Voltage-clamp analysis of synaptic transmission at the Drosophila larval neuromuscular junction.
The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Although intracellular recording is particularly valuable in revealing membrane potential changes, it has several 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 minis (spontaneous miniature synaptic potentials) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only at low release conditions when nonlinear summation is not a major issue. The voltage-clamp technique can overcome these limitations by using negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. 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 NMJ preparation.
Zhang
Bing
B
Stewart
Bryan
B
D000818:Animals; D004330:Drosophila / Q000502:physiology*; D009469:Neuromuscular Junction / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D009435:Synaptic Transmission