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BACKGROUND: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline (NA) stress system is implicated in AUD relapse. METHODS: Here we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure (CIE) to induce dependence, or withdrawn from CIE for two weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by HPLC), in situ hybridization and behavioral brain-site specific pharmacology studies were performed. We also used qRT-PCR to assess gene expression of the α1(B), β1, and β2 adrenergic receptor in human post-mortem brain tissue from men diagnosed with AUD and matched controls. RESULTS: We found that α1 receptors potentiate CeA GABAergic transmission and drive moderate alcohol intake in control rats. In dependent rats, β receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, though there are some long-lasting differences in the cellular patterns of adrenergic receptor mRNA expression. Additionally, post-mortem brain analyses reveal increased α1B receptor mRNA in the amygdala of humans with AUD. CONCLUSIONS: Thus, CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, support ongoing new medication development for AUD.

Loss-of-function mutations in SLC30A10 induce hereditary manganese (Mn)-induced neuromotor disease in humans. We previously identified SLC30A10 to be a critical Mn efflux transporter that controls physiological brain Mn levels by mediating hepatic and intestinal Mn excretion in adolescence/adulthood. Our studies also revealed that in adulthood, SLC30A10 in the brain regulates brain Mn levels when Mn excretion capacity is overwhelmed (e.g. after Mn exposure). But, the functional role of brain SLC30A10 under physiological conditions is unknown. We hypothesized that, under physiological conditions, brain SLC30A10 may modulate brain Mn levels and Mn neurotoxicity in early postnatal life because body Mn excretion capacity is reduced in this developmental stage. We discovered that Mn levels of pan-neuronal/glial Slc30a10 knockout mice were elevated in specific brain regions (thalamus) during specific stages of early postnatal development (postnatal day 21), but not in adulthood. Furthermore, adolescent or adult pan-neuronal/glial Slc30a10 knockouts exhibited neuromotor deficits. The neuromotor dysfunction of adult pan-neuronal/glial Slc30a10 knockouts was associated with a profound reduction in evoked striatal dopamine release without dopaminergic neurodegeneration or changes in striatal tissue dopamine levels. Put together, our results identify a critical physiological function of brain SLC30A10—SLC30A10 in the brain regulates Mn levels in specific brain regions and periods of early postnatal life, which protects against lasting deficits in neuromotor function and dopaminergic neurotransmission. These findings further suggest that a deficit in dopamine release may be a likely cause of early-life Mn-induced motor disease.

BACKGROUND: Norepinephrine has been suggested to regulate ethanol-related behaviors, but little is known about the effects of ethanol on norepinephrine release in mesocortical and mesolimbic brain areas that are targets of ethanol actions. METHODS: We used in vivo microdialysis to examine the effects of ethanol on extracellular norepinephrine concentrations in mesocorticolimbic brain regions of male Long Evans rats. We determined effects of intravenous infusion of saline or ethanol in the medial prefrontal cortex (mPFC), and the basal forebrain. We also measured dialysate norepinephrine concentrations during operant self-administration of ethanol in the mPFC. RESULTS: Intravenous infusion (1 or 0.25 mL/min) of 1.0 g/kg ethanol stimulated an increase in dialysate norepinephrine in mPFC and in basal forebrain. In the basal forebrain an infusion of 0.5 g/kg ethanol did not stimulate dialysate norepinephrine concentrations. In both regions saline infusions did not increase dialysate norepinephrine concentrations. In the behavioral experiment, one week of experience with operant self-administration of sweetened ethanol resulted in an apparent reduction in basal dialysate norepinephrine concentrations in the mPFC relative to the sucrose control. Dialysate norepinephrine increased during the transfer from home cage to the operant chamber in all groups. CONCLUSIONS: We conclude that acute ethanol stimulates both the locus coeruleus (which projects to the mPFC) and the nucleus tractus solitarius (which projects to the basal forebrain) noradrenergic neurons. Additionally, limited ethanol self-administration experience alters dialysate norepinephrine in the mPFC in a manner consistent with a decrease in tonic norepinephrine release. Further studies are necessary to elucidate the mechanisms by which ethanol exerts these variable effects.

The ability of environmental cues to trigger alcohol-seeking behaviors is believed to facilitate problematic alcohol use. We previously showed that the development of this cue-evoked alcohol approach reflects cue-alcohol learning and memory in the adult male rat; however, we do not know whether the same is true for similarly aged female rats. Consequently, adult Long-Evans female rats were allowed to drink unsweetened alcohol in the home cage (Monday, Wednesday, Friday; 24-h two-bottle choice; 5 weeks) and were subsequently split into two experimental groups: Paired and Unpaired. Groups were matched for ingested doses and alcohol bottle preference across the pre-conditioning home cage period. Both groups were trained in conditioning chambers using a Pavlovian procedure. For the Paired group, the chamber houselight was illuminated to signal access to an alcohol sipper. Houselight onset was yoked for the Unpaired group, but access to the alcohol sipper was scheduled to occur only during the intervening periods (in the absence of light). We found that in the Paired, but not Unpaired group, an alcohol approach reaction was conditioned to houselight illumination, and the level of cue-conditioned reactivity predicted drinking behavior within trials. Groups experienced equivalently low but non-negligible blood alcohol concentrations over the course of conditioning sessions. We conclude that cue-triggered alcohol-seeking behavior in adult female rats reflects associative learning about the relationship between alcohol availability and houselight illumination.

Although alcohol (i.e., ethanol) is a major drug of abuse, the acute functional effects of ethanol on the reward circuitry are not well defined in vivo. In freely moving rats, we examined the effect of intravenous ethanol administration on neuronal unit activity in the posterior ventral tegmental area (VTA), a central component of the mesolimbic reward system. VTA units were classified as putative dopamine (DA) neurons, fast-firing GABA neurons, and unidentified neurons based on a combination of electrophysiological properties and DA D2 receptor pharmacological responses. A gradual infusion of ethanol significantly altered the firing rate of DA neurons in a concentration-dependent manner. The majority of DA neurons were stimulated by ethanol and showed enhanced burst firing activity, but a minority was inhibited. Ethanol also increased the proportion of DA neurons that exhibited pacemaker-like firing patterns. In contrast, ethanol mediated a variety of effects in GABA and other unidentified neurons that were distinct from DA neurons, including a nonlinear increase in firing rate, delayed inhibition, and more biphasic activity. These results provide evidence of discrete electrophysiological effects of ethanol on DA neurons compared with other VTA cell types, suggesting a complex role of the VTA in alcohol-induced responses in freely moving animals.

Implicit learning about antecedent stimuli and the unconditional stimulus (US) properties of alcohol may facilitate the progressive loss of control over drinking. To model this learning, Cofresi et al. (2017) developed a procedure in which a discrete, visual conditional stimulus (houselight illumination; CS) predicted the availability of a retractable sipper that rats could lick to receive unsweetened alcohol [Alcoholism: Clinical and Experimental Research, 41, 608–617]. Here we investigated the possibility that houselight illumination, sipper presentation, and oral alcohol receipt might each exert control over alcohol seeking and drinking. We also determined the relationship between ingested dose and blood alcohol concentration, in order to validate the idea that the US is a post-ingestive action of alcohol. Finally, we tested a major prediction from the conditioning account of problematic drinking [Tomie, A., & Sharma, N. (2013). Current Drug Abuse Reviews, 6, 201–219], which is that once learned, responses elicited by a CS will promote drinking. We found that despite having constrained opportunities to drink alcohol during the conditioning procedure, ingested doses produced discriminable blood concentrations that supported cue conditioning. Based on our analysis of the dynamics of cue reactivity in well-trained rats, we found that houselight illumination triggered conditioned approach, sipper presentation evoked licking behavior, and alcohol receipt promoted drinking. Reactivity to these cues, which varied in terms of their temporal proximity to the alcohol US, persisted despite progressive intoxication or satiety. Additionally, rats with the greatest conditioned reactivity to the most distal alcohol cue were also the fastest to initiate drinking and drank the most. Our findings indicate that the post-ingestive effects of alcohol may condition multiple cues simultaneously in adult rats, and these multiple cues help to trigger alcohol seeking and drinking. Moreover, identification and characterization of these cues should be helpful for designing interventions that attenuate the power of these cues over behavior.

The response to drugs of abuse is a combination of aversive and reinforcing reactions. While much is known about the role of dopamine in mammalian drug reinforcement, we know little about the brain circuits mediating drug aversion. Here we show that two distinct dopaminergic circuits mediate reinforcing and acute aversive responses to alcohol consumption in Drosophila. Protocerebral anterior medial dopamine neurons projecting to the mushroom bodies are required for flies to acquire alcohol preference. Conversely, a bilateral pair of dopamine neurons projecting to the dorsal fan-shaped body (dFSB) mediates acute alcohol avoidance. Alcohol consumption can be reduced by decreasing the activity of the appetitive reinforcement-circuit to the mushroom bodies, or by increasing activity in the dopamine neurons projecting to the dFSB. Thus, distinct dopaminergic pathways can be targeted to reduce the intake of harmful drugs.

Background Conditioned responses to alcohol-associated cues can hinder recovery from alcohol use disorder (AUD). Cue exposure (extinction) therapy (CET) can reduce reactivity to alcohol cues, but its efficacy is limited by phenomena such as spontaneous recovery and reinstatement that can cause a return of conditioned responding after extinction. Using a preclinical model of alcohol cue reactivity in rats, we evaluated whether the efficacy of alcohol CET could be improved by conducting CET during the memory reconsolidation window after retrieval of cue-alcohol associations. Methods Rats were provided with intermittent access to unsweetened alcohol. Rats were then trained to predict alcohol access based on a visual cue. Next, rats were treated with either standard extinction (n = 14) or postretrieval extinction (n = 13). Rats were then tested for long-term memory of extinction and susceptibility to spontaneous recovery and reinstatement. Results Despite equivalent extinction, rats treated with postretrieval extinction exhibited reduced spontaneous recovery and reinstatement relative to rats treated with standard extinction. Conclusions Postretrieval CET shows promise for persistently attenuating the risk to relapse posed by alcohol cues in individuals with AUD.

Heightened emotional states increase impulsive behaviors such as excessive ethanol consumption in humans. Though positive and negative affective states in rodents can be monitored in real-time through ultrasonic vocalization (USV) emissions, few animal studies have focused on the role of emotional status as a stimulus for initial ethanol drinking. Our laboratory has recently developed reliable, high-speed analysis techniques to compile USV data during multiple-hour drinking sessions. Since High Alcohol Drinking (HAD-1) rats are selectively bred to voluntarily consume intoxicating levels of alcohol, we hypothesized that USVs emitted by HAD-1 rats would reveal unique emotional phenotypes predictive of alcohol intake and sensitive to alcohol experience. In this study, male HAD-1 rats had access to water, 15% and 30% EtOH or water only (i.e., Controls) during 8 weeks of daily 7-hr drinking-in-the-dark (DID) sessions. USVs, associated with both positive (i.e., 50–55 kHz frequency-modulated or FM) and negative (i.e., 22–28 kHz) emotional states, emitted during these daily DID sessions were examined. Findings showed basal 22–28 kHz USVs were emitted by both EtOH-Naïve (Control) and EtOH-experienced rats, alcohol experience enhanced 22–28 kHz USV emissions, and USV acoustic parameters (i.e., mean frequency in kHz) of both positive and negative USVs were significantly suppressed by chronic alcohol experience. These data suggest that negative affective status initiates and maintains excessive alcohol intake in selectively bred HAD-1 rats and support the notion that unprovoked emissions of negative affect-associated USVs (i.e., 22–28 kHz) predict vulnerability to excessive alcohol intake in distinct rodent models.

Striatal dopamine plays a critical role in the expression of goal-directed behaviors and in mediating the reinforcing effects of various drugs of abuse, including alcohol. Techniques such as microdialysis and fast-scan voltammetry, which enable monitoring molecules in the extracellular space of freely behaving animals, have permitted the study of ethanol’s effects on dopamine activity in striatal subregions. Animal models have demonstrated that alcohol produces differential effects on extracellular dopamine, depending on the route of administration, dose, and subregion. Clinical studies using positron emission tomography have generally supported the findings from animal models, showing that the striatal dopamine response to acute alcohol and alcohol-related cues varies by subregion and drinking status (e.g., social, heavy, or alcoholic). While decades of research have provided an abundance of data, the implications of alcohol’s effects on extracellular striatal dopamine continues to be investigated.

Background Among the evidence implicating neuroimmune signaling in alcohol use disorders are increased levels of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the brains of human alcoholics and animal models of alcohol abuse. However, it is not known whether neuroimmune signaling can directly increase ethanol (EtOH) consumption, and whether MCP-1 is involved in that mechanism. We designed experiments to determine whether MCP-1 signaling itself is sufficient to accelerate or increase EtOH consumption. Our hypothesis was that increasing MCP-1 signaling by directly infusing it into the brain would increase operant EtOH self-administration. Methods We implanted osmotic minipumps to chronically infuse either one of several doses of MCP-1 or vehicle into the cerebral ventricles (intracerebroventricular) of Long-Evans rats and then tested them in the operant self-administration of a sweetened EtOH solution for 8 weeks. Results There was a significant interaction between dose of MCP-1 and sweetened EtOH consumed across the first 4 weeks (while pumps were flowing) and across the 8-week experiment. Animals receiving the highest dose of MCP-1 (2 μg/d) were the highest consumers of EtOH during weeks 3 through 8. MCP-1 did not influence the acquisition of self-administration (measured across the first 5 days), the motivation to consume EtOH (time to lever press or progressive ratio), withdrawal-induced anxiety, or the consumption of sucrose alone. Conclusions We provide novel evidence that neuroimmune signaling can directly increase chronic operant EtOH self-administration, and that this increase persists beyond the administration of the cytokine. These data suggest that EtOH-induced increases in MCP-1, or increases in MCP-1 due to various other neuroimmune mechanisms, may further promote EtOH consumption. Continued research into this mechanism, particularly using models of alcohol dependence, will help determine whether targeting MCP-1 signaling has therapeutic potential in the treatment of alcohol use disorders.

The development of alcohol-use disorders is thought to involve a transition from casual alcohol use to uncontrolled alcohol-seeking behavior. This review will highlight evidence suggesting that the shift toward inflexible alcohol seeking that occurs across the development of addiction consists, in part, of a progression from goal-directed to habitual behaviors. This shift in "response strategy" is thought to be largely regulated by corticostriatal network activity. Indeed, specific neuroanatomical substrates within the prefrontal cortex and the striatum have been identified as playing opposing roles in the expression of actions and habits. A majority of the research on the neurobiology of habitual behavior has focused on non-drug reward seeking. Here, we will highlight recent research identifying corticostriatal structures that regulate the expression of habitual alcohol seeking and a comparison will be made when possible to findings for non-drug rewards.

Background Emotional states are often thought to drive excessive alcohol intake and influence the development of alcohol use disorders. To gain insight into affective properties associated with excessive alcohol intake, we utilized ultrasonic vocalization (USV) detection and analyses to characterize the emotional phenotype of selectively bred alcohol-preferring (P) rats; an established animal model of excessive alcohol intake. USVs emitted by rodents have been convincingly associated with positive (50-55 kHz frequency-modulated [FM]) and negative (22-28 kHz) affective states. Therefore, we hypothesized that 50-55 and 22-28 kHz USV emission patterns in P rats would reveal a unique emotional phenotype sensitive to alcohol experience. Methods 50-55 kHz FM and 22-28 kHz USVs elicited from male P rats were assessed during access to water, 15 and 30% EtOH (v/v). Ethanol (EtOH; n = 12) or water only (Control; n = 4) across 8 weeks of daily drinking-in-the-dark (DID) sessions. Results Spontaneous 22-28 kHz USVs are emitted by alcohol-naive P rats and are enhanced by alcohol experience. During DID sessions when alcohol was not available (e.g., “EtOH OFF” intervals), significantly more 22-28 kHz than 50-55 kHz USVs were elicited, while significantly more 50-55 kHz FM than 22-28 kHz USVs were emitted when alcohol was available (e.g., “EtOH ON” intervals). In addition, USV acoustic property analyses revealed chronic effects of alcohol experience on 22-28 kHz USV mean frequency, indicative of lasting alcohol-mediated alterations to neural substrates underlying emotional response. Conclusions Our findings demonstrate that acute and chronic effects of alcohol exposure are reflected in changes in 22-28 and 50-55 kHz FM USV counts and acoustic patterns. These data support the notion that initiation and maintenance of alcohol intake in P rats may be due to a unique, alcohol-responsive emotional phenotype and further suggest that spontaneous 22-28 kHz USVs serve as behavioral markers for excessive drinking vulnerability.

Alcohol self-administration produces brain and behavior adaptations that facilitate a progressive loss of control over drinking and contribute to relapse. One possible adaptation is the ability of antecedent environmental stimuli that are consistently paired with alcohol to trigger alcohol-seeking behaviors. We previously modeled this adaptation in rats using a Pavlovian conditioning procedure in which illumination of a houselight preceded the presentation of a sipper tube that produced unsweetened alcohol when licked. However, in our previous work we did not demonstrate whether this adaptation represented a consequence of repeated exposure to alcohol or the houselight, or whether it was the consequence of associative learning and memory. Thus, in the present study, we tested the associative basis of alcohol seeking in response to houselight illumination in our task using adult male rats that were not food- or water-deprived and were not dependent on alcohol. Separate groups of rats received houselight illumination that was explicitly paired or unpaired with presentation of the retractable sipper that provided access to unsweetened alcohol. Our primary dependent variable was appetitive alcohol-directed behavior: the frequency of movement toward and interaction with the hole in the wall of the chamber through which the sipper was presented during the period of houselight illumination trial before each sipper presentation. However, we also analyzed consummatory sipper licking behavior and blood ethanol concentration in the same rats. Finally, we explored the brain basis of cue-elicited alcohol seeking using c-Fos immunohistochemistry. Our findings confirmed the associative basis of cue-elicited alcohol seeking in our paradigm and mapped these onto the insular cortex, suggesting a role for this brain region in early stages of brain and behavior adaptation to regular alcohol use.

This chapter reviews the growing body of research focused on understanding the relationship between emotion and alcohol vulnerability in animal models of alcohol use disorders. Emotional status is directly modeled in rodents through analyses of ultrasonic vocalizations (USVs) with 22-kHz calls signaling negative affect and 50-kHz calls signaling positive affect. We review work with selectively bred high-alcohol drinking “P” and “HAD-1” rat lines that reveals high spontaneous rates of negative-affect USV emissions. We make the case that a focus on USV counts underutilizes the rich multidimensional acoustic properties associated with USVs. We argue that algorithms such as our WAAVES algorithm make the application of advanced statistics to the multidimensional USV acoustic properties feasible, and will allow more accurate characterization of emotional phenotypes. We briefly review some of our unpublished data that takes this approach. We show that advanced statistical techniques such as linear mixed modeling and linear discriminant function analysis reveal important properties of the data previously unexamined using call counts. For example, across three data sets (alcohol-naive male P vs. NP, alcohol-naive female vs. male HAD-1, and alcohol-naive male P vs. Long Evans rats) we show that individual acoustic characteristics as well as the full complement of acoustic characteristics taken together differ across groups when the focus is on negative-affect calls but not positive-affect calls. The implications of this work for human alcohol research are discussed.

In vivo monitoring of dopamine via microdialysis has demonstrated that acute, systemic ethanol increases extracellular dopamine in regions innervated by dopaminergic neurons originating in the ventral tegmental area and substantia nigra. Simultaneous measurement of dialysate dopamine and ethanol allows comparison of the time courses of their extracellular concentrations. Early studies demonstrated dissociations between the time courses of brain ethanol concentrations and dopaminergic responses in the nucleus accumbens (NAc) elicited by acute ethanol administration. Both brain ethanol and extracellular dopamine levels peak during the first 5 min following systemic ethanol administration, but the dopamine response returns to baseline while brain ethanol concentrations remain elevated. Post hoc analyses examined ratios of the dopamine response (represented as a percent above baseline) to tissue concentrations of ethanol at different time points within the first 25–30 min in the prefrontal cortex, NAc core and shell, and dorsomedial striatum following a single intravenous infusion of ethanol (1 g/kg). The temporal patterns of these “response ratios” differed across brain regions, possibly due to regional differences in the mechanisms underlying the decline of the dopamine signal associated with acute intravenous ethanol administration and/or to the differential effects of acute ethanol on the properties of subpopulations of midbrain dopamine neurons. This Review draws on neurochemical, physiological, and molecular studies to summarize the effects of acute ethanol administration on dopamine activity in the prefrontal cortex and striatal regions, to explore the potential reasons for the regional differences observed in the decline of ethanol-induced dopamine signals, and to suggest directions for future research.

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λex=220 nm and λem=385 nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 24 hours. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15°C significantly improved sensitivity with a detection limit of approximately 1 nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n=4), basal GABA concentrations were 12.9±2.2 and 14.5±2.2 nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=3) were 4.6±1.1 and 5.1±0.6 nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4 ± 1.9 nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19 ± 0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50 μM) significantly increased extracellular GABA concentration to 170 ± 4 nM and reduced in vivo extraction fraction to 0.112 ± 0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.

Naltrexone, a non-selective opioid antagonist, decreases the euphoria and positive subjective responses to alcohol in heavy drinkers. It has been proposed that the μ-opioid receptor plays a role in ethanol reinforcement through modulation of ethanol-stimulated mesolimbic dopamine release.To investigate the ability of naltrexone and β-funaltrexamine, an irreversible μ-opioid specific antagonist, to inhibit ethanol-stimulated and morphine-stimulated mesolimbic dopamine release, and to determine whether opioid receptors on mesolimbic neurons contribute to these mechanisms.Ethanol-naïve male Long Evans rats were given opioid receptor antagonists either intravenously, subcutaneously, or intracranially into the ventral tegmental area (VTA), followed by intravenous administration of ethanol or morphine. We measured extracellular dopamine in vivo using microdialysis probes inserted into the nucleus accumbens shell (n = 114).Administration of naltrexone (intravenously) and β-funaltrexamine (subcutaneously), as well as intracranial injection of naltrexone into the VTA did not prevent the initiation of dopamine release by intravenous ethanol administration, but prevented it from being as prolonged. In contrast, morphine-stimulated mesolimbic dopamine release was effectively suppressed.Our results provide novel evidence that there are two distinct mechanisms that mediate ethanol-stimulated mesolimbic dopamine release (an initial phase and a delayed phase), and that opioid receptor activation is required to maintain the delayed-phase dopamine release. Moreover, μ-opioid receptors account for this delayed-phase dopamine response, and the VTA is potentially the site of action of this mechanism. We conclude that μ-opioid receptors play different roles in the mechanisms of stimulation of mesolimbic dopamine activity by ethanol and morphine.

Ethanol (EtOH) affects prefrontal cortex functional roles such as decision making, working memory, and behavioral control. Yet, the pharmacological effect of EtOH on dopamine, a neuromodulator in the medial prefrontal cortex (mPFC), is unclear. Past studies exploring this topic produced conflicting outcomes; however, a handful of factors (temporal resolution, method of drug administration, estrous cycle) possibly contributed to these discrepancies. We sought to mitigate these factors in order to elucidate EtOH's pharmacological effects on mPFC dopamine in Long-Evans rats.We administered experimental solutions via an intravenous (iv), handling-free route, monitored dopamine in the mPFC via microdialysis (10-minute samples), and used male rats to avoid estrous cycle/EtOH interactions. First, we rapidly (approximately 2.7 ml/min) or slowly (approximately 0.6 ml/min) administered 1.0 g/kg EtOH and saline infusions, showing that the experimental methods did not contribute to dopamine changes. Then, a cumulative dosing protocol was used to administer 0.25, 0.75, 1.50, and 2.25 g/kg iv EtOH doses to evaluate dose-response. Finally, we monitored dialysate EtOH levels during an oral EtOH self-administration session to compare the dialysate EtOH levels achieved during the pharmacological experiments to those seen during self-administration.IV administration of a rapid or slow 1.0 g/kg EtOH infusion resulted in similar significant 55 ± 9 and 63 ± 15% peak dialysate dopamine increases, respectively. The 0.25, 0.75, 1.50, and 2.25 g/kg EtOH doses produced a nonsignificant 17 ± 5% and significant 36 ± 15, 68 ± 19, and 86 ± 20% peak dialysate dopamine increases, respectively. Self-administration dialysate EtOH concentrations fell within the range of concentrations noted during the EtOH dose-response curve.These experiments show that, using experimental methods that minimize possibly confounding factors, acute iv EtOH increases extracellular dopamine in the mPFC in a dose-dependent manner, thereby clarifying EtOH's pharmacological effects on the mesocortical dopamine system.

Reversed-phase HPLC with derivatization using o-phthalaldehyde (OPA) and sulfite allows electrochemical detection of γ-aminobutyric acid (GABA) in microdialysis samples. However, OPA/sulfite derivatives have been reported to produce lower fluorescent yield than OPA derivatives using organic thiols as the nucleophile. To overcome this limitation we examined excitation and emission spectra, reaction time, pH, and concentration of reagents in the derivatization solution. Optimal detection parameters were determined as λex=220nm and λem=385nm for maximal fluorescence. The derivatization reaction occurred immediately and the product was stable up to 24 h [corrected]. A pH of 10.4 for the borate buffer used in the derivatization solution was significantly better than lower pH. Increasing the amount of sulfite combined with diluting the derivatization solution in borate buffer resulted in complete separation of the GABA peak from contaminants without any loss in signal. Controlling the temperature of the detector at 15°C significantly improved sensitivity with a detection limit of approximately 1nM. To validate this assay, we performed microdialysis in the dorsal striatum and ventral tegmental area (VTA) of adult Long Evans rats. GABA concentrations in dialysates were determined using external standards and standard additions, in order to further confirm interfering peaks were not present in biological samples. Within the dorsal striatum (n=4), basal GABA concentrations were 12.9±2.2 and 14.5±2.2nM (external and additions, respectively). Respective basal GABA concentrations in the VTA (n=3) were 4.6±1.1 and 5.1±0.6nM. Thus, we have developed a novel, sensitive fluorescence method to determine GABA in microdialysates using HPLC of an OPA/sulfite derivative.

Background: The mu opioid receptor (MOR) has previously been found to regulate ethanol-stimulated dopamine release under some, but not all, conditions. A difference in ethanol-evoked dopamine release between male and female mixed background C57BL/6J-129SvEv mice led to questions about its ubiquitous role in these effects of ethanol. Using congenic C57BL/6J MOR knockout (KO) mice and C57BL/6J mice pretreated with an irreversible MOR antagonist, we investigated the function of this receptor in ethanol-stimulated dopamine release. Methods: Microdialysis was used to monitor dopamine release and ethanol clearance in MOR -/-, +/+, and +/− . male and female mice after intraperitoneal (i.p.) injections of 1.0, 2.0, and 3.0 g/kg ethanol (or saline). We also measured the increase in dopamine release after 5 mg/kg morphine (i.p.) in male and female MOR+/+ and −/− mice. In a separate experiment, male C57BL/6J mice were pretreated with either the irreversible MOR antagonist beta funaltrexamine (BFNA) or vehicle, and dopamine levels were monitored after administration of 2 g/kg ethanol or 5 mg/kg morphine. Results: Although ethanol-stimulated dopamine release at all the 3 doses of alcohol tested, there were no differences between MOR+/+, −/−, and +/− mice in these effects. Female mice had a more prolonged effect compared to males at the 1 g/kg dose. Administration of 2 g/kg ethanol also caused a similar increase in dopamine levels in both saline-pretreated and BFNA-pretreated mice. Five mg/kg morphine caused a significant increase in dopamine levels in MOR+/+ mice but not in MOR−/− mice and in saline-pretreated mice but not in BFNA-pretreated mice. Intraperitoneal saline injections had a significant, albeit small and transient, effect on dopamine release when given in a volume equivalent to the ethanol doses, but not in a volume equivalent to the 5 mg/kg morphine dose. Ethanol pharmacokinetics were similar in all genotypes and both sexes at each dose and in both pretreatment groups. Conclusions: MOR is not involved in ethanol-stimulated dopamine release in the ventral striatum of C57BL/6J mice.

Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release. In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABA(A) and GABA(B) receptors (Picrotoxin (75 μM) and SCH50911 (20 μM)) disinhibited VTA-DA firing rate whereas a GABA(A) agonist (muscimol, 1 μM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 μM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 μM) had no effect on VTA-DA firing rate. Naltrexone (60 μM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 μM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.

After publication of this paper, the authors determined an error in the calculation of the norepinephrine standard concentrations for the HPLC calibration curves.

Dopamine concentrations in the nucleus accumbens fluctuate on phasic (subsecond) and tonic (over minutes) timescales in awake rats. Acute ethanol increases tonic concentrations of dopamine, but its effect on subsecond dopamine transients has not been fully explored.We measured tonic and phasic dopamine fluctuations in the nucleus accumbens of rats in response to ethanol (within-subject cumulative dosing, 0.125 to 2 g/kg, i.v.).Microdialysis samples yielded significant tonic increases in dopamine concentrations at 1 to 2 g/kg ethanol in each rat, while repeated saline infusions had no effect. When monitored with fast scan cyclic voltammetry, ethanol increased the frequency of dopamine transients in 6 of 16 recording sites, in contrast to the uniform effect of ethanol as measured with microdialysis. In the remaining 10 recording sites that were unresponsive to ethanol, dopamine transients either decreased in frequency or were unaffected by cumulative ethanol infusions, patterns also observed during repeated saline infusions. The responsiveness of particular recording sites to ethanol was not correlated with either core versus shell placement of the electrodes or the basal rate of dopamine transients. Importantly, the phasic response pattern to a single dose of ethanol at a particular site was qualitatively reproduced when a second dose of ethanol was administered, suggesting that the variable between-site effects reflected specific pharmacology at that recording site.These data demonstrate that the relatively uniform dopamine concentrations obtained with microdialysis can mask a dramatic heterogeneity of phasic dopamine release within the accumbens.

Dopamine increases in the nucleus accumbens after ethanol administration in rats, but the contributions of the core and shell subregions to this response are unclear. The goal of this study was to determine the effect of various doses of intravenous (i.v.) ethanol infusions on dopamine in these two subregions of the nucleus accumbens. Male Long-Evans rats were infused with either acute i.v. ethanol (0.5, 1.0, 1.5 g/kg), repeated i.v. ethanol (four 1.0 g/kg infusions resulting in a cumulative dose of 4.0 g/kg), or saline as a control for each condition. Dopamine and ethanol were measured in dialysate samples from each experiment. The in vivo extraction fraction for ethanol of probes was determined using i.v. 4-methylpyrazole, and was used to estimate peak brain ethanol concentrations after the infusions. The peak brain ethanol concentrations after the 0.5, 1.0, and 1.5 g/kg ethanol infusions were estimated to be 20, 49, and 57 mM, respectively. A significant dopamine increase was observed for the 0.5 g/kg ethanol group when collapsed across subregions. However, both the 1.0 g/kg and 1.5 g/kg ethanol infusions produced significant increases in dopamine levels in the shell that were significantly higher than those in the core. An ethanol dose-response effect on dopamine in the shell was observed when saline controls, 0.5, 1.0, and 1.5 g/kg groups were compared. For the cumulative-dosing study, the first, second, and fourth infusions resulted in significant increases in dopamine in the shell. However, these responses were not significantly different from one another. The results of this study show that the shell has a stronger response than the core to intravenous ethanol, and that dopamine in the shell increases in a dose-dependent manner between 0.5 -1.0 g/kg doses, but that the response to higher ethanol doses reaches a plateau.

The initiation phase of ethanol self-administration is difficult to study using the well-established, sucrose-fading procedure due to the changing concentrations of ethanol in the first few days. The purpose of this experiment was to test whether a modified sucrose-substitution procedure in which rats are initially exposed to high concentrations of ethanol and sucrose for three days would successfully initiate ethanol self-administration. Male Long-Evans rats were trained to lever-press with a 10% sucrose solution in which 4 or 20 responses allowed 20-min access to the solution. Subsequently, rats were exposed to a three-day period of operant self-administration of 10% sucrose + 10% ethanol. This constant-concentration exposure was followed by the standard procedure in which sucrose is completely faded out. The establishment of ethanol self-administration was determined by ethanol intake, pre- and post-procedure 2-bottle choice preference tests, and extinction trials. The mean ethanol intake was 2.2 times higher on day 2 compared with day 1 on the 10% sucrose + 10% ethanol solution. After fading out the sucrose, the daily intake of 10% ethanol solution over 5 days was stable at approximately 0.57 g/kg. Ethanol preference was approximately 3-fold higher after the modified sucrose-fading procedure. Responding during a single session extinction test was dramatically increased from 4 to 61 ± 13 or 20 to 112 ± 22 responses in 20 min. Similar to the standard sucrose-fading method, we did not observe a significant relationship between extinction responding and ethanol intake. Blood alcohol concentrations were 4.5 mM 20 min after consumption began. We conclude that initiation and establishment of ethanol self-administration will occur using this modified sucrose-fading procedure.

Dopamine plays a critical role in striatal and cortical function, and depletion of the dopamine precursors phenylalanine and tyrosine is used in humans to temporarily reduce dopamine and probe the role of dopamine in behavior. This method has been shown to alter addiction-related behaviors and cognitive functioning presumably by reducing dopamine transmission, but it is unclear what specific aspects of dopamine transmission are altered.We performed this study to confirm that administration of an amino acid mixture omitting phenylalanine and tyrosine (Phe/Tyr[-]) reduces tyrosine tissue content in the prefrontal cortex (PFC) and nucleus accumbens (NAc), and to test the hypothesis that Phe/Tyr[-] administration reduces phasic dopamine release in the NAc.Rats were injected with a Phe/Tyr[-] amino acid mixture, a control amino acid mixture, or saline. High-performance liquid chromatography was used to determine the concentration of tyrosine, dopamine, or norepinephrine in tissue punches from the PFC and ventral striatum. In a separate group of rats, phasic dopamine release was measured with fast-scan cyclic voltammetry in the NAc core after injection with either the Phe/Tyr[-] mixture or the control amino acid solution.Phe/Tyr[-] reduced tyrosine content in the PFC and NAc, but dopamine and norepinephrine tissue content were not reduced. Moreover, Phe/Tyr[-] decreased the frequency of dopamine transients, but not their amplitude, in freely moving rats.These results indicate that depletion of tyrosine via Phe/Tyr[-] decreases phasic dopamine transmission, providing insight into the mechanism by which this method modifies dopamine-dependent behaviors in human imaging studies.

Background: The C57BL/6 mouse model has been used extensively in alcohol drinking studies, yet significant differences in ethanol preference between substrains exist. Differences in ethanol-induced dopamine release in the ventral striatum could contribute to this variability in drinking behavior as dopamine has been implicated in the reinforcing properties of ethanol. Methods: A 2-bottle choice experiment investigated the difference in ethanol preference between C57BL/6J and C57BL/6NCrl animals. Microdialysis was used to determine dopamine release and ethanol clearance in these 2 substrains after intraperitoneal injections of 1.0, 2.0 and 3.0 g/kg ethanol or saline. Results: C57BL/6J mice exhibited significantly greater ethanol preference and less ethanol-stimulated dopamine release compared with C57BL/6NCrl mice. The intraperitoneal injections of ethanol caused a significant increase in dopamine in both substrains at all 3 doses with significant differences between substrains at the 2 highest alcohol doses. Saline injections had a significant effect on dopamine release when given in a volume equivalent to the 3 g/kg ethanol dose. Ethanol pharmacokinetics were similar in the 2 substrains at all 3 doses. Conclusions: Ethanol-induced dopamine release in the ventral striatum may contribute to the differences in alcohol preference between C57BL/6J and C57BL/6NCrl mice.

Background The objective of this study was to characterize the acute pharmacological effects of ethanol (EtOH) on extracellular dopamine in the dorsomedial and dorsolateral striata. This is the first study to quantify and directly compare the effects of acute EtOH on dopamine in these subregions. Therefore, we also tested the nucleus accumbens as a positive control. We hypothesized that while EtOH may increase extracellular dopamine in the dorsomedial striatum and dorsolateral striatum, the magnitude of this increase and the temporal profiles of extracellular dopamine concentrations would differ among the dorsomedial striatum, dorsolateral striatum, and nucleus accumbens. Methods We performed in vivo microdialysis in adult, male Long Evans rats as they received a single (experiment 1) or repeated (experiment 2) doses of EtOH. Results The results of our positive control study validate earlier work by our laboratory demonstrating that acute intravenous EtOH immediately and robustly increases extracellular dopamine in the nucleus accumbens (Howard et al., 2008). In contrast, a single 1-g/kg dose of intravenous EtOH did not significantly affect extracellular dopamine in the dorsomedial striatum or the dorsolateral striatum. However, following a cumulative EtOH dosing protocol, we observed a ramping up of tonic dopamine activity in both the dorsomedial striatum and dorsolateral striatum over the course of the experiment, but this effect was more robust in the dorsomedial striatum. Conclusions These results suggest that distinct mechanisms underlie the stimulating effects of acute EtOH on extracellular dopamine in striatal subregions. Additionally, our findings suggest a role for the dorsomedial striatum and minimal-to-no role for the dorsolateral striatum in mediating the intoxicating effects of acute moderate to high doses of EtOH.

The mechanisms underlying ethanol self-administration are not fully understood; however, it is clear that ethanol self-administration stimulates nucleus accumbens dopamine release in well-trained animals. During operant sweetened ethanol self-administration behavior, an adaptation in the nucleus accumbens dopamine system occurs between the first and second exposure, paralleling a dramatic increase in sweetened ethanol intake, which suggests a single exposure to sweetened ethanol may be sufficient to learn the association between sweetened ethanol cues and its reinforcing properties. In the present experiment, we test the effects of blockade of nucleus accumbens dopamine D1 receptors on operant sweetened ethanol self-administration behavior during the first 2 days of exposure. Adult male Long-Evans rats were first trained to self-administer 10% sucrose (10S) across 6 days in an appetitive and consummatory operant model (appetitive interval: 10-min pre-drinking wait period and a lever response requirement of 4; consummatory interval: 20-min access to the drinking solution). After training on 10S, the drinking solution was switched to 10% sucrose plus 10% ethanol (10S10E); control rats continued drinking 10S throughout the experiment. Bilateral nucleus accumbens microinjections of the dopamine D1 antagonist, SCH-23390 (0, 1.0, or 3.0 μg/side), immediately preceded the first two sessions of drinking 10S10E. Results show that blocking nucleus accumbens dopamine D1 receptors has little or no influence on consumption during the first 2 days of exposure to the sweetened ethanol solution or maintenance of sucrose-only drinking. Furthermore, the high dose of SCH-23390, 3.0 μg/side, reduced open-field locomotor activity. In conclusion, we found no evidence to suggest that nucleus accumbens D1 receptor activation is involved in consumption of a sweetened ethanol solution during the first 2 days of exposure or maintenance of sucrose drinking, but rather D1 receptors seem necessary for general locomotor activity that contributes to initiation of appetitive behavior.

Repeated exposure to drugs of abuse results in an increased sensitivity to their behavioral effects, a phenomena referred to as behavioral sensitization. It has been suggested that the same neuroadaptations underlying behavioral sensitization contribute to the maintenance and reinstatement of addiction. Dysregulation of dopamine (DA) neurotransmission in the mesoaccumbens system is one neuroadaptation that is thought to lead to the compulsive drug-seeking that characterizes addiction. Evidence that sensitization to psychostimulants and opiates is associated with an enhancement of drug-evoked DA levels in the nucleus accumbens has also been obtained. Like other drugs of abuse, the acute administration of ethanol (ETOH) stimulates DA release in this brain region. Moreover, repeated ETOH experience results in an enhanced behavioral response to a subsequent ethanol challenge. Data regarding the influence of repeated ethanol intoxication and withdrawal upon mesoaccumbal DA neurotransmission is limited. Studies examining ETOH-evoked alterations in mesoaccumbal DA neurotransmission as a function of withdrawal duration are lacking. The present experiments quantified basal and ethanol-evoked DA levels 14 days and 24 h following the cessation of a repeated ETOH intoxication protocol, which results in sensitization to the locomotor activating effects of ethanol. Locomotor activity was assessed in parallel groups of animals. Studies were conducted in two mouse strains, C57BL/6J and DBA/2J, which differ in their behavioral responses to ETOH. The results indicate the development of transient tolerance to both ETOH-induced behavioral activation and evoked accumbens DA release at early withdrawal. Moreover, no enhanced DA response to a subsequent ETOH challenge could be demonstrated in ETOH experienced animals 2 weeks after withdrawal, in spite of the observation of clear behavioral sensitization at this time point. These results suggest that, at least in the case of ethanol, sensitization of the DA mesolimbic system may not be necessary for the development of behavioral sensitization.

Although operant ethanol self-administration can increase accumbal dopamine activity, the relationship between dopamine and ethanol levels during consumption remains unclear. We trained Long-Evans rats to self-administer escalating concentrations of ethanol (with 10% sucrose) over 7 days, during which two to four lever presses resulted in 20 min of access to the solution with no further response requirements. Accumbal microdialysis was performed in rats self-administering 10% ethanol (plus 10% sucrose) or 10% sucrose alone. Most ethanol (1.6 +/- 0.2 g/kg) and sucrose intake occurred during the first 10 min of access. Sucrose ingestion did not induce significant changes in dopamine concentrations. Dopamine levels increased within the first 5 min of ethanol availability followed by a return to baseline, whereas brain ethanol levels reached peak concentration more than 40 min later. We found significant correlations between intake and dopamine concentration during the initial 10 min of consumption. Furthermore, ethanol-conditioned rats consuming 10% sucrose showed no effect of ethanol expectation on dopamine activity. The transient rise in dopamine during ethanol ingestion suggests that the dopamine response was not solely due to the pharmacological properties of ethanol. The dopamine response may be related to the stimulus properties of ethanol presentation, which were strongest during consumption.

The goal of the current study was to determine the effect of operant self-administration of (1) 10% sucrose and (2) a first-time solution of 10% sucrose with 5% or 10% ethanol, on dopamine concentration in the nucleus accumbens. We used an operant procedure that distinguished lever pressing (an appetitive behavior) from drinking to better assess the effect of fluid consumption on accumbal dopamine activity. Male Long-Evans rats were trained to bar press by using 10% sucrose reinforcement, and they were required to emit an escalating number of bar presses across daily sessions. Completion of the response requirement resulted in 20 min of access to the solution. Microdialysis samples were collected before, during, and after bar pressing and drinking, and content of ethanol and dopamine was determined. Dopamine concentration in the dialysate was slightly, but significantly, increased in both groups during lever pressing. However, after consumption began, dopamine concentration increased in the sucrose, but not in the sucrose with ethanol, group, followed by a return to baseline values. Ethanol consumption was low (0.27 +/- 0.02 g/kg) and corresponded to low dialysate ethanol concentrations, which appeared within 5 min of drinking. These results demonstrate that operant self-administration of sucrose increases accumbal dopamine concentration during consummatory phases of behavior, but that a similar increase is not apparent when a novel, perhaps aversive, solution (sucrose with ethanol) is presented. This difference may be due to the sensory-related stimulus properties of each solution. In addition, oral self-administration of ethanol at 0.27 +/- 0.02 g/kg over 20 min is not sufficient for stimulation of dopamine activity in the nucleus accumbens.

The neurobiological processes by which ethanol seeking and consumption are established and maintained are thought to involve areas of the brain that mediate motivated behavior, such as the mesolimbic dopamine system. The mesolimbic dopamine system is comprised of cells that originate in the ventral tegmental area (VTA) and project to several forebrain regions, including a prominent terminal area, the nucleus accumbens (NAcc). The NAcc has been subdivided into core and shell subregions. Both areas receive converging excitatory input from the cortex and amygdala and dopamine input from the VTA, with the accumbal medium spiny neuron situated to integrate the signals. Although forced ethanol administration enhances dopamine activity in the NAcc, conclusions regarding the role of mesolimbic dopamine in ethanol reinforcement cannot be made from these experiments. Behavioral experiments consistently show that pharmacological manipulations of the dopamine transmission in the NAcc alter responding for ethanol, although ethanol reinforcement is maintained after lesions of the accumbal dopamine system. Additionally, extracellular dopamine increases in the NAcc during operant self-administration of ethanol, which is consistent with a role of dopamine in ethanol reinforcement. Behavioral studies that distinguish appetitive responding from ethanol consumption show that dopamine is important in ethanol-seeking behavior, whereas neurochemical studies suggest that accumbal dopamine is also important during ethanol consumption before pharmacological effects occur. Cellular studies suggest that ethanol alters synaptic plasticity in the mesolimbic system, possibly through dopaminergic mechanisms, and this may underlie the development of ethanol reinforcement. Thus, anatomical, pharmacological, neurochemical, cellular, and behavioral studies are more clearly defining the role of mesolimbic dopamine in ethanol reinforcement.

Background: This present study was designed to clarify the role of dopamine in the nucleus accumbens during operant ethanol self-administration by separating bar pressing (ethanol seeking) from ethanol consumption. Furthermore, we sought to define the relationship between ethanol in the brain and the accumbal dopamine response after oral self-administration of ethanol. Methods: Two separate groups of male Long-Evans rats were trained to bar press with 10% ethanol or water. Rats were trained to elicit an escalating number of bar presses across daily sessions before gaining access to the drinking solution for 20 min. Microdialysis was performed before (during a waiting period), during, and after bar pressing and drinking. A handling control group was included, but did not receive training. Results: A significant increase in dopamine occurred during placement of the rats into the operant chamber in trained rats and handling controls. The lever-pressing period did not produce an increase in dialysate dopamine. Accumbal dopamine was increased in the first 5 min of ethanol, but not water, consumption. Ethanol appeared in the dialysate sample following ethanol availability, and peak concentrations were reached at 10 min. Most of the ethanol and water consumption occurred within 5 min of fluid access. The probes were distributed in the core (32%), shell (32%), and core plus shell (36%) regions of the nucleus accumbens. Conclusions: The enhancement of dopamine during transfer into the operant chamber does not depend on anticipation or operant training with ethanol or water reinforcement. Furthermore, the difference between the time course of accumbal dopamine and ethanol in dialysates suggests that the dopamine response is not solely due to pharmacological effects of ethanol. The dopamine response may be associated with the stimulus properties of ethanol presentation, which would be strongest during consumption.

Two quantitative methods, the Lönnroth (no-net-flux) and variation of perfusion flow rate methods, were used to investigate the influence of the probe and tissue on dopamine microdialysis measurements. In vivo measurements were made in the nucleus accumbens of awake, freely moving rats on two consecutive days of dialysis. The results of the no-net-flux study showed that there was no statistically significant difference in extraction fraction at a perfusion flow rate of 2.0 microl/min between in vitro in a well-stirred solution and in vivo measured during 2 days of continuous dialysis. Also, varying the perfusate flow rate over the range 0.25-2.0 microl/min produced a variation in the extraction fraction that was the same in vitro and in vivo. These results indicate that the extraction fraction for dopamine over the 2 days was dominated by the properties of the probe. The negligible influence of the tissue on dopamine extraction fraction was probably due to the high basal activity of the dopamine transporter in vivo. Therefore, the extraction fraction is unlikely to be sensitive to increases in dopamine uptake in the vicinity of the probe. The apparent extracellular dopamine concentration increased by 37% on the second day of dialysis while the calcium-dependence of basal dialysate dopamine levels declined by 20%. These findings are consistent with a decrease in physiological viability of the dopamine nerve terminals surrounding the probe during a long-term experiment.

The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.

Background: Aspects of ethanol pharmacokinetics in the blood, such as elimination, are known to vary by gender in the rat. Ethanol concentrations reaching the brain, which are relevant in affecting behavior, have not been measured rigorously by gender. This study used quantitative microdialysis to measure ethanol pharmacokinetics in the nucleus accumbens after a moderate dose of ethanol, comparing males with females and females across the estrous cycle. Methods: We administered 1 g/kg ethanol intravenously or intragastrically to male and female rats. We measured ethanol concentrations in the nucleus accumbens by use of microdialysis with in vivo probe calibration and compared them with concentrations in jugular venous blood. Results: After intravenous delivery, apparent ethanol elimination was approximately 15% faster in females than males from both brain and blood. After intragastric delivery, blood ethanol concentrations peaked faster in females than males by approximately 20 min, suggesting faster absorption or diffusion from the stomach. Likewise, accumbal ethanol concentrations peaked faster in females than males by approximately 25 min, indicating faster distribution to the brain. These pharmacokinetic parameters did not vary across the estrous cycle, nor were they influenced significantly by body water. Conclusions: This study provides the first quantitative measurement of ethanol concentrations and, thus, pharmacokinetics in the brain of awake rats. If ethanol distribution to the brain varies between genders, as these results suggest, then differences in the pharmacological response to ethanol can be expected, providing a rationale for studying the pharmacodynamics of ethanol in the brain of the self-administering rat.

Background Little is known regarding mechanisms regulating ethanol (EtOH) self-administration during adolescence or if the mechanisms differ from adults. One of the best models of abuse liability is operant self-administration. Therefore, we characterized operant sweetened EtOH self-administration behavior in adolescent and adult rats. Methods Adolescent (36 days old at first EtOH exposure) and adult male Long-Evans rats were first trained to self-administer 10% sucrose (10S) in an appetitive/consummatory operant model for 1 week, and then the drinking solution was switched to 10% sucrose plus 10% EtOH (10S10E) for 2 weeks. Next, rats were switched to a fixed ratio 2 schedule, and this was followed by 1 session using a progressive ratio schedule of reinforcement. Last, rats were tested for cue-induced reinstatement of lever-pressing behavior under extinction conditions after 13 days of abstinence. Blood EtOH concentration (BEC) of sweetened EtOH (self-administered or intragastric [IG] administration of 1 g/kg) was determined via gas chromatography. Control rats drank only 10S. Results Consumption of sweetened EtOH was not different between adolescents and adults under any schedule tested, reaching 1 g/kg in 20 minutes in the appetitive/consummatory model. Appetitive behavior directed at sweetened EtOH was less focused in adolescents versus adults. No age differences were found in motivation for sweetened EtOH. Cue-induced reinstatement of EtOH-seeking behavior after abstinence also did not differ by age. In control groups, no age difference was found in appetitive behavior or the amount of sucrose consumed, although adults exhibited greater cue-induced reinstatement. BEC after self-administration or IG administration of sweetened EtOH was higher in adults than adolescents. Conclusions Consumption and motivation for sweetened EtOH are similar in adolescents and adults, although adolescents are more vulnerable to the effects of EtOH consumption on appetitive behavior. The IG results suggest larger volume of distribution and higher first-pass metabolism of sweetened EtOH in adolescents versus adults, which may limit the reinforcing effects of EtOH in some adolescents. Overall, we have begun to establish an operant sweetened EtOH self-administration model in adolescent rats.

Amphetamine increases extracellular dopamine and induces locomotor and stereotypical behaviors in rats. This study examined the effect of the dopamine D2/D3 receptor antagonist sulpiride (50 mg/kg s.c.) on the dopaminergic response to amphetamine (0.5, 2.0, or 8.0 mg/kg i.p.) in male Sprague-Dawley rats. Extracellular dopamine in the striatum was monitored using in vivo microdialysis and high performance liquid chromatography with electrochemical detection. Dopamine concentration curves were analyzed using non-linear regression and residual F-testing. Amphetamine enhanced extracellular dopamine in a dose-dependent manner. Sulpiride augmented the increase in dopamine induced by 0.5 and 2 mg/kg amphetamine by decreasing the rate of dopamine concentration fall off in the extracellular space (P

Background: Dopamine release in the nucleus accumbens has been linked to the reinforcing effects of ethanol, but the time course or relationship of this response to ethanol concentrations in the brain has not been studied. Methods: Various doses of ethanol (0-2.0 g/kg) were administered intraperitoneally to male Sprague Dawley® rats, and dopamine and ethanol were simultaneously analyzed in dialysate samples from the nucleus accumbens. A separate study to compare the ethanol-induced dopamine response in male and female rats was carried out by using a 1 g/kg intraperitoneal dose of ethanol. Results: In male rats, 1 and 2 g/kg ethanol significantly increased dialysate dopamine by 40% over basal, whereas 0.25 and 0.5 g/kg ethanol produced a nonsignificant 20% increase. Dialysate ethanol concentrations exhibited a curvilinear decline after reaching peak levels for the lower doses but showed a linear decrease after 1 and 2 g/kg. There was a dissociation between the time courses of extracellular dopamine and ethanol after 1 and 2 g/kg ethanol treatment. The dopamine response returned to basal within 90 min, whereas the ethanol concentrations remained elevated. In a separate study that compared male and female rats, the ratio of the dopamine response over basal to the dialysate ethanol concentrations was significantly decreased at 60 min after an injection of I g/kg. However, there were no differences between males and females. Conclusions: The dissociation between dopamine and ethanol levels may reflect the development of acute tolerance to ethanol-induced dopamine release in the nucleus accumbens within the time course of a single acute injection. Given the strong links between dopamine and ethanol reinforcement, our findings may be relevant for understanding the time course of ethanol's reinforcing effects in vivo.

Female rodents tend to drink more alcohol than males, a difference that emerges at puberty and appears to vary over the female estrous cycle. In addition, male and female rodents display different responses to alcohol; for example, female rats are reported to have faster elimination rates than males. We were interested in whether circulating ovarian hormones influence alcohol distribution to or elimination from the brain of rats, which might explain observed differences in drinking behavior. We administered 0.8 g/kg of ethanol via intraperitoneal injection to age-matched male and female Sprague-Dawley rats. Extracellular brain ethanol was sampled using microdialysis, and vascular ethanol concentrations were determined via tail blood collection, in two separate experiments. Ethanol pharmacokinetic parameters were calculated for both compartments. There were no differences in pharmacokinetic parameters due to gender or estrous cycle stage in brain ethanol concentration profiles. There were, however, differences in blood ethanol profiles: females showed faster elimination rates and a smaller area under the ethanol concentration versus time curve than males. In addition, the maximum concentration varied significantly across the estrous cycle. These results suggest that (1) circulating ovarian hormones do not influence alcohol distribution to the brain, but do influence distribution to more peripheral tissues such as the tail; and (2) apparent differences in tail blood alcohol levels may not reflect differences in brain levels.

The opiate antagonist naltrexone suppresses ethanol-reinforced behavior in animals and decreases ethanol intake in humans. However, the mechanisms underlying these actions are not well understood. Experiments were designed to test the hypothesis that naltrexone attenuates the rewarding properties of ethanol by interfering with ethanol-induced stimulation of dopamine activity in the nucleus accumbens (NAcc). Simultaneous measures of the effects of naltrexone on dialysate dopamine levels in the NAcc and on operant responding for oral ethanol were used. Male Wistar rats were trained to self-administer ethanol (10–15%, w/v) in 0.2% (w/v) saccharin during daily 30 min sessions and were surgically prepared for intracranial microdialysis. Experiments began after reliable self-administration was established. Rats were injected with naltrexone (0.25 mg/kg, s.c.) or saline and 10 min later were placed inside the operant chamber for a 20 min waiting period with no ethanol available, followed by 30 min of access to ethanol. A transient rise in dialysate dopamine levels was observed during the waiting period, and this effect was not altered by naltrexone. Ethanol self-administration reliably increased dopamine levels in controls. Naltrexone significantly suppressed ethanol self-administration and prevented ethanol-induced increases in dialysate dopamine levels. Subsequent dose–effect analyses established that the latter effect was not merely a function of reduced ethanol intake but that naltrexone attenuated the efficacy of ethanol to elevate dialysate dopamine levels. These results suggest that suppression of ethanol self-administration by opiate antagonists is the result of interference with dopamine-dependent aspects of ethanol reinforcement, although possible additional effects via nondopaminergic mechanisms cannot be eliminated as a factor in opiate antagonist-induced reduction of ethanol intake.

To determine the site of action of systemic ethanol on dopaminergic function in the nucleus accumbens, we compared the effect of intraperitoneal (ip) and local administration of ethanol on interstitial dopamine concentration using microdialysis in freely moving rats. The ip administration of 1 g/kg of ethanol significantly increased the dialysate dopamine (DA) concentrations (∼40% above basal), compared with saline treatment. The concentration-time profile of DA and ethanol in dialysates was similar after two ethanol injections 4 hr apart. Local perfusion with several ethanol concentrations showed that 510 and 860 mM of ethanol caused a significant concentration-related increase in extracellular DA concentrations in the nucleus accumbens (510 mM, 28% increase; 860 mM, 62% increase). However, lower ethanol concentrations, 170 mM or below, failed to change basal DA concentrations. Stimulation with high potassium (50 mM) in artificial cerebrospinal fluid preceding local ethanol treatment increased dialysate DA concentrations to 523 ± 83% of basal levels, confirming that the DA terminals were responsive to pharmacological manipulation. Basal DA levels in dialysates were ∼70% calcium-dependent when tested at the end of the local perfusion experiments. Stereological examination of the nucleus accumbens revealed probe-induced damage, but did not detect additional damage by local perfusion of ethanol. When ethanol concentrations in the DA sampling area around the probe are taken into account in both systemic and local administration experiments, this study suggests that concentrations of ethanol associated with moderate intoxication do not directly affect the function of DA terminals in the nucleus accumbens. Therefore, the systemic effects of ethanol on nucleus accumbens DAergic function is more likely due to an interaction with sites other than the nucleus accumbens.