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.

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">needles 0.3 x 13 mm [30G 1/2&#34;]&#160;</td>
			<td style="width:85px;">BD Microlance</td>
			<td style="width:119px;">304000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">green tattoo paste</td>
			<td style="width:85px;">Ketchum Manufacturing Inc., Ottawa, Canada</td>
			<td style="width:119px;">329AA</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">thermometer</td>
			<td style="width:85px;">Fisherbrand, Waltham, USA</td>
			<td style="width:119px;">4126 (W255NA)</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">self-made soundproof chamber (pups)</td>
			<td style="width:85px;">Institut Pasteur, Paris</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">acoustic foam + plexiglas; inside dimensions (W x H x D): 32 x 33 x 32 cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">small surface thermister + single probe thermocouple</td>
			<td style="width:85px;">Harvard Apparatus</td>
			<td style="width:119px;">599814 + 601956</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">smell-less pen</td>
			<td style="width:85px;">for instance: Giotto</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">ink made with water, washable: these pens are designed for babies</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Ethanol absolute (100%)</td>
			<td style="width:85px;">Sigma Aldrich,&#160; Saint-Quentin Fallavier, France</td>
			<td style="width:119px;">24103</td>
			<td style="width:167px;">diluted 1/10</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Condenser ultrasound microphone Avisoft-Bioacoustics CM16/CMPA</td>
			<td style="width:85px;">Avisoft Bioacoustics, Berlin, Germany</td>
			<td style="width:119px;">#40011</td>
			<td style="width:167px;">furnished with extension cables by the Avisoft company</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ultrasound Gate 416H</td>
			<td style="width:85px;">Avisoft Bioacoustics, Berlin, Germany</td>
			<td style="width:119px;">#34163</td>
			<td style="width:167px;">sound card</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Avisoft Recorder USGH</td>
			<td style="width:85px;">Avisoft Bioacoustics, Berlin, Germany</td>
			<td style="width:119px;">#10301; #10302</td>
			<td style="width:167px;">recording software for Windows Vista, 7 and 8</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Avisoft SASLab Pro</td>
			<td style="width:85px;">Avisoft Bioacoustics, Berlin, Germany</td>
			<td style="width:119px;">#10101, 10111; #10102, 10112;&#160;</td>
			<td style="width:167px;">Windows 10, 8.1, 8, 7 or Vista including Intel-based Apple Macintosh running Boot Camp, Parallels or similar virtualization software.</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Laptop or Apple Macintosh running Boot Camp</td>
			<td style="width:85px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">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 height="126">
			<td height="126" style="height:126px;width:216px;">plastic recipient (pup recordings)</td>
			<td style="width:85px;">Lock &#38; Lock, Chatswood, USA</td>
			<td style="width:119px;">HPL932D</td>
			<td style="width:167px;">Lock &#38; Lock Stackable Airtight Container Round 700ml; use without the cover; dimensions: 9 cm diameter, 10 cm height</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS 1X (pH=7.4)</td>
			<td style="width:85px;">Gibco (Life Technologies)</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">slides</td>
			<td style="width:85px;">Menzel-Gl&#228;ser, Thermo Scientific</td>
			<td style="width:119px;">J1800AMNZ</td>
			<td style="width:167px;">Superfrost Plus</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">May-Gr&#252;nwald solution 500 ml</td>
			<td style="width:85px;">RAL R&#233;actifs, Martillac, France</td>
			<td style="width:119px;">320070-0500</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Giemsa R 500 ml</td>
			<td style="width:85px;">RAL R&#233;actifs, Martillac, France</td>
			<td style="width:119px;">720-1107</td>
			<td style="width:167px;">diluted 1/20 in phosphate buffer solution</td>
		</tr>
		<tr height="117">
			<td height="117" style="height:117px;width:216px;">phosphate buffer solution (self-made)</td>
			<td style="width:85px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">pH=7, 0.1 M: 39 ml NaH2PO4 0.2 M + 61 ml Na2HPO4 0.2 M + 100 ml H2O (final volume: 200 ml)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">test cage</td>
			<td style="width:85px;">Institut Pasteur, Paris</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">50&#160;x&#160;25&#160;cm, 30&#160;cm height; Plexiglas</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">self-made soundproof chamber (adult recordings)</td>
			<td style="width:85px;">Institut Pasteur, Paris</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">acoustic foam + PVC; inside dimensions (W x H x D): 66 x 90 x 46 cm</td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:216px;">video camera</td>
			<td style="width:85px;">From Noldus Information Technologies, Wageningen, The Netherlands</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">high-resolution CamTech Super-Hi-Res video camera; 25 fps&#160;</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">EthoVision XT</td>
			<td style="width:85px;">Noldus Information Technology, Wageningen, The Netherlands</td>
			<td style="width:119px;">http://www.noldus.com/animal-behavior-research/products/ethovision-xt</td>
			<td style="width:167px;">video acquisition software</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Mice Profiler Tracker plugin from the ICY platform</td>
			<td style="width:85px;">Bio Image Analysis, Institut Pasteur, Paris</td>
			<td style="width:119px;">http://icy.bioimageanalysis.org/plugin/Mice_Profiler_Tracker</td>
			<td style="width:167px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

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 ...

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 ...

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children &#8211; Working Memory (CABC-WM)

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of working memory in young school-age children. Working memory deficits have been identified in children with language-based learning disabilities, including dyslexia1,2 and language impairment3,4, but it is not clear whether these children exhibit deficits in subcomponents of working memory, such as visuospatial or phonological working memory. The CABC-WM is administered on a desktop computer with a touchscreen interface and was specifically developed to be engaging and motivating for children. Although the long-term goal of the CABC-WM is to provide individualized working memory profiles in children, the present study focuses on the initial success and utility of the CABC-WM for measuring central executive, visuospatial, phonological loop, and binding constructs in children with typical development. Immediate next steps are to administer the CABC-WM to children with specific language impairment, dyslexia, and comorbid specific language impairment and dyslexia.

Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children.

The Comprehensive Assessment Battery for Children - Working Memory (CABC-WM) is a computer-based battery designed to assess different components of ...

Use of a Video Scoring Anchor for Rapid Serial Assessment of Social Communication in Toddlers

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and social competency&#8212;is hypothesized to be disrupted in autism spectrum disorder (ASD). The ability to quantify the full range of RSB during the toddler period, when core symptoms of ASD often arise, is pivotal for evaluating early risk for ASD, characterizing social development, and tracking response to early interventions. However, important parameters of variation in RSB&#8212;especially prior to the development of verbal language&#8212;can be nuanced and difficult to characterize using questionnaire-based methods. To address this challenge, we developed a system for measuring quantitative variation in RSB in toddlers (ages 18 - 30 months) that incorporated not only standard questionnaire data from caregivers but also a novel set of video-referenced items, through which a respondent compares the behavior of a subject to that observed in a short video of a young child manifesting a highly competent level of social communication. Testing of this measure in a general population sample of twins confirmed that both the video-referenced items and the RSB Total Score (video-referenced items plus non-video-referenced items) displayed unimodal, continuous distributions, strong internal consistency, marked preservation of individual differences, and extremely high heritability. In addition, video-referenced items were particularly sensitive to quantifying incremental changes in social communication, a major element of RSB, over the course of early childhood development. Scores on the vrRSB clearly differentiated children with and without ASD and these data comprise an initial validation of this promising method for quantifying early RSB&#8212;cross-sectionally, over time, and as a function of early intervention.

Here we present a protocol for administration of the video-referenced rating of reciprocal social behavior (vrRSB) using a paper or online version. This survey quantifies RSB, a prerequisite for social competence, in toddlers through &#34;video-referenced&#34; items whereby a subject is compared to a reference child shown in a three-minute video.

Reciprocal social behavior (RSB), an early-emerging capacity to engage in social contingency&#8212;which is foundational for both social learning and ...

A Semantic Priming Event-related Potential (ERP) Task to Study Lexico-semantic and Visuo-semantic Processing in Autism Spectrum Disorder

Individuals with autism spectrum disorder (ASD) have characteristic deficits in understanding the meaning of language, or semantic processing. However, some evidence indicates that semantic processing of non-linguistic stimuli is intact, suggesting that semantic deficits may be language-specific. To appropriately characterize semantic processing deficits in individuals with ASD, comparison of within-modality linguistic (e.g., written words) and non-linguistic (e.g., pictures) stimuli is required. This paper describes such a methodology that makes use of a semantic priming paradigm during concurrent recording of electroencephalographic (EEG) data. EEG provides a dynamic measure of brain activity that is well-suited to characterize subtle differences in semantic processing that may not be observable at the behavioral level. The semantic priming paradigm presents a prime picture or word (e.g., dog) followed by a target picture or word that is either related (e.g., cat) or unrelated (e.g., pencil) to the prime. This paradigm can thus be used to evaluate semantic processing across different modalities, and to compare lexico-semantic and visuo-semantic processing abilities in individuals with ASD and how they might differ from TD individuals. The specific steps involved in creating the stimuli, performing the EEG testing, and analyzing the EEG data are discussed. Representative results illustrate how the N400 component of the event-related potential (ERP) is reduced following semantically-related prime-target pairs compared to unrelated pairs. Comparisons of the N400 between conditions, modalities, and groups can provide estimates of the success of semantic processing, and can thereby be used to characterize semantic deficits in individuals with ASD or other clinical populations.

A Semantic Priming Event-related Potential ERP Task to Study Lexico-semantic and Visuo-semantic Processing in Autism Spectrum Disorder

This paper describes a semantic priming ERP task using within-modality pairs of pictures and words to investigate semantic processing in individuals with autism spectrum disorder (ASD).

Individuals with autism spectrum disorder (ASD) have characteristic deficits in understanding the meaning of language, or semantic processing. However, ...

Using Eye Movements Recorded in the Visual World Paradigm to Explore the Online Processing of Spoken Language

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded via an eye tracker as the participant produces or comprehends a spoken language describing the concurrent visual world. This paradigm has high versatility, as it can be used in a wide range of populations, including those who cannot read and/or who cannot overtly give their behavioral responses, such as preliterate children, elderly adults, and patients. More importantly, the paradigm is extremely sensitive to fine grained manipulations of the speech signal, and it can be used to study the online processing of most topics in language comprehension at multiple levels, such as the fine grained acoustic phonetic features, the properties of words, and the linguistic structures. The protocol described in this article illustrates how a typical visual world eye tracking study is conducted, with an example showing how the online processing of some semantically complex statements can be explored with the visual world paradigm.

The visual world paradigm monitors participants' eye movements in the visual workspace as they are listening to or speaking a spoken language. This paradigm can be used to investigate the online processing of a wide range of psycholinguistic questions, including semantically complex statements, such as disjunctive statements.

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded ...

A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies have described the behaviors connected with disease defense, and the associated use of chemicals and antimicrobials, no common visual reference has been made. The main aim of this study was to record short clips of behaviors involved in disease defense, both prophylactically and directly targeted towards an antagonist of the colony following infection. To do so we used an infection experiment, with sub-colonies of the leaf-cutting ant species Acromyrmex echinatior, and the most significant known pathogenic threat to the ants&#39; fungal crop (Leucoagaricus gongylophorus), a specialized pathogenic fungus in the genus Escovopsis. We filmed and compared infected and uninfected colonies, at both early and more advanced stages of infection. We quantified key defensive behaviors across treatments and show that the behavioral response to pathogen attack likely varies between different castes of worker ants, and between early and late detection of a threat. Based on these recordings we have made a library of behavioral clips, accompanied by definitions of the main individual defensive behaviors. We anticipate that such a guide can provide a common frame of reference for other researchers working in this field, to recognize and study these behaviors, and also provide greater scope for comparing different studies to ultimately help better understand the role these behaviors play in disease defense.

We present a visual guide to disease defense behaviors in leaf-cutting ants, with individual clips and accompanying definitions, illustrated in an experimental infection scenario. Our main aim is to help other researchers recognize key defensive behaviors and to provide a common understanding for future research in this field.

The complex lifestyle, evolutionary history of advanced cooperation, and disease defenses of leaf-cutting ants are well studied. Although numerous studies ...