Immunolabeling of the rat central nervous system with antibodies partiallyselective of the short form of the 5-HT3 receptor

Polyclonal antibodies were raised against a synthetic hexadecapeptide corre sponding to the portion of the second intracytoplasmic loop of the short fo rm of the mouse 5-hydroxytryptamine-3(A) receptor subunit (5-HT(3)A-S), whi ch differs from the long form (5-HT(3)A-L) by the removal of six amino acid s. Antibodies were detected by enzyme-linked immunosorbent assay as soon as two months after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoblot detection of fusion proteins comprisi ng glutathione-S-transferase and the second intracellular loop of 5-HT(3)A- S or 5-HT(3)A-L, and immunoprecipitation of cloned receptors showed that an tibodies exhibited some selectivity for the short variant. Affinity chromat ography allowed the purification of selective anti-5-HT(3)A-S antibodies wh ich yielded a strong positive labeling of plasma membrane, reticulum and Go lgi apparatus of COS-7 cells expressing murine 5-HT(3)A-S. In contrast, COS -7 cells expressing similar levels of 5-HT(3)A-L exhibited only a very weak labeling. Selectivity was also observed on immunoblots of cloned receptors transiently expressed in COS-7 cells, or stably expressed in CHO cells, bo th systems showing an immunolabeled component at 53,000-54,000 mel. wt. Imm unoautoradiographic labeling of central nervous system sections showed that 5-HT(3)A-S-like immunoreactivity was found mostly within the nucleus of th e solitary tract, the nucleus of the spinal tract of the trigeminal nerve, and the dorsal horn of the the spinal cord in the rat. After unilateral abl ation of the nodose ganglion, 5-HT(3)A-S-like immunoreactivity decreased ma rkedly in the ipsilateral part of the nucleus of the solitary tract, as exp ected of the presynaptic localization of 5-HT3 receptors. Finally, immunohi stochemistry at the light and electron microscope levels revealed that 5-HT (3)A-S-like immunoreactivity was associated essentially with terminals and axonal profiles. All these results demonstrate that the immunolabeling exhibited by these an tibodies is consistent with a specific and partially selective recognition of the short isoform of the 5-HT(3)A subunit. Because the pattern of immuno autoradiographic labeling matches the distribution previously established w ith selective radioligands, it can be inferred that these antibodies probab ly recognized the same fully assembled form of the 5-HT(3)A-S receptor subu nit. (C) 1999 IBRO. Published by Elsevier Science Ltd.