Techniques for the simultaneous demonstrations of neuropeptide y gene expression and peptide storage in single neurons of the human brain.

Abstract

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons. A combination of the methods of in situ hybridization for mRNA encoding Neuropeptide Y (NPY) using a 32P-labeled complementary RNA probe and immunocytochemistry using polyclonal antibodies against NPY were applied on human cortical brain samples. The manipulation of these two techniques allowed successful simultaneous demonstration of NPY gene expression and peptide storage in single neurons of the human brain.