Emission Of Ultrasonic Vocalizations Research Articles

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

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

Testosterone regulates the emission of ultrasonic vocalizations and mounting behavior during different developmental periods in mice.

Testosterone masculinizes male sexual behavior by providing organizational and activational effects during the perinatal and peripubertal periods and during adulthood, respectively. We revealed that the emission of ultrasonic vocalizations (USVs) and mounting behavior was regulated by different neural circuits. However, the detailed testosterone effects on these two behaviors have not been fully elucidated. Here, we evaluated the time-dependent effects of testosterone on USVs and mounting behavior in mice using a testosterone treatment model, in which females were treated with testosterone to assess the "gain-of-function" and a "loss-of-function" model. In the loss-of-function model, we used Ad4BP/SF-1ΔFLC/- male mice, in which testosterone production was abolished in prenatal and postnatal stages, and Ad4BP/SF-1ΔFLC/ΔFLC mice, in which testosterone production was markedly reduced only in prenatal stages. When testosterone was administered to female mice during the neonatal and peripubertal periods, but not during adulthood, USV emissions increased. Conversely, testosterone treatment in adult female mice increased the mounting behavior, but not USVs. In Ad4BP/SF-1ΔFLC/- mice, USVs and mounting behavior was completely absent. Ad4BP/SF-1ΔFLC/ΔFLC male mice displayed equivalent levels of USVs but less mounting behavior. Collectively, these results suggest that testosterone has dual regulatory roles in USV emissions and mounting behavior.

Ultrasonic courtship vocalizations of male house mice contain distinct individual signatures

Acoustic communication mediates many types of social interactions; however, few studies have investigated whether courtship vocalizations contain distinctive individual signatures necessary for individual recognition. Male house mice, Mus musculus, produce spectrally complex ultrasonic vocalizations (USVs) during courtship and mating, which appear to attract females and increase male reproductive success. Our goals were to (1) describe quantitative and qualitative changes in the vocalizations of wild-derived male house mice, M. m. musculus, induced by a female odour stimulus; (2) measure individual variation and consistency in male USV emission over time; (3) test whether the variation in USVs is greater between than within individuals; and (4) identify individual signatures in spectrotemporal features using univariate statistics, multivariate models and machine learning methods. We recorded males once per week for 3 weeks, used an automated method (A-MUD) to detect USVs, and manually classified them into distinct syllables. We found that most males did not vocalize until they encountered female scent, and then most males dramatically increased the number and the types of different vocalizations (repertoire size and composition). Male USVs showed high interindividual variation and most showed intraindividual consistency, and we found greater inter- than intraindividual variation for both USV count and repertoire size. Male USVs contained individual signatures in most spectrotemporal features, regardless of the method of analysis. Males sometimes produced few, if any, vocalizations when presented with female scent, but consistent nonvocalizers were rare. Our study provides the first evidence that individual signatures in USVs of male house mice are stable over time and across recording trials, although studies are still needed to investigate repeatability across social contexts and to test whether USVs mediate individual recognition.

Ultrasonic vocalizations and individual differences in rats performing a Pavlovian conditioned approach task

Rats emit distinct types of ultrasonic vocalizations (USV), including high-frequency 50-kHz USV, which occur in appetitive situations. Such 50-kHz USV are thought to reflect positive affective states, for example in case of reward anticipation, and are linked to dopamine signaling. The present study was conducted to investigate whether rats emit 50-kHz USV during a Pavlovian conditioned approach task and whether trait-like differences in 50-kHz USV emission are associated with sign- versus goal-tracking. We hypothesize that individuals engaging more with a cue predicting a food reward will also elicit more 50-kHz USV. In order to test this, we investigated 34 female rats and gauged USV while they underwent a Pavlovian conditioned approach training and extinction paradigm. For one, we found a high subject-dependent variability in the emission of 50-kHz calls. These were not largely affected by state differences, since these 50-kHz USV were observed throughout the task. During task progress and in most subjects, there was a rather complete shift toward goal-tracking, but subjects engaging more with the cue predicting a reward also emitted higher numbers of appetitive 50-kHz calls. This supports the hypothesis that sign-tracking is positively associated with the emission of 50-kHz USV. The high subject-dependent variability in the emission of 50-kHz calls warrants special attention in future appetitive studies.

Effects of pubertal exposure to low doses of di-(2-ethylexyl)phthalate on reproductive behaviors in male mice

Reproductive behaviors are tightly regulated by sex steroid hormones. Interference with these hormones or their neural signaling pathways leads to behavioral alterations. We have previously shown that oral exposure of adult male mice to di(2-ethylhexyl) phthalate (DEHP), an organic environmental endocrine disruptor, altered sexual behavior. In this study, we examined the effects of pubertal exposure to DEHP and analyzed whether pubertal and adult exposures to DEHP trigger long-term effects. For pubertal exposure, male mice were exposed orally to the vehicle or DEHP at 5 or 50 μg/kg/d from postnatal day (PND) 30 to PND60. Exposure was arrested and animals were analyzed on PND120. They exhibited normal olfactory preference but showed modified emission of ultrasonic vocalizations. DEHP exposure also affected partner preference and mating components. These modifications were associated with normal circulating testosterone levels and weight of androgen-sensitive tissues. In contrast, androgen receptor (AR) protein amount was reduced in the hypothalamic preoptic area in particular for the DEHP-50 group. Pubertal exposure also increased the anxiety-state level without changing circadian activity. When adult male mice were exposed to DEHP at the same doses from PND60 to PND105 and analyzed two months later, no effects of treatment on reproductive and anxiety-related behaviors or hypothalamic AR protein amount were seen.Our data show that pubertal exposure of male mice to DEHP induces long-term behavioral changes in contrast to the adult exposure. This highlights the sensitivity of the nervous system to low doses of DEHP during the critical period of puberty.

Reduced emission of alarm 22-kHz ultrasonic vocalizations during fear conditioning in rats lacking the serotonin transporter

Rats display a rich social behavioral repertoire. An important component of this repertoire is the emission of whistle-like calls in the ultrasonic range, so-called ultrasonic vocalizations (USV). Long low-frequency 22-kHz USV occur in aversive situations, including aggressive interactions, predator exposure, and electric shocks during fear conditioning. They are believed to reflect a negative affective state akin to anxiety and fear. A prominent theory suggests that 22-kHz USV function as alarm calls to warn conspecifics. Serotonin (5-hydroxytryptamine, 5-HT) is strongly implicated in the regulation of affective states, particularly anxiety and fear. A key component of the system is the 5-HT transporter (5-HTT, also known as SERT), regulating 5-HT availability in the synaptic cleft. In the present experiment, we studied the effects of SERT deficiency on overt fear-related behavior and alarm 22-kHz USV during fear conditioning in male and female rats. While overt fear-related behavior was not affected by SERT deficiency and sex, the emission of alarm 22-kHz USV was clearly reduced in homozygous SERT−/− but not heterozygous SERT+/− mutants, as compared to their wildtype SERT+/+ littermate controls. Genotype effects were particularly prominent in females. Females in general emitted fewer alarm 22-kHz USV than males. This supports the view that 22-kHz USV are, at least partly, independently regulated from anxiety or fear and as socially mediated alarm calls do not simply express a negative affective state. Reduced 22-kHz USV emission in rats lacking SERT might be due to social deficits in the use of 22-kHz USV as a socio-affective signal to warn conspecifics about threats.

Effects of prenatal exposure to valproic acid or poly(I:C) on ultrasonic vocalizations in rat pups: The role of social cues

Sociocommunicative deficits commonly observed in autism spectrum disorder (ASD) can be experimentally modeled using rodents’ ultrasonic vocalizations (USVs). For example, USVs emitted by pups, separated from their mothers and nest, serve as a useful tool to identify autistic-like behaviors during the early period of development. Being sensitive to social context, these neonatal calls may help to reveal reduced social attachment or abnormal processing of social information.The aim of the present study was to characterize quantitative and structural changes in USVs emitted during isolation by male and female rat pups prenatally exposed to either valproic acid (VPA) or poly(I:C). To determine whether those pups differed from controls in sensitivity to social stimuli, isolation-induced USVs were recorded under two bedding conditions, i.e., novel bedding and soiled bedding from their home cages.Our results demonstrated early communication deficits in both models of autism. We reported a reduced number of USVs emitted by both VPA- and poly(I:C)-exposed males and females. Moreover, compared to the controls, VPA (but not poly(I:C)) pups emitted shorter calls with a higher peak frequency. While VPA offspring demonstrated fewer USVs on the “safe” bedding imbued with maternal/nest odors, this calming effect was not observed in poly(I:C) males, suggesting a more specific deficit in social communication.The present results demonstrate that qualitative along with quantitative analyses of neonatal vocalizations are a useful tool for assessing early sociocommunicative deficits in ASD models. Notably, more specific changes in USV emission may be detected when introducing social context.

Social deprivation substantially changes multi-structural neurotransmitter signature of social interaction: Glutamate concentration in amygdala and VTA as a key factor in social encounter-induced 50-kHz ultrasonic vocalization

Ultrasonic vocalizations are important for coordinating social behavior in rats. Examination of the neurochemical mechanisms that govern social behavior and ultrasonic vocalization emission is crucial for understanding the social impairments that occur in many neuropsychiatric disorders. To elucidate neurochemical changes in the brain structures related to social behavior and their mutual relationships, we conducted three-phase experiment. Neurochemicals were measured in the following behavioral situations: without social encounter, with short social encounter, with long social encounter in isolated and non-isolated rats. The aims of this study were to: (1) extract the most important neurotransmitters and their metabolites that are involved in social encounter-induced emission of 50 kHz calls; (2) to elucidate mutual relationships among the neurochemical changes in the selected, six brain structures, and analyze compound relationships by step analysis; (3) create a model of all-to-all neurotransmitter correlations; (4) find the neurochemical basis of 50-kHz USVs emission during social encounter. Our behavioral and neurochemical analysis indicated that social encounter was a triggering factor of the glutamatergic neurotransmission in the ventral tegmental area (VTA), hippocampus, and amygdala; serotonergic neurotransmission in the NAcc, CPu, and amygdala; the dopaminergic neurotransmission in the caudate putamen (CPu) and hippocampus; GABAergic neurotransmission in the hippocampus and VTA. Social encounter-induced 50-kHz USVs were bound up with changes in glutamate in amygdala and VTA, glycine in the amygdala, VTA, hippocampus, nucleus accumbens and CPu, and dopamine metabolites in VTA and CPu.

Effects of nicotine, nornicotine and cotinine, alone or in combination, on locomotor activity and ultrasonic vocalization emission in adult rats.

The behavioral effects of the nicotine metabolites nornicotine and cotinine have not been investigated extensively. To evaluate the effects of nicotine, cotinine, and nornicotine, given alone or in combination, on locomotor activity and emission of ultrasonic vocalizations in male adult rats. Rats were first given home cage nicotine injections to make them tolerant to the drug's locomotor depressant effects. On subsequent days, locomotor activity (LMA) and ultrasonic vocalizations were recorded in an open field, for 60min after challenge injection, using repeated measures designs. In single-drug experiments, subjects were tested with nicotine 0.05-0.4mg/kg, cotinine 0.03-3mg/kg, or nornicotine 0.1-10mg/kg. In drug-combination experiments, saline or nicotine 0.2mg/kg challenge was preceded by cotinine (0, 0.3, 3mg/kg) or nornicotine (0, 0.1, 0.3, 1, 3mg/kg) injection. High doses of nornicotine increased LMA and blunted the locomotor stimulant effect of nicotine. Less consistently, nicotine and high doses of nornicotine decreased the 50-kHz call rate, with no clear evidence of a nornicotine × nicotine interaction. Cotinine, given alone or before nicotine injection, altered neither LMA nor the call rate. No drug altered the relative prevalence of flat vs. trill 50-kHz call subtypes, except that the highest dose of nornicotine promoted flat calls over trills. No drug evoked 22-kHz calls. Nornicotine can exert an acute anti-nicotine effect in vivo, as previously reported in vitro. The finding that nicotine did not detectably alter the 50-kHz call profile appears consistent with this drug's mild subjective effects in human subjects.

Sex differences in the acoustic features of social play-induced 50-kHz ultrasonic vocalizations: A detailed spectrographic analysis in wild-type Sprague-Dawley and Cacna1c haploinsufficient rats.

Sexual dimorphisms are widespread in the animal kingdom. At the behavioral level, there is evidence for sex differences in social play behavior. In rats, males typically engage more in rough-and-tumble play than females. One prominent component of the rough-and-tumble play repertoire in rats is the emission of 50-kHz ultrasonic vocalizations (USV). Such 50-kHz USV reflect the rewarding nature of play and serve as socioaffective signals. Here, we provide evidence for sexual dimorphisms within rough-and-tumble play-induced 50-kHz USV in juvenile rats. Specifically, females displayed reduced 50-kHz USV emission during playful interactions. This reduction was associated with changes in 50-kHz USV emission rates and subtype profiles during specific rough-and-tumble components, i.e., pinning, wrestling, and chasing, as well as differences in acoustic parameters. Interestingly, sex differences were modulated by Cacna1c, a gene strongly implicated in major neuropsychiatric disorders, often characterized by prominent sex biases, most notably autism. Specifically, Cacna1c haploinsufficiency affected the emission of 50-kHz USV during rough-and-tumble play in female rats and we provide evidence supporting the notion that such effects of Cacna1c haploinsufficiency are driven by male-typical features of 50-kHz USV emission. This is in line with the hypermasculinized social play repertoire previously observed in juvenile Cacna1c haploinsufficient females.

Ultrasonic vocalizations in house mice depend upon genetic relatedness of mating partners and correlate with subsequent reproductive success

BackgroundCourtship vocalizations are used by males of many species to attract and influence the behavior of potential mating partners. Our aim here was to investigate the modulation and reproductive consequences of courtship ultrasonic vocalizations (USVs) in wild-derived house mice (Mus musculus musculus). The courtship USVs of male mice are surprisingly complex and are composed of a variety of different syllable types. Our specific aims were to test whether (1) the emission of courtship USVs depends upon the kinship of a potential mating partner, and (2) whether USV emission during courtship affects the pairs’ subsequent reproductive success.ResultsWe experimentally presented males with an unfamiliar female that was either genetically related or unrelated, and we recorded USV emission, first while the sexes were separated by a perforated partition and then during direct interactions, after removing the partition. USVs were detected by the Automatic Mouse Ultrasound Detector (A-MUD) and manually classified into 15 syllable types. The mice were kept together to test whether and how courtship vocalizations predict their subsequent reproductive success. We found that the mice significantly increased their amount of vocalizations (vocal performance) and number of syllable types (vocal repertoire) after the partition was removed and they began interacting directly. We show that unrelated pairs emitted longer and more complex USVs compared to related pairs during direct interactions. Unrelated pairs also had a greater reproductive success compared to related pairs, and in addition we found a negative correlation between the mean length and amount of vocalizations with the latency to their first litter.ConclusionOur study provides evidence that house mice modulate the emission of courtship USVs depending upon the kinship of potential mating partners, and that courtship USVs correlate with reproductive success.

Social context increases ultrasonic vocalizations during restraint in adult mice.

Adult mice emit many ultrasonic vocalizations (USVs) during social interaction tasks, but only a few studies have yet reported USVs in stressed adult mice. Our aim was to study which experimental conditions favor USV emission during behaviors associated with different emotional states. As USVs likely mediate social communication, we hypothesized that temporary social isolation followed by exposure to a novel social congener would promote USV emission. USVs were recorded in three different behavioral paradigms: restraint, free moving in a new environment, and during a social interaction task. We compared USV emission, with or without the presence of a social congener, in animals socially isolated during different periods (0, 6 or 21days). Social isolation decreased the number of USVs during free moving, whereas it increased during restraint. During the social interaction task, animals produced high-frequency USVs (median: 72.6kHz, 25-75% range: 67.6-78.2kHz), especially when the social partner was active and social motivation was high. During restraint, presence of a social congener increased the call rate of low-frequency USVs (median: 52.4kHz, 25-75% range: 44.8-56.5kHz). USV frequency followed two unimodal distributions that distinguished low-frequency USVs (≤ 60kHz) mainly emitted during free-moving (90.9% of total USVs) and restraint (93.1%) conditions, from high-frequency USVs (> 60kHz) mainly emitted during the social interaction task (85.1% of total USVs). The present study confirms that USV call rate and frequency depend on behavioral states, and provides evidence that the presence of a congener promotes ultrasonic vocalizations in restrained adult mice.

Influence of Perinatal Polychlorinated Biphenyl or Thiouracil on Dopamine Function

Objective: Dopamine function is sensitive to toxin exposure. The environmental toxin polychlorinated biphenyl (PCB) alters dopamine physiology in several, harmful ways. Intermediary signals include thyroid hormones as PCB exposure alters thyroid function leading to changes in dopamine neurochemistry. To compare PCB and thyroid alterations directly, we exposed rats perinatally to either PCB (25 ppm) or thiouracil (THIO; 0.625%) from gestational day 1 to separation from the dam at postnatal day (PND) 25 and examined a set of tests sensitive to dopamine function. Methods: We measured behaviors including prepulse inhibition (PPI) of the acoustic startle response (ASR) in adulthood (60-90 PND) and ultrasonic vocalizations (USVs) during pup and adult periods. Dopamine levels in striatum were estimated using tyrosine hydroxylase immunohistochemistry and thyroid hormone levels were assayed in adulthood. Results: Animals exposed to thiouracil had significantly elevated startle response and impaired PPI relative to controls and PCB exposed animals. This group also had significant reductions in both USV emission during PPI and tyrosine hydroxylase staining within striatum. Conversely, PCB exposure during the perinatal period did not lead to alterations in gating or dopamine levels in adulthood. Similar to previous studies using perinatal exposure to PCB or THIO, thyroid hormone levels were unchanged (THIO group) relative to controls or actually increased (PCB group) by adulthood. Conclusion: The findings support an important role for thyroid function in dopamine-dependent behaviors. Results suggest PCB exposure leads to different outcomes in comparison to direct thyroid manipulations suggesting PCB exposure has distinct mechanisms of developmental hormone dysfunction and potential compensatory changes in response to endocrine disruption.

Early Life Maternal Separation and Maternal Behaviour Modulate Acoustic Characteristics of Rat Pup Ultrasonic Vocalizations

Early separation of preterm infants from their mothers has adverse, long-term neurodevelopmental consequences. We investigated the effects of daily maternal separation (MS) of rat pups from postnatal days 2–10 (PND2–10) on neurobehavioural responses to brief isolation at PND12 compared with pups receiving controlled handling without MS. Ultrasonic vocalizations (USV) were measured at PND12 during two, 3-minute isolations occurring immediately before and after a 3-minute maternal reunion. There were no significant differences in acoustic characteristics between MS and control animals in the first isolation. However, in the second isolation, MS pups produced a greater proportion of high (~60 kHz) vs low (~40 kHz) frequency calls. During this isolation, control pups made longer and louder low frequency calls compared to the first isolation, whereas MS pups did the opposite. Maternal behaviour of control and MS mothers modulated pup acoustic characteristics in opposite directions; higher maternal care was associated with more low frequency calls in control pups but more high frequency calls in MS pups. We hypothesize that MS results in USV emission patterns reflective of a greater stress response to isolation. This translational model can be used to identify mechanisms and interventions that may be exploited to overcome the negative, long-term effects of MS.

Influence of dopamine transmission in the medial prefrontal cortex and dorsal striatum on the emission of 50-kHz ultrasonic vocalizations in rats treated with amphetamine: Effects on drug-stimulated and conditioned calls

Rat ultrasonic vocalizations (USVs) of 50 kHz are increasingly being evaluated as a behavioral marker of the affective properties of drugs. Studies in amphetamine-treated rats have shown that activation of dopamine transmission in the nucleus accumbens (NAc) initiates the emission of 50-kHz USVs, but little is known on how dopamine transmission in other brain regions modulates the effects of drugs on calling behavior. To clarify this issue, we evaluated 50-kHz USV emissions in rats subjected to dopaminergic denervation of either the medial prefrontal cortex (mPFC) or the dorsal striatum (DS) and treated with amphetamine. Rats received amphetamine (1 mg/kg, i.p. × 5) on alternate days in a test cage; 7 days later, they were re-exposed to the test cage, to measure calling behavior that may reflect drug conditioning, and then challenged with amphetamine (1 mg/kg, i.p.). The numbers of total and categorized 50-kHz USVs emitted were evaluated, along with immunofluorescence for Zif-268 in the NAc. Dopamine-denervated and sham-operated rats displayed comparable patterns of calling behavior during amphetamine treatment and after amphetamine challenge. Conversely, rats that were dopamine-denervated in the mPFC, but not DS, emitted low numbers of 50-kHz USVs on test cage re-exposure. Finally, dopamine-denervated rats displayed a less marked increase in Zif-268-positive neurons in the NAc shell after amphetamine challenge, compared with sham-operated rats. These results may be relevant to identify the neuronal circuits that modulate 50-kHz USV emissions in rats treated with amphetamine, as well as the interplay between calling behavior and affective properties of drugs.

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.

Functional Ontogeny of Hypothalamic Agrp Neurons in Neonatal Mouse Behaviors

Hypothalamic Agrp neurons regulate food ingestion in adult mice. Whether these neurons are functional before animals start to ingest food is unknown. Here, we studied the functional ontogeny of Agrp neurons during breastfeeding using postnatal day 10 mice. In contrast to adult mice, we show that isolation from the nursing nest, not milk deprivation or ingestion, activated Agrp neurons. Non-nutritive suckling and warm temperatures blunted this effect. Using invivo fiber photometry, neonatal Agrp neurons showed a rapid increase in activity upon isolation from the nest, an effect rapidly diminished following reunion with littermates. Neonates unable to release GABA from Agrp neurons expressed blunted emission of isolation-induced ultrasonic vocalizations. Chemogenetic overactivation of these neurons further increased emission of these ultrasonic vocalizations, but not milk ingestion. We uncovered important functional properties of hypothalamic Agrp neurons during mouse development, suggesting these neurons facilitate offspring-to-caregiver bonding.

The antagonistic relationship between aversive and appetitive emotional states in rats as studied by pharmacologically-induced ultrasonic vocalization from the nucleus accumbens and lateral septum

Rats can emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) in negative, as well as positive contexts which index their emotional state. 22-kHz USVs are emitted during aversive contexts and can be initiated by activation of the ascending cholinergic pathways originating from the laterodorsal tegmental nucleus or initiated pharmacologically by injection of cholinergic agonists into target areas of these pathways (medial cholinoceptive vocalization strip). Conversely, 50-kHz USVs are emitted during positive pro-social contexts and can be initiated by stimulation of ascending dopaminergic pathways originating from the ventral tegmental area or by injection of dopamine agonists into target areas of these pathways (nucleus accumbens shell). Recently, we have shown an inhibitory effect a positive emotional state has on the emission of carbachol-induced 22-kHz USVs from the anterior hypothalamic/medial preoptic area. However, this structure is a fragment of that cholinoceptive vocalization strip. We wanted to examine if we could observe similar effect when the aversive state is induced from the lateral septum, the most rostral division of the cholinoceptive vocalization strip. The results supported previous findings. First, microinjection of the dopamine agonist R-(-)-apomorphine into the nucleus accumbens shell resulted in increased emission of frequency modulated (FM) 50-kHz USVs that are regarded as signals expressing a positive emotional state in rats. Second, FM 50-kHz USVs and not flat (F) 50-kHz USVs were able to decrease 22-kHz USVs induced by microinjections of carbachol into the lateral septum. This research provides further support to the hypothesis that the initiation of a positive emotional state functionally antagonizes initiation of a negative emotional state in rats.

Effects of Cacna1c haploinsufficiency on social interaction behavior and 50-kHz ultrasonic vocalizations in adult female rats

The risk gene CACNA1C is strongly implicated in the etiology of all major psychiatric disorders, such as depressive disorder, bipolar disorder, autism spectrum disorder, and schizophrenia. These disorders feature high levels of comorbidity and share an overlap of symptoms; in particular, deficits in social functioning are common. Intriguingly, sex-dependent effects of CACNA1C single nucleotide polymorphisms on prevalence, health outcomes, and psychological traits have been reported, typically suggesting that women are more affected by CACNA1C mutations than men. In rodents, genetic modifications specifically targeting Cacna1c have repeatedly been linked to deficits in social behavior in male mice and rats but many studies neglect the sex-dependent effects observed in humans. Our study focused on the role of Cacna1c in regulating social behavior and communication in adult female rats. We compared social and non-social behavior together with concomitant emission of pro-social 50-kHz ultrasonic vocalizations (USV) associated with positive affect in constitutive heterozygous (Cacna1c+/−) rats to wildtype (Cacna1c+/+) littermate controls. Our results indicate that partial Cacna1c depletion leads to strongly reduced emission of 50-kHz USV and mild social deficits during female direct reciprocal social interaction. Detailed temporal analyses revealed most prominent reductions of 50-kHz USV during non-social behavior, suggesting that reduced positive affect occurs in a social context in Cacna1c+/− rats but is not specifically linked to social behavior. Finally, we observed increased self-grooming behavior in Cacna1c+/− rats, consistent with an autism-like phenotype. Our findings in rats thus support a role of Cacna1c in regulating behavioral phenotypes with relevance for several neuropsychiatric disorders.