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We have previously described long-term survival of isolated bovine chromaffin cell suspension grafts in the periaqueductal gray of adult rats. Electron microscopic analysis of the graft sites revealed synapses on the transplanted chromaffin cells. The origin of these synapses is not known, but they are probably derived from the host since the initial grafts were suspensions of chromaffin cells that were essentially free of other cell types. In order to determine the origin of the observed synapses, retrograde and anterograde tracer analyses were performed on grafted rats at 4 and 8 weeks after transplantation. Following injection of the retrograde tracer (Fluoro-Gold) into graft sites, four major host sites were labeled: hindbrain reticular formation, substantia nigra, lateral hypothalamus, and cingulate cortex. Injection of anterograde tracer (rhodamine-conjugated dextranamine) into the substantia nigra, lateral hypothalamus, and cingulate cortex produced labeled fibers and terminals in and around 4 and 8 weeks old chromaffin cell graft sites. An increase in both the number of retrogradely labeled cells, as well as in the density of anterogradely labeled fibers and terminals within the graft site, was observed from 4 to 8 weeks. This study shows that graft innervation from the host is primarily from areas that normally project afferent fibers to the periaqueductal gray. The increase in labeled fibers and terminals over 8 weeks suggests that de novo synapse formation on grafted bovine chromaffin cells is a continuous process that is dependent on the regenerative capacity and plasticity of the host neuronal network and the grafted bovine chromaffin cells.

Origin of Afferent Projections into Bovine Chromaffin Cell Implants in the Rat Periaqueductal Gray Determined by Retrograde and Anterograde Tracing