A primary culture system of rat olfactory bulb forming many synapses similar to intact ones and spontaneously generating synchronous intracellular calcium oscillations

Previously, several studies attempting to analyze olfactory functions using dissociated culture systems of the olfactory bulb (OB) have been reported. Reciprocal dendrodendritic synapses between secondary neurons (mitral/tuft ed cells) and interneurons (periglomerular/granule cells) are considered to play the most important role in signal processing in the OB. However, it i s unclear whether these reciprocal synapses are formed in vitro in the same way as they are in the intact OB. Thus, we synaptologically investigated t he nature of cultured OB neurons. These neurons from embryonic rats were cl assified into four groups based on the size of their somata and their gluta mic acid decarboxylase (GAD) immunoreactivity At 14 days in vitro, most of the neurons synchronously showed spontaneous intracellular Ca2+ oscillation s that were reversibly inhibited by application of D-APV and CNQX, Moreover , the frequency of the oscillations decreased and their amplitude became la rger following application of bicuculline. These results suggest functional glutamatergic synaptic coupling and inhibitory GABAergic synaptic modulati on. Immunocytochemical staining revealed many dot-like products (puncta) th at were immunoreactive to GAD as well as to synaptophysin surrounding the c ultured neurons. These results strongly indicate the presence of GABAergic synapses. The existence of synaptic contacts in OB neuron cultures was also confirmed by electron microscopy. Two types of synapses, symmetrical and a symmetrical, were morphologically recognizable. Moreover, we could also ide ntify peculiar synapses resembling the in vivo reciprocal dendrodendritic s ynapses. The use of these primary culture systems will facilitate the eluci dation of mechanisms underlying olfactory functions.