DISTRIBUTION OF THE 5-HYDROXYTRYPTAMINE(2C) RECEPTOR PROTEIN IN ADULT-RAT BRAIN AND SPINAL-CORD DETERMINED USING A RECEPTOR-DIRECTED ANTIBODY - EFFECT OF 5,7-DIHYDROXYTRYPTAMINE

A synthetic peptide, corresponding to the N-terminal decapeptide (+(YC 12)-C-11) of the rat 5-hydroxytryptamine(2C) (5-HT2C) receptor protein was used to produce a sheep polyclonal antiserum. Western blot analys is showed that the resultant antibody G241 recognised two membrane pro teins, one (55 kDa) approximating the molecular mass of the 5-HT2C rec eptor (52 kDa) and a second (63 kDa), which may be a glycosylated form of the receptor protein. HEK 293 cells transfected with human 5-HT2C cDNA displayed intense cell surface immunoreactivity with the 5-HT2C a ntiserum, which was completely prevented by incubating the antibody wi th the synthetic 5-HT2C peptide (10 mu M), whilst neither nonimmune se rum nor untransfected cells displayed any immunoreactivity. A radioimm unoassay was developed to quantify the regional distribution of 5-HT2C -like immunoreactivity (LI) in the adult rat brain. The choroid plexus contained five-fold higher levels of 5-HT2C-LI than any brain region but high levels were found in the frontal cortex, septum, hypothalamus , and striatum, intermediate levels in the thalamus and midbrain, and lower levels in brainstem, cerebellum, and spinal cord. In rat cortica l membranes, the B-max value from [H-3]-mesulergine binding was ten-fo ld, lower than 5-HT2C-LI levels determined by radioimmunoassay, which may reflect measurement of internalised receptor protein by radioimmun oassay which is not detected with conventional 5-HT2C ligands. Ten day s after depletion of 5-HT with the serotonergic neurotoxin 5,7-dihydro xytryptamine (5,7-DHT), there was a significant increase in 5-HT2C-LI in the choroid plexus and the ventral cervical spinal cord, suggesting that receptors therein are located post-synaptic to destroyed seroton ergic nerve terminals. In contrast, the significant reduction in 5-HT2 C-LI observed in the midbrain, brainstem, and dorsal thoracic spinal c ord following 5,7-DHT implies that 5-HT2C receptors may be located on 5-HT nerve terminals in these regions. (C) 1997 Wiley-Liss, Inc.