Neurons in the sacral parasympathetic nucleus that project to the hypothalamus do not also project through the pelvic nerve--a double labeling study combining Fluoro-gold and cholera toxin B in the rat.

We recently reported that neurons in the sacral parasympathetic nucleus (SPN) project directly to the hypothalamus. In the present study, we examined the possibility that individual neurons in SPN send both an axon into the pelvic nerve and an ascending projection to the hypothalamus. We used a new double-labeling technique in which two sensitive retrograde tracers (Fluoro-gold and cholera toxin subunit B immunocytochemically stained with rhodamine-labeled antibodies) were combined. The effectiveness of this combination for singly and doubly labeling neurons was established in experiments in which both tracers were injected into overlapping areas of the tongue or ventrobasal thalamus. These injections doubly labeled large numbers of neurons in the hypoglossal or dorsal column nuclei, respectively. In studies of the projections of neurons in the SPN, injection of one tracer into the hypothalamus and the other into the pelvic nerve and/or pelvic ganglion singly labeled many neurons (more than 3300 in the 7 examined cases). However, no SPN neurons were doubly labeled. These findings indicate that the SPN in the rat consists of at least two distinct groups of cells, parasympathetic preganglionic neurons and neurons that project to the hypothalamus.