Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT1A binding sites

We describe the production and characterization of a specific anti-5-HT1A r eceptor antibody made against a fusion protein consisting of glutathione-S- transferase (GST) coupled to a 75-amino acid sequence from the middle porti on of the third intracellular loop (5-HT1A-m3i, serine(253)-arginine(327)) of the rat 5-HT1A receptor protein. This region was chosen to avoid putativ e phosphorylation and glycosylation sites and regions of known homology wit h other 5-HT receptors. Western blot analysis indicated that the polyclonal anti-5-HT1A-m3i antibody accurately recognized the fusion protein expresse d in bacteria and labeled a prominent 67 kDa protein band in the hippocampu s, cortex, brainstem, cerebellum and kidney with a density profile correspo nding to the relative abundance of the 5-HT1A receptor in these tissues. No protein was detected in liver or muscle tissue preparations, and no protei n bands were labeled in any of the above tissues following preabsorption of the antibody with the 5-HT1A-m3i fusion protein. Immunohistochemistry reve aled prominent labeling in limbic structures including the hippocampus, amy gdala, entorhinal cortex, and septum as well as in raphe nuclei. In the hip pocampus, 5-HT1A-m3i labeling revealed a characteristic laminar pattern tha t coincided with that seen by autoradiographic binding of the 5-HT1A agonis t [H-3]-8-OH-DPAT in all strata of the hippocampal formation. In the dorsal and medial raphe nuclei, anti-5-HT1A-m3i antibodies labeled the somatodend ritic membranes of 5-HT neurons, consistent with its role as an autorecepto r. The detailed matching of the anti-5-HT1A-m3i antibody with [H-3]-8-OH-DP AT binding suggests that the antibody recognizes a functionally active form of the 5-HT1A receptor protein capable of binding 5-HT1A agonist ligands. These anti-5-HT1A antibodies mg therefore be useful tools in localizing fun ctional 5-HT1A receptors in specific regions of the brain as well as in stu dying the plasticity and ontogeny of the 5-HT1A receptor at the cellular an d subcellular level. (C) 1999 Elsevier Science B.V. All rights reserved.