Name: assessment of sexual behavior of male mice
Uploaded: 2020-03-05T12:00:00
Description: Watch this Scientific Journal Video about Assessment of Sexual Behavior of Male Mice at JoVE.com

We thank Lu Cong, Zhang Hongxia, Zhang Beiyue, and Hu Mi for their suggestions for the experiments.

All experiments were performed in compliance with the guidelines of the Principles of Laboratory Animal Care (NIH Publication No. 80-23, revised 1996) and under the approval and supervision of the Academy of Experimental Animal Centre of the Institute of Medicinal Plant Development (China).
1. Animal husbandry
<ol>
	<li>House female and male mice at 25 &#176;C for 12 h light/12 h dark cycles.</li>
	<li>Provide free access to water and a standard pelleted diet.</li>
	<li>Allow mice to acclima...

<ol>
	<li>The staff of the Jackson laboratory. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of the Laboratory Mouse. , (2007).</li><li>Burns-Cusato, M., Scordalakes, E. M., Rissman, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+missing+data:+what+we+know+(and+need+to+learn)+about+male+sexual+behavior.">Of mice and missing data: what we know (and need to learn) about male sexual behavior.</a> Physiology &amp; Behavior. 83 (2), 217-232 (2004).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's Wrong With My Mouse?. , (2006).</li><li>Whishaw, I. Q., Haun, F., Kolb, B., Windhorst, U., Johansson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Behavior+in+Laboratory+Rodents.">Analysis of Behavior in Laboratory Rodents.</a> Modern Techniques in Neuroscience Research. , (1999).</li><li>Sachs, B., Barfield, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Analysis of Masculine Copulatory Behavior in the Rat. 7, (1976).</li><li>Hull, E. M., Dominguez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+behavior+in+male+rodents.">Sexual behavior in male rodents.</a> Hormones and Behavior. 52 (1), 45-55 (2007).</li><li>Mosig, D. W., Dewsbury, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+copulatory+behavior+of+house+mice+(Mus+musculus).">Studies of the copulatory behavior of house mice (Mus musculus).</a> Behavioral Biology. 16 (4), 463-473 (1976).</li><li>Park, J. H., Gould, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Male+Sexual+Behavior+in+Mice.">Assessment of Male Sexual Behavior in Mice.</a> Mood and Anxiety Related Phenotypes in Mice. 63, (2011).</li><li>Bonthuis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+rats:+key+species+variations+in+the+sexual+differentiation+of+brain+and+behavior.">Of mice and rats: key species variations in the sexual differentiation of brain and behavior.</a> Frontiers in Neuroendocrinology. 31 (3), 341-358 (2010).</li><li>Levine, L., Barsel, G. E., Diakow, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+behaviour+of+two+inbred+strains+of+mice.">Mating behaviour of two inbred strains of mice.</a> Animal Behavior. 14 (1), 1-6 (1966).</li><li>McGill, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+Behavior+in+Three+Inbred+Strains+of+Mice.">Sexual Behavior in Three Inbred Strains of Mice.</a> Behaviour. 19 (4), 341 (1962).</li><li>James, P. J., Nyby, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testosterone+rapidly+affects+the+expression+of+copulatory+behavior+in+house+mice+(Mus+musculus).">Testosterone rapidly affects the expression of copulatory behavior in house mice (Mus musculus).</a> Physiology &amp; Behavior. 75 (3), 287-294 (2002).</li><li>Arteaga-Silva, M., Rodriguez-Dorantes, M., Baig, S., Morales-Montor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+castration+and+hormone+replacement+on+male+sexual+behavior+and+pattern+of+expression+in+the+brain+of+sex-steroid+receptors+in+BALB/c+AnN+mice.">Effects of castration and hormone replacement on male sexual behavior and pattern of expression in the brain of sex-steroid receptors in BALB/c AnN mice.</a> Comparative Biochemistry and Physiology - Part A: Molecular &amp; Integrative Physiology. 147 (3), 607-615 (2007).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Type+3+Adenylyl+Cyclase+Perturbs+the+Postnatal+Maturation+of+Olfactory+Sensory+Neurons+and+Olfactory+Cilium+Ultrastructure+in+Mice.">Deletion of Type 3 Adenylyl Cyclase Perturbs the Postnatal Maturation of Olfactory Sensory Neurons and Olfactory Cilium Ultrastructure in Mice.</a> Frontiers in Cellular Neuroscience. 11, 1 (2017).</li><li>Mandiyan, V. S., Coats, J. K., Shah, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+sexual+and+aggressive+behaviors+in+Cnga2+mutant+mice.">Deficits in sexual and aggressive behaviors in Cnga2 mutant mice.</a> Nature Neurosciemce. 8 (12), 1660-1662 (2005).</li><li>Choi, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+deletion+of+floxed+genes+mediated+by+CaMKIIalpha-Cre+in+the+brain+and+in+male+germ+cells:+application+to+conditional+and+conventional+disruption+of+Goalpha.">Simultaneous deletion of floxed genes mediated by CaMKIIalpha-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goalpha.</a> Experimental &amp; Molecular Medicine. 46, 93 (2014).</li><li>Pelch, K. E., Sharpe-Timms, K. L., Nagel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+surgically-induced+endometriosis+by+auto-transplantation+of+uterine+tissue.">Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue.</a> Journal of Visualized Experiments. (59), e3396 (2012).</li><li>McLean, A. C., Valenzuela, N., Fai, S., Bennett, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+vaginal+lavage,+crystal+violet+staining,+and+vaginal+cytological+evaluation+for+mouse+estrous+cycle+staging+identification.">Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.</a> Journal of Visualized Experiments. (67), e4389 (2012).</li><li>Liu, Z. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postweaning+Isolation+Rearing+Alters+the+Adult+Social,+Sexual+Preference+and+Mating+Behaviors+of+Male+CD-1+Mice.">Postweaning Isolation Rearing Alters the Adult Social, Sexual Preference and Mating Behaviors of Male CD-1 Mice.</a> Frontiers in Behavioral Neuroscience. 13, 21 (2019).</li><li>TIan, E. P., Long, T., Qin, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+applications+of+mating+model+in+male+rat.">Establishment and applications of mating model in male rat.</a> Chinese Journal of Andrology. 22 (1), 7-10 (2008).</li><li>. Anesthesia Guidelines: Mice Available from: <a target="_blank" href="https://www.researchservices.umn.edu/services-name/research-animal-resources/research-support/guidelines/anesthesia-mice">https://www.researchservices.umn.edu/services-name/research-animal-resources/research-support/guidelines/anesthesia-mice</a> (2019)</li><li>Leypold, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+sexual+and+social+behaviors+in+trp2+mutant+mice.">Altered sexual and social behaviors in trp2 mutant mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (9), 6376-6381 (2002).</li></ol>

There are a few critical steps in the presented protocol. Regarding the ovariectomy of females, the surgery incision opening from the back is less detrimental than that from the abdomen. Given that the position of the ovary is deep, pulling other organs when the incision is cut open from the abdomen often leads to bleeding and results in unclear surgical vision20. We performed the incision on the back to reach the ovary easily and shorten the surgical time, as well as to ensure the safety of the s...

Sexual behavior in mature male mice results from the interaction of a series of related and interdependent hormonal systems and neural systems in different brain circuits1. It also requires developmental experiences, learning, context, and an appropriate partner. Behavioral analysis is an important reflection on neural or neurocrine function. Hence, sexual behavior study on animal models has been widely used in behavioral neuroscience and other related research2. The ethogram of sexual behaviors in rodents has been explained in many articles and books1,3,4. For instance, Scahs and Barfield&#39;s description of sexual behavior in the rat5 has helped understand a similar behavioral pattern in mice5. The mouse is one of the most commonly used subjects for behavioral studies. Hull et al.6 gave a detailed introduction of male mouse sexual behaviors: When a male mouse encounters a female, it starts to investigate the anogenital region of the female. Then, the male presses his front paws against the female&#39;s flanks to mount the female from the rear. The female exhibits a characteristic sexually receptive posture, bending its spine down into a bow and moving its tail to one side of the body, exposing an opening introitus for the sexual penetration of the male (i.e., lordosis). Following the mounting, the male makes rapid, shallow pelvic thrusts, followed by slow and deep vaginal thrusts. After numerous intromissions, a long-lasting thrust results in the ejaculation of semen, during which the male mouse may freeze for about 25 s before dismounting or falling off from the female6. At ejaculation the male mouse accessory glands may produce a mixture containing semen that hardens to form the copulatory plug. Finally, following ejaculation, the male begins genital grooming and displays a lack of interest in the female. In brief, the basic sequence of male sexual behavior consists of sniffing, following, mounting, intromission, ejaculation, and post-ejaculation grooming. Mouse sexual behavior exhibits strain differences. For instance, ejaculation latencies range from 594 to 6943 s, and the numbers of intromissions range from 5 to more than 100. Post-ejaculation latencies range from 17 to 60 min. However, the introduction of a novel female can decrease this time interval. In some cases, the male ejaculates on the first intromission with the new female7.
The major events for the evaluation of sexual behavior are mounting, intromission, and ejaculation. Behavioral scientists have recommended the measurement of not only the frequency of each action, but also its latency and time interval5,8. Some major measurement indicators in past studies include: number of mounts, number of intromissions, mount latency, intromission latency, ejaculation latency, post-ejaculatory mount latency (or post-ejaculatory intervals), post-ejaculatory intromission latency, number of copulatory series, and duration of copulatory series. Park et al.8 and Sachs et al.5 described how to identify each action of mounting, intromission, and ejaculation of rodents. Mounting is defined as the male mounting the female from the rear, palpating her flanks with his forelegs, and thrusting his penis rapidly and repeatedly without penile insertion. Intromission, also known as penile insertion, is identified by one or more of the following acts: a long, deep thrust after rapid shallow thrusts, a rapid kick with one hindleg, and a marked lateral withdrawal of the male from the female. Ejaculation is identified by a terminal pelvic thrust that is slower and deeper than that of an intromission and a reduction in the elevation of the hindleg. A copulatory series is identified by each sequence from mounting to ejaculation. The definitions of behavioral parameters used in the present study are listed as follows: 1) Mounting latency: the time from the introduction of the female to the first mounting of the male; 2) Intromission latency: the time from the introduction of the female to first intromission; 3) Ejaculation latency: the time from the first intromission to first ejaculation (generally following the last pelvic thrust); 4) Post-ejaculatory mount latency: the time from ejaculation to the next mounting; 5) Post-ejaculatory intromission latency: the time from ejaculation and the next intromission; 6) Number of mounts: the number of mounting times before first ejaculation; 7) Number of intromissions: the number of intromissions before the first ejaculation; 8) Number of copulatory series: the number of copulatory series during the observation period; 9) Duration of copulatory series: the time of all copulatory series during the observation period.
Sexual behavior and related behavior can be conducted in either the male&#39;s home cage or in an enclosed arena, among which an apparatus called Rissman&#39;s &#34;No Secrets&#34; mirrored box is introduced to observe the mating behavior3. A video camera is placed in front of the box to simultaneously record the action of the mice from a lateral view and through an inclined mirror from a ventral view. However, this method requires bright lights, which inevitably leads to longer habituation in order to eliminate environmental stress in mice. As for the measuring method, video-based behavioral analysis is recommended to record and quantify behavior4. A video recorder that has a frame-by-frame video advance option with recommended shutter speeds greater than 1/1000 s can be used to record rapid mouse movements. The high resolution infrared camera is necessary when recording in a dark environment. To analyze the film, a computer with a frame grabber to allow the individual frames of behavior to be captured for computer manipulation is needed. Mice are extremely versatile and can display compensatory behavior after almost any treatment. Ambiguity can exist about every moving body part4. Hence, the analysis of some behaviors may require still greater resolution and higher speed cameras.
Male sexual behaviors in mice are affected by many factors, including strain differences, hormone changes, and gene mutantions1,3,9,10. McGill and Blight11 illustrated the strain differences in mouse mating behaviors. For example, C57BL/6 males typically gain intromission quickly and ejaculate in about 20 min11. DBA/2 males are slow to gain intromission but ejaculate rapidly. BALB/c males are slow to achieve ejaculation (average latency of 1 h) due to a long period of courtship11. Testosterone facilitates and maintains male sexual behavior2, and changes in testosterone levels can alter sexual behavior performance12. Both surgical castration and antiandrogen treatment can reduce the level of testosterone and result in a rapid decline of sexual behaviors and even sexual motivation and sexual arousal13. Administered testosterone can restore precopulatory and copulatory behaviors in castrated mice. Lastly, knockout and knockdown mice display differences in facets of sexual behaviors compared to wild type mice. For example, male mice with targeted mutations of Adcy3, Cnga2, and Gnao exhibit a reduced ability to detect pheromones, whereas Trpc2 knockout mice show altered partner preference14,15,16. Other effects of transgenics and knockouts on the sexual behavior of mice are explained by Crawley3.
Here, one of the most common procedures to assess sexual behavior in the pairing of a male mouse with an ovariectomized female that has been hormonally primed to be receptive is described. An experimental protocol is presented for conducting sexual behavior experiments in mice. In addition, an example of changing sexual behavior patterns resulting from social isolation in CD-1 mice is shown.

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			<td>Coated VICRYL Plus Sutures</td>
			<td>Ethicon, Inc.</td>
			<td>missing</td>
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			<td>Estradiol benzoate</td>
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			<td>Beijing Chemical Works Co., Ltd.</td>
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			<td>Ibuprofen (Children&#39;s Motrin)</td>
			<td>Shanghai Johnson &#38; Johnson Co., Ltd.</td>
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			<td>Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
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			<td>HebeI Tiancheng Pharmacreutical Co., Ltd.</td>
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			<td>Male and female CD-1 mice</td>
			<td>Vital River Beijing</td>
			<td>SCXK(<img src="/files/ftp_upload/60154/SCXK.jpg" />)2013-0023</td>
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			<td>Penicillin sodium</td>
			<td>North China Pharmaceutical Co., Ltd.</td>
			<td>F5126420</td>
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			<td>Progesterone</td>
			<td>J&#38;K Scientific, Ltd.</td>
			<td>LR50Q07</td>
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			<td>Sony digital camera</td>
			<td>Sony Corporation</td>
			<td>HDR-CX290E</td>
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			<td>Test box</td>
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			<td>DIY</td>
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			<td>ThinkStation Computer</td>
			<td>Lenovo</td>
			<td>S/N PCOGLQKG</td>
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			<td>Vaporizer for Isoflurane</td>
			<td>RWD Life Science Co., Ltd.</td>
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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.

A comparison of sexual behavior between CD-1 mice reared in isolation and group-housed CD-1 mice is shown. Male CD-1 mice were randomly assigned into an isolation-reared group (IS, one mouse per cage, n = 30) and a group-housed group (GH, five mice per cage, n = 15). The mice underwent isolation rearing from postnatal day 23 to day 93. Then, both groups of mice were assessed for sexual behavior. Our study found that the success rate of copulation tended to be lower in the IS group than in the GH group (IS: 80.0%, GH: 86....

Watch this Scientific Journal Video about Assessment of Sexual Behavior of Male Mice at JoVE.com

Assessment of Sexual Behavior of Male Mice

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 ...

Sexual behavior is a dynamic and a complex process that's influenced by biologic, social, and the environment factors. The study of mouse sexual behavior can provide more information about this interactions. This procedure facilitates the recording of the number and the duration of male mice sexual behaviors to allow the identification and the characterization of mouse sexual behavior interactions and the patterns.48 hours before the sexual behavior test, inject 20 micrograms of Estradiol benzoate in 100 microliters of sterilized olive oil intraperitoneally into a six to eight week old female mouse to induce estrus. On the same day, place the male mice to be tested in the center of a 40 by 40 by 40 centimeter square open field box with three black plexiglass walls and one transparent front wall. Allow the animals to explore the environment freely for 30 minutes per day for the next two days.On the day of the test, four hours before the experiment inject 500 micrograms of progesterone in 100 microliters of sterile olive oil intraperitoneally into the female mice and set the room lighting to 650 lux. Then set up a digital camera linked to a computer to videotape the movement and behavior of the mice. To conduct the behavioral assay, during the first hours of the dark cycle turn on the camera and place the male mouse to be tested in the center of the open field in the test box for five minutes.When the mouse has acclimated to the test environment, place a female mouse in hormone induced estrus into the test box and record the social and mating behaviors and interactions between the male and female mice for 30 minutes. At the end of the test period, confirm that the video has been saved and turn off the camera. Return the mice to their individual home cages and clean any urine, feces, and padding within the apparatus.Then remove the smell of the tested animals with 75%ethanol and begin the test for the next male mouse as just demonstrated. To analyze the behavioral data, when all of the mice have been tested play back the video recording and record the number of mounts and the number of intromissions that occurred during the 30 minute experimental period. Record the time from the introduction of the female to the first mounting as the mounting latency, the time from the introduction of the female to the first intromission as the intromission latency, and the time from the first intromission to the first ejaculation as the ejaculation latency.Then record the number and the time of all the copulatory series in 30 minutes. The success rate of copulation tends to be lower in isolation reared groups than in group housed groups, likely due, at least in part, to the longer mounting latency observed in isolation reared groups compared to the group housed groups. The intromission latency is also longer in isolation reared groups indicating that these animals require a longer time to perform the insertion of the penis into the female's vagina.No statistically significant difference between the two groups is observed in terms of ejaculation latency or post-ejaculatory mount latency. The duration of the copulatory series is shorter in the isolation housed group than in the group housed group. No statistically significant differences between the two groups are observed in terms of the number of mounts, number of intromissions, or number of copulatory series.Sexual behavior is a refraction of neural and the neurocrine function. Understanding mouse sexual behavior patterns can help further our understanding of human sexual behaviors and their interactions.

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Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

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 ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...