이 작품은 MH084494 (CJL), 그리고 MH079276 및 NS060658 (CAP)에 의해 지원되었다.

1. 슬라이스 준비 <ol><li> 진동 마이크로톰를 사용하여 뇌의 슬라이스 컷. 우리는 산 안토니오 기관 애니멀 케어 및 사용위원회 텍사스 대학에 따라 진동 마이크로톰 (Microm HM 650V)를 사용하여 isoflurane - anethetized 스프 라그 - 돌리 쥐 (찰스 리버 연구소)에서 240 μm의에게 수평 midbrain의 조각을 준비했습니다. </li><li> 당신이 기록 준비가 될 때까지 배양 챔버의 조각 유지. (aCSF, MM)을 우리는 32 ° C로 가열하고 인공 뇌척수 가득 부화 컨테이너를 사용하여 : 126 NaCl, 2.5 KCl, 1.25 아니 2 PO 4, 4 MgCl 2, 2 CaCl 2, 10 덱스 트로 오스, 25 NaHCO 3, 1.3 아스코르비 산, 2.4 나트륨 pyruvate와 0.05 글루타티온. </li></ol> 2. Electrophysiological 녹음 <ol><li> 35 인공 뇌척수 (aCSF)가 ° C가 perfused되고있는 세포 녹음 장비에 대한 슬라이스를 전송합니다. 우리는 2mM MgCl 2가 사용되었고 글루타티온가 생략되었습니다 제외 1.2와 같은 aCSF을 사용합니다. 수평으로 준비 조각을 위해, 우리는 일반적으로 정중선을 따라 조각을 이등분. </li><li> 대상 신경 세포를 시각화. 우리는 기울기 콘트라스트 이미징 시스템과 개별 substantia 니그라 dopaminergic의 뉴런을 시각. </li><li> 전자 전극 풀러를 사용하여 전극을 당기세요. 우리는 MΩ P97 Micropipette 풀러 (서터 악기 회사)를 사용 40-10의 팁 저항과 전극을 가져옵니다. </li><li> 원하는 내부 솔루션 전극을 입력합니다. 138 K - 글루 콘 산, 10 HEPES, 2 MgCl 2, 0.2 EGTA, 0.0001 CaCl 2, 4 나 - ATP, 0.4 나 - GTP : 우리는이 포함된 솔루션을 (MM)을 사용합니다. 내부 솔루션은 1M 코와 270-275 mOsms의 osmolarity를​​ 사용하여 7.3의 산도 조정되었습니다. </li><li> 원하는 신경 세포에 gigaohm 인감을 확인합니다. 파열 흡입과 인감. 이것은 전체 휴대폰 녹음을 구성합니다. Multiclamp 700B 앰프는 우리의 구성에 사용되었습니다. 증폭기는 다음의 &#39;= 0&#39;현재 클램프 모드에 배치되어야합니다. </li></ol> 3. 다이나믹 클램프와 전도성 신청 <ol><li> RTXI (www.rtxi.org)은 동적 클램프 컴퓨터에서 실행되었습니다. NMDA 수용체를 포함하는 사용자 정의로 작성된 모델은 메모리에로드되었습니다. 실시간으로 세포에 주입되는 전류는 다음 공식에 의해 계산됩니다 : I NMDA = - g NMDA * [1 / (1 ​​+ ([MG] / 3.57) * E (- V m * 0.062))] * (V m - E NMDA) 여기서 g NMDA 원하는 전도성 (NS입니다; 기본적으로) 0 NS로 설정 [MG]는, E NMDA는 NMDA 수용체 (0 MV으로 설정)에 대한 반전 가능성 마그네슘 농도 (아래의 예제에서 1.5 mm까지 세트)입니다, 그리고 V M은 막 잠재력이다 (millivolts)를 앰프에서 측정된 세포의. </li><li> 동적 클램프 컴퓨터에서 출력은 아날로그 - 디지털 변환기를 통해 앰프의 명령 입력에 연결되었습니다. </li><li> 앰프는 &#39;IC&#39;현재 클램프 모드에 배치되었다. </li><li> RTXI (예 : 40nS)에 원하는 NMDA 수용체의 전도성을 입력합니다. 당신은 행동 잠재력의 phasic 버스트가 나타납니다. 또한, 전도성은 아날로그 출력 (그림 1A, &#39;g (t)&#39;)를 통해 RTXI에 주어진 수 있습니다. 적절한 스케일링 계수는 볼트에서 지멘스 신호를 변환하는 RTXI 이내에 사용해야합니다. </li></ol> 4. 대표 결과 동적 클램프를 사용하여 전도성의 응용 프로그램에 대한 성공적인 설정은 그림 1A에 표시됩니다. 이 설정을 사용하여, 우리는 substantia 니그라 갈 거예요 compacta의 dopaminergic 신경 세포에서 전체 세포 체세포의 녹음을했다. Dopaminergic 세포는 일반적으로 맥박 조정기와 같은 패턴으로 낮은 속도에서 저절로 불이났다. 액션 잠재력의 파열은 동적 클램프 (그림 1B)와 NMDA 수용체의 전도성의 phasic 응용 프로그램에 의해 evoked 수 있습니다. <img src="/files/ftp_upload/2275/2275fig1.jpg" alt="그림 1" /> 그림 1 : 동적 클램프 기법을 사용하여 NMDA 수용체의 전도성의 응용. A. 하드웨어 설치 세포내 녹음 장비 및 동적 클램프 컴퓨터 사이의 연결을 보여주는. 액션 잠재력의 B. 파열은 substantia 니그라 갈 거예요 compacta dopaminergic 신경 세포에서 전체 셀 녹음 40nS에서 NMDA 수용체의 전도성의 응용 프로그램에 의해 evoked입니다.

<ol>
	<li>Robinson, H. P., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> J Neurosci Methods. 49, 157-165 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp:+artificial+conductances+in+biological+neurons.">The dynamic clamp: artificial conductances in biological neurons.</a> Trends Neurosci. 16, 389-394 (1993).</li><li>Sharp, A. A., O'Neil, M. B., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> J Neurophysiol. 69, 992-995 (1993).</li><li>Prinz, A. A., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+clamp+comes+of+age.">The dynamic clamp comes of age.</a> Trends Neurosci. 27, 218-224 (2004).</li><li>Deister, C. A., Teagarden, M. A., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intrinsic+neuronal+oscillator+underlies+dopaminergic+neuron+bursting.">An intrinsic neuronal oscillator underlies dopaminergic neuron bursting.</a> J Neurosci. 29, 15888-15897 (2009).</li><li>Lobb, C. J., Wilson, C. J., Paladini, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+role+for+GABA+receptors+on+the+firing+pattern+of+midbrain+dopaminergic+neurons.">A dynamic role for GABA receptors on the firing pattern of midbrain dopaminergic neurons.</a> J Neurophysiol. 104, 403-413 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기 시연 동적 클램프 기술은 실험자이 수용체의 활성화의 전기 효과를 모방 수 있도록함으로써 직류 분사의 전통적인 기법에이 향상됩니다. 이 비디오에서는, 우리는 dopaminergic 신경 세포의 자발적인 활동에 NMDA 수용체의 활성화의 evoked 아르 행동 잠재력의 버스트 즉 하나는 효과를 추가할 수 것으로 나타났습니다. 하드웨어 / 소프트웨어 구현의 유연성으로 인해, 확장의 다양한 사용하실 수 있습니다. 주입 전류의 서명이 활성화 수용체의 영향이 신경 세포에서 제거됩니다 시나리오를 대표하는, 긍정을 부정에서 전환하실 수 있습니다. 양식 미분 방정식의 일련의 대표 모델 뉴런은 또한 숫자 해결하고 실험자들이 소규모 네트워크를 조사하도록 할 수 있습니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>K-gluconate anhydrous</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2 X 2H2O</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis(B-aminoethyl ether)-N,N,N&#x2019;,N&#x2019;-tetraacetic acid</td>
<td>Reagent</td>
<td>EGTA; Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgATP</td>
<td>Reagent</td>
<td>MP Biomedicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaGTP</td>
<td>Reagent</td>
<td>MP Biomedicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaH2PO4, Anhydrous</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CholCl</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ascorbic Acid</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX51WI Microscope (with 40x objective)</td>
<td>Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2 A/D converters</td>
<td>Equipment</td>
<td>e.g. Heka Instruments Inc. ITC-18, National Instruments BNC-2090A</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Multiclamp 700B with CV-7B headstage</td>
<td>Equipment</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>Equipment</td>
<td>Sutter Instrument Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microfil syringe needles</td>
<td>Equipment</td>
<td>World Precision Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>Equipment</td>
<td>Siskiyou, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monitor</td>
<td>Equipment</td>
<td>Triview</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

application of a nmda receptor conductance in rat midbrain dopaminergic neurons using the dynamic clamp technique

Neuroscientists는 두뇌의 뉴런 통신하는 방법을 조사하여 뇌의 기능을 연구합니다. 많은 조사는 실험적으로 제어 입력에 대한 응답으로 하나 이상의 뉴런의 전기 활동의 변화보세요. 뉴런의 전기 활동은 유리 micropipettes와 패치 클램프 기법을 사용하여 절연 뇌 슬라이스에 기록하실 수 있습니다. 전통적으로, 경험이 기록된 세포의의 연결을 막에있는 수용체에 의해 슬라이스, 또는 약리 조작에있는 다른 셀 또는 남은 axonal 연결 피펫, 전기 자극을 통해 현재의 직접 분사로의 연결을 입력을 모방 수 있습니다. 직류 주사 녹음이 (보통 소마)의 사이트에서 높은 시간적 정밀도로 만들어진 전류 파형을 전달하는 장점이 있습니다. 그러나, 이것은 이온 채널이 개설되지 않습니다 물리적으로의 연결을 막의 저항을 변경하지 않습니다. 현재 분사는 일반적으로 직사각형 펄스를 고용하기 때문에 이온 채널의 반응 속도론을 모델하지 않습니다. 마지막으로, 현재 분사는 이온 채널의 개통과 함께 발생하는 세포에서 화학적 변화를 모방 수 없습니다. 수용체는 물리적 또는 전기적 자극에 의​​해 약리 활성화할 수 있습니다. 실험자들은 조각의 전기 자극과 수용체 활성화 잘 시간적 정밀도를했다. 수용체 활성화 자극시 활성화되어 어떤의 정확한 성격 그러나,이 제한됩니다 공간적 정밀도 알 수 있습니다. 이 후자의 문제는 부분적으로 특정 약리 에이전트가 완화된 수 있습니다. 불행히도, 약리 대리인의 활성화 시간 과정은 일반적으로 속도가 느린되고 기록된 세포에 입력의 공간적 정밀도 알 수 있습니다. . 검토를 위해, 프린츠 동부 표준시를 참조하고, 동적 클램프 기술은 실험자의 세포의 막 전위 (로빈슨과 카와이 1993, 샤프 외, 1993a, b의 실시간 피드백을 기반으로 세포에 직접 전달되는 전류를 변경할 수 있습니다 알. 2004). 이것은 실험자이 수용체의 활성화에 대한 응답으로 녹화 현장에서 발생하는 전기적 변화를 모방 수 있습니다. 적용 전류에 실시간으로 변경되며, 변경된 사항은 하드웨어에서 구현 수학적 방정식에 의해 결정됩니다. 우리는 최근 substantia 니그라 갈 거예요 compacta (.; 로브 외, 2010. Deister 외, 2009)의 dopaminergic 뉴런에서 NMDA 수용체의 phasic 활성화하여 실천 가능성의 파열의 생성을 조사하기 위해 동적 클램프 기법을 사용했습니다. 이 비디오에서는, 우리는 dopaminergic 신경 세포에 NMDA 수용체의 전도성을 적용하는 데 필요한 절차를 보여줍니다.

Watch this Scientific Journal Video about Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique at JoVE.com

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

이 비디오에서는, 우리는 쥐의 뇌 조각의 전체 세포 구성에 기록된 dopaminergic 신경 세포로 전도성을 적용하는 방법을 보여줍니다. 이 기법은 동적 클램프라고합니다.

동적 클램프 기법을 사용 랫 Midbrain Dopaminergic 뉴런에서 NMDA 수용체의 전도의 응용

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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12.0K 조회수.  University of Texas San Antonio - UTSA. 이 비디오에서는, 우리는 쥐의 뇌 조각의 전체 세포 구성에 기록된 dopaminergic 신경 세포로 전도성을 적용하는 방법을 보여줍니다. 이 기법은 동적 클램프라고합니다.

비디오: Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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신경과학

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic 신경 세포 개발에서 유전자 기능을 유학 칙의 Midbrain에서 비켜 Electroporation에

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

마우스 망막 신경절 세포의 시냅스와 인공 컨덕턴스와 동적 클램프 구현

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

마우스 도파민 뉴런의 차 문화

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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, ...

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 ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

RNA 시퀀싱 및 TH-프로모터 중심 eGFP는 표현을 사용하여 중간 뇌 문화에서 생활 도파민 뉴런의 신뢰성 확인

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

대뇌 혈류와의 광 산란 특성을 동시에 평가<em&gt; 생체</em&gt; 다중 스펙트럼 확산 반사 이미징을 이용한 쥐 뇌

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

뇌 조각에서 뉴런 연구를위한 자동 이미지 유도 패치 클램프의 적용

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...