Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT 1A binding sites

Abstract

We describe the production and characterization of a specific anti-5-HT 1A receptor antibody made against a fusion protein consisting of glutathione- S-transferase (GST) coupled to a 75-amino acid sequence from the middle portion of the third intracellular loop (5-HT 1A-m3i, serine 253–arginine 327) of the rat 5-HT 1A receptor protein. This region was chosen to avoid putative phosphorylation and glycosylation sites and regions of known homology with other 5-HT receptors. Western blot analysis indicated that the polyclonal anti-5-HT 1A-m3i antibody accurately recognized the fusion protein expressed in bacteria and labeled a prominent 67 kDa protein band in the hippocampus, cortex, brainstem, cerebellum and kidney with a density profile corresponding to the relative abundance of the 5-HT 1A receptor in these tissues. No protein was detected in liver or muscle tissue preparations, and no protein bands were labeled in any of the above tissues following preabsorption of the antibody with the 5-HT 1A-m3i fusion protein. Immunohistochemistry revealed prominent labeling in limbic structures including the hippocampus, amygdala, entorhinal cortex, and septum as well as in raphe nuclei. In the hippocampus, 5-HT 1A-m3i labeling revealed a characteristic laminar pattern that coincided with that seen by autoradiographic binding of the 5-HT 1A agonist [ 3 H ]-8-OH-DPAT in all strata of the hippocampal formation. In the dorsal and medial raphe nuclei, anti-5-HT 1A-m3i antibodies labeled the somatodendritic membranes of 5-HT neurons, consistent with its role as an autoreceptor. The detailed matching of the anti-5-HT 1A-m3i antibody with [ 3 H ]-8-OH-DPAT binding suggests that the antibody recognizes a functionally active form of the 5-HT 1A receptor protein capable of binding 5-HT 1A agonist ligands. These anti-5-HT 1A antibodies may therefore be useful tools in localizing functional 5-HT 1A receptors in specific regions of the brain as well as in studying the plasticity and ontogeny of the 5-HT 1A receptor at the cellular and subcellular level.