Rodent ultrasonic vocalizations as biomarkers of future alcohol use: A predictive analytic approach.

Excessive alcohol consumption has a vast, negative impact on society. Rodent models have been successful in furthering our understanding of the biological underpinnings that drive alcohol consumption. Rodents emit ultrasonic vocalizations (USVs) that are each composed of several acoustic characteristics (e.g., frequency, duration, bandwidth, power). USVs reflect neurotransmitter activity in the ascending limb of the mesolimbic dopaminergic and cholinergic neurotransmitter systems and serve as noninvasive, real-time biomarkers of dopaminergic and cholinergic neurotransmission in the limbic system. In the present study, we recorded spontaneously emitted USVs from alcohol-naïve Long-Evans (LE) rats and then measured their alcohol intake. We compared the USV acoustic characteristics and alcohol consumption data from these LE rats with previously published data from selectively bred high-alcohol (P and HAD-1) and low-alcohol (NP and LAD-1) drinking lines from studies with the same experimental method. Predictive analytic techniques were applied simultaneously to this combined data set and revealed that (a) USVs emitted by alcohol-naïve rats accurately discriminated among high-alcohol consuming, LE, and low-alcohol consuming rat lines, and (b) future alcohol consumption in these same rat lines was reliably predicted from the USV data collected in an alcohol-naïve state. To our knowledge, this is the first study to show that alcohol consumption is predicted directly from USV profiles of alcohol-naïve rats. Because USV acoustic characteristics are sensitive to underlying neural activity, these findings suggest that baseline differences in mesolimbic cholinergic and dopaminergic tone could determine the propensity for future alcohol consumption in rodents.

Negative Affect-Associated USV Acoustic Characteristics Predict Future Excessive Alcohol Drinking and Alcohol Avoidance in Male P and NP Rats.

BACKGROUND: Negative emotional status and adverse emotional events increase vulnerability to alcohol abuse. Ultrasonic vocalizations (USVs) emitted by rats are a well-established model of emotional status that can reflect positive or negative affective responses in real time. Most USV studies assess counts, yet each USV is a multidimensional data point characterized by several acoustic characteristics that may provide insight into the neurocircuitry underlying emotional response. METHODS: USVs emitted from selectively bred alcohol-naïve and alcohol-experienced alcohol-preferring and nonpreferring rats (P and NP rats) were recorded during 4-hour sessions on alternating days over 4 weeks. Linear mixed modeling (LMM) and linear discriminant analysis (LDA) were applied to USV acoustic characteristics (e.g., frequency, duration, power, and bandwidth) of negative affect (22 to 28 kilohertz [kHz])- and positive (50 to 55 kHz) affect-related USVs. RESULTS: Hundred percent separation between alcohol-naïve P and NP rats was achieved through a linear combination (produced by LDA) of USV acoustic characteristics of 22- to 28-kHz USVs, whereas poor separation (36.5%) was observed for 50- to 55-kHz USVs. 22- to 28-kHz LDA separation was high (87%) between alcohol-experienced P and NP rats, but was poor for 50- to 55-kHz USVs (57.3%). USV mean frequency and duration were the highest weighted characteristics in both the naïve and experienced 22- to 28-kHz LDA representations suggesting that alcohol experience does not alter the representations. LMM analyses of 22- to 28-kHz USV acoustic characteristics matched the LDA results. Poor LDA separation was observed between alcohol-naïve and alcohol-experienced P rats for both 22- to 28-kHz and 50- to 55-kHz USVs. CONCLUSIONS: Advanced statistical analysis of negative affect-associated USV data predicts future behaviors of excessive alcohol drinking and alcohol avoidance in selectively bred rats. USV characteristics across rat lines reveal affect-related motivation to consume alcohol and may predict neural pathways mediating emotional response. Further characterization of these differences could delineate particular neurocircuitry and methods to ameliorate dysregulated emotional states often observed in human alcohol abusers.

Alcohol-preferring P rats emit spontaneous 22-28 kHz ultrasonic vocalizations that are altered by acute and chronic alcohol experience.

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-naïve 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.

Acoustilytix™: A Web-Based Automated Ultrasonic Vocalization Scoring Platform.

Ultrasonic vocalizations (USVs) are known to reflect emotional processing, brain neurochemistry, and brain function. Collecting and processing USV data is manual, time-intensive, and costly, creating a significant bottleneck by limiting researchers' ability to employ fully effective and nuanced experimental designs and serving as a barrier to entry for other researchers. In this report, we provide a snapshot of the current development and testing of Acoustilytix™, a web-based automated USV scoring tool. Acoustilytix implements machine learning methodology in the USV detection and classification process and is recording-environment-agnostic. We summarize the user features identified as desirable by USV researchers and how these were implemented. These include the ability to easily upload USV files, output a list of detected USVs with associated parameters in csv format, and the ability to manually verify or modify an automatically detected call. With no user intervention or tuning, Acoustilytix achieves 93% sensitivity (a measure of how accurately Acoustilytix detects true calls) and 73% precision (a measure of how accurately Acoustilytix avoids false positives) in call detection across four unique recording environments and was superior to the popular DeepSqueak algorithm (sensitivity = 88%; precision = 41%). Future work will include integration and implementation of machine-learning-based call type classification prediction that will recommend a call type to the user for each detected call. Call classification accuracy is currently in the 71-79% accuracy range, which will continue to improve as more USV files are scored by expert scorers, providing more training data for the classification model. We also describe a recently developed feature of Acoustilytix that offers a fast and effective way to train hand-scorers using automated learning principles without the need for an expert hand-scorer to be present and is built upon a foundation of learning science. The key is that trainees are given practice classifying hundreds of calls with immediate corrective feedback based on an expert's USV classification. We showed that this approach is highly effective with inter-rater reliability (i.e., kappa statistics) between trainees and the expert ranging from 0.30-0.75 (average = 0.55) after only 1000-2000 calls of training. We conclude with a brief discussion of future improvements to the Acoustilytix platform.

Spontaneous Ultrasonic Vocalization Transmission in Adult, Male Long-Evans Rats Is Age-Dependent and Sensitive to EtOH Modulation.

Ultrasonic vocalizations (USVs) are well-established markers of motivational and emotional status. Recent work from our lab has provided novel evidence for a role of USVs in models of ethanol (EtOH) use. For instance, USV acoustic characteristics can be used to accurately discriminate between rats selectively bred for high EtOH intake (e.g., alcohol-preferring (P) and high-alcohol-drinking (HAD)) versus EtOH-avoiding (e.g., alcohol-non-preferring (NP) and low-alcohol-drinking (LAD)) strains, as well as differentiate between male and female rats. In the present study we sought to explore the effect of age and alcohol availability on spontaneously emitted 50-55 kHz frequency modulated (FM) and 22-28 kHz USVs in adult, male Long-Evans rats. With the hypothesis that age and alcohol experience influence spontaneous USV emissions, we examined USV data collected across a 24-week intermittent EtOH access experiment in male Long-Evans rats. USV counts and acoustic characteristic (i.e., mean frequency, duration, bandwidth and power) data revealed distinct age-dependent phenotypes in both 50-55 kHz FM and 22-28 kHz USV transmission patterns that were modulated by EtOH exposure. These results highlight the influence of age and EtOH experience on the unique emotional phenotypes of male Long-Evans rats.

The recreational drug ecstasy disrupts the hypothalamic-pituitary-gonadal reproductive axis in adult male rats.

Reproductive function involves an interaction of three regulatory levels: hypothalamus, pituitary, and gonad. The primary drive upon this system comes from hypothalamic gonadotropin-releasing hormone (GnRH) neurosecretory cells, which receive afferent inputs from other neurotransmitter systems in the central nervous system to result in the proper coordination of reproduction and the environment. Here, we hypothesized that the recreational drug (+/-)-3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy'), which acts through several of the neurotransmitter systems that affect GnRH neurons, suppresses the hypothalamic-pituitary-gonadal reproductive axis of male rats. Adult male Sprague-Dawley rats self-administered saline or MDMA either once (acute) or for 20 days (chronic) and were euthanized 7 days following the last administration. We quantified hypothalamic GnRH mRNA, serum luteinizing hormone concentrations, and serum testosterone levels as indices of hypothalamic, pituitary, and gonadal functions, respectively. The results indicate that the hypothalamic and gonadal levels of the hypothalamic-pituitary-gonadal axis are significantly altered by MDMA, with GnRH mRNA and serum testosterone levels suppressed in rats administered MDMA compared to saline. Furthermore, our finding that hypothalamic GnRH mRNA levels are suppressed in the context of low testosterone concentrations suggests that the central GnRH neurosecretory system may be a primary target of inhibitory regulation by MDMA usage.

Certain or uncertain cocaine expectations influence accumbens dopamine responses to self-administered cocaine and non-rewarded operant behavior.

Uncertainty and errors in predicting natural rewards influence associative learning and dopamine activity. The present study was conducted to determine the influence of cue-induced cocaine uncertainty, certainty and prediction error on nucleus accumbens dopamine (NAcc DA) in rats. For Certainty training, distinctive sensory cues were present during cocaine availability and alternate cues were paired with non-reinforced (saline) operant sessions. For Uncertainty training, all cues were equally associated with both cocaine and non-reinforcement. After training, animals self-administered cocaine or saline in the presence of conditioned cues while NAcc DA responses were assessed using in vivo microdialysis. Findings revealed cocaine-stimulated NAcc DA increased significantly less in Certainty--compared to Uncertainty-trained animals, and cocaine-paired cues in the absence of cocaine (Negative Prediction Error) resulted in a significant depression of baseline NAcc DA. These findings provide support for enhanced DA activity during cocaine uncertainty or the development of conditioned cocaine tolerance in subjects certain of a cocaine outcome.

Diazepam alters cocaine self-administration, but not cocaine-stimulated locomotion or nucleus accumbens dopamine.

Cocaine is known to enhance nucleus accumbens dopamine (NAcc DA), to serve as a positive reinforcer and to produce negative effects, such as anxiety. The influence of diazepam on cocaine intake, cocaine-stimulated behavioral activity and NAcc DA was investigated using self-administration and experimenter-administered intravenous (i.v.) cocaine. In Experiment 1, rats were pretreated with diazepam (0.25 mg/kg) or saline (0.1 ml) 30 min prior to 20 daily 1-hour cocaine (0.75 mg/kg/injection) self-administration sessions. Cocaine intake increased for all animals across sessions, but was highest in diazepam-pretreated animals. Diazepam rats also self-administered their first cocaine injection of each session faster than controls. Experiment 2 utilized in vivo microdialysis to assess NAcc DA levels before and after experimenter-administered i.v. cocaine injections (0.75 mg/kg/injection x 2; 10-min interval) in diazepam- and saline-pretreated rats. Group differences were not revealed across basal and cocaine-stimulated NAcc DA assessments, indicating that diazepam did not decrease NAcc DA during cocaine self-administration. Findings that diazepam enhances cocaine self-administration and decreases cocaine response latency support the notion that cocaine-induced anxiety limits voluntary cocaine intake. It is further suggested that individual variations in cocaine-induced aversive effects may determine whether cocaine use is avoided or repeated.

Effects of the Endocrine-Disrupting Chemicals, Vinclozolin and Polychlorinated Biphenyls, on Physiological and Sociosexual Phenotypes in F2 Generation Sprague-Dawley Rats.

BACKGROUND: Exposure to endocrine-disrupting chemicals (EDCs) during gestation influences development of the F1 generation offspring and can result in disease and dysfunction in adulthood. Limited evidence suggests consequences on the F2 generation, exposed as germ cells within the F1 fetus. These F2s provide a unique window into the programming effects of EDCs. OBJECTIVE: This study assessed intergenerational effects of EDC exposure on adult physiology and behavior in Sprague-Dawley rats. METHODS: Pregnant rats were exposed to either a polychlorinated biphenyl (PCB) mixture, Aroclor 1,221 (A1221), the fungicide vinclozolin (VIN), or the vehicle (VEH) (6% dimethylsulfoxide in sesame oil) alone. A1221 is weakly estrogenic, while VIN is antiandrogenic, enabling us to compare different classes of EDCs. The F1 male and female offspring were bred to generate the paternal- and maternal-lineage F2 generation. This F2 generation was assessed for physiological outcomes, ultrasonic vocalizations (USVs), and sexual behavior in adulthood. RESULTS: Each EDC caused phenotypic effects in a sex- and lineage-dependent manner. The most robustly affected group was the paternal-lineage males. F2 VIN paternal male descendants had increased body weight throughout the lifespan, lower concentrations of circulating estradiol, and lower adrenal and testicular indices. Both VIN and A1221 paternal-lineage males also exhibited the greatest number of changes in the characteristics of USVs in response to an opposite-sex animal and changes in sexual behaviors in a mating test. CONCLUSION: Exposure of rats to EDCs at the germ cell stage led to differences in the physiological and behavioral phenotype later in life, especially in males. This finding has implications for multigenerational physiological and reproductive health in wildlife and humans. https://doi.org/10.1289/EHP3550.

Alcohol-naïve USVs distinguish male HAD-1 from LAD-1 rat strains.

Ultrasonic vocalizations (USVs) are mediated through specific dopaminergic and cholinergic neural pathways and serve as real-time measures of positive and negative emotional status in rodents. Although most USV studies focus primarily on USV counts, each USV possesses a number of characteristics shown to reflect activity in the associated neurotransmitter system. In the present study, we recorded spontaneously emitted USVs from alcohol-naïve high alcohol drinking (HAD-1) and low alcohol drinking (LAD-1) rats. Using our recently developed WAAVES algorithm, we quantified four acoustic characteristics (mean frequency, duration, power, and bandwidth) from each 22-28 kHz and 50-55 kHz frequency-modulated (FM) USV. This rich USV representation allowed us to apply advanced statistical techniques to identify the USV acoustic characteristics that distinguished HAD-1 from LAD-1 rats. Linear mixed models (LMM) examined the predictability of each USV characteristic in isolation and linear discriminant analysis (LDA), and binomial logistic regression examined the predictability of linear combinations of the USV characteristics as a group. Results revealed significant differences in acoustic characteristics between HAD-1 and LAD-1 rats in both 22-28 kHz and 50-55 kHz FM USVs. In other words, these rats selectively bred for high- and low-alcohol consumption can be identified as HAD-1 or LAD-1 rats with high classification accuracy (approximately 92-100%) exclusively based on their emitted 22-28 kHz and 50-55 kHz FM USV acoustic characteristics. In addition, acoustic characteristics of 22-28 kHz and 50-55 kHz FM USVs emitted by alcohol-naïve HAD-1 and LAD-1 rats significantly correlate with their future alcohol consumption. Our current findings provide novel evidence that USV acoustic characteristics can be used to discriminate between alcohol-naïve HAD-1 and LAD-1 rats, and may serve as biomarkers in rodents with a predisposition for, or against, excessive alcohol intake.

Experience-dependent effects of cocaine self-administration/conditioning on prefrontal and accumbens dopamine responses.

Experiments were performed to examine the effects of cocaine self-administration and conditioning experience on operant behavior, locomotor activity, and nucleus accumbens (NAcc) and prefrontal cortex (PFC) dopamine (DA) responses. Sensory cues were paired with alternating cocaine and nonreinforcement during 12 (limited training) or 40 (long-term training) daily operant sessions. After limited training, NAcc DA responses to cocaine were significantly enhanced in the presence of cocaine-associated cues compared with nonreward cues and significantly depressed after cocaine-paired cues accompanied a nonreinforced lever response. PFC DA levels were generally nonresponsive to cues after the same training duration. However, after long-term training, cocaine-associated cues increased the magnitude of cocaine-stimulated PFC DA levels significantly over levels observed with nonreinforcement cues. Conversely, conditioned cues no longer influenced NAcc DA levels after long-term training. In addition, cocaine-stimulated locomotor activity was enhanced by cocaine-paired cues after long-term, but not after limited, training. Findings demonstrate that cue-induced cocaine expectation exerts a significant impact on dopaminergic and behavioral systems, progressing from mesolimbic to mesocortical regions and from latent to patent behaviors as cocaine and associative experiences escalate.

Increased accumbens Cdk5 expression in rats after short-access to self-administered cocaine, but not after long-access sessions.

Upregulation of cyclin-dependent kinase 5 (Cdk5) after chronic cocaine administration has led to speculation that Cdk5 plays an important role in drug addiction. However, as Cdk5 involvement is implicated in a variety of neural events, including neuronal development, synaptic plasticity and learning, a specific role in drug abuse is yet to be determined. The present study utilized cocaine self-administration and food-reinforced operant procedures to assess possible relationships between cocaine intake, food-reinforced operant responding, behavioral activity, and Cdk5 levels in the nucleus accumbens (NAcc), ventral tegmental area (VTA), and prefrontal cortex (PFC) in rats. In Experiment 1, animals undergoing daily cocaine self-administration (1-h/30 days) or food-reinforced operant sessions (20-min/30 days) showed significant between-group differences in operant responding and behavioral activity, but no significant differences in NAcc, VTA or PFC Cdk5 levels compared to a Handled Control group. In Experiment 2, animals that had self-administered cocaine in 10 daily 1-h sessions (Short-Access Cocaine) showed significantly greater NAcc Cdk5 expression compared to an Unhandled Control group, and no evidence of cocaine-induced behavioral sensitization. Animals given 4-h daily access to cocaine over the same number of sessions (Long-Access Cocaine) showed significantly enhanced cocaine-reinforced responding and locomotor activation by the end of the sessions, but no significant differences in Cdk5 expression compared to Control animals. These findings suggest that overexpression of Cdk5 may be a transient adaptation to cocaine experience that subsides with increased cocaine exposure and does not correspond with measures of cocaine-induced behavioral sensitization.

Prefrontal cortex D1 modulation of the reinforcing properties of cocaine.

The involvement of the dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) in the reinforcing properties of many drugs of abuse is well established. Though the prefrontal cortex (PFC) exhibits significant influence over activity in this pathway, its role in drug abuse is less defined. The present experiment investigated the impact of PFC D1 activity on cocaine self-administration (0.25, 0.75 mg/kg/inj) under progressive (PR) and fixed ratio (FR) schedules of reinforcement by assessing immediate and delayed effects of bilateral intra-PFC infusions of a D1 agonist (SKF 38393; 0.23 microg/side) and antagonist (SCH 23390; 0.25 microg/side). Immediately following infusion of dopaminergic agents or vehicle, no significant changes in self-administration occurred under any tested condition. However, 24 h after intra-PFC antagonist treatment, significantly lower PR breakpoints were observed for low (0.25 mg/kg), but not moderate (0.75 mg/kg) unit doses of self-administered cocaine. Locomotor activity levels during these assessments were unaffected by intra-PFC treatments. On an FR-3 schedule of reinforcement, the 0.25 cocaine unit dose elicited higher total cocaine intake and hyperlocomotor activation during a shorter session, but intra-PFC treatment had no significant effects on the number of reinforced responses or behavioral activity. The observation of decreased cocaine breakpoints after intra-PFC DA antagonist treatment reflects decrements in cocaine reinforcement efficacy. This finding corresponds temporally with previous work showing increased NAcc DA levels after similar treatment. Current findings demonstrate that transient changes in PFC DA neurotransmission can specifically influence reinforced behaviors without affecting overall behavioral activation.

Comparing nucleus accumbens and dorsal striatal dopamine responses to self-administered cocaine in naïve rats.

Dopamine (DA) responses in the nucleus accumbens (NAcc) and dorsal striatum (DS) are commonly associated with different aspects of cocaine effects. Enhanced NAcc DA has been most convincingly linked with the positive reinforcing effects of cocaine, while DS DA is thought to mediate cocaine-induced motoric effects. Though several studies have shown NAcc DA enhancement following cocaine self-administration, very little work has examined the effects of cocaine self-administration on DS DA. In this study, DA levels in the NAcc and DS, and locomotor responses to a single self-administered cocaine injection (1.5mg/kg) were assessed in operant-trained, drug-naïve Sprague-Dawley rats. Locomotor activity, NAcc and DS DA levels increased significantly over baseline activity immediately after cocaine injection. However, while basal and cocaine-stimulated NAcc DA concentrations (nM) were significantly greater than DS DA levels, the magnitude of response was statistically comparable between brain regions. These findings indicate that, though both the NAcc and DS are importantly involved in the dopaminergic response to self-administered cocaine in drug-naïve rats, basal DA differences in dialysis data are obscured by statistical conversions to baseline percentages.

Experience-dependent changes in temperature and behavioral activity induced by MDMA.

Hyperthermia and hyperlocomotor activity are commonly reported acute effects of high dose, experimenter-delivered 3,4-methylenedioxymethamphetamine (MDMA). The current investigation was performed to determine short- to long-term physiological and behavioral changes induced by moderate intake MDMA self-administration. In the present study, rats self-administered MDMA (approx. 2.0-7.0 mg/kg/day) across 20 days during daily 2-h operant sessions. Locomotor activity was assessed during MDMA self-administration sessions and core temperatures were recorded before and after each session. Findings of the first several sessions showed core temperatures significantly decreased after MDMA self-administration compared to baseline and to a control group that self-administered saline during operant sessions. As sessions proceeded, the MDMA-induced hypothermic response diminished and core temperatures normalized, then increased during the last few sessions. Also, locomotor activity during MDMA self-administration sessions was initially equivalent to saline level activity, but increased by day 8 to significantly greater levels. Our findings demonstrate experience-dependent changes after voluntary administration of MDMA that are clearly observable in temperature regulation and behavioral activity.

Alcohol enhances unprovoked 22-28 kHz USVs and suppresses USV mean frequency in High Alcohol Drinking (HAD-1) male rats.

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-h 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.

Age-dependent (+)MDMA-mediated neurotoxicity in mice.

In the present study the effects of a neurotoxic regimen of (+)-MDMA (20 mg/kgx4, s.c.) in 4- and 10-week-old C57Bl/6J mice during treatment and 7 days post-treatment were examined. Rectal temperatures monitored between (+)-MDMA injections (30 min post-injection/2 h intervals) revealed hyperthermic responses in both age groups, with the magnitude of the response significantly greater in older mice. Seven days post-treatment, immunoblot analyses of the vesicular monoamine transporter 2 (VMAT2), and tyrosine hydroxylase (TH) revealed significant reductions (-37 and -58%, respectively) in the older animals, but not in the younger group, compared to age-matched controls. Dopamine transporter (DAT) expression was significantly reduced in both 4- and 10-week-old animals (26 and 69.7%, respectively). (+)-MDMA-treated animals also exhibited significantly lower levels of striatal dopamine, and 3,4-dihydroxyphenylacetic acid than controls, again the effect being more pronounced in the older animals. Although both age groups showed evidence of (+)-MDMA-induced toxicity, our data revealed that older animals exhibited a greater hyperthermic response to (+)-MDMA and were also are more susceptible to subsequent dopaminergic damage than the younger animals.

Nucleus accumbens and medial prefrontal cortex dopaminergic response to self-administered cocaine in naive rats.

Cocaine reinforcement is strongly associated with increased nucleus accumbens dopamine (NAcc DA). The involvement of medial prefrontal cortex (mPFC) DA in cocaine reward is less defined, but substantial evidence indicates that increased mPFC DA may suppress NAcc DA levels. Using in vivo microdialysis, NAcc or mPFC DA was determined in cocaine-naive rats after a self-administered cocaine injection (3.0 mg/kg). Extracellular levels of NAcc DA were dramatically enhanced 10 min post-cocaine injection, but dropped significantly at each subsequent assessment. mPFC DA also increased significantly, but to a lesser extent than observed in the NAcc. Findings of prominent DA increases in both the NAcc and mPFC terminals during the test session indicate that NAcc DA responses do not appear to be inhibited by increased mPFC DA during cocaine self-administration.

Chronic D1 agonist and ethanol coadministration facilitate ethanol-mediated behaviors.

Separate lines of evidence suggest that neuroadaptations associated with ethanol (EtOH) reinforcement can be initiated by chronic EtOH preexposure and a signaling pathway activated by dopamine (DA) D1 receptor stimulation. We have previously shown that rewarding and locomotor effects of EtOH alone [Pharmacol. Biochem. Behav. 72 (2002) 787] are enhanced after chronic exposure to self-administered EtOH/cocaine combinations. To determine the importance of chronic EtOH exposure, dopamine D1 receptor activation and mode of drug administration in EtOH reward, animals were given daily intravenous infusions of experimenter-administered saline, EtOH (2.0 g/kg), the DA D1 receptor agonist, SKF81297 (0.2 mg/kg), or EtOH+SKF81297 over a 4-week period. Compared to other groups, animals preexposed to EtOH+SKF81297 self-administered significantly greater amounts of intravenous EtOH and showed greater enhancement and less suppression of locomotor activity in response to a range of intravenous EtOH dosages (0.125, 0.25, 0.5, 1.0 and 1.5 g/kg). Since chronic treatment with EtOH alone did not enhance EtOH-induced reinforcement or locomotor activity, it is unlikely that these effects were due to EtOH tolerance. These findings suggest that chronic D1 receptor activation combined with EtOH administration alters neural responsiveness to EtOH and support the notion that D1 activation is important to EtOH reward.

Intravenous ethanol/cocaine self-administration initiates high intake of intravenous ethanol alone.

Evidence suggests that ethanol (EtOH) preexposure influences the rewarding valence of subsequent EtOH use. This study was conducted to determine if EtOH preexposure through EtOH/cocaine self-administration facilitates the motivational effects of EtOH alone. Rats self-administered intravenous (iv) EtOH/cocaine combinations (EtOH/Cocaine Fading group; EtOH 125.0 mg/kg/inj+Cocaine 0.1-0.75 mg/kg/inj) during a preexposure period. Consequently, these rats self-administered intravenous EtOH alone (62.5, 125.0, 250.0 and 500.0 mg/kg/inj) significantly more than a control group with prior cocaine self-administration experience (0.1-0.75 mg/kg/inj). In addition, at equal EtOH intake levels, locomotor activity was significantly enhanced in the EtOH/Cocaine Fading group but not the Cocaine Control animals (P=.01). The amount of EtOH self-administered in the EtOH/Cocaine Fading group during 1-h sessions (approximately 0.5-2.0 g/kg) corresponded with blood alcohol levels (BAL) ranging from 44 to 221 mg/dl. The highest BALs reported here have not previously been demonstrated after voluntary EtOH intake through any route of administration. These data suggest that preexposure to EtOH during EtOH/cocaine self-administration sessions modified neural substrates underlying both the reinforcing and locomotor responses to EtOH alone. Further studies utilizing intravenous EtOH self-administration will allow identification of various long-term behavioral and neural consequences of voluntary high EtOH intake.