Characterization and regional distribution of glutamatergic and cholinergic ligand binding sites in goldfish brain

Abstract

The binding of several ligands that selectively interact with glutamate receptor subtypes was characterized in extensively washed synaptosomal membrane preparations from goldfish brain. The binding affinity (K(d)), an estimate of the number of sites (B(max)), the rank-order potency of glutamatergic ligands at inhibiting binding, and the regional localization of binding sites were determined. In whole brain preparations, 3H-kainate had a K(d) of 136 nM and a B(max) of 63 pmol/mg protein, 3H-AMPA had a K(d) of 26 nM and a B(max) of 0.4 pmol/mg protein, and 3H-L-glutamate bound with an apparent affinity of 323 nM and a B(max) of 5 pmol/mg protein. Most of the binding sites for each of the glutamate analogs were present in the cortex and the fewest were in the cerebellum, except for 3H-kainate binding sites, which were most prevalent in the cerebellum and least abundant in the cortex. The proposed neuronal nicotinic acetylcholine receptor (nAChR) ligands 3H(-)nicotine and 125I-α-Bgt were also investigated. 125I-α-Bgt had a K(d) of 0.08 nM and a B(max) of 132 fmol/mg protein. 3H-(-)nicotine did not bind to the extensively washed membrane preparations, so a less stringently washed P2 tissue fraction was used. In this tissue preparation, 3H-(-)nicotine had a K(d) of 9 nM and a B(max) of 84 fmol/mg protein. Eye removal resulted in a time-dependent decrease in the number of 3H-(-)nicotine and 125I-α-Bgt binding sites in the contralateral optic tectum, but no reduction in the number of binding sites for any of the glutamatergic ligands. The results suggest that both 3H-(-)nicotine and 125I-α-Bgt binding sites are similarly regulated in the optic tectum, which supports previously reported data indicating that the binding sites are located on closely related proteins or may, at least partially, be colocalized on the same protein (Henley and Oswald, 1987). The data for glutamatergic ligands do not preclude a role for glutamatergic receptors in the retinotectal system of goldfish, but indicate that the receptor numbers are not regulated by removal of visually evoked neuronal stimulation.