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Ultrasonic vocalizations (USVs) have been established as an animal model of emotional status and are often utilized in drug abuse studies as motivational and emotional indices. Further USV functionality has been demonstrated in our recent work showing accurate identification of selectively-bred high versus low alcohol-consuming male rats ascertained exclusively from 22 to 28 kHz and 50–55 kHz FM USV acoustic parameters. With the hypothesis that alcohol-sensitive sex differences could be revealed through USV acoustic parameters, the present study examined USVs and alcohol consumption in male and female selectively bred high-alcohol drinking (HAD-1) rats. For the current study, we examined USV data collected during a 12-week experiment in male and female HAD-1 rats. Experimental phases included Baseline (2 weeks), 4-h EtOH Access (4 weeks), 24-h EtOH Access (4 weeks) and Abstinence (2 weeks). Findings showed that both male and female HAD-1 rats spontaneously emitted a large number of 22–28 kHz and 50–55 kHz FM USVs and that females drank significantly more alcohol compared to males over the entire course of the experiment. Analyses of USV acoustic characteristics (i.e. mean frequency, duration, bandwidth and power) revealed distinct sex-specific phenotypes in both 50–55 kHz FM and 22–28 kHz USV transmission that were modulated by ethanol exposure. Moreover, by using a linear combination of these acoustic characteristics, we were able to develop binomial logistic regression models able to discriminate between male and female HAD-1 rats with high accuracy. Together these results highlight unique emotional phenotypes in male and female HAD-1 rats that are differentially modulated by alcohol experience.

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.

Background Human emotion is a crucial component of drug abuse and addiction. Ultrasonic vocalizations (USVs) elicited by rodents are a highly translational animal model of emotion in drug abuse studies. A major roadblock to comprehensive use of USV data is the overwhelming burden to attain accurate USV assessment in a timely manner. One of the most accurate methods of analyzing USVs, human auditory detection with simultaneous spectrogram inspection, requires USV sound files to be played back 4% normal speed. New method WAAVES (WAV-file Automated Analysis of Vocalizations Environment Specific) is an automated USV assessment program utilizing MATLAB's Signal and Image Processing Toolboxes in conjunction with a series of customized filters to separate USV calls from background noise, and accurately tabulate and categorize USVs as flat or frequency-modulated (FM) calls. In the current report, WAAVES functionality is demonstrated by USV analyses of cocaine self-administration data collected over 10 daily sessions. Results WAAVES counts are significantly correlated with human auditory counts ( r (48) = 0.9925; p Comparison with existing method WAAVES output is highly accurate and provides tabulated data in approximately 0.3% of the time required when using human auditory detection methods. Conclusions The development of a customized USV analysis program, such as WAAVES streamlines USV assessment and enhances the ability to utilize USVs as a tool to advance drug abuse research and ultimately develop effective treatments.

There is a concern that hot environments enhance adverse effects of 3,4-methylenedioxymethamphetamine (MDMA or "Ecstasy"). In this study, long-term (4-weeks) daily MDMA self-administration sessions and an MDMA Challenge test were conducted with rats under normal and high thermal conditions (23° or 32°C). During MDMA self-administration sessions, activity and body temperature were increased by heat or MDMA experience, while MDMA self-administration rates increased with experience, but were comparable between thermal conditions. At the MDMA Challenge test (3.0 mg/kg, i.v.), in vivo microdialysis showed that nucleus accumbens serotonin (NAcc 5-HT) and dopamine (DA) responses were significantly increased in both thermal conditions. In the heated environment, MDMA-stimulated 5-HT responses and core temperature (but not DA) were significantly greater than at room temperature. Though the heated environment did not acutely boost MDMA intake, exaggerated NAcc 5-HT responses to MDMA may result in 5-HT depletion; a condition associated with Ecstasy use escalation and neural dysfunctions altering mood and cognition.

In drug dependence studies, rats are often tested daily with short breaks (such as weekends) spent untested in their home cages. Research on alcohol models has suggested that breaks from continuous testing can transiently enhance self-administration (termed the “alcohol deprivation effect”). The present study explored whether the salience of cocaine-access cues is increased after skipping weekend cocaine and cue exposures. Ultrasonic vocalizations (USVs) of the 50-kHz class are emitted by rats exposed to intravenous cocaine and have been shown to increase with repeated drug exposure at the same dose level (sensitization). The present study found that over the course of several weeks of cocaine self- or yoked-administration pre-drug cues signaling forthcoming access or delivery of cocaine elicited marked amounts of anticipatory 50-kHz USVs, and that weekend deprivation from cues and cocaine exaggerated further the level of calling (more calls on Mondays compared to Fridays). Anticipatory USVs extinguished less rapidly when weekend access to unreinforced cues was denied. The results may have clinical implications, in that intermittently avoiding cues or context may enhance drug cue salience and resistance to extinction.

Ultrasonic vocalization (USV) in the 50-kHz range occurs in rats immediately upon first-time exposure to cocaine or amphetamine, and rapidly increases with repetitive drug exposure at the same dose. This sensitized positive-affect response to these drugs of abuse is persistent in that the peak level of USVs again appears when the drug is reintroduced after several weeks of drug discontinuation. The present study explored whether with enough experience USVs might be elicited, and gradually escalate, in anticipation of impending drug delivery. Rats were trained to self-administer (SA) cocaine intravenously by lever pressing 5 days per week for 4 weeks. Yoked rats received experimenter-delivered cocaine matching that of SA rats. USVs and locomotor activity were recorded during each 10-min period prior to 60-min drug access sessions. Extinction trials in which drug access was denied were then carried out over an additional 4-week period. After about a week of cocaine experience, both the SA and Yoked groups began to progressively increase USVs when placed in an environment that predicted forthcoming drug exposure. Extinction of anticipatory calls and locomotion occurred over days after drug access ended. USVs may be a useful model for specifically investigating the neural basis of drug anticipation and aid in developing and assessing new addiction treatment strategies for reducing craving and relapse.

Short 50-kilohertz (kHz) range frequency-modulated ultrasonic vocalizations (USVs) produced by rats and mice are unconditionally elicited by drugs of abuse or electrical stimulation that increase dopamine activity in the nucleus accumbens, and it has been suggested that they reflect “positive affect” or incentive motivational states associated with appetitive behavior. The repeated administration of amphetamine is known to not only produce “psychomotor” sensitization, but also to facilitate a number of appetitive behaviors, including conditioned drug pursuit behavior. We were interested, therefore, in whether amphetamine-induced 50-kHz USVs would also increase with repeated drug exposure. USV recordings were made during 5-min sessions immediately after a saline infusion, and again 4-5 hours later, after 1.0 mg/kg intravenous amphetamine exposure. These sessions took place every other day over a 5-day period. A challenge dose of 1.0 mg/kg amphetamine was administered 2 weeks later to determine whether sensitization would persist. The initial amphetamine infusion increased 50-kHz USVs relative to the saline infusion. This effect was enhanced over trials and during the amphetamine challenge two weeks later. Classification of 50-kHz range call types revealed that complex frequency-modulated trill calls were sensitized by amphetamine, but not flat 50-kHz calls. It is possible that 50-kHz USV recordings could provide a potentially valuable behavioral measure of sensitization linked to enhanced incentive salience and increased tendency to self-administer drugs of abuse.

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.

MDMA (3,4-methylenedioxymethamphetamine), also known as ecstasy, is a popular drug often taken in environments rich in audio and visual stimulation, such as clubs and dance parties. The present experiments were conducted to test the notion that auditory stimulation influences the rewarding effects of MDMA. In Experiment 1, a conditioned place preference (CPP) procedure was conducted in which rats received MDMA (1.5 mg/kg, s.c.) in a distinctive environment accompanied by music (65–75 dB), white noise (70 dB), or no added sound. Animals were pretreated with saline on alternating days in an alternate environment. Results revealed CPP in animals exposed to white noise during MDMA trials. For Experiment 2, rats from Experiment 1 had access to operant levers that delivered intravenous MDMA (0.5 mg/kg/inj) or saline (0.1 ml) on alternate days in the presence or absence of the same types of auditory stimuli as previously experienced. After three each of MDMA and non-reinforced (saline) sessions, animals were tested for NAcc DA and 5-HT responses to MDMA (1.5 mg/kg) or saline under the same stimulus conditions. Findings revealed that NAcc DA and 5-HT increased after an MDMA injection, and both DA and 5-HT were significantly highest in animals exposed to music during the test session. These results indicate that paired sensorial stimuli can engage the same systems activated during drug use and enhance neurochemical and behavioral responses to MDMA administration.

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.

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.

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-hr/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 ten daily 1-hr 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-hr 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 cocaineinduced behavioral sensitization.

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.

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 μg/side) and antagonist (SCH 23390; 0.25 μg/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.

This is a commentary on article Barker DJ. Making WAAVES in the vocalization community, but how big is the splash? J Neurosci Methods. 2014 Jan 15;221:228-9.

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.

The dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) is well known to be involved in the reinforcing properties of many drugs of abuse. The medial prefrontal cortex (mPFC) has been shown to exhibit significant influence over activity in this pathway, and has also been implicated in drug abuse. The present experiment investigated the ability of D1 activity in the mPFC to influence accumbal dopamine levels. NAcc dopamine (DA) was monitored before, immediately after, and 24 h following mPFC infusion of a D1 agonist (SKF 38393), D1 antagonist (SCH 23390), or a vehicle solution. Immediately following infusion of dopaminergic agents or vehicle, no significant changes in accumbal DA were observed. However, 24 h following infusion of the antagonist but not the agonist, significant elevations of accumbal DA were observed. Since elevated NAcc DA was only observed 24 h after treatment, these results provide evidence that long-term neural adaptations can be induced by transient neuropharmacological treatment.

Ventral tegmental area (VTA) stimulation produced conditioned place preferences for stimulation-paired environments the magnitudes of which were dose-dependently reduced by systemic application of the dopamine antagonist, haloperidol (0.0, 0.15, 0.3 mg/kg). Bilateral microinjections of cis-flupenthixol (FLU) into the nucleus accumbens (0.0, 1.0, 5.0 or 10.0 micrograms) also resulted in reductions in the size of stimulation-induced place preferences as well as reductions in the magnitude of the hyperlocomotor response to 1.5 mg/kg (s.c.) D-amphetamine. Comparable microinjections of FLU into the medial prefrontal cortex (PFC) produced diametrically opposite effects: the size of VTA stimulation-induced place preferences was either unaffected (1.0 and 5.0 microgram groups) or slightly increased (10 micrograms group) and amphetamine-stimulated hyperlocomotion was dose-dependently potentiated. These behavioral findings suggest a dopamine-mediated modulatory role for the PFC over reward relevant elements within the nucleus accumbens.

A Conditioned Place Preference test procedure [Ettenberg and Duvauchelle (13)] was used to investigate the effects of dopamine antagonist challenge on the rewarding properties of medial prefrontal cortex (MPFC) electrical stimulation. Rats exhibited strong preferences for the side of a two-compartment test apparatus in which they experienced sessions of experimenter-administered 0.5-s trains of MPFC sine-wave 60-Hz stimulation. Pretreatment with the neuroleptic dopamine antagonist drug, haloperidol (0.0, 0.15, or 0.3 mg/kg IP), resulted in a dose-dependent reduction in the magnitude of observed place preferences. Preference tests were conducted 24 hours after drug-conditioning trials and, hence, were not subject to motoric or other nonspecific actions of the neuroleptic treatments. In a control experiment, haloperidol did not block the place aversions produced by dorsomedial tegmental stimulation. Animals can, therefore, recall place-associations formed in the presence of haloperidol, a result which challenges “state-dependent learning” explanations of the drug's actions. Together, these results are consistent with the view that dopamine neurotransmission is involved in the rewarding consequences of electrical stimulation in the medial prefrontal cortex.