NIH 그랜트 SBC COBRE 1P20GM121310-01에 의해 지원.

여기에 설명 된 모든 메서드는 기관 동물 관리 및 사용 위원회 (IACUC)의 와이오밍 대학에 의해 승인 되었습니다. Electrophysiological 녹음을 포함 한 모든 절차는 실 온, 약 23 ° c.에서 실시 여기에 설명 된 모든 방법은 tadpoles 발달 단계 42 및 49 (Neiuwkoop와 페이 버15에 따라 개최) 사이에서 tectal 신경 기록 최적화 되어 있다.
1. Vivo에서 준비
<ol><li>올챙이 anesthetize<ol>
<li>0.01% 스 테 인 버그의 솔루션을 포함 하는 작은 페 트리 접시에는 올챙이 놓고 약 5 분 동안 MS-222. 참고: MS-222 일명 "Tricaine"는 일반적인 물고기와 양서류 마 취. Tadpoles는 m m에서 스 테 인 버그의 솔루션에서 발생 하는: 0.067 4.9 HEPES 5.8 NaCl, KCl, 0.034 Ca (3)2•4H2O, 0.083 MgSO4•7H2O.</li>
		<li>올챙이 깊이 anaesthetized (무응답과 이상 수영) 2 단계를 진행 하기 전에 확인 합니다.</li>
	</ol>
</li>
	<li>해 부/녹음 접시의 바닥에 잠긴된 실리콘 블록을 마 취 올챙이 보안 합니다.<ol>
<li>처분할 수 있는 이동 피 펫을 사용 하 여 외부 기록 솔루션을 포함 해 부/녹음 접시 anaesthetized 올챙이 이동 (115 m m의 NaCl, KCl, 2mm CaCl23 m m, MgCl23 m m, 5mm HEPES, 1mm 포도 당; 조정 7.25 사용 하 여 pH 10 N NaOH, osmolarity 255 mOsm의).<ol>
<li>자발적인 근육을 최소화 하기 위해 기, 아 세 틸 콜린 수용 체 차단제, tubocurarine (100 µ M) 외부 솔루션에 추가 합니다. (일반적으로,이 이루어집니다 외부 솔루션의 10 mL를 100 µ L 10 mM tubocurarine 주식의 추가 200 µ L 피 펫을 사용 하 여).</li>
		</ol>
</li>
		<li>곤충 핀을 사용 하 여 보안 올챙이, 등 쪽, 실리콘 탄성 중합체의 침수 블록 (예: Sylgard 184 참조 자료 테이블 세부 정보에 대 한), 어떤 해 접시 바닥에 붙어 있다. 참고: 핀의 위치는 중요 한: 눈에서 그 프로젝트 뇌, 성의 RGC 축 삭을 impaling 피하기 위해 충분히 꼬리의 양쪽에 그들을 배치 하 고 그냥 눈 tectum (그림 1A) 앞쪽 뇌를 입력.</li>
	</ol>
</li>
	<li>중간 선 따라 뇌를 모 깎기.<ol>
<li>두뇌의 명확한 보기에 대 한 살 균 25 G 바늘을 사용 하 여 중간에 따라 표면 절 개 하 여 뇌를 overlying 피부를 제거 합니다.</li>
		<li>신경 관에 바늘을 삽입 하 고 당겨 부드럽게 위쪽으로 (dorsally) 등 튜브의 등 쪽 부분 깔끔하게 절단 (절단) 바닥 플레이트 그대로 (그림 1B)를 떠나 있는 동안 그 같은 중간 축 따라 뇌를 모 깎기. 참고: 때문에 구심 성 감각 입력 입력은 tectum는 바닥에 접시를 통해 신경 튜브의 바닥 접시 그대로 남아 있을 중요 하다.</li>
	</ol>
</li>
	<li>Tectal 신경 세포를 포함 하는 투명 한 심 실 막을 제거 합니다.<ol>
<li>전기 생리학 장비에 기록 접시를 이동 하 고 깨진된 유리 피 펫 팁을 사용 하 여 심 실 막 overlying tectal 신경 셀 시체를 제거. 가볍게 섬세 한 작업와이 퍼에 걸쳐 그것을 드래그 하 여 팁을 휴식.</li>
		<li>피 펫 홀더에 피펫으로 나사와 micromanipulator 통해 광섬유 tectum 내려 낮은.</li>
		<li>깨진된 피 펫을 사용 하 여 껍질은 tectum에서 심 실 막. 참고: 그것은 전체 tectum;에서 멤브레인을 제거 하는 데 필요한 작은 창이 충분 하 고 충분 한 뉴런에 대 한 액세스를 제공.</li>
	</ol>
</li>
	<li>패치 클램프 기록을 전체 셀을 가져옵니다. 참고:이 시점에서 접근이 비슷합니다 Segev, 외. 에 설명 된 대로 마우스 뇌 조각에서 전체 셀 패치 클램프 녹음을 실시 16 (그림 1C).</li>
	<li>망막에 투영 하는 빛의 전체 필드 플래시 tectal 신경 응답을 기록 합니다.<ol>
<li>망막에 빛의 전체 필드 플래시 프로젝트, 광학 섬유를 올챙이 눈에 인접 한 장소. 광학 섬유의 다른 끝에 LED 빛 광도 제어할 수 있도록 가변 저항 라인 이다. 앰프의 디지털 출력으로 LED를 트리거하십시오. 이 방법에서는, 섬광 빛의 다양 한 농도의 수는 전체 필드(그림 4)를기록 했다.</li>
	</ol>
</li>
</ol>2. 뇌 준비
<ol><li>1.1 1.3 단계를 수행 합니다. 참고: tubocurarine이이 준비를 위한 외부 솔루션에 대 한 필요가 있다.</li>
	<li>뇌를 격리 합니다.<ol>
<li>25 G 바늘을 사용 하 여 서버 hindbrain (그림 2A).</li>
		<li>올챙이에서 뇌를 분리 하려면 부드럽게 모든 측면 및 복 부 결합 조직 및 신경 섬유를 절단 하는 꼬리-rostral 방향에서 뇌 밑 바늘을 실행 합니다.</li>
	</ol>
</li>
	<li>실리콘 엘라 스토 머의 블록에 두뇌를 보호 합니다.<ol>
<li>일단 완전히 해제 후 각 전구 중 하나를 통해 하나의 핀 hindbrain (그림 2B)를 통해 또 다른 핀을 배치 하 여 실리콘 탄성 중합체의 블록에 뇌를 보안 합니다. Tectal 신경에서 기록에 대 한 최적의 구성입니다.</li>
	</ol>
</li>
	<li>전기 생리학 장비에 해 범위에서 고정 된 뇌 준비를 포함 하는 접시를 이동 합니다. 1.4 단계에서 설명한 대로 깨진된 유리 피 펫을 사용 하 여 심 실 멤브레인을 제거 합니다.</li>
	<li>바이 폴라 자극 전극에 배치 시 chiasm (어디 각 눈에서 축 삭 책자는 midbrain에 크로스) RGC 축 삭을 직접 활성화 하.<ol>
<li>바이 폴라 자극 전극 rostral, 그리고 거의 옆에, 큰 중간 심 장소. 광학 chiasm는 즉시 rostral 큰 가운데 심 (그림 2B)에 있습니다.<ol>
<li>부드럽게 낮은 자극 전극 다운 시 chiasm에 되도록 작은 덴트 직물에서 형성 된다. 양극 전극은 정밀 하 게 제어 하는 자극의 강도 수 있는 펄스 자극에 의해 구동 됩니다.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Segev, 그 외 여러분 에 의해 설명 된 대로 수행 하는 전체 셀 패치 클램프 기록 (그림 2C) 16
</li>
</ol>3. 수평 뇌 슬라이스 준비
<ol><li>2.1 2.3 단계를 수행 합니다.</li>
	<li>면도날을 사용 하 여 하나의 광섬유 tectum의 한쪽의 가장 측면 4 (는 비보에 해당 하는 가장 등 4) 소비 세. 이 컷 평면에 평행 rostral 꼬리 그림 3A와 같이 이루어집니다.</li>
	<li>두뇌는 슬라이스 된 실리콘 엘라 스토 머의 측면에 다시 핀 사이드 (somata 및 neuropil는 기록에 대 한 직접 액세스할 수 있습니다)을 향하도록 하 고 실리콘 고무 블록 (그림 3 멀리 직면 하는 뇌의 심 실 표면 B) (있도록 양극 전극 광학 chiasm에 놓일 수 있다).</li>
	<li>전체 셀 패치 클램프 또는 로컬 출원된 잠재적인 기록 (그림 3C) Segev, 그 외 여러분 에 의해 설명 된 대로 수행 16
</li>
</ol>

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저자는 공개 없다.

이 작품에서 설명 하는 모든 방법 tadpoles 발달 단계 42 및 49 (Neiuwkoop와 페이 버15에 따라 개최) 사이에서 tectal 신경 기록 최적화 되어 있다. 무대 42는 tadpoles는 충분히 크고 충분히 개발 곤충 핀 vivo에서 녹음 한 뇌 해 부를 수행 하기 위한 뇌의 양쪽에 배치 될 수 있도록. 이전 단계에서는 tadpoles는 근본적으로 2 차원 (즉, 평면), 여기서 설명 하는 접근은 최적이 아닙니다...

Retinotectal 회로 양서류 비주얼 시스템의 주요 구성 요소. 그것은 광섬유 tectum postsynaptic tectal 신경 세포와 시 냅 스 연결을 형성 하는 어디에 그들의 축 삭을 프로젝트 눈에서 RGCs 구성 됩니다. Xenopus 올챙이 retinotectal 회로 신경 회로 형성 기능을 공부 하 고 인기 있는 개발 모델입니다. 그것은 강력한 실험 모델,12,3렌더링이 올챙이 retinotectal 회로의 많은 특성이 있습니다. 한 주요 특성, 그리고이 문서의 초점 tectal 신경, vivo에서 또는 뇌의 준비를 사용 하 여 전체 셀 패치 클램프 기록 수행 하는 기능입니다. 전압 및 전류 클램프 녹음 모드를 지원 하는 앰프와 복 전기 생리학 장비, 전체 셀 패치 클램프 기록 수 높은 해상도에서 특징에 뉴런의 전기 생리학. 그 결과, 전체 셀 패치 클램프 기록 retinotectal 회로 형성의 주요 단계에 걸쳐 tectal 뉴런에서 개발의 상세 하 고 포괄적인 이해 하 고 내장4,5 의 소성 제공 , 6 , 7 고 시 냅 스8,9,,1011 속성. 전체 셀 패치 클램프 tectal 신경 녹음을 결합, 유전자 또는 morpholinos 이러한 뉴런12, 그리고 설립된 시각적 회피 테스트13 통해 시각적 가이드 동작을 평가 하는 방법에 관심을 표현 하는 능력을 촉진 합니다 분자, 회로 기능 및 행동 사이 연결의 id입니다.
데이터 전체 셀 패치 클램프 기록에서 취득 불가능 유전 칼슘 표시기 GCaMP6, 같은 새로운 이미징 접근을 사용 하 여 있기 때문에 높은 해상도의 종류 칼슘 표시기를 사용 하 여 비록 허가 이미징 주의 하는 것이 중요 하다 칼슘의 신경 세포의 큰 인구에 걸쳐 역학 동시에, 아니 직접 또는 확실 한 방법은 특정 전기 매개 변수 somata에서 델타 형광을 측정 하 여 얻을 수 있습니다 그리고 거기 방법은 전압 클램프 측정 하 신경 전류-전압 관계입니다. 명확 하 게이 두 가지 접근, electrophysiological 녹음 및 칼슘 이미징, 겹치지 않는 강점가지고 고 다른 유형의 데이터를 생성. 따라서, 가장 좋은 방법은 해결 되 고 특정 실험적인 질문에 따라 달라 집니다.
여기, 우리가 올챙이 눈 tectum는 vivo에서 준비, 뇌 준비를 사용 하 여의 뉴런에서 전체 셀 패치 클램프 기록 인수에 대 한 우리의 방법을 설명 하 고 새로운 수정14 연구소에서 개발 된 뇌 준비 . 대표적인 결과 섹션에서 각 준비와 얻을 수 있는 데이터의 종류의 실험적인 장점을 보여 줍니다. 문제 해결을 위한 팁 뿐만 아니라 다른 준비의 강점과 한계 토론 섹션에 포함 됩니다.

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preparations and protocols for whole cell patch clamp recording of xenopus laevis tectal neurons

Xenopus 올챙이 retinotectal 회로, 어떤 형태로 광섬유 tectum에 신경에 직접 synapses 눈에서 망막 신경 절 세포 (RGCs)의 구성 어떻게 신경 회로 공부 하는 인기 모델은 자기 조립. 전체 수행 하는 기능 패치 클램프 기록 중 vivo에서 기록 RGC 갖는 응답을 tectal 뉴런에서 셀 또는 전체 두뇌 준비를 사용 하 여 일반 기본 메커니즘에 대 한 고해상도 데이터의 큰 몸 생성 했다 비정상적인, 회로 형성 및 기능. 여기 우리가 준비 vivo에서 , 원래 뇌 준비를 수행 하는 방법을 설명 하 고 더 최근 개발 tectal 뉴런에서 전체 셀 패치 클램프 녹음을 얻기 위해 수평 뇌 조각을 준비. 각 준비 독특한 실험 장점이 있습니다. Vivo에서 준비 tectal 신경의 눈에 투영 하는 시각적 자극에 직접 응답의 녹음 수 있습니다. 뇌 준비 매우 제어 방식에서 활성화 RGC 축 삭에 대 한 허용 하며 수평 두뇌 슬라이스 준비에서 녹음은 tectum의 모든 레이어에서

빛 갖는 응답를 빛의 전체 필드 플래시 결과 응답 개별 tectal 뉴런(그림 4)에서 기록 하는 동안 망막에 예상 된다. 이 특정 프로토콜은 모두 빛 ("에" 응답) 켜기 한 다음 15 신경의 응답을 측정 하도록 설계 되어 나중 "에서 응답." 측정 하는 s Tectal 신경은 일반적으로 응답에 이따금 강력한 전시 (표시 여기에 기록 된 전압 클램프 모드, 신경?...

Watch this Scientific Journal Video about Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons at JoVE.com

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

이 종이 전체 셀 패치 클램프 기록 Xenopus laevis tadpoles의 retinotectal 회로 공부 하는 데 사용 하는 세 뇌 준비를 다루겠습니다. 그것의 자신의 특정 이점 각 준비 신경 회로 기능을 공부 하는 모델로 Xenopus 올챙이의 실험 추적성에 기여 한다.

준비 및 Xenopus laevis Tectal 신경의 전체 셀 패치 클램프 기록에 대 한 프로토콜

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

preparations-protocols-for-whole-cell-patch-clamp-recording-xenopus

Research

JoVE Journal

Neuroscience

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

9.3K 조회수.  University of Wyoming. 이 종이 전체 셀 패치 클램프 기록 Xenopus laevis tadpoles의 retinotectal 회로 공부 하는 데 사용 하는 세 뇌 준비를 다루겠습니다. 그것의 자신의 특정 이점 각 준비 신경 회로 기능을 공부 하는 모델로 Xenopus 올챙이의 실험 추적성에 기여 한다.

비디오: Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

연구

신경과학

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

적외선 신경 자극의 메커니즘을 조사하는 전체 세포 패치 클램프

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

녹화 패치 클램프 기술을 사용하여 어두운 적응 마우스 망막 슬라이스 준비에 신경 세포의 시냅스 응답 빛-유발

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

본래 Neuroepithelium에서 마우스 강한 감각 뉴런의 천공 패치 클램프 녹음 : 확인 된 부 취제 수용체를 발현 뉴런의 기능 분석

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

뇌 조각에서 전체 셀 패치 클램프 녹음

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

의 후각 망울에서 칼슘의 변경 사항을 모니터링을 통해 온도에 의한 신경 세포의 활동을 기록<em&gt; Xenopus의의 laevis의</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.