This work was supported by the Howard Hughes Medical Institute funds to EDJ. We thank Pr. Sylvie Granon (NeuroPSI - University Paris south XI - FRANCE) for lending us the speaker hardware. We also thank members of the Jarvis Lab for their support, discussions, corrections and comments on this work, especially Joshua Jones Macopson for help with figures and testing. We thank Dr. Gustavo Arriaga for help with the Mouse Song Analyzer software, upgrading it for us to V1.3, and other aspects of this protocol. v1.0 of the software was developed by Holy and Guo, and v1.1 and v1.3 by Arriaga.

<ol>
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The authors have nothing to disclose.

This protocol provides approaches to collect, quantify, and study male mice courtship vocalizations in the laboratory across a variety of mostly female-related stimuli. As presented previously in Chabout, et al. 23and in the representative results, the use of this method allowed us to discover context-dependent vocalizations and syntax that matter for the receiving females. The standardization of these stimuli will provide the collection of a reliable number of USVs and allow detailed analyses of the male&#39;s courtship songs and repertoires.
When a live female is present with the male, the protocol does not allow us to clearly identify the emitter of the vocalizations. However, previous studies showed that the majority of the vocalizations emitted in such context were by the male26,29. Most of the studies using a conspecific (male or female) as a stimulus for the males believe that the amount of female&#39;s vocalizations in these contexts is negligible4,5,22,30. However a recent paper used triangulation to localize the vocalization&#39;s of the emitter in group housed conditions31, and showed that within a dyad, the female contributes to ~10% of the USVs. In the present protocol the use of the anesthetized female allows the user to study the male vocalizations in the presence of a female without her vocalizing. In contrast to expectations of this recent study31, we found no difference in the number of syllables emitted between the FE and AF conditions23. It is possible that live females did not significantly contribute to the recordings or that the males vocalized less in the presence of live females versus anaesthetized females. Nevertheless, we believe that future experiments should consider the use of this triangulation method to assess the potential effect of the female contribution.
There are other software available that can do some the steps we have outlined, although we do not believe in a manner sufficient for the questions we asked using a combination of three programs: Software A, Mouse Song Analyzer software script C using software B, the syntax analysis software using a custom spreadsheet software D+E calculations, and syntax decorder using R. For example, a recent paper proposed a software named VoICE that allows the user to extract acoustic variables automatically from the sonograms or directly on units that had been manually selected by the user32. But, the automated or semi-automated sequence analyses are not as detailed as our approach. Some commercial software can automatically analyze the acoustic features, but do not provide an automatic classification of the syllables; the user has to sort the different syllables afterwards. Grimsley, Gadziola, et al. 33 developed a Table-based virtual mouse vocal organ program that clusters syllables based on shared acoustic features, but does not provide automatic detection of the syllables. Their program34 is unique in that it creates novel sequences from recorded songs using Markov models, and thus has more advanced features than simple editing.
Most prior communication studies on mice have focused on the emitter&#39;s side35,36. Few studies have explored the receiver&#39;s side30,37,38. Playback and discrimination protocols provide a simple test to study the receiver&#39;s side, such as the one also recently described by Asaba, Kato, et al. 39. In that study, the authors used a two-choice test box separated with acoustic foam instead of the Y-maze box described here. Both choice setups have advantages and disadvantages. First, the Y-maze doesn&#39;t isolate the sound from one arm to the other, but the two-choice box does. However, by using the Y-maze design, the animal can quickly evaluate the two songs that are played simultaneously, and move towards the preferred one. Nevertheless, playback experiments in general help experimenters determine the meaning and thus functions of the vocalizations generated for conspecific animals. In conclusion, after mastering the techniques of this protocol and analyses, readers should be able to address many questions that influence the context, genetics, and neurobiology of mouse USVs.
Using B6D2F1/J mice, the female associated stimuli almost always trigger USVs from the males we have tested in our lab. It is critical to collect enough syllables (&#62; 100 in 5 min) to be able to obtain strong statistically analysis. For troubleshooting, if no USVs are recorded (or not enough), check the configuration to make sure sounds are recorded. Do a live inspection of what is happening in the cage during recording by looking at the real-time sonogram on the computer screen after the introduction of the stimulus. Otherwise try to re-expose the male to a sexually mature/receptive female overnight and then house them alone for several days or up to a week before recording again. Based on anecdotal observations, we find that some males sing a lot on one day (for almost the full 5 min), and not much the next day, and then again another day. We do not know the reason why such within subject variability occurs, but we surmise it is probably a motivational or seasonal for the males, and the estrus state for female urine. If no USVs are recorded, try to record the animal on multiple days to pick up these variable effects. Unlike in songbirds, we have not noted overt differences in amount of singing based on time of day. We find that the males do not sing much (&#60; 100 syllables in 5 min) before they are 7 weeks old.
The detection methods presented here can extract thousands of syllables and all the acoustic parameters in a few minutes. But as any automatic detection method, it is very sensitive to background noise. Using Mouse Song Analyzer detection software with noisy recordings (for example from animals recorded with bedding) may require adjustment of the detection &#34;threshold&#34; to allow more flexibility. However, this will also increase the number of false positive syllables and the automatic detection might fail. Under such circumstances, manual coding can be used.
As stated earlier, the number, repertories, and latency of vocalizations are widely variable depending on the strain, thus one may have to change parameters (recording length, stimulus, automated syllable detection, etc.) for some strains to ensure optimal recordings for statistical analyses.

Relative to humans, mice produce both low and high frequency vocalizations, the later known as ultrasonic vocalizations (USVs) above our hearing range. The USVs are produced in a variety of contexts, including from mother-pup retrieval, juvenile interactions, to opposite or same sex adult interactions4,12. These USVs are composed of a diverse multisyllabic repertoire which can be categorized manually9 or automatically10,11. The role of these USVs in communication has been under increasing investigation in recent years. These include using the USVs as readouts of mouse models of neuropsychiatric, developmental or behavioral disorders7,8, and internal motivational/emotional states13. The USVs are thought to convey reliable information on the emitter&#39;s state that is useful for the receiver14,15.
In 2005, Holy and Guo 11, advanced the idea that adult male mice USVs were organized as a succession of multisyllabic call elements or syllables similar to songbirds. In many species, a multisyllabic repertoire allows the emitter to combine and order syllables in different ways to increase the potential information carried by the song. Variation in this syntax is believed to have an ethological relevance for sexual behavior and mate preferences16,17. Subsequent studies showed that male mice were able to change the relative composition of syllable types they produce before, during, and after the presence of a female5,18. That is, the adult male mice use their USVs for courtship behavior, either to attract or maintain close contact with a female, or to facilitate mating19,20,21. They are also emitted in male-male interactions, probably to convey social information during interactions4. To capture these changes in repertoires, scientists usually measure the spectral features (acoustic parameters, such as amplitude, frequencies, etc.), number of USVs syllables or calls, and latency to the first USV. However, few actually look at the sequence dynamics of these USVs in detail22. Recently our group developed a novel method to measure dynamic changes in the USV syllable sequences23. We showed that syllable order within a song about (namely syntax) is not random, that it changes depending on social context, and that the listening animals detect these changes as ethologically relevant.
We note that many investigators studying animal communication do not attach to the term &#39;syntax&#39; the same exact meaning as syntax used in human speech. For animal communication studies, we simply mean an ordered, non-random, sequence of sounds with some rules. For humans, in addition, specific sequences are known to have specific meanings. We do not know if this is the case for mice.
In this paper and associated video, we aim to provide reliable protocols to record male mice&#39;s courtship USVs across various contexts, and perform playbacks. The use of three sequentially used software for: 1) automated recordings; 2) syllable detection and coding; and 3) in-depth analysis of the syllable features and syntax is demonstrated (Figure 1). This allows us to learn more about male mice USV structure and function. We believe that such methods ease data analyses and may open new horizons in characterizing normal and abnormal vocal communication in mouse models of communication and neuropsychiatric disorders, respectively.

<table><tbody><tr><td>Sound proof beach cooler</td><td>See Gus paper has more info on specific kind</td><td></td><td>Inside dimensions (L 27 x W 23 x H 47 cm):</td></tr><tr><td>Condenser ultrasound microphone CM16/CMPA</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#40011</td><td>Includes extension cable&amp;nbsp;</td></tr><tr><td>Ultrasound Gate 1216H sound card</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#34175</td><td>12 channel sound card</td></tr><tr><td>Ultrasound Gate Player 216H</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#70117</td><td>2 channels playback player</td></tr><tr><td>Ultrasonic Electrostatic Speaker ESS polaroid</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#60103</td><td>2 playback speakers</td></tr><tr><td>Test cage&amp;nbsp;</td><td>Ace</td><td>#PC75J</td><td>30 x 8 x 13 cm height; plexiglas</td></tr><tr><td>plexiglas separation</td><td>home made</td><td>-</td><td>4 x 13 cm plexiglas with 1 cm holes</td></tr><tr><td>Video camera</td><td>Logitech</td><td>C920</td><td>logitech HD Pro webcam C920</td></tr><tr><td>Heat pad&amp;nbsp;</td><td>Sunbeam</td><td>722-810-000</td><td></td></tr><tr><td>Y-maze&amp;nbsp;</td><td>Home made</td><td>-</td><td>Inside dimensions (L 30 x W 11 x H 29 cm):</td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Software&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Avisoft Recorder (Software A)</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10101, #10111, #10102, #10112</td><td>http://www.avisoft.com</td></tr><tr><td>MATLAB R2013a (Software B)</td><td>MathWorks</td><td>-</td><td>MATLAB R2013a (8.1.0.604)</td></tr><tr><td>Mouse Song Analyzer v1.3 (Software C)</td><td>Custom designed by Holy, Guo, Arriaga, &amp;amp; Jarvis; Runs with software B</td><td></td><td>http://jarvislab.net/wp-content/uploads/2014/12/Mouse_Song_Analyzer_&lt;br /&gt;v1.3-2015-03-23.zip</td></tr><tr><td>Microsoft Office Excel 2013 (Software D)</td><td>Microsoft</td><td>-</td><td>Microsoft Office Excel</td></tr><tr><td>Song Analysis Guide v1.1 (Software E)</td><td>Custom designed by Chabout &amp;amp; Jarvis. Excel calculator sheets, runs with software D</td><td></td><td>http://jarvislab.net/wp-content/uploads/2014/12/Song-analysis_Guided.xlsx</td></tr><tr><td>Syntax decorder v1.1 (Software F)</td><td>Custom designed by Sakar, Chabout, Dunson, Jarvis - in R studio</td><td></td><td>https://www.rstudio.com/products/rstudio/download/</td></tr><tr><td>Graphiz (Software G)</td><td>AT&amp;amp;T Research and others&amp;nbsp;</td><td></td><td>http://www.graphviz.org</td></tr><tr><td>Avisoft SASLab (Software H)</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10101, #10111, #10102, #10112</td><td>http://www.avisoft.com</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Reagents&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Xylazine (20 mg/mL)</td><td>Anased</td><td>-</td><td></td></tr><tr><td>Ketamine HCl (100 mg/mL)</td><td>Henry Schein</td><td>#045822</td><td></td></tr><tr><td>distilled water</td><td></td><td></td><td></td></tr><tr><td>Eye ointment&amp;nbsp;</td><td>Puralube Vet Ointment&amp;nbsp;</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Cotton tips</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td></td></tr></tbody></table>

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.

In present protocol, changes in vocal behavior and syntax of male B6D2F1/J mice were characterized. Generally, using this protocol we were able to record, on average per male per 5 min session, 675 &#177; 98.5 classified syllables in response to female UR, 615.6 &#177; 72 in FE, 450 &#177; 134 in AF, 75.6 &#177; 38.9 in AM and 0.2 &#177; 0.1 in male UR (n = 12 males). The rates were ~ 130 syllables/min for female UR, ~ 120 syllables/min for FE, or ~ 100 syllables/min for AF contexts (Figure 5A). Males produce a much larger amount of syllables in response to freshly collected urine relative to overnight collected urine10,23. Males also sing considerably less in presence of an anesthetized male or fresh male urine Figure 5A). Males also change their repertoire across context23. For example, B6D2F1/J males significantly increase production of multiple pitch jump &#34;m&#34; category syllables in the female urine condition (Figure 5B). They also change acoustic features of individual syllables across context. For example, B6D2F1/J males sing syllables at a higher amplitude and bandwidth in the female urine context, and higher spectral purity in the awake female context compared to the others (Figure 6)23.
This protocol also provides a means for measuring dynamic features of sequence and thus syntax changes. Using an adapted method from Ey, et al. 22, we use the ISI to define the gaps between sequences (Figure 7A)23, and then use the gaps to distinguish and analyze temporal patterns of syllable sequences. We showed longer sequence lengths are produced in the awake female context (Figure 7B)23. This technique allows us to calculate the ratio of complex sequences (composed by at least 2 occurrences of the &#34;m&#34; syllable type) versus simple sequences (composed of one or no &#34;m&#34; type, and thus mostly by &#34;s&#34; type). We found that with B6D2F1/J males the female urine context triggered a higher ratio compared to the others (Figure 7C)23, indicating they produced more complex syllables in the female urine condition, but also that such syllables are distributed over more sequences.
We are also able to calculate the conditional transition probabilities from one syllable type to another (24 transition types total including the transitions from and to the &#34;silence&#34; state)23. We found that in different contexts, the mice&#39;s choice of transition types for given starting syllables differs, and that there is more syntax diversity in the female urine condition (Figure 8)23. These observations are consistent with previous reports that show that males can change the acoustic features or repertoire composition of their vocalizations in response to different stimuli and experiences4,5,24.
Finally, the present protocol provides guidance to test female preferences with playbacks. We found that B6D2F1/J females prefer more complex songs (containing 2 or more &#34;m&#34; syllables) relative to simple songs23. Most females chose to stay more often on the side of the Y-maze that played the complex songs (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/54137/54137fig1.jpg" /> 
Figure 1: Flow Chart of Software Use and Analyses. Each program and associated code is given a letter name to help explain their identity and use in the main text. In ( ) are the specific programs we use in our protocol. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54137/54137fig2.jpg" /> 
Figure 2: Set up for Recording Male Mice Songs. (A) Picture of a sound attenuation recording box and set up to record USV vocalizations. (B) Example sonogram of a recording made with Software A (Table 1), including detailed spectral features calculated by &#34;Mouse Song Analyzer v1.3&#34;: duration, inter-syllable interval (ISI), peak frequency min (Pf min), peak frequency max (Pf max), peak frequency start (Pf start), peak frequency end (Pf end), and bandwidth. (C) Sonograms of another male singing to a live female, inside the sound attenuation box and outside of the box on the lab bench in the same room. Our anecdotal observations indicate that the recordings in the box of the same animal show larger volume (stronger intensity) and less harmonics, but no evidence of echoes in the box without sound foam. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54137/54137fig3.jpg" /> 
Figure 3: Screenshot of the &#34;Mouse Song Analyzer v1.3&#34; whis_gui Window Showing the Different Options Available for Analyses. The parameters shown are ones used to record male USVs in the figures and data analyses presented (except min note duration was 3 ms). <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54137/54137fig4.jpg" /> 
Figure 4: Female Choices Between Playbacks of Complex and Simple Songs. (A) Picture of the Y-maze apparatus used and dimension measurements. (B) Time spent by the females in each arm, playing either from a complex (female urine elicited) or more simple (awake female elicited) song from the same male. Data are presented for n = 10 B6D2F1J female mice as mean &#177; SE, with individual values also shown; 9 of the 10 females showed a preference for the more complex syllable/sequence song. * p &#60; 0.05 paired student t-test. Figure modified from Chabout, et al. 23 with permission. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54137/54137fig5.jpg" /> 
Figure 5: Number of Syllables Emitted and Repertoire Across Conditions. (A) Syllable production rate of males in different contexts. (B) Repertoire compositions of males when in the presence of female urine (UR), anesthetized female (AF), awake female (FE), and anesthetized male (AM) contexts. Data are presented as mean &#177; SEM. * p&#60;0.03; ** p&#60;0.005; *** p&#60;0.0001 for post-hoc paired student t-test after Benjamini and Hochberg correction (n = 12 males). Figure from Chabout, et al. 23 with permission. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54137/54137fig6.jpg" /> 
Figure 6: Examples of Spectral Features in Different Context. (A) Amplitude. * p&#60; 0.025 for post-hoc paired student t-test after correction. (B) Frequency range or Bandwidth. *: p &#60; 0.041; **: p &#60; 0.005; ***: p &#60; 0.0001 after correction. (C) Spectral purity of the syllables. * p: &#60; 0.025; **: p &#60; 0.005; ***: p &#60; 0.0001 after correction. Abbreviations: female urine (UR), anesthetized female (AF), awake female (FE), and anesthetized male (AM). Figures modified from Chabout, et al. 23 with permission. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54137/54137fig7.jpg" /> 
Figure 7: Sequence Measurements. (A) Usage of the ISI to separate the sequence. Short ISI (SI) and medium ISI (MI) are used to separate syllables within a sequence, and long ISI over 250 ms (LI) separate two sequences. (B) Length of the sequences, measured as number of syllables per sequence, produced by males in different contexts. *: p &#60; 0.025; ** p &#60; 0.005; *** p &#60; 0.0001 after correction. (C) Ratio of complex songs over simple songs produced by males in different contexts. * p &#60; 0.041; ** p &#60; 0.005; *** p &#60; 0.0001 after correction. Data are presented as mean &#177; SEM (n = 12 males). Figure from Chabout, et al. 23 with permission. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54137/54137fig8.jpg" /> 
Figure 8: Syllable Syntax Diagrams of Sequences Based on Conditional Probabilities for Each Contexts. Arrow thickness is proportional to the conditional probability occurrence of a transition type in each context averaged from n = 12 males: P (occurrence of a transition given the starting syllable). For clarity, rare transitions below a probability of 0.05 are not shown. Figure from Chabout, et al. 23 with permission. <a href="http://cloudflare.jove.com/files/ftp_upload/54137/54137fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs at JoVE.com

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.

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 ...

eliciting-and-analyzing-male-mouse-ultrasonic-vocalization-usv-songs

Research

JoVE Journal

Behavior

15.7K Views.  Duke University. 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.

Video: Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.

The distractor-response binding paradigm is described. It can be used to shed light on the influence irrelevant stimuli, competing with targets for a response, can have on human action. Both response retrieval effects and distractor inhibition effects can be analyzed within the paradigm.

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

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, ...

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.

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 ...

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 ...

Neuroscience

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

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Tickling, a Technique for Inducing Positive Affect When Handling Rats

Assessment of Sexual Behavior of Male Mice