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Keywords:

  • alternative splicing;
  • lateral hypothalamus;
  • lateral septal nucleus;
  • melanin-concentrating hormone gene;
  • periventricular nucleus

Abstract

The rat melanin-concentrating hormone (MCH) gene may produce, through alternative splicing, either the precursor of MCH and neuropeptide EI, two neuropeptides coexpressed in the zona incerta (ZI) and lateral hypothalamus (LHA), or a putative protein we named previously MCH-gene-overprinted-polypeptide (MGOP). First, we investigated the distribution and relative expression of MCH and MGOP mRNA in the rat brain by Northern blotting, RT-PCR and in situ hybridization. MGOP gene transcripts were detected mainly in the hypothalamus only by RT-PCR. Second, different antisera were raised toward the C-terminus of MGOP and used to identify the translational products. In the rat brain, no MGOP-processed peptide could be detected based on RP-HPLC coupled to specific RIA. A polypeptide of 14 kDa was found in the secretory pathway of transfected monkey COS7 cells expressing recombinant MGOP. In the rat hypothalamus, a specific protein of 12 kDa was identified by Western blot analysis. Finally, distribution of MGOP-immunoreactivity (IR) was investigated in the rat brain. Colocalization studies demonstrated that 98% of the MGOP-expressing perikarya in ZI/LHA also synthesized MCH. In addition, numerous, strongly stained MGOP-containing neurons were encountered in the hypothalamic periventricular nucleus. Perikarya labelled with MGOP antiserum were also found scattered in the cortex, caudate putamen, amygdala and lateral septal nucleus. MCH was not detected in these MGOP-containing neurons. Strikingly, dense staining of terminals was observed with MGOP antiserum but not with MCH antibodies in the suprachiasmatic, ventromedial and arcuate nuclei, and also in the external layer of the median eminence. These results demonstrated that MGOP and MCH-IR overlapped in LHA/ZI but displayed a differential distribution in other areas. Based on this cerebral distribution, MGOP may act as a new secreted protein in regulating many neuroendocrine functions, such as nursing, feeding and growth control in associated behavioural components.