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Background: The Duchenne and Becker muscular dystrophies (DMD, BMD) are neuromuscular disorders commonly associated with diverse cognitive and behavioral comorbidities. Genotype–phenotype studies suggest that severity and risk of central defects in DMD patients increase with cumulative loss of different dystrophins produced in CNS from independent promoters of the DMD gene. Mutations affecting all dystrophins are nevertheless rare and therefore the clinical evidence on the contribution of the shortest Dp71 isoform to cognitive and behavioral dysfunctions is limited. In this study, we evaluated social, emotional and locomotor functions, and fear-related learning in the Dp71-null mouse model specifically lacking this short dystrophin. Results: We demonstrate the presence of abnormal social behavior and ultrasonic vocalization in Dp71-null mice, accompanied by slight changes in exploratory activity and anxiety-related behaviors, in the absence of myopathy and alterations of learning and memory of aversive cue-outcome associations. Conclusions: These results support the hypothesis that distal DMD gene mutations affecting Dp71 may contribute to the emergence of social and emotional problems that may relate to the autistic traits and executive dysfunctions reported in DMD. The present alterations in Dp71-null mice may possibly add to the subtle social behavior problems previously associated with the loss of the Dp427 dystrophin, in line with the current hypothesis that risk and severity of behavioral problems in patients increase with cumulative loss of several brain dystrophin isoforms. [ABSTRACT FROM AUTHOR]

Studying ultrasonic vocalizations (USVs) plays a crucial role in understanding animal communication, particularly in the field of ethology and neuropharmacology. Communication is associated with social behaviour; so, USVs study is a valid assay in behavioural readout and monitoring in this context. This paper delved into an investigation of ultrasonic communication in mice treated with Cannabis sativa oil (CS mice), which has been demonstrated having a prosocial effect on behaviour of mice, versus control mice (vehicle‐treated, VH mice). To conduct this study, we created a dataset by recording audio‐video files and annotating the duration of time that test mice spent engaging in social activities, along with categorizing the types of emitted USVs. The analysis encompassed the frequency of individual sounds as well as more complex sequences of consecutive syllables (patterns). The primary goal was to examine the extent and nature of diversity in ultrasonic communication patterns emitted by these two groups of mice. As a result, we observed statistically significant differences for each considered pattern length between the two groups of mice. Additionally, the study extended its research by considering specific behaviours, aiming to ascertain whether dissimilarities in ultrasonic communication between CS and VH mice are more pronounced or subtle within distinct behavioural contexts. Our findings suggest that while there is variation in USV communication between the two groups of mice, the degree of this diversity may vary depending on the specific behaviour being observed. [ABSTRACT FROM AUTHOR]

Memories are thought to be stored within sparse collections of neurons known as engram ensembles. Neurons active during a training episode are allocated to an engram ensemble ('engram neurons'). Memory retrieval is initiated by external sensory or internal cues present at the time of training reactivating engram neurons. Interestingly, optogenetic reactivation of engram ensemble neurons alone in the absence of external sensory cues is sufficient to induce behaviour consistent with memory retrieval in mice. However, there may exist differences between the behaviours induced by natural retrieval cues or artificial engram reactivation. Here, we compared two defensive behaviours (freezing and the syllable structure of ultrasonic vocalizations, USVs) induced by sensory cues present at training (natural memory retrieval) and optogenetic engram ensemble reactivation (artificial memory retrieval) in a threat conditioning paradigm in the same mice. During natural memory recall, we observed a strong positive correlation between freezing levels and distinct USV syllable features (characterized by an unsupervised algorithm, MUPET (Mouse Ultrasonic Profile ExTraction)). Moreover, we observed strikingly similar behavioural profiles in terms of freezing and USV characteristics between natural memory recall and artificial memory recall in the absence of sensory retrieval cues. Although our analysis focused on two behavioural measures of threat memory (freezing and USV characteristics), these results underscore the similarities between threat memory recall triggered naturally and through optogenetic reactivation of engram ensembles. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'. [ABSTRACT FROM AUTHOR]

Rodent pups use a variety of ultrasonic vocalizations (USVs) to facilitate maternal care. Importantly, infant USV repertoires are dependent on both the age and early life experiences of the pups. We have shown that an adverse rearing environment modeled with the maternal separation (MS) paradigm alters caregiving behavior but little is known about how pup USVs differentially elicit maternal attention. In the present study, maternal approach towards a vocalizing pup over a non‐vocalizing pup was tested in a Y‐maze apparatus at two developmental time points over the course of MS. At postnatal day (P)10, MS dams engaged in longer interaction times with vocalizing pups compared to non‐vocalizing pup, and this effect was strongest in male pups. As expected at P20, dams did not show a preference for either the vocalizing or non‐vocalizing pups regardless of rearing environment; however, MS dams spent a greater amount of time in the center of the apparatus as compared to control dams, which can be interpreted as a measure of uncertainty or indecision. These effects of MS on dam USV sensitivity are important considering the sex specific effects of MS exposure across all developmental stages. Our novel findings support the hypothesis that sex‐specific pup‐dam interactions may drive later life outcomes following adversity. [ABSTRACT FROM AUTHOR]

Abstract Background The Duchenne and Becker muscular dystrophies (DMD, BMD) are neuromuscular disorders commonly associated with diverse cognitive and behavioral comorbidities. Genotype–phenotype studies suggest that severity and risk of central defects in DMD patients increase with cumulative loss of different dystrophins produced in CNS from independent promoters of the DMD gene. Mutations affecting all dystrophins are nevertheless rare and therefore the clinical evidence on the contribution of the shortest Dp71 isoform to cognitive and behavioral dysfunctions is limited. In this study, we evaluated social, emotional and locomotor functions, and fear-related learning in the Dp71-null mouse model specifically lacking this short dystrophin. Results We demonstrate the presence of abnormal social behavior and ultrasonic vocalization in Dp71-null mice, accompanied by slight changes in exploratory activity and anxiety-related behaviors, in the absence of myopathy and alterations of learning and memory of aversive cue-outcome associations. Conclusions These results support the hypothesis that distal DMD gene mutations affecting Dp71 may contribute to the emergence of social and emotional problems that may relate to the autistic traits and executive dysfunctions reported in DMD. The present alterations in Dp71-null mice may possibly add to the subtle social behavior problems previously associated with the loss of the Dp427 dystrophin, in line with the current hypothesis that risk and severity of behavioral problems in patients increase with cumulative loss of several brain dystrophin isoforms.

Background The proliferation of novel psychoactive substances (NPS) in the drug market raises concerns about uncertainty on their pharmacological profile and the health hazard linked to their use. Within the category of synthetic stimulant NPS, the phenethylamine 2-Cl-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA) has been linked to severe intoxication requiring hospitalization. Thereby, the characterization of its pharmacological profile is urgently warranted. Methods By in vivo brain microdialysis in adolescent and adult male rats we investigated the effects of 2-Cl-4,5-MDMA on dopamine (DA) and serotonin (5-HT) neurotransmission in two brain areas critical for the motivational and rewarding properties of drugs, the nucleus accumbens (NAc) shell and the medial prefrontal cortex (mPFC). Moreover, we evaluated the locomotor and stereotyped activity induced by 2-Cl-4,5-MDMA and the emission of 50-kHz ultrasonic vocalizations (USVs) to characterize its affective properties. Results 2-Cl-4,5-MDMA increased dialysate DA and 5-HT in a dose-, brain area-, and age-dependent manner. Notably, 2-Cl-4,5-MDMA more markedly increased dialysate DA in the NAc shell and mPFC of adult than adolescent rats, while the opposite was observed on dialysate 5-HT in the NAc shell, with adolescent rats being more responsive. Furthermore, 2-Cl-4,5-MDMA stimulated locomotion and stereotyped activity in both adolescent and adult rats, although to a greater extent in adolescents. Finally, 2-Cl-4,5-MDMA did not stimulate the emission of 50-kHz USVs. Conclusions This is the first pharmacological characterization of 2-Cl-4,5-MDMA demonstrating that its neurochemical and behavioral effects may differ between adolescence and adulthood. These preclinical data could help understanding the central effects of 2-Cl-4,5-MDMA by increasing awareness on possible health damage in users. [ABSTRACT FROM AUTHOR]

Introduction: Empathic behaviors are driven by the ability to understand the emotional states of others along with the motivation to improve it. Evidence points towards forms of empathy, like targeted helping, in many species including rats. There are several variables that may modulate targeted helping, including sex, sensory modalities, and activity of multiple neural substrates. Methods: Using a model of social contact-independent targeted helping, we first tested whether sex differences exist in helping behavior. Next, we explored sex differences in sensory and affective signaling, including direct visualization and an analysis of ultrasonic vocalizations made between animal pairs. Finally, we examined the neural activity in males and females of multiple regions of interest across time. Here, we aim to examine any behavioral differences in our lab's social contact independent targeted helping task between males and females. Results and Discussion: These findings are the first to intimate that, like other prosocial behaviors, males and females may exhibit similar social-independent targeted helping behavior, but the underlying sensory communication in males and females may differ. In addition, this is the first set of experiments that explore the neural correlates of social-independent targeted helping in both males and females. These results lay the groundwork for future studies to explore the similarities and differences that drive targeted helping in both sexes. [ABSTRACT FROM AUTHOR]

Abstract Background Pup-dam ultrasonic vocalizations (USVs) are essential to cognitive and socio-emotional development. In autism and Fragile X Syndrome (FXS), disruptions in pup-dam USV communication hint at a possible connection between abnormal early developmental USV communication and the later emergence of communication and social deficits. Methods Here, we gathered USVs from PND 10 FXS pups during a short period of separation from their mothers, encompassing animals of all possible genotypes and both sexes (i.e., Fmr1-/y vs. Fmr1+/y males and Fmr1+/+, +/-, and -/- females). This allowed comparing the influence of sex and gene dosage on pups’ communication capabilities. Leveraging DeepSqueak and analyzing vocal patterns, intricate vocal behaviors such as call structure, duration, frequency modulation, and temporal patterns were examined. Furthermore, homing behavior was assessed as a sensitive indicator of early cognitive development and social discrimination. This behavior relies on the use of olfactory and thermal cues to navigate and search for the maternal or nest odor in the surrounding space. Results The results show that FMRP-deficient pups of both sexes display an increased inclination to vocalize when separated from their mothers, and this behavior is accompanied by significant sex-specific changes in the main features of their USVs as well as in body weight. Analysis of the vocal repertoire and syntactic usage revealed that Fmr1 gene silencing primarily alters the USVs’ qualitative composition in males. Moreover, sex-specific effects of Fmr1 silencing on locomotor activity and homing behavior were observed. FMRP deficiency in females increased activity, reduced nest-reaching time, and extended nest time. In males, it prolonged nest-reaching time and reduced nest time without affecting locomotion. Conclusions These findings highlight the interplay between Fmr1 gene dosage and sex in influencing communicative and cognitive skills during infancy.

Background: Pup-dam ultrasonic vocalizations (USVs) are essential to cognitive and socio-emotional development. In autism and Fragile X Syndrome (FXS), disruptions in pup-dam USV communication hint at a possible connection between abnormal early developmental USV communication and the later emergence of communication and social deficits. Methods: Here, we gathered USVs from PND 10 FXS pups during a short period of separation from their mothers, encompassing animals of all possible genotypes and both sexes (i.e., Fmr1-/y vs. Fmr1+/y males and Fmr1+/+, +/-, and -/- females). This allowed comparing the influence of sex and gene dosage on pups' communication capabilities. Leveraging DeepSqueak and analyzing vocal patterns, intricate vocal behaviors such as call structure, duration, frequency modulation, and temporal patterns were examined. Furthermore, homing behavior was assessed as a sensitive indicator of early cognitive development and social discrimination. This behavior relies on the use of olfactory and thermal cues to navigate and search for the maternal or nest odor in the surrounding space. Results: The results show that FMRP-deficient pups of both sexes display an increased inclination to vocalize when separated from their mothers, and this behavior is accompanied by significant sex-specific changes in the main features of their USVs as well as in body weight. Analysis of the vocal repertoire and syntactic usage revealed that Fmr1 gene silencing primarily alters the USVs' qualitative composition in males. Moreover, sex-specific effects of Fmr1 silencing on locomotor activity and homing behavior were observed. FMRP deficiency in females increased activity, reduced nest-reaching time, and extended nest time. In males, it prolonged nest-reaching time and reduced nest time without affecting locomotion. Conclusions: These findings highlight the interplay between Fmr1 gene dosage and sex in influencing communicative and cognitive skills during infancy. Summary: In this study, we investigated ultrasonic vocalizations (USVs) and homing behavior in a mouse model of Fragile X Syndrome (FXS), a leading genetic cause of autism spectrum disorder (ASD) caused by a mutation of the X-chromosome linked Fmr1 gene. Disruptions in pup-dam USV communication and cognitive skills may be linked to the later emergence of communication and social deficits in ASD. USVs were collected from 10-day-old FXS pups of all possible genotypes and both sexes during a short period of separation from their mothers. We utilized DeepSqueak, an advanced deep learning system, to examine vocal patterns and intricate vocal behaviors, including call structure, duration, frequency modulation, and their temporal patterns. Homing, a sensitive indicator of early cognitive development and social discrimination was assessed at P13. The results showed that FXS pups of both sexes displayed an increased inclination to vocalize when separated from their mothers. Examination of the vocal repertoire and its syntactic usage revealed that the silencing of the Fmr1 gene primarily alters the qualitative composition of ultrasonic communication in males. The sex-specific changes observed in USVs were accompanied by modifications in body weight. Regarding homing behavior, the deficiency of FMRP led to opposite deficits in activity, time to reach the nest, and nesting time depending on sex. Taken together, these findings highlight the interplay between Fmr1 gene dosage and sex in shaping communication and cognition during infancy. Highlights: We investigated early life communicative and cognitive abilities in a mouse model of Fragile X Syndrome (FXS), a prominent genetic cause of autism spectrum disorder resulting from a mutation in the Fmr1 gene on the X-chromosome. Ultrasonic vocalizations (USVs) and homing behavior were collected respectively from 10- and 13-day-old FXS pups, encompassing all possible genotypes and both sexes, during a brief separation from their mothers. Both male and female FXS pups exhibited an increased inclination to vocalize when separated from their mothers, along with significant sex-specific alterations in the primary characteristics of their USVs, homing behavior, and body weight. Silencing of the Fmr1 gene primarily influenced the qualitative composition of ultrasonic communication in males. In homing behavior, FMRP's deficiency led to contrary deficits in activity, time to reach the nest, and nesting time depending on sex. The interaction between Fmr1 gene dosage and sex impacted on communication and cognition during early infancy. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a progressive neurologic disease and the most common cause of dementia. Classic pathology in AD is characterized by inflammation, abnormal presence of tau protein, and aggregation of ß-amyloid that disrupt normal neuronal function and lead to cell death. Deficits in communication also occur during disease progression and significantly reduce health, well-being, and quality of life. Because clinical diagnosis occurs in the mid-stage of the disease, characterizing the prodrome and early stages in humans is currently challenging. To overcome these challenges, we use the validated TgF344-AD (F344-Tg(Prp-APP, Prp-PS1)19/Rrrc) transgenic rat model that manifests cognitive, behavioral, and neuropathological dysfunction akin to AD in humans. Objectives: The overarching goal of our work is to test the central hypothesis that pathology and related behavioral deficits such as communication dysfunction in part manifest in the peripheral nervous system and corresponding target tissues already in the early stages. The primary aims of this study are to test the hypotheses that: (1) changes in ultrasonic vocalizations (USV) occur in the prodromal stage at 6 months of age and worsen at 9 months of age, (2) inflammation as well as ADrelated pathology can be found in the thyroarytenoid muscle (TA) at 12 months of age (experimental endpoint tissue harvest), and to (3) demonstrate that the TgF344-AD rat model is an appropriate model for preclinical investigations of early AD-related vocal deficits. Methods: USVs were collected from male TgF344-AD (N = 19) and wildtype (WT) Fischer-344 rats (N = 19) at 6 months (N = 38; WT: n = 19; TgF344-AD: n = 19) and 9 months of age (N = 18; WT: n = 10; TgF344-AD: n = 8) and acoustically analyzed for duration, mean power, principal frequency, low frequency, high frequency, peak frequency, and call type. RT-qPCR was used to assay peripheral inflammation and AD-related pathology via gene expressions in the TA muscle of male TgF344-AD rats (n = 6) and WT rats (n = 6) at 12 months of age. Results: This study revealed a significant reduction in mean power of ultrasonic calls from 6 to 9 months of age and increased peak frequency levels overtime in TgF344-AD rats compared to WT controls. Additionally, significant downregulation of AD-related genes Uqcrc2, Bace2, Serpina3n, and Igf2, as well as downregulation of pro-inflammatory gene Myd88 was found in the TA muscle of TgF344-AD rats at 12 months of age. Discussion: Our findings demonstrate early and progressive vocal deficits in the TgF344-AD rat model. We further provide evidence of dysregulation of ADpathology-related genes as well as inflammatory genes in the TA muscles of TgF344-AD rats in the early stage of the disease, confirming this rat model for early-stage investigations of voice deficits and related pathology. [ABSTRACT FROM AUTHOR]

IntroductionEmpathic behaviors are driven by the ability to understand the emotional states of others along with the motivation to improve it. Evidence points towards forms of empathy, like targeted helping, in many species including rats. There are several variables that may modulate targeted helping, including sex, sensory modalities, and activity of multiple neural substrates.MethodsUsing a model of social contact-independent targeted helping, we first tested whether sex differences exist in helping behavior. Next, we explored sex differences in sensory and affective signaling, including direct visualization and an analysis of ultrasonic vocalizations made between animal pairs. Finally, we examined the neural activity in males and females of multiple regions of interest across time. Here, we aim to examine any behavioral differences in our lab’s social contact independent targeted helping task between males and females.Results and DiscussionThese findings are the first to intimate that, like other prosocial behaviors, males and females may exhibit similar social-independent targeted helping behavior, but the underlying sensory communication in males and females may differ. In addition, this is the first set of experiments that explore the neural correlates of social-independent targeted helping in both males and females. These results lay the groundwork for future studies to explore the similarities and differences that drive targeted helping in both sexes.

Abstract Positive and negative emotional states in rats can be studied by investigating ultrasonic vocalizations (USVs). Positive affect in rats is indexed by 50 kHz hedonic USVs, and negative affect is indexed by 22 kHz aversive calls. We examined the relationship of emotional states in rats using medial prefrontal cortex (MPFC) quantitative electroencephalograms (qEEG) and found that hedonic USVs were associated with active wake qEEG (high alpha/low delta power), and aversive USVs occurred with groggy wake qEEG (low alpha/high delta). Further, alpha frequency electrical stimulation of the MPFC induces hedonic calls and reward-seeking behavior, whereas delta frequency stimulation produces aversive calls and avoidance behavior. The brain region responsible for generating motor output for USVs, the periaqueductal gray (PAG), shows a motor-evoked potential that is temporally locked to the alpha (hedonic) and delta (aversive) motor-evoked potential. Closed-loop alpha frequency electrical stimulation could prevent delta qEEG and aversive USVs. At the neuronal circuit level, the alpha rhythm was associated with synaptic long-term potentiation (LTP) in the cortex, whereas the delta rhythm was associated with synaptic depotentiation (LTD) in the cortex. At the pharmacological level, NMDAR and growth factor modulation regulated these forms of neuroplasticity. At the single neuron level, excitatory neurons show increased activity in response to alpha frequencies and decreased activity during delta frequencies. In humans, the feeling of joy increased alpha and decreased delta power in frontal scalp qEEG, and the opposite response was seen for sadness. Thus, the synchronization of alpha/delta oscillations through the neuronal circuit responsible for emotional expression coordinates emotional behavior, and the switch between active wake/positive affect and groggy wake/negative affect is under the control of an LTP- LTD synaptic plasticity mechanism.

Opioid use disorder has become an epidemic in the United States, fuelled by the widespread availability of fentanyl, which produces rapid and intense euphoria followed by severe withdrawal and emotional distress. We developed a new preclinical model of fentanyl seeking in outbred male and female rats using volitional oral self‐administration (SA) that can be readily applied in labs without intravascular access. Using a traditional two‐lever operant procedure, rats learned to take oral fentanyl vigorously, escalated intake across sessions, and readily reinstated responding to conditioned cues after extinction. Oral SA also revealed individual and sex differences that are essential to studying substance use risk propensity. During a behavioural economics task, rats displayed inelastic demand curves and maintained stable intake across a wide range of fentanyl concentrations. Oral SA was also neatly patterned, with distinct 'loading' and 'maintenance' phases of responding within each session. Using our software DeepSqueak, we analysed ultrasonic vocalizations (USVs), which are innate expressions of current emotional state in rats. Rats produced 50 kHz USVs during loading then shifted quickly to 22 kHz calls despite ongoing maintenance of oral fentanyl taking, reflecting a transition to negative reinforcement. Using fibre photometry, we found that the lateral habenula differentially processed drug cues and drug consumption depending on affective state, with potentiated modulation by drug cues and consumption during the negative affective maintenance phase. Together, these results indicate a rapid progression from positive to negative reinforcement occurs even within an active drug taking session, revealing a within‐session opponent process. [ABSTRACT FROM AUTHOR]

Positive and negative emotional states in rats can be studied by investigating ultrasonic vocalizations (USVs). Positive affect in rats is indexed by 50 kHz hedonic USVs, and negative affect is indexed by 22 kHz aversive calls. We examined the relationship of emotional states in rats using medial prefrontal cortex (MPFC) quantitative electroencephalograms (qEEG) and found that hedonic USVs were associated with active wake qEEG (high alpha/low delta power), and aversive USVs occurred with groggy wake qEEG (low alpha/high delta). Further, alpha frequency electrical stimulation of the MPFC induces hedonic calls and reward-seeking behavior, whereas delta frequency stimulation produces aversive calls and avoidance behavior. The brain region responsible for generating motor output for USVs, the periaqueductal gray (PAG), shows a motor-evoked potential that is temporally locked to the alpha (hedonic) and delta (aversive) motor-evoked potential. Closed-loop alpha frequency electrical stimulation could prevent delta qEEG and aversive USVs. At the neuronal circuit level, the alpha rhythm was associated with synaptic long-term potentiation (LTP) in the cortex, whereas the delta rhythm was associated with synaptic depotentiation (LTD) in the cortex. At the pharmacological level, NMDAR and growth factor modulation regulated these forms of neuroplasticity. At the single neuron level, excitatory neurons show increased activity in response to alpha frequencies and decreased activity during delta frequencies. In humans, the feeling of joy increased alpha and decreased delta power in frontal scalp qEEG, and the opposite response was seen for sadness. Thus, the synchronization of alpha/delta oscillations through the neuronal circuit responsible for emotional expression coordinates emotional behavior, and the switch between active wake/positive affect and groggy wake/negative affect is under the control of an LTP- LTD synaptic plasticity mechanism. [ABSTRACT FROM AUTHOR]

Joseph the MoUSE — Mouse Ultrasonic Sound Explorer (MoUSE) software aims to address the issue of manual analysis of recordings from experiments on rodents by introducing automatic techniques for ultrasonic vocalization (USV) detection. It combines deep learning (DL) methods with classical pattern recognition and computer graphics algorithms. During development, we used a dataset that consisted of recordings from real-world experiments in the open field. Recordings like these pose obstacles to automatic USV detection, one of which is the noise produced by mice in the experimental area or in nearby cages. Therefore, additionally, we conducted research and implemented de-noising methods along with detection algorithms. The project includes Python packages with algorithms for sound noise removal and USV detection, and provides a user-friendly graphical interface.

BackgroundAlzheimer’s disease (AD) is a progressive neurologic disease and the most common cause of dementia. Classic pathology in AD is characterized by inflammation, abnormal presence of tau protein, and aggregation of β-amyloid that disrupt normal neuronal function and lead to cell death. Deficits in communication also occur during disease progression and significantly reduce health, well-being, and quality of life. Because clinical diagnosis occurs in the mid-stage of the disease, characterizing the prodrome and early stages in humans is currently challenging. To overcome these challenges, we use the validated TgF344-AD (F344-Tg(Prp-APP, Prp-PS1)19/Rrrc) transgenic rat model that manifests cognitive, behavioral, and neuropathological dysfunction akin to AD in humans.ObjectivesThe overarching goal of our work is to test the central hypothesis that pathology and related behavioral deficits such as communication dysfunction in part manifest in the peripheral nervous system and corresponding target tissues already in the early stages. The primary aims of this study are to test the hypotheses that: (1) changes in ultrasonic vocalizations (USV) occur in the prodromal stage at 6 months of age and worsen at 9 months of age, (2) inflammation as well as AD-related pathology can be found in the thyroarytenoid muscle (TA) at 12 months of age (experimental endpoint tissue harvest), and to (3) demonstrate that the TgF344-AD rat model is an appropriate model for preclinical investigations of early AD-related vocal deficits.MethodsUSVs were collected from male TgF344-AD (N = 19) and wildtype (WT) Fischer-344 rats (N = 19) at 6 months (N = 38; WT: n = 19; TgF344-AD: n = 19) and 9 months of age (N = 18; WT: n = 10; TgF344-AD: n = 8) and acoustically analyzed for duration, mean power, principal frequency, low frequency, high frequency, peak frequency, and call type. RT-qPCR was used to assay peripheral inflammation and AD-related pathology via gene expressions in the TA muscle of male TgF344-AD rats (n = 6) and WT rats (n = 6) at 12 months of age.ResultsThis study revealed a significant reduction in mean power of ultrasonic calls from 6 to 9 months of age and increased peak frequency levels over time in TgF344-AD rats compared to WT controls. Additionally, significant downregulation of AD-related genes Uqcrc2, Bace2, Serpina3n, and Igf2, as well as downregulation of pro-inflammatory gene Myd88 was found in the TA muscle of TgF344-AD rats at 12 months of age.DiscussionOur findings demonstrate early and progressive vocal deficits in the TgF344-AD rat model. We further provide evidence of dysregulation of AD-pathology-related genes as well as inflammatory genes in the TA muscles of TgF344-AD rats in the early stage of the disease, confirming this rat model for early-stage investigations of voice deficits and related pathology.

A central question in sensory neuroscience is how neurons represent complex natural stimuli. This process involves multiple steps of feature extraction to obtain a condensed, categorical representation useful for classification and behaviour. It has previously been shown that central auditory neurons in the starling have composite receptive fields composed of multiple features. Whether this property is an idiosyncratic characteristic of songbirds, a group of highly specialized vocal learners or a generic property of sensory processing is unknown. To address this question, we have recorded responses from auditory cortical neurons in mice, and characterized their receptive fields using mouse ultrasonic vocalizations (USVs) as a natural and ethologically relevant stimulus and pitch‐shifted starling songs as a natural but ethologically irrelevant control stimulus. We have found that these neurons display composite receptive fields with multiple excitatory and inhibitory subunits. Moreover, this was the case with either the conspecific or the heterospecific vocalizations. We then trained the sparse filtering algorithm on both classes of natural stimuli to obtain statistically optimal features, and compared the natural and artificial features using UMAP, a dimensionality‐reduction algorithm previously used to analyse mouse USVs and birdsongs. We have found that the receptive‐field features obtained with both types of the natural stimuli clustered together, as did the sparse‐filtering features. However, the natural and artificial receptive‐field features clustered mostly separately. Based on these results, our general conclusion is that composite receptive fields are not a unique characteristic of specialized vocal learners but are likely a generic property of central auditory systems. Key points: Auditory cortical neurons in the mouse have composite receptive fields with several excitatory and inhibitory features.Receptive‐field features capture temporal and spectral modulations of natural stimuli.Ethological relevance of the stimulus affects the estimation of receptive‐field dimensionality. [ABSTRACT FROM AUTHOR]

Simple Summary: Mammals provide information about their emotional state and physical characteristics through vocal signals. Due to a similar vocal production apparatus across mammals, common rules for encoding this information are proposed. With increasing age/body weight, it is suggested that mammals utter elongated calls of lower frequency. With increasing arousal, it is proposed that mammal vocalizations become higher in frequency and longer in duration. However, in recent years an increasing number of mammalian species have showed discrepancies with these common rules. In this study, we investigated whether developing Mongolian gerbil pups fulfill the proposed common rules by recording vocalizations in a low and high arousal condition in four age groups. We discovered they produce three call types covering the audible (<20 kHz) and ultrasonic (>20 kHz) frequency ranges. Interestingly, the call types differed in the encoding of arousal state as well as physical characteristics and only partly fulfilled the common rules suggested for mammals. Thus, our results show that divergent encoding patterns do not only differ between species but also between call types within a species, indicating that rules to transmit information can be shaped by social, developmental, and environmental factors or different production mechanisms for vocalizations in certain frequency ranges. In mammals, common rules for the encoding of arousal and physical characteristics of the sender are suggested based on a similar vocal production apparatus. In this study, we want to investigate to what extent vocalizations of developing Mongolian gerbil pups fulfill these rules. We recorded vocalizations of 28 Mongolian gerbil pups in four developmental stages using a separation paradigm, suggested to induce different arousal levels. For low arousal, a pup was placed in an arena isolated from its siblings and parents; for high arousal, the pup was additionally stressed through the simulation of a predator. An unsupervised cluster analysis revealed three call types: ultrasonic (USV), audible vocalizations (ADV), and transitions between both (USV-ADV). The USV and USV-ADV rate showed an age-dependent decrease, contrasting an age-dependent increase for ADVs. Vocal correlates for the encoding of arousal were found for USVs and of physical characteristics for USVs and ADVs. However, the pattern of encoding these cues differed between call types and only partly confirmed the common rules suggested for mammals. Our results show that divergent encoding patterns do not only differ between species but also between call types within a species, indicating that coding rules can be shaped by socio-ecological factors or call type specific production mechanisms. [ABSTRACT FROM AUTHOR]

Introduction: The lateral hypothalamus (LH) plays an important physiological role in brain function and also plays an important role in substance abuse. The neuropeptides called orexin (or hypocretins) have been identified as being located exclusively in the cell bodies of the LH. Our previous studies have demonstrated that mechanical stimulation (MS) of the ulnar nerve produces strong inhibitory effects on cocaine addiction-like behaviors through activation of LH projection to the lateral habenula (LHb). Methods: Therefore, the present study hypothesized that ulnar MS would suppress the psychomotor responses induced by cocaine through the orexinergic LH-to-LHb pathway. Results: Ulnar MS attenuated cocaine enhancement of locomotor activity and 50-kHz ultrasonic vocalizations, which was prevented by antagonism of orexinreceptor type 2 (OX2R) in the LHb. Injection of orexin-A into the LHb reduced the cocaine-induced psychomotor responses. MS of the ulnar nerve excited LH orexinergic neurons. In addition, the excitation of LHb neurons by MS was blocked by the systemic administration of an OX2R antagonist. Discussion: These findings suggest that MS applied to the ulnar nerve recruits an orexinergic LH-to-LHb pathway to suppress the psychomotor responses induced by cocaine. [ABSTRACT FROM AUTHOR]

Pursuing rewards while avoiding danger is an essential function of any nervous system. Here, we examine a new mechanism helping rats negotiate the balance between risk and reward when making high-stakes decisions. Specifically, we focus on GABA neurons within an emerging mesolimbic circuit nexus: the ventral pallidum (VP). These neurons play a distinct role from other VP neurons in simple motivated behaviors in mice, but their role in more complex motivated behaviors is unknown. Here, we interrogate the behavioral functions of VPGABA neurons in male and female transgenic GAD1:Cre rats (and WT littermates), using a reversible chemogenetic inhibition approach. Using a behavioral assay of risky decision-making, and of the food-seeking and shock-avoidance components of this task, we show that engaging inhibitory Gi/o signaling specifically in VPGABA neurons suppresses motivation to pursue highly salient palatable foods, and possibly also motivation to avoid being shocked. In contrast, inhibiting these neurons did not affect seeking of low-value food, free consumption of palatable food, or unconditioned affective responses to shock. Accordingly, when rats considered whether to pursue food despite potential for shock in a risky decision-making task, inhibiting VPGABA neurons caused them to more readily select a small but safe reward over a large but dangerous one, an effect not seen in the absence of shock threat. Together, results indicate that VPGABA neurons are critical for high-stakes adaptive responding that is necessary for survival, but which may also malfunction in psychiatric disorders.SIGNIFICANCE STATEMENT In a dynamic world, it is essential to implement appropriate behaviors under circumstances involving rewards, threats, or both. Here, we demonstrate a crucial role for VPGABA neurons in high-stakes motivated behavior of several types. We show that this VPGABA role in motivation impacts decision-making, as inhibiting these neurons yields a conservative, risk-averse strategy not seen when the task is performed without threat of shock. These new roles for VPGABA neurons in behavior may inform future strategies for treating addiction, and other disorders of maladaptive decision-making.

Ultrasonic vocalizations (USVs) fulfill an important role in communication and navigation in many species. Because of their social and affective significance, rodent USVs are increasingly used as a behavioral measure in neurodevelopmental and neurolinguistic research. Reliably attributing USVs to their emitter during close interactions has emerged as a difficult, key challenge. If addressed, all subsequent analyses gain substantial confidence. We present a hybrid ultrasonic tracking system, Hybrid Vocalization Localizer (HyVL), that synergistically integrates a high-resolution acoustic camera with high-quality ultrasonic microphones. HyVL is the first to achieve millimeter precision (~3.4–4.8 mm, 91% assigned) in localizing USVs, ~3× better than other systems, approaching the physical limits (mouse snout ~10 mm). We analyze mouse courtship interactions and demonstrate that males and females vocalize in starkly different relative spatial positions, and that the fraction of female vocalizations has likely been overestimated previously due to imprecise localization. Further, we find that when two male mice interact with one female, one of the males takes a dominant role in the interaction both in terms of the vocalization rate and the location relative to the female. HyVL substantially improves the precision with which social communication between rodents can be studied. It is also affordable, open-source, easy to set up, can be integrated with existing setups, and reduces the required number of experiments and animals.

Background. Animals rely on sound for daily activities, and anthropogenic noise is a pollutant that alters the natural soundscape within which they are active. As human infrastructure expands, broadband anthropogenic noise increases, which can affect behaviors of nocturnal animals. Mice are nocturnal animals that produce ultrasonic calls as part of their behavioral repertoire. Methods. We assessed effects of anthropogenic and natural noise on the behaviors of wild deer mice (Peromyscus maniculatus) and woodland jumping mice (Napaeozapus insignis), two species of mice that produce ultrasonic calls. We measured activity, foraging behavior at a foraging tray, and calling behavior to broadcasts of natural and anthropogenic noise, compared to a baseline with no broadcasting, at 25 focal areas in the Southern Appalachian Mountain Range of North Carolina, USA. Results. Deer mice exposed to anthropogenic noise spent less time in focal areas with broadcasted anthropogenic noise. Mice took longer to begin foraging in the presence of anthropogenic noise, they spent less time at the foraging tray, and left fewer husks but consumed the same number of seeds as mice exposed to natural noise. Deer mice were less likely than woodland jumping mice to be the first to enter the focal area and approach food when in the presence of anthropogenic noise. Both species produced few ultrasonic calls in the presence of broadcasted natural and anthropogenic noise compared to their baseline level of calling. We present the first calls recorded from woodland jumping mice. Conclusion. Anthropogenic noise affects activity, foraging behavior, and calling behavior of nocturnal mice. Natural noise also affects the calling behavior of mice. Mouse species respond differently to anthropogenic noise, with deer mice appearing more sensitive to anthropogenic noise than woodland jumping mice. Responses to noise could have important effects on the ecology of mice and these two species respond differently. Species differences should be considered when mitigating the effects of noise in conservation ecology. [ABSTRACT FROM AUTHOR]

Highlights Brain maturation of the nigrostriatal pathway is sex- and age- dependent. Exposure to hypoxia in late pregnancy impacts brain maturation of the nigrostriatal pathway that can be observed during puberty and young adulthood. Gestational hypoxia impacted female offspring during puberty more than males, whereas it impacted male offspring during young adulthood more than females. These novel findings demonstrate that hypoxic insults during pregnancy mediate developmental programming of the cortical and subcortical pathways, in which male offspring exhibit long-term adverse effects compared to female offspring. Long-term adverse effects of gestational hypoxia in offspring can occur in the absence of pregnancy complications, especially if they occur within critical embryological developmental periods.

Abstract Background Various mammalian species emit ultrasonic vocalizations (USVs), which reflect their emotional state and mediate social interactions. USVs are usually analyzed by manual or semi-automated methodologies that categorize discrete USVs according to their structure in the frequency-time domains. This laborious analysis hinders the effective use of USVs as a readout for high-throughput analysis of behavioral changes in animals. Results Here we present a novel automated open-source tool that utilizes a different approach towards USV analysis, termed TrackUSF. To validate TrackUSF, we analyzed calls from different animal species, namely mice, rats, and bats, recorded in various settings and compared the results with a manual analysis by a trained observer. We found that TrackUSF detected the majority of USVs, with less than 1% of false-positive detections. We then employed TrackUSF to analyze social vocalizations in Shank3-deficient rats, a rat model of autism, and revealed that these vocalizations exhibit a spectrum of deviations from appetitive calls towards aversive calls. Conclusions TrackUSF is a simple and easy-to-use system that may be used for a high-throughput comparison of ultrasonic vocalizations between groups of animals of any kind in any setting, with no prior assumptions.

Vital early-life dyadic interaction in mice requires a pup to signal its needs adequately, and a dam to recognize and respond to the pup's cues accurately and timely. Previous research might have missed important biological and/or environmental elements of this complex bidirectional interaction, because it often focused on one dyadic member only. In laboratory rodents, the Pup Retrieval Test (PRT) is the leading procedure to assess pup-directed maternal care. The present study describes BAMBI (Bidirectional Automated Mother-pup Behavioral Interaction test), a novel automated PRT methodology based on synchronous video recording of maternal behavior and audio recording of pup vocalizations, which allows to assess bidirectional dam-pup dyadic interaction. We were able to estimate pup retrieval and pup vocalization parameters accurately in 156 pups from 29 dams on postnatal days (PND) 5, 7, 9, 11, and 13. Moreover, we showed an association between number of emitted USVs and retrieval success, indicating dyadic interdependency and bidirectionality. BAMBI is a promising new automated home-cage behavioral method that can be applied to both basic and preclinical studies investigating complex phenotypes related to early-life social development. [ABSTRACT FROM AUTHOR]

The cholinergic system is a critical regulator of Pavlovian fear learning and extinction. As such, we have begun investigating the cholinergic system's involvement in individual differences in cued fear extinction using a transgenic ChAT::Cre rat model. The current study extends behavioral phenotyping of a transgenic ChAT::Cre rat line by examining both freezing behavior and ultrasonic vocalizations (USVs) during a Pavlovian cued fear learning and extinction paradigm. Freezing, 22 kHz USVs, and 50 kHz USVs were compared between male and female transgenic ChAT::Cre+ rats and their wildtype (Cre‐) littermates during fear learning, contextual and cue‐conditioned fear recall, cued fear extinction, and generalization to a novel tone. During contextual and cued fear recall ChAT::Cre+ rats froze slightly more than their Cre‐ littermates, and displayed significant sex differences in contextual and cue‐conditioned freezing, 22 kHz USVs, and 50 kHz USVs. Females showed more freezing than males in fear recall trials, but fewer 22 kHz distress calls during fear learning and recall. Females also produced more 50 kHz USVs during exposure to the testing chambers prior to tone (or shock) presentation compared with males, but this effect was blunted in ChAT::Cre+ females. Corroborating previous studies, ChAT::Cre+ transgenic rats overexpressed vesicular acetylcholine transporter immunolabeling in basal forebrain, striatum, basolateral amygdala, and hippocampus, but had similar levels of acetylcholinesterase and numbers of ChAT+ neurons as Cre‐ rats. This study suggests that variance in behavior between ChAT::Cre+ and wildtype rats is sex dependent and advances theories that distinct neural circuits and processes regulate sexually divergent fear responses. [ABSTRACT FROM AUTHOR]

In mammals, ivermectin acts as a GABAA receptor agonist and stimulates GABA release. Previous studies showed that ivermectin (IVM) reduces sexual performance, impairing the latency to the first mount and intromission. These parameters are usually considered motivational parameters of sexual behavior. However, IVM increases GABAergic activity leading to motor incoordination. Thus, it is reasonable to propose that IVM affects sexual performance via motor incoordination pathways. The present study analyzed ultrasonic vocalization in rats to verify whether IVM impairs sexual behavior via motivational mechanisms or motor impairment. Because sexual experience attenuates the impairment of motor performance, rats with sexual experience were also studied. Sexually naive and experienced rats were administered a therapeutic IVM dose and saline. The rats were exposed to receptive females, and the latency to the first mount was evaluated, followed by the 50-kHz USV test. IVM treatment in naïve rats increased the latency to first to mount relative to Saline naïve rats, while no differences were observed between saline and experienced rats. In naïve-IVM rats, a reduced frequency and total calls and increased mean time of calls occur relative to SAL-naïve rats. Experienced IVM rats did not show differences in the frequency, mean, and maximal calls close to Saline experienced rats. However, an increase in the total calls and the dominant frequency of calls were observed in IVM-experienced rats compared to Saline experienced rats. A negative and positive correlation occurred between the latency to the first mount and USVs in groups with and without ivermectin exposure. Hence, we propose that ivermectin increased the sexual motivation of rats exposed to a female in estrous based in USVs despite an increased latency to the first mount that occurred. The increased latency to the first mount resulted from motor incoordination, as previously observed and proposed by our group.

Vital early-life dyadic interaction in mice requires a pup to signal its needs adequately, and a dam to recognize and respond to the pup’s cues accurately and timely. Previous research might have missed important biological and/or environmental elements of this complex bidirectional interaction, because it often focused on one dyadic member only. In laboratory rodents, the Pup Retrieval Test (PRT) is the leading procedure to assess pup-directed maternal care. The present study describes BAMBI (Bidirectional Automated Mother-pup Behavioral Interaction test), a novel automated PRT methodology based on synchronous video recording of maternal behavior and audio recording of pup vocalizations, which allows to assess bidirectional dam-pup dyadic interaction. We were able to estimate pup retrieval and pup vocalization parameters accurately in 156 pups from 29 dams on postnatal days (PND) 5, 7, 9, 11, and 13. Moreover, we showed an association between number of emitted USVs and retrieval success, indicating dyadic interdependency and bidirectionality. BAMBI is a promising new automated home-cage behavioral method that can be applied to both basic and preclinical studies investigating complex phenotypes related to early-life social development.

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. [ABSTRACT FROM AUTHOR]

Social play in rats is a highly rewarding, energetic form of social interaction and important for development of the brain and social skills. The 50 kHz ultrasonic vocalizations (USV) emitted during social play are thought to be an expression of a positive affective state (laughter), which in some situations may also function as communication signals. Heterospecific play, 'tickling' by an experimenter, is thought to simulate conspecific play, and has been used to improve welfare and to study the neurobiology of positive affect. Given that tickling evokes substantial amounts of USV, we investigated whether heterospecific play is simulating conspecific play by comparing USV-behaviour associations in both contexts. If the 50 kHz calls are merely an expression of 'laughter' then the pattern and type of emission in both contexts should be similar. By contrast, as playing with a conspecific involves a two-way exchange of signalling, the additional demands on communication should lead to a different pattern of calling. While calling was prevalent in both types of play, how the different types of 50 kHz calls are used in the two contexts differed markedly. The findings suggest that while conspecific and heterospecific play are positive experiences, tickling is not the equivalent of conspecific play. This article is part of the theme issue 'Cracking the laugh code: laughter through the lens of biology, psychology and neuroscience'. [ABSTRACT FROM AUTHOR]

Mechanisms underlying psychostimulant euphoria remain poorly understood. In adult rats, positive emotional states are associated with alterations in 50-kHz ultrasonic vocalizations (USVs): specifically, “trill” calls are promoted over “flat” calls. Here, we investigated the effects of acute and repeated cocaine administration, and—based on previous findings with amphetamine—their possible dependence on beta-adrenergic receptors.Adult male Long-Evans rats received intraperitoneal drug or saline injections before daily USV recording. Fourteen 50-kHz call subtypes were analyzed. In Experiments 1 and 2, cocaine (1–10 mg/kg) and propranolol (10 mg/kg) were tested alone. In Experiment 3, propranolol/cocaine interactions were sought within a conditioned place preference (CPP) procedure. Experiment 4 investigated acute and chronic cocaine effects (Phase 1), and propranolol/cocaine interactions either in an open field (Phase 2) or within a CPP procedure (Phase 3).In drug-naïve animals, cocaine increased the 50-kHz call rate, with sensitization developing rapidly. After more extended exposure, cocaine now also increased the relative prevalence of trill versus flat calls; effects on other subtypes were also revealed. The beta-blocker propranolol prevented neither cocaine CPP nor cocaine effects on USV emission or locomotion but exerted significant USV-related effects when given alone. CPP magnitude and USV-related measures were uncorrelated.With long-term intraperitoneal administration, cocaine can alter the relative prevalence of several 50-kHz call subtypes; its ability to promote trill versus flat calls, in particular, is consistent with a positive affect interpretation. Cocaine’s behavioral effects (i.e., USV-related, locomotor, CPP) appear independent of beta-adrenergic receptor activity. [ABSTRACT FROM AUTHOR]

Neonatal hypoxic-ischemic (HI) brain injury leads to cognitive impairments including social communication disabilities. Current treatments do not sufficiently target these impairments, therefore new tools are needed to examine social communication in models for neonatal brain injury. Ultrasonic vocalizations (USVs) during early life show potential as a measurement for social development and reflect landmark developmental stages in neonatal mice. However, changes in USV emission early after HI injury have not been found yet. Our current study examines USV patterns and classes in the first 3 days after HI injury. C57Bl/6 mice were subjected to HI on postnatal day (P)9 and USVs were recorded between P10 and P12. Audio files were analyzed using the VocalMat automated tool. HI-injured mice emitted less USVs, for shorter durations, and at a higher frequency compared to control (sham-operated) littermates. The HI-induced alterations in USVs were most distinct at P10 and in the frequency range of 50–75 kHz. At P10 HI-injured mouse pups also produced different ratios of USV class types compared to control littermates. Moreover, alterations in the duration and frequency were specific to certain USV classes in HI animals compared to controls. Injury in the striatum and hippocampus contributed most to alterations in USV communication after HI. Overall, neonatal HI injury leads to USV alterations in newborn mice which could be used as a tool to study early HI-related social communication deficits. [Display omitted] • Neonatal brain injury reduces USV numbers and duration, and increases USV frequency. • USV class types are altered after neonatal brain injury. • USVs provide a tool to assess early social communication after neonatal brain injury. [ABSTRACT FROM AUTHOR]

Background: Hypoxia is associated with pregnancy complications, such as preeclampsia, placental abruption, and gestational sleep apnea. Hypoxic insults during gestation can impact the brain maturation of cortical and subcortical pathways, such as the nigrostriatal pathway. However, the long-term effects of in utero hypoxic stress exposure on brain maturation in offspring are unclear, especially exposure during late gestation. The purpose of this study was to determine the impact of gestational hypoxia in late pregnancy on developmental programming of subcortical brain maturation by focusing on the nigrostriatal pathway. Methods: Timed pregnant Long–Evans rats were exposed to chronic intermittent hypoxia or room air normoxia from gestational day (GD) 15–19 (term 22–23 days). Male and female offspring were assessed during two critical periods: puberty from postnatal day (PND) 40–45 or young adulthood (PND 60–65). Brain maturation was quantified by examining (1) the structural development of the nigrostriatal pathway via analysis of locomotor behaviors and the substantia nigra dopaminergic neuronal cell bodies and (2) the refinement of the nigrostriatal pathway by quantifying ultrasonic vocalizations (USVs). Results: The major findings of this study are gestational hypoxia has age- and sex-dependent effects on subcortical brain maturation in offspring by adversely impacting the refinement of the nigrostriatal pathway in the absence of any effects on the structural development of the pathway. During puberty, female offspring were impacted more than male offspring, as evidenced by decreased USV call frequency, chirp USV call duration, and simple call frequency. In contrast, male offspring were impacted more than female offspring during young adulthood, as evidenced by increased latency to first USV, decreased simple USV call intensity, and increased harmonic USV call bandwidth. No effects of gestational hypoxia on the structural development of the nigrostriatal pathway were observed. Conclusions: These novel findings demonstrate hypoxic insults during pregnancy mediate developmental programming of the cortical and subcortical pathways, in which male offspring exhibit long-term adverse effects compared to female offspring. Impairment of cortical and subcortical pathways maturation, such as the nigrostriatal pathway, may increase risk for neuropsychiatric disorders (e.g., mood disorders, cognitive dysfunction, brain connectivity dysfunction). Highlights: Brain maturation of the nigrostriatal pathway is sex- and age- dependent. Exposure to hypoxia in late pregnancy impacts brain maturation of the nigrostriatal pathway that can be observed during puberty and young adulthood. Gestational hypoxia impacted female offspring during puberty more than males, whereas it impacted male offspring during young adulthood more than females. These novel findings demonstrate that hypoxic insults during pregnancy mediate developmental programming of the cortical and subcortical pathways, in which male offspring exhibit long-term adverse effects compared to female offspring. Long-term adverse effects of gestational hypoxia in offspring can occur in the absence of pregnancy complications, especially if they occur within critical embryological developmental periods. [ABSTRACT FROM AUTHOR]

Sensory signals are critical to perform adaptive social behavior. During copulation, male mice emit ultrasonic vocalizations (USVs). Our previous studies have shown that female mice exhibit approach behavior toward sound sources of male USVs and that, after being exposed to a male pheromone, exocrine gland-secreting peptide 1 (ESP1), female mice exhibited a preference toward a particular type of male USVs. These findings suggest that male USVs modulate female courtship behavior. However, it remains unclear which brain regions and what cell types of neurons are involved in neuronal processing of male USVs. To clarify this issue, immediate early gene analysis, behavioral analysis, and neurochemical analysis were performed. The in situ hybridization analysis of c-fos mRNA in multiple brain regions showed that neurons in the prelimbic cortex were responsive to presentation of male USVs in the presence of ESP1. Furthermore, this study found that activity of prelimbic cortex was correlated with the duration of female exploration behavior toward a sound source of the USVs. Finally, by using double immunohistochemistry, the present study showed that the prelimbic neurons responding to the presentation of male USVs were presumably excitatory glutamatergic neurons. These results suggest that the prelimbic cortex may facilitate female courtship behavior in response to male USVs. [ABSTRACT FROM AUTHOR]

Rats emit 50‐kHz ultrasonic vocalizations (USV) in appetitive situations, reflecting a positive affective state. Particularly high rates of 50‐kHz USV are elicited by the psychostimulant d‐amphetamine. Exaggerated 50‐kHz USV emission evoked by d‐amphetamine is modulated by dopamine, noradrenaline and 5‐hydroxytyrptamine receptor ligands and inhibited by the mood stabilizer lithium, the gold standard anti‐manic drug for treating bipolar disorder. This indicates that exaggerated 50‐kHz USV emission can serve as a reliable and valid measure for assessing mania‐like elevated mood in rats with sufficient translational power for gaining a better understanding of relevant pathophysiological mechanisms and the identification of new therapeutic targets. The improved capacity to study the effects of anti‐manic pharmacological interventions on a broader range of behaviours by including exaggerated 50‐kHz USV emission as preclinical outcome measure complementary to locomotor hyperactivity will refine rodent models for mania. LINKED ARTICLES: This article is part of a themed issue on New discoveries and perspectives in mental and pain disorders. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.17/issuetoc [ABSTRACT FROM AUTHOR]

Background: Various mammalian species emit ultrasonic vocalizations (USVs), which reflect their emotional state and mediate social interactions. USVs are usually analyzed by manual or semi-automated methodologies that categorize discrete USVs according to their structure in the frequency-time domains. This laborious analysis hinders the effective use of USVs as a readout for high-throughput analysis of behavioral changes in animals. Results: Here we present a novel automated open-source tool that utilizes a different approach towards USV analysis, termed TrackUSF. To validate TrackUSF, we analyzed calls from different animal species, namely mice, rats, and bats, recorded in various settings and compared the results with a manual analysis by a trained observer. We found that TrackUSF detected the majority of USVs, with less than 1% of false-positive detections. We then employed TrackUSF to analyze social vocalizations in Shank3-deficient rats, a rat model of autism, and revealed that these vocalizations exhibit a spectrum of deviations from appetitive calls towards aversive calls. Conclusions: TrackUSF is a simple and easy-to-use system that may be used for a high-throughput comparison of ultrasonic vocalizations between groups of animals of any kind in any setting, with no prior assumptions. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a neurodevelopmental disease caused by mutations in the X-linked CDKL5 gene and characterized by early-onset epilepsy, intellectual disability, and autistic features. To date, the etiological mechanisms underlying CDD are largely unknown and no effective therapies are available. The Cdkl5 knock-out (KO) mouse has been broadly employed in preclinical studies on CDD; Cdkl5-KO mice display neurobehavioral abnormalities recapitulating most CDD symptoms, including alterations in motor, sensory, cognitive, and social abilities. However, most available preclinical studies have been carried out on adult Cdkl5-KO mice, so little is known about the phenotypic characteristics of this model earlier during development. Furthermore, major autistic-relevant phenotypes, for example, social and communication deficits, have been poorly investigated and mostly in male mutants. Here, we assessed the autistic-relevant behavioral phenotypes of Cdkl5-KO mice during the first three post-natal weeks and in adulthood. Males and females were tested, the latter including both heterozygous and homozygous mutants. Cdkl5 mutant pups showed qualitative and quantitative alterations in ultrasonic communication, detected first at 2 weeks of age and confirmed later in adulthood. Increased levels of anxiety-like behaviors were observed in mutants at 3 weeks and in adulthood, when stereotypies, reduced social interaction and memory deficits were also observed. These behavioral effects of the mutation were evident in both sexes, being more marked and varied in homozygous than heterozygous females. These findings provide novel evidence for the autistic-relevant behavioral profile of the Cdkl5 mouse model, thus supporting its use in future preclinical studies investigating CDD pathology and autism spectrum disorders., (© 2024 The Author(s). Autism Research published by International Society for Autism Research and Wiley Periodicals LLC.)

Rationale: Opioid use during pregnancy can lead to negative infant health outcomes, including neonatal opioid withdrawal syndrome (NOWS). NOWS comprises gastrointestinal, autonomic nervous system, and neurological dysfunction that manifest during spontaneous withdrawal. Variability in NOWS severity necessitates a more individualized treatment approach. Ultrasonic vocalizations (USVs) in neonatal mice are emitted in isolation as a stress response and are increased during opioid withdrawal, thus modeling a negative affective state that can be utilized to test new treatments., Objectives: We sought to identify the behavioral and USV profile, brainstem transcriptomic adaptations, and role of kappa opioid receptors in USVs during neonatal opioid withdrawal., Methods: We employed a third trimester-approximate opioid exposure model, where neonatal inbred FVB/NJ pups were injected twice-daily with morphine (10mg/kg, s.c.) or saline (0.9%, 20 ul/g, s.c.) from postnatal day(P) 1 to P14. This protocol induces reduced weight gain, hypothermia, thermal hyperalgesia, and increased USVs during spontaneous morphine withdrawal., Results: On P14, there were increased USV emissions and altered USV syllables during withdrawal, including an increase in Complex 3 syllables in FVB/NJ females (but not males). Brainstem bulk mRNA sequencing revealed an upregulation of the kappa opioid receptor ( Oprk1) , which contributes to withdrawal-induced dysphoria. The kappa opioid receptor (KOR) antagonist, nor-BNI (30 mg/kg, s.c.), significantly reduced USVs in FVB/NJ females, but not males during spontaneous morphine withdrawal. Furthermore, the KOR agonist, U50,488h (0.625 mg/kg, s.c.), was sufficient to increase USVs on P10 (both sexes) and P14 (females only) in FVB/NJ mice., Conclusions: We identified an elevated USV syllable, Complex 3, and a female-specific recruitment of the dynorphin/KOR system in increased USVs associated with neonatal opioid withdrawal severity., Competing Interests: CONFLICTS OF INTERESTS The authors have nothing to disclose.

Thousands of species use vocal signals to communicate with one another.Vocalisations carry rich information, yet characterising and analysing these high-dimensional signals is difficult and prone to human bias. Moreover, animal vocalisations are ethologically relevant stimuli whose representation by auditory neurons is an important subject of research in sensory neuroscience. A method that can efficiently generate naturalistic vocalisation waveforms would offer an unlimited supply of stimuli to probe neuronal computations. While unsupervised learning methods allow for the projection of vocalisations into low-dimensional latent spaces learned from the waveforms themselves, and generative modelling allows for the synthesis of novel vocalisations for use in downstream tasks, there is currently no method that would combine these tasks to produce naturalistic vocalisation waveforms for stimulus playback. Here, we demonstrate BiWaveGAN: a bidirectional Generative Adversarial Network (GAN) capable of learning a latent representation of ultrasonic vocalisations (USVs) from mice. We show that BiWaveGAN can be used to generate, and interpolate between, realistic vocalisation waveforms. We then use these synthesised stimuli along with natural USVs to probe the sensory input space of mouse auditory cortical neurons. We show that stimuli generated from our method evoke neuronal responses as effectively as real vocalisations, and produce receptive fields with the same predictive power. BiWaveGAN is not restricted to mouse USVs but can be used to synthesise naturalistic vocalisations of any animal species and interpolate between vocalisations of the same or different species, which could be useful for probing categorical boundaries in representations of ethologically relevant auditory signals.

Recognizing conspecifics is vitally important for differentiating kin, mates, offspring and social threats. 1 Although often reliant upon chemical or visual cues, individual recognition across the animal kingdom is also facilitated by unique acoustic signatures in vocalizations. 2-4 However, amongst the large Muroidea superfamily of rodents that encompasses laboratory species amenable to neurobiological studies, there is scant behavioral evidence for individual vocal recognition despite individual acoustic variation. 5-10 Playback studies have found evidence for coarse communicative functions like mate attraction and territorial defense, but limited finer ability to discriminate known individuals' vocalizations. 11-17 Such a capacity would be adaptive for species that form lifelong pair bonds requiring partner identification across timescales, distances and sensory modalities, so to improve the chance of finding individual vocal recognition in a Muroid rodent, we investigated vocal communication in the prairie vole ( Microtus ochrogaster ) - one of the few socially monogamous mammals. 18 We found that the ultrasonic vocalizations of adult prairie voles can communicate individual identity. Even though the vocalizations of individual males change after cohabitating with a female to form a bond, acoustic variation across individuals is greater than within an individual so that vocalizations of different males in a common context are identifiable above chance. Critically, females behaviorally discriminate their partner's vocalizations over a stranger's, even if emitted to another stimulus female. These results establish the acoustic and behavioral foundation for individual vocal recognition in prairie voles, where neurobiological tools 19-22 enable future studies revealing its causal neural mechanisms., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Male predominance is a known feature of autism spectrum disorder (ASD). Although ASD mouse models can be useful for elucidating mechanisms underlying abnormal behaviors relevant to human ASD, suitable models to analyze sex differences in ASD pathogenesis remain insufficient. Herein, we used collapsin response mediator protein 4 (Crmp4)-knockout (KO) mice exhibiting ASD-like phenotypes in a male-predominant manner and analyzed ultrasonic vocalizations (USVs) to detect potential differences between genotypes and sexes during the early postnatal period. We recorded isolation-induced USVs emitted from wild-type (WT) and Crmp4-KO littermates and compared the total number of USVs between genotypes and sexes. We classified USVs into 10 types based on internal pitch changes, lengths, and shapes and compared the number of USVs in each type by genotypes and sex. Male Crmp4-KO mice exhibited a reduction in the total number of USVs. Crmp4-KO decreased the number of USVs in 7 out of 10 USV types, and male KO mice exhibited a greater reduction than females in 3 of the 7 types. This study offers a suitable ASD animal model and tool for assessing sex-based communication deficits during the early postnatal period, both of which would be valuable for elucidating the underlying mechanism. [ABSTRACT FROM AUTHOR]

To investigate flexibility in vocal signaling by rodent pups, we examined whether olfactory stimuli influence characteristics of pup calls and how these calls may be affected by sex and litter size in California mice (Peromyscus californicus). Pups were isolated and recorded during a 3‐min baseline period followed by a 5‐min exposure to bedding containing scent from their home cage, scent from the home cage of an unfamiliar family, coyote urine, or no scent (control). Latency to call, call rate, and call characteristics (duration, frequency, and amplitude) were compared between the baseline and scent‐exposure periods and among olfactory conditions. Compared with the control condition, pups from two‐pup litters called more quietly when exposed to odor from a predator, whereas pups from three‐pup litters called more loudly. Additionally, pups showed nonsignificant tendencies to reduce call rates in response to odors from their home cage and to increase call rates when exposed to predator urine. Last, males produced higher‐frequency calls and more ultrasonic vocalizations than females. These results indicate that pup calling behavior in this species can be influenced by acute olfactory stimuli as well as litter size and sex. The flexibility of pup calling in response to these three variables potentially increases the communication value of pup calls and helps shape the parents' responses. [ABSTRACT FROM AUTHOR]

Objectives Mutations in the human IQSEC2 gene are associated with drug-resistant epilepsy and severe behavioral dysfunction. We have focused on understanding one human IQSEC2 missense mutation (A350V) for which we have created a corresponding A350V IQSEC2 mouse model by CRISPR which demonstrates seizures when the mice are 15–20 days old and impaired social vocalizations in adulthood. We observed that a child with the A350V mutation stops having seizures when experiencing a fever of greater than 38 °C. In this study, we first sought to determine if we could recapitulate this phenomenon in A350V 15–20 day old mice using a previously established protocol to raise body temperature to 39 °C achieved by housing the mice at 37 °C. We then sought to determine if mice in whom seizure activity had been prevented as pups would develop social vocalization activity in adulthood. Methods 15–20 day old A350V male mice were housed either at 37 °C or 22 °C. Ultrasonic vocalizations of these mice were assessed at 8–10 weeks in response to a female stimulus. Results Housing of 15–20 day old A350V mice at 37 °C resulted in a reduction in lethal seizures to 2% (1/41) compared to 45% (48/108) in mice housed at 22 °C, p = 0.0001. Adult A350V mice who had been housed at 37 °C as pups displayed a significant improvement in the production of social vocalizations. Conclusion Raising the body temperature by raising the ambient temperature might provide a means to reduce seizures associated with the A350V IQSEC2 mutation and thereby allow for an improved neurodevelopmental trajectory.

Rats produce ultrasonic vocalizations (USVs) for conspecific communication. These USVs are valuable biomarkers for studying behavioral and mechanistic changes in a variety of diseases and disorders. Previous work has demonstrated operant conditioning can progressively increase the number of USVs produced by rats over multiple weeks. This operant conditioning paradigm is a useful model for investigating the effects of increased laryngeal muscle use on USV acoustic characteristics and underlying central and peripheral laryngeal sensorimotor mechanisms. Previous USV operant conditioning studies relied on manual training to elicit USV productions, which is both time and labor intensive and can introduce human variability. This manuscript introduces a semi-automated method for training rats to increase their rate of USV production by pairing commercially available operant conditioning equipment with an ultrasonic detection system. USV training requires three basic components: elicitation cue, detection of the behavior, and a reward to reinforce the desired behavior. With the semi-automated training paradigm, indirect exposure to the opposite sex or an olfactory cue can be used to elicit USV production. The elicited USV is then automatically detected by the ultrasonic acoustic system, which consequently triggers the release of a sucrose pellet reward. Our results demonstrate this semi-automated procedure produces a similar increase in USV production as the manual training method. Through automation of USV detection and reward administration, staffing requirements, human error, and subject behavioral variability may be minimized while scalability and reproducibility are increased. This automation may also result in greater experimental flexibility, allowing USV training paradigms to become more customizable for a wider array of applications. This semi-automated USV behavioral training paradigm improves upon manual training techniques by increasing the ease, speed, and quality of data collection. [ABSTRACT FROM AUTHOR]

Mice use ultrasonic vocalizations (USVs) to convey a variety of socially relevant information. These vocalizations are affected by the sex, age, strain, and emotional state of the emitter and can thus be used to characterize it. Current tools used to detect and analyze murine USVs rely on user input and image processing algorithms to identify USVs, therefore requiring ideal recording environments. More recent tools which utilize convolutional neural networks models to identify vocalization segments perform well above the latter but do not exploit the sequential structure of audio vocalizations. On the other hand, human voice recognition models were made explicitly for audio processing; they incorporate the advantages of CNN models in recurrent models that allow them to capture the sequential nature of the audio. Here we describe the HybridMouse software: an audio analysis tool that combines convolutional (CNN) and recurrent (RNN) neural networks for automatically identifying, labeling, and extracting recorded USVs. Following training on manually labeled audio files recorded in various experimental conditions, HybridMouse outperformed the most commonly used benchmark model utilizing deep-learning tools in accuracy and precision. Moreover, it does not require user input and produces reliable detection and analysis of USVs recorded under harsh experimental conditions. We suggest that HybrideMouse will enhance the analysis of murine USVs and facilitate their use in scientific research. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rats produce ultrasonic vocalizations (USVs) for conspecific communication. These USVs are valuable biomarkers for studying behavioral and mechanistic changes in a variety of diseases and disorders. Previous work has demonstrated operant conditioning can progressively increase the number of USVs produced by rats over multiple weeks. This operant conditioning paradigm is a useful model for investigating the effects of increased laryngeal muscle use on USV acoustic characteristics and underlying central and peripheral laryngeal sensorimotor mechanisms. Previous USV operant conditioning studies relied on manual training to elicit USV productions, which is both time and labor intensive and can introduce human variability. This manuscript introduces a semi-automated method for training rats to increase their rate of USV production by pairing commercially available operant conditioning equipment with an ultrasonic detection system. USV training requires three basic components: elicitation cue, detection of the behavior, and a reward to reinforce the desired behavior. With the semi-automated training paradigm, indirect exposure to the opposite sex or an olfactory cue can be used to elicit USV production. The elicited USV is then automatically detected by the ultrasonic acoustic system, which consequently triggers the release of a sucrose pellet reward. Our results demonstrate this semi-automated procedure produces a similar increase in USV production as the manual training method. Through automation of USV detection and reward administration, staffing requirements, human error, and subject behavioral variability may be minimized while scalability and reproducibility are increased. This automation may also result in greater experimental flexibility, allowing USV training paradigms to become more customizable for a wider array of applications. This semi-automated USV behavioral training paradigm improves upon manual training techniques by increasing the ease, speed, and quality of data collection.