Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets

Abstract

Previously we developed a co-culture system of cultured accessory olfactory bulb (AOB) neurons with partially dissociated vomeronasal neurons, referred as vomeronasal pockets (VP), to explore mechanisms of specific synapse formation between sensory and first relay station of the vomeronasal system. However, it was unclear whether fibers from VP formed functional synaptic contacts with AOB neurons. To address this issue, we examined the synapse formation between cultured VP and AOB neurons using a calcium imaging technique combined with electrical stimulation.
After 7 days in culture, a few bundles of fibers from a spherical structure of VP extending to the AOB neurons were observed. After 21 days in culture, significant changes in the fluorescence intensity of AOB neurons loaded with a calcium indicator, fluo-4, were observed when VP was electrically stimulated with a bipolar electrode. When a VP was stimulated, some AOB neurons immediately showed transient Ca²⁺ increases. These responses of AOB neurons were reversibly suppressed by the application of 10μM CNQX; the Ca²⁺ transients disappeared in some neurons and diminished in a few neurons. The application of 25μM APV resulted in the suppression of spontaneous Ca²⁺ increases observed repetitively in many cultured AOB neurons and partially affected evoked Ca²⁺ transients. The application of both CNQX and APV completely blocked Ca²⁺ transients evoked by VP stimulation. These results suggest the formation of functional synapses between the VP and AOB neurons in the culture. [Jpn J Physiol 54 Suppl:S161 (2004)]