The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

모든 마우스 프로토콜은 건강 지침 국립 연구소에 따라이었고, 의학의 워싱턴 대학의 동물 연구위원회 (세인트 루이스, MO)에 의해 승인되었다. 1. 테스트 판 및 인클로저 준비 <ol><li> 유리 표면을 청소합니다. </li><li> 실험실 테이프로 유리판의 중앙에 표면에 T 형 필라멘트 열전쌍 프로브를 고정. </li><li> 플레이트의 중앙을 따라 한 줄로 유리 접시에 동물 인클로저를 놓습니다. </li><li> 중앙 동물 인클로저를 통해 열전쌍 프로브를 끼 우고 데이터 로거에 연결합니다. 자동 종료 기능을 비활성화하는 동안의 데이터 로거를 켜고 제공된 케이블로 컴퓨터에 데이터 로거를 연결합니다. <ol><li> 실험 기간 동안 판의 온도를 기록하는 경우, 플레이트 온도 기록을 시작하기 위해 데이터 로거 소프트웨어를 엽니 다. </li><li> 필요하다면, 다시 소프트웨어를 조정코드 플레이트 온도 매초마다. </li><li> 열 데이터 로거와 함께 패키지 소프트웨어를 사용하여 온도를 기록 시작합니다. </li></ol></li><li> 마우스 사이의 시각적 상호 작용을 방지하기 위해 검은 삽입 인클로저를 분리합니다. </li><li> 위치는 인클로저의 밑면이 편안한 장착 위치에서 볼 수 있도록 유리 아래에 반영한다. </li></ol> 2. 온난화 / 유리 접시를 냉각 <ol><li> 가온 물에 젖은 얼음, 드라이 아이스 알루미늄 상자를 채우고 유리판 상에 적절하게 위치 (드라이 아이스로 채워진 알루미늄 호일 패킷과 같은 유리를 냉각하기 위해 사용될 수있다;도 1) 2. <ol><li> 30 ° C에서 테스트를 위해, 멀리 동물 인클로저 (그림 2B) 2에서 &#39;약 0.25 알루미늄 상자를 배치합니다. <ol><li> 유리 기판의 양측에 가열 된 물이 순환 장치를 설정한다. 45 서큘 레이터 설정 - 60 ° C가, 우리뜨거운 물 (그림 1C) 2의 꾸준히 알루미늄 상자를 채우기 위해 전자. </li><li> 서큘 레이터의 위치를 같은 알루미늄 상자에서 뜨거운 물이 다시 직접 각면 (그림 1C)에 순환의 저수지에 배수 것을 2. </li></ol></li><li> 실온에서 테스트를 위해 상자를 빈 (그림 2) (2)를 둡니다. </li><li> (그림 2) 17 ° C에서 테스트를 위해, 거리 양쪽에 동물 우리에서 &#39;약 0.25 상자를 놓고 젖은 얼음 채우기 2. </li><li> (그림 2) 12 ° C에서 테스트를 위해, 거리 양쪽에 인클로저에서 &#39;약 1.25 상자를 놓고 드라이 아이스 채우기 2. </li><li> (그림 2) 5 ° C에서 테스트를 위해 약 0.25 &#39;&#39;거리 양쪽에 인클로저에서 상자를 놓고 드라이 아이스 채우기 2.<ol><li> 드라이 아이스와 유리를 냉각 할 때, 방에 이산화탄소 발생을 방지하기 위해 통풍이 있는지 확인하십시오. </li></ol></li></ol></li><li> 원하는 온도 범위에 도달하는 유리 기다립니다. </li><li> 접시에 인클로저에 마우스를 추가합니다. 주 : 백색 잡음 발생기 잡음 교란을 감소하기 위해 사용될 수있다. </li><li> 마우스 적응 기다립니다. 참고 : 우리 시설에서이 약 2.5 시간이 필요하지만이 동물 하우징 및 취급 조건에 따라 상당히 다를 수 있습니다. </li><li> 상자를 따뜻하게 물, 젖은 얼음 또는 드라이 아이스의 전체 유지하도록하여 원하는 온도 범위에서 유리를 유지한다. 참고 : 우리의 장치와 함께 상자 얼음 대략 매 90 분 리필 할 필요가있다. 주 : 17 ° C 조건의 경우,이 얼음을 보충하기 전에 배수 구멍을 통해 알루미늄 상자에서 대부분의 물을 비우는 것이 도움이된다. 이것은 더 나은 온도, 및 PR을 안정화이벤트 오버 플로우 주 : 드라이 아이스의 정확한 양은 계절에 따라 다르지만, 일반적으로 온도를 일정하게 유지한다 상자의 전체 길이를 따라 완전 이상 박스 ¼ 유지한다. </li></ol> 고정 온도에서 3. 테스트 마우스 <ol><li> 행동 방 밖에서, 드라이 아이스의 전체 절반에 대한 얼음 양동이를 입력합니다. </li><li> 망치 또는 망치를 사용하여 미세 분말로 드라이 아이스를 분쇄. 참고 : 버킷을 너무 가득 채우면하는 것은 어려운 완전히 분말로 드라이 아이스 호감을 만들 것입니다. </li><li> 직선 면도날이나 가위를 사용하여, 3 ML의 주사기를 상단을 잘라. </li><li> 21 G 바늘을 사용하여, 주사기 (6 구멍 전체)에 대향하는 측면 (3) 구멍을 찌를. 주 : 드라이 아이스를 압축하면서이 구멍은 승화에 의해 생성 된 압력이 감소한다. 컷오프 주사기 여러 실험을 위해 재사용 될 수있다. </li><li> 주사기, 드라이 아이스 분말, 및 행동 방에 휴대용 스톱워치를 가져 가라. </li><li> 드라이 아이스 분말의 절반 전체 주사기 챔버를 입력합니다. 평면 객체와 주사기의 절단 끝을 잡고 단단히 플런저를 사용하여 분말을 압축합니다. 주의; 플라스틱 플런저는 구부리거나 압력에서 중단 될 수 있습니다. 이 경우, 새로운 주사기에서 플런저를 교체합니다. </li><li> 주사기의 가장자리를지나 압축 된 드라이 아이스 펠릿의 끝을 확장합니다. </li><li> 휴식을 충분히입니다 테스트 마우스. <ol><li> 30 ° C, 23 ° C, 17 ° C에서 4 유리에 발 및하지를 테스트 마우스는 이동하지만, 19 완전히 잠이 없습니다. </li><li> 12 ° C 5 ° C에서, 이동하거나 점프 2 발, 4 발과하지에있는 테스트 마우스. </li></ol></li><li> 부드럽게, 타겟팅을 위해 거울을 사용하여 단단히 마우스 뒷발 (그림 1A)이 아래 유리 표면에 평평 펠릿 플러시를 누릅니다. 손으로 타이머를 시작합니다. </li><li> 타이머를 중지하고 마우스가 떨어져 냉각 된 유리에서 이동할 때 펠렛을 제거합니다. 참고 : 철수 운동이 수직 또는 수평이 될 수 있습니다. <ol><li> 마우스가 매우 간략하게 발을 이동 한 후 냉각 표면에 반환하는 경우 마우스가 떨어져 영구적 인 이동을하게 될 때까지, 타이밍과 자극을 계속합니다. 참고 : 우리 연구소는 대부분의 경우에 마우스 20 초 최대 자극 시간을 사용합니다. </li></ol></li><li> 각 동물의 각 발에 3 개 이상의 값을 수집 할 때까지이 테스트 절차를 반복합니다. 적어도 15 분에 의해 단일 ​​발에 적어도 7 분, 별도의 연속 시험하여 동일한 마우스에 반대 발을 테스트 별도의 시험. </li><li> 필요한 경우, 냉각 (그림 3) (1)의 서로 다른 속도를 생성하는 유리의 다른 두께를 사용합니다. 주 : 냉각 속도가 반비례 유리의 두께와 상관된다. </li></ol> 4. 콜드 적응하는 동안 마우스를 테스트 주 :이 작은지면 유리로 테스트 할 수 대체 프로토콜전자는 오히려 판을 안정화하고 마우스가 완전히 추운 환경에 적응 한 번 이상, 냉각시킨다. <ol><li> 장치를 설정하는 제 1 절에 나와있는 지침을 따르십시오. </li><li> RT (그림 7A) 2에서 기준 측정을 수행하기 위해 제 3 절에 나와있는 지침을 따르십시오. </li><li> 드라이 아이스와 알루미늄 상자를 사전 냉각. </li><li> 기준 철수 대기 시간을 측정 한 후, 멀리 인클로저의 양쪽에 접시 약 1.25 &#39;&#39;의 미리 냉각 된 상자의 위치 (그림 7a는, 화살표가 표시된 &quot;드라이 아이스 추가&quot;) 2. </li><li> 유리 접시가 냉각됨에 따라 가능한 한 자주 측정을, 측정을 수행 할 제 3 절에 나와있는 지침을 따르십시오. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+in+the+spinal+cord+by+oxidized+linoleic+acid+metabolites+contributes+to+inflammatory+hyperalgesia.">Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18820-18824 (2009).</li><li>Fujita, F., Uchida, K., Takaishi, M., Sokabe, T., Tominaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Temperature+Affects+the+Temperature+Threshold+for+TRPM8+Activation+through+Interaction+of+Phosphatidylinositol+4,5-Bisphosphate.">Ambient Temperature Affects the Temperature Threshold for TRPM8 Activation through Interaction of Phosphatidylinositol 4,5-Bisphosphate.</a> Journal of Neuroscience. 33 (14), 6154-6159 (2013).</li><li>Rohacs, T., Lopes, C. M., Michailidis, I., Logothetis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI(4,5)P2+regulates+the+activation+and+desensitization+of+TRPM8+channels+through+the+TRP+domain.">PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain.</a> Nature neuroscience. 8 (5), 626-634 (2005).</li><li>Daniels, R. L., Takashima, Y., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+the+neuronal+cold+sensor+TRPM8+is+regulated+by+phospholipase+C+via+the+phospholipid+phosphoinositol+4,5-bisphosphate.">Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate.</a> The Journal of biological chemistry. 284 (3), 1570-1582 (2009).</li><li>Zhang, 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=Neurokinin-1+receptor+enhances+TRPV1+activity+in+primary+sensory+neurons+via+PKCepsilon:+a+novel+pathway+for+heat+hyperalgesia.">Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 27 (44), 12067-12077 (2007).</li><li>Wang, H., Zylka, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mrgprd-expressing+polymodal+nociceptive+neurons+innervate+most+known+classes+of+substantia+gelatinosa+neurons.">Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 29 (42), 13202-13209 (2009).</li></ol>

The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

설치류 냉 반응성 측정은 모두 정상 및 병리학 적 조건 하에서 인간 감기 감도의 전위 메커니즘에 대한 이해를 향상시키는 것이 중요하다. 콜드 발바닥 분석 (CPA)는 원래 전 1 몇 년이, RT에 전달 차가운 자극에 재현, 모호하지 않은 쥐의 행동 반응을 생성하도록 설계 개발했다. 이러한 분석의보다 최근의 개선이 온도의 넓은 범위에서 감기 감도 측정 재현성있게되었다. 두 버전은 상대적으로 높은 처리량, 사용이 저렴하도록 설계되어 있습니다. 진보는 많은 다른 방법을 이용하여 행동 민감성 감기 메커니즘을 이해에서했다. 하나의 방법은 dabbing 또는 마우스 발에 분사하고 아세톤 마우스 발바닥 3,4 플릭 소비하는 시간의 양을 측정하는 것을 포함 아세톤 증발 시험이다. 불행하게도,아세톤의 증발에 대한 응답은 젖은 감각과 아세톤의 냄새에 의해 혼동된다. 또한, 아세톤 증발 시험에서인가되는 냉 자극을 적용 아세톤의 양을 기준으로 정량화하기 어렵다 변할 수있다. 마지막으로, 손상되지 않은 마우스는 기준선 아세톤 최소한 응답이 불가능이 방법 과민증의 부재 진통을 측정 할 수있다. 차가운 반응에 대한 또 다른 고전적인 분석은 꼬리가 차가운 물 5,6에 침지 한 후 철수 대기 시간을 측정 꼬리 영화 분석이다. 이 분석에서 행동 반응가 명확한 및 분석은 특정 온도에 대한 반응을 측정하는 동안, 동물은 잘 설명 스트레스 유발 진통 메커니즘 (7)를 통해 차가운 응답을 변경할 수있는, 테스트 기간 동안 억제해야합니다. 일반적으로 사용되는 또 다른 도구는 행동을 측정하는 냉각 판 테스트입니다마우스의 응답은 그들이 펠티어 냉각 판 8-10에 배치 된 후. 이 도구는 특정 온도에서 동물의 응답에 대한 정보를 제공하지만, 그것은 또한 일관성없이 사용되어왔다; 상이한 그룹 점프 8,11 수, 제 13 8,11- 응답 대기 시간을 포함한 다른 종류의 반응을 측정하고, 발과의 수는 매우 다른 결과 11,13,14 리프트. 콜드 플레이트 분석은 한번에 시험 할 수 하나만 동물로 비교적 낮은 스루풋이며 비싸고 깨지기 펠티에 소자를 필요로한다. 2 판 온도 환경 시험 동물이 서로 다른 온도 9,15- (17)이 연결 판에 보내는 시간의 상대적인 양을 측정 냉각 판 시험의 일반적으로 사용되는 파생 상품이다. 또 다른 유사한 분석은 일반적으로 사용되는 열 구배 분석 생쥐으로 다른 온도 대에서 보내는 시간의 양긴 금속 접시에 5 ° C와 45 ° C 사이의 범위는 (16)을 측정한다. 이러한 분석은 온도의 비교를 할 수 있지만, 그것은 행동이 온도 회피 또는 온도 취향을 나타내는 지 여부를 명확하지 않다. 마지막으로, 냉각 판 동적 분석은 마우스 (18) 주변의 온도 변화에 어떻게 반응하는지 측정하기 위해 사용되었다. 이 방법은 RT의 펠티어 장치에 마우스를 놓고 마우스 점프 또는 다른 판의 온도에서 자신의 발을 핥고 얼마나 측정하면서 1 ° C로 낮아진다 포함한다. 이 마우스는 냉각 환경에 적응하는 방법을 테스트하는 동안, 마우스는 냉각기 주위 온도의 환경에서 냉 자극에 반응하는 방법을 테스트하기위한 방법을 제공하지 않는다. 또한, 그것을 수행하기 위해 고가의 장비를 필요로하며, 그 감기 감도를 측정하기 전에 테스트 장비에 쥐를 적응 방법을 제공하지 않는다. 이러한 분석을 보완하기 위해, CPA는 ACCLIMA을 테스트다양한 온도 범위에서 잘 정의 냉 자극에 응답 테드 또는 차가운 주변 온도에 적응하는 과정. 그것은 저렴한 비용으로 높은 처리량 테스트를 위해 확장 할 수있는 가능성과 함께, 우리의 현재 장치에 한 번에 14 마우스까지 테스트 할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

30 ° C, 23 ° C, 17 ° C, 12 ° C에서 시작 마우스에서 유발 행동 반응이 높은 재현성 (그림 4A) (20)이다. 뒷발에 따라 발생되는 냉 자극을 측정하기 위하여, 마우스를 케타민 / 자일 라진 / 아세 프로 마진 칵테일로 마취하고 그의 발은 T 형 필라멘트 열전대 (도 4b) (20)의 상부에 유리에 고정 하였다. 유리 냉각하거나 원하는 테스트 범위로 가온 하였다. 판은 판 <str...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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14.6K 조회수.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

비디오: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

베르 미 첼리와 Capellini 처리 시험 : 쥐 및 생쥐의 손재주가 앞발 기능의 단순 양적 측정

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

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A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

파리에서 에탄올 민감도를 측정하는 간단한 방법

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

의 장기 강한 적응의 분자 판독<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

예 자궁 Electroporation 및 전체 반구의 Explants : 초기 두피 개발 연구 간단한 실험 방법

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

에탄올 진정 감도 및 신속한 공차 측정을위한 저렴하고 확장 가능한 행동 분석<em&gt; 초파리</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

Method는 목표와 마우스의 격리기도 - 근에 분포하는 감각 뉴런하기

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

콜드 통각에 대한 소설 분석<em&gt; 초파리</em&gt; 애벌레

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivo에서경작-Murine 이다 빠르고, 간단 하 고 저렴 한 AWESAM 프로토콜을 사용 하 여 같은

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...