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.

<table><tbody><tr><td>K-gluconate anhydrous</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>HEPES</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; X 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ethylene glycol-bis(B-amin&amp;#8211;thyl ether)-N,N,N&amp;rsquo;,N&amp;rsquo;-tetraacetic acid</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>MgATP</td><td>Reagent</td><td>MP Biomedicals</td><td></td><td></td></tr><tr><td>NaGTP</td><td>Reagent</td><td>MP Biomedicals</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>KCl</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Anhydrous</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Glucose</td><td>Reagent</td><td>Acros Organics</td><td></td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>CholCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Reagent</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Ascorbic Acid</td><td>Reagent</td><td>Acros Organics</td><td></td><td></td></tr><tr><td>Glutathione</td><td>Reagent</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Olympus BX51WI Microscope (with 40x objective)</td><td>Microscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>2 A/D converters</td><td>Equipment</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Multiclamp 700B with CV-7B headstage</td><td>Equipment</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>P-97 Flaming/Brown Micropipette Puller</td><td>Equipment</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Microfil syringe needles</td><td>Equipment</td><td>World Precision Instruments, Inc.</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Equipment</td><td>Siskiyou, Inc.</td><td></td><td></td></tr><tr><td>Monitor</td><td>Equipment</td><td>Triview</td><td></td><td></td></tr></tbody></table>

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 ...

application-nmda-receptor-conductance-rat-midbrain-dopaminergic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

성인 쥐에서 본래 등쪽 뿌리 신경절에 패치 클램프 녹음

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

연구

생화학

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

동시 전압 클램프 및 Amperometry과 도파민 릴리스의 단일 세포 측정

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

신경과학

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 ...

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

표현 이온 채널 연구에 대한 특정 감각 신경 세포 인구의 식별

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

흑색질 도파민 뉴런의 Somatodendritic 도메인에서 세포 내 패치 클램프 녹음

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

조각 패치 클램프 기술 학습 유도 소성을 분석 하기 위한

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

1 채널의 세포 부착 된 패치 클램프 녹음

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

생물학

Patch-Clamp Recordings from the Dendrite of a Dopaminergic Neuron in a Brain Slice

Source: Engel, D. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54601">Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for patch-clamp recording from the dendritic membrane surface. This helps to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels in dopaminergic neurons.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Source: Kinnavane, L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63077">Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for studying synaptic efficiency in the lateral entorhinal cortex (LEC) by optogenetically stimulating medial prefrontal cortex (mPFC) axons and recording the responses of pyramidal neurons in the LEC. This method combines patch-clamp recordings with synaptic activation by light stimulation to study synaptic plasticity.

쥐 뇌 절편에서 시냅스 사건의 광유전학적 자극 및 전기생리학적 기록

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology and ...

Stimulation and Modulation Techniques

Applying Noise Currents to Medial Vestibular Nucleus Neurons Using Whole-Cell Patch-Clamp Technique

For whole-cell patch clamp electrophysiology, first, pull micropipettes with a final resistance that will range from 3 to 5 megaohms when filled with a potassium-based internal solution, and place in the bath. To obtain whole-cell patch clamp recordings from individual neurons in the MVN, transfer a single tissue slice from the incubation chamber to the recording chamber of a standard electrophysiological setup, and use a nylon thread on a U-shaped weight to secure the slice.
Continuously perfuse the recording chamber with carbogenated aCSF at 25 degrees Celsius at a flow rate of three milliliters per minute, and fill a micropipette with internal solution. Apply a small amount of positive pressure using a pipette to push debris away from the pipette tip. Using a lower power objective, locate the MVN before switching to a high power to locate individual neurons within the MVN.
Before breaching the tissue with the pipette, apply a small amount of positive pressure to push debris away from the pipette tip, and use the micromanipulator to move the pipette toward the selected neuron. A small dimple should form on the neuronal membrane. Release the positive pressure and apply a small amount of negative pressure.
Once a one gigaohm seal has been achieved, apply gentle, short, and sharp negative pressure to the pipette holder through the suction port to rupture the membrane and to create a whole-cell configuration. Then, obtain whole-cell current clamp recordings according to standard protocols.
To apply stochastic and sinusoidal noise to individual medial vestibular nucleus neurons, set the range of amplitudes from 3 to 24 picoamps to determine the neuronal threshold and firing rate, and group the lower and higher stimulus intensities to determine the sensory threshold.
Next, calculate the average firing rate over the 10-second period during which the depolarizing current step will be injected for each individual current level. Use the average firing rate values to generate a firing rate versus current plot. Then, perform a linear regression analysis to determine the gradient of the line of best fit to determine the neuronal gain.