Acetylcholinesterase and luteinizing hormone-releasing hormone distinguish separate populations of terminal nerve neurons

The terminal nerve is composed of a morphologically heterogeneous population of unipolar, bipolar and multipolar neurons located in the nasal and intracranial cavities of vertebrates. The question has arisen as to whether these neurons are neurochemically heterogeneous and therefore possibly functionally different as well. Among the substances localized in the terminal nerve are acetylcholinesterase and luteinizing hormone-releasing hormone-like immunoreactive material. We have developed a double-label procedure, combining immunocytochemistry and enzyme histochemistry to determine whether these two substances are localized within different populations of terminal nerve neurons. Compatibility of the two procedures was accomplished by modifications of the fixative and primary antibody solutions. In the immunocytochemical step, the avidin-biotin-peroxidase complex coupled to a new chromogen, Chromo-red, produced a bright red reaction product in neurons containing luteinizing hormone-releasing hormone-like material. This reaction product was easily differentiated from the black silver-intensified acetylcholinesterase label. In both neonatal and adult preparations, a large population of terminal neurons contained the acetylcholinesterase label only, whereas a smaller population contained both acetylcholinesterase and luteinizing hormone-releasing hormone-like material. The acetylcholinesterase-containing population of neurons was concentrated peripherally and included multipolar neurons. In contrast neurons with the two substances co-localized were unipolar or bipolar and were concentrated centrally. The simultaneous visualization of acetylcholinesterase and luteinizing hormone-releasing hormone-like material in the same tissue section enable the differentiation of two separate neurochemically defined populations of terminal neurons. The distribution of these two neuronal types was the same in neonatal and adult animals. These data provide support for a functional diversity of terminal neurons.