Name: determining ultrasonic vocalization preferences in mice using a two-choice playback test
Uploaded: 2015-09-03T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

עבודה זו נתמכה על ידי גרנט ב- סיוע לJSPs עמיתים לAA; על ידי מענקים ב-סיוע למחקר מדעי על תחומי חדשניים לעמיתי JSPs (מס &#39;4501 ומס&#39; 25,132,712) לTK; ועל ידי מענק מחקר מטעם אוניברסיטת Azabu. איור 2 הודפס מחדש מKihara, ט, Harada, ט, וKoshida, ייצור תואם ופל נ ומאפיינים של קרינת אולטרא-סאונד מושרה תרמית סיליקון nanocrystalline. ב: חיישנים ומפעילים: פיסיים, נפח 125, Elsevier, עמ &#39;. 426, (2006), באישור Elsevier.

<html> כל ההליכים אושרו על ידי ועדת האתיקה של אוניברסיטת Azabu. כל הניסויים בוצעו בתא אטום לרעש. 1. הכנת בעלי החיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זכרים להקלטה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג עכברי זכרים מגיעים לבגרות מינית חוו בהזדווגות. </li></ol></li><li style=";text-align:right;direction:rtl"> נושאים נקבה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג נקבות עכברי בתולה שכבר שוכנו עם 2 עד 5 להמלטה לכלוב (בדרך כלל 8 - 12 שבועות). </li><li style=";text-align:right;direction:rtl"> להשיג מריחות נרתיק יומי כדי לקבוע את השלב של מחזור הייחום לפני הבדיקה, לפי מקלין 21. יש להקפיד למזער את הגירוי בנרתיק כדי למנוע pseudopregnancy. </li><li style=";text-align:right;direction:rtl"> להכתים את המריחות עם Giemsa, ולקבוע שלב של מחזור הייחום מבוסס על הנוכחות או עדר של כדוריות דם לבנות, תאי האפיתל cornified, ותאי אפיתל גרעיני, על פי 22 נלסון. </li></ol></li></ol> 2. מכשירים (איור 1) _content &quot;&gt; <img alt="איור 1" src="/files/ftp_upload/53074/53074fig1.jpg" /> איור 1. סכמטי של מבחן התיבה והתקנים דו בחירה. עכברים יכולים לגשת חדרים ו- B דרך השערים הקטנים הממוקמים ביניהם לבין האזור הניטרלי. התיבה דו-הבחירה ושני משדרי אולטרסאונד מונחים על רצפת חדר אטום לרעש. המיקרופון מושעה בתוך החדר אטום לרעש. קיצור:. מחשב, מחשב אישי <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקלטה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חבר מיקרופון הקבל קולי למחשב באמצעות ממיר A / D USB. </li><li style=";text-align:right;direction:rtl"> הגדר את רמת הרווח של ממיר A / D ל&quot; 7 &quot;. </li><li style=";text-align:right;direction:rtl"> נתונים אקוסטיים רשומות בקבצים בפורמט של 16 סיביות עם קצב דגימה של 400 kHz באמצעות תוכנה ללכידה אודיו. </li></ol></li><li style=";text-align:right;direction:rtl"> עבור השמעה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חבר D / ממיר למחשב באמצעותממשק USB. </li><li style=";text-align:right;direction:rtl"> חיבור שני מגברים לD / ממיר באמצעות מחליש. (המכשיר האחרון משמש כדי למנוע עומס יתר.) </li><li style=";text-align:right;direction:rtl"> חבר פולט אולטרסאונד לכל מגבר. </li></ol></li></ol> 3. USV הקלטה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח עכבר זכר ב( 17 x 10 x 11 סנטימטר) קטן או כלוב בגודל בינוני (21 x 14 x 13 סנטימטרים) עם חור בקוטר 6 סנטימטר בדפנות והחור מכוסה ברשת תיל 0.5 סנטימטר. בעת שימוש בכלוב בגודל בינוני, לבנות מחיצה מעבר למרכז הכלוב לצמצם את השטח. </li><li style=";text-align:right;direction:rtl"> אין להשתמש במצעים בכלוב, כדי למנוע רעשי רשרוש שיכול לזהם את ההקלטות. </li><li style=";text-align:right;direction:rtl"> מניחים מיקרופון ליד הרשת. </li><li style=";text-align:right;direction:rtl"> בגין ניטור ניקוד קולי עם הגדרת ההקלטה מוצגת בשלב 2.1. </li><li style=";text-align:right;direction:rtl"> הנח עכבר נקבה לבגרות מינית בשני metestrus או diestrus בכלוב עם עכבר הזכר. </li><li style=";text-align:right;direction:rtl"> שיא USVs במשך 3 - 5 דקות. </li></ol> 4. Test תיבה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבנות תיבת הבדיקה באמצעות לוח אקריליק (איור 1). הערה: תיבת הבדיקה (35 x 20, וx 20 סנטימטר) מחולקת בשלושה מדורים: אזור ניטראלי (15 x 20 סנטימטרים), וחדרים ו- B (20 x 10 סנטימטר, כל אחת). חדרים ו- B כל יש חור 4 סנטימטר בקוטר שמכוסה ברשת תיל 0.5 ס&quot;מ. החורים והרשת נמצאים בכל קצה של חדרים וב הפוך שערים (5 X 5 סנטימטרים כל אחת) בין האזור הניטרלי וחדרי A ו- B. </li><li style=";text-align:right;direction:rtl"> כדי למנוע דליפת קול לבודד כל פולט אולטרסאונד, פרט לצד הפונה לרשת, באמצעות צלחות גומי, איטום אותם לתיבה מסביב לכל רשת. </li><li style=";text-align:right;direction:rtl"> חותמות להוביל מקום בשני הצדדים של הקיר בין החדרים ו- B לבידוד קול. </li></ol> 5. הכנה לקול ניגון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יצירת צלילי פלייבק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ללכוד קטע 20 שניות של USV מהקובץ המוקלט באמצעות תוכנה לעריכת אודיו דיגיטלית. </li><li style=";text-align:right;direction:rtl"> בחר וללכוד רקע לאISE (0.35 שניות) מקובץ 20 שניות קטע USV. צור קובץ רעשי רקע 20 שניות על ידי חזרה על קטע הרעש. </li><li style=";text-align:right;direction:rtl"> כדי לנגן את שני קבצי הקול בו זמנית, לייצא אותם כקובץ צליל סטריאו אחת, עם קובץ אחד ל&quot; אוזן שמאל &quot;וקובץ האחר ל&quot; אוזן ימין.&quot; </li><li style=";text-align:right;direction:rtl"> סנן את קבצי קול באמצעות כמסנן גבוה לעבור עם הפסקת של 40 קילוהרץ. </li><li style=";text-align:right;direction:rtl"> להפחית את הרעש באמצעות הכלי להפחתת רעשים בתוכנה שלאחר עיבוד. בחר וללכוד קטע של הקובץ שמכיל לא USVs כפרופיל להפחתת רעשים, ולהפעיל הפחתת רעש עם רמה 40. </li></ol></li><li style=";text-align:right;direction:rtl"> כיול של צלילי פלייבק <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא שהמרחב בין קרינת אולטרא-סאונד ומיקרופון הוא 10 סנטימטר. </li><li style=";text-align:right;direction:rtl"> כדי לכייל את רמות לחץ קול מקרינת אולטרא-סאונד, לבצע ניטור קולי באמצעות התצורה המתוארת בשלב 2.1. מדוד את רמת לחץ קול על ידי המיקרופון בדציבלים (dB). </li><li style=";text-align:right;direction:rtl"> Using המחליש ומגברים, להתאים את עוצמת הקול של USVs שנוצר על ידי קרינת אולטרא-סאונד לאותה רמת לחץ הקול כUSVs הזכר שנרשם בשלב 3. </li><li style=";text-align:right;direction:rtl"> בעת השימוש בשני משדרי אולטרסאונד וקובץ קול מורכב מUSVs ורעשי רקע, לאשר כי שני משדרי אולטרסאונד תערוכה באותה רמת לחץ קול באמצעות קול כיול (למשל, צליל טהור 75-kHz) לפני ביצוע הבדיקה התנהגותית. </li><li style=";text-align:right;direction:rtl"> כאשר מתרבה קובץ קול מורכב משני זרמים של USVs, להתאים שתי רמות לחץ קול שנוצרו באותה הרמה, לפני הבדיקה. </li></ol></li></ol> 6. מבחן דו-בחירה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את קרינת אולטרא-סאונד מאחורי משתלב בתיבת הבדיקה. </li><li style=";text-align:right;direction:rtl"> סגור את השערים לחדרים ו- B עם לוח אקריליק, ולהרגיל את הנקבה בכפוף לאזור הניטרלי למשך 30 דקות. לכסות את התיבה עם אקריליק לוח כדי למנוע שהעכבר יכול לברוח. </li><li style=";text-align:right;direction:rtl"> התחל הקלטת וידאו באמצעות CCהמצלמה D רכובה מעל לתיבה. המצלמה (לא מוצג באיור 1) משתרעת על השטח של כל שלושת התאים. </li><li style=";text-align:right;direction:rtl"> הסר את השער וכיסוי לוח ולאפשר הנקבה לחקור באופן חופשי את תיבת הבדיקה, כולל אזורי הצליל. </li><li style=";text-align:right;direction:rtl"> ברגע שהנקבה חקרה שני רשתות שינוי, וחזרה לאזור הניטרלי, להתחיל לשחק קובץ קול 20 שניות שוב ושוב במשך 5 או 10 דקות. </li><li style=";text-align:right;direction:rtl"> לנהל מעקב התנהגות במשך 5 או 10 דקות. </li><li style=";text-align:right;direction:rtl"> כדי להפחית את רמזי חוש הריח לא רצויים שהופקדו מהנושא הקודם, לנקות את כלובי המבחן בין הבדיקות עם 70% אתנול. </li><li style=";text-align:right;direction:rtl"> לעבור המקום של קרינת אולטרא-סאונד ו- B באופן אקראי, כדי למנוע השפעת הטיה צד טבועה כלפי חדרי A או B. </li></ol> 7. ניתוח סטטיסטי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש בתוכנת ניקוד אירוע התנהגותיות, לנתח את הפרמטרים הבאים: מספר כולל של ערכים בכל חדר; משך זמן כולל של שהייה בכל אזור מרכז; משך זמן כולל של שהייה בכל אזור צליל; ומשך הזמן הכולל של חיפוש של כל רשת מול פולט אולטרסאונד. איור 1 מדגים מה הכוונה &quot;אזור מרכז&quot; ו- &quot;אזור צליל.&quot; הערה: במקרים בהם העכבר נשאר על הקו המפריד בין שני האזורים (איור 1, קו מקווקו) להחליט מיקום על ידי הכיוון של הראש. כאשר הראש פונה לצד הרשת, הוא הבקיע כמו שהייה באזור הקול. </li><li style=";text-align:right;direction:rtl"> לכל פרמטר התנהגות, להשוות את תגובות לחדרים ו- B באמצעות בדיקה חתמה דרגה Wilcoxon, או -tests לא זיווג עם רמה מובהקת של 0.05. </li></ol>

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A-Phys. 125 (2), 422-428 (2006).</li><li>Kikusui, T., 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=Cross+fostering+experiments+suggest+that+mice+songs+are+innate.">Cross fostering experiments suggest that mice songs are innate.</a> PloS One. 6 (3), e17721 (2011).</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> J. Vis. Exp. (67), e4389 (2012).</li><li>Nelson, J. F., Felicio, L. S., Randall, P. K., Sims, C., Finch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+longitudinal+study+of+estrous+cyclicity+in+aging+C57BL/6J+mice:+I.+Cycle+frequency,+length+and+vaginal+cytology.">A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology.</a> Biol. Reprod. 27 (2), 327-339 (1982).</li><li>Tomihara, K., 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=Effect+of+ER-beta+gene+disruption+on+estrogenic+regulation+of+anxiety+in+female+mice.">Effect of ER-beta gene disruption on estrogenic regulation of anxiety in female mice.</a> Physiol. Behav. 96 (2), 300-306 (2009).</li><li>Zheng, Q. Y., Johnson, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+loss+associated+with+the+modifier+of+deaf+waddler+(mdfw)+locus+corresponds+with+age-related+hearing+loss+in+12+inbred+strains+of+mice.">Hearing loss associated with the modifier of deaf waddler (mdfw) locus corresponds with age-related hearing loss in 12 inbred strains of mice.</a> Hear. Res. 154 (1-2), 45-53 (2001).</li><li>Haga, S., 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=The+male+mouse+pheromone+ESP1+enhances+female+sexual+receptive+behaviour+through+a+specific+vomeronasal+receptor.">The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.</a> Nature. 466 (7302), 118-122 (2010).</li><li>Grimsley, J. M., Monaghan, J. J., Wenstrup, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+social+vocalizations+in+mice.">Development of social vocalizations in mice.</a> PLoS One. 6 (3), e17460 (2011).</li><li>Sugimoto, H., 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=A+role+for+strain+differences+in+waveforms+of+ultrasonic+vocalizations+during+male-female+interaction.">A role for strain differences in waveforms of ultrasonic vocalizations during male-female interaction.</a> PLoS ONE. 6 (7), (2011).</li><li>Hanson, J. L., Hurley, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+presence+and+estrous+state+influence+mouse+ultrasonic+courtship+vocalizations.">Female presence and estrous state influence mouse ultrasonic courtship vocalizations.</a> PLoS One. 7 (7), e40782 (2012).</li><li>Wang, H., Liang, S., Burgdorf, J., Wess, J., Yeomans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic+vocalizations+induced+by+sex+and+amphetamine+in+M2,+M4,+M5+muscarinic+and+D2+dopamine+receptor+knockout+mice.">Ultrasonic vocalizations induced by sex and amphetamine in M2, M4, M5 muscarinic and D2 dopamine receptor knockout mice.</a> PLoS One. 3 (4), (2008).</li><li>Moles, A., Kieffer, B. L., D'Amato, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficit+in+attachment+behavior+in+mice+lacking+the+mu-opioid+receptor+gene.">Deficit in attachment behavior in mice lacking the mu-opioid receptor gene.</a> Science. 304 (5679), 1983-1986 (2004).</li><li>Hiramoto, T., 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=Tbx1:+identification+of+a+22q11.2+gene+as+a+risk+factor+for+autism+spectrum+disorder+in+a+mouse.">Tbx1: identification of a 22q11.2 gene as a risk factor for autism spectrum disorder in a mouse.</a> Hum. Mol. Genet. 20 (24), 4775-4785 (2011).</li><li>Ey, E., 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=Absence+of+deficits+in+social+behaviors+and+ultrasonic+vocalizations+in+later+generations+of+mice+lacking+neuroligin4.">Absence of deficits in social behaviors and ultrasonic vocalizations in later generations of mice lacking neuroligin4.</a> Genes Brain Behav. , (2012).</li><li>Roy, S., Watkins, N., Heck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+ultrasonic+vocalizations+in+a+mouse+model+of+fragile+X+syndrome+reveals+limited,+call+type+specific+deficits.">Comprehensive analysis of ultrasonic vocalizations in a mouse model of fragile X syndrome reveals limited, call type specific deficits.</a> PLoS One. 7 (9), e44816 (2012).</li></ol>

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, possib...

בעלי חיים רבים משתמשים בקולות לתקשורת intraspecific. במאסכאלאס העכבר, סוג אחד חשוב של אות תקשורת הוא קולות קוליים (USVs), שיש לי תדרים גבוהים יותר מ -20 קילו-הרץ. USVs נפלט על ידי עכברים נחשב מרכיב של הכרה חברתית בזכר-נקבת 1-4, נקבה-נקבה 1, 5, וגבר-גבר 1, 6 אינטראקציות. USVs גם נפלטים על ידי גורים כשהם מבודדים מאמם, אשר מגדיל אחזור-גור התנהגותה, ולכן כלבון הישרדות 7. למרות דיווחים רבים ניתחו ומסווג USVs עכבר 8, 9, תגובות התנהגותיות ומנגנונים עצביים של בעלי החיים שקיבלו כבר פחות מתועד 10, 11. האחרון הוא צורך להבהיר את המשמעות הביולוגית של המאפיינים השונים של USVs. לחשוף מנגנונים אלה, ניסוי ההשמעה הוא שיטה יעילה. מחקרי השמעה לאחרונה עולים כי נקבהעכברים נמשכים 12 USVs, וכי הם מעדיפים USVs מזכרים שהם שונים מהוריהם 13, 14. מאמר זה מסביר את מבחן ההשמעה משמש להערכת העדפת USV בעכברים. תיבת מבחן דו-בחירה פותחה באשר ניתן לשחק שתי USVs שונה בחזרה בו זמנית בשני תאים של מתחם בדיקה, כפי שמוצג באיור 1. סוג זה של תיבת המבחן מונע זיהום צליל על ידי חלוקת אזור הבדיקה בשלושה תת-תאים , באמצעות קירות עופרת. קרינת אולטרא-סאונד נמצאות מחוץ לכל חדר. בקיר שבין החדרים וקרינת אולטרא-סאונד חורים מכוסים ברשת תיל. עכברים יכולים לנוע בחופשיות בשלושה חדרים, ולהראות התנהגות &quot;חיפוש ברשת&quot;, כאילו כדי להגיב לUSVs שיחק בחזרה על ידי קרינת אולטרא-סאונד. בבדיקה זו, עכברים להישאר לתקופות של משך שונה קרוב לפולט קול אחד או אחר. יכולים להיות מחוברים פרמטרים אלה כדי להשיג measu רגיש<html> מחדש של העדפת קול. כדי שתוכל לשחק USVs בחזרה, קרינה תרמו-אקוסטי סיליקון nanocrystalline (כלומר, &quot;NC-סי פולט&quot;) שימשה כבמחקרים קודמים 15-17. התקנים אלה מורכבים מהאלקטרודה סרט דק דוד, שכבת סיליקון ננו-נקבובי, ופרוסות סיליקון חד גבישיים. קובץ הקול הדיגיטלי מומר לאות אנלוגי ולאחר מכן עבר דרך אלקטרודה דוד. המכשיר ממיר את אותות תרמית מתח תלוי וכתוצאה מכך ללחץ קול משמעותי עם עיוות נמוכה. מכשיר זה הוא ייחודי בכך ש, שלא כמו מחוללי צליל משותפים שתלויות בתנודות מכאניות, זה יכול להתרבות קול ללא הצורך בסרעפת. הפולט מציג רמת לחץ קול שטוחה בתדרי 20-160 קילו-הרץ (איור 2), ויכול להתרבות USVs עכברי נרשם דיגיטלי מאוד מדויק במונחים של זמן, תדירות, ורמת צליל לחץ 15, 18, ​​19. ve_content &quot;&gt; בניסוי נציג מוצג באיור 3, / 6 נקבות C57BL (B6) הורשו לבחור בין BALB / ג (BALB) USVs זכר ורעשי רקע. בנוסף, איור 4 מראה את הבחירה של נקבות B6 וBALB בין פלייבקים USV בו זמנית מBALB וזכר B6, כפי שדווחו במחקר קודם 14. המאפיינים של USVs הגברי שונים בין B6 וBALB זני 20. כפי שניתן לראות בתוצאות אלה, את האטרקטיביות של USVs ניתן להעריך בפרוטוקול הנוכחי, שבו צלילים נרשמים מאדם חי, ניתחו אקוסטית, ושיחקו בחזרה לאנשים אחרים.

<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.

USVs נרשם מBALB-זכר אחד (161 הברות לכל 20 שניות), כמו גם רעש רקע שמשו השמעת צלילים בניסוי הנציג מוצג באיור 3. בניסוי זה, 7 עכברים B6 נשיים שמשו בשעת 9 שבועות של גיל . כדי לקבוע את משך הזמן הטוב ביותר של בדיקות של תגובה נשית להשמעת צלילים, הפרמטרים התנהגותיים נותחו בנפרד במ...

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.

קביעת העדפות Ultrasonic ניקוד בעכברים באמצעות מבחן השמעה דו בחירה

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

Universidad San Sebastian

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.

הערכת קולות Ultrasonic במהלך והתרופות עצמית הממשל חולדות

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).

הקלטת עכבר Ultrasonic קולות של להערכת תקשורת חברתית

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.

אימון ממוקד של קולות Ultrasonic בחולדות ומעלה פרקינסון

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.

Eliciting וניתוח זכר העכבר Ultrasonic ווקאליזציה (USV) שירים

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.

היכרות הדרכת Clicker בתור העשרה קוגניטיבית עבור עכברי מעבדה

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.

הרגלה ו Prepulse עיכוב של רתיעה אקוסטית במכרסמים

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.

וזמינותו התנהגותית עבור חוקרים את התפקיד של זיכרון מרחבי במהלך ההגנה אינסטינקטיבית בעכברים

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.

הגדרת עלות נמוכה לaudiometry התנהגות במכרסמים

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

חקר התנהגויות הגנתיות אקטיביות בעכברים עם התניה שונה של פחד

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.

התניה שונה של פחד לגרימת התנהגויות טיסה בעכברים

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

קביעת העדפות Ultrasonic ניקוד בעכברים באמצעות מבחן השמעה דו בחירה

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הערכת קולות Ultrasonic במהלך והתרופות עצמית הממשל חולדות

הקלטת עכבר Ultrasonic קולות של להערכת תקשורת חברתית

אימון ממוקד של קולות Ultrasonic בחולדות ומעלה פרקינסון

Eliciting וניתוח זכר העכבר Ultrasonic ווקאליזציה (USV) שירים

היכרות הדרכת Clicker בתור העשרה קוגניטיבית עבור עכברי מעבדה

הרגלה ו Prepulse עיכוב של רתיעה אקוסטית במכרסמים

וזמינותו התנהגותית עבור חוקרים את התפקיד של זיכרון מרחבי במהלך ההגנה אינסטינקטיבית בעכברים

הגדרת עלות נמוכה לaudiometry התנהגות במכרסמים

התניה שונה של פחד לגרימת התנהגויות טיסה בעכברים

התניה שונה של פחד לגרימת התנהגויות טיסה בעכברים