Name: determining ultrasonic vocalization preferences in mice using a two-choice playback test
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Description: Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

This work was supported by a Grant-in-Aid for JSPS Fellows to A.A.; by Grants-in-Aid for Scientific Research on Innovative Areas for JSPS Fellows (No. 4501 and No. 25132712) to T.K.; and by a research project grant awarded by Azabu University. Figure 2 is reprinted from Kihara, T., Harada, T., &#38; Koshida, N. Wafer-compatible fabrication and characteristics of nanocrystalline silicon thermally induced ultrasound emitters. In: Sensors and Actuators A: Physical, volume 125, Elsevier, p. 426, (2006), with permission from Elsevier.

All procedures were approved by the Ethics Committee of Azabu University. All experiments were carried out in a soundproof chamber.
1. Animal Preparation
<ol>
	<li>Males for Recording
	<ol>
		<li>Obtain sexually mature male&#160;mice experienced in mating.</li>
	</ol>
	</li>
	<li>Female Subjects
	<ol>
		<li>Obtain virgin female mice that have been housed with 2 to 5 littermates per cage (usually 8 - 12 weeks old).</li>
		<li>Obtain vaginal smears daily to determine the phase of the estrous cycle before the test, according to McLean21. Care should be taken to minimize the vaginal stimulation to avoid pseudopregnancy.</li>
		<li>Stain the smears with Giemsa, and determine phase of the estrous cycle based on the presence or absence of leukocytes, cornified epithelial cells, and nucleated epithelial cells, according to Nelson22.</li>
	</ol>
	</li>
</ol>
2. Devices (Figure 1)
<img alt="Figure 1" src="/files/ftp_upload/53074/53074fig1.jpg" /> 
Figure 1.&#160;Schematic of the Two-choice Test Box and Devices. Mice can access rooms A and B through the small gates located between them and the neutral zone. The two-choice box and two ultrasound emitters are placed on the floor of a soundproof chamber. The microphone is suspended inside the soundproof chamber. Abbreviation: PC, personal computer. <a href="https://www.jove.com/files/ftp_upload/53074/53074fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>For Recording
	<ol>
		<li>Connect an ultrasonic condenser microphone to a PC using a USB A/D converter.</li>
		<li>Set the gain level of the A/D converter to &#8220;7&#8221;.</li>
		<li>Record acoustic data in 16-bit format files with a sampling rate of 400 kHz using audio capture software.</li>
	</ol>
	</li>
	<li>For Playback
	<ol>
		<li>Connect a D/A converter to the PC using a USB interface.</li>
		<li>Connect two amplifiers to the D/A converter through an attenuator. (The latter device serves to prevent overload.)</li>
		<li>Connect an ultrasound emitter to each amplifier.</li>
	</ol>
	</li>
</ol>
3. USV Recording
<ol>
	<li>Place a male mouse in a small- (17 x 10 x 11 cm) or medium-sized (21 x 14 x 13 cm) cage with a 6 cm-diameter hole in the sidewall and the hole covered with a 0.5 cm wire mesh. When using a medium-sized cage, construct a partition across the center of the cage to reduce the area.</li>
	<li>Do not use bedding in the cage to avoid rustling noises that could contaminate the recordings.</li>
	<li>Place a microphone beside the mesh.</li>
	<li>Begin ultrasonic vocalization monitoring with the recording setup shown in step 2.1.</li>
	<li>Place a sexually mature female mouse in either metestrus or diestrus in the cage with the male mouse.</li>
	<li>Record USVs for 3 &#8211; 5 min.</li>
</ol>
4. Test Box
<ol>
	<li>Build the test box using acrylic board (Figure 1). 
	Note: The test box (35 x 20, and x 20 cm high) is divided in three compartments: a neutral zone (15 x 20 cm), and rooms A and B (20 x 10 cm, each). Rooms A and B each have a 4 cm-diameter hole that is covered with a 0.5cm wire mesh. The holes and mesh are located at each end of rooms A and B. Make gates (5 x 5 cm each) between the neutral zone and rooms A and B.</li>
	<li>To prevent sound leakage insulate each ultrasound emitter, except for the side facing the mesh, using rubber plates, sealing them to the box around the perimeter of each mesh.</li>
	<li>Place lead seals on both sides of the wall between rooms A and B for sound insulation.</li>
</ol>
5. Preparing for Sound Playback
<ol>
	<li>Creation of Playback Sounds
	<ol>
		<li>Capture a 20 sec segment of USV from the recorded file using digital audio editing software.</li>
		<li>Select and capture background noise (0.35 sec) from the 20 sec USV segment file. Create a 20 sec background noise file by repeating the noise segment.</li>
		<li>To play back the two sound files simultaneously, export them as a single stereo sound file, with one file for the &#8220;left ear&#8221; and the other file for the &#8220;right ear.&#8221;</li>
		<li>Filter the sound files using as high-pass filter with a cutoff of 40 kHz.</li>
		<li>Reduce noise using the noise-reduction tool in post-processing software. Select and capture a segment of the file that contains no USVs as the noise-reduction profile, and run noise reduction with level 40.</li>
	</ol>
	</li>
	<li>Calibration of Playback Sounds
	<ol>
		<li>Ensure that the space between ultrasound emitters and microphone is 10 cm.</li>
		<li>To calibrate sound pressure levels from the ultrasound emitters, perform ultrasonic monitoring using the configuration described in Step 2.1. Measure the sound pressure level by the microphone in decibels (dB).</li>
		<li>Using the attenuator and amplifiers, adjust the volume of the USVs generated by the ultrasound emitters to the same sound pressure level as the male USVs recorded in Step 3.</li>
		<li>When using the two ultrasound emitters and a sound file composed of USVs and background noise, confirm that the two ultrasound emitters exhibit the same sound pressure level using a calibration sound (e.g., a 75-kHz pure tone) before performing the behavioral test.</li>
		<li>When reproducing a sound file composed of two streams of USVs, adjust the two generated sound pressure levels to the same level, before testing.</li>
	</ol>
	</li>
</ol>
6. Two-choice Test
<ol>
	<li>Place the ultrasound emitters behind the meshes of the test box.</li>
	<li>Close the gates to rooms A and B with an acrylic board, and habituate the female subject to the neutral zone for 30 min. Cover the box with acrylic board to avoid that the mouse can escape.</li>
	<li>Start video recording using a CCD camera mounted above the box. The camera (not shown in Figure 1) covers the area of all three compartments.</li>
	<li>Remove the gate and cover-board and allow the female to explore freely the test box, including the sound zones.</li>
	<li>Once the female has investigated both meshes, and has returned to the neutral zone, start playing a 20 sec sound file repeatedly for 5 or 10 min.</li>
	<li>Conduct behavioral monitoring for 5 or 10 min.</li>
	<li>To reduce undesired olfactory cues deposited from the previous subject, clean the test cages between the tests with 70% ethanol.</li>
	<li>Switch the place of ultrasound emitters A and B randomly to avoid inherent side bias effect towards rooms A or B.</li>
</ol>
7. Statistical Analyses
<ol>
	<li>Using behavioral event-scoring software, analyze the following parameters: total number of entries in each room; total duration of stay in each middle zone; total duration of stay in each sound zone; and total duration of search of each mesh in front of ultrasound emitter. Figure 1 illustrates what is meant by &#8220;middle zone&#8221; and &#8220;sound zone.&#8221; 
	Note: In cases where the mouse stays on the dividing line between the two zones (Figure 1, dashed line) decide the location by the direction of the head. When the head faces the mesh side, it is scored as a stay in the sound zone.</li>
	<li>For each behavioral parameter, compare responses for rooms A and B using a Wilcoxon signed-rank test, or paired t-tests with a significance level of 0.05.</li>
</ol>

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The authors declare that they have no competing interests.

Here, the results of a representative test showed that female mice can discriminate between artificial male USVs and background noise (Figure 3). The conclusion to be drawn from these results is that the discrimination signal is reflected in the duration of stay in the room and sound zone, and in the duration of searching the mesh in the first 5 min of testing, but not in the second 5 min (Figure 3C, E and F). These data indicate that mice become habituated to the playback sounds, possibly because of lack of social contact occurring near the ultrasound emitter. Therefore, the optimal testing time is 5 min. 
These findings might be accounting for the fact that USVs are just &#8220;more novel sounding than noise.&#8221; However, a previous playback study showed that female mice were attracted to the sounds of male USVs, but not to novelty artificial sounds12. It is assumed that merely novel sounds are not affective to female mice.
As expected, USV preference was dependent on the phase of the estrous cycle, since social investigation towards a male is enhanced by moderate levels of estrogen23. A previous study has revealed that females in pro-estrus or estrus do not show the preference for USVs of males of a different strain observed here14. Therefore, only females in diestrus were used in the present study; in general, when using females for this test, the phase of the estrous cycle should be known.
The limitation of this test is that USVs generated by the ultrasound emitter are one-way playback sounds. This test is not appropriate to evaluate the role of USVs in complex social interactions.In addition, inbred strains of mice such as B6, can exhibit a severe hearing loss after 12 weeks of age24. Therefore, it is recommended that all mice used in this test are less than 12 week-old.
In case of failure to observe differences in response related to the playback sounds, the addition of other social cues could help make the subject more sensitive to the sounds. For instance, it is recommended that the test subject be exposed to mouse odor during the habituation time in the neutral zone. In a previous study, the preference of females for some USVs over others was potentiated when they were exposed to male-soiled bedding, or to the male pheromone ESP125 before behavioral testing. In the absence of male-odor stimuli, females did not show a preference for USVs of males of a different strain14.
This model allows comparing the various features of murine USVs. The characteristics of mouse USVs change with maturation from pup to adult26, and differ across strains1, 20, 27. In addition, the female estrous cycle influences male courtship USVs28. Moreover, the rate of male production of USVs, and the incidence of a specific syllable type show a temporal increase related to male mounting behavior28, 29. Research using models of communication disorders have demonstrated unusual repertoires of USV profiles2, 29-33. However, the question of whether the receiver mice can discriminate and show a specific response to these different USVs remains to be elucidated. The present protocol increases the usefulness of mouse USVs for understanding the context-dependence of social signal production and auditory reaction, and for uncovering the characteristics of attractive vocal communications in mice.

Many animals use vocalizations for intraspecific communication. In the mouse Mus musculus, one important type of communication signal is ultrasonic vocalizations (USVs), which have frequencies higher than 20 kHz. USVs emitted by mice are considered a component of social recognition in male-female1-4, female-female1, 5, and male-male1, 6 interactions. USVs are also emitted by pups when they are isolated from their mother, which increases her pup-retrieving behavior, and therefore pup survival 7. Although many reports have analyzed and categorized mouse USVs8,9, the behavioral responses and neural mechanisms of the receiving animal have been less documented10, 11. The latter is necessary to clarify the biological significance of the various characteristics of USVs. To reveal these mechanisms, the playback experiment is an efficient method. Recent playback studies have revealed that female mice are attracted to USVs12, and that they prefer USVs from males that are different from their parents13, 14.
This article explains the playback test used to evaluate USV preference in mice. A two-choice test box was developed in which two different USVs can be played back simultaneously in two compartments of a test enclosure, as shown in Figure 1. This type of test box prevents sound contamination by dividing the test area in three sub-chambers, using lead walls. The ultrasound emitters are located outside each room. In the wall between the rooms and ultrasound emitters are holes covered with wire mesh. Mice can move freely in the three rooms, and show a &#8220;searching the mesh&#8221; behavior, as if to respond to USVs played back by the ultrasound emitters. In this test, mice stay for periods of different duration close to one sound emitter or the other. These parameters can be logged to obtain a sensitive measure of sound preference.
To play the USVs back, nanocrystalline silicon thermo-acoustic emitters (i.e., &#8220;nc-Si emitter&#8221;) were used as in previous studies15-17. These devices are composed of a thin-film heater electrode, a nano-porous silicon layer, and a single-crystalline silicon wafer. The digital sound file is converted to an analog signal and then passed through the heater electrode. The device converts the resulting voltage-dependent thermal signals into significant sound pressure with low distortion. This device is unique in that, unlike common sound generators that depend on mechanical vibrations, it can reproduce sound without the need of a diaphragm. The emitter exhibits a flat sound pressure level at frequencies from 20 to 160 kHz (Figure 2), and can reproduce digitally recorded murine USVs very accurately in terms of duration, frequency, and sound pressure level15, 18, 19.
In a representative experiment shown in Figure 3, C57BL/6 (B6) females were allowed to choose between BALB/c (BALB) male USVs and background noise. In addition, Figure 4 shows the choice of B6 and BALB females between simultaneous USV playbacks from a BALB and a B6 male, as reported in a previous study14. The characteristics of male USVs differ between B6 and BALB strains20. As shown by these results, the attractiveness of USVs can be assessed with the present protocol, in which sounds are recorded from a live individual, acoustically analyzed, and played back to other individuals.

<table><tbody><tr><td>Soundproof chamber</td><td>Muromachi Kikai</td><td></td><td></td></tr><tr><td>Small cage</td><td>CLEA Japan</td><td>CL-0113-1</td><td></td></tr><tr><td>Middle cage</td><td>CLEA Japan</td><td>CL-0103-1</td><td></td></tr><tr><td>Ultrasound condenser microphones</td><td>Avisoft Bioacoustics</td><td>CM16/CMPA</td><td></td></tr><tr><td>A/D converter</td><td>Avisoft Bioacoustics</td><td>UltraSoundGate116H</td><td></td></tr><tr><td>Audio software</td><td>Avisoft Bioacoustics</td><td>RECORDER USGH</td><td></td></tr><tr><td>Adobe Audition 3.0 / Audio editing software</td><td>Adobe Systems</td><td>Adobe Audition 3.0</td><td></td></tr><tr><td>Nc-Si emitter</td><td>Original</td><td></td><td>not commercially available but it is planned to be so in near future</td></tr><tr><td>D/A converter</td><td>National Instruments</td><td>NI USB-6251 BNC</td><td></td></tr><tr><td>Attenuator</td><td>Original</td><td></td><td></td></tr><tr><td>Amplifier</td><td>Yamatake</td><td></td><td></td></tr><tr><td>PC</td><td>Windows 7 professional</td><td></td><td>Intel&amp;reg; core i7-2600K CPU @ 3.4GHz, 8GB RAM, 64-bit operating system</td></tr><tr><td>Event recorder Excel-macro / Event-scoring software</td><td>original</td><td></td><td>Programmed by Naoto Akagawa &amp;amp; Takeru Yamamoto</td></tr><tr><td>CCD Camera</td><td></td><td></td><td></td></tr><tr><td>Rubber plates (made of elastomer resin)</td><td>Tokyo bouon</td><td>TI-75BK B4</td><td>Cut them to the proper size http://www.piano-bouon.jp/shopping/?pid=1329272401-447630&amp;amp;ca=6&amp;amp;p=3</td></tr><tr><td>Giemsa&#039;s azur eosin methylene blue solution</td><td>Merck Millipore</td><td>1.09204.0500</td><td></td></tr></tbody></table>

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.

The USVs recorded from one BALB-male (161 syllables per 20 sec), as well as background noise were used as playback sounds in the representative experiment shown in Figure 3. In this experiment, 7 female B6 mice were used at 9 weeks of age. To determine the best duration of testing of female response to playback sounds, the behavioral parameters were analyzed separately for the first and last five min of the total 10 min test time.
First, there was no significant difference in total duration of stay in rooms A or B during investigation time before playback (Figure 3A), demonstrating that there was no side bias.The results also showed that there was no difference between the background-noise side and the male-USVs side in the number of room entries, analyzing the first 5 min, the last 5 min, and total testing time (Figure 3B). Figure 3C shows cumulative seconds of duration of stay in each room during playback (i.e., total duration of stay in the middle zone, and in the sound zone). The subjects spent significantly longer time in the BALB male-USV side than in the background-noise side when analyzing the first 5 min (p = 0.043), or the total testing time (p = 0.043). There were no significant differences in these parameters when analyzing by last 5 min. There were no significant differences between the background-noise side and the male-USVs side in the middle zone (Figure 3D). However, B6 females spent significantly longer time in the sound zone and searching the mesh in the BALB male-USV side, compared to the background-noise side, when analyzing the first 5 min (Sound zone, p = 0.018, Figure 3E; Mesh, p = 0.018, Figure 3F) or total testing time (Sound zone, p = 0.028, Figure 3E; Mesh, p = 0.043, Figure 3F). The results clearly demonstrate that a B6 female approaches more the reproduced male-USVs than the background noise, particularly in the first 5 min.
This two-choice test is useful to compare the characteristics of USVs. In a previous study, this test was used to determine USV preference of B6 and BALB female mice for USVs from males of these strains11. When using a playback combination of USVs from a B6 (133 syllables per 20 sec) and a BALB (108 syllables per 20 sec) male, female mice of each of these strains showed longer searching times for the USVs of males of the other strain than of their own one (Figure 4).
<img alt="Figure 2" src="/files/ftp_upload/53074/53074fig2.jpg" /> 
Figure 2.&#160;Measured Sound Pressure Level (SPL) Generated by the nc-Si Emitter as a Function of Frequency. An almost constant SPL of approximately 80 &#8211; 90 dB is observed between 40 and 160 kHz. AC input power is 1.3 W in this case. The emitter was placed at a distance of 20 mm from a high-frequency condenser microphone and aligned with its center. This figure has been modified from Kihara19. <a href="https://www.jove.com/files/ftp_upload/53074/53074fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53074/53074fig3.jpg" /> 
Figure 3.&#160;B6 Female Response to Background Noise and BALB Male USVs. (A) Total duration of stay in rooms A or B during investigation time before playback. (B) Number of room entries during playback. (C) Total duration of stay in the rooms. (D) Total duration of stay in the middle zone. (E) Total duration of stay in the sound zone. (F) Total duration of searching the meshes.&#160;Measurements of each individual are given in dot plots; horizontal bars in each plot indicate the mean;&#160;n =&#160;7; * indicate significant differences (p &#60;0.05) between background noise and male USVs for the parameters analyzed; Wilcoxon signed-rank tests. <a href="https://www.jove.com/files/ftp_upload/53074/53074fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53074/53074fig4.jpg" /> 
Figure 4.&#160;Female Response to USVs of Males from a Different Strain vs. the Same Strain. B6 (n = 6) and BALB (n = 10) females in diestrus exposed to male-soiled bedding before testing showed longer duration search times for the other strain USVs. Values shown are mean + standard error; * indicate significant differences (p &#60;0.05); Wilcoxon signed-rank tests. This figure has been modified from Asaba14. <a href="https://www.jove.com/files/ftp_upload/53074/53074fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

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

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, ...

determining-ultrasonic-vocalization-preferences-mice-using-two-choice

Nanyang Technological University

Research

JoVE Journal

Behavior

11.1K Views.  Azabu University. 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.

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

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

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

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

Neuroscience

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 ...

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 ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...