Characterization of metorphamide-like immunoreactivity in the zona incerta and lateral hypothalamus: co-localization with alpha-melanocyte-stimulating hormone-like immunoreactivity.

Double-staining in either vibratome or paraffin sections using contrasting chromogens revealed an alpha-melanocyte-stimulating hormone (alpha-MSH)-containing cell group in the arcuate nucleus, a metorphamide-containing cell group in the paraventricular hypothalamus, and an extensive group of magnocellular perikarya in the zona incerta (ZI) and the lateral hypothalamus (LH) that appeared to contain both antigens. Staining of adjacent paraffin sections also suggested that most (and perhaps all) of the magnocellular perikarya in the ZI and LH that contained metorphamide-like immunoreactivity also contained alpha-MSH-like immunoreactivity. Metorphamide-like immunoreactivity in the ZI and the LH was abolished by absorption of the antiserum with metorphamide but was unaffected by absorption with alpha-MSH. alpha-MSH-like immunoreactivity in the ZI and LH was abolished by absorption of the antiserum with alpha-MSH but was unaffected by absorption with metorphamide. Antisera directed against [Met5]-enkephalin (Met-ENK), [Met5]-enkephalin-Arg6,Gly7,Leu8 (ENK-8), [Met5]-enkephalin-Arg6,Phe7 (ENK-7), neuropeptide Y, and FMRF-amide did not stain magnocellular perikarya in the ZI and LH. Pretreatment of paraffin sections with trypsin resulted in the appearance of [Met5]-enkephalin-Arg6-like immunoreactivity in the ZI and LH. Pretreatment of paraffin sections with trypsin did not reveal any occult Met-ENK-, ENK-7- or ENK-8-like immunoreactivity in either the ZI or the LH. These observations indicate that magnocellular neurons in the ZI and LH contain both a metorphamide-like and an alpha-MSH-like peptide but do not express either the preproenkephalin or the prepro-opiomelanocortin48 gene.