHRP‐6 reduced the induction of c‐Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c‐Fos immunopositive cells after D‐Lys3‐GHRP‐6 administration, but the antagonist had no effect on ethanol‐induced expression of c‐Fos. D‐Lys3‐GHRP‐6‐pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline‐pretreated mice ( Experiment 4 ). Conclusions: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.     

Nicotine stimulation on extracellular glutamate levels in the nucleus accumbens of ethanol-withdrawn rats in vivo

Background: Nicotine can release glutamate in the limbic system. Presynaptic activation of glutamate receptors might be relevant for the subsequent firing of excitatory postsynaptic potentials. This might be relevant in early ethanol withdrawal. The effects and differences of nicotine stimulation on glutamate response measured by microdialysis in the nucleus accumbens (NAC) between ethanol‐withdrawn rats (EW group) and ethanol‐naïve rats (control group) were investigated. Methods: Rats were ethanol‐intoxicated according to a binge‐drinking model: recurrent cycle of 4 days of intoxication (EW group) or 5% sucrose (control group), followed by a 3‐day recovery. This was followed by a 2‐day intoxication period and subsequent abstinence. After the last oral intake, microdialysis was performed in the left NAC for a 16‐hour withdrawal period. At the end of the withdrawal period, a rated withdrawal score (RWS) was documented. Then, nicotine was given subcutaneously at a dose of 0.5 mg/kg and amino acid levels determined by microdialysis were followed for an additional 3 hours. Results: The RWS was not correlated to the last amount of ethanol received, but was correlated to the total amount of ethanol administered during the pretreatment period: the basal values of extracellular glutamate were found to be decreased in the EW group before withdrawal. Cessation of ethanol significantly increased glutamate levels with a peak between 4 and 10 hours after the last oral intake. Sixteen hours after ethanol withdrawal, the same level as in the control group was achieved. Nicotine significantly increased glutamate levels in the NAC of the EW group but not in ethanol‐naïve rats. Conclusions: This study showed that withdrawal of ethanol was associated with an increase in extracellular glutamate levels. Systemic administration of nicotine in vivo produced an increase in extracellular levels of glutamate in the core region of the NAC during ethanol withdrawal. This might be a relevant pathomechanism for increased craving either for alcohol or for nicotine after ethanol withdrawal.     

Effects of microinjection of the D2 dopamine antagonist raclopride into the ventral tegmental area on ethanol and sucrose self-administration

From previous microinjection studies, a reciprocal feedback between the nucleus accumbens and the ventral tegmental area (VTA) has been implicated in the reinforcing stimulus actions of ethanol and sucrose. In these studies, the effects of self administration of ethanol or sucrose solutions on maintained responding were similar when a dopamine antagonist was injected in the nucleus accumbens or a dopamine agonist was injected into the VTA. Our study was performed to determine if the effects on responding that had been observed when a dopamine agonist was injected into the nucleus accumbens would occur after an injection of a dopamine antagonist into the VTA. Male, Long-Evans rats were initially trained to lever press using either 10% ethanol or 75% sucrose solutions as the reinforcers. Bilateral guide cannulae were implanted to allow microinjection into the VTA of differing doses of the dopamine D2 antagonist, raclopride. Only at the highest dose tested (10 microg) was any effect observed on responding maintained by either reinforcer. The effect was minimal and different from that observed after the microinjection of a dopamine agonist into the nucleus accumbens. This suggests that either the actions of the nucleus accumbens agonist manipulation involved other processes or that the level of enhanced dopamine release in the nucleus accumbens from the VTA antagonist injection was not sufficient to mimic the effect of the nucleus accumbens agonist injections.     

Relationship between ethanol's acute locomotor effects and ethanol self-administration in male Long-Evans rats

Background: Human studies have suggested an important relationship between ethanol sensitivity and risk of alcoholism. These studies have led some to hypothesize that a low initial sensitivity to ethanol’s depressant effects and/or an elevated response to ethanol’s stimulant effects may represent important risk factors associated with the development of abusive drinking behavior. Unfortunately, elucidating neurobiologic mechanisms that may underlie these relationships between ethanol sensitivity and ethanol drinking have been hampered by difficulties in modeling some of these interactions in animals. In this study, we re‐examined some of these relationships in an outbred strain of rats using continuous access two‐bottle choice drinking and a limited‐access operant procedure that engenders pharmacologically relevant levels of ethanol intake and permits the discrete assessment of appetitive and consummatory measures of ethanol drinking behavior. Methods: Twenty‐three male Long‐Evans rats were habituated to a locomotor activity box and then tested for their response to a stimulant (0.5 g/kg) and depressant (1.5 g/kg) ethanol dose. Rats were then trained to complete a lever pressing requirement to gain access to 10% ethanol for 20‐minute sessions conducted 5 d/wk for 5 weeks. Appetitive behavior was assessed after 2.5 and 4.5 weeks using 20‐minute extinction trials in which ethanol was not presented and lever responses were recorded. Home‐cage ethanol preference was also assessed prior to and immediately following the 5‐week self‐administration regimen using a continuous access, two‐bottle choice procedure. Results: A significant increase in home‐cage ethanol preference was observed following the self‐administration procedure, however, neither measure of ethanol preference correlated with average daily ethanol intake during the operant self‐administration sessions or with initial sensitivity to ethanol’s stimulant or depressant effects. Notably, a significant negative correlation was observed between sensitivity to ethanol’s locomotor depressant effect and daily intake during the operant self‐administration sessions. No significant relationships were noted between sensitivity to ethanol’s locomotor effects and extinction responding. Conclusions: The results of these studies suggest that the well‐established relationship between a low level of response to ethanol and increased ethanol consumption reported in human studies can be observed in an outbred rodent strain using a limited‐access operant self‐administration procedure, but not with home‐cage ethanol drinking.     

Pregnenolone and ganaxolone reduce operant ethanol self-administration in alcohol-preferring p rats

Background: Neuroactive steroids modulate ethanol intake in several self‐administration models with variable effects. The purpose of this work was to examine the effects of the long‐acting synthetic GABAergic neurosteroid ganaxolone and the endogenous neurosteroid pregnenolone, a precursor of all GABAergic neuroactive steroids, on the maintenance of ethanol self‐administration in an animal model of elevated drinking—the alcohol‐preferring (P) rats. Methods: P rats were trained to self‐administer ethanol (15% v/v) versus water on a concurrent schedule of reinforcement, and the effects of ganaxolone (0 to 30 mg/kg, subcutaneous [SC]) and pregnenolone (0 to 75 mg/kg, intraperitoneal [IP]) were evaluated on the maintenance of ethanol self‐administration. After completion of self‐administration testing, doses of the neuroactive steroids that altered ethanol self‐administration were assessed on spontaneous locomotor activity. Finally, the effect of pregnenolone administration on cerebral cortical levels of the GABAergic neuroactive steroid (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone, 3α,5α‐THP) was determined in both ethanol‐experienced and ethanol‐inexperienced P rats because pregnenolone is a precursor of these steroids. Results: Ganaxolone produced a dose‐dependent biphasic effect on ethanol reinforcement, as the lowest dose (1 mg/kg) increased and the highest dose (30 mg/kg) decreased ethanol‐reinforced responding. However, the highest ganaxolone dose also produced a nonspecific reduction in locomotor activity. Pregnenolone treatment significantly reduced ethanol self‐administration (50 and 75 mg/kg), without altering locomotor activity. Pregnenolone (50 mg/kg) produced a significant increase in cerebral cortical allopregnanolone levels. This increase was observed in the self‐administration trained animals, but not in ethanol‐naïve P rats. Conclusions: These results indicate that pregnenolone dose‐dependently reduces operant ethanol self‐administration in P rats without locomotor impairment, suggesting that it may have potential as a novel therapeutic for reducing chronic alcohol drinking in individuals that abuse alcohol.     

Selective presynaptic enhancement of the prefrontal cortex to nucleus accumbens pathway by cocaine

The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.     

Effects of ethanol on the rat glutamate excitatory amino acid transporter type 3 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase

Background: Glutamate is a major excitatory neurotransmitter in the central nervous system. Glutamate transporters play a critical role in maintaining extracellular glutamate concentrations. We investigated the effects of ethanol on a neuronal glutamate transporter, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3 K) in mediating these effects. Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 messenger RNA. By using a two‐electrode voltage clamp, membrane currents were recorded after the application of l‐glutamate. Responses were quantified by integration of the current trace and reported as microcoulombs. Data are mean ± SEM. Results: Ethanol enhanced EAAT3 activity in a concentration‐dependent manner. At 25, 50, 100, and 200 mM of ethanol, the responses were significantly increased compared with control values. Kinetic study demonstrated that ethanol (50 mM) significantly increased V_max (3.48 ± 0.2 μC for control versus 4.16 ± 0.24 μC for ethanol; n= 19; p < 0.05) without a significant change in the K_m (65.6 ± 11.1 μM for control versus 55.8 ± 9.6 μM for ethanol; n= 19; p > 0.05) of EAAT3 for glutamate. Preincubation of the oocytes with phorbol‐12‐myristate‐13‐acetate (PMA) significantly increased EAAT3 activity (0.98 ± 0.08 μC for control versus 1.28 ± 0.09 μC for ethanol; n= 19; p < 0.05). However, there was no statistical difference among the responses of EAAT3 to PMA, ethanol, or PMA plus ethanol. Although the PKC inhibitors chelerythrine and staurosporine did not decrease the basal EAAT3 activity, they abolished the enhancement of EAAT3 activity by ethanol. Pretreatment with wortmannin, a PI3 K inhibitor, also abolished the ethanol‐enhanced EAAT3 activity. Conclusions: These results suggest that acute ethanol exposure increases EAAT3 activity at clinically relevant concentrations and that PKC and PI3 K may be involved in mediating these ethanol effects.