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

이 작품은에 의해 지원되었다 그랜트 - 에이드에서 AA에 JSPS 휄로우; 보조금 인 원조 과학 연구에 대한 JSPS 휄로우위한 혁신적인 분야 (제 4501 호 및 제 25132712) TK에에 의해; 와 아자 부 대학. 그림 2에 의해 수여 연구 프로젝트 부여하여 키하라, 토니, 하라다, T., Koshida, N. 웨이퍼 호환 제조 및 나노 결정 실리콘 열 유도 초음파 이미 터의 특성에서 다시 인쇄됩니다. 센서 및 액추에이터 :에서 물리적 볼륨 (125), 엘스 비어, P. (426), (2006), 엘스 비어의 허가.

<html> 모든 절차가 아자 부 대학의 윤리위원회에 의해 승인되었다. 모든 실험은 방음 실에서 실시 하였다. 1. 동물 준비 <ol><li> 녹음을위한 남성 <ol><li> 짝짓기 경험이 성적으로 성숙한 수컷 마우스를 가져옵니다. </li></ol></li><li> 여성 주제 <ol><li> (- 오래된 12 주 보통 8) 케이지 당 2-5 한배 새끼와 함께 보관 된 처녀 암컷 마우스를 가져옵니다. </li><li> 매 클린 (21)에 따라, 검사 전 발정주기의 위상을 결정하기 위해 매일 질 얼룩을 얻습니다. 주의 pseudopregnancy 않도록 질 자극을 최소화하기 위해주의해야한다. </li><li> 넬슨 (22)에있어서, 김사와 얼룩 얼룩 및 존재 또는 백혈구, 각질화 된 상피 세포, 상피 세포 및 핵의 유무에 기초하여 발정주기의 위상을 결정한다. </li></ol></li></ol> 2. 장치 (그림 1) _content &quot;&gt; <img alt="그림 1" src="/files/ftp_upload/53074/53074fig1.jpg" /> 그림 두 선택 테스트 상자 및 장치의 도식. 마우스는 그들과 중립 지역 ​​사이에있는 작은 문을 통해 방 A와 B에 액세스 할 수 있습니다. 두 선택 상자와 두 초음파 방사체는 방음 실의 바닥에 배치된다. 마이크는 방음 실 내부에 일시 중단됩니다. 약기 :. PC, 개인용 컴퓨터 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol><li> 기록 <ol><li> USB의 A / D 컨버터를 이용하여 PC에 초음파 콘덴서 마이크로폰을 연결한다. </li><li> &quot;7&quot;에 대한 A / D 컨버터의 이득 레벨을 설정한다. </li><li> 오디오 캡처 소프트웨어를 사용하여 400 kHz의 샘플링 레이트를 갖는 16 비트 포맷 파일의 레코드 데이터 탄성. </li></ol></li><li> 재생 <ol><li> D / 사용하여 PC에 컨버터를 연결USB 인터페이스. </li><li> 감쇠기를 통해 D에 두 앰프 / A 컨버터를 연결합니다. (후자의 디바이스는 과부하를 방지하는 역할을한다.) </li><li> 각 앰프에 초음파 이미 터를 연결합니다. </li></ol></li></ol> 3. USV 기록 <ol><li> 측벽에 6cm 직경의 구멍과 0.5 CM 철망으로 덮여 구멍에 소형 (17 × 10 X 11cm)에 남성 마우스 또는 중간 크기 (21 X 14 X 13cm) 케이지를 놓습니다. 중형 케이지를 사용할 때, 면적을 감소시키는 케이지의 중심을 가로 질러 파티션을 구성. </li><li> 녹음을 오염시킬 수 부스럭 거리는 소음을 방지하기 위해 새장에 침대를 사용하지 마십시오. </li><li> 메시 옆에 마이크를 배치합니다. </li><li> 2.1 단계에 표시된 녹화 설정과 초음파 발성 모니터링을 시작합니다. </li><li> 남성 마우스와 케이지에 metestrus 또는 diestrus 중 하나의 성적으로 성숙한 여성 마우스를 놓습니다. </li><li> 5 분 - 3 녹음 USVs. </li></ol> 4. TEST 박스 <ol><li> 아크릴 보드 (그림 1)를 사용하여 테스트 상자를 구축 할 수 있습니다. 참고 : (× 20 ​​(35), 및 X 20cm 높이)를 세 구획으로 나누어 져 시험 상자 : 중립 지역 (15 X 20cm), 객실 A와 B (20 × 10cm, 각각). 방 및 B는 각각 0.5 ㎝ 철망으로 덮여 4cm 직경 홀을 갖는다. 구멍 메시는 중립 지역 및 객실 A와 B 사이의 객실 A와 B의 각 끝에 확인 게이트 (5 × 5cm 각)에 위치 </li><li> 사운드 누설은 각 메시의 둘레 상자로 밀봉 고무 플레이트를 사용하여, 메쉬에 대향 측면을 제외한 각 초음파 방사기를 절연 방지한다. </li><li> 방음을위한 객실 A와 B 사이의 벽의 양쪽에 배치 리드 씰. </li></ol> 5. 사운드 재생을위한 준비 <ol><li> 재생 사운드의 창조 <ol><li> 디지털 오디오 편집 소프트웨어를 사용하여 기록 된 파일로부터 USV의 20 초 세그먼트를 캡처. </li><li> 선택하지 않고 배경을 캡처이세 20 초 USV 세그먼트 파일에서 (0.35 초). 소음 세그먼트를 반복하여 20 초 배경 잡음 파일을 만듭니다. </li><li> &quot;왼쪽 귀&quot;를 위해 다른 파일을 하나의 파일로, 하나의 스테레오 사운드 파일로 내보낼, 동시에 두 개의 사운드 파일을 재생하려면 &quot;오른쪽 귀.&quot; </li><li> 40 kHz의 컷오프와 같은 고역 통과 필터를 사용하여 사운드 파일을 필터링. </li><li> 후 처리 소프트웨어의 노이즈 감소 도구를 사용하여 소음을 줄일 수 있습니다. 선택하고 노이즈 감소 프로파일과 더 USVs가없는 파일의 세그먼트를 캡처하고, 40 레벨과 노이즈 감소를 실행합니다. </li></ol></li><li> 재생 사운드의 교정 <ol><li> 초음파 이미 터와 마이크 사이의 공간이 10cm 있는지 확인합니다. </li><li> 에미 터에서 초음파의 음압 레벨을 교정하는 단계 2.1에 기재된 구성을 사용하여 초음파 모니터링을 수행한다. 데시벨 마이크 (dB)로 음압 레벨을 측정합니다. </li><li> USI감쇠기 및 증폭기 겨, 3 단계에서 기록한 남성 USVs 동일한 음압 레벨 초음파 에미 터에 의해 발생 USVs의 볼륨을 조정한다. </li><li> 두 초음파 에미 터 및 USVs 및 배경 잡음으로 구성된 사운드 파일을 사용하는 경우, 두 초음파 방출기 행동 시험을 수행하기 전에 보정 사운드 (예를 들어, 75 kHz의 순음)를 사용하여 동일한 음압 레벨을 나타내는 것을 확인한다. </li><li> USVs 두 스트림으로 구성된 사운드 파일을 재생하는 경우, 실험 전에, 동일한 레벨로 생성 된 두 음압 레벨을 조정한다. </li></ol></li></ol> 6. 두 선택 테스트 <ol><li> 시험 상자의 메쉬 뒤에 초음파 이미 터를 놓습니다. </li><li> 아크릴 보드와 함께 객실 A와 B에 문을 닫고 30 분 동안 중립 영역에 여성의 주제를 길들. 마우스를 탈출 할 수있는 방지하기 위해 아크릴 보드 상자를 커버. </li><li> CC를 사용하여 비디오 녹화를 시작합니다D 카메라는 상자 위에 장착. 카메라 (하지 그림 1)은 세 개의 구획의 영역을 다루고 있습니다. </li><li> 게이트와 커버 보드를 제거하고 여성의 사운드 영역을 포함하여 자유롭게 테스트 상자를 탐색 할 수 있습니다. </li><li> 여성 모두 메쉬를 조사하였으며, 중성 영역으로 돌아 오면, 5 또는 10 분 동안 20 초 반복 사운드 파일을 재생 시작한다. </li><li> 5 또는 10 분 동안 행동 모니터링을 실시한다. </li><li> 이전 대상에서 침착 원하지 않는 후각 신호를 감소 70 % 에탄올과 시험 사이의 테스트 새장을 청소합니다. </li><li> 초음파의 이미 터의 위치를​​ 랜덤하게 전환 B 실 또는 B. 향해 고유 측 바이어스 효과를 피하기 </li></ol> 7. 통계 분석 <ol><li> 다음 매개 변수를 분석, 행동 이벤트 득점 소프트웨어를 사용하여 각 방에있는 항목의 총 수를; 각각의 중간 영역에서 체류 전체 기간; 각 사운드 영역에서 체류 전체 기간; 과초음파 방출의 앞에 각 메시의 검색의 총 시간. 그림 1은 &quot;중간 영역&quot;과 무엇을 의미하는지 설명 &quot;사운드 영역을 설정합니다.&quot; 참고 경우 마우스 두 영역 사이의 분할 선 (도 1, 점선)의 머리 방향으로의 위치 결정을 유지한다. 헤드 측 메시에 직면했을 때, 그것은 사운드 존에서 머물 얻었된다. </li><li> 각각의 행동 매개 변수에 대해 0.05의 유의 수준을 갖춘 객실에 대한 반응과 B 윌 콕슨 부호 순위 테스트를 사용하여, 또는 쌍 T의 -tests를 비교합니다. </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.

많은 동물 종자 간 통신을위한 발성을 사용합니다. 마우스 뮤스의 musculus에서 통신 신호의 하나의 중요한 유형은 20 kHz의보다 높은 주파수가 초음파 발성 (USVs)이다. 마우스에 의해 방출 USVs는 남성 여성, 1-4 여성 - 여성 1, 5, 남성 - 남성 (1), (6) 상호 작용의 사회 인식의 구성 요소로 간주됩니다. USVs는 그녀의 강아지 - 검색 행동을 증가 어머니로부터 분리되는 경우도 새끼에 의해 방출, 따라서 생존 7 강아지 있습니다. 많은 보고서가 적은 10에 설명 된 분석 마우스 USVs 8, 9, 행동 반응 및 수신 동물의 신경 메커니즘을 분류하고 있지만 11. 후자 USVs의 다양한 특성의 생물학적 의미를 명확하게 할 필요가있다. 이러한 메커니즘을 나타 내기 위해, 재생 실험 효율적인 방법이다. 최근 재생 연구는 여성의 것으로 밝혀졌다마우스는 USVs (12)에 매료되어, 그들은 그들의 부모 (13), (14)에서 다른 남성에서 USVs을 선호. 이 문서에서는 마우스의 USV 환경을 평가하는 데 사용되는 재생 테스트를 설명합니다. 두 선택 테스트 박스가도 1에 도시 된 두 개의 다른 USVs는 테스트 케이스의 두 구획을 동시에 재생할 수있는 개발되었다. 시험 상자의이 유형은 세 개의 서브 - 챔버에서 검사 영역을 분할하여 음성 오염을 방지 납 벽을 사용하여. 초음파 방출은 각 객실 외부에 있습니다. 객실과 초음파 이미 터 사이의 벽에서 철망으로 덮여 구멍이 있습니다. 마우스는 3 방에서 자유롭게 이동하고, 초음파 에미 터에 의해 재생 USVs에 응답하는 것처럼, &quot;메시 검색&quot;동작을 보여줄 수있다. 이 시험에서, 생쥐는 하나의 사운드 이미 터 또는 서로 근접 상이한 지속 기간까지. 이러한 매개 변수는 민감한 measu를 얻을 기록 할 수 있습니다<html> 사운드 환경 설정의 재. 다시 USVs를 재생하려면, 나노 결정 실리콘 열 음향 방출 (즉, &quot;NC-시 터&quot;)는 이전의 연구 15-17에서 사용 하였다. 이러한 장치는 박막 가열기 전극, 나노 다공성 실리콘 층과 단결정 실리콘 웨이퍼로 구성된다. 디지털 사운드 파일을 아날로그 신호로 변환 한 후 히터 전극을 통과한다. 장치는 낮은 왜곡과 상당한 음압으로 인한 전위 의존성 열 신호를 변환한다. 기계적 진동에 의존하는 일반적인 소리 발생기,이 진동판의 필요없이 음향을 재생 달리이 장치는 점에서 독특하다. 이미 터 (20)로부터 160 kHz에서 (도 2)까지의 주파수에서 평면 음압 레벨을 나타내고, 그리고 시간, 주파수 및 음압 레벨 15, 18, ​​19의 측면에서 매우 정확하게 디지털 기록 뮤린 USVs를 재생할 수있다. 도 3에 도시 된 대표적인 실험에서 ve_content은 &quot;&gt;, C57BL / 6 (B6) 암컷 BALB / C (BALB) 사이 남성 USVs 및 배경 잡음을 선택하는 것이 허용되었다. 또한,도 4는 사이 B6 및 BALB 암컷의 선택을 도시 BALB 및 B6 남성에서 동시 USV의 플레이는 이전의 연구에서보고 된 14. 남성 USVs의 특성은 B6 사이의 차이와 BALB 20 종자. 이러한 결과에 의해, USVs의 매력은 본 프로토콜로 평가 될 수있다 바와 같이, 하는 소리, 라이브 개인으로부터 기록 된 음향 분석 및 기타 개인 재생됩니다.

<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.

USVs는도 3에 도시 된 대표적인 실험에서 사운드 재생으로 사용 하였다 한 BALB-남성 (20 초 당 161 음절)뿐만 아니라 배경 잡음에서 기록 하였다.이 실험에서, 7 여성 B6 마우스는 9 주령에 사용 하였다 . 사운드를 재생하기 위해 여성의 응답 테스트의 가장 시간을 결정하기 위해, 행동 파라미터는 총 10 분의 시험 시간의 처음과 마지막 다섯 분 동안 별도로 분석 하였다. 먼저, 어떤 측면 bias.The 결과는 배경 잡음 측면 사이에 차이가 없다고 없었다 있다는 것을 보여주는, 재생 (도 3a) 전의 조사 시간 동안 룸 또는 B의 체류 전체 기간에서 유의 한 차이가 없었다 및 제 5 분, 마지막 5 분, 총 시험 시간 (도 3b)을 분석 룸 항목 수에 남성 USVs 측은.도 3c는 durat 누적 초를 나타낸다재생하는 동안 각 방에 숙박 이온 (즉, 총 중간 영역에서 숙박 기간, 사운드 영역에서). 처음 5 분 (P = 0.043), 또는 전체 시험 시간 (p는 = 0.043)을 분석 할 때 대상은 배경 잡음 측에 비해 BALB 남성 USV 측에서 상당히 긴 시간을 보냈다. 이러한 매개 변수 마지막 5 분에 의해 분석에서 유의 한 차이가 없었다. 중간 영역 (그림 3D)에서 배경 잡음 측과 남성 USVs 측 사이에 유의 한 차이가 없었다. 그러나, B6 암컷 사운드 존에 상당히 긴 시간을 소비 및 백그라운드 잡음 측면, 제 5 분 (음성 구역, p = 0.018을 분석하고,도 3e에 비해 BALB 남성 USV 측에 메시를 검색하는 단계; 메쉬 , P = 0.018, 그림 3 층) 또는 총 시험 시간 (사운드 영역, P = 0.028, 그림 3E, 메쉬, P = 0.043, 그림 3 층). 결과는 명확 B6 암컷이 입증특히, 제 5 분에, 배경 잡음보다 재생 남성 USVs 접근한다. 이 두 선택 시험은 USVs의 특성을 비교하는 데 유용합니다. 이전 연구에서,이 검사는 이들 균주의 11 대 남성에서 USVs 및 B6 암컷 마우스 BALB USV의 선호도를 결정하는 데 사용되었다. B6 (133 음절 초 20 당)와 BALB 남성 (20 초 당 108 음절), 이들 균주의 각각의 암컷 마우스에서 USVs의 재생 조합을 사용하는 경우 다른 변형보다 수컷의 USVs을위한 시간을 검색 이상 보여 주었다 자신의 하나의 (그림 4). <img alt="그림 2" src="/files/ftp_upload/53074/53074fig2.jpg" /> 그림 2. 측정 된 음압 레벨 (SPL) 주파수의 함수로 NC - 네 에미 터에 의해 생성 된 약 80의 거의 일정 SPL -. 90 dB의 40과 160 kHz의 사이에 관찰된다. AC 입력 펑어이 경우 1.3 W입니다. 에미 터는 고주파 콘덴서 마이크로폰에서 20mm의 거리에 배치되고 그 중앙에 정렬되었다. 이 그림은 키하라 (19)에서 수정되었습니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53074/53074fig3.jpg" /> 그림 3. 배경 잡음 및 BALB 남성 USVs에 B6 여성 응답. (A)를 재생하기 전에 조사 기간 동안 객실 A 또는 B에 체류 전체 기간. (B) 재생 중에 방 항목의 수입니다. (C) 객실에서 숙박 총 기간. (D) 중간 영역에서 체류 전체 기간 . (E)의 사운드 존에서의 총 체류 시간. (F) 망을 검색하는 총 지속. 각각의 측정개인은 도트 플롯에 나와있다; 각 플롯의 수평 막대는 평균을 나타냅니다; N = 7; * 배경 잡음 분석 매개 변수에 대한 남성 USVs 사이에 유의 한 차이 (P &lt;0.05)를 나타냅니다; 윌 콕슨 부호 순위 테스트합니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53074/53074fig4.jpg" /> 그림 4. 여성 같은 변형 대 다른 균주로부터 남성의 USVs에 응답. B6 (N = 6)과 BALB (N = 10) 시험 전에 남성 오염 된 침구에 노출 diestrus에 여성이 보였다 더 긴 기간 검색 시간 다른 변형 USVs. 표시된 값은 평균 ± 표준 오차이다; * 유의 한 차이 (P &lt;0.05)를 나타냅니다; 윌 콕슨 부호 순위 테스트합니다. 이 수치는 아사바에서 수정되었습니다 <s&gt; 14까지. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53074/53074fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

두 선택 재생 테스트를 사용하여 마우스의 초음파 발성 환경 설정을 결정

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

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JoVE Journal

Behavior

11.1K 조회수.  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.

비디오: 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.

쥐에서 의약품 자체 관리하는 동안 초음파 Vocalizations 평가

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

연구

신경과학

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).

마우스 초음파 발성을 녹화 사회 커뮤니케이션을 평가하는

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

행동분석학

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.

세 Parkinsonian 쥐의 초음파 Vocalizations의 타겟 트레이닝

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.

수컷 마우스 초음파 발성 (USV) 노래 추출 및 분석

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.

실험실 쥐에 대한인지 심화로 리모콘 교육 소개

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.

설치류의 어쿠스틱 놀라게의 요법 이니 및 Prepulse 억제

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.

생쥐의 본능적 인 방어 하는 동안 공간 메모리의 역할을 조사에 대 한 행동 분석 결과

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.

쥐의 행동 Audiometry를위한 저비용 설정

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

수정된 공포 조건화를 이용한 생쥐의 적극적인 방어 행동 연구(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.

저자 스포트라이트: Exploring Neural Correlate of Defensive Behaviors in Fear Learning and Extinction (공포 학습과 소멸에서 방어적 행동의 신경 상관관계 탐구)

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.

생쥐의 비행 행동을 유도하기 위한 수정된 공포 조건화

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

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