Name: recording mouse ultrasonic vocalizations to evaluate social communication
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This work was supported by the Fondation de France; by the ANR FLEXNEURIM [ANR09BLAN034003]; by the ANR [ANR- 08-MNPS-037-01-SynGen]; by Neuron-ERANET (EUHF-AUTISM); by the Fondation Orange; by the Fondation FondaMentale; by the Fondation de France; by the Fondation Bettencourt-Schueller. The research leading to this article has also received support from the Innovative Medicine Initiative Joint Undertaking under grant agreement no. 115300, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007-2013) and EFPIA companies' in kind contribution. We thank Julie L&#233;vi-Strauss for helpful comments on the manuscript and six anonymous reviewers whose comments noticeably improved the manuscript.

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The authors have nothing to disclose.

The protocol presented here provides standardized and reliable ways to collect mouse ultrasonic vocalizations in the laboratory. These very constrained situations present the advantage of standardization. They are used with success to compare strains or genotypes within strains18,19,26,27. As presented in the representative results, these methods allow the identification of atypical social communication in mice mutated for Shank2, a gene associated with autism spectrum disorders. Comparisons between mouse strains, between different contexts or even between laboratories will be triggered by the availability of larger datasets on the mouseTube database. This tool should boost studies on mouse ultrasonic vocalizations by allowing multivariate analyzes.
The protocols described here are optimized to test mice of different genotypes within a strain, as it is done in the majority of studies modeling the genetic contribution to neuropsychiatric disorders. It is recommended to experimentally design each study to have the best controls possible. Indeed, litter effects might mask or artificially inflate genetic effects28,29. It is therefore advisable to include littermate controls for each genotype. Breeding heterozygous parents should therefore be favored, since it will allow the correct matching of mutant and control mice within a litter. This justifies the paw tattoo marking of all pups (blinded to genotype) to track individuals throughout the recordings every two days. Genotyping is done at weaning, by taking tail samples. When recording pup isolation calls from P2 on, we would not recommend taking tail samples already in pups, since this operation includes supplementary manipulation and stress very close in time to a recording session.
The protocols suggested here to elicit ultrasonic vocalizations in adults does not allow clear identification of the emitter of the vocalizations. This explains why we manipulate the motivation of the test animal. Indeed, the test mice are isolated and not the new-comer and the test animals habituate for a long time to the test cage during same-sex interactions. In male-female interactions, the introduced female is not isolated and the test male habituates for shorter time since motivation might be higher in this sexual context. These manipulations of motivation should maximize the probability of the test mouse emitting the vocalizations and not the introduced one. To record male ultrasonic vocalizations in a sexual context, a simple cotton swab with fresh (i.e., not frozen) urine of an estrus female can also be introduced in the cage30. This method allows the assignment of ultrasonic vocalizations to the test male with 100% certainty but it prevents collecting any specific information about the actual social context of emission of these vocalizations. Therefore, we favor the protocol described here (with a freely-moving estrus female). We also recommend to always use introduced mice from the same strain when testing mice from a mutant strain and to analyze the data as a pair of mice vocalizing. One recent study promotes the use of triangulation to localize the emitter31. In this study, females were found to also emit ultrasonic vocalizations during encounters with a male. This might be explained by the fact that they were isolated for at least two weeks before the recording session. The generalization of the use of the triangulation proposed in this study should nevertheless allow identification of the emitter of the vocalizations in most cases if video recordings are properly synced.
The isolation calls from pups recorded during development are not disturbed by background noise from the bedding. Usually an automatic analysis works very well to extract the main variables. In contrast, vocalizations recorded from adults are disturbed by background noise from the animals moving in the bedding. Automatic analysis might fail, and therefore manual analysis should be used. Nevertheless, adding bedding in the test cage should provide conditions that are less stressful for the animals than bare soil (that mice do not like). Further efforts in the community are concentrated on improving the automatic detection of ultrasonic vocalizations under various conditions, even those implying background noises. For instance, the VoICE software allows to analyze vocalizations that had been manually selected for the absence of background noise32. In this software, the extraction of the acoustic variables is automatic but needs the initial manual selection.
It should be noted that the inter-individual variability is very important in the vocal behavior of mice. For instance, the call rate of adult males in presence of an estrus female is very distributed (Figure 1). We suggest these standardized protocols to elicit ultrasonic vocalizations already to limit the variability related to the experimental context. Nevertheless, we would like to point out the importance of presenting not only the mean and SEM for the data, but most importantly the individual points in samples of small size33. It is also very relevant &#8212; if not necessary&#160;&#8212; to record at least 12 individuals of each group/genotype to gather representative data. In many cases, the inter-individual variability should not be hidden (usually it cannot be), and it might be of high importance to identify individuals carrying the genetic mutation studied but not displaying any atypical phenotype. Such individuals could provide clues about compensations, which might open new pathways for therapies of genetic disorders.
In most behavioral characterizations of mouse models for neuropsychiatric disorders, vocal behavior and social contacts are considered apart (e.g., 19,27,34,35). Recent analysis methods now provide a semi-automatic detailed characterization of the social events and sequences of events during an interaction (using MiceProfiler for instance)36, as well as the possibility to combine this analysis with data from audio recordings. The main advantage of this method is to provide a comprehensive view of the social communication in mouse models of ASD, to more precisely identify which aspects of social communication are affected. In the present protocol the synchronization is still manual but this can be improved by triggering the video recording through the audio recording software. This type of analyses should become the standard to provide a more comprehensive view of social communication deficits in mouse models of neuropsychiatric disorders. In addition, up to now, vocal signals are mostly analyzed from the emitter side (i.e., tests are built to favor the emission of vocal signals by the tested mouse, as in the present protocols). The focus should now also be set on the receiver of these signals, to better identify the functions of these acoustic signals. This should be done by evaluating also the behavior of the new-comer mice in the present protocols in adults (using MiceProfiler for instance)36, by using playback experiments16, or by setting up new protocols. Indeed, the present protocols provide very constrained situations that might not reflect the exact ethological conditions of vocalization emission in mice. The spontaneous emission of ultrasonic vocalizations will have to be better-characterized using continuous audio and video recordings to shed more light on the spontaneous vocal behavior of mice.

Patients with neuropsychiatric disorders usually display deficits in social communication (e.g., patients with autism spectrum disorders, schizophrenia, or Alzheimer disease)1. Genetically engineered mice are more and more frequently used to model genetic causes of these disorders2. Studying social communication in these mouse models is of high interest for understanding the mechanisms of genetic mutations leading to atypical social dysfunctions and for testing new therapies. Since mice are social animals and communicate with each others using olfactory, tactile, visual and acoustic signals, they are suitable models to evaluate social communication.
Mouse ultrasonic vocalizations are now currently used as a proxy for modeling the genetic bases of vocal communication deficits3,4 (but the existence of vocal learning in this species is still debated5,6, even if most recent studies argue for the absence of vocal learning7). Laboratory mice have been found to emit ultrasonic vocalizations in mother-infant relationships, in male-female socio-sexual interactions, in same-sex social interactions (reviewed in reference8) and in juvenile-juvenile social interactions9. Mouse pups emit isolation calls during their first two weeks of life when isolated from dam and littermates10. Males emit ultrasonic vocalizations when in presence of an estrus female (or urinary cues from her)11,12. Males and females emit ultrasonic vocalization when interacting with an unknown conspecific of the same sex13,14. The organization and functions of these vocalizations are not completely clear and need further investigations. Current knowledge on the functional aspect is limited to the elicitation of retrieval behavior in mothers hearing pup isolation calls, the facilitation of proximity of adult females toward adult male vocalizations15 and the increased exploratory behavior of adult males hearing adult female vocalizations16.
Characterizing the abnormalities in vocal communication in mouse models of neuropsychiatric disorders should be conducted in standardized conditions to rule out major contribution of the experimental conditions. Such characterizations, combined with the evaluation of simultaneous social interactions and neurobiological studies, in various genetic models should improve our knowledge on the genetic contribution to the different aspects of mouse ultrasonic communication. Over a long term, it should give further light on some neurobiological bases of social communication in humans. We presently aim at providing simple protocols to reliably elicit ultrasonic vocalizations during development and in adulthood for both male and female mice in the laboratory. Such protocols should ease the standardization of recordings to more reliably compare ultrasonic vocalization emissions between strains and laboratories. It should also facilitate the setting up of such recordings in laboratories having no prior experience with mouse ultrasonic vocalizations recordings. We also highlight the current possibility to combine ultrasonic vocalizations data with detailed behavioral data collected simultaneously during social interactions in adult mice, to obtain crucial information on social impairments as well as on the context of emission of ultrasonic vocalizations. Such analyses will shed new light on the organization and functions of mouse ultrasonic vocalizations. Finally, we also advertise the possibility to share ultrasonic vocalization recordings with the whole scientific community on the mouseTube database (http://mousetube.pasteur.fr). Open access to audio recording data should boost knowledge on mouse ultrasonic communication by allowing scientists to compare their own data with ultrasonic vocalizations recorded in other laboratories (with similar or different strains/protocols), and/or to challenge their analysis methods with files recorded under different conditions.

<table><tbody><tr><td>needles 0.3mm x 13 mm [30&amp;nbsp;G 1/2&quot;]</td><td>BD Microlance</td><td>304000</td><td>-</td></tr><tr><td>green tattoo paste</td><td>Ketchum Manufacturing Inc., Ottawa, Canada</td><td>329AA</td><td>-</td></tr><tr><td>thermometer</td><td>Fisherbrand, Waltham, USA</td><td>4126 (W255NA)</td><td>-</td></tr><tr><td>self-made soundproof chamber (pups)</td><td>Institut Pasteur, Paris</td><td>-</td><td>acoustic foam + plexiglas; inside dimensions (W x H x D): 32 cm x 33 cm x 32 cm</td></tr><tr><td>small surface thermister + single probe thermocouple</td><td>Harvard Apparatus</td><td>599814 + 601956</td><td>-</td></tr><tr><td>smell-less pen</td><td>for instance: Giotto</td><td>-</td><td>ink made with water, washable: these pens are designed for babies</td></tr><tr><td>Ethanol absolute (100%)</td><td>Sigma Aldrich,&amp;nbsp; Saint-Quentin Fallavier, France</td><td>24103</td><td>diluted 1/10</td></tr><tr><td>Condenser ultrasound microphone Avisoft-Bioacoustics CM16/CMPA</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#40011</td><td>furnished with extension cables by the Avisoft company</td></tr><tr><td>Ultrasound Gate 416H</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#34163</td><td>sound card</td></tr><tr><td>Avisoft Recorder USGH</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10301; #10302</td><td>recording software for Windows Vista, 7 and 8</td></tr><tr><td>Avisoft SASLab Pro</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10101, 10111; #10102, 10112;&amp;nbsp;</td><td>Windows 10, 8.1, 8, 7 or Vista including Intel-based Apple Macintosh running Boot Camp, Parallels or similar virtualization software.</td></tr><tr><td>Laptop or Apple Macintosh running Boot Camp</td><td>-</td><td>-</td><td>running Windows 10, 8.1, 8, 7 or Vista; for the Apple Macintosh, Boot Camp is preferred to virtualizations softwares such as Parallels due to memory constraints</td></tr><tr><td>plastic recipient (pup recordings)</td><td>Lock &amp;amp; Lock, Chatswood, USA</td><td>HPL932D</td><td>Lock &amp;amp; Lock Stackable Airtight Container Round 700&amp;nbsp;ml; use without the cover; dimensions: 9&amp;nbsp;cm diameter, 10 cm height</td></tr><tr><td>PBS 1x (pH = 7.4)</td><td>Gibco (Life Technologies)</td><td>10010-023</td><td>-</td></tr><tr><td>slides</td><td>Menzel-Gl&amp;auml;ser, Thermo Scientific</td><td>J1800AMNZ</td><td>Superfrost Plus</td></tr><tr><td>May-Gr&amp;uuml;nwald solution 500 ml</td><td>RAL R&amp;eacute;actifs, Martillac, France</td><td>320070-0500</td><td>-</td></tr><tr><td>Giemsa R 500 ml</td><td>RAL R&amp;eacute;actifs, Martillac, France</td><td>720-1107</td><td>diluted 1/20 in phosphate buffer solution</td></tr><tr><td>phosphate buffer solution (self-made)</td><td>-</td><td>-</td><td>pH = 7, 0.1 M: 39 ml NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 0.2 M + 61 ml Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 0.2 M + 100&amp;nbsp;ml H&lt;sub&gt;2&lt;/sub&gt;O (final volume: 200&amp;nbsp;ml)</td></tr><tr><td>test cage</td><td>Institut Pasteur, Paris</td><td>-</td><td>50&amp;nbsp;x&amp;nbsp;25&amp;nbsp;cm, 30&amp;nbsp;cm height; Plexiglas</td></tr><tr><td>self-made soundproof chamber (adult recordings)</td><td>Institut Pasteur, Paris</td><td>-</td><td>acoustic foam + PVC; inside dimensions (W x H x D): 66 cm x 90 cm x 46 cm</td></tr><tr><td>video camera</td><td>From Noldus Information Technologies, Wageningen, The Netherlands</td><td>-</td><td>high-resolution CamTech Super-Hi-Res video camera; 25 fps&amp;nbsp;</td></tr><tr><td>EthoVision XT</td><td>Noldus Information Technology, Wageningen, The Netherlands</td><td>http://www.noldus.com/animal-behavior-research/products/ethovision-xt</td><td>video acquisition software</td></tr><tr><td>Mice Profiler Tracker plugin from the ICY platform</td><td>Bio Image Analysis, Institut Pasteur, Paris</td><td>http://icy.bioimageanalysis.org/plugin/Mice_Profiler_Tracker</td><td>tracking software to analyse behavioral events during social interactions</td></tr></tbody></table>

recording mouse ultrasonic vocalizations to evaluate social communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

With the present protocols, we characterized the vocal behavior of mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders (ASD)23-25. ASD are characterized by deficits in social communication and stereotyped behaviors1. Our Shank2-/- mice displayed hyperactivity, increased anxiety and atypical vocal communication18,26. Indeed, we noted that Shank2-/- mice displayed an atypical developmental profile in their emission rate of pup isolation calls in comparison with the typical inverted U-shaped curve in their wild-type littermates. Shank2-/- mice displayed an increased call rate at P4 and decreased call rate at P6 in comparison with their wild-type littermates (Figure 2). We also observed a decreased call rate in female interactions involving a Shank2-/- female in comparison with interactions involving a wild-type littermate (Figure 2). We examined the repertoire of the 5 different call categories. It appeared to be different between pups (for instance here P2, P6 and P10) and adults (Figure 3). Genotype-related differences were significant mostly in adulthood. During social interactions involving adult Shank2-/- males or females with a C57BL/6N female, more short calls and unstructured calls were recorded in comparison with interactions involving their wild-type littermates (Figure 3D and E). Less complex calls and frequency jumps calls were also recorded during interactions with a C57BL/6N female involving adult Shank2-/- females in comparison with interactions involving Shank2+/+ females (Figure 3E). Finally, we also measured manually acoustic variables. There was no significant genotype-related difference during development. In contrast, the duration of calls recorded during interactions involving adult Shank2-/- females were shorter than those recorded during interactions involving their wild-type littermates (Figure 4A). We also highlighted that the peak frequency of ultrasonic vocalizations increased during pup development without significant genotype-related difference26. During interactions involving Shank2-/- males or females with a C57BL/6N female, ultrasonic vocalizations had a lower peak frequency in comparison with calls recorded during interactions involving their wild-type littermates (Figure 4B).
In addition, the present protocol also allowed to study the context of emission of ultrasonic vocalizations by combining the data from audio recordings to the behavioral data extracted from MiceProfiler (ICY software, Institut Pasteur, Paris). For instance, in female-female interactions, most ultrasonic vocalizations were emitted when animals were in contact and more specifically the occupant sniffing the new-comer&#39;s ano-genital region, or at least the occupant being behind the new-comer. Mice also emitted many ultrasonic vocalizations when the occupant approached the new-comer (Figure 5, upper panel). Less vocalizations were recorded when the occupant Shank2-/- mice were in physical contact with the new-comer (e.g., sniffing the ano-genital region of the new-comer) than when the occupant was a wild-type mouse. Less vocalizations were triggered when the occupant behind the new-comer was a Shank2-/- mouse than when it was a wild-type mouse. More vocalizations were also recorded when the new-comer was in the visual field of the occupant mouse, and more so in the wild-types than in the mutants (Figure 5, lower panel).
<img alt="Figure 2" src="/files/ftp_upload/53871/53871fig2.jpg" /> 
Figure 2: Emission rate of ultrasonic vocalizations during development and in adult male and female Shank2-/- mice and wild-type littermates. Call rate of pups (every two days from P2 to P12, n = 18-19 Shank2+/+, n = 15-16 Shank2-/-) and adults during male-estrus female interactions (n = 15 Shank2+/+, n = 16 Shank2-/-) and female-female interactions (n = 15 Shank2+/+, n = 13 Shank2-/-) in wild-type mice (left panel) and Shank2-/- mice (right panel). Data are presented as mean+/-SEM and individual points (non-paired Wilcoxon tests: *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53871/53871fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53871/53871fig3.jpg" /> 
Figure 3: Vocal repertoire of Shank2-/- mice and wild-type littermates. Proportions of the five different call types emitted by P2 pups (A; n = 20 Shank2+/+, n = 18 Shank2-/-), P6 pups (B; n = 19 Shank2+/+, n = 18 Shank2-/-), P10 pups (C; n = 20 Shank2+/+, n = 18 Shank2-/-), adult males with an estrus female (D; n = 16 Shank2+/+, n=16 Shank2-/-) and adult females with another female (E; n = 15 Shank2+/+, n=13 Shank2-/-) in wild-type mice (left panels) and Shank2-/- mice (right panels). Data are presented as mean+/-SEM and individual points (chi-squared tests: *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53871/53871fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53871/53871fig4.jpg" /> 
Figure 4: Acoustic variables extracted from ultrasonic vocalizations in Shank2-/- mice and wild-type littermates. (A) Duration of all call types confounded emitted by P2 pups (n = 20 Shank2+/+, n = 18 Shank2-/-), P6 pups (n = 19 Shank2+/+, n = 18 Shank2-/-), P10 pups (n = 20 Shank2+/+, n = 18 Shank2-/-), adult males with an estrus female (n = 16 Shank2+/+, n = 16 Shank2-/-) and adult females with another female (n = 15 Shank2+/+, n = 13 Shank2-/-) in wild-type mice (left panel) and Shank2-/- mice (right panel). (B) Maximum peak frequency measured on all call types confounded in P2 pups, P6 pups, P10 pups, adult males with an estrus female and adult female with another female (same Ns as above). Data are presented as mean+/-SEM and individual points (non-paired Wilcoxon tests: *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53871/53871fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53871/53871fig5.jpg" /> 
Figure 5: Contexts of emission of mouse ultrasonic vocalizations in female-female adult social interactions. Proportion of ultrasonic vocalizations emitted by pairs involving a Shank2+/+ with a C57BL/6N mouse (n = 16, A) and pairs involving a Shank2-/- with a C57BL/6N mouse (n = 13, B) during the following types of behavioral events (red: occupant, green: new-comer): social contacts, oro-oral contact, ano-genital sniffing from the occupant mouse, ano-genital sniffing from the new-comer mouse, occupant behind new-comer, new-comer behind occupant, immobility of occupant, immobility of new-comer, approach from the occupant &#38; escape from the new-comer, approach from the new-comer &#38; escape from the occupant, approach &#38; escape from the occupant, approach &#38; escape from the new-comer, occupant following the new-comer, new-comer in the vision field of occupant, occupant in the vision field of new-comer. Data are presented as mean+/-SEM and individual points (non-paired Wilcoxon tests: *p &#60;0.05, **p &#60;0.01). Unpublished data. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53871/53871fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Call types</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>short</td>
			<td>duration &#8804;5&#160;msec and frequency range &#8804;6.25&#160;kHz</td>
		</tr>
		<tr>
			<td>simple</td>
			<td>duration &#62;5&#160;msec and frequency range &#8804;6.25&#160;kHz (flat), or frequency modulation in only one direction (upward or downward) with frequency range &#62;6.25&#160;kHz</td>
		</tr>
		<tr>
			<td>complex</td>
			<td>frequency modulations in more than one direction and frequency range &#62;6.25&#160;kHz (modulated), or inclusion of one or more additional frequency component (harmonic or non-linear phenomena, but no saturation) but no constraint on frequency range (complex)</td>
		</tr>
		<tr>
			<td>frequency jumps</td>
			<td>inclusion of one jump (one frequency jump) or more jumps (frequency jumps, others) in frequency without time gap between the consecutive frequency components, with (mixed) or without any noisy part within the pure tone call</td>
		</tr>
		<tr>
			<td>unstructured</td>
			<td>no pure tone component identifiable; &#8220;noisy&#8221; calls</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of five types of mouse ultrasonic vocalizations. Examples of criteria of duration, frequency range, frequency modulations and frequency jumps used to determine 5 different call types within mouse ultrasonic vocalizations.

Watch this Scientific Journal Video about Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication at JoVE.com

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

recording-mouse-ultrasonic-vocalizations-to-evaluate-social

Nanyang Technological University

Research

JoVE Journal

Behavior

22.2K Views.  University Paris Diderot, CNRS UMR 3571, Institut Pasteur. Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

Video: Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Neuroscience

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Experimental Research Examining How People Can Cope with Uncertainty Through Soft Haptic Sensations

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has focused on the cognitive strategies people adopt to deal with uncertainty. However, especially when uncertainty is due to unpredictable societal events (e.g., economical crises, political revolutions, terrorism threats) of which one is unable to judge the impact on one&#8217;s future live, cognitive strategies (like seeking additional information) is likely to fail to combat uncertainty. Instead, the current paper discusses a method demonstrating that people might deal with uncertainty experientially through soft haptic sensations. More specifically, because touching something soft creates a feeling of comfort and security, people prefer objects with softer as compared to harder properties when feeling uncertain. Seeking for softness is a highly efficient and effective tool to deal with uncertainty as our hands are available at all times. This protocol describes a set of methods demonstrating 1) how environmental (un)certainty can be situationally activated with an experiential priming procedure, 2) that the quality of the softness experience (what type of softness and how it is experienced) matters and 3) how uncertainty can be reduced using different methods.

To date research has focused on cognitive strategies people adopt to cope with uncertainty. This research examines instead an experiential way of dealing with uncertainty and introduces a set of experimental methods showing how the experience of haptic softness can serve as a tool to deal with uncertainty.

Human beings are constantly surrounded by uncertainty and change. The question arises how people cope with such uncertainty. To date, most research has ...

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...