PRODUCTION AND CHARACTERIZATION OF POLYCLONAL ANTIBODIES RECOGNIZING THE INTRACYTOPLASMIC 3RD-LOOP OF THE 5-HYDROXYTRYPTAMINE(1A) RECEPTOR

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine,, (serotonin) receptor was subclon ed into the vector pGEX-KG and expressed in Escherichia coli as a fusi on protein coupled with the glutathione S transferase of Schistosoma j aponicum. The fusion protein was purified on a glutathione-agarose aff inity column and used to immunize rabbits for the production of polycl onal anti-5-hydroxytryptamine(1A) receptor antibodies. Enzyme-linked i mmunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster inje ction. These antibodies only marginally affected the specific binding of [H-3]8-hydroxy-2-(di-n-propylamino) tetralin to solubilized and mem brane bound 5-hydroxytryptamine(1A) receptors, and did not interfere w ith serotonin-induced inhibition of forskolin-stimulated adenylate cyc lase negatively coupled to 5-hydroxytryptamine(1A) receptors in rat hi ppocampal membranes. However, antibodies were able to immunoprecipitat e 5-hydroxytryptamine(1A) receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labe lling and immunohistochemical staining of rat brain sections exposed t o the antibodies raised against the fusion protein superimposed to tha t of 5-hydroxytryptamine(1A) receptor binding sites labelled by specif ic radioligands, with marked enrichment in the limbic areas (dentate g yrus and CA1 area in the hippocampus, lateral septum, entorhinal corte x) and the anterior raphe nuclei. The differential cellular location o f immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucl eus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine(1A). receptors mig ht differ from one neuronal cell type to another.