A classic improved: minor tweaks yield major benefits in crayfish slow-flexor preparations

Action potentials and the postsynaptic potentials they evoke fill the pages of neuroscience textbooks, but students have relatively few opportunities to record these phenomena on their own. However, the act of making such recordings can be key events in a student's scientific education. The crayfish abdominal slow flexor muscle system is a well-established platform for recording spikes and PSPs. It enables students to see nerves and the muscles they innervate, record spontaneous spikes from several motor axons in these nerves as well as PSPs in their postsynaptic muscle fibers, and interpret these recordings quantitatively. Here we describe an improved method for preparing the slow-flexor system for recording that employs transmitted illumination through the stereo microscope's conventional substage lighting. Oblique transmitted lighting allows students to see the nerve and muscles fibers in each segment clearly and position recording electrodes accurately under visual control. Because students can see the nerves, muscles, and recording electrodes, broken electrode tips are relatively uncommon and the first successful recordings come more quickly. Many kinds of neurons in the CNS have the same pattern of multineuronal, multiterminal innervation that occurs on these muscle fibers. To visualize these innervation patterns on these fibers, we describe an immunohistochemical protocol that labels the GABAergic inhibitory motor axon and all the synaptic vesicles in the synaptic terminals on these muscle fibers. Dual-color images reveal extensive branching of the axons and fields of presynaptic terminals, only some of which are double-labeled for GABA.