Ultrasonic Vocalizations Research Articles

Acute Effects of Hypothermia and Inhalant Anesthesia on Ultrasonic Vocalizations and Neuroendocrine Markers in Neonatal Rats

Neonatal rodents undergo anesthesia for numerous procedures and for euthanasia by anesthetic overdose. However, data regarding whether neonatal anesthesia is humane are limited. Hypothermia (cryoanesthesia) is the most commonly used anesthetic protocol for neonatal rats 10 d of age or younger. However, hypothermia has recently been restricted in several countries due to perceived painful effects, including pain on rewarming. Minimizing the potential pain and distress of neonates in research is imperative, although very challenging. Traditional validated and nonvalidated behavioral and physiologic outcome measures used for adult rats undergoing anesthesia are unsuitable for evaluating neonates. Therefore, we investigated the effects of several anesthetic methods on neonatal rats by using the innovative objective approaches of noninvasive ultrasonic vocalizations and more invasive neuroendocrine responses (i.e., serum corticosterone, norepinephrine, glucose). Our results show that hypothermia leads to heightened acute distress in neonatal rats as indicated by prolonged recovery times, increased duration of vocalizations, and elevated corticosterone levels, as compared with neonates undergoing inhalational anesthesia. We demonstrate that inhalational anesthesia is preferable to cryoanesthesia for neonatal rats, and researchers using hypothermia anesthesia should consider using inhalational anesthesia as an alternative method.

Rodent ultrasonic vocalizations as emotional expressions: Vocal communication in mice and rats

Rodent ultrasonic vocalizations as emotional expressions: Vocal communication in mice and rats

Altered ultrasonic vocalizations and impaired social behaviors in a mouse model of copy number variation in the neuropeptide receptor VIPR2 gene

Altered ultrasonic vocalizations and impaired social behaviors in a mouse model of copy number variation in the neuropeptide receptor VIPR2 gene

Molecular pattern of a decrease in the rewarding effect of cocaine after an escalating-dose drug regimen

BackgroundLong-term cocaine exposure leads to dysregulation of the reward system and initiates processes that ultimately weaken its rewarding effects. Here, we studied the influence of an escalating-dose cocaine regimen on drug-associated appetitive behavior after a withdrawal period, along with corresponding molecular changes in plasma and the prefrontal cortex (PFC).MethodsWe applied a 5 day escalating-dose cocaine regimen in rats. We assessed anxiety-like behavior at the beginning of the withdrawal period in the elevated plus maze (EPM) test. The reinforcement properties of cocaine were evaluated in the Conditioned Place Preference (CPP) test along with ultrasonic vocalization (USV) in the appetitive range in a drug-associated context. We assessed corticosterone, proopiomelanocortin (POMC), β-endorphin, CART 55–102 levels in plasma (by ELISA), along with mRNA levels for D2 dopaminergic receptor (D2R), κ-receptor (KOR), orexin 1 receptor (OX1R), CART 55–102, and potential markers of cocaine abuse: miRNA-124 and miRNA-137 levels in the PFC (by PCR).ResultsRats subjected to the escalating-dose cocaine binge regimen spent less time in the cocaine-paired compartment, and presented a lower number of appetitive USV episodes. These changes were accompanied by a decrease in corticosterone and CART levels, an increase in POMC and β-endorphin levels in plasma, and an increase in the mRNA for D2R and miRNA-124 levels, but a decrease in the mRNA levels for KOR, OX1R, and CART 55–102 in the PFC.ConclusionsThe presented data reflect a part of a bigger picture of a multilevel interplay between neurotransmitter systems and neuromodulators underlying processes associated with cocaine abuse.

Intrauterine Inflammation Leads to Select Sex- and Age-Specific Behavior and Molecular Differences in Mice.

Sex-specific differences in behavior have been observed in anxiety and learning in children exposed to prenatal inflammation; however, whether these behaviors manifest differently by age is unknown. This study assesses possible behavioral changes due to in utero inflammation as a function of age in neonatal, juvenile, and adult animals and presents potential molecular targets for observed differences. CD-1 timed pregnant dams were injected in utero with lipopolysaccharide (LPS, 50 μg/animal) or saline at embryonic day 15. No differences in stress responses were measured by neonatal ultrasonic vocalizations between LPS- and saline-exposed groups of either sex. By contrast, prenatal inflammation caused a male-specific increase in anxiety in mature but not juvenile animals. Juvenile LPS-exposed females had decreased movement in open field testing that was not present in adult animals. We additionally observed improved memory retrieval after in utero LPS in the juvenile animals of both sexes, which in males may be related to a perseverative phenotype. However, there was an impairment of long-term memory in only adult LPS-exposed females. Finally, gene expression analyses revealed that LPS induced sex-specific changes in genes involved in hippocampal neurogenesis. In conclusion, intrauterine inflammation has age- and sex-specific effects on anxiety and learning that may correlate to sex-specific disruption of gene expression associated with neurogenesis in the hippocampus.

Rates of ultrasonic vocalizations are more strongly related than acoustic features to non-vocal behaviors in mouse pups.

Mouse pups produce. ultrasonic vocalizations (USVs) in response to isolation from the nest (i.e., isolation USVs). Rates and acoustic features of isolation USVs change dramatically over the first two weeks of life, and there is also substantial variability in the rates and acoustic features of isolation USVs at a given postnatal age. The factors that contribute to within age variability in isolation USVs remain largely unknown. Here, we explore the extent to which non-vocal behaviors of mouse pups relate to the within age variability in rates and acoustic features of their USVs. We recorded non-vocal behaviors of isolated C57BL/6J mouse pups at four postnatal ages (postnatal days 5, 10, 15, and 20), measured rates of isolation USV production, and applied a combination of pre-defined acoustic feature measurements and an unsupervised machine learning-based vocal analysis method to examine USV acoustic features. When we considered different categories of non-vocal behavior, our analyses revealed that mice in all postnatal age groups produce higher rates of isolation USVs during active non-vocal behaviors than when lying still. Moreover, rates of isolation USVs are correlated with the intensity (i.e., magnitude) of non-vocal body and limb movements within a given trial. In contrast, USVs produced during different categories of non-vocal behaviors and during different intensities of non-vocal movement do not differ substantially in their acoustic features. Our findings suggest that levels of behavioral arousal contribute to within age variability in rates, but not acoustic features, of mouse isolation USVs.

Sex-specific neurobehavioral and prefrontal cortex gene expression alterations following developmental acetaminophen exposure in mice

Acetaminophen (N-acetyl-p-aminophenol (APAP), also known as paracetamol) is one of the most common medications used by the general population, including pregnant people. Although many human observational and animal model studies have shown associations between prenatal and early postnatal APAP exposure and attention deficit hyperactivity disorder, autism spectrum disorders, and altered neurodevelopment, the existing literature is limited. In particular, no mouse studies of prenatal APAP exposure have investigated offspring attention deficits in behavioral tasks specifically designed to measure attention, and no prior rodent studies have utilized ‘omics’ technologies, such as transcriptomics, for an untargeted exploration of potential mechanisms. We randomly assigned pregnant mice (starting embryonic day 4–10) to receive APAP (150 mg/kg/day) or vehicle control through postnatal day 14. We evaluated 111 mouse offspring in a battery of behavioral tests, including pup ultrasonic vocalizations, elevated plus-maze, open field test, CatWalk (gait), pre-pulse inhibition, and the automated 5-choice serial reaction time task. Prefrontal cortex was collected at birth from 24 pups for RNA sequencing. Developmental APAP treatment resulted in increased and hastened separation-induced pup vocalizations between postnatal days 2 and 11, as well as decreased ambulation and vertical rearings in the open field in male but not female adult offspring. APAP treatment was also associated with altered sex-specific prefrontal cortex gene expression relating to glutathione and cytochrome p450 metabolism, DNA damage, and the endocrine and immune systems. This study provides additional evidence for the neurodevelopmental harm of prenatal APAP exposure and generates hypotheses for underlying molecular pathways via RNA sequencing.

Reactivity to conditioned threat cues is distinct from exploratory drive in the elevated plus maze.

Fear and anxiety are adaptive states that allow humans and animals alike to respond appropriately to threatening cues in their environment. Commonly used tasks for studying behaviour akin to fear and anxiety in rodent models are Pavlovian threat conditioning and the elevated plus maze (EPM), respectively. In threat conditioning the rodents learn to associate an aversive event with a specific stimulus or context. The learnt association between the two stimuli (the 'memory') can then be recalled by re-exposing the subject to the conditioned stimulus. The elevated plus maze is argued to measure the agoraphobic avoidance of the brightly lit open maze arms in crepuscular rodents. These two tasks have been used extensively, yet research into whether they interact is scarce. We investigated whether recall of an aversive memory, across contextual, odour or auditory modalities, would potentiate anxiety-like behaviour in the elevated plus maze. The data did not support that memory recall, even over a series of time points, could influence EPM behaviour. Furthermore, there was no correlation between EPM behaviour and conditioned freezing in independent cohorts tested in the EPM before or after auditory threat conditioning. Further analysis found the production of 22 kHz ultrasonic vocalisations revealed the strongest responders to a conditioned threat cue. These results are of particular importance for consideration when using the EPM and threat conditioning to identify individual differences and the possibility to use the tasks in batteries of tests without cross-task interference.

Male scent but not courtship vocalizations induce estrus in wild female house mice

Exposure to males or male urinary scent can induce and accelerate the rate of female estrous cycling in house mice ("Whitten effect"), and this response has been replicated many times since its discovery over 60 years ago. Here, we tested whether exposing female mice to recordings of male courtship ultrasonic vocalizations (USVs) induces estrous cycling, and whether exposure to both male scent and USVs has a stronger effect than to either of these stimuli alone. We conducted our study with 60 wild-derived female house mice (Mus musculus musculus). After singly housing females for 14 days, we monitored estrous stages via vaginal cytology for two weeks while isolated from males or male stimuli. We continued monitoring estrus for two more weeks during experimental exposure to one of four different types of stimuli: (1) clean bedding and background noise playback (negative control); (2) recordings of male USVs (16 min per day) and clean bedding (male USV treatment); (3) soiled male bedding and background noise playback (male odor treatment; positive control); or (4) male USVs and soiled male bedding (male odor and USV treatment). Females were then paired with males to test whether any of the four treatments influenced female reproduction (especially latency to birth). We confirmed that exposure to male odor increased female cycling, as expected, but exposure to recordings of male USVs had no effect on estrus. Females exposed to both USVs and odor went through more cycles compared to controls, but did not differ significantly from exposure to male odor (and background noise). After pairing females with a male, females showing male odor-induced cycling produced their first litter sooner than controls, whereas USVs did not have such an effect. This is the first study to our knowledge to show that male odor induces estrus in wild house mice and to show functional effects on reproduction. Our results do not support the hypothesis that male vocalizations induce female estrus, although we suggest other approaches that could be used to further test this hypothesis.

Changes in ultrasonic vocalizations after unilateral cerebral ischemia in a rat stroke model

Stroke frequently results in communication impairments that negatively impact quality of life and overall recovery, yet the biological mechanisms underlying these changes are not well understood. Ultrasonic vocalizations (USVs) in rodent models of disease and aging have been used to improve our understanding of the biological mechanisms that underlie vocal deficits and their response to interventions. Changes in USVs after middle cerebral artery occlusion (MCAO) in mice have been reported, yet rat models have significant anatomical and behavioral advantages over mice, including the ability to vocally train rats with an established paradigm. We sought to determine whether a unilateral MCAO rat stroke model provides a biologically and behaviorally relevant way to study post stroke vocalization deficits. We hypothesized that left MCAO would be associated with changes in USVs. Six weeks after MCAO or sham-control surgery, USVs were recorded in rats using an established mating paradigm. Stroke was associated with differences in USV acoustics including more frequent use of simple calls characterized by shorter durations and restricted bandwidths. These parameters were also found to correlate with post stroke lingual weakness. This is the first study to describe changes to rat USVs using a stroke model. These results suggest the unilateral MCAO rat stroke model is a biologically and behaviorally relevant model to understand how stroke affects vocal behaviors.

An IGFBP2-derived peptide promotes neuroplasticity and rescues deficits in a mouse model of Phelan-McDermid syndrome.

We developed an IGFBP2-mimetic peptide fragment, JB2, and showed that it promotes basal synaptic structural and functional plasticity in cultured neurons and mice. We demonstrate that JB2 directly binds to dendrites and synapses, and its biological activity involves NMDA receptor activation, gene transcription and translation, and IGF2 receptors. It is not IGF1 receptor-dependent. In neurons, JB2 induced extensive remodeling of the membrane phosphoproteome. Synapse and cytoskeletal regulation, autism spectrum disorder (ASD) risk factors, and a Shank3-associated protein network were significantly enriched among phosphorylated and dephosphorylated proteins. Haploinsufficiency of the SHANK3 gene on chromosome 22q13.3 often causes Phelan-McDermid Syndrome (PMS), a genetically defined form of autism with profound deficits in motor behavior, sensory processing, language, and cognitive function. We identified multiple disease-relevant phenotypes in a Shank3 heterozygous mouse and showed that JB2 rescued deficits in synaptic function and plasticity, learning and memory, ultrasonic vocalizations, and motor function; it also normalized neuronal excitability and seizure susceptibility. Notably, JB2 rescued deficits in the auditory evoked response latency, alpha peak frequency, and steady-state electroencephalography response, measures with direct translational value to human subjects. These data demonstrate that JB2 is a potent modulator of neuroplasticity with therapeutic potential for the treatment of PMS and ASD.

In utero exposure to cannabidiol disrupts select early-life behaviors in a sex-specific manner

Cannabidiol (CBD), one of the main components of cannabis, is generally considered safe. CBD crosses the placenta and its use during pregnancy is steadily increasing, the impact of gestational CBD’s effects on prenatal life and neurodevelopment are poorly understood. Here, we combined behavioral approaches and deep learning analysis to assess the sex-dependent neonatal behavior of CBD exposed progeny. Gestating C57BL6/J dams were exposed daily with vehicle or CBD (3 mg/Kg, s.c.), from gestational day 5 to 18. Body weight, pup ultrasound vocalizations (USVs, PND 10) and homing behavior (PND 13) were quantified in the progeny. Thus, male (but not female) pups from CBD-treated dams gained more weight than sham. There were sex-dependent differences in the coarse characteristics of ultrasonic vocalizations. Prenatally-CBD exposed male pups emitted shorter calls, whereas CBD females made more high frequency calls when compared with their control counterparts. There were significant qualitative changes in the syllabic USV repertoire reflected in call typologies and communication patterns. Finally, the homing behavior test showed that CBD-exposed females presented a greater vulnerability to gestational CBD than males. Only CBD-exposed female pups showed reduced motor and discriminatory abilities. Together the results suggest a sexual divergence in the consequences of in utero CBD exposure on neonates at early developmental ages, which may be predictive of adult psychopathology. Given the extent of cannabis and CBD use worldwide, these findings challenge the idea that CBD is a universally safe compound and reveal the need for additional studies on the effect of perinatal CBD exposure.

Microbe–Immune–Stress Interactions Impact Behaviour during Postnatal Development

Decades of research have established the role of microbiota-brain communication in behaviour and brain function. Studies have shown that microbiota composition and diversity are influenced by a variety of factors including host genetics, diet, and other environmental exposures, with implications for the immunological and neurobiological development of the host organism. To further understand early-life interactions between environment, genetic factors, the microbiome and the central nervous system, we investigated the impact of postnatal stress in C57Bl/6 wild type and T-cell deficient mice on microbe-brain interactions and behaviour. Mice were exposed to immune challenge with lipopolysaccharide (LPS) at postnatal day (P) 3 and maternal separation at P9 (16 h overnight). Behavioural assessment of growth and development as well as behaviour (righting reflex, ultrasonic vocalizations in response to brief maternal separation, open field, sociability, and grooming) was conducted. Microbiota diversity and composition of fecal samples collected at P24 revealed reduced alpha diversity in T-cell-deficient mice as well as genotype- and stress-related taxa. Notably, integrated analyses of microbiota and behaviour in the context of immunocompromise revealed key behavioural related taxa that may be important to brain development. These findings are important to determining the influence of genetic and environmental factors on gut microbiota and advances our understanding microbiome-brain signaling pathways on neurodevelopment and behaviour.

Introduction of gloved hand to cage induces 22-kHz ultrasonic vocalizations in male albino rats.

Rodents emit ultrasonic vocalizations (USVs) above the human hearing threshold of ~ 20 kHz to communicate emotional states and to coordinate their social interactive behavior. Twenty-two kHz USVs emitted by adult rats have been reported in a variety of aversive social and behavioral situations. They occur not only under painful or restraining conditions but can also be evoked by gentle cutaneous touch or airflow. This study aimed to test if placement of a human hand in a cage can evoke 22-kHz USVs. It was found that 36% of the adult male Sprague-Dawley and 13% of the adult male Wistar Han rats emitted 22-kHz USVs when a gloved hand was introduced into the cages. Average vocalization onset latencies were 5.0 ± 4.4 s (Sprague-Dawley) and 7.4 ± 4.0 s (Wistar Han) and the USVs had a stable frequency (22 kHz) across the calls, ranging from 0.1 to 2.3 seconds in duration. Surprisingly, no 22-kHz USVs were found in any female Wistar Han rats tested. To further explore the mechanisms underlying this observation, we compared retinal function, basal serum corticosterone, and testosterone levels between the 22-kHz USV responders and non-responders. None of these parameters or endpoints showed any significant differences between the two cohorts. The results suggest that the introduction of a gloved-hand inside the cage can trigger adult male albino rats to emit 22-kHz ultrasonic vocalizations. This response should be considered in USV studies and animal welfare.

Stage 1 Registered Report: Refinement of tickling protocols to improve positive animal welfare in laboratory rats.

Rat tickling is a heterospecific interaction for experimenters to mimic the interactions of rat play, where they produce 50 kHz ultrasonic vocalisations (USV), symptoms of positive affect; tickling can improve laboratory rat welfare. The standard rat tickling protocol involves gently pinning the rat in a supine position. However, individual response to this protocol varies. This suggests there is a risk that some rats may perceive tickling as only a neutral experience, while others as a positive one, depending on how tickling is performed. Based on our research experiences of the standard tickling protocol we have developed a playful handling (PH) protocol, with reduced emphasis on pinning, intended to mimic more closely the dynamic nature of play. We will test whether our PH protocol gives rise to more uniform increases in positive affect across individuals relative to protocols involving pinning. We will compare the response of juvenile male and female Wistar rats as: Control (hand remains still against the side of the test arena), P0 (PH with no pinning), P1 (PH with one pin), P4 (PH with four pins). P1 and P4 consist of a background of PH, with treatments involving administration of an increasing dosage of pinning per PH session. We hypothesise that rats exposed to handling protocols that maximise playful interactions (where pinning number per session decreases) will show an overall increase in total 50 kHz USV as an indicator of positive affect, with less variability. We will explore whether behavioural and physiological changes associated with alterations in PH experience are less variable. We propose that maximising the numbers of rats experiencing tickling as a positive experience will reduce the variation in response variables affected by tickling and increase the repeatability of research where tickling is applied either as a social enrichment or as a treatment.

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.

Assessment of the effects of sex, age, and rearing condition on ultrasonic vocalizations elicited by pups during the maternal potentiation paradigm in C57BL/6J mice.

Isolation-induced ultrasonic vocalizations (USVs) are important to elicit parental retrieval. This behavior is critical for the animal's survival and can be altered in models of developmental disorders. The potentiation of vocalizations in response to reunion with the dam, also called maternal potentiation, has been extensively studied in rats. However, the assessment of this paradigm in mice is scarce. In rats, the potentiation of vocalizations is dependent on rearing conditions. Since mice are the main species used for genetic models of diseases, we aimed to investigate how different factors such as age, sex, and rearing conditions can affect the potentiation of vocalizations in the maternal potentiation paradigm in mice. We carried out experiments using biparental (dam and sire) or uniparental rearing (dam). Pups were tested on postnatal days (PD) 9 or 12. Pups showed increased potentiation in both sexes at PD9 with uniparental rearing. Both rearing conditions and ages changed the repertoire from the first to the second isolation. Spectral parameters were affected by sex, rearing condition and reunion at PD9. At PD12, only duration was altered by reunion. We conclude that the performance of the pups in the maternal potentiation paradigm is dependent on age, sex, and rearing condition.

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.

Sounding the Alarm: Sex Differences in Rat Ultrasonic Vocalizations during Pavlovian Fear Conditioning and Extinction.

Pavlovian fear conditioning is a prevalent tool in the study of aversive learning, which is a key component of stress-related psychiatric disorders. Adult rats can exhibit various threat-related behaviors, including freezing, motor responses, and ultrasonic vocalizations (USVs). While these responses can all signal aversion, we know little about how they relate to one another. Here we characterize USVs emitted by male and female rats during cued fear acquisition and extinction, and assess the relationship between different threat-related behaviors. We found that males consistently emitted >22 kHz calls (referred to here as "alarm calls") than females, and that alarm call frequency in males, but not females, related to the intensity of the shock stimulus. Interestingly, 25% of males and 45% of females did not emit any alarm calls at all. Males that did make alarm calls had significantly higher levels of freezing than males who did not, while no differences in freezing were observed between female Alarm callers and Non-alarm callers. Alarm call emission was also affected by the predictability of the shock; when unpaired from a tone cue, both males and females started emitting alarm calls significantly later. During extinction learning and retrieval sessions, males were again more likely than females to emit alarm calls, which followed an extinction-like reduction in frequency. Collectively these data suggest sex dependence in how behavioral readouts relate to innate and conditioned threat responses. Importantly, we suggest that the same behaviors can signal sex-dependent features of aversion.

Contingent Social Interaction Does Not Prevent Habituation towards Playback of Pro-Social 50-kHz Calls: Behavioral Responses and Brain Activation Patterns.

Rats, which are highly social animals, are known to communicate using ultrasonic vocalizations (USV) in different frequency ranges. Calls around 50 kHz are related to positive affective states and promote social interactions. Our previous work has shown that the playback of natural 50-kHz USV leads to a strong social approach response toward the sound source, which is related to activation in the nucleus accumbens. In male Wistar rats, the behavioral response habituates, that is, becomes weaker or is even absent, when such playback is repeated several days later, an outcome found to be memory-dependent. Here, we asked whether such habituation is due to the lack of a contingent social consequence after playback in the initial test and whether activation of the nucleus accumbens, as measured by c-fos immunohistochemistry, can still be observed in a retest. To this end, groups of young male Wistar rats underwent an initial 50-kHz USV playback test, immediately after which they were either (1) kept temporarily alone, (2) exposed to a same-sex juvenile, or (3) to their own housing group. One week later, they underwent a retest with playback; this time not followed by social consequences but by brain removal for c-fos immunohistochemistry. Consistent with previous reports, behavioral changes evoked by the initial exposure to 50-kHz USV playback included a strong approach response. In the retest, no such response was found, irrespective of whether rats had experienced a contingent social consequence after the initial test or not. At the neural level, no substantial c-fos activation was found in the nucleus accumbens, but unexpected strong activation was detected in the anterior cingulate cortex, with some of it in GABAergic cells. The c-fos patterns did not differ between groups but cell numbers were individually correlated with behavior, i.e., rats that still approached in response to playback in the retest showed more activation. Together, these data do not provide substantial evidence that the lack of a contingent social consequence after 50-kHz USV playback accounts for approach habituation in the retest. Additionally, there is apparently no substantial activation of the nucleus accumbens in the retest, whereas the exploratory findings in the anterior cingulate cortex indicate that this brain area might be involved when individual rats still approach 50-kHz USV playback.