Cold Spring Harbor protocols最新文献

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.

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.

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.

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.

The Drosophila larval body wall muscle preparation was first used for electrophysiological analysis in the 1970s. This preparation has become the "gold standard" for studying neuronal excitability as well as synaptic transmission. Here, we first describe the steps for performing intracellular recording from fly larval body wall muscles and then explain how to record and analyze spontaneous and evoked synaptic potentials. Methods used include larval dissection (filleting), identification of muscle fibers and their innervating nerves, the use of the micromanipulator and microelectrode in penetrating the muscle membrane, and nerve stimulation to evoke synaptic potentials.

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.

The Maize Genetics and Genomics Database (MaizeGDB) is the community resource for maize researchers, offering a suite of tools, informatics resources, and curated data sets to support maize genetics, genomics, and breeding research. Here, we provide an overview of the key resources available at MaizeGDB, including maize genomes, comparative genomics, and pan-genomics tools. This review aims to familiarize users with the range of options available for maize research and highlights the importance of MaizeGDB as a central hub for the maize research community. By providing a detailed snapshot of the database's capabilities, we hope to enable researchers to make use of MaizeGDB's resources, ultimately assisting them to better study the evolution and diversity of maize.

Chromatin immunoprecipitation (ChIP) is a technique used to study specific protein-DNA interaction. Briefly, in this technique, antibodies to proteins of interest are used to isolate regions of DNA where these proteins bind. ChIP samples can be processed and analyzed in different ways. One of the approaches for assessing the results of ChIP experiments is quantitative PCR (qPCR). qPCR is used to quantitatively measure the amount of DNA fragments that have been isolated, reflecting the signal of specific proteins interacting with these fragments. This protocol describes both the "percent input" method and the "fold enrichment" method for ChIP-qPCR data analysis using Drosophila tissues as an example. The "percent input" method measures signals of DNA fragments against the input measurement. In contrast, the "fold enrichment" method quantifies the amplified signal strength relative to a background control. Because the quality of primers is critical for the reliability of ChIP-qPCR results, this protocol also describes how to measure primer amplification efficiency using Drosophila genomic DNA.

In vitro antibody evolution is a powerful technique for improving monoclonal antibodies. This can be achieved by generating artificial diversity on an antibody template, which can be done using different in vitro diversification techniques. The resulting libraries consist of single- or multimutant variants of a defined antibody template that are screened for improved function using antibody display. Here, we describe a bioinformatic protocol for tracking synthetic antibody variants using high-throughput sequencing across screening rounds, enabling efficient high-throughput interpretation of the function of individual mutations in sorted antibody display libraries. The protocol enables a user to achieve precision analysis and interpretation of clonal antibody variant data for discovery purposes, especially for high-throughput antibody engineering or optimization against target antigens.

Antibody functional screening studies and next-generation sequencing require careful processing and interpretation of sequence data for optimal results. Here, we provide a detailed protocol for the functional analysis of antibody gene data, including antibody repertoire quantification and functional mapping of high-throughput screening data based on enrichment ratio values, which are a simple way to determine the enrichment of each sequenced antibody after sorting a display library against desired antigens. This protocol enables a user to apply a set of simple yet powerful bioinformatic tools for high-throughput analysis and interpretation of antibody data that is especially well suited for display library screening and for antibody discovery applications.