이 작품은 근육질 영양 장애 협회, NIH (NS062320)에 의해 지원되었다.

1. muscarinic 아세틸콜린 수용체 (mAChR) antagonists의 피하 관리 <ol><li> 1.5mL 반응 관에 멸균 생리 식염수에 마약을 분해하여 무균 조건 mAChR 길항제의 적절한 복용량 (참조하라, 테이블)에서 준비합니다. 다음 antagonists 사용되었습니다 아트로 핀, Methoctramine, 4 축축한, AFDX - 116, AFDX - 384, MT 7. </li><li> a1cc 인슐린 주사기로 솔루션의 50μl를 그려 각 마우스에 대해 별도의 주사기를 사용합니다. 또한 전용 컨트롤 그룹에있는 각 마우스에 대한 생리 식염수가 들어있는 주사기가 준비합니다. 주입 준비까지 얼음에 주사기 보관하십시오. </li><li> 핀치를 발끝에 응답의 부족을 관찰하여 적절한 마취 심도를 확인 케타민을 - xylazine 칵테일 (120 MG / kg 마취제, 8 MG / kg xylazine)와 생쥐를 마취. </li><li> 적절한 진정제를 맞았 후, 목 꼬리 영역에 머리의 주동이의 영역을 다루는 면도를 70 % 에탄올로 떨어져 머리를 나머지 닦으십시오. </li><li> 플레이스 stereomicroscope 아래의 마우스, 그리고 주사기 (그림 1A, B)의 바늘에 가볍게 위로 당기면서 랄 근육에 주사 바늘 평행를 삽입합니다. 이것은 바늘이 결합 조직을 overlying에 삽입하고 직접 랄 근육 자체를 손상하지 않는 것을 보장합니다. 팁이 랄 근육의 꼬리 부분을 커버 있도록 바늘을 삽입합니다. </li><li> 아주 천천히 주사기를 철수하면서 오른쪽 랄 근육 이상의 솔루션을 주입하기 시작하며, 총 주입 시간은 약 일분해야합니다. 제대로 주입하면, 솔루션은 하나 이상의 시간 동안 유지됩니다 overlying 피부에 직경 약 1cm입니다 &quot;돔&quot;을 형성한다. 균일하게 주입 솔루션은 바로 랄 근육의 전체 주동이의 꼬리와 지역을 커버합니다. </li><li> 1~7일에 대한 12 시간마다 절차를 반복합니다. 기타 studies1 들어, 우리는 최대 21일하는 성장 요인에게 매 12 시간을 삽입하기 위해 동일한 프로토콜을 사용합니다. 더 이상 응답은 최대 21일하는 마취의 2X 하루 투여 마우스에서 관찰되지 않았습니다. </li></ol> 2. 랄 근육의 해부 <ol><li> pentobarbital 나트륨의 과다 (390 MG / kg)와 쥐를 안락사. 죽음은 thoracotomy를 수행한 후에는 심장 박동의 부족을 평가하여 보증합니다. </li><li> 각 포 1 핀과 코로 1 핀, 해부 접시에 등의 측면을 마우스를 아래로 핀. </li><li> 왼쪽 견갑골 주변 지역에 귀를 단 근위 지역에서 왼쪽 랄을 따라 잘라 작은 봄 가위를 사용하여, 오직 피부를 통해 첫 절개를합니다. </li><li> 조심스럽게이 지역에있는 피부를 제거하지만, 이것이 바로 랄 근육에 대한 첨부 파일의 포인트 중 하나이기 때문입니다 오른쪽 귀에 너무 가까이 절단하지 마십시오. </li><li> 좌우 랄 근육이 만나는 머리 표면 표면에 정중선을 따라 지질의 중앙 위치에 지방 조직의 스트립을, 찾습니다. 작은 스프링 가위를 사용하여 어깨 (그림 1C)를 도달하기 전까지 왼쪽 랄 근육의 근위 가장자리에서 지방 조직 (왼쪽 귀 방향)의 오른쪽에 1cm 했네요. </li><li> 일단 절개는 근육의 복부 측면을 폭로 작은 집게로 다시 오른쪽 랄 근육 껍질, 이루어집니다. 포셉 (그림 1C)와 오른쪽 랄 근육을 신중하게 뽑고하면서 결합 조직과 근막을 낸다. </li><li> (内耳)의 기지에서 오른쪽 랄의 꼬리 끝에 주위 컷. , 절삭 오른쪽 귀의 주동이의 끝을 향해 움직이고, 오른쪽 랄은 (그림 1C) 인계 유지를 계속합니다.. 그것은 자체 랄을 망가뜨릴 이상이 단계에서 더 많은 조직을 포함하는 것이 좋습니다. 근육은 근육을 통해 피펫 1X PBS를 건조하기 시작하면. </li><li> 장소가 1X PBS, 등의 측면을 포함 30mm 문화 Sylgard 요리의 오른쪽 랄 해부 했어요. 작은 곤충 핀과 근육의 네 귀퉁이를 핀. </li><li> 오른쪽 랄 근육의 지느러미와 복부 표면에서 작은 집게와 봄 가위, 깨끗한 결합 조직을 사용합니다. 복부 표면을 청소하기 위해 근육을 뒤집어 다시 핀. 2-3 근육 같은 30mm 접시에 게재될 수 있습니다. </li></ol> 3. 랄 NMJs의 트리플 immunostaining : 1 일 <ol><li> 를 시작 immunostaining 전에 모든 필요한 시약을 준비합니다. 1X PBS, 2% 소 혈청 알부민 BSA) / PBS, 2 %에 0.1M 글리신 BSA / PBS, 2퍼센트 BSA / PBS 및 1X PBS에 4 % paraformaldehyde (PFA)의 0.2 % 트리톤의 X100이 필요합니다. -20 ° C의 냉장고에 미리 냉각 메탄올. </li><li> 이 과정에서 접시 등의 측면에 고정된 랄 근육을 둡니다. 1X PBS 솔루션을 무시하고 근육을 감추기에 충분 할만큼 4퍼센트 (PFA)를 추가합니다. 쉐이크에 플레이스 요리 20 분 동안 속도를 2 또는 3으로 설정합니다. 별도의 언급이없는 한, 접시는 다음 단계를 흔드는에 배치해야합니다. </li><li> 4퍼센트 PFA를 버리고 1X PBS 3 회, 10 &#39;각 근육을 씻으십시오. </li><li> 마지막 1X PBS 30 &#39;을위한 2 %의 BSA / PBS 용액을 0.1M 글리신을 씻고 추가 삭제. </li><li> 0.1M 글리신 솔루션을 삭제하시겠습니까에게D 추가 1X PBS 15 &#39;에 대한 1:200의 농도에 rhodamine 복합 α - bungarotoxin를 포함. </li><li> α - bungarotoxin 솔루션을 버리고 1X PBS, 3 번 10 &#39;각각의 근육을 씻으십시오. </li><li> 사전 냉각 메탄올을 추가하여 조직을 Permeabilize하고 정확하게 5 분 -20 ° C의 냉장고에 음식을 배치, 아무 필요 떨고 없습니다. </li><li> 메탄올을 제거하고 3 회 10 &#39;1X PBS로 각각 씻으십시오. 지난 세척하고 차단 조직을 폐기 0.2 % 트리톤 X - 100 2퍼센트에 BSA / PBS 1 시간. 세탁과 차단 단계는 실온에서 수행됩니다. </li><li> 4, 흔들림, 밤새 근육을 품어 ° 차단 솔루션 (참조하라, 표)에 희석 기본 항체의 칵테일 C. </li></ol> 4. 랄 NMJs의 트리플 immunostaining : 날 2 <ol><li> 1X PBS 3 회, 10 &#39;상온에서 각 근육을 씻으십시오. </li><li> 실온에서 1 시간 (참조하라, 테이블)에 대한 솔루션을 차단에서 이차 항체의 칵테일을 추가합니다. 알렉사 - 플루어 647 형광의 사진 표백을 방지하기 위해 근육이 단계 이후의 빛으로부터 보호되어야합니다. </li><li> 1X PBS에 3 번, 10 &#39;각 근육을 씻으십시오. </li><li> 1X PBS에 남아있는 결합 조직을 청소, 랄 근육의 측면 가장자리를 잘라 슬라이드에있는 특정 측면을 탑재, 유리 커버 - 풀렸는데 장착 미디어를 사용합니다. 장소에 커버 슬립을 확보하기 위해 명확한 매니큐어를 사용하십시오. </li><li> 분석을위한 준비까지 -20 ° C에서 빛과 가게에서 슬라이드를 보호할 수 있습니다. </li></ol> 5. 랄 NMJs의 공촛점 이미징 <ol><li> 고해상도 공촛점 이미지는 Leica TCS 4D 공촛점 현미경에 Leica 계획 APO 63X 오일 목적 (1.4NA)를 얻을 수 있습니다. </li><li> 플루오레신와 알렉사 647 신호를 동시에 각각의 레이저 라인 488 NM (아르곤, 488 NM, 20 MW)와 633 nm의 (HeNe, 633 NM, 10 MW)로 스캔했다. 순차적으로, Rhodamine 신호는 다음 레이저 라인 561 NM (DPSS, 561, 20 MW)를 스캔합니다. 광학 섹션은 0.3 μm의 간격으로 수집하고, Z - 비행기에서 이미지의 숫자는 60-80에서 변화했다. 핀홀은 에어리 1 (107.97 음)으로 조정됩니다. 이미지 포맷 1,024 X 1,024 (X, Y), 234.32 μm의 X 234.32 μm의 및 229.05 NM의 픽셀 크기를 X 229.05 NM의 이미지 크기 400Hz 속도 결과 1의 줌 계수와. </li><li> Leica TCS - NT 수집 소프트웨어는 다음 최대 강도 예측에 Z - 시리즈 이미지를 재구성하는 데 사용됩니다. </li><li> Multipanel 이미지는 어도비 포토샵을 사용하여 밝기 및 대비를 위해 조정됩니다. </li><li> 전형적인 랄 근육 들어, 하나는 약 75-100 EN의 얼굴 NMJs를 분석할 수 있어야합니다. 시냅스 안정성 등 사전, 페리 및 postsynaptic 요소들의 공간적 배열 (즉, 신경, Schwann 세포와 근육)과 같은 기능 및 신경 영역의 측정 (예 : 버렸네 크기)에 의해 결정됩니다. </li></ol> 대표 결과 : 정확하게 nicotinic AChRs의 postsynaptic 클러스터 (; 레드 그림 2) apposed 하나의 근육 섬유에, 어른 포유류 NMJ에서 하나의 모터 축삭은 신경 단말기의 매우 차별 아보스 (그린 그림 2) 양식 멋진 가지를 elaborates . Perisynaptically 터미널 Schwann 세포가 밀접하게 presynaptic 신경 터미널의 모든 지점 (; 블루 그림 2) 커버. 이 삼자 간의 조직의 구조와 기능 무결성이 심각하게 subtype 특정 mAChR 억제제의 일상 응용 프로그램에 의해 불안정하게된다 는걸 알수 있습니다. 여기에 제시된 예제 (그림 3)에서 4 축축한, M1, M3, M4와 M5 mAChR의 subtypes에 대한 높은 친화력을 가진 mAChR 길항제는 (그림 3B, 근육의 표면에 걸쳐 많은 NMJs에서 신경 단말기의 선택 제거를 끝 C). 처리 확장하지 않고 밝은 S100 라벨 (그림 3B &quot;3C&quot;)에 의해 입증로서뿐만 아니라, 터미널 Schwann 세포는 8 비정상적으로 정지됩니다. Postsynaptically, 근육 섬유는 정상이며 nAChRs에 대한 손실 (그림 3B &#39;, 3C&#39;는) 없습니다. <img src="/files/ftp_upload/3124/3124fig1.jpg" alt="그림 1"/> 그림 1. 랄 근육의 해부 학적 위치 및 조직입니다. 주동이의 (rLAL), 꼬리 (cLAL) 및 랄 신경 (LALn) 및 해당 endplate 밴드의 위치 (A, 삽입된 페이지) 표시됩니다. 랄 근육에 대하여에있는 바늘의 위치와 방향은 사출 절차 (B)에 표시됩니다. 절개 포인트는 절개 절차 (C)에 대해 표시됩니다. <img src="/files/ftp_upload/3124/3124fig2.jpg" alt="그림 2"/> 그림 2. NMJ의 삼자 간의 조직입니다. 마우스 NMJ의 높은 배율 공촛점 전망. 하나의 축삭은 단단히 터미널 Schwann 세포와 그 프로세스 (터미널 Schwann 세포 기관을 &#39;별표 점)이 적용되는 고급 터미널 지점 (A), elaborates. Postsynaptically 근육 섬유에 nicotinic AChRs의 클러스터가 정확하게 신경 termi의 지점에 apposed입니다nals 및 단말 Schwann 세포. <img src="/files/ftp_upload/3124/3124fig3.jpg" alt="그림 3"/> 그림 3. 랄 근육이 4 습기, mAChR 길항제로 치료. 랄 근육의 낮은 높은 배율 공촛점 전망 차량 또는 4 - 습기 취급. 차량 취급 근육 (4 습기 - 대우 근육 부족 신경 터미널 (의 A - &#39;&#39;&#39;), 수많은 NMJs 대조적으로 B - B &#39;&#39;C - C &#39;&#39;&#39;). 그림 2B의 박스 영역을 그림 2C에 확대한 것입니다.

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+cranial+muscles+to+investigate+neuromuscular+pathology+in+vivo.">Using mouse cranial muscles to investigate neuromuscular pathology in vivo.</a> Neuromuscul Disord. 20, 740-743 (2009).</li><li>Dorje, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonist+binding+profiles+of+five+cloned+human+muscarinic+receptor+subtypes.">Antagonist binding profiles of five cloned human muscarinic receptor subtypes.</a> J Pharmacol Exp Ther. 256, 727-733 (1991).</li><li>Caulfield, M. P., Birdsall, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+XVII.+Classification+of+muscarinic+acetylcholine+receptors.">International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.</a> Pharmacol Rev. 50, 279-290 (1998).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 제시하는 방법은 포유 동물 NMJs의 안정성과 유지 보수에 신호 subtype 특정 mAChR의 이전에 인식되지 않는 역할의 조사를 허용합니다. 이 방법은 또한 neurotrophic 요인과 약리 대리인의 효과를 테스트하기 위해 도움이 될 것입니다. 예를 들어, 저희 연구실은 Ciliary Neurotrophic 팩터 (CNTF)는 성인 생쥐의 거의 모든 랄 신경 터미널에서 돋아 elicited 것으로 나타났습니다 <a>1</a> . 이 결과는 CA에서 돋아 ...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 마취제 </td><td> Hospira </td><td> NDC0409 - 2051-05 </td><td> 복용량 : 120mg/kg </td></tr><tr><td> xylazine </td><td> 로이드 레버 토리스 </td><td> LA33806 </td><td> 복용량 : 8mg/kg </td></tr><tr><td> 아트로 핀 </td><td> 시그마 - 알드리치 </td><td> A0132 </td><td> (&gt; 98 % 순도); 복용 : 0.2mg/kg - 20mg/kg </td></tr><tr><td> 아트로 핀 </td><td> 보이트 글로벌 유통 </td><td> AT105 </td><td> 제약 학년 </td></tr><tr><td> Methoctramine </td><td> 시그마 - 알드리치 </td><td> M105 </td><td> 복용량 : 100 - 400M </td></tr><tr><td> 4 - 습기 </td><td> 시그마 - 알드리치 </td><td> D142 </td><td> 복용량 : 2.5mg/kg </td></tr><tr><td> AFDX - 116 </td><td> Bioscience Tocris </td><td> 1105 </td><td> 250M </td></tr><tr><td> AFDX - 384 </td><td> Bioscience Tocris </td><td> 1345 </td><td> 50M - 500 미터 </td></tr><tr><td> MT 7 </td><td> 펩티드 국제 </td><td> PMT - 4340 - S </td><td> 0.1M - 1M </td></tr><tr><td> 1X 인산은 살린, 산도 7.4 버퍼 </td><td> Invitrogen </td><td> 10010049 </td><td></td></tr><tr><td> Paraformaldehyde </td><td> 어부 </td><td> T353 - 500 </td><td> 10g/100mL 드 이온화 증류수를 용해하여 처음 10 %의 솔루션을 만들어, 1X PBS로 4 % 수 있도록, 7.4으로 산도를 조절 </td></tr><tr><td> pentobarbitol 나트륨 </td><td> Virbac 동물 보건 </td><td> NDC - 051311 - 050-01 </td><td> 복용량 : 390mg/kg </td></tr><tr><td> Sylgard </td><td> 다우 코닝 </td><td> 부품 # 184 </td><td> 필요에 따라 (30mm, 60mm, 100mm) 크기의 여러 문화 요리를 사용할 수 키트와 함께 지시 사항을 따르십시오 </td></tr><tr><td> 0.1M 글리신 </td><td> 시그마 - 알드리치 </td><td> G - 7126 </td><td> 추가 2% BSA / PBS의 0.185g을 25mL </td></tr><tr><td> 2퍼센트 소 혈청 알부민 (2 % BSA) </td><td> 시그마 - 알드리치 </td><td> A3059 - 100g </td><td> 1X PBS의 100mL에 2g BSA를 디졸브 </td></tr><tr><td> 2퍼센트에서 0.2 % 트리톤 X100 BSA / PBS (차단 버퍼) </td><td> 시그마 - 알드리치 </td><td> T9284 - 100mL </td><td> 0.2ml/100mL 2% BSA / PBS를 디졸브 </td></tr><tr><td> α - bungarotoxin </td><td> Invitrogen </td><td> T1175 </td><td> 1:200의 농도에서 사용 </td></tr><tr><td> SMI - 312 </td><td> Sternberger의 Monoclonals </td><td> SMI312 </td><td> 1:1000의 농도에서 사용 </td></tr><tr><td> SV2 </td><td> 발달 연구 브리 도마 뱅크 </td><td> SV2 - 뜨는 </td><td> 1시 10분의 농도에서 사용 </td></tr><tr><td> S100 </td><td> Dako </td><td> Z0311 </td><td> 1:400의 농도에서 사용 </td></tr><tr><td> FITC - 염소 방지 마우스 IgG1 </td><td> 로체 </td><td> 03117731001 </td><td> 1:200의 농도에서 사용하지만, 배경이 높으면, 1:400을 시도 </td></tr><tr><td> 알렉사 - 플루어 647 복합 염소 안티 - 토끼 </td><td> Invitrogen </td><td> A21244 </td><td> 1:200의 농도에서 사용 </td></tr><tr><td> Vectashield 형광 설치 미디어 </td><td> 벡터 실험실 </td><td> H - 1000 </td><td> 이것은 하드 세트 미디어 아니라, 당신이 투명 매니큐어로 커버 슬립을 확보해야합니다. </td></tr><tr><td> 소형 스프링 가위 </td><td> 파인 과학 도구 </td><td> 15002-08 </td><td></td></tr><tr><td> 해부의 포셉 </td><td> 파인 과학 도구 </td><td> 11295-51 </td><td></td></tr></tbody></table>

subcutaneous administration of muscarinic antagonists and triple-immunostaining of the levator auris longus muscle in mice

이러한 gastrocnemius 및 정강뼈 앞쪽에 같은 설치류 동물, 뒷다리의 사지 근육은 자주 포유 동물 NMJs의 형성 및 유지에 필수적인 신호의 생체내 약리 연구에 사용됩니다. 그러나, 피하 또는 근육 관리 후이 근육에 약물 침투가 종종 불완전하거나 고르지이며 많은 NMJs는 영향을받지 남아있을 수 있습니다. 이러한 미니 펌프와 같은 장치와 함께 체계적인 관리가 spatiotemporal 효과를 향상시킬 수 있지만,이 방법의 침략 자연 혼란함을 주죠 염증 반응 및 / 또는 직접 근육 손상을 일으킬 수 있습니다. 그것이 시간이 많이 걸리는 시리얼 sectioning 및 광범위한 immunostaining을 필요로하기 때문에 또한 뒷다리 근육에 NMJs 완벽하게 분석 도전이다. 마우스 랄은 목 dorsum에 피상적으로있는 근육의 얇은 평면 시트입니다. 이 기능은 날개를 이동하는 빠른 트위치 근육이다. 이것은 두개골의 정중선에서 발생한 각 날개의 연골 부분에 옆으로 확장 주동이의와 꼬리 부분을 포함하고 있습니다. 근육은 그것이 종료 stylomastoid 구멍을 같은 caudally 프로젝트 안면 신경의 한 분과로 제공됩니다. 우리와 다른 NMJs 및 근육에 약물의 생체내 효과의 단기 및 장기 모두의 조사에 대한 이점을 제공하는 편리 준비하는 랄 발견했다. 첫째, 그 표면 위치는 가벼운 마취 약품의 여러 지역의 애플 리케이션을 용이하게합니다. 둘째, 그 두께 (근육 섬유로부터 2-3 층)은 근육 내의 거의 모든 NMJs의 시각화 및 분석을 수 있습니다. 셋째, 그 innervation의 패턴과 함께 자사의 신경 손상으로 해부의 용이성은 체외 9,5에 보충 electrophysiological 분석이 이루어질 수 있습니다. 아마도 마지막으로, 그리고 가장 중요한, 작은 볼륨 적용 (~ 50μl)는 쉽게 전체 근육의 표면을 커버 약물에 모든 NMJs의 유니폼 및 장기 노출을 제공하고 체계적인 접근 1,8의 필요성을 없애줍니다.

Watch this Scientific Journal Video about Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice at JoVE.com

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

우리는 levator auris의 longus (랄) 젊은 성인 마우스의 근육을 muscarinic 신호의 억제제의 반복 관리와 wholemounts 년에 신경근육학 분기점의 후속 immunostaining (NMJs)에 대한 절차를 설명합니다. 랄 근육이 공개에 대해 고유한 장점이 있습니다<em&gt; 생체내에</emNMJs에&gt; 약리 효과.

피하 Muscarinic Antagonists의 관리 및 마우스에 Levator Auris의 Longus 근육의 트리플 - Immunostaining

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

subcutaneous-administration-muscarinic-antagonists-triple

Research

JoVE Journal

Neuroscience

13.6K 조회수.  Arcadia University. 우리는 levator auris의 longus (랄) 젊은 성인 마우스의 근육을 muscarinic 신호의 억제제의 반복 관리와 wholemounts 년에 신경근육학 분기점의 후속 immunostaining (NMJs)에 대한 절차를 설명합니다. 랄 근육이 공개에 대해 고유한 장점이 있습니다 생체내에 약리 효과.

비디오: Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

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

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

만성 니코틴 관리와 마우스 노크 인의 Fluorescently 태그를 Nicotinic 수용체의 분광 공촛점 이미징

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

가스 크로마토 그래피에 의한 operant 에탄올 자기 관리 및 에탄올 결정시 에탄올의 Microdialysis

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

게 사지에서 고유 감각과 긴장 수용체 : 학생 실험실 연습

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

성인 마우스의 중간 뇌 도파민 뉴런의 수의 환경 변조

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

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.

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 ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

비디오 이미징 및 시공간지도는 마우스에서 위장관 운동성을 분석하는

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

관리의 실시간 fMRI Neurofeedback 훈련에 대 한 프로토콜

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus 포유류 신경 근육 학 전송 전압 클램프 조건에서의 시험을 위한 준비

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...