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>

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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><tbody><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>Micr&amp;#8211;lectrode 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 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1.2 MgCl&lt;sub&gt;2&lt;/sub&gt;; 10 HEPES </td></tr><tr><td>Electrode solution (loose-patch) </td><td></td><td></td><td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1.3 MgCl&lt;sub&gt;2&lt;/sub&gt;; 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 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1 MgCl&lt;sub&gt;2&lt;/sub&gt;; 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

Universidad de Cartagena UDEC

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

애벌레의 Zebrafish의 Forebrain Electrophysiological 기록

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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 ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

생물학

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish의<em&gt; 시츄</em&gt; 척수 척추 감각과 운동 뉴런 전기 생리 녹음 준비

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

형질전환 제브라피시 유충에서 먹이가 유발하는 뉴런 활동 분석

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Adult Zebrafish Electrophysiology: A Method to Measure the Extracellular Electrical Activity of Neurons In Vivo

Source: Johnston, L., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/51065/" target="_blank">Electrophysiological Recording in the Brain of Intact Adult Zebrafish</a>. &#160;J. Vis. Exp. (2013)
The protocol describes the procedure to perform electrophysiology in adult zebrafish. This method can be used for measuring neural activity in vivo.

성체 제브라피시 전기생리학: 생체 내 뉴런의 세포 외 전기 활동을 측정하는 방법

Source: Johnston, L., et al. Electrophysiological Recording in the Brain of Intact Adult Zebrafish. &#160;J. Vis. Exp. (2013)
The protocol describes the ...

형질 전환 생선의 본래 뇌에서 발견 뉴런의 전기적 활동을 기록

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