Implanted embryonic sensory neurons project axons toward adult auditory brainstem neurons in roller drum and Stoppini co-cultures

Previously we have shown in vivo the survival, migration and integration of embryonic dorsal root ganglion (DRG) neurons that were grafted into the inner ear and peripheral auditory nervous system. In order to evaluate relevant factors determining integration of sensory neurons further into the central auditory nervous system, complementary in vitro techniques are necessary. The advantages of in vitro systems are that a large number of factors including various grafts and different conditions can be efficiently examined for. Hence, we co-cultured 300 μm thick postnatal rat brainstem slices containing the cochlear nucleus including the central part of the 8th cranial nerve with mouse embryonic DRG neurons. The organotypic co-cultures were either grown on coverslips using the roller drum method described by Gähwiler or on membranes according to the interface method described by Stoppini. Neurons in the cochlear nucleus were labeled with DiI. The results demonstrate that (1) brainstem slices survive for up to 5 weeks in culture, and that (2) co-cultures of embryonic sensory neurons and brainstem show a high degree of neuronal survival, and that (3) survival and axonal outgrowth from the implanted embryonic neurons are dependant on the presence of the brainstem slice rather than on exogenous NGF and that (4) implanted embryonic neurons send axons toward neurons in the cochlear nucleus.