Cold Spring Harbor protocols最新文献

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction. 果蝇幼虫神经肌肉接头处突触传递的电压钳分析

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108132

Bing Zhang, Bryan Stewart

{"title":"Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108132","DOIUrl":"10.1101/pdb.prot108132","url":null,"abstract":"Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a \"ceiling effect,\" because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108132"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Electrophysiological Recording from a "Model" Cell. 模型 "细胞的电生理记录

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108130

Bing Zhang, Bryan Stewart

{"title":"Electrophysiological Recording from a \"Model\" Cell.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108130","DOIUrl":"10.1101/pdb.prot108130","url":null,"abstract":"The muscle cell or neuron membrane is functionally equivalent to a resistor-capacitor (RC) circuit with the membrane resistance and capacitor in parallel. Once inserted inside the membrane, an electrode introduces a serial resistance and small capacitance to the RC circuit. Through a narrow opening at its tip (∼0.1-μm), current can pass through the electrode, into the cell, and back to the outside (ground) across the membrane to complete the circuit. This arrangement enables a voltage difference between the outside and inside of the cell membrane to be recorded. To determine cell membrane properties, a current can be injected into the cell through the electrode. One complication with this approach, however, is that the voltage difference measured with the electrode includes the voltage drop across the cell membrane and that across the electrode. Furthermore, a small amount of current is drawn by the electrode capacitor, thereby slowing the current flow across the membrane. Fortunately, most amplifiers are equipped with bridge balance and capacitance compensation functions so that the effects of the electrode on cell membrane properties can be canceled out or minimized. This protocol describes the basics of setting up and conducting electrophysiological experiments using a model cell. For the novice, a model cell provides a way to learn the operation of electrophysiology equipment and software without the anxiety of damaging living cells. This protocol also illustrates passive membrane properties such as the input resistance, capacitance, and time constant.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108130"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording in Drosophila. 用于果蝇电生理记录的微电极、抽吸电极和病灶电极的制作。

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108134

Bing Zhang, Bryan Stewart

{"title":"Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes for Electrophysiological Recording in Drosophila.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108134","DOIUrl":"10.1101/pdb.prot108134","url":null,"abstract":"Electrophysiological recording is a group of techniques used to record electrical field potentials generated by cells. These techniques rely on several types of electrodes, which can be manufactured in the laboratory. In intracellular recording, glass microelectrodes are used to pierce the cell membrane, and then to measure the electrical potential difference between the inside and the outside of the cell. Another technique, called loose patch or focal recording, is similar to intracellular recording but the electrode tip does not pierce into the cell membrane. Rather, the electrode tip is placed near a nerve or the postsynaptic side of the neuromuscular junction (NMJ) to record extracellular changes in local potentials. A third technique involves a suction electrode, which is used to draw part of the motor nerve into the electrode so that electrical pulses can be applied to elicit action potentials of the nerve. Suction electrodes are specifically used to evoke synaptic potentials at the Drosophila larval NMJ. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108134"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Synaptic Electrophysiology of the Drosophila Neuromuscular Junction. 果蝇神经肌肉接头的突触电生理学

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.top107820

Bing Zhang, Bryan Stewart

{"title":"Synaptic Electrophysiology of the Drosophila Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.top107820","DOIUrl":"10.1101/pdb.top107820","url":null,"abstract":"Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal experiences an influx of calcium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neurotransmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SVs at presynaptic terminals. The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic features of synaptic transmission, as well as the molecular mechanisms regulating the SV cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ is an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Here, we describe the theory and practice of electrophysiology as applied to the Drosophila larval NMJ preparation. We introduce the basics of membrane potentials, with an emphasis on the resting potential and synaptic potential. We also describe the equipment and methods required to set up an electrophysiology rig.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107820"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Recording from Drosophila Larval Body Wall Muscles: Passive Membrane Properties and Basic Features of Synaptic Transmission. 果蝇幼虫体壁肌肉的记录：被动膜特性和突触传递的基本特征。

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108131

Bing Zhang, Bryan Stewart

引用次数: 0

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction. 果蝇幼虫神经肌肉接头处单个突触电流的聚焦记录

Cold Spring Harbor protocols Pub Date : 2025-02-03 DOI: 10.1101/pdb.prot108133

Bing Zhang, Bryan Stewart

{"title":"Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Larval Neuromuscular Junction.","authors":"Bing Zhang, Bryan Stewart","doi":"10.1101/pdb.prot108133","DOIUrl":"10.1101/pdb.prot108133","url":null,"abstract":"Focal recording is an extracellular method for studying synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all the boutons as occurs with intracellular recording or two-electrode voltage-clamp. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons. A compound microscope with epifluorescent imaging capability is very helpful but not essential; any GFP Drosophila strain that labels the nerve terminal or synaptic boutons can be used to locate the boutons. A particularly useful strain is Mhc-CD8-Sh-GFP, containing a GFP molecule that is expressed in muscle, localizes to the postsynaptic apparatus, and outlines boutons. Vital fluorescent dyes (such as 4-Di-2-Asp) may also be applied to the dissected preparation to help locate boutons. The microscope should be equipped for differential interference contrast (DIC or Nomarski) optics if fluorescence is not used.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108133"},"PeriodicalIF":0.0,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140189535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tools and Resources at the Maize Genetics and Genomics Database (MaizeGDB). 玉米遗传学和基因组学数据库（MaizeGDB）的工具和资源。

Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.over108430

Margaret R Woodhouse, Ethalinda K Cannon, John L Portwood, Jack M Gardiner, Rita K Hayford, Olivia Haley, Carson M Andorf

引用次数: 0

Performing Quantitative PCR after Chromatin Immunoprecipitation (ChIP) of Drosophila Antennal and Brain Samples. 在果蝇触角和大脑样本的染色质免疫沉淀 (ChIP) 之后进行定量 PCR。

Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108143

Chengcheng Du, Pelin Volkan

引用次数: 0

Clonal Variant Analysis of Antibody Engineering Libraries. 抗体工程库的克隆变异分析。

Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108626

Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky

引用次数: 0

Antibody Data Analysis from Diverse Immune Libraries. 来自不同免疫库的抗体数据分析

Cold Spring Harbor protocols Pub Date : 2025-01-02 DOI: 10.1101/pdb.prot108627

Ahmed S Fahad, Matías F Gutiérrez-Gonzalez, Bharat Madan, Brandon J DeKosky

引用次数: 0