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The dynorphin (DYN)/kappa-opioid receptor (KOR) system is involved in dysphoria and negative emotional states. Dysregulation of KOR function promotes maladaptive behavioral regulation during withdrawal associated with alcohol dependence. Mesolimbic dopaminergic (DA) projections from the ventral tegmental area (VTA) innervate the extended amygdala circuitry and presynaptic KORs attenuate DA in these regions leading to an excessive alcohol consumption and negative affective-like behavior, whereas mesocortical KOR-regulated DA projections have been implicated in executive function and decision-making. Thus, the neuroadaptations occurring in DYN/KOR systems are important aspects to consider for the development of personalized therapeutic solutions. Herein, we study the contribution of the VTA DA neuron Oprk1 (KOR gene) in excessive alcohol consumption, negative emotional state, and executive function. To do so, Oprk1 mRNA expression and KOR function were characterized to confirm alcohol dependence-induced dysregulation in the VTA. Then, a transgenic Cre-Lox rat model (male and female TH::Cre rats) was used to allow for conditional and inducible overexpression of Oprk1 in VTA DA neurons. The effect of this overexpression was evaluated on operant alcohol self-administration, negative emotional states, and executive function. We found that VTA Oprk1 overexpression recapitulates some phenotypes of alcohol dependence including escalated alcohol self-administration and depressive-like behavior. However, working memory performance was not impacted following VTA Oprk1 overexpression in TH::Cre rats. This supports the hypothesis that dysregulated KOR signaling within the mesolimbic DA system is an important contributor to symptoms of alcohol dependence and shows that understanding Oprk1-mediated contributions to alcohol use disorder (AUD) should be an important future goal.

Compulsive alcohol use, or drinking that persists despite negative or aversive consequences, is a defining characteristic of alcohol use disorder. Here, chemogenetic technology (i.e. Designer Receptors Exclusively Activated by Designer Drugs; DREADDs) was used to inhibit or excite the NAc core or selectively inhibit D1-or D2 receptor-expressing neurons in the NAc core to understand the role of the NAc core and how these subpopulations of neurons may influence compulsive-like ethanol (EtOH) drinking using C57BL/6J, Drd1-cre, and Drd2-cre male and female mice. Compulsive-like EtOH drinking was modeled with a two-bottle choice, drinking in the dark paradigm. The major finding of this study was that mice decreased compulsive-like EtOH intake when the NAc core was inhibited and there was no change of EtOH + quinine intake when the NAc core was excited. Interestingly, inhibition of D1-or D2 receptor-expressing neurons did not alter compulsive-like EtOH intake. Control experiments showed that NAc core excitation and selective inhibition of D1-or D2-receptor-expressing neurons had no effect on baseline EtOH drinking, intake of water, or intake of quinine-adulterated water. CNO reduced amphetamine-induced locomotion in the D1-CRE+ (but not the D2CRE+) group in a control experiment. Finally, pharmacological antagonism of D1 and D2 receptors together, but not separately, reduced quinine-resistant EtOH drinking. These results suggest that the NAc core is a critical region involved in compulsive-like EtOH consumption, and that both D1-and D2 receptor-expressing medium spiny neurons participate in controlling this behavior.

The endogenous cannabinoid (eCB) system has been suggested to play a key role in ethanol preference and intake, the acute effects of ethanol, and in the development of withdrawal symptoms following ethanol dependence. Ethanol-dependent alterations in glutamatergic signaling within the lateral/basolateral nucleus of the amygdala (BLA) are critical for the development and expression of withdrawal-induced anxiety. Notably, the eCB system significantly regulates both glutamatergic and GABAergic synaptic activity within the BLA. Chronic ethanol exposure significantly alters eCB system expression within regions critical to the expression of emotionality and anxiety-related behavior, including the BLA. Here, we investigated specific interactions between the BLA eCB system and its functional regulation of synaptic activity during acute and chronic ethanol exposure. In tissue from ethanol naïve-rats, a prolonged acute ethanol exposure caused a dose dependent inhibition of glutamatergic synaptic activity via a presynaptic mechanism that was occluded by CB1 antagonist/inverse agonists SR141716a and AM251. Importantly, this acute ethanol inhibition was attenuated following 10 day chronic intermittent ethanol vapor exposure (CIE). CIE exposure also significantly down-regulated CB1-mediated presynaptic inhibition at glutamatergic afferent terminals but spared CB1-inhibition of GABAergic synapses arising from local inhibitory-interneurons. CIE also significantly elevated BLA N-arachidonoylethanolamine (AEA or anandamide) levels and decreased CB1 receptor protein levels. Collectively, these data suggest a dynamic regulation of the BLA eCB system by acute and chronic ethanol.

Caffeine and ethanol are two commonly overused psychoactive dietary components. The purpose of this study was to assess the effects of acute, chronic, oral (p.o.) and intravenous (i.v.) caffeine, ethanol and their combination on infarct volume following focal ischemia in rats. Rats received treatment either p.o. 3 h and 1 h before, or by i.v. infusion for 2.5 h beginning 30-180 min after, ischemia. There were six acute treatment groups. (1) oral dH2O (control); (2) oral caffeine (10 mg/kg); (3) oral ethanol (0.65 g/kg total); (4) oral ethanol plus caffeine; (5) intravenous saline; and (6) intravenous ethanol (0.65 g/kg) plus caffeine (10 mg/kg) in saline. A 7th group received oral ethanol plus caffeine for three weeks prior to ischemia. After 3 h of left MCA/CCA occlusion and 24 h reperfusion, infarct volume was determined. Control animal infarct volume was 102.4+/-42.0 mm3. Oral caffeine alone had no effect (122.4+/-30.2 mm3). Oral ethanol alone exacerbated infarct volume (177.2+/-27.8 mm3). Oral caffeine plus ethanol almost entirely eliminated the damage (17.89+/-10.41 mm3). When i.v. treatment with ethanol plus caffeine was initiated at 30, 60, 90 and 120 minutes post-ischemia the infarct volume was reduced by 71.7%, 49.8%, 64.8% and 47.1%, respectively. Chronic daily oral ethanol plus caffeine prior to ischemia eliminated the neuroprotection seen with acute treatment. These studies indicate that ethanol, which by itself aggravates cerebral ischemia, and caffeine, when combined together immediately before or for 2 h after focal stroke, reduces ischemic damage.

Physical dependence on ethanol results in an ethanol withdrawal (ETX) syndrome including susceptibility to audiogenic seizures (AGS) in rodents after abrupt cessation of ethanol. Chronic ethanol administration and ETX induce functional changes of neurons in several brain regions, including the amygdala. Amygdala neurons are requisite elements of the neuronal network subserving AGS propagation during ETX induced by a subacute "binge" ethanol administration protocol. However, the effects of chronic ethanol administration on amygdala neuronal firing and ETX seizure behaviors are unknown. In the present study ethanol (5g/kg) was administered intragastrically in Sprague-Dawley rats once daily for 28days [chronic intermittent ethanol (CIE) protocol]. One week later the rats began receiving ethanol intragastrically three times daily for 4days (binge protocol). Microwire electrodes were implanted prior to CIE or on the day after CIE ended to record extracellular action potentials in lateral amygdala (LAMG) neurons. The first dose of ethanol administered in the binge protocol following CIE treatment did not alter LAMG neuronal firing, which contrasts with firing suppression seen previously in the binge protocol alone. These data indicate that CIE induces neuroadaptive changes in the ETX network which reduce LAMG response to ethanol. LAMG neuronal responses to acoustic stimuli prior to AGS were significantly decreased during ETX as compared to those before ethanol treatment. LAMG neurons fired tonically throughout the tonic convulsions during AGS. CIE plus binge treatment resulted in a significantly greater mean seizure duration and a significantly elevated incidence of death than was seen previously with the binge protocol alone, indicating an elevated seizure severity following chronic ethanol administration.

Several studies have shown that ethanol (EtOH) can enhance the activity of GABAergic synapses via presynaptic mechanisms, including in hippocampal CA1 neurons. The serotonin type 3 receptor (5-HT3-R) has been implicated in the neural actions of ethanol (EtOH) and in modulation of GABA release from presynaptic terminals. In the present study, we investigated EtOH modulation of GABA release induced by 5-HT3-R activation using the mechanically isolated neuron/bouton preparation from the rat CA1 hippocampal subregion. EtOH application before and during exposure to the selective 5-HT3 receptor agonist, m-chlorophenylbiguanide (mCPBG) potentiated the mCPBG-induced increases in the peak frequency and charge transfer of spontaneous GABAergic inhibitory postsynaptic currents. Interestingly, the potentiation was maintained even after EtOH was removed from the preparation. A protein kinase A inhibitor reduced the magnitude of EtOH potentiation. Fluorescent Ca2+ imaging showed that Ca2+ transients in the presynaptic terminals increased during EtOH exposure. These findings indicate that EtOH produces long-lasting potentiation of 5-HT3-induced GABA release by modulating calcium levels, via a process involving cAMP-mediated signaling in presynaptic terminals.

Alcohol use disorder (AUD) places a tremendous burden on society, with approximately two billion alcohol users in the world. While most people drink alcohol recreationally, a subpopulation (3-5%) engages in reckless and compulsive drinking, leading to the development of AUD and alcohol dependence. The Ventral Tegmental Area (VTA)-Nucleus Accumbens (NAc) circuit has been shown to encode rewarding stimuli and drive individual alcohol drinking behavior. Our previous work successfully separated C57BL/6J isogenic mice into high or low alcohol drinking subgroups after a 12-day, two-bottle choice voluntary alcohol access paradigm. Electrophysiological studies revealed that low alcohol drinking mice exhibited elevated spontaneous and burst firing properties of their VTA dopamine (DA) neurons and specifically mimicking this pattern of activity in VTA-NAc neurons in high alcohol drinking mice using optogenetics decreased their alcohol preference. It is also known that VTA DA neurons encode the salience and rewarding properties of external stimuli while also regulating downstream dopamine concentrations. Here, as a follow-up to this study, we utilized Fast Scan Cyclic Voltammetry (FSCV) to examine dopamine release in the NAc shell and core between alcohol drinking groups. We observed dynamic changes of dopamine release in the core of high drinking mice, but failed to see widely significant differences of dopamine release in the shell of both groups, when compared with ethanol-naive controls. Overall, the present data suggest subregion-specific differences of evoked dopamine release in the NAc of low and high alcohol drinking mice, and may provide an anatomical substrate for individual alcohol drinking behavior. This article is part of the special issue on Stress, Addiction and Plasticity.

This review highlights literature relating the anatomy, physiology, and behavioral contributions by projections between rodent prefrontal cortical areas and the basolateral amygdala. These projections are robustly modulated by both environmental experience and exposure to drugs of abuse including ethanol. Recent literature relating optogenetic and chemogenetic dissection of these circuits within behavior both compliments and occasionally challenges roles defined by more traditional pharmacological or lesion-based approaches. In particular, cortico-amygdala circuits help control both aversive and reward-seeking. Exposure to pathology-producing environments or abused drugs dysregulates the relative 'balance' of these outcomes. Modern circuit-based approaches have also shown that overlapping populations of neurons within a given brain region frequently govern both aversion and reward-seeking. In addition, these circuits often dramatically influence 'local' cortical or basolateral amygdala excitatory or inhibitory circuits. Our understanding of these neurobiological processes, particularly in relation to ethanol research, has just begun and represents a significant opportunity. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.