50-kHz Calls Research Articles

Comparison of milnacipran, duloxetine and pregabalin in the formalin pain test and in a model of stress-induced ultrasonic vocalizations in rats

Milnacipran and duloxetine, serotonin/noradrenalin reuptake inhibitors, and pregabalin, a α 2-δ 1 Ca 2+ channel blocker, are efficacious against fibromyalgia, a condition characterized by diffuse chronic pain and associated with stress. We compared these compounds (i.p. route), in rat models of acute/inflammatory pain (2.5% intraplantar formalin) and stress-induced ultrasonic vocalization (USV: 22 kHz calls following presentation of a conditioned stimulus previously associated with foot-shocks). In the formalin test, milnacipran dose-dependently attenuated paw elevation and licking (minimal effective dose, MED: 2.5 mg/kg for licking/late phase). Duloxetine was slightly more potent (MED = 0.63). Pregabalin also reduced paw licking/late phase (MED = 0.63), but was inactive up to 160 mg/kg for paw elevation (both phases) and paw licking (early phase). Milnacipran dose-dependently reduced USV (MED = 10, near total inhibition at 20 mg/kg); duloxetine was less potent (MED = 20). Pregabalin (2.5–80 mg/kg) was only significantly active at 40 mg/kg. Milnacipran, duloxetine and pregabalin possess analgesic activity in the formalin test on paw licking/late phase (corresponding to inflammatory pain with a central sensitization component). In the stress-induced USV model, milnacipran was the most potent and efficacious compound. To summarize, reduction of formalin-induced paw licking/late phase might constitute a useful indicator of potential activity against inflammatory/centrally sensitized pain, as might be expressed in fibromyalgia.

Ultrasonic communication in rats: Effects of morphine and naloxone on vocal and behavioral responses to playback of 50-kHz vocalizations

Rats emit ultrasonic vocalizations and it was hypothesized that these vocalizations have an important role in intra-specific communication. Recently, we demonstrated that playback of 50-kHz ultrasonic vocalizations can induce social approach, indicating that 50-kHz calls can serve to (re)establish or to maintain social contact. It is known that endogenous opioids are implicated in the regulation of social behavior, particularly in rough and tumble play. Here, we tested whether administration of opioid ligands can affect social approach in response to playback of 50-kHz calls in juvenile and adult rats. Rats were either treated with 1 mg/kg naloxone, 1 mg/kg morphine, or with saline vehicle. Administration of opioid ligands affected social approach at both ages. Specifically, in juvenile and adult rats, social approach displayed in response to playback of 50-kHz calls was reduced in case of naloxone treatment, but enhanced with morphine. Furthermore, juvenile rats treated with saline or morphine emitted a substantial amount of ultrasonic vocalizations in response to the playback of 50-kHz calls. Such ultrasonic calling was not seen in naloxone treated rats. Importantly, these drug-dependent differences were stimulus-specific, i.e. seen only in response to playback of 50-kHz calls and not in response to playback of background noise. The present finding that opioid ligands can affect social approach and ultrasonic vocalizations induced by playback of 50-kHz calls, indicates that an important feature of social interaction in rats, namely ultrasonic communication, is at least partially regulated by endogenous opioids.

Reduction of dopamine synaptic activity: Degradation of 50-khz ultrasonic vocalization in rats.

Vocal deficits are prevalent and debilitating in Parkinson's disease. These deficits may be related to the initial pathology of the nigrostriatal dopamine neurons and resulting dopamine depletion, which contributes to dysfunction of fine motor control in multiple functions. Although vocalization in animals and humans may differ in many respects, we evaluated complex (50-kHz) ultrasonic mate calls in 2 rat models of Parkinson's disease, including unilateral infusions of 6-hydroxydopamine to the medial forebrain bundle and peripheral administration of a nonakinesia dose of the dopamine antagonist haloperidol. We examined the effects of these treatments on multiple aspects of the acoustic signal. The number of trill-like (frequency modulated) 50-kHz calls was significantly reduced, and appeared to be replaced by simpler (flat) calls. The bandwidth and maximum intensity of simple and frequency-modulated calls were significantly decreased, but call duration was not. Our findings suggest that the nigrostriatal dopamine pathway is involved to some extent in fine sensorimotor function that includes USV production and complexity.

Communication of Adult Rats by Ultrasonic Vocalization: Biological, Sociobiological, and Neuroscience Approaches

Rats have developed antipredator defensive adaptations to protect themselves from the large number of animals that prey on them. One such adaptation is the ability to communicate by ultrasonic vocalization, which decreases the likelihood of detection by a predator. Almost all rat vocalizations are inaudible to the human ear as well, so laboratory studies of ultrasonic vocalization require specialized electronic equipment. The most popular of these is the bat detector, which lowers ultrasonic frequency to a humanly audible frequency. Adult rats emit two types of ultrasonic calls: alarm (22 kHz) calls, in aversive and dangerous situations, and appetitive (50 kHz) calls, in appetitive or "friendly" (i.e., nonaggressive) behavioral situations. These two types of calls differ in all acoustic parameters, and their initiation depends on activity in different ascending tegmental pathways to the forebrain: 22 kHz calls require activity in the cholinergic system, and 50 kHz calls in the mesolimbic dopaminergic system. The release of acetylcholine in the diencephalon and forebrain is associated with the emission of 22 kHz vocalizations, and the release of dopamine in the nucleus accumbens with 50 kHz calls. Thus the two calls are reliable predictors of increased cholinergic or dopaminergic activity in the brain. The calls serve as indices of the rat's state, including its affective state, induced by activity of one or the other neurochemical system.

When Love Comes Calling: Measuring Sexual Selection on Sagebrush Crickets

Male sagebrush crickets exhibit differential mating success based on their previous mating experience: virgin males have a higher probability of obtaining a mating than do non-virgin males. Measures of lifetime mating success in male sagebrush crickets have revealed that the median mating frequency is one, with many males failing to secure a mate at all and a small minority obtaining two to four mates. The purpose of this study was to investigate the acoustic and morphological characteristics that make male sagebrush crickets attractive to females. Male crickets were captured from Deadman’s Bar in Grand Teton National Park and their songs were recorded on subsequent evenings. Five song characteristics were measured including pulse duration, interpulse duration, dominant frequency, train duration, and intertrain duration. Multivariate selection analysis revealed significant linear and nonlinear selection on male song, with each of the five measured song characters contributing to male attractiveness. There was significant directional selection favoring longer pulse durations and shorter interpulse durations, which could be an honest indicator of male quality because these song characters likely impose high energetic costs. Significant stabilizing selection favored males with ~ 13.2 kHz calls and intermediate intertrain durations, which may be imposed by the auditory sensitivity of females.

The effects of selective breeding for differential rates of 50‐kHz ultrasonic vocalizations on emotional behavior in rats

Fifty-kHz ultrasonic vocalizations have previously been shown to be positively correlated with reward and appetitive social behavior in rats, and to reflect a positive affective state. In this study, rats selectively bred for high and low rates of 50-kHz vocalizations as juveniles were tested as adults in a battery of behavioral tests for social/emotional behaviors. We found that animals selectively bred for high rates of 50-kHz vocalizations exhibited more crosses into the center area of the open field apparatus, were more likely to show a preference for a dilute sucrose solution (.8%) compared to tap water, and were less aggressive than randomly bred animals. Conversely, animals bred for low rates of 50-kHz calls produced more fecal boli during both open field testing and "tickling" stimulation, and made less contact with conspecifics in a social interaction test compared to randomly bred animals. We also observed that low line rats have elevated brain levels of cholecystokinin (CCK) in the cortex, which is consistent with literature showing that CCK content in the cortex is positively correlated with rates of aversive 22-kHz USVs. Conversely, high line animals had elevated levels of met-enkephalin in several brain regions, which is consistent with the role of endogenous-opioids in the generation 50-kHz USVs and positive affect. These results suggest that animals bred for high rates of 50-kHz may show a stress resilient phenotype, whereas low line rats may show a stress prone phenotype. As such these animals could provide novel insights into the neurobiology of emotion.

Repeated intravenous amphetamine exposure: Rapid and persistent sensitization of 50-kHz ultrasonic trill calls in rats

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 h 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 2 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.

Ultrasonic calling during fear conditioning in the rat: no evidence for an audience effect

Rats, Rattus norvegicus, produce 22 kHz ultrasonic vocalizations in aversive situations such as during exposure to cats or fear conditioning. These calls are considered to be part of the animal's defensive repertoire and might serve as alarm signals for conspecifics. The aim of the present study was to test whether the production of 22 kHz calls is affected by the social context during fear conditioning. Animals were tested in one of three experimental conditions: either alone, with an anaesthetized conspecific, or with an active conspecific. In line with the hypothesized alarming function of 22 kHz calls, we expected the presence of a conspecific to potentiate the production of 22 kHz calls. The results, however, show that the production of 22 kHz calls during fear conditioning is clearly not potentiated by the presence of a conspecific; if at all, its presence had a mild attenuating effect on call rate. Also, call characteristics were similar in all three experimental conditions. Therefore, our findings do not support the hypothesis that the sender actively produces 22 kHz calls to warn conspecifics about danger.

Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: Behavioral concomitants, relationship to reward, and self-administration of playback..

Rats (Rattus norvegicus) emit a variety of ultrasonic vocalizations throughout their lifespan that reflect different forms of emotional arousal and accompanying affective states. In this study, high frequency recordings of ultrasonic vocalizations were made during mating, aggression, and both conspecific and heterospecific (dubbed "tickling") rough-and-tumble play behavior. We found that frequency modulated 50-kHz calls (trills and step calls) were positively correlated with positively valenced appetitive behavior during mating, play, and aggression. These calls were also positively correlated with the reward value of these social encounters. However, constant frequency (i.e., flat) 50-kHz calls were not related to appetitive behaviors or reward. In contrast, 22-kHz calls were positively related to aversive/withdrawal behaviors during mating, play, and aggression. Finally, we found that rats self-administered playback of frequency modulated 50-kHz trill calls and avoided playback of 22-kHz calls. Playback of flat 50-kHz calls or tape hiss was neutral. These results suggest that frequency modulated 50-kHz calls index a positively valenced, appetitive, social-emotional state in rats.

Ultrasonic Communication in Rats: Can Playback of 50-kHz Calls Induce Approach Behavior?

Rats emit distinct types of ultrasonic vocalizations, which differ depending on age, the subject's current state and environmental factors. Since it was shown that 50-kHz calls can serve as indices of the animal's positive subjective state, they have received increasing experimental attention, and have successfully been used to study neurobiological mechanisms of positive affect. However, it is likely that such calls do not only reflect a positive affective state, but that they also serve a communicative purpose. Actually, rats emit the highest rates of 50-kHz calls typically during social interactions, like reproductive behavior, juvenile play and tickling. Furthermore, it was recently shown that rats emit 50-kHz calls after separation from conspecifics. The aim of the present study was to test the communicative value of such 50-kHz calls. In a first experiment, conducted in juvenile rats situated singly on a radial maze apparatus, we showed that 50-kHz calls can induce behavioral activation and approach responses, which were selective to 50-kHz signals, since presentation of 22-kHz calls, considered to be aversive or threat signals, led to behavioral inhibition. In two other experiments, we used either natural 50-kHz calls, which had been previously recorded from other rats, or artificial sine wave stimuli, which were identical to these calls with respect to peak frequency, call length and temporal appearance. These signals were presented to either juvenile (Exp. 2) or adult (Exp. 3) male rats. Our data clearly show that 50-kHz signals can induce approach behavior, an effect, which was more pronounced in juvenile rats and which was not selective to natural calls, especially in adult rats. The recipient rats also emitted some 50-kHz calls in response to call presentation, but this effect was observed only in adult subjects. Together, our data show that 50-kHz calls can serve communicative purposes, namely as a social signal, which increases the likelihood of approach in the recipient conspecific.

Effects of experience and context on 50-kHz vocalizations in rats

Rats can emit ultrasonic 50-kHz vocalizations which are generally assumed to reflect the animals' positive emotional state. However, some aspects question the reliability of 50-kHz calls as indicators of positive affective states. Firstly, rats also emit them in novel environments containing scents of other rats, or even while being victims of intra-species aggression. Secondly, huge inter-individual variability in call production can be observed. The present two studies were conducted to further determine factors other than reward, which may influence or even induce calling. Experiment A showed that 50-kHz calls were emitted in relatively high numbers during short isolation in test cages, and, to a lesser extent, also during testing in an open field and an elevated plus maze. Despite inter-individual variability, calling behavior was individually stable over days and occurred irrespective of whether rats were tested in a cage with or without familiar rat scents. These data indicate that 50-kHz calling is not necessarily a response to the presence of pleasurable or social stimuli. Additionally, it was observed that call emission during isolation is strongly affected by prior experience. Rats that had been trained repeatedly in an appetitive discrimination task emitted only few calls during short isolation in test cages, whereas naïve rats emitted high numbers of 50-kHz calls which decreased over time. The most likely explanation is that rats call in response to separation from the cage mate, as the first group was trained before the recordings, while the naïve rats were recorded immediately after separation. This explanation was supported by Experiment B, which showed that the rats that remained alone in the home cage also called at 50 kHz after separation from the cage mates. In both experiments, most of the 50-kHz calls were not frequency modulated, which lend support for the suggestion that this subtype has a social-coordinating function. The present findings urge sophisticated spectrographic analysis of ultrasonic vocalizations and caution when interpreting 50-kHz vocalizations, since specific subtypes of these calls can occur in contexts that are not necessarily pleasurable to rats, and are affected by prior experience and huge individual differences.

Are rats predisposed to learn 22 kHz calls as danger-predicting signals?

Alarm calls are widely used in mammals. Their biological function is to deter predators and warn relatives of danger. Despite this important function of alarm calls, the development of alarm call recognition is poorly understood. Using laboratory rats, the present study investigated in a first experiment whether alarm calls are recognized innately. In experimentally naive animals, we found significantly increased freezing if stimuli in the 22 kHz range were presented but this response was not specific to conspecific 22 kHz calls. Therefore, a second experiment addressed the hypothesis whether recognition of conspecific 22 kHz calls can be learned and whether this learning is facilitated by a preparedness to acquire defensive responses to alarm calls. Our data show that rats learned quickly to associate the 22 kHz calls with aversive stimuli. Interestingly, the animals were more reluctant to extinguish this memory, and this information retained longer in memory than in the case of other types of calls and ultrasonic stimuli. We, therefore, conclude that rats are predisposed to acquire adaptive defensive behaviour in response to alarm calls. In particular, our data indicate that better encoding of such learning in rats leads to a stable memory which better resists extinction.

Neurobiology of 50-kHz ultrasonic vocalizations in rats: Electrode mapping, lesion, and pharmacology studies

Fifty-kHz ultrasonic vocalizations have been proposed to reflect a positive appetitive affective state in rats, being consistently linked to the positive appetitive behavior. In the first study, we examined the brain substrates of 50-kHz ultrasonic vocalizations (USVs) by using localized electrical stimulation of the brain (ESB) at various sites that are known to mediate reward. We found that the brain areas that produced ESB-induced 50-kHz calls are the areas that have previously been shown to support the most vigorous self-stimulation behavior (prefrontal cortex, nucleus accumbens, ventral pallidum, lateral preoptic area, lateral hypothalamus, ventral tegmental area, and raphe). Importantly, all animals that showed repeatable ESB-induced 50-kHz USVs demonstrated self-stimulation behavior. In the second study, conditioned place preference was assessed following microinjection of the μ-opiate agonist Tyr- d-Ala-Gly- N-methyl-Phe-Gly-ol (DAMGO) directly into the ventral tegmental area (VTA) at a dose previously found to be rewarding. Animals that showed more 50-kHz USVs in response to drug injections compared to vehicle injections showed significant place preferences, whereas animals that did not show elevated vocalization to DAMGO did not show place preference. In experiment 3, we examined the effect of VTA electrolytic lesions, 6-OHDA lesions, and the effect of the D1/D2 dopamine antagonist flupenthixol (0 and 0.8 mg/kg, i.p.) on 50-kHz ultrasonic vocalizations. We found that these manipulations all selectively reduced 50-kHz ultrasonic vocalizations, and that these effects could be disassociated from any side effects. These data are consistent with the proposition that 50-kHz calls are tightly linked to reward in rats and that the neural circuit of 50-kHz calls closely overlaps that of ESB self-stimulation reward, drug reward, and the mesolimbic dopamine system.

Qualitative changes in ultrasonic vocalization in rats after unilateral dopamine depletion or haloperidol: A preliminary study

The sensorimotor speech/voice deficits associated with Parkinson disease have been well documented in humans. They are largely resistant to pharmacological and surgical treatment, but respond to intensive speech therapy. The mechanisms underlying this phenomenon are not well understood and are difficult to systematically test in humans. Thus, we turn to the rat as a model. The purpose of this study is to compare the ultrasonic vocalization (USV) of rats in three conditions: control, haloperidol-induced transient dopamine depletion, and unilateral 6-hydroxydopamine (6-OHDA) induced moderately-severe degeneration of dopamine neurons. It was hypothesized that both dopamine-altered conditions would lead to a change in the features of the USV acoustic signal. Results demonstrated that bandwidth decreased in the dopamine-altered rats. This is the first study to document a degradation of the acoustic signal of frequency-modulated 50-kHz calls as a result of interfering with dopamine synaptic transmission in rats. The data suggest that mild transient dopamine depletion with haloperidol or even unilateral degeneration of dopamine neurons is associated with changes in the USV acoustic signal. Dopaminergic dysfunction influences USV quality without reducing the number of calls. This study provides a foundation to examine the role of dopamine in sensorimotor processes underlying USV production and potentially to explore treatments for dopamine deficiency-related impaired vocal outcome.

Situational factors, conditions and individual variables which can determine ultrasonic vocalizations in male adult Wistar rats

The fact that rats emit different types of ultrasonic vocalizations in a variety of contexts has received increasing experimental attention, since such calls might serve as indices of the animal's subjective state, and/or as social signals in various types of interactions with other rats. Here, we present two experiments in adult male Wistar rats where we tested several different situations and conditions with respect to the occurrence of high-frequency (50-kHz) and low-frequency (22-kHz) calls. These experiments showed that rats emitted high-frequency calls when tested singly in a housing cage, which was situated in a room with no other rats present. Calling did not habituate with repeated testing, and occurred in the animal's own home cage, or a fresh housing cage, and irrespective of whether the animal's motivational status was high or low, that is, irrespective of whether they were food-deprived or fed ad libitum. Furthermore, high- and low-frequency calls were observed when applying a standardized new tickling procedure, which provided evidence for effective types of tickling. Most, but not all, young adult rats still accepted this stimulation as play. Therefore, this procedure might be a useful method to elicit high-frequency calls in adult rats. Overall, substantial evidence for inter-individual variability and intra-individual stability in vocalization was provided both, within and between housing cage and tickle tests. This variability seems to depend at least partly on dispositions or traits, which can be gauged by specific screening tests, like measuring risk-assessment in the elevated plus-maze, since animals with more risk-assessment were more likely to emit high-frequency calls during cage and tickle tests. These findings are discussed with respect to the major hypotheses concerning the functional significance of ultrasonic vocalizations, namely the social/communicatory and the motivational/emotional hypothesis.

Carbachol injections into the nucleus accumbens induce 50 kHz calls in rats

In rats, different types of vocalization can be observed. High frequency vocalizations (so called 50 kHz calls) are believed to indicate an appetitive state of the emitting animal. This is supported by studies demonstrating that infusions of the dopamine agonist amphetamine into the nucleus accumbens (NAC), a key structure for appetitive behaviors, induce 50 kHz calls. Several studies during the last years demonstrated that not only infusions of dopamine agonists such amphetamine but also infusions of acetylcholine receptor agonists into the NAC stimulate the appetitive system. In present study, we tested whether infusions of the unspecific cholinergic agonist carbachol into the NAC are able to induce 50 kHz calls. Indeed, we observed a high number of 50 kHz calls after intra-NAC infusions of carbachol. The main frequency of the these calls was between 40 and 70 kHz, and the duration was mainly between 10 and 50 ms. We hypothesize that acetylcholine transmission within the NAC plays an important role in the induction of those ultrasonic calls indicating an appetitive state.

Amphetamine-induced 50 kHz calls from rat nucleus accumbens: A quantitative mapping study and acoustic analysis

Emission of 50 kHz ultrasonic calls in rats is known to be associated with appetitive behavioural situations and positive social interactions. The purpose of the study was to pharmacologically characterize amphetamine-induced 50 kHz calls and to perform quantitative mapping of this response in the nucleus accumbens. Injections of amphetamine into the nucleus accumbens induced species-typical 50 kHz calls in adult rats. The acoustic parameters of the calls were not affected by different amphetamine doses or combination of agents. The increase in the number of calls occurred predominantly from the accumbens shell and to a lesser degree from the core region. This effect was dose-dependent within the range of 1–20 μg of amphetamine and was reversed by pretreatment with D 1 or D 2 dopamine antagonists (SKF-83566 or raclopride) administered to the same brain site. However, another D 2 dopamine receptor antagonist, haloperidol, which is known to increase the accumbens dopamine level, was ineffective in reversing the increase in call number at the dose studied. On the contrary, intraacumbens haloperidol, when injected alone, caused an increase in 50 kHz calls. It is concluded that the release of dopamine, predominantly in the accumbens shell region, is responsible for production of 50 kHz calls and the calls may indicate an appetitive state compatible with anticipation of reward and positive affect. Both D 1 and D 2 subtypes of dopamine receptors may be necessary to induce 50 kHz calls and signal the appetitive state.

Overt behavior and ultrasonic vocalization in a fear conditioning paradigm: A dose–response study in the rat

The behavioral analysis of laboratory rats is usually confined to the level of overt behavior, like locomotion, behavioral inhibition, instrumental responses, and others. Apart from such visible outcome, however, behaviorally relevant information can also be obtained when analyzing the animals’ ultrasonic vocalization, which is typically emitted in highly motivational situations, like 22-kHz calls in response to acute or conditioned threat. To further investigate such vocalizations and their relationship with overt behavior, we tested male Wistar rats in a paradigm of Pavlovian fear conditioning, where a tone stimulus (CS) was preceding an aversive foot-shock (US) in a distinct environment. Importantly, the shock dose was varied between groups (0–1.1 mA), and its acute and conditioned outcome were determined. The analysis of visible behavior confirms the usefulness of immobility as a measure of fear conditioning, especially when higher shock doses were used. Rearing and grooming, on the other hand, were more useful to detect conditioned effects with lower shock levels. Ultrasonic vocalization occurred less consistently than changes in overt behavior; however, dose–response relationships were also observed during the phase of conditioning, for example, in latency, call rate and lengths, intervals between calls, and sound amplitude. Furthermore, total calling time (and rate) were highly correlated with overt behavior, namely behavioral inhibition as measured through immobility. These correlations were observed during the phase of fear conditioning, and the subsequent tests. Importantly, conditioned effects in overt behavior were observed, both, to the context and to the CS presented in this context, whereas conditioned vocalization to the context was not observed (except for one rat). In support and extent of previous results, the present data show that a detailed analysis of ultrasonic vocalization can substantially broaden and refine the spectrum of analysis in behavioral work with rats, since it can provide information about situational-, state-, and subject-dependent factors which are partly distinct from what is visible to the experimenter.

Acoustic characteristics of air puff-induced 22-kHz alarm calls in direct recordings

Alarm calls were induced in adult Wistar rats by an air puff. Emitted calls were digitized and directly recorded on a computer hard drive. The long-duration 22-kHz calls were emitted almost exclusively in series. Initial calls in the series tended to have the longest durations, higher frequency range, and the highest degree of frequency modulation, as compared to other calls. The frequency modulation always appeared as a downward sweep and seemed to represent a tuning of individual calls to a 3kHz communicatory band. Regardless of the maximum frequency, rats always reached approximately the same minimum frequency, common to all calls. Thus, the broader was the frequency range of a given call, the longer the call duration. It is postulated, therefore, that rats emit 22-kHz calls at the minimum possible ultrasonic frequency they are able to produce, which is synonymous with peak frequency. It is further postulated that production of alarm calls in series, with long call duration and the invariably low ultrasonic frequency, maximizes successful communication in dangerous situations. Exceptions to this rule were observed immediately following air puffs, suggesting that acoustic parameters of the initial calls may differ from the alarming properties of the remaining 22-kHz calls.

Breeding for 50-kHz Positive Affective Vocalization in Rats

Adolescent and adult rats exhibit at least two distinct ultrasonic vocalizations that reflect distinct emotional states. Rats exhibit 22-kHz calls during social defeat, drug withdrawal, as well as in anticipation of aversive events. In contrast, 50-kHz calls are exhibited in high rates during play behavior, mating, as well as in anticipation of rewarding events. The neurochemistry of 22-kHz and 50-kHz calls closely matches that of negative and positive emotional systems in humans, respectively. The aim of this study was to replicate and further evaluate selective breeding for 50-kHz vocalization, in preparation for the analysis of the genetic underpinnings of the 50-kHz ultrasonic vocalization (USV). Isolate housed adolescent rats (23-26 days old) received experimenter administered tactile stimulation (dubbed "tickling"), which mimicked the rat rough-and-tumble play behavior. This stimulation has previously been shown to elicit high levels of 50-kHz USVs and to be highly rewarding in isolate-housed animals. Each tickling session consisted of 4 cycles of 15 seconds stimulation followed by 15 seconds no stimulation for a total of 2 min, and was repeated once per day across 4 successive days. Rats were then selected for either High or Low levels of sonographically verified 50-kHz USVs in response to the stimulation, and a randomly selected line served as a control (Random group). Animals emitted both 22-kHz and 50-kHz types of calls. After 5 generations, animals in the High Line exhibited significantly more 50-kHz and fewer 22-kHz USVs than animals in the Low Line. Animals selected for low levels of 50-kHz calls showed marginally more 22-kHz USVs then randomly selected animals but did not differ in the rate of 50-kHz calls. These results extend our previous findings that laboratory rats could be bred for differential rates of sonographically verified 50-kHz USVs.