이 작품은 SFB (604), DFG MO 1421/2-1 및 Krebshilfe 107089 (HM까지)에 의해 지원되었다. 아이들의 종양 재단 (뉴욕, 미국)에서 젊은 연구자 상을받는 사람이 때문이다.

본 연구는 독일 연방 공화국 (Tierschutzgesetz 데르 Bundesrepublik Deutschland의)의 동물 보호법에 따라 수행하고, 식품 안전과 소비자 보호 (링거 Landesamt FÜR Lebensmittelsicherheit 싶게 Verbraucherschutz)의 튀 링겐 주 사무소에 의해 승인되었습니다. 1. 측정 설정 <ol><li> 이소 플루 란 / O 2 흡입 쥐를 마취 - 마취 3 %의 개시를 위해, 100 % 산소의 유지 보수 2 % 이소 플루 란 (그림 1)를 참조하십시오. 이러한 운동 반사와 낮은 수준의 통증에 대한 민감도 테스트와 같은 간단한 반사 신경을 테스트하여 충분한 마취를 확인합니다. 절차는 일반적으로 총 약 30 분 / 동물 걸리기 때문에 마취 동안 건조를 방지하기 위해 눈에 연고의 사용을 권장하지만 필수되지 않습니다. 생존 실험의 경우, 수술 후 통증 관리를 위해 적시 이상 작용하는 진통제를 투여. </li><li> 전기 면도기로 두 다리를 덮고있는 털을 면도하고 동물이 마취에서 이미 동안 시중에서 판매하는 제모 크림으로 제모를 수행합니다. 절차를 통해 무균 상태를 유지하기 위해, 무균 장갑을 착용하고 항상 조심스럽게 70 % 에탄올로 세척 장비를 사용합니다. </li><li> 피드백 제어 가열 플레이트와 직장 열 프로브 (그림 2)에 의한 제어 체온 안정성. 필요한 경우, 멸균 실험 환경을 유지하기 위해 동물들 사이 가열판을 덮 멸균 드레이프 사용해주세요. 더욱이, 전자적으로 조직 손상을 방지하기 위해 40 ° C ≤를 위해 가열 온도를 제한하는 통합 된 센서를 통해 제어되는 가열판을 사용하는 것이 추천된다. <img alt="그림 1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> 그림 1. 전문가에게면도 뒷다리로 마취 된 쥐를 보여주는 erimental 설치. </li><li> (ECG) 레코딩 중요한 매개 변수로 심장 박동을 모니터링하는 심전도를 가져 가라. 목 부분의 피부 아래의 각 앞다리와 하나의 전극의 피부 아래에 하나의 전극을 다음과 같이 ECG 녹음을 세 전극을 설치합니다. </li><li> 최적의 전도도 / 전사 저항 접촉 겔을 사용하는 장소 링 전극. (블랙 표시)에 감지 전극은 비복근이 최대 직경을 갖는 위치에 배치된다. (빨간색으로 표시됨) 기준 전극은 단지 감지 전극 아래에 배치된다. 참고 : 장비에 대한 자세한 내용은 &#39;자료 표&#39;를 참조하십시오. </li><li> 근위 및 원위 좌골 신경의 자극과 리드 전극 사이에 미리 정해진하지만 일관된 거리와 측정의 정확한 위치를 표시합니다. 실험 계획안 : 감지 전극 내지 4 ㎜의 거리에서, 원위 자극이 열린다.감지 전극에 16mm의 거리에서, 인접한 자극 (도 2)을 실시한다. </li></ol><img alt="그림 2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> 도 2. 직전 측정의 시작 실험 상황을 도시 대표 화면. 흰색 화살표는 감지 (블랙) 및 참조 (레드) 왼쪽 뒷다리의 비복근에서 전극의 위치를 나타낸다. 니들 전극에 의해 자극 검정 감지 전극과 관련하여 정의 된 위치에서 수행 될 것이다. 말초 자극의 포인트는 (왼쪽 뒷다리에서 &quot;DS&quot;블랙 마크) 감지 전극에서 4mm의 거리가; 근위 자극 ( &quot;PS&quot;블랙 마크)의 장소는 거리 16mm입니다. 오른쪽 뒷다리에 빨간색 라인은 약을 보여줍니다<html> 좌골 신경의 해부학 적 과정으로 iMate. 또한, 관련 뒷다리 근육의 거친 위치는 관광 명소로 표시됩니다. 별표는 직장 온도 프로브를 나타냅니다. 2. 측정 <ol><li> 원리 : 모노 일회용 28 G 바늘 전극 0.1 밀리 기간 반복적으로 생성 된 하나의 구형파 펄스와 신경 자극의 일련의 수행 (반복 속도 200 밀리 초를, 그림 3A 참조). 오프라인 데이터 분석을 위해, 그것은 동시에 인한 신경 자극에 신경근 함수 응답 곡선 (도 3b)와 함께 자극 신호를 획득하도록 권장한다. 후속 데이터 분석을위한 최대 응답의 시리즈 ( &quot;대표 신경근 함수 응답을&quot;) 보통. 신뢰할 수있는 데이터 기록 및 자극 사이트 및 동물 당 저장 보통​​ 적어도 세 독립적 최적 반응 곡선을 생성하기 위해. p_upload/51181/51181fig3highres.jpg &quot;SRC =&quot;/ files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; 데이터 수집 및 분석 (개략적으로 표현) 그림 3. 절차는. 반복적으로 생성 된 펄스는 바늘 전극 (그림 3a에 상단 행)를 통해 좌골 신경에 적용됩니다. 동시에 여러 대응하는 신경 근육 학 반응 곡선은 (그림 3A 낮은 행)가 기록되어있다. 신호 응답의 지연뿐만 아니라, 재생 시간과 신호의 진폭을 표시 할 수있다 : 평균 및 확대하면 인해 자극 펄스 (도 3b에 위쪽 열)에 신경근 반응 곡선은 다음과 같은 특징적인 특성 (도 3b에 아래쪽 열)를 보여주는 이후의 계산 및 통계를 얻을 수. 불규칙한 신호 전도 및 / 또는 차선의 기록은 보통 하나의 긍정적와 음수 편향 또는 신호 변형보다 더 다양한 신호 변형을 초래할감소 된 진폭 (그림 3C)와 (바이 모달 모양). </li><li> 결정된 위치에 바늘 전극 (그림 2의 &quot;PS&quot;)와 근위 자극을 수행합니다. <ol><li> 최대 진폭 최상의 촬영 조건을 달성하기 위해, 자극 프로세스 동시 실제 응답 곡선을 시각화. 이렇게함으로써, 실험자 즉시 반응 곡선의 형상뿐만 아니라 진폭의 크기를 평가할 수있다. </li><li> 필요하다면, 약간 조심스럽게 위치 및 / 또는 좌골 신경에 대한 자극 바늘의 각도를 조작. 자극 조건이 부드러운 최적화는 최대 값과 전형적인 바이 페이 모양의 응답 곡선 (그림 4)와 일정한 진폭에 도달 할 수 있습니다. </li></ol></li><li> 결정된 위치 (그림 2에서 &quot;DS&quot;)에서 바늘 전극과 말초 자극을 수행합니다. </li><li> completio 후이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 N 측정, 별도의 케이지에 테스트 마우스를 전송합니다. 완전히 마취에서 회복 될 때까지 무인과 다른 동물의 회사에서 동물을 방치하지 마십시오. 수술 후 통증 관리를위한 적절한 행동을 더 이상 진통제를 관리 할 수​​ 있습니다. 비 스테로이드 성 항 염증성 약물 (NSAIDs)와 아편의 전신 투여는 1-3 일을 권장합니다. </li><li> 또한, 목 탈구에 의해 예를 들어 동물에 대한 더 이상의 고통없이 신속하고 인도적인 방식으로 여전히 마취 마우스를 희생. </li></ol><img alt="그림 4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> 그림 4. 최대 진폭 CMAP 녹음을 결정하는 그림. 전체 등록 시리즈가 제공됩니다. ( </st최소한의 CMAP의 응답 룽&gt;) 삽입 포인트. 최대 진폭 CMAP 녹화에서 (B) 약간의 자극 바늘 운동의 결과. (C) 바늘 위치의 추가 변경은 거의 최대 진폭 등 다양한 진폭 CMAP 녹음을 생산하고 있습니다. 가까운 최대 진폭의 시리얼 CMAP 녹화와 (d)에 자극 바늘 교체. 참고 : CMAP 진폭의 일반적인 감소는 최적의 자극 사이트 (12, 13)에 반복적 인 자극하는 동안 발생할 수 있습니다. 별표는 평균에 묘사 된 최대 진폭과 CMAP 녹음을 나타냅니다. 3. 분석 <ol><li> 적절한 소프트웨어 패키지 (예 AtisaPro)를 사용하여 대표적인 신경 근육 기능 응답 데이터 세트에 기초하여 신경 전도 파라미터를 추출한다. 참고 : 특별한주의 시간 관련 데이터의 결정을 처리하십시오 때문에 화합물 모터 동작 냄비의 변곡점 결정ential (CMAP) 개시 및 종료가 어려울 수 있습니다. 확인 재현성 절차 개시 후 및 종료하기 전에 신호 편향에 접선을 사용하는 그림 (b)에서 제공됩니다. </li><li> &#39;신호 대기&#39;와 &#39;CMAP&#39;값을 계산합니다. <ol><li> CMAP 기간 &#39;대기 시간&#39;초기 부정적인 편향의 CAMP 발병 사이의 시간 간격이 기준이 호출로 돌아 최초 반면, 자극과 CMAP의 발병 사이의 시간 지연을 나타내는 &#39;. 원위 및 근위 자극 위치 사이의 거리와 함께 전도 속도를 계산하는 근위 지연 사이의 차이를 사용한다. 자극 사이트 간의 전도 속도 = 지연 시간 / 거리 </li><li> CMAP &#39;(복합 운동 활동 전위) 진폭 (MV에 주어진) CMAP 신호의 최대 양 및 음의 턴어라운드 시점 사이의 크기를 나타냅니다. CMAP = 값 &#39;긍정적 인 턴어라운드 시점&#39;- 값 &#39;부정적인 소요 포INT &#39; </li></ol></li></ol>

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저자가 공개하는 게 없다.

설명 프로토콜은 관심의 신경을 노출 할 필요없이 마취 된 쥐의 좌골 신경의 전도 특성을 측정 할 수있는 쉽고 신뢰할 수있는 방법을 제공합니다. 그럼에도 불구하고,이 실험 과정은 바늘 구멍에 의해 조직 손상의 원인이됩니다. 따라서 녹음을 마친 후 동물을 희생 할 수있는 합리적인 선택이 될 것입니다. 그러나, 이전에 녹음 신경의 노출을 필요로하는 다른 추가 침습 절차에 비해 조직 손상은 <...

전기 생리학 측정은 임상 및 실험실 환경 모두에서 말초 신경의 기능 무결성을 조사를위한 필수 도구입니다. 인간에서는, 신경근 장애 및 신경 병증의 다수 진단 학적 측정 전기 생리에 의존한다. 전도 속도 또는 신호의 전위 진폭 같이 신경 특성을 측정함으로써, 말초 신경 질환의 거친 원점을 특성화 할 수있다. 신경 전도 속도는 수초에 의해 활성화 빠른 신호 전달에 매우 의존적이다. 따라서 탈수 초성 프로세스는 일반적으로 전도 속도 4 감소합니다. 복합 운동 활동 전위 (CMAP) - 기능 축삭의 수와의 상관 관계는 - 크게 5 감소 축삭 손상에 대한 지표이다. 따라서, 전기 생리 방법에 의한 말초 신경 손상의 병인이러한 유전성 신경 병증 6,7, 당뇨병 성 신경 병증 8,9, 만성 염증성 탈수 초성 다발성 신경 병증 (CIDP) 10, 또는 신진 대사 신경 병증 11로 구별 할 수 있습니다. 일반적으로 인간의 응용 프로그램에서 수랄 또는 척골 신경에 비 침습적 녹음이 바람직하다. 및 행등 및 감각계의 소 구경 축삭 - 생쥐에서는, 좌골 신경의 신경 속성, 대용량을 모두 포함하는 말초 신경계 (PNS)의 규모 신경을 분석하기 쉽다. 여기에 설명 된대로 절차는 그대로 쥐의 말초 신경에 전기 생리학에 대한 관련 모든 표준 값을 측정 할 수있는, 쉽고 빠르고 안정적​​인 방법입니다. 보존 유기체에서 녹음을 복용함으로써, 신경 환경의 생리적 조건은 보장된다.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

전기 생리학 연구는 기본 병태 생리 (1)의 이해, 함께 신경 병리학 기술과, 다양한 신경 근육 질환의 합리적인 분류를 허용하고 도움이됩니다. 여기에서 우리는 생체 내에서 마우스 좌골 신경에 전기 생리학 연구를 수행하는 방법을 설명합니다. 동물 실험군에 진통을 보장하기 위해 이소 플루 란으로 마취 약 30 분 / 동물을 측정하는 동안 작업 환경을 방해받지 않습니다. 37 ° C의 일정한 체온은 가열 플레이트에 의해 유지되고 지속적으로 직장 열 프로브 (2)에 의해 측정된다. 또한, 심전도 (ECG)를 정기적으로 계속해서 조사 동물의 생리 학적 상태를 모니터링하기 위해 측정 중에 기록된다. 전기 생리학 기록은 좌골 신경, 일의 가장 큰 신경에서 수행됩니다행등과 감각 모두 섬유 책자와 마우스의 뒷다리를 공급하는 전자 말초 신경계 (PNS). 우리의 프로토콜에서, 좌골 신경은 현장에 남아 있으므로 추출 또는 노출 할 필요가 없습니다, 실제 녹음과 함께 어떤 불리한 신경 자극없이 측정이 가능. 적절한 바늘 전극 3을 사용하여 우리는 비복근 근육에서 감지 전극과 함께 전송 된 잠재력을 등록, 근위 및 말초 신경 자극을 모두 수행합니다. 데이터 처리, 신뢰성과 신경 전도 속도 (NCV) 및 복합 모터 활동 전위 (CMAP) 고도로 일관된 값, 총 말초 신경 기능의 정량화를위한​​ 주요 파라미터가 달성 될 수 후.

<html> 각 성별의 6 동물 : 우리는이 연구를 위해 총 12 쥐의 좌골 신경에 생체 전기 생리학 일련의 측정을 실시했다. 측정은 제시된 프로토콜을 수행하고 다음과 같은 결과를 제공 하였다 : 둘 다 남성과 여성 마우스는 약 20 M / sec의 평균 좌골 신경의 전도 속도 (그림 5)를 표시합니다. 이 문헌의 다른 측정과 일치한다. 또한, 남성과 여성 사이의 신경 전도 속도...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

생체 내에서 신경의 전도 특성의 측정은 신경 근육 질환의 다양한 동물 모델을 특성화 할 수있는 강력한 도구를 예시. 여기서, 우리는 마취 된 쥐의 좌골 신경의 전기 생리 분석이 수행 될 수있는 간단하고 신뢰성있는 프로토콜을 제시한다.

마우스 좌골 신경에 생체 전기 생리 측정에서

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

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Neuroscience

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.1K 조회수.  Fritz Lipmann Institute.  생체 내에서 신경의 전도 특성의 측정은 신경 근육 질환의 다양한 동물 모델을 특성화 할 수있는 강력한 도구를 예시. 여기서, 우리는 마취 된 쥐의 좌골 신경의 전기 생리 분석이 수행 될 수있는 간단하고 신뢰성있는 프로토콜을 제시한다. 

비디오: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

모쓰 강한 시스템에서 Electrophysiological 측정

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

연구

신경과학

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Electrophysiological 측정과 인간 유아의 Nociception 분석

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Electroporation에 의해 쥐 좌골 신경의 슈반 세포로 생체 내 유전자 전달에

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

단일 좌 골 신경 및 Cytometry 사용 하 여 단일 마우스에서 지 루트 신경 절에 면역 세포의 분석

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Vivo에서 모터 신경 퇴보의 마우스 모델에서 앞 발에서 복합 근육 활동 전위의 electrophysiological 측정

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

애벌레 Zebrafish의 측면 라인 세포의 생체 내 칼슘 이미징

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...