<ol>
	<li>Papazian, D. M., Schwarz, T. L., Tempel, B. L., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+genomic+and+complementary+DNA+from+Shaker,+a+putative+potassium+channel+gene+from+Drosophila.">Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.</a> Science. 237, 749-753 (1987).</li><li>Tempel, B. L., Papazian, D. M., Schwarz, T. L., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+a+probable+potassium+channel+component+encoded+at+Shaker+locus+of+Drosophila.">Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.</a> Science. 237, 770-775 (1987).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Cell+Recordings+from+Brain+of+Adult+Drosophila.">Whole Cell Recordings from Brain of Adult Drosophila.</a> J. Vis. Exp. (6), e248 (2007).</li><li>Gu, H., Jiang, S. A., Campusano, J. M., Iniguez, J., Su, H., Hoang, A. A., Lavian, M., Sun, X., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cav2-type+calcium+channels+encoded+by+cac+regulate+AP-independent+neurotransmitter+release+at+cholinergic+synapses+in+adult+Drosophila+brain.">Cav2-type calcium channels encoded by cac regulate AP-independent neurotransmitter release at cholinergic synapses in adult Drosophila brain.</a> J. Neurophysiol. 101, 42-53 (2009).</li><li>Baines, R. A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+development+of+central+neurons+in+the+Drosophila+embryo.">Electrophysiological development of central neurons in the Drosophila embryo.</a> J. Neurosci. 18, 4673-4683 (1998).</li><li>Lin, W. H., Wright, D. E., Muraro, N. I., Baines, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+in+the+voltage-gated+sodium+channel+DmNav+regulates+activation,+inactivation,+and+persistent+current.">Alternative splicing in the voltage-gated sodium channel DmNav regulates activation, inactivation, and persistent current.</a> J. Neurophysiol. 102, 1994-2006 (2009).</li><li>Worrell, J. W., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+voltage-dependent+Ca2++currents+in+identified+Drosophila+motoneurons+in+situ.">Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ.</a> J. Neurophysiol. 100, 868-878 (2008).</li><li>Srinivasan, S., Lance, K., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+differences+in+firing+properties+and+potassium+currents+in+Drosophila+larval+motoneurons.">Segmental differences in firing properties and potassium currents in Drosophila larval motoneurons.</a> J. Neurophysiol. , (2011).</li><li>Choi, J. C., Park, D., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+identified+motor+neurons+in+the+Drosophila+third+instar+larva+central+nervous+system.">Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system.</a> J. Neurophysiol. 91, 2353-2365 (2004).</li><li>Pulver, S. R., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spike+integration+and+cellular+memory+in+a+rhythmic+network+from+Na+/K++pump+current+dynamics.">Spike integration and cellular memory in a rhythmic network from Na+/K+ pump current dynamics.</a> Nat. Neurosci. 13, 53-59 (2010).</li><li>Fayyazuddin, A., Zaheer, M. A., Hiesinger, P. R., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nicotinic+acetylcholine+receptor+Dalpha7+is+required+for+an+escape+behavior+in+Drosophila.">The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila.</a> PLoS Biol. 4, e63 (2006).</li><li>Duch, C., Vonhoff, F., Ryglewski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrite+elongation+and+dendritic+branching+are+affected+separately+by+different+forms+of+intrinsic+motoneuron+excitability.">Dendrite elongation and dendritic branching are affected separately by different forms of intrinsic motoneuron excitability.</a> J. Neurophysiol. 100, 2525-2536 (2008).</li><li>Ryglewski, S., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaker+and+Shal+mediate+transient+calcium-independent+potassium+current+in+a+Drosophila+flight+motoneuron.">Shaker and Shal mediate transient calcium-independent potassium current in a Drosophila flight motoneuron.</a> J. Neurophysiol. 102, 3673-3688 (2009).</li><li>Ryglewski, S., Lance, K., Levine, R. B., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cav2+Channels+Mediate+LVA+and+HVA+Calcium+Currents+in+Drosophila+Motoneurons.">Cav2 Channels Mediate LVA and HVA Calcium Currents in Drosophila Motoneurons.</a> J. Physiol. , (2011).</li><li>Ikeda, K., Koenig, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+identification+of+the+motor+neurons+innervating+the+dorsal+longitudinal+flight+muscle+of+Drosophila+melanogaster.">Morphological identification of the motor neurons innervating the dorsal longitudinal flight muscle of Drosophila melanogaster.</a> J. Comp. Neurol. 1273, 436-444 (1988).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Mount+Preparation+of+the+Adult+Drosophila+Ventral+Nerve+Cord+for+Giant+Fiber+Dye+Injection.">Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection.</a> J. Vis. Exp. (52), e3080 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

GFP 같은 형광 단백질과 세포를 시각화 할 때, 너무 많은 빛에 준비를 과잉 노출하지 않는 것이 중요합니다. 이 사진 손상 될 수 있습니다. 우리는 조명을위한 100W HBO 짧은 아크 수은 전구를 사용하며, 우리는 또한 0.8 (채도 ND 필터 0.3과 0.5)의 중간 밀도 (ND)을 ​​사용합니다. 청소 좋은 가시성의 품질을 판단 할 수 있도록하는 것은 매우 중요합니다. 따라서, ND 필터는 여러 번에 20 s의 짧은 기간 동?...

<table border="1"><tbody><tr><td> 에이전트 / 항목 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 단백 분해 효소 타입 XIV </td><td> 시그마 알드리치 USA </td><td> P5147 </td></tr><tr><td> Microfil 유연한 주입 바늘 </td><td> 세계 정밀 계측기 USA </td><td> MF28G-5 </td></tr><tr><td> 붕규산 유리 모세 혈관, OD 1.5 mm, ID 1.0 mm, 아니 필라멘트 </td><td> 세계 정밀 계측기 USA </td><td> PG52151-4 </td></tr><tr><td> DiI </td><td> Invitrogen USA </td><td> D3899 </td></tr><tr><td> Sylgard 엘라스토머 키트 184 (다우 코닝) </td><td> <a href="http://www.ellsworth.com" target="_blank">www.ellsworth.com</a> </td><td> 184 SIL ELAST KIT </td></tr><tr><td> ND 필터 세트 (언 마운트) </td><td> 크로마 </td><td> 22000b 시리즈 </td></tr><tr><td> 전극 홀더 1-HL-U </td><td> 모lecular 장치 </td><td> 1-HL-U </td></tr></tbody></table>

preparation of drosophila central neurons for in situ patch clamping

짧은 세대 시간 및 손쉬운 유전자 기술은 과일 파리 Drosophila melanogaster 기초 신경 과학 연구의 훌륭한 유전자 모델을 확인합니다. 그들은 neuronal 흥분을 중재 이후 이온 채널은 모든 행동의 기초입니다. 복제 제 1 전압 개폐 이온 채널은 Drosophila 전압 개폐 칼륨 채널 쉐이커 1,2했다. 신경계 기능에 대한 이온 채널과 막 흥분의 역할을 이해하는 방향으로는 현장 패치 클램프 녹음에서와 Drosophila에서 강력한 유전 도구를 사용할 수 결합 할 때 유용합니다. 수년 동안 이러한 녹음은 Drosophila의 CNS의 작은 크기에 방해되었습니다. 또한, glia 및 콜라겐으로 만든 강력한 집은 중앙 뉴런에 패치 피펫 액세스에 대한 장애물을 형성. 이 집의 제거는 성인 Drosophila의 CNS에서 뉴런의 패치 클램프 녹음에 필요한 전제 조건이다. 최근 몇 년과학자들은 성인 뇌 3,4 및 배아 5,6, 애벌레의 7,8,9,10, 그리고 성인 Drosophila 11,12,13,14의 복부 신경 코드를 뉴런에서 현장 패치 클램프 녹음에서 수행 할 수있었습니다. 안정적인 기가 - 씰은 좋은 패치의 주요 전제 조건이며, 누설 전류를 방지하기 위해 세포막과 패치 피펫의 깨끗한 접촉에 따라 달라집니다. 따라서 성인 Drosophila의 뉴런에서 현장 패치 클램프 녹음에서 전체 셀 철저하게 세척해야합니다. 첫 번째 단계에서, ganglionic 집은 효소 처리 할 수​​ 있으며 기계적으로 대상 셀에 액세스 할 수 있도록하기 위해 제거. 두 번째 단계에서, 세포막은 glia, 콜라겐 또는 기타 자료의 어떤 항목은 기가 - 씰 형성을 방해하지 수 있도록 연마되어야한다. 이 문서는 체세포의 전체 C의 Drosophila 복부 신경 코드, 비행 motoneuron 5 (MN5 15)에서 확인 중앙 신경 세포를 준비하는 방법에 대해 설명합니다예쁜 패치 클램프 녹음. 신경 세포의 식별 및 가시성이 MN5에서 GFP의 대상 표현에 의해 달성된다. 우리는 패치 클램프 기술 자체를 설명하는 것을 목표로하지 않습니다.

Watch this Scientific Journal Video about Preparation of Drosophila Central Neurons for in situ Patch Clamping at JoVE.com

Preparation of Drosophila Central Neurons for in situ Patch Clamping

현장 패치에 클램프 녹음은 그대로 회로의 뉴런의 electrophysiological 특성에 사용됩니다. CNS는 작고 강력한 피복에 둘러싸여 있기 때문에 Drosophila 유전자 모델 패치를 고정으로 어렵습니다. 이 문서는 이후의 패치 클램프 녹음의 집과 깨끗한 뉴런을 제거 할 수있는 절차에 대해 설명합니다.

의 작성<em&gt; Drosophila</em에 대한&gt; 중부 뉴런<em&gt; 현장에서</em&gt; 패치 클램핑

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

preparation-of-drosophila-central-neurons-for-in-situ-patch-clamping

Research

JoVE Journal

Neuroscience

Preparation of Drosophila Central Neurons for in situ Patch Clamping

14.0K 조회수.  Arizona State University. 현장 패치에 클램프 녹음은 그대로 회로의 뉴런의 electrophysiological 특성에 사용됩니다. CNS는 작고 강력한 피복에 둘러싸여 있기 때문에 Drosophila 유전자 모델 패치를 고정으로 어렵습니다. 이 문서는 이후의 패치 클램프 녹음의 집과 깨끗한 뉴런을 제거 할 수있는 절차에 대해 설명합니다.

비디오: Preparation of Drosophila Central Neurons for in situ Patch Clamping

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

연구

신경과학

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

어두운 적응 슬라이스 준비에 마우스 망막 뉴런에서 패치 클램프 레코딩

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

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 ...

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

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

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

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

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

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

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

급성 척수 조각 Substantia Gelatinosa 신경에 있는 전체 셀 패치 클램프 기록에 대 한 준비

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

시투 혼성화에서 멀티플렉스를 사용하여 마우스 Vagal afferent 뉴런에서 G 단백질 결합 수용체 발현의 검출

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

Drosophila 중앙 두뇌에 있는 성인 신경 발생을 자극하고 분석합니다

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...