Chloride‐dependent binding sites for L‐[ 3 H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

Dendrodendritic synapses occur between granule cell dendrites and secondary dendrites of mitral cells within the olfactory bulb and are attainable in a subcellular fraction (DDS). Since the mitral cells are thought to utilize an excitatory amino acid as a neurotransmitter, we determined the pharmacologic specificity of Na + ‐independent L‐[ 3 H]glutamate binding to fresh membrances of DDS in 50 mM Tris‐HCI, pH 7.1. Binding of L‐glutamate to membrances of DDS was specific, CI − dependent, and saturable. Scatchard plots were analyzed by nonlinear regression analysee using the computer program LIGAND, and the data was best‐fitted to a one‐site model with K D of 0.56 ± 0.04 μM and an apparent B max of 48 ± 5 pmol/mg protein. Hill plots also indicated the presence of one site and no cooperativity (n H = 0.99 ± 0.03). However, the relative effictiveness of several compounds in inhibiting L‐glutamate binding to membranes of DDS clearly demonstrated the presence of more than one site. Electrophysiological studies suggest that 2‐amino‐4‐phosphonobutyrate (APB) is a potent antagonist of evoked responses elicited by simulation of mitral cell axons and that quisqualate is a potent agonist; both of these compounds were highly effective inhibitors of L‐glutamate binding to DDS membrances. APB displaced about 70% of tke sites labeled with 200 nM L‐glutamate with a K 1 of 1.6 μM, whereas quisqualate inhibition of L‐glutamate binding yielded a line that was curvilinear in the Scatchard plot and was resolved into two sites of relatively high affinity (K 1 values of 0.02 and 0.65 μM). Several other compounds, including N‐methyl‐D‐aspartate and kainate, were relatively ineffective inhibitors. Although a small amount of CI − ‐independent L‐glutamate binding was demonstrable in the preparation, CI − ‐dependent sites predominated and were stimulated by clacium. These results suggest an important role of L‐glutamate, or a closely related compound, at dendrodendritic synapses of the olfactory bulb.