Emission Of Ultrasonic Vocalizations Research Articles

Involvement and regulation of the left anterior cingulate cortex in the ultrasonic communication deficits of autistic mice

IntroductionAutism spectrum disorder (ASD) is a group of diseases often characterized by poor sociability and challenges in social communication. The anterior cingulate cortex (ACC) is a core brain region for social function. Whether it contributes to the defects of social communication in ASD and whether it could be physiologically modulated to improve social communication have been poorly investigated. This study is aimed at addressing these questions.MethodsFragile X mental retardation 1 (FMR1) mutant and valproic acid (VPA)-induced ASD mice were used. Male–female social interaction was adopted to elicit ultrasonic vocalization (USV). Immunohistochemistry was used to evaluate USV-activated neurons. Optogenetic and precise target transcranial magnetic stimulation (TMS) were utilized to modulate anterior cingulate cortex (ACC) neuronal activity.ResultsIn wild-type (WT) mice, USV elicited rapid expression of c-Fos in the excitatory neurons of the left but not the right ACC. Optogenetic inhibition of the left ACC neurons in WT mice effectively suppressed social-induced USV. In FMR1−/−- and VPA-induced ASD mice, significantly fewer c-Fos/CaMKII-positive neurons were observed in the left ACC following USV compared to the control. Optogenetic activation of the left ACC neurons in FMR1−/− or VPA-pretreated mice significantly increased social activity elicited by USV. Furthermore, precisely stimulating neuronal activity in the left ACC, but not the right ACC, by repeated TMS effectively rescued the USV emission in these ASD mice.DiscussionThe excitatory neurons in the left ACC are responsive to socially elicited USV. Their silence mediates the deficiency of social communication in FMR1−/− and VPA-induced ASD mice. Precisely modulating the left ACC neuronal activity by repeated TMS can promote the social communication in FMR1−/− and VPA-pretreated mice.

Divergent Acute and Enduring Changes in 50-kHz Ultrasonic Vocalizations in Rats Repeatedly Treated With Amphetamine and Dopaminergic Antagonists: New Insights on the Role of Dopamine in Calling Behavior.

Rats emit 50-kHz ultrasonic vocalizations (USVs) in response to nonpharmacological and pharmacological stimuli, with addictive psychostimulants being the most effective drugs that elicit calling behavior in rats. Earlier investigations found that dopamine D1-like and D2-like receptors modulate the emission of 50-kHz USVs stimulated in rats by the acute administration of addictive psychostimulants. Conversely, information is lacking on how dopamine D1-like and D2-like receptors modulate calling behavior in rats that are repeatedly treated with addictive psychostimulants. We evaluated the emission of 50-kHz USVs in rats repeatedly treated (×5 on alternate days) with amphetamine (1mg/kg, i.p.) either alone or together with (1) SCH 23390 (0.1-1mg/kg, s.c.), a dopamine D1 receptor antagonist; (2) raclopride (0.3-1mg/kg, s.c.), a selective dopamine D2 receptor antagonist; or (3) a combination of SCH 23390 and raclopride (0.1+0.3mg/kg, s.c.). Calling behavior of rats was recorded following pharmacological treatment, as well as in response to the presentation of amphetamine-paired cues and to amphetamine challenge (both performed 7 days after treatment discontinuation). Amphetamine-treated rats displayed a sensitized 50-kHz USV emission during repeated treatment, as well as marked calling behavior in response to amphetamine-paired cues and to amphetamine challenge. Antagonism of D1 or D2 receptors either significantly suppressed or attenuated the emission of 50-kHz USVs in amphetamine-treated rats, with a maximal effect after synergistic antagonism of both receptors. These results shed further light on how dopamine transmission modulates the emission of 50-kHz USVs in rats treated with psychoactive drugs.

Simultaneous recording of ultrasonic vocalizations and sniffing from socially interacting individual rats using a miniature microphone

Vocalizations are pivotal in mammalian communication, especially in humans. Rodents accordingly rely on ultrasonic vocalizations (USVs) that reflect their internal state as a primary channel during social interactions. However, attributing vocalizations to specific individuals remains challenging, impeding internal state assessment. Rats emit 50-kHz USVs to indicate positive states and intensify sniffing during alertness and social interactions. Here, we present a method involving a miniature microphone attached to the rat nasal cavity that allows to capture both male and female individual rat vocalizations and sniffing patterns during social interactions. We found that while the emission of 50-kHz USVs increases during close interactions, these signals lack specific behavioral associations. Moreover, a previously unreported low-frequency vocalization type marking rat social interactions was uncovered. Finally, different dynamics of sniffing and vocalization activities point to distinct underlying internal states. Thus, our method facilitates the exploration of internal states concurrent with social behaviors.

Acute anxiogenic effects of escitalopram are associated with mild alterations in D-amphetamine-induced behavior and social approach evoked by playback of 50-kHz ultrasonic vocalizations in rats

Rats communicate through auditory signals in the ultrasonic range, so-called ultrasonic vocalizations (USV). Short, high-frequency 50-kHz USV are associated with positive affective states and are emitted in appetitive situations, often rewarding social interactions, such as rough-and-tumble play and mating. Exaggerated levels of 50-kHz USV emission can be observed in response to psychostimulants, most notably d-amphetamine (AMPH). There is robust evidence suggesting that 50-kHz USV serve as affiliative signals and help to maintain or re-establish social proximity. A key neurotransmitter involved in behavioral regulation is serotonin (5-hydroxytryptamine, 5-HT). This includes both, the regulation of anxiety-related behavior and ultrasonic communication. Here, we show that acute treatment with the selective 5-HT reuptake inhibitor (SSRI) escitalopram (ESC) leads to increased anxiety-related behavior in the elevated plus maze and tested whether such acute anxiogenic effects of ESC result in alterations in ultrasonic communication in sender and/or receiver. To this aim, we conducted a dose-response study in male rats and assessed AMPH-induced hyperactivity and 50-kHz ultrasonic calling in the sender and social approach behavior evoked by playback of pro-social 50-kHz USV in the receiver. Acute ESC treatment affected both, sender and receiver. This was reflected in a lack of AMPH-induced changes in acoustic features of 50-kHz USV and absence of social exploratory behavior evoked by 50-kHz USV playback, respectively. Albeit the SSRI effects were relatively mild, this supports the notion that the 5-HT system is involved in the regulation of a key aspect of the social behavior repertoire of rodents, namely socio-affective communication through 50-kHz USV.

Group I and group II metabotropic glutamate receptors are upregulated in the synapses of infant rats prenatally exposed to valproic acid

RationaleAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction and restricted/stereotyped behavior. Prenatal exposure to valproic acid (VPA) is associated with an increased risk of developing ASD in humans and autistic-like behaviors in rodents. Increasing evidence indicates that dysfunctions of glutamate receptors at synapses are associated with ASD. In the VPA rat model, an involvement of glutamate receptors in autism-like phenotypes has been suggested; however, few studies were carried out on metabotropic glutamate (mGlu) receptors.ObjectivesWe examined the protein expression levels of group I (mGlu1 and mGlu5) and group II (mGlu2/3) mGlu receptors in rats prenatally exposed to VPA and evaluated the effect of mGlu receptor modulation on an early autism-like phenotype in these animals.MethodsWe used western blotting analysis on synaptosomes obtained from forebrain of control and VPA rats at different ages (postnatal day P13, 35, 90) and carried out ultrasonic vocalization (USV) emission test in infant control and VPA rats.ResultsThe expression levels of all these receptors were significantly increased in infant VPA rats. No changes were detected in adolescent and adult rats. An acute treatment with the preferential mGlu2/3 antagonist, LY341495, attenuated the impairment in the USV emission in VPA rats. No effect was observed after a treatment with the mGlu5 selective antagonist, MTEP.ConclusionsOur findings demonstrate that the expression of group I and group II mGlu receptors is upregulated at synapses of infant VPA rats and suggest that mGlu2/3 receptor modulation may have a therapeutic potential in ASD.

2-Chloro-3,7,8-tribromodibenzofuran as a new environmental pollutant inducing atypical ultrasonic vocalization in infant mice.

Epidemiological and experimental studies indicate that maternal exposure to environmental pollutants impairs the cognitive and motor functions of offspring in humans and laboratory animals. Infant ultrasonic vocalizations (USVs), the communicative behavior of pups toward caregivers, are impaired in rodent models of neurodevelopmental disorders, suggesting a useful method to evaluate the developmental neurotoxicity of environmental pollutants. Therefore, we investigated USVs emitted by mouse pups of dams exposed to 2-chloro-3,7,8-tribromodibenzofuran (TeXDF) and 1,2,3,7,8-pentabromodibenzofuran (PeBDF), which are detected in the actual environment. The USV duration and number in the pups born to dams administered with TeXDF 40μg/kg body weight (b.w.), but not 8μg/kg b.w., on gestational day (GD) 12.5, were significantly lower than those in the corresponding pups on postnatal days 3-9. Conversely, there was no statistical change in the USVs emitted by the pups of dams administered with PeBDF 35 or 175μg/kg b.w. on GD 12.5. To examine whether maternal exposure leads to behavioral impairments in adulthood, we analyzed exploratory behaviors in a novel environment using IntelliCage, a fully automated testing apparatus for group-housed mice. Neither TeXDF nor PeBDF exposure induced significant differences in offspring exploration. Considered together, our findings revealed that TeXDF induces atypical USV emission in infant mice, suggesting the importance of further studies on the risk assessment of mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans.

Wildtype peers rescue social play and 50-kHz ultrasonic vocalization deficits in juvenile female Cacna1c heterozygous rats.

Healthy brain development depends on early social practices and experiences. The risk gene CACNA1C is implicated in numerous neuropsychiatric disorders, in which key characteristics include deficits in social functioning and communication. Recently, we reported sex-dependent impairments in social behavior and ultrasonic vocalizations (USV) in juvenile heterozygous Cacna1c+/- (HET) rats. Specifically, HET females displayed increases in rough-and-tumble play that eliminated the typically observed sex difference between male and female rats. Interestingly, female wild-type Cacna1c+/+ (WT) pairs also showed a similar increase in social play when housed with HET females, suggesting their behavior may be influenced by HET cage mates. This indicates that the genetic makeup of the social environment related to Cacna1c can influence social play, yet systematic studies are lacking. In the present study, we housed juvenile females in MIXED- or SAME-genotype cages and tested them in a social play paradigm with a same- and opposite-genotype partner. The results show that the early social environment and the genotype of the play partner influence social play and 50-kHz USV emission. Experience with a WT play partner appears necessary for HET females to show comparable levels of play and 50-kHz USV emission. Same-genotype HET pairs played less and emitted fewer 50-kHz USV than same-genotype WT or opposite-genotype pairs; however, we found that the decrease in social play and 50-kHz USV in HET pairs can be rescued by playing with a WT partner. The effect was particularly prominent when the first play partner was WT, as we found it increased play and 50-kHz USV emission in all subsequent interactions with ensuing partners. These findings suggest that the genetic makeup related to the social environment and/or social peers influences social play in Cacna1c+/- haploinsufficient rats. Specifically, our results show that WT peers can rescue behavior and communication alterations in Cacna1c female rats. Our findings have important implications because they show that the genetic makeup of the social environment can divulge phenotypic changes in genetic rat models of neuropsychiatric disorders.

Detailed spectrographic analysis of rat ultrasonic vocalizations emitted during the acoustic startle response test

Rats emit ultrasonic vocalizations (USV). During aversive situations, rats emit 22-kHz USV, which are considered "alarm calls" and supposed to reflect a negative affective state of the sender. During appetitive situations, rats emit 50-kHz USV, which are believed to reflect a positive affective state. Here, we recorded USV emission in adult male rats during the acoustic startle response test. Our results indicate varied USV emission in both the 22- and 50-kHz USV ranges. Enhanced startle responses were observed in rats with a predominant 22-kHz call profile, supporting the notion that 22-kHz USV emission is associated with a negative affective state.

Behavior, Neural Structure, and Metabolism in Adult Male Mice Exposed to Environmentally Relevant Doses of Di(2-ethylhexyl) Phthalate Alone or in a Phthalate Mixture.

We have previously shown that chronic exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate (DEHP) altered male sexual behavior and induced down-regulation of the androgen receptor (AR) in the neural circuitry controlling this behavior. The cellular mechanisms induced by chronic exposure of adult male mice to low doses of DEHP alone or in an environmental phthalate mixture were studied. Two-month-old C57BL/6J males were exposed orally for 8 wk to DEHP alone (0, 5, or ) or to DEHP () in a phthalate mixture. Behavior, dendritic density per length, pre-/postsynaptic markers, synapse ultrastructure, and bioenergetic activity were analyzed. Mice exposed to DEHP either alone or in a phthalate mixture differed in mating, emission of ultrasonic vocalizations, and the ability to attract receptive females in urinary preference tests from control mice. Analyses in the medial preoptic area, the key hypothalamic region involved in male sexual behavior, showed lower dendritic spine density and protein levels of glutamate receptors and differences in other postsynaptic components and presynaptic markers between the treated groups. Ultrastructural observation of dendritic synapses by electron microscopy showed comparable morphology between the treated groups. Metabolic analyses highlighted differences in hypothalamic metabolites of males exposed to DEHP alone or in a phthalate mixture compared to control mice. These differences included lower tryptophan and higher levels, respectively, a precursor and end product of the kynurenine pathway of tryptophan metabolism. The protein amounts of the xenobiotic aryl hydrocarbon receptor, one of the targets of this metabolic pathway and known negative regulator of the AR, were higher in the medial preoptic area of exposed male mice. Differences in behavior of male mice exposed to environmental doses of phthalates were associated with differences in neural structure and metabolism, with possibly a key role of the kynurenine pathway of tryptophan metabolism in the effects mediated by these substances. https://doi.org/10.1289/EHP11514.

Sociability versus empathy in adolescent mice: Different or distinctive?

In recent years, a growing number of pre-clinical studies have made use of the social abilities of mice, asking how gene variants (e.g., null, transgenic or mutant alleles) give rise to abnormalities in neurodevelopment. Two distinct courses of research provide the foundation for these studies. One course has mostly focused on how we can assess “sociability” using metrics, often automated, to quantitate mouse approach and withdrawal responses to a variety of social stimuli. The other course has focused on psychobiological constructs that underlie the socio-emotional capacities of mice, including motivation, reward and empathy. Critically, we know little about how measures of mouse sociability align with their underlying socio-emotional capacities. In the present work, we compared the expression of sociability in adolescent mice from several strains versus a precisely defined behavioral model of empathy that makes use of a vicarious fear learning paradigm. Despite substantial strain-dependent variation within each behavioral domain, we found little evidence of a relationship between these social phenotypes (i.e., the rank order of strain differences was unique for each test). By contrast, emission of ultrasonic vocalizations was highly associated with sociability, suggesting that these two measures reflect the same underlying construct. Taken together, our results indicate that sociability and vicarious fear learning are not manifestations of a single, overarching social trait. These findings thus underscore the necessity for a robust and diverse set of measures when using laboratory mice to model the social dimensions of neuropsychiatric disorders.

Mouse and rat ultrasonic vocalizations in neuroscience and neuropharmacology: State of the art and future applications.

Mice and rats emit ultrasonic vocalizations (USVs), which may express their arousal and emotional states, to communicate with each other. There is continued scientific effort to better understand the functions of USVs as a central element of the rodent behavioral repertoire. However, studying USVs is not only important because of their ethological relevance, but also because they are widely applied as a behavioral readout in various fields of biomedical research. In mice and rats, a large number of experimental models of brain disorders exist and studying the emission of USVs in these models can provide valuable information about the health status of the animals and the effectiveness of possible interventions, both environmental and pharmacological. This review (i) provides an updated overview of the contexts in which ultrasonic calling behaviour of mice and rats has particularly high translational value, and (ii) gives some examples of novel approaches and tools used for the analysis of USVs in mice and rats, combining qualitative and quantitative methods. The relevance of age and sex differences as well as the importance of longitudinal evaluations of calling and non-calling behaviour is also discussed. Finally, the importance of assessing the communicative impact of USVs in the receiver, that is, through playback studies, is highlighted.

Vocal signals with different social or non-social contexts in two wild rodent species (Mus caroli and Rattus losea).

The ultrasonic vocalizations (USVs) of rodents play a substantial role in the communication and interaction between individuals; exhibit a high degree of complexity; and are influenced by a multitude of developmental, environmental, and phylogenetic factors. The functions of USVs are mainly studied in laboratory mice or rats. However, the behavioral relevance of USVs in wild rodents is poorly studied. In this work, we systematically investigated the vocal repertoire of the wild mouse Mus caroli and wild rat Rattus losea in multiple social or non-social contexts, e.g., pup-isolation, juvenile-play, paired opposite-sex encounter, female-female interaction, social-exploring, or foot-shock treatment. Unlike the laboratory mice, M. caroli, whose USVs were recorded during pup-isolation and courtship behaviors, did not produce any vocal sounds during juvenile-play and female-female interactions. R. losea, similar to laboratory rats, emitted USVs in all test situations. We found higher peak frequencies of USVs in both these two wild rodent species than in laboratory animals. Moreover, the parameters and structures of USVs varied significantly across different social or non-social contexts even within each species, confirming the context-sensitivity and complexity of vocal signals in rodents. We also noted a striking difference in call types between these two species: no downward type occurred in M. caroli, but no upward type occurred in R. losea, thereby highlighting the interspecific difference of vocal signals among rodents. Thus, the present study presents behavioral foundations of the vocalization context in wild mice and wild rats, and contributes to revealing the behavioral significance of widely used USVs in rodents.

The Effects of Positive Allosteric Modulators of α7-nAChR on Social Play Behavior in Adolescent Rats Prenatally Exposed to Valproic Acid.

There is still no effective treatment that addresses the core symptoms of autism spectrum disorders (ASD), including social and communication deficits. A comprehensive body of evidence points to the cholinergic system, including alpha7-nicotinic acetylcholine receptors (α7-nAChRs), as a potential target of pharmacotherapy. A promising approach is based on positive allosteric modulators (PAMs) of these receptors due to their advantages over direct agonists. Nevertheless, α7 n-AChR ligands have not been widely studied in the context of autism. Therefore, using one of the most widely used rodent models of ASD, that is, prenatal exposure to valproic acid (VPA), we examined the impact of α7-nAChR PAMs (PNU-120596 and CCMI) on socio-communicative behavior during social play in adolescent male and female rats. The current study demonstrated that PAM treatment affected certain aspects of socio-communicative behavior in adolescent rats. Accordingly, PNU-120596 ameliorated deficient play abilities in VPA-exposed males, as revealed by increased play time during a social encounter. In addition, this compound enhanced the emission of ultrasonic vocalizations that accompanied playful interactions. Moreover, we observed the overall effect of PNU-120596 on non-playful forms of social behavior (i.e., social exploration) and acoustic parameters (i.e., the duration) of emitted calls. The present results suggest the ability of α7-nAChR PAMs to facilitate socio-communicative behavior in adolescent rats.

Moderate ethanol exposure during early ontogeny of the rat alters respiratory plasticity, ultrasonic distress vocalizations, increases brain catalase activity, and acetaldehyde-mediated ethanol intake.

Early ontogeny of the rat (late gestation and postnatal first week) is a sensitive period to ethanol's positive reinforcing effects and its detrimental effects on respiratory plasticity. Recent studies show that acetaldehyde, the first ethanol metabolite, plays a key role in the modulation of ethanol motivational effects. Ethanol brain metabolization into acetaldehyde via the catalase system appears critical in modulating ethanol positive reinforcing consequences. Catalase system activity peak levels occur early in the ontogeny. Yet, the role of ethanol-derived acetaldehyde during the late gestational period on respiration response, ultrasonic vocalizations (USVs), and ethanol intake during the first week of the rat remains poorly explored. In the present study, pregnant rats were given a subcutaneous injection of an acetaldehyde-sequestering agent (D-penicillamine, 50 mg/kg) or saline (0.9% NaCl), 30 min prior to an intragastric administration of ethanol (2.0 g/kg) or water (vehicle) on gestational days 17-20. Respiration rates (breaths/min) and apneic episodes in a whole-body plethysmograph were registered on postnatal days (PDs) 2 and 4, while simultaneously pups received milk or ethanol infusions for 40-min in an artificial lactation test. Each intake test was followed by a 5-min long USVs emission record. On PD 8, immediately after pups completed a 15-min ethanol intake test, brain samples were collected and kept frozen for catalase activity determination. Results indicated that a moderate experience with ethanol during the late gestational period disrupted breathing plasticity, increased ethanol intake, as well brain catalase activity. Animals postnatally exposed to ethanol increased their ethanol intake and exerted differential affective reactions on USVs and apneic episodes depending on whether the experience with ethanol occur prenatal or postnatally. Under the present experimental conditions, we failed to observe, a clear role of acetaldehyde mediating ethanol's effects on respiratory plasticity or affective states, nevertheless gestational acetaldehyde was of crucial importance in determining subsequent ethanol intake affinity. As a whole, results emphasize the importance of considering the participation of acetaldehyde in fetal programming processes derived from a brief moderate ethanol experience early in development, which in turn, argues against "safe or harmless" ethanol levels of exposure.

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.

Exposure to environmentally relevant doses of plasticizers alters maternal behavior and related neuroendocrine processes in primiparous and multiparous female mice

Phthalates are organic pollutants frequently detected in the environment. The effects of these substances on male reproduction have been extensively studied but their potential impact on female reproductive behaviors in particular at environmental doses still remains to be documented. In the present study, we examined the effects of chronic exposure to di (2-ethylhexyl) phthalate (DEHP) alone at 5 or 50 μg/kg/d, or in an environmental phthalate mixture on maternal behavior of lactating female mice after a first (primiparous) and a second gestation (multiparous). Exposure of DEHP alone or in a phthalate mixture reduced pup-directed behaviors, increased self-care and forced nursing behaviors and altered nest quality for both primiparous and multiparous dams. In pup-retrieval test, primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture retrieved their pups more rapidly, probably due to a higher emission of ultrasonic vocalizations by the pups. At lactational day 2 following the third and last gestational period, the neural circuitry of maternal behavior was analyzed. A lower number of oxytocin-immunoreactive neurons in the paraventricular and anterior commissural nuclei was found in dams exposed to DEHP alone or in a phthalate mixture, while no changes were observed in the number of arginine-vasopressin immunoreactive cells. In the medial preoptic area, exposure to DEHP alone or in a phthalate mixture reduced ERα-immunoreactive cell number. Dendritic spine density assessed for DEHP at 5 μg/kg/d was also reduced. Thus, exposure to DEHP alone or in a phthalate mixture altered maternal behavior probably through a neuroendocrine mode of action involving oxytocin and estrogen through ERα, key pathways necessary for neuroplasticity and behavioral processing.

Trigeminal neuropathic pain reduces 50-kHz ultrasonic vocalizations in rats, which are restored by analgesic drugs

Trigeminal neuralgia (TN) is a severe form of neuropathic pain frequently associated with anxiety. The chronic constriction injury of the infraorbital nerve (CCI-ION) of rodents is a well-established model to study sensory alterations related to TN. However, few studies have addressed the emotional component of pain, which is fundamental to increase its translational capability. Emission of ultrasonic vocalization (USV) is considered a reliable measure of the emotional state of rats. Rats emit 50-kHz USVs in social and appetitive situations, whereas 22-kHz USVs may index a negative state. Studies suggest that persistent pain causes reduction in 50-kHz calls, but this may also indicate anxiety-like behavior. Thus, we hypothesize that CCI-ION would decrease 50-kHz calls and that pharmacological pain relief would restore USVs, without interfering with anxiety-like behavior. On day 15 after surgery, male rats were treated with local lidocaine, midazolam or carbamazepine to determine their effect on facial mechanical hyperalgesia, USV and anxiety-like behavior. The results showed that CCI-ION induced hyperalgesia, which was attenuated by lidocaine or carbamazepine, developed anxiety-like behavior, which was reduced only by midazolam, and displayed a reduced number of 50-kHz calls, compared to sham. Lidocaine and carbamazepine increased 50-kHz calls emitted by CCI-ION rats, but midazolam failed to change them. These data add information on the translational aspects of CCI-ION model and carbamazepine treatment for trigeminal neuropathic pain. Furthermore, they suggest that the reduction of USV in persistent pain conditions is related to spontaneous pain and reinforce the idea that it reflects the emotional component of pain.

Response Calls Evoked by Playback of Natural 50-kHz Ultrasonic Vocalizations in Rats.

Rats are highly social animals known to communicate with ultrasonic vocalizations (USV) of different frequencies. Calls around 50 kHz are thought to represent a positive affective state, whereas calls around 22 kHz are believed to serve as alarm or distress calls. During playback of natural 50-kHz USV, rats show a reliable and strong social approach response toward the sound source. While this response has been studied in great detail in numerous publications, little is known about the emission of USV in response to natural 50-kHz USV playback. To close this gap, we capitalized on three data sets previously obtained and analyzed USV evoked by natural 50-kHz USV playback in male juvenile rats. We compared different rat stocks, namely Wistar (WI) and Sprague-Dawley (SD) and investigated the pharmacological treatment with the dopaminergic D2 receptor antagonist haloperidol. These response calls were found to vary broadly inter-individually in numbers, mean peak frequencies, durations and frequency modulations. Despite the large variability, the results showed no major differences between experimental conditions regarding call likelihood or call parameters, representing a robust phenomenon. However, most response calls had clearly lower frequencies and were longer than typical 50-kHz calls, i.e., around 30 kHz and lasting generally around 0.3 s. These calls resemble aversive 22-kHz USV of adult rats but were of higher frequencies and shorter durations. Moreover, blockade of dopamine D2 receptors did not substantially affect the emission of response calls suggesting that they are not dependent on the D2 receptor function. Taken together, this study provides a detailed analysis of response calls toward playback of 50-kHz USV in juvenile WI and SD rats. This includes calls representing 50-kHz USV, but mostly calls with lower frequencies that are not clearly categorizable within the so far known two main groups of USV in adult rats. We discuss the possible functions of these response calls addressing their communicative functions like contact or appeasing calls, and whether they may reflect a state of frustration. In future studies, response calls might also serve as a new read-out in rat models for neuropsychiatric disorders, where acoustic communication is impaired, such as autism spectrum disorder.

Trigeminal neuropathic pain causes changes in affective processing of pain in rats.

Trigeminal neuropathic pain has been modeled in rodents through the constriction of the infraorbital nerve (CCI-ION). Sensory alterations, including spontaneous pain, and thermal and mechanical hyperalgesia are well characterized, but there is a notable lack of evidence about the affective pain component in this model. Evaluation of the emotional component of pain in rats has been proposed as a way to optimize potential translational value of non-clinical studies. In rats, 22 and 50 kHz ultrasonic vocalizations (USVs) are considered well-established measures of negative and positive emotional states, respectively. Thus, this study tested the hypothesis that trigeminal neuropathic pain would result, in addition to the sensory alterations, in a decrease of 50 kHz USV, which may be related to altered function of brain areas involved in emotional pain processing. CCI-ION surgery was performed on 60-day-old male Wistar rats. 15 days after surgery, von Frey filaments were applied to detect mechanical hyperalgesia, and USV was recorded. At the same timepoint, systemic treatment with d,l-amphetamine (1 mg/kg) allowed investigation of the involvement of the dopaminergic system in USV emission. Finally, brain tissue was collected to assess the change in tyrosine hydroxylase (TH) expression in the nucleus accumbens (NAc) and c-Fos expression in brain areas involved in emotional pain processing, including the prefrontal cortex (PFC), amygdala, and NAc. The results showed that CCI-ION rats presented mechanical hyperalgesia and a significant reduction of environmental-induced 50 kHz USV. Amphetamine caused a marked increase in 50 kHz USV emission in CCI-ION rats. In addition, TH expression was lower in constricted animals and c-Fos analysis revealed an increase in neuronal activation. Taken together, these data indicate that CCI-ION causes a reduction in the emission of environmental-induced appetitive calls concomitantly with facial mechanical hyperalgesia and that both changes may be related to a reduction in the mesolimbic dopaminergic activity.

LMT USV Toolbox, a Novel Methodological Approach to Place Mouse Ultrasonic Vocalizations in Their Behavioral Contexts-A Study in Female and Male C57BL/6J Mice and in Shank3 Mutant Females.

Ultrasonic vocalizations (USVs) are used as a phenotypic marker in mouse models of neuropsychiatric disorders. Nevertheless, current methodologies still require time-consuming manual input or sound recordings clean of any background noise. We developed a method to overcome these two restraints to boost knowledge on mouse USVs. The methods are freely available and the USV analysis runs online at https://usv.pasteur.cloud. As little is currently known about usage and structure of ultrasonic vocalizations during social interactions over the long-term and in unconstrained context, we investigated mouse spontaneous communication by coupling the analysis of USVs with automatic labeling of behaviors. We continuously recorded during 3 days undisturbed interactions of same-sex pairs of C57BL/6J sexually naive males and females at 5 weeks and 3 and 7 months of age. In same-sex interactions, we observed robust differences between males and females in the amount of USVs produced, in the acoustic structure and in the contexts of emission. The context-specific acoustic variations emerged with increasing age. The emission of USVs also reflected a high level of excitement during social interactions. We finally highlighted the importance of studying long-term spontaneous communication by investigating female mice lacking Shank3, a synaptic protein associated with autism. While the previous short-time constrained investigations could not detect USV emission abnormalities, our analysis revealed robust differences in the usage and structure of the USVs emitted by mutant mice compared to wild-type female pairs.