Crustacean Motor Neuron Connections Traced by Backfilling for Electron Microscopy

Individual neurons can be marked by injection through a microelectrode of the fluorescent dye Procion yellow (17) or of cobalt chloride (13). These materials can also be used to study neurons which have peripheral connections by allowing the markers to move up the cut axons into the cell bodies and central processes (5, 11). This "backfilling" method is facilitated by application of electrical current through the cut ends of the axons (5). So far, both Procion yellow and cobalt chloride have been more useful for studies carried out by light microscopy than for those requiring electron microscopy, although attempts have been made to trace Procion yellow-injected cells with the electron microscope (14). The microinjection of Procion brown instead of Procion yellow (2) and a modification of the cobalt chloride method (3) have made electron micrscope studies of injected neurons more feasible. In addition, the modified cobalt chloride method can be used to trace backfilled motor neurons in the central nervous system and to study their central connections with other neurons. The present paper was written for three purposes: (a) to show that the modified cobalt chloride method can be combined with the simple backfilling technique to trace motor neurons into the neuropile; (b) to show that both light and electron microscopy can be employed on a specimen to trace the motor neurons; (c) to demonstrate the