50-kHz Vocalizations Research Articles

Effects of repeated morphine on ultrasonic vocalizations in adult rats: increased 50-kHz call rate and altered subtype profile.

Adult rat 50-kHz vocalizations have been proposed to indicate a positive affective state, putatively revealed by a predominance of trill calls over flat calls. However, short-term exposure to non-sedative doses of the euphorigen morphine suppresses calling, with no discernible shift in trill or flat call prevalence. This study aimed to determine whether morphine acutely increases 50-kHz call rates or alters the relative prevalence of trill or flat calls, after long-term morphine exposure or acute pharmacological pretreatment. Experiment 1 comprised 10 once-daily tests, alternating between saline and morphine, 1mg/kg SC, followed by dose-response testing (0, 0.3, 1, and 3mg/kg). Experiment 2 was similar but included additional testing with morphine in combination with the antinausea drug ondansetron or the peripheral opioid antagonist methylnaltrexone. In experiment 3, morphine was again combined with ondansetron or methylnaltrexone but in rats that were initially drug naïve. In animals that were initially drug naïve, morphine tended to suppress calling and did not alter the 50-kHz call subtype profile. In morphine-experienced rats, morphine acutely increased the 50-kHz call rate and promoted trills over flat calls; short calls were also inhibited. Neither ondansetron nor methylnaltrexone detectably altered any effect of morphine on calling, nor did these two drugs affect 50-kHz calling when given alone. With chronic exposure, morphine acutely enhances 50-kHz calling and differentially promotes trill calls, mainly at the expense of flat calls. These effects appear consistent with a positive affect interpretation of 50-kHz vocalizations.

Rats Synchronize Locomotion with Ultrasonic Vocalizations at the Subsecond Time Scale.

Acoustic signals have the potential for transmitting information fast across distances. Rats emit ultrasonic vocalizations of two distinct classes: “22-kHz” or “alarm” calls and “50-kHz” calls. The latter comprises brief sounds in the 30–80-kHz range, whose ethological role is not fully understood. We recorded ultrasonic vocalizations from pairs of rats freely behaving in neighboring but separated arenas. 50-kHz vocalizations in this condition were tightly linked to the locomotion of the emitter at the subsecond time scale, their rate sharply increasing and decreasing prior to the onset and offset of movement respectively. This locomotion-linked vocalization behavior showed a clear “audience effect,” as rats recorded alone displayed lower vocal production than rats in social settings for equivalent speeds of locomotion. Furthermore, calls from different categories across the 50 and 22-kHz families displayed markedly different correlations with locomotor activity. Our results show that rat vocalizations in the high ultrasonic range are social signals carrying spatial information about the emitter and highlight the possibility that they may play a role in the social coordination of spatial behaviors.

The effects of repeated forced ethanol consumption during adolescence on reproductive behaviors in male rats.

Adolescence is a sensitive period of brain development when changes in hormone levels may have long-lasting effects on synaptic connections and behavior. In humans, alcohol consumption frequently begins during this critical period, although the impact of early exposure has not been fully examined. The current study was designed to investigate short- and long-term effects of repeated forced ethanol consumption during adolescence on emerging reproductive behaviors. Twenty-six young male Long-Evans rats were assigned to ethanol (Young EtOH, n=12) or water (Young Control, n=14) groups at postnatal day (P) 32, receiving a modified binge protocol of 3g/kg of solution via gavage twice per week from P32 to P80. For comparison, another cohort of rats received a similar treatment paradigm in adulthood from P75-P133 (Adult EtOH, n=8; Adult Control, n=10). Reproductive behavior was assessed with tests for copulation, partner preference, and 50-kHz vocalizations during forced consumption (intoxication) and again after a 4-5 week period of abstinence. During forced consumption, the Young EtOH group showed significantly longer latencies on copulation tests than Young Controls, but these differences did not persist after abstinence. Different patterns were observed in Adult animals, who only showed significant, delayed impairments in the post-ejaculatory interval. Preference for sexually receptive females increased with sexual experience in both adolescent and adult rats, regardless of treatment during the forced consumption phase. However, after abstinence, the Young EtOH group showed a significantly reduced partner preference compared to the Young Control group, which may indicate long-term effects on sexual motivation. Additionally, during forced consumption the Young EtOH group tended to emit fewer ultrasonic vocalizations, perhaps reflecting impairments in sexual communication. Adult groups showed no differences in partner preference or vocalization tests at any time. Taken together, these findings indicate that repeated, intermittent ethanol exposure may have moderate effects on reproductive behavior that vary as a function of age. After abstinence, differences were only observed in the younger group, suggesting that the adolescent brain and behavior are more sensitive to ethanol exposure than the adult brain for sexual motivation and performance.

Rats showing low and high sensitization of frequency-modulated 50-kHz vocalization response to amphetamine differ in amphetamine-induced brain Fos expression

Individuals predisposed to addiction constitute a minority of drug users, in both humans and animal models of the disorder, but there are no established characteristics that would allow identifying them beforehand. Our studies demonstrate that sensitization of rat 50-kHz ultrasonic vocalization (USV) response to amphetamine shows marked inter-individual diversity but substantial intra-individual stability. Low sensitization of the response shows relevance to the acquisition of self-administration of this drug and hence might be of predictive value regarding the risk of addiction. We compared amphetamine-induced Fos expression in 16 brain regions considered important for the development of addiction between rats preselected for low and high sensitization of the response and next given nine daily amphetamine doses followed by a 2-week withdrawal and final amphetamine challenge. Ventral tegmental area and nucleus accumbens shell Fos-positive nuclei counts correlated positively with 50-kHz USV response to the challenge in high-sensitized rats. Compared to those in amphetamine-untreated controls, Fos-positive nuclei counts were significantly and markedly (2–6 times) higher in 12 regions in high-sensitized rats, whereas in low-sensitized rats they were significantly higher in the cingulate cortex and dorsomedial striatum only. The difference in the counts between the latter two subsets reached statistical significance in dorsomedial and dorsolateral striatum and three out of four cortical regions studied. The fact that the diversification was most distinct in dorsal striatum that plays a critical role in the transition from controlled to compulsive drug intake suggests that the USV-based categorization may be related to divergent vulnerability of rats to AMPH addiction.

Modulation of 22-khz postejaculatory vocalizations by conditioning to new place: Evidence for expression of a positive emotional state.

It has been assumed that the 22-kHz ultrasonic vocalizations (USVs) are emitted by adult rats as a result of a negative emotional state. However, emission of the 22-kHz vocalizations by male rats has been also observed following ejaculation, which has a high rewarding value as shown by a conditioned place preference test. These observations suggest that 22-kHz USVs may also occur in response to a positive emotional state. The aim of this study was to determine whether the postejaculatory 22-kHz USVs are related to conditioning processes. The 22 kHz USVs were recorded in Sprague-Dawley males in the postejaculatory refractory period during conditioning processes to a new chamber unrelated to copulation. During the first session in the clean recording chamber, males vocalized marginally and exhibited intensive rearing behavior. From the second to fourth sessions, vocalization duration increased and the number of rearing decreased. Following established conditioning process, odor cues from foreign males, but not the familiar ones, resulted in decreased duration of 22-kHz USVs and increased the number of rearing. On the other hand, in the presence of mating cues (copulatory chamber and presence of the female), males exhibited increased duration of postejaculatory 22-kHz USVs and reduced number of rearing. These results demonstrated that the conditioning to the cues, both unrelated and related to copulation, is important for evoking postejaculatory 22-kHz USVs as well as the relaxation state. Furthermore, these results confirmed the postejaculatory 22-kHz USVs' involvement in expression of the positive emotional state. (PsycINFO Database Record

Poor sensitization of 50-kHz vocalization response to amphetamine predicts rat susceptibility to self-administration of the drug.

RationaleOur previous studies showed promise for using sensitization of the frequency-modulated 50-kHz vocalization response to amphetamine (AMPH) as an index of rat vulnerability to AMPH addiction.ObjectiveThis study aimed to test the utility of sensitizing frequency-modulated (FM) 50-kHz vocalization in the AMPH self-administration paradigm as well as the ability of N-acetylcysteine to prevent self-administration relapse.MethodsRats were subjected to the so-called two-injection protocol of sensitization (TIPS) using AMPH and were categorized as low-sensitized callers (LCTIPS) or high-sensitized callers (HCTIPS) based on the individual outcomes. Then, they were given 44 sessions of AMPH self-administration followed by a 17-session N-acetylcysteine-aided extinction course and a single session of AMPH-primed self-administration reinstatement.ResultsLCTIPS compared to HCTIPS rats showed no considerable difference in the FM 50-kHz vocalization rate during the self-administration training or extinction course, but they were considerably more likely to acquire AMPH self-administration and experience drug-induced reinstatement of this trait. Moreover, the LCTIPS rats were more likely than HCTIPS rats to have a markedly higher FM 50-kHz vocalization rate after AMPH reinstatement. N-acetylcysteine did not affect the course of self-administration extinction or the instrumental or FM 50-kHz vocalization responses to AMPH reinstatement.ConclusionsThere is no link between the FM 50-kHz vocalization and key characteristics of AMPH self-administration. Additionally, N-acetylcysteine does not help prevent AMPH self-administration relapse. However, there is a high predictive value for poor sensitization of the FM 50-kHz vocalization response to AMPH with respect to the acquisition and maintenance of self-administration of this psychostimulant.

Effects of the phencyclidine model of schizophrenia and nicotine on total and categorized ultrasonic vocalizations in rats

Patients with schizophrenia smoke cigarettes at a higher rate than the general population. We hypothesized that a factor in this comorbidity is sensitivity to the reinforcing and reinforcement-enhancement effects of nicotine. Phencyclidine (PCP) was used to model behavioral changes resembling negative symptoms of schizophrenia in rats. Ultrasonic vocalizations (USVs) in rats have been used to measure emotional states, with 50 kHz USVs indicating positive states and 22 kHz USVs indicating negative states. Total and categorized numbers of 22 and 50 kHz USVs and USVs during a visual stimulus (e.g. a potential measure of reinforcement-enhancement) were examined in rats following injection of PCP (2.0 mg/kg) and/or nicotine (0.2 or 0.4 mg/kg) daily for 7 days. PCP was then discontinued and all rats received nicotine (0.2 and 0.4 mg/kg) and PCP (2.0 mg/kg) on three challenge days. PCP acutely decreased 50 kHz vocalizations, whereas repeated nicotine potentiated rates of vocalizations, with similar patterns during light presentations. Rats in the PCP and nicotine combination groups made more 50 kHz vocalizations compared with rats in the control groups on challenge days. We conclude that PCP may produce a reward deficit, which is shown by decreased 50 kHz USVs, and behaviors post-PCP exposure may best model the comorbidity between schizophrenia and nicotine.

Expression of c-Fos in rat auditory and limbic systems following 22-kHz calls

In the present study, adult Long-Evans rats were exposed either to natural conspecific aversive 22-kHz vocalizations or to artificial call-like stimuli with comparable frequency-temporal features, followed by c-Fos immunohistochemistry. The natural 22-kHz vocalizations was either played from a recording or produced by a foot-shocked animal located nearby (live vocalizations). In comparison with controls (non-exposed animals), c-Fos immunoreactivity was significantly increased in the inferior colliculus (IC), auditory cortex (AC), periaqueductal grey (PAG), basolateral amygdala (BA), and hippocampus (Hip) of rats exposed to either live or recorded 22-kHz natural vocalizations. Exposure to live natural vocalizations of the foot-shocked animal resulted in a similar pattern of c-Fos activity, as did exposure to the playback of the natural vocalizations. In contrast to this, foot-shocked rats (emitting the 22-kHz vocalizations) had the c-Fos positivity increased markedly in the PAG and only slightly in the AC. The expression of c-Fos also increased in the IC, AC, and in the PAG in animals exposed to the artificial call-like stimuli, when compared to controls; however, the increase was much less pronounced. In this case, c-Fos expression was not increased in the hippocampus or basolateral amygdala. Interestingly, almost no c-Fos expression was found in the medial nucleus of the geniculate body in any of the experimental groups. These findings suggest that differences exist between the processing of important natural conspecific vocalizations and artificial call-like stimuli with similar frequency-temporal features, and moreover they suggest the specific role of individual brain structures in the processing of such calls.

Cat‐Exposure Results in Significantly More Elicited Alarm Calls (22kHz Ultrasonic Vocalizations, USVs) Compared to Snake‐, Ferret‐, or Sham‐Exposure During a Rodent Model of Traumatic Stress

Ultrasonic vocalizations (USVs) in rodents can be used as a method of measuring the affective state of the animal during a wide range of experiences. Adult rats generally emit two types of USVs: “alarm” calls (22kHz) and calls during “nonaggressive” behavioral situations (50kHz). Rat USVs can therefore be measured and classified when a rat is in the presence of a natural predator. In humans, PTSD results from exposure to a traumatic event(s) which evoke a level of fear, helplessness, or horror with a threat of physical harm. In this study, a rodent model of traumatic stress was used to elicit a traumatic event via (a single protected) exposure to a predator species (snake, ferret, or cat). To determine the impact of these animals as a natural predator species, we examined each one individually and assessed USVs to determine the impact on ultrasonic communication by these predator species. Rats (~11–12 weeks old) were behaviorally assessed pre‐ and post‐predator exposure on several behavioral tasks. USVs were recorded during the open field task and on predator exposure days (pre‐ and post‐exposure, in transfer cages, and during predator exposure, in the predator home cage). To record the USVs, an Avisoft‐Ultra SoundGate electret 116Hnb microphone and Recorder USGH program (Avisoft Bioacoutistc, Germany) were used. Rats were placed in a protective predator tube, and then placed in the predator home cage. These tubes allow for visual and olfactory cues; however, prevent the rat from gross physical contact. The rats were exposed to one of the predators or sham (using a predator tube free of predator scent). Sham‐exposed rats emitted significantly more 50kHz USVs than any of the predator‐ exposed rats (F(3,44)= 9.12, p < .0001). Cat‐exposed rats did not emit any 50kHz USVs during exposure, but emitted the most 22kHz USVs, significantly more than any other predator or sham (F(3,44)= 3.29, p < .05). 22kHz USVs were emitted by 75% of cat‐exposed rats and only 2 of 12 snake‐exposed rats. Rats exposed to the ferret and sham did not emit any fear‐induced 22kHz vocalizations, only 50kHz vocalizations. Based on emitted ultrasonic calls, the cat appears to be the most impactful, eliciting the most fear‐induced vocalizations. To the contrary, the ferret did not cause any fear‐inducing vocalizations. This study has provided the ground work for the recording of USVs during our laboratory's rodent model of traumatic stress. For future research, this method can be used as an additional measure, along with assessing overt behavior measures, to identify candidate therapeutic compounds for the prevention and/or treatment of PTSD.Support or Funding InformationMaterial has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted in an AAALACi accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition. Supported by the Military Operational Medicine Research Program, US Army Medical Research and Materiel Command.

Discomfort-related changes in pup ultrasonic calls of fat-tailed gerbils Pachyuromys duprasi

Rodent pups vocalize when placed in social isolation. We apply a method of “joint calls” for examining discomfort in rodent pup ultrasonic (>20 kHz) calls. Previously, this method has been developed for audible calls of fur farm mammals. Using a repeated measures design to exclude effects of individual identity and age on the analysed variables, we compared the ultrasonic call variables produced by 8–40-day pups of fat-tailed gerbils Pachyuromys duprasi during two subsequent experimental stages, the Isolation Stage and the Handling Stage. We considered that discomfort-related negative emotional arousal increased towards the Handling Stage compared to the Isolation Stage because of cumulative effects of handling and time of pup isolation from the nest. At the Isolation Stage, the call rate (calls/s) was higher from 10 to 18 days of age, whereas both the maximum amplitude frequency and power quartiles of joint calls were lower than at the Handling Stage from 20 to 32 days of age. At the same time, in audible (<20 kHz) vocalizations of a wide range of mammalian species, both the higher call rate and the upward shift of the maximum amplitude frequency and power quartiles indicate the discomfort-related increase of negative emotional arousal. We discuss the advantages of the method of joint calls for express-analyses of power variables for large sequences of ultrasonic vocalizations of complex acoustic structure during experimental trials.

Air puff-induced 22-kHz calls in F344 rats

Air puff-induced ultrasonic vocalizations in adult rats, termed “22-kHz calls,” have been applied as a useful animal model to develop psychoneurological and psychopharmacological studies focusing on human aversive affective disorders. To date, all previous studies on air puff-induced 22-kHz calls have used outbred rats. Furthermore, newly developed gene targeting technologies, which are essential for further advancement of biomedical experiments using air puff-induced 22-kHz calls, have enabled the production of genetically modified rats using inbred rat strains. Therefore, we considered it necessary to assess air puff-induced 22-kHz calls in inbred rats. In this study, we assessed differences in air puff-induced 22-kHz calls between inbred F344 rats and outbred Wistar rats. Male F344 rats displayed similar total (summed) duration of air puff-induced 22kHz vocalizations to that of male Wistar rats, however, Wistar rats emitted fewer calls of longer duration, while F344 rats emitted higher number of vocalizations of shorter duration. Additionally, female F344 rats emitted fewer air puff-induced 22-kHz calls than did males, thus confirming the existence of a sex difference that was previously reported for outbred Wistar rats. The results of this study could confirm the reliability of air puff stimulus for induction of a similar amount of emissions of 22-kHz calls in different rat strains, enabling the use of air puff-induced 22-kHz calls in inbred F344 rats and derived genetically modified animals in future studies concerning human aversive affective disorders.

Female rats are resistant to developing the depressive phenotype induced by maternal separation stress.

Many stress-related psychiatric disorders are more common in women than in men. We aimed to determine how female rats respond to maternal separation (MS; removal of the dam from the litter for 3 h/day from postnatal day (P) 2-14)). A subset of MS females were also exposed to chronic constant light for 3 weeks during adolescence (P42-63) to investigate whether the antidepressant effect of light treatment, previously observed in male rats, could be seen in female rats. Ultrasonic vocalizations (22 kHz) were recorded and the forced swim test was conducted immediately after light exposure (P65-67) and 33 days later (P98-99) to determine depressive-like behaviour. Key proteins in the MAPK signal transduction pathway (MKP-1, phospho-ERK, total ERK) and a synaptosomal marker (synaptophysin) were measured in the ventral hippocampus. We found that MS decreased the duration of 22 kHz vocalizations at P65 which was reversed by subsequent light. Light exposure increased time spent in the inner zone of the open field and the number of 22 kHz calls in response to novelty at P98. MS decreased the time females spent immobile and increased time actively swimming in the forced swim test at P67 but not at P99. MKP-1 and synaptophysin levels remained unchanged while MS decreased phospho-ERK levels in the ventral hippocampus. In contrast to clinical findings, the results suggest that female rats may be resistant to MS-induced depression-like behaviour. The behavioural effects of MS and light treatment in female rats may involve the MAPK/ERK signal transduction pathway.

Pharmacology of Ultrasonic Vocalizations in adult Rats: Significance, Call Classification and Neural Substrate.

Pharmacological studies of emotional arousal and initiation of emotional states in rats measured by their ultrasonic vocalizations are reviewed. It is postulated that emission of vocalizations is an inseparable feature of emotional states and it evolved from mother-infant interaction. Positive emotional states are associated with emission of 50 kHz vocalizations that could be induced by rewarding situations and dopaminergic activation of the nucleus accumbens and are mediated by D1, D2, and partially D3 dopamine receptors. Three biologically significant subtypes of 50 kHz vocalizations have been identified, all expressing positive emotional states: (1) flat calls without frequency modulation that serve as contact calls during social interactions; (2) frequencymodulated calls without trills that signal rewarding and significantly motivated situation; and (3) frequency-modulated calls with trills or trills themselves that are emitted in highly emotional situations associated with intensive affective state. Negative emotional states are associated with emission of 22 kHz vocalizations that could be induced by aversive situations, muscarinic cholinergic activation of limbic areas of medial diencephalon and forebrain, and are mediated by M2 muscarinic receptors. Two biologically significant subtypes of 22 kHz vocalizations have been identified, both expressing negative emotional sates: (1) long calls that serve as alarm calls and signal external danger; and (2) short calls that express a state of discomfort without external danger. The positive and negative states with emission of vocalizations are initiated by two ascending reticular activating subsystems: the mesolimbic dopaminergic subsystem as a specific positive arousal system, and the mesolimbic cholinergic subsystem as a specific negative arousal system.

Repeated amphetamine administration and long-term effects on 50-kHz ultrasonic vocalizations: Possible relevance to the motivational and dopamine-stimulating properties of the drug

Ultrasonic vocalizations (USVs) of 50kHz are thought to indicate positive affective states in rats, and are increasingly being used to investigate the motivational properties of drugs of abuse. However, previous studies have observed that only dopaminergic psychostimulants of abuse, but not other addictive drugs, stimulate 50-kHz USVs immediately after their administration. This would suggest that 50-kHz USVs induced by addictive dopaminergic psychostimulants might reflect rewarding dopaminergic effect, rather than motivational effect. To elucidate this issue, our study compared the effects of the psychostimulant of abuse amphetamine and the dopamine receptor agonist apomorphine on 50-kHz USVs. Rats that received five drug administrations on alternate days in a novel test-cage were first re-exposed to the test-cage 7 days after treatment discontinuation to assess drug-conditioning, and then received a drug challenge. USVs were recorded throughout the experiments together with locomotor activity. To further clarify how amphetamine and apomorphine influenced 50-kHz USVs, rats were subdivided into “low” and “high” vocalizers, and time-dependence of drug effects was assessed. Amphetamine and apomorphine stimulated both 50-kHz USVs and locomotor activity, though they elicited dissimilar changes in these behaviors, depending on drug dose, rats׳ individual predisposition to vocalize, and time. Moreover, only amphetamine-treated rats displayed both sensitized 50-kHz USVs emission and conditioned vocalizations on test-cage re-exposure. These results indicate that the effects of amphetamine on 50-kHz USVs are not mimicked by a dopaminergic agonist with a low abuse potential, and may further support the usefulness of 50-kHz USVs in the study of the motivational properties of psychoactive drugs.

100 kHz Bandwidth Ultrasonic Recording System

We have developed a complete 100 kHz ultrasonic recording system. The primary components of the system are a SensComp Series 600 Instrument Grade Electrostatic Transducer, custom printed circuit board (PCB), wide-bandwidth instrumentation amplifier, linear-phase filter, fully differential 16-bit analog to digital converter (ADC) sampling at 840 kHz, 168 MHz STM32F4 32-bit microcontroller, and a direct 100 Mbps Ethernet connection. A host computer running a custom application written in C++ provides a unified computer interface that allows the user to control all aspects of the system and provides a real time spectrogram. The system is designed for general use but has been initially used to study the ultrasonic vocalizations of rodents. We demonstrate the system by recording a well-established call made by Long-Evans rats termed 50-kHz vocalizations. The system was also designed with possible physical measurements in mind, e.g., distance, temperature, humidity, and gas mixture ratios.

N-acetyl cysteine does not modify the sensitization of the rewarding effect of amphetamine as assessed with frequency-modulated 50-kHz vocalization in the rat

A satisfactory pharmacological cure for addictions to psychostimulants has not yet been developed. Because of the well-known role of changes in the corticoaccumbal and corticostriatal glutamatergic system(s) in drug seeking and relapses in psychostimulant addiction, much hope is presently linked to the use of agents that restore glutamate homeostasis. In this regard, one of the most promising agents is N-acetyl cysteine, which has been shown to reverse some changes in neuroplasticity associated with psychostimulant addiction/dependence. In this study, we used the enhancement of locomotor activity and the induction of frequency-modulated 50-kHz ultrasonic vocalization (FM 50-kHz USV) to test the possible stimulant properties of N-acetyl cysteine itself in various experimental settings (acute and subchronic administration in amphetamine-naïve and amphetamine-pretreated rats) and the capacity of N-acetyl cysteine to attenuate both the rewarding effects of amphetamine and the behavioral sensitization to this stimulant in rats showing considerable differences in their susceptibility to the FM 50-kHz USV sensitization. Our data showed no stimulant properties of N-acetyl cysteine and no acute effect of the drug on the rewarding properties of amphetamine. Moreover, no effect of N-acetyl cysteine on the pre-existing sensitization of the FM 50-kHz USV and locomotor activity responses to amphetamine were observed, independent of the susceptibility of the rats to the FM 50-kHz USV sensitization. Hence, N-acetyl cysteine seems to be ineffective at reversing the neurobiological changes underlying the sensitization of these responses to amphetamine in rats.

Sexual performance and precontact 50-kHz ultrasonic vocalizations in WAG/Rij rats: Effects of opioid receptor treatment

WAG/Rij rats are genetically selected animals that model absence epilepsy in rats. Ultrasonic vocalizations and sexual behavior – both ethologically relevant markers of reward system functioning – are poorly described in this strain. The aim of our experiment was to investigate reward-dependent precontact 50-kHz vocalizations (PVs) and copulatory behavior as well as the effects of opioid receptor treatment on such behaviors in sexually experienced WAG/Rij males and rats from two control strains: Sprague–Dawley and Crl: Han Wistar. We analyzed the effects of the opioid receptor antagonist naltrexone (3mg/kg) and the agonist morphine (1mg/kg) administration. Additionally, we analyzed the initiation of copulation in sexually naïve males before drug treatment. A significantly lower number of sexually naïve WAG/Rij rats initiated copulation. Sexually experienced WAG/Rij males differed at the control session (after physiological saline treatment) compared with Sprague–Dawley rats: WAG/Rij rats displayed more 50-kHz precontact vocalizations and had longer mount and intromission latencies, longer ejaculation latency, longer postejaculatory latency to exploration, longer 22-kHz vocalization duration after ejaculation, and longer postejaculatory intromission latency. Compared with Crl: Han Wistar rats, WAG/Rij males displayed longer mount latency and shorter 22-kHz vocalization duration. Neither naltrexone nor morphine affected PVs in all groups. On the other hand, opioid receptor treatment differently influenced the number of intromissions required to achieve ejaculation and 22-kHz postejaculatory vocalization duration in WAG/Rij rats than in both control groups. This suggests functional differences in the opioid system in this strain. As a result of the number of males that initiated copulation as well as the number of intromissions to ejaculation and 22-kHz postejaculatory vocalizations which all depend on D1 receptor activation, we suggest that the proportion of opioid receptor to D1 receptors in WAG/Rij rats is different when compared with the control strains. The reward system of Wag/Rij rats with absence epilepsy is sensitive to social rewards (high level of precontact 50-kHz ultrasounds) although this strain displays a lower level of sexual motivation (longer mount latency) compared with other control strains. A lower number of sexually naïve rats initiating copulation and longer mount latency in sexually experienced males could suggest a moderate depressive-like syndrome in this strain of rats.

Are 50-kHz calls used as play signals in the playful interactions of rats? I. Evidence from the timing and context of their use

During playful interactions, rats emit increased levels of 50-kHz vocalizations. It is possible that these vocalizations are used as play signals that promote and maintain playful contact. The study investigated this possibility. It was predicted that if these vocalizations are used as play signals, they should be more prevalent (1) before an attack, (2) in attacks leading to wrestling, and (3) in males compared to females, as males play more roughly. Moreover, given that there are at least 15 different subtypes of 50-kHz calls, it is possible that different calls are used in different contexts. Therefore, our prediction (4) was that different subtypes would be used for initiating and terminating playful contact. Pairs of same-sex juveniles were tested so that video recordings of their play and audio recordings of their vocalizations were synchronized. 50-kHz vocalizations occur more often before an attack and in male pairs. Specific calls were associated with specific types of behaviors and these associations differed between male and female rats. However, calls were not more frequent in attacks leading to wrestling than in attacks leading to withdrawal. The data provide qualified support for the hypothesis that 50-kHz vocalizations function as play signals.

Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment

Behavioral sensitization and tolerance to repetitive exposure to addictive drugs are commonly used for the assessment of the early stages of the drug dependence progress in animals. The orchestra of tools for studying the progress of drug dependence in laboratory rodents has been considerably enriched in the 1980s by the introduction of ultrasonic vocalization (USV) detection and characterization. However, the relationship between the results of this technology and those of traditional behavioral tests is not clear. We attempted to elucidate some of the respective ambiguities by comparing the effects of an intermittent amphetamine treatment, which was aimed both at the induction of sensitization and tolerance to this drug and at testing the persistence of these effects, on the locomotor activity and 50-kHz USV responses to both the drug and the context of drug exposure in adult male rats showing diverging susceptibility for sensitization to amphetamine. Categorization of the rats into low and high responders/callers based on sensitization of their frequency-modulated 50-kHz USV responsiveness showed some correspondence with conditioned place preference effects, but not with responses to amphetamine. The study showed distinct changes in the rate and latency of the frequency-modulated 50-kHz USV responses to repetitive amphetamine treatment, which were reminiscent of classical behavioral signs of sensitization and tolerance. These results show the utility of the appetitive USV for monitoring of early phases of complex processes leading to drug dependence. However, USV, locomotor activity and conditioned place preference seem to reflect different aspects of these phenomena.

The Sweet Drive Test: refining phenotypic characterization of anhedonic behavior in rodents

Measuring anhedonic behavior in rodents is a challenging task as current methods display only moderate sensitivity to detect anhedonic phenotype and, consequently, results from different labs are frequently incongruent. Herein we present a newly-developed test, the Sweet Drive Test (SDT), which integrates food preference measurement in a non-aversive environment, with ultrasonic vocalizations (USVs) recording. Animals were placed in a soundproofed black arena, under red light illumination, and allowed to choose between regular and sweet food pellets. During the test trials, 50 KHz USVs, previously described to be associated with positive experiences, were recorded. In a first experimental approach, we demonstrate the ability of SDT to accurately characterize anhedonic behavior in animals chronically exposed to stress. In a subsequent set of experiments, we show that this paradigm has high sensitivity to detect mood-improving effects of antidepressants. The combined analysis of both food preference and the number of 50 KHz vocalizations in the SDT provides also a valuable tool to discriminate animals that responded to treatment from non-responder animals.