우리는 기술 지원을위한 박사 멩 턱 린 양 위안 동 감사합니다. 이 작품은 건강 HD053767의 국립 연구소 (NLW에 하청)에서 교부금에 의해 캘리포니아 로스 앤젤레스의 연구 대학 (NLW)를위한 부총장의 생리학 및 Office의 부서에서 기금에 의해 지원되었다.

1. 성인 송사리의 두뇌의 해부 <ol><li> 에 침지하여 (그림 1A) 남성 또는 여성 성인 마취 5 ML MS-222 (150 ㎎ / L, 산도 7.4), 아가미의 움직임이 해고되기 전에 중지 한 후 몇 분을 기다립니다. 모든 절차는 캘리포니아 대학교 로스 앤젤레스 캠퍼스의 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다. </li><li> 60 mm 직경 페트리 접시에 가위로 아가미의 꼬리 끝에 물고기 식염수에있는 물고기의 목을 베십시오. </li><li> 35 mm 직경 페트리 접시에 전송 생선 머리는 반 염수 물고기로 가득 뇌 해부에 대한 머리의 위치 및 보안에있는 우울증이 실 가드와 함께 늘어서있다. 위치 및 곤충 핀으로 머리 등쪽면이 위로를 고정합니다. </li><li> 신중하게 잘 가위로 그 주위에 두개골의 지느러미 부분을 잘라 조심스럽게 완전히 척수의 앞쪽 부분을 포함한 뇌의 등쪽 부분을 노출, 집게로 제거합니다. </li> &lt;리&gt; 미세 팁 집게로 조심스럽게 척수를 들어 올리고 뾰족한 가위로 상호 결합 신경을 절단 : 두뇌의 복부 측면에 뇌신경, 척수 신경, 그리고 시신경, 그리고 앞쪽 부분에있는 후각 신경 뇌. </li><li> 물고기 식염수로 가득 신선한 페트리 접시에 완전 해부 두뇌를 배치, 전체 뇌가 어떤 상처 나 구멍없는 (뇌하수체 포함) 그대로 확인하기 위해 현미경으로주의 깊게 확인합니다. 손상된 두뇌가 실험에 사용되지 않습니다. </li></ol> 2. 녹음 회의소 뇌 장착 <ol><li> 시아 노 아크릴 레이트의 작은 방울 또는 녹음 챔버의 중앙에 바늘로 다른 속효성 접착제를 놓고 1cm 약 2 접착제를 확산. </li><li> 빠르게 복부면이 위치에 뇌와 접착제 아래로 전송할 수 있습니다. </li><li> 이 기록 챔버를 채우고 머리를 덮을 수 있도록 물고기 식염수 1 ML을 추가합니다. 지속적으로 붓는다최소 200 μL / 분의 속도로 통기 물고기 식염수 뇌. </li><li> 좋은 팁을 사용하여 조심스럽게 뇌의 녹화 영역의 표면에 수막을 제거 포셉. </li></ol> 3. 전기 생리학 <ol><li> 활동 전위 사격 느슨한 패치 기록 <ol><li> 유리 미세 전극 (MΩ 6-10)은 전극 풀러 (예를 들어 셔터 P-87)를 사용하여 붕규산 유리 피펫 (1B150F-4, 세계 정밀 계측기)에서 생성, 다시 절반 방법까지 느슨한 - 패치 녹음 용액으로 채워져 전극. </li><li> 냉각 전하 결합 소자 (CCD) 카메라를 똑바로 형광 현미경을 사용하여 GFP 발현 배를 통해 TN-GnRH3 뉴런 (그림 1B), 다음 40X 물 침지 목표 (그림 1C)를 찾을 수 있습니다. 그들은 telencephalon 바로 앞쪽에 후각 전구의 복부 표면 또는 근처에 위치하고 있습니다. </li><li> 제공하는 비디오 모니터로 전환현미경 실시간 영상 TN-GnRH3 신경 세포 클러스터 (그림 1C)를 찾습니다. </li><li> 전극의 끝이 신경 세포 표적 위에 있도록 욕조에 미세 전극을 배치합니다. 가가 막혀, 부드럽게 전극의 끝 표적 신경 세포에 접근하지 않도록 미세 상수 약간의 양압을 적용합니다. </li><li> AxoGraph 소프트웨어 및 Axograph 소프트웨어 (액슨 악기)와 앰프 Axoclamp 200B를 사용하여 전압 클램프 모드에서 전극의 저항을 점검하고 모니터링 할 수 있습니다. 당신은 전극의 끝이 신경 세포의 표면과 저항이 약간 변경하는 AxoGraph 디스플레이에서인지 비디오 모니터에서 볼 수있는 경우에 낮은 저항 씰을 만들기 위해 긍정적 인 압력을 해제하고 필요한 경우 약간의 부정적인 압력을 적용 신경 세포 (&lt;100 MΩ). </li><li> 전류 클램프 모드로 전환하고 규모를 조정하는 현재의 입력없이 지속적으로 막 전압을 기록전압 및 녹음 필요한 경우의 비율. 데이터가 PowerLab의 소프트웨어 (ADInstruments 주식 회사)에 의해 수집되고 분석됩니다. </li><li> 어떤 처리 (그림 2A) 전에 약 5 분 동안 정상 물고기 식염수에 기록 기준선 전기 활동. 약물, 호르몬, 펩타이드 등의 목욕 관류. 물고기 식염수에 추가하는 것은 보통 물고기 식염수 5, 6, 11 유실 뒤에 마침표 200 μL / 분의 속도로 계속됩니다. </li></ol></li><li> 막 잠재력의 전체 세포 녹음 3.1.4 - 전체 세포 패치 클램프 전기 생리학 절차는 단계 3.1.1에서 설명한 세포 느슨한 - 패치 녹음 절차와 동일합니다. 다음 절차에 따라 편차 : <ol><li> 전압 클램프 모드에서 전극의 저항을 점검하고 모니터링 할 수 있습니다. 당신은 저항 변화가 약간 긍정적 풀어 전극의 끝이 신경 세포의 표면에와​​ 오실로스코프에서인지 비디오 모니터에서 볼 수있는 경우압력과 신경 (2 ~ 3 기가 Ω)에 높은 저항 인감을 만들기 위해 약간의 부정적인 압력을 적용합니다. 파열 패치 막 입으로 부드러운 흡입을 적용, 당신은 MΩ 약 12​​0-250에 대한 저항의 급격한 저하를 볼 수 있습니다. </li><li> 전류 클램프 모드로 전환, 녹화 필요한 경우의 전압과 속도의 규모를 조정하는 현재의 입력없이 지속적으로 막 전압을 기록한다. </li><li> 어떤 치료를하기 전에 약 5 분 동안 정상 물고기 식염수에 기록 기준선 전기 활동이 아니라 3.1.7 (그림 2B)에서 설명한. </li></ol></li></ol>

<ol>
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Physiol. 297 (1), R135-R141 (2009).</li><li>Abe, H., Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neuromodulation+by+a+nonhypophysiotropic+GnRH+system+controlling+motivation+of+reproductive+behavior+in+the+teleost.">Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost.</a> 57 (6), 665-674 (2011).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+persistent+Na++current+underlying+pacemaker+potentials+of+fish+gonadotrophin-releasing+hormone+neurones.">Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones.</a> J. Physiol. 482 (Pt. 1), 1-6 (1995).</li><li>Zhao, Y., Wayne, N. 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저자는 공개 아무것도 없어.

GnRH의 3 GFP 유전자 변형 물고기는 신경 세포의 통합과 직간접 적으로 재생 3, 8-10에 관여 둘 다 행동의 중앙 제어 규제의 근간이되는 신경 생리 학적 메커니즘을 연구하는 독특한 모델을 제공합니다. 이 모델 시스템의 중요한 장점 중 하나는 GFP를 표현하는 많은 GnRH3 뉴런은 신경 회로 6, 9, 11, 12 (그림 방해하지 않고 전기 생리학 기록을 위해 신경을 비교적 쉽게 ?...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>BX50W (Upright)</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axoclamp 200B </td>
 </tr>
 <tr>
 <td>A-D converter</td>
 <td>Computer Interference Corp.</td>
 <td></td>
 <td>Digidata ITC-18 </td>
 </tr>
 <tr>
 <td>Cooled CCD camera</td>
 <td>PCO Computer Optics </td>
 <td></td>
 <td>Sensicam</td>
 </tr>
 <tr>
 <td>Xenon lamp</td>
 <td>Sutter Instruments Co.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>GFP filter set</td>
 <td>Chroma Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging Software</td>
 <td>Intelligent Imaging Innovations</td>
 <td></td>
 <td>Slidebook software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>Axograph software </td>
 </tr>
 <tr>
 <td>Electrophysiology Data Acquisition Software</td>
 <td>AD Instruments Inc.</td>
 <td></td>
 <td>PowerLab </td>
 </tr>
 <tr>
 <td>Headstage for electrophysiology</td>
 <td>Axon Instruments</td>
 <td></td>
 <td>CV 203BU </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument Co </td>
 <td>MP-285 </td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Chamber Platform</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>P1 </td>
 </tr>
 <tr>
 <td>Recording Chamber</td>
 <td>Warner Instrument Corp.</td>
 <td></td>
 <td>RC-26G </td>
 </tr>
 <tr>
 <td>Electrode Puller</td>
 <td>Sutter instruments</td>
 <td></td>
 <td>P87 </td>
 </tr>
 <tr>
 <td>Filament for electrode puller</td>
 <td>Sutter Instruments</td>
 <td>FB330B</td>
 <td>3.0 mm wide trough filament</td>
 </tr>
 <tr>
 <td>1.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B150-4</td>
 <td>Microelectrode for recording</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309586</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>MS-222 </td>
 <td>Sigma</td>
 <td>E10521-10G</td>
 <td>Ethyl 3-aminobenzoate methanesulfonate salt</td>
 </tr>
 <tr>
 <td>Fish saline</td>
 <td></td>
 <td></td>
 <td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl2; 1.2 MgCl2; 10 HEPES </td>
 </tr>
 <tr>
 <td>Electrode solution (loose-patch) </td>
 <td></td>
 <td></td>
 <td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl2; 1.3 MgCl2; 10 HEPES; 10 glucose </td>
 </tr>
 <tr>
 <td>Electrode solution (whole-cell patch) </td>
 <td></td>
 <td></td>
 <td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl2; 1 MgCl2; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td>
 </tr>
 </tbody>
</table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

복잡한 행동을 조절하는 신경 회로의 세포 생리학을 이해하는 것은이 작품은 CNS의 신경 회로가 그대로 남아 그대로 뇌의 준비에서 수행 할 수있는 모델 시스템을 사용하여 크게 향상됩니다. 우리는 성선 자극 호르몬 분비 호르몬 (GnRH의) 신경 세포가 유전자 손상 뇌의 식별을 위해 녹색 형광 단백질로 태그 된 유전자 변형 물고기를 사용합니다. 물고기 GnRH의 뉴런의 여러 인구를 가지고 있고, 그 기능은 뇌의 위치와 그들이 1을 표현하는 GnRH의 유전자에 따라 달라집니다. 우리는 유전자 변형 송사리의 손상 뇌 (그림 1B 및 C)를 사용하여 후각 전구에 연결된 터미널 신경 (TN)에 위치한 GnRH3 신경에 우리의 시범 초점을 맞추고있다. 연구는 송사리 TN-GnRH3 뉴런은 중추 신경계에 외부 환경으로부터 정보를 송신기 역할을 neuromodulatory 것을 제안, 티셔츠헤이로 잘 알려진 시상 하부 GnRH1 신경 세포 2, 3을 수행합니다. TN-GnRH3 뉴런의 자발적인 활동 전위 사격 토닉 패턴이 고유의 속성을 4-6, 주파수, 뇌하수체 - 생식선 기능 조절에 직접적인 역할을하지 않는다 어느 것이 conspecifics 2 신경 펩타이드 kisspeptin 5에서 시각적으로 변조된다. 이 비디오에서는, 우리는 TN-GnRH3 뉴런이 어떻게 뉴런을 식별하고 뇌 전체에 자신의 전기 활동을 모니터링하는 방법을 보여 강화 된 녹색 형광 단백질 7에 연결 Gnrh3의 프로모터 부위를 포함하는 유전자를 표현하는 형질 전환 송사리의 안정적인 회선을 사용 준비 6.

송사리의 절제 뇌에서 GFP - 라벨 TN-GnRH3 신경의 양측 클러스터의 예는 그림의 1B 및 1C에 표시됩니다. 각 클러스터는 8-10 GnRH의 뉴런을 포함하고 있습니다. 대상 TN-GnRH3의 자발적인 신경 활동은 0.5-6 Hz의 전형적인 발사 속도와 전류 클램프 모드 (I = 0)에 기록 하였다. 활동 전위 사격 패턴은 매우 일반적인 interspike 간격으로, 일반적 토닉이나 구타 패턴이다. 샘플 트레이스는 <s...

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

이 비디오에서는, 우리는 복잡한 신경 회로를 유지하는 뇌 전체 준비에서 확인 된 하나의 뉴런에서 전기 활동을 기록하는 방법을 보여줍니다. 우리는 성선 자극 호르몬 분비 호르몬 (GnRH의) 신경 세포가 유전자 손상 뇌의 준비 식별을위한 형광 단백질로 태그 된 유전자 변형 물고기를 사용합니다.

형질 전환 생선의 본래 뇌에서 발견 뉴런의 전기적 활동을 기록

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

recording-electrical-activity-from-identified-neurons-intact-brain

Research

JoVE Journal

Neuroscience

13.4K 조회수.  University of California, Los Angeles. 이 비디오에서는, 우리는 복잡한 신경 회로를 유지하는 뇌 전체 준비에서 확인 된 하나의 뉴런에서 전기 활동을 기록하는 방법을 보여줍니다. 우리는 성선 자극 호르몬 분비 호르몬 (GnRH의) 신경 세포가 유전자 손상 뇌의 준비 식별을위한 형광 단백질로 태그 된 유전자 변형 물고기를 사용합니다.

비디오: Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

연구

신경과학

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Axons, 수석 및 뇌 슬라이스의 개별 뉴런의 수지상 쪽의 전압에 민감한 염료 기록

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

세포 내 기록, 감각이 필드를 매핑하고, 거머리의 배양 확인 된 뉴런, Hirudo의 medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

온전한 성인 Zebrafish의의 뇌에 전기 생리학 기록

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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 ...

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. ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Thy1를 사용 하 여 기본 Somatosensory 피 질에서 신경 활동을 이미징-GCaMP6s 유전자 변형 쥐

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

자유롭게 수영 물고기에서 이동 식 Tetrodes에 의해 뉴런의 무선 전기 생리적 기록

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...