DEXTRAN BACKFILL TRACERS COMBINED WITH LUCIFER-YELLOW INJECTIONS FOR NEUROANATOMICAL STUDIES OF THE LEECH HEAD GANGLION

Several neuronal tracing substances were applied to the cut ends of le ech cephalic nerves and the resulting backfills into the subesophageal ganglion (sbEG) were mapped. A 12 h incubation in 3 kDa dextrans conj ugated either to a fluorochrome or to biotin (subsequently tagged with peroxidase) was satisfactory. In separate experiments, possible targe ts of cephalic nerve afferents (R3 Retzius neurons) were injected with Lucifer Yellow (LY) to visualize their projections. Comparison of the LY-R3 Retzius neuron map with that of the dextran-backfilled D-1 nerv e revealed extensive overlap in the sbEG. Experiments were performed c ombining the two protocols. confirming this observation. Moreover, con focal microscopy placed D-1 nerve processes in close proximity to R3 R etzius neuron processes, suggesting that they could make synaptic cont act with one another in the sbEG. With modifications, this method coul d be used to identify such contacts using electron microscopy.