50-kHz Vocalizations Research Articles

Ultrasonic vocalizations near 30 kHz may indicate excitement rather than distress in female Wistar rats

Rats emit ultrasonic vocalizations (USVs), sometimes referred to as 50-kHz vocalizations, during activities such as play and lower-frequency USVs, sometimes referred to as 22-kHz vocalizations, when experiencing distress. Definitions of 22-kHz vocalizations vary in terms of which frequencies should be included, with differing upper limits (e.g., 25 kHz, 30 kHz, or 33 kHz). In the current experiments, we examined the extent to which USVs near the upper frequency limit may represent milder anxiety than lower-frequency vocalizations. In Experiment 1, rats received both gentle tickling and simulated rough-and-tumble play with a human hand, while low-frequency (i.e.,18–33 kHz) and high-frequency (i.e., 34–90 kHz) USVs were recorded; rough-and-tumble play elicited significantly more low-frequency USVs (median 29.5 kHz), but also significantly more high-frequency USVs (median 50.7 kHz). In Experiment 2, rats were presented with 23-kHz, 32-kHz, and 52-kHz USVs while foraging in a T-maze. The 23-kHz USV elicited the most freezing, while the 32-kHz USV elicited significantly less freezing than either the 23- or the 52-kHz USV. Taken together, these results suggest that within the range of frequencies referred to as 22-kHz vocalizations, lower-frequency USVs may indicate a more negative affective state than higher-frequency ones. As USV production can serve as an indicator of rat welfare, an understanding of what frequencies represent positive versus negative affective states is crucial.

Stable long-term individual differences in 50-kHz vocalization rate and call subtype prevalence in adult male rats: Comparisons with sucrose preference.

Sucrose preference (SP) is a widely used measure of anhedonia in rat models of depression, yet depressed patients do not reliably show an analogous deficit. As an alternative affect-related measure, adult rat ultrasonic vocalizations (USVs) are attracting interest, but it is unclear whether SP and USVs provide independent measures. Here, we have assessed whether SP and USV emission are correlated in the absence of a depressogenic procedure. To this end, 24 male Long-Evans rats were tested daily for 24 days, with alternating SP tests and USV recordings; after a 3-month hiatus, USV emission was re-evaluated for 6 more days. SP was measured in simultaneous two-bottle choice tests, and USVs were recorded in an open field. The main measures were: SP, 50-kHz call rate, and relative prevalence of trill and flat call subtypes. These measures showed temporally-stable individual differences across the initial 24-day testing period, and at the 3-month USV follow-up tests. Correlational analysis revealed no significant relationships between SP and the three main USV measures. Rats differed consistently, not only in their 50-kHz call rates but also in their 50-kHz call profiles (i.e., the relative prevalence of 14 call subtypes); most rats preferentially emitted either trill or flat calls. Several inter-call subtype associations were detected, including a strong negative relationship between the relative prevalence of flat and trill calls. The 50-kHz call rate was correlated with the relative prevalence of only one call subtype (short calls, negative correlation), but was positively correlated with absolute emission rates for almost all subtypes. In conclusion, adult rats exhibited temporally-stable individual differences over weeks (SP) or months (USVs) of testing. This trait-like stability helped to reveal a lack of relationship between SP and the USV-related variables under study, suggesting that these measures may capture different constructs of possible relevance to animal models of depression.

Multimodal cues displayed by submissive rats promote prosocial choices by dominants

Animals often display prosocial behaviors, performing actions that benefit others. Although prosociality is essential for social bonding and cooperation, we still know little about how animals integrate behavioral cues from those in need to make decisions that increase their well-being. To address this question, we used a two-choice task where rats can provide rewards to a conspecific in the absence of self-benefit and investigated which conditions promote prosociality by manipulating the social context of the interacting animals. Although sex or degree of familiarity did not affect prosocial choices in rats, social hierarchy revealed to be a potent modulator, with dominant decision-makers showing faster emergence and higher levels of prosocial choices toward their submissive cage mates. Leveraging quantitative analysis of multimodal social dynamics prior to choice, we identified that pairs with dominant decision-makers exhibited more proximal interactions. Interestingly, these closer interactions were driven by submissive animals that modulated their position and movement following their dominants and whose 50-kHz vocalization rate correlated with dominants' prosociality. Moreover, Granger causality revealed stronger bidirectional influences in pairs with dominant focals and submissive recipients, indicating increased behavioral coordination. Finally, multivariate analysis highlighted body language as the main information dominants use on a trial-by-trial basis to learn that their actions have effects on others. Our results provide a refined understanding of the behavioral dynamics that rats use for action-selection upon perception of socially relevant cues and navigate social decision-making.

Prenatal exposure to valproic acid and treatment with intranasal oxytocin have sex-specific effects on behavior in Long Evans rats.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social behaviors and communication. In rodents and humans, prenatal exposure to antiepileptic valproic acid is associated with an increased risk for autistic-like characteristics. One potential treatment is oxytocin, a prosocial neuropeptide that can be delivered intranasally. However, the sex-specific effects of valproic acid exposure and intranasal oxytocin treatment on behavior have not been fully explored. Pregnant Long Evans rats were administered valproic acid (500 mg/kg) or saline midday on gestational day 12, and after weaning, male and female pups were assigned to control (saline-saline), valproic acid-saline, or valproic acid-oxytocin groups. Oxytocin (0.8 IU/kg) or saline was delivered intranasally 30-60 min before tests for anxiety-like behaviors (elevated plus maze), social interactions (sociability) and sociosexual behaviors (partner preference, 50 kHz vocalizations and scent marking). Prenatal exposure to valproic acid resulted in sex-specific differences in behavior. When compared to controls, valproic acid males showed enhanced anxiety-like behaviors in adolescence and fewer scent marks in adulthood, while valproic acid females showed reduced sexual (partner) preference as adults. Intranasal oxytocin was anxiolytic for valproic acid males, but moderately anxiogenic for valproic acid females, and in both sexes it surprisingly impaired social interactions in the sociability test. Furthermore, intranasal oxytocin failed to improve sociosexual deficits in valproic acid rats. These findings highlight the importance of conducting preclinical studies in both sexes, and suggest that oxytocin may be an effective treatment in animal models with heightened anxiety-like behaviors.

Rat 50 kHz Trill Calls Are Tied to the Expectation of Social Interaction.

Rats emit a variety of calls in the 40–80 kHz range (50 kHz calls). While these calls are generally associated with positive affect, it is unclear whether certain calls might be used selectively in certain contexts. To examine this, we looked at ultrasonic calls in 30–40 day old male rats during the expectation of either play or food, both of which are reinforcing. Behavior and vocalizations were recorded while rats were in a test chamber awaiting the arrival of a play partner or food over seven days of testing. Control groups were included for the non-specific effects of food deprivation and social isolation. Play reward led to an increase in 50 kHz vocalizations, generally, with specific increases in trill and “trill with jump” calls not seen in other groups. Expectation of food reward did not lead to a significant increase in vocalizations of any type, perhaps due to the young age of our study group. Further, rats that were food deprived for the food expectation study showed markedly lower calls overall and had a different profile of call types compared to rats that were socially isolated. Taken together, the results suggest that trill-associated calls may be used selectively when rats are socially isolated and/or expecting a social encounter.

Distinct Profiles of 50 kHz Vocalizations Differentiate Between Social Versus Non-social Reward Approach and Consumption.

Social animals tend to possess an elaborate vocal communication repertoire, and rats are no exception. Rats utilize ultrasonic vocalizations (USVs) to communicate information about a wide range of socially relevant cues, as well as information regarding the valence of the behavior and/or surrounding environment. Both quantitative and qualitative acoustic properties of these USVs are thought to communicate context-specific information to conspecifics. Rat USVs have been broadly categorized into 22 and 50 kHz call categories, which can be further classified into subtypes based on their sonographic features. Recent research indicates that the 50 kHz calls and their various subtype profiles may be related to the processing of social and non-social rewards. However, only a handful of studies have investigated USV elicitation in the context of both social and non-social rewards. Here, we employ a novel behavioral paradigm, the social-sucrose preference test, that allowed us to measure rats’ vocal responses to both non-social (i.e., 2, 5, and 10% sucrose) and social reward (interact with a Juvenile rat), presented concurrently. We analyzed adult male Long-Evans rats’ vocal responses toward social and non-social rewards, with a specific focus on 50 kHz calls and their 14 subtypes. We demonstrate that rats’ preference and their vocal responses toward a social reward were both influenced by the concentration of the non-social reward in the maze. In other words, rats showed a trade-off between time spent with non-social or social stimuli along with increasing concentrations of sucrose, and also, we found a clear difference in the emission of flat and frequency-modulated calls in the social and non-social reward zones. Furthermore, we report that the proportion of individual subtypes of 50 kHz calls, as well as the total USV counts, showed variation across different types of rewards as well. Our findings provide a thorough overview of rat vocal responses toward non-social and social rewards and are a clear depiction of the variability in the rat vocalization repertoire, establishing the role of call subtypes as key players driving context-specific vocal responses of rats.

Biological Functions of Rat Ultrasonic Vocalizations, Arousal Mechanisms, and Call Initiation.

This review summarizes all reported and suspected functions of ultrasonic vocalizations in infant and adult rats. The review leads to the conclusion that all types of ultrasonic vocalizations subserving all functions are vocal expressions of emotional arousal initiated by the activity of the reticular core of the brainstem. The emotional arousal is dichotomic in nature and is initiated by two opposite-in-function ascending reticular systems that are separate from the cognitive reticular activating system. The mesolimbic cholinergic system initiates the aversive state of anxiety with concomitant emission of 22 kHz calls, while the mesolimbic dopaminergic system initiates the appetitive state of hedonia with concomitant emission of 50 kHz vocalizations. These two mutually exclusive arousal systems prepare the animal for two different behavioral outcomes. The transition from broadband infant isolation calls to the well-structured adult types of vocalizations is explained, and the social importance of adult rat vocal communication is emphasized. The association of 22 kHz and 50 kHz vocalizations with aversive and appetitive states, respectively, was utilized in numerous quantitatively measured preclinical models of physiological, psychological, neurological, neuropsychiatric, and neurodevelopmental investigations. The present review should help in understanding and the interpretation of these models in biomedical research.

Active space of grasshopper mouse vocalizations (Onychomys) in relation to woody plant encroachment

Abstract The efficacy of animal acoustic communication depends on signal transmission through an oft-cluttered environment. Anthropogenic-induced changes in vegetation may affect sound propagation and thus habitat quality, but few studies have explored this hypothesis. In the southwestern United States, fire suppression and cattle grazing have facilitated displacement of grasslands by pinyon-juniper woodlands. Northern grasshopper mice (Onychomys leucogaster) inhabit regions impacted by juniper encroachment and produce long-distance vocalizations to advertise their presence to conspecifics. In this study, we coupled acoustic recordings and electrophysiological measurements of hearing sensitivity from wild mice in the laboratory with sound transmission experiments of synthesized calls in the field to estimate the active space (maximum distance that stimuli are detected) of grasshopper mouse vocalizations. We found that mice can detect loud (85 dB SPL at 1 m) 11.6 kHz vocalizations at 28 dB SPL. Sound transmission experiments revealed that signal active space is approximately 50 m. However, we found no effect of woody plant encroachment on call propagation because juniper and woody plant density were inversely associated and both present barriers to a 9 cm mouse advertising at ground level. Our data indicate that woody plant encroachment does not directly impact the efficacy of grasshopper mouse communication, but vegetation shifts may negatively impact mice via alternative mechanisms. Identifying the maximum distance that vocalizations function provides an important metric to understand the ecological context of species-specific signalling and potential responses to environmental change.

Treatment of electrical wrist stimulation reduces chemotherapy-induced neuropathy and ultrasound vocalization via modulation of spinal NR2B phosphorylation

Docetaxel, a chemotherapeutic agent used to treat breast cancer, produces a robust painful neuropathy that is aggravated by mechanical and thermal stimuli. This study was undertaken to investigate the analgesic effects of electrical stimulation on docetaxel-induced neuropathic pain in mice and to identify associated changes in ultrasound vocalizations. Peripheral neuropathy was induced with intraperitoneally injected docetaxel (5 mg/kg) on 3 times every 2 days in male ICR mice. Electrical wrist stimulation was administered and pain behavior signs were evaluated by von Frey filaments and thermal stimulation on the hind paw. Ultrasound vocalizations were measured using ultrasound microphones, after electrical stimulation. After mice developed docetaxel-induced neuropathic pain behavior, an electrical stimulation temporarily attenuated mechanical allodynia and thermal hyperalgesia. In formalin and NMDA test, pain-induced mice showed increases in 10−30 kHz ultrasound vocalizations, but not in 30–50 and 50−80 kHz vocalizations. Treatment with docetaxel selectively increased 10−30 kHz ultrasound vocalizations, whereas electrical stimulation caused a meaningful decrease. Moreover, electrical stimulation suppressed the docetaxel-enhanced phosphorylation of the NMDA receptor NR2B subunit in the spinal dorsal horn. These results of the analgesic effect of electrical stimulation in chemotherapy-induced neuropathy could potentially provide a new method to treat and manage peripheral neuropathy in patients with cancer.

Dissimilar interaction between dopaminergic and cholinergic systems in the initiation of emission of 50-kHz and 22-kHz vocalizations

Rats emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) to signal their emotional state to other conspecifics. The 22-kHz USVs signal a negative emotional state while 50-kHz USVs reflect a positive affective state. The initiation of 22-kHz USVs is dependent on the activity of cholinergic neurons within the laterodorsal tegmental nucleus that release acetylcholine along the medial cholinoceptive vocalization strip. Emission of 50-kHz USVs is dependent upon the activation of dopaminergic neurons located within the ventral tegmental area that release dopamine into the medial shell of the nucleus accumbens. There have been reports that showed an antagonistic interaction between acetylcholine and dopamine during the expression of emotional states, and dopamine agonists decreased carbachol-induced emission of 22-kHz USVs. The current study tests the hypothesis that initial antagonism of dopamine receptors by systemic haloperidol or intraacumbens raclopride should increase the subsequent emission of 22 kHz USVs induced by carbachol from the lateral septum. Our findings showed that antagonism of dopaminergic signaling either via systemic haloperidol or via intracerebral raclopride did not alter the number of emitted 22-kHz USVs. Thus, inhibition of the mesolimbic dopamine system did not increase the magnitude of a negative emotional state. It was found, however, that prolonged emission of 22-kHz USVs initiated by carbachol caused a delayed rebound emission (R) of 50-kHz USVs appearing after 300 s of emission of 22-kHz USVs, i.e., when the response was subsiding. The R-50-kHz USVs were predominantly frequency modulated (FM) USVs and their number was directly proportional to the number of recorded 22-kHz USVs. The emission of R-50-kHz USVs was reversed by systemic pretreatment with haloperidol or intraacumbens injection of raclopride. It is argued that the R-50-kHz USVs represent a rebound emotional state that is opposite in valence and arousal induced by carbachol. Importantly, prolonged emission of amphetamine-induced 50 kHz USVs failed to show any vocalization rebound effect.

Association of medial corticostriatal regions with amphetamine-induced emission of 50 kHz vocalizations as studied by Zif-268 expression in the rat brain

Behavioural sensitization of locomotion and 50 kHz ultrasonic vocalizations (USVs) following repeated amphetamine (AMPH) injections in rats has been extensively demonstrated. These two behaviours appear dissociable in their sensitization patterns and are thought to be reflective of underlying emotional states of the organism. Although AMPH is often used to induce 50 kHz USVs there is little research to date on the extent of cortical and subcortical forebrain region involvement in 50 kHz call production associated with the drug. Nor has general ergometric activity (a measure that in addition to locomotor activity includes all major muscular activity of the body) been investigated in such a framework. The present study sought to address this by performing a minimal sensitization protocol, utilizing only two injections, to investigate expression of the inducible transcription factor Zif-268 (Zif) among brain regions thought to be associated with 50 kHz USV emission. It was found that animals that spent a longer time emitting 50 kHz calls after a second AMPH injection showed statistically significant correlative patterns of Zif expression in medial prefrontal and striatal regions. These associations were not significant in animals that spent a shorter period of time calling after AMPH. There was also no significant correlation between any ergometric activity and time spent calling. The results provide evidence that the medial prefrontal cortices (prelimbic and infralimbic regions) of the rat may be involved with 50 kHz USV emission induced by AMPH in association with medial portions of the ventral and dorsal striatum.

Alterations of acoustic features of 50 kHz vocalizations by nicotine and phencyclidine in rats.

Ultrasonic vocalizations are widely used to examine affective states in rats, yet relatively few studies explore the acoustic features of vocalizations, especially in relation to drug exposure, and no studies have explored alterations in acoustic features over time. The goal of this study was to examine nicotine- and phencyclidine-induced alterations of bandwidth, duration, and frequency of 50 kHz vocalizations. The minimum and maximum frequency, bandwidth, and duration of calls were examined after 7 days of daily subcutaneous administration of phencyclidine (2.0 mg/kg) and nicotine (0.2 and 0.4 mg/kg) in male Sprague-Dawley rats. Bandwidth was significantly decreased in rats treated with both nicotine (0.2 and 0.4 mg/kg) and phencyclidine. Maximum frequency was lowest on the first day of exposure compared with all other days and was not altered by drug exposure. Call duration was not affected by time or drug exposure. These findings suggest the importance of studying alterations in acoustic features in time, especially those induced by drug exposure.

Effects of anxiogenic drugs on the emission of 22- and 50-kHz ultrasonic vocalizations in adult rats.

Adult rat 22-kHz vocalizations are often associated with alarm or distress, whereas a subset of 50-kHz calls is preferentially emitted in response to amphetamine and other rewarding stimuli. Whether any 50-kHz calls reflect anxiety is unknown. To determine the effects of anxiogenic drugs on 50-kHz call rate and call subtype profile, in comparison with D-amphetamine. Adult male rats received systemic amphetamine (1mg/kg) three times several days before testing. Ultrasonic vocalizations were then recorded after acute intraperitoneal injection of amphetamine or one of five anxiogenic drugs: yohimbine (2.5mg/kg), N-methyl-β-carboline-3-carboxamide (FG 7142, 5mg/kg), pentylenetetrazol (PTZ, 20mg/kg), m-chlorophenylpiperazine (mCPP, 1mg/kg), caffeine (25mg/kg), or vehicle. The duration of immobility was increased by FG 7142, PTZ, and mCPP; this measure was unchanged by yohimbine and reduced by the locomotor stimulant drugs amphetamine and caffeine. Conversely, the 50-kHz call rate was reduced by FG 7142, PTZ and mCPP, and increased by caffeine and amphetamine. Overall, the most common 50-kHz call subtypes were flat, trill, step-up, and complex. Consistent with previous reports, amphetamine increased the relative prevalence of trill calls while reducing the relative prevalence of flat calls. Yohimbine and caffeine reduced flat call prevalence, whereas mCPP reduced trill call prevalence. No other shifts in the call profile were observed, and no anxiogenic drug induced 22-kHz calls. Anxiogenic drugs, as a class, did not uniformly alter the 50-kHz call rate or subtype profile. Amphetamine-induced effects on 50-kHz call rate and profile do not reflect anxiety.

Chronic stress sensitizes amphetamine-elicited 50-kHz calls in the rat: Dependence on positive affective phenotype and effects of long-term fluoxetine pretreatment

High level of positive affectivity acts as a protective factor against adverse effects of stress and decreases vulnerability to mood disorders and drug abuse. Fifty-kHz ultrasonic vocalizations (50-kHz USV) index the level of positive affect in the rat, whereas stable, trait-like inter-individual differences in terms of vocalization activity exist. Previously we have demonstrated that chronic stress can alter the effect of repeated amphetamine administration on 50-kHz vocalizations, and this effect is different in rats with high and low positive affectivity. In the present study it was tested whether the chronic stress effect on amphetamine-induced 50-kHz USV activity is altered by inhibition of serotonin reuptake. Male Wistar high (HC) and low (LC) 50-kHz vocalizing rats were subjected to 43-day chronic variable stress (CVS) regimen. On day 17 of the CVS, the four-week once a day fluoxetine (10 mg/kg) treatment was started. After the CVS and fluoxetine treatment, amphetamine (1 mg/kg) was daily administered for ten days and again nine days after withdrawal. Chronically stressed rats developed cross-sensitization of 50-kHz USV-s with repeated administration of amphetamine except the stressed LC rats that had not received fluoxetine. Amphetamine treatment decreased serotonin turnover in the fluoxetine-treated HC rats, but increased it in fluoxetine-treated LC rats. The effect of amphetamine on levels of amino acids in frontal cortex and hippocampus also depended on previous experience with chronic stress, repeated treatment with fluoxetine, and positive affectivity. Hence, this study provides further evidence the effects of chronic stress, psychostimulants, and a selective serotonin reuptake inhibitor are influenced by the inherent positive affectivity.

Do juvenile rats use specific ultrasonic calls to coordinate their social play?

Play fighting in juvenile rats is associated with a high occurrence of 50 kHz vocalizations. These calls are varied in form, ranging from long, flat calls to short, frequency-modulated ones. We hypothesize that at least some types of calls serve as play signals to facilitate play. In the present study, pairs of juvenile male rats that were unfamiliar with one another were paired in a neutral test enclosure to which they had been habituated. Video and audio records were made of the encounters. Pairs were of two types: both pairs could vocalize or only one partner could do so. There were some differences between the play of pairs containing a devocalized partner, but overall, the pattern of play, the frequency and types of calls were similar between the two types of pairs. We used a Monte Carlo shuffling technique to analyse the correlations between the playful actions performed and the types and frequencies of various 50 kHz calls that were produced. The analyses revealed that there were strong associations between types of calls and types of social contact: an approach followed by playful nape contact was associated with calls, but an approach followed by nonplayful contact (e.g. anogenital sniffing) was not. Similarly, different calls were associated with different actions, such as nape contact, evade and wrestling, with most of these calls being uttered by the initiator of the action, not the recipient. However, coordinating calls reciprocally with complementary calls uttered by participants as they engaged in complementary actions (e.g. attacking, being attacked) appeared to be a way in which calls could potentially be used as play signals to influence the ongoing cooperation needed to sustain play fights.

Specific 50-kHz vocalizations are tightly linked to particular types of behavior in juvenile rats anticipating play.

Rat ultrasonic vocalizations have been suggested to be either a byproduct of physical movement or, in the case of 50-kHz calls, a means to communicate positive affect. Yet there are up to 14 distinct types of 50-kHz calls, raising issues for both explanations. To discriminate between these theories and address the purpose for the numerous 50-kHz call types, we studied single juvenile rats that were waiting to play with a partner, a situation associated with a high number of 50-kHz calls. We used a Monte-Carlo shuffling procedure to identify vocalization-behavior correlations that were statistically different from chance. We found that certain call types (“split”, “composite” and “multi-step”) were strongly associated with running and jumping while other call types (those involving “trills”) were more common during slower movements. Further, non-locomotor states such as resting and rearing were strongly predictive of a lack of vocalizations. We also found that the various sub-types of USVs can be clustered into 3–4 categories based on similarities in the way they are used. We did not find a one-to-one relationship between any movements and specific vocalizations, casting doubt on the motion byproduct theory. On the other hand, the use of specific calls during specific behaviors is problematic for the affect communication hypothesis. Based on our results, we suggest that ultrasonic calls may serve to coordinate moment-to-moment social interactions.

The effects of 100 dB 1‐kHz and 22‐kHz tones as punishers on lever pressing in rats

Aversive control is an important yet understudied process of learning. One reason aversive control may be relatively understudied is ethical concerns about painful stimuli (e.g., electric shock). High decibel broad-band noise and 22-kHz vocalizations both demonstrably affect rodent behavior while not necessarily being painful. The goal of this study was to determine if 100-dB 22-kHz-pure tones were differentially more effective in reducing operant response rates in rats. We examined whether 22-kHz pure tones would function as aversive stimuli, specifically as positive punishers. The effects of response-dependent as well as continuously presented 22-kHz and 1-kHz tones on rate of response maintained by variable interval 30-s food deliveries were assessed across several conditions. We found that response rates were lower when tones were presented response dependently than when tones were presented continuously throughout a session. We also found that the lower response rates obtained with response-dependent 22-kHz tones were not significantly different from response rates obtained with response-dependent 1-kHz tones. The primary conclusion of this experiment is that both 1-kHz and 22-kHz tones functioned as punishers, but that the 22-kHz tones were not differentially more effective in reducing response rate.

Affective valence of neurons in the vicinity of the rat amygdala: Single unit activity in response to a conditioned behavior and vocal sound playback

We recorded single unit activity within and around the rat amygdala while rats were engaged in an operant task (which included both reward and aversive trials) and during playback of ultrasonic vocalizations (USVs) to determine if there existed neurons which responded to two different types of either positive contexts (i.e., water reward and positively associated 50kHz vocalizations) or negative contexts (i.e., white noise and negatively associated 22kHz vocalizations). Ultimately, we wanted to determine if these two contexts (operant condition task and vocal sounds) could be represented as either positive or negative in a single neuron. Neural activity in 90% of cells was modulated in response to one or more of those events. A small number of those cells showed neural responses to both the aversive operant trials and 22kHz USVs, but did not show responses to reward operant trials or 50kHz USVs, suggesting the activity of these neurons encodes for similar negative emotion in response to these two contexts. Some cells showed responses to either the reward trials or 50kHz USVs, but no cells showed responses to both, suggesting that these cells do not show a common response to events associated with positive emotion. This might mean that 50kHz vocal sounds and the operant rewards were segregated into two different categories within the neural representation at the level of the amygdala, even though it appeared that both events were associated with positive emotions in rats.

Differential patterns of constant frequency 50 and 22 khz usv production are related to intensity of negative affective state.

Adult rat ultrasonic vocalizations (USVs) are a valuable tool for noninvasively assessing an animal's emotional state. USVs are produced in 1 of 2 frequency ranges labeled as 22 kHz or 50 kHz vocalizations. One USV subtype within the 50 kHz call category, constant frequency 50 kHz (CF 50 kHz) calls, is not viewed as signaling an emotional state. The current study tested the hypothesis that CF 50 kHz calls are related to a mild negative affective state. In Experiment 1, diazepam (1, 2.5, or 5 mg/kg), or control injections were administered prior to receiving a sequence of mild footshocks (0.5 mA, 0.5 s). Subjects transitioned from producing CF 50 to 22 kHz USVs as footshocks were repeated; a pattern paralleled by a shift from rearing to increased time freezing. USV production was largely absent in the higher diazepam dose groups, whereas the 1 mg/kg dose attenuated CF 50 kHz USV production prior to and immediately following initial footshocks. The higher doses of diazepam similarly reduced rearing activity and overall freezing behavior. In Experiment 2, pre-exposure to the testing environment with or without access to palatable food elicited CF 50 kHz calls and rearing. During re-exposure to the test chamber the following day, CF 50 kHz USV production was reduced prior to footshock onset compared to the prior test day. The pattern of results support an association between CF 50 kHz USVs and a mild negative affective state; dissociating this call type may increase the sensitivity of behavioral measurements of emotion. (PsycINFO Database Record

Neurotensin NTS1 and NTS2 receptor agonists produce anxiolytic-like effects in the 22-kHz ultrasonic vocalization model in rats

Neurotensin is a neuropeptide neurotransmitter that interacts with multiple neurotransmitter systems, including those regulating amygdalar function, via NTS1 and NTS2 receptors. Both receptors are expressed in the amygdala and agonists for NTS1 or NTS2 receptors have exhibited anxiolytic effects in animal models. Systemic adminstration of NTS1 receptor agonist PD149163 was recently shown to reduce footshock conditioned 22-kHz ultrasonic vocalizations in rats, suggesting that PD149163 produced an anxiolytic effect. The effects that neurotensin may have or a selective NTS2 receptor agonist may have on 22-kHz vocalizations has yet to be examined. The current study evaluated the effects of intracerebroventricularly administered neurotensin (0.1–10.0μg), PD149163 (0.1–10.0ng), or the NTS2 receptor agonist JMV-431 (0.1–1.0μg) on footshock conditioned 22-kHz vocalizations in male Wistar rats. Neurotensin, PD149163, and JMV-431 all significantly reduced the number 22-kHz calls. No changes in call duration were found, suggesting that non-specific drug effects do not account for the reductions in 22-kHz calls. These data support anxiolytic effects produced by activation of NTS1 or NTS2 receptors, and suggest that neurotensin plays a natural role in the expression of conditioned USVs. These data suggest that both receptor subtypes are putative pharmacologic targets.