A PRIMARY-CELL CULTURE SYSTEM OF LUTEINIZING-HORMONE-RELEASING HORMONE NEURONS DERIVED FROM EMBRYONIC OLFACTORY PLACODE IN THE RHESUS-MONKEY

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH c ells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 wer e delivered surgically and the area including the olfactory placode (P L) and the areas that encompass the migratory pathway (MP) were dissec ted out. The tissues were cut into small pieces and plated on collagen - or poly-L-lysine-coated glass coverslips in medium M199. Cultures we re maintained for up to 33 days and immunostained for LHRH, GnRH-assoc iated peptide, neurofilament protein, neuron-specific enolase, and gli al fibrillary acidic protein. LHRH positive cells were also positive f or neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the fi rst week of culture, LHRH cells remained within the explants of PL, we re rounded (average dimensions: 13.0 x 11.3 mum) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 mum) with neuri tes started to migrate out from PL explants, whereas some still remain ed in the PL. By the third week a large number of LHRH cells (19.3 x 9 .4 mum) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 mum in len gth. A few ''pioneer'' LHRH cells appeared to lead the migration of 10 0-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants . LHRH cells from the ventral part of the MP, which is close to the PL , migrated out by 1-2 weeks and formed several migratory paths, wherea s LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were ext ended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) pri mary LHRH neurons can be obtained from the embryonic PL and their migr atory pathway, 2) these neurons migrate and mature in culture and 3) t hey are accessible for cellular and molecular studies.