Dextran backfill tracers combined with Lucifer Yellow injections for neuroanatomic studies of the leech head ganglion

Abstract

Several neuronal tracing substances were applied to the cut ends of leech cephalic nerves and the resulting backfills into the subesophageal ganglion (sbEG) were mapped. A 12 h incubation in 3 kDa dextrans conjugated either to a fluorochrome or to biotin (subsequently tagged with peroxidase) was satisfactory. In separate experiments, possible targets 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 nerve revealed extensive overlap in the sbEG. Experiments were performed combining the two protocols, confirming this observation. Moreover, confocal microscopy placed D 1 nerve processes in close proximity to R3 Retzius neuron processes, suggesting that they could make synaptic contact with one another in the sbEG. With modifications, this method could be used to identify such contacts using electron microscopy.