An immunohistochemical and chromatographic analysis of the distribution and processing of proneuropeptide Y in the rat suprachiasmatic nucleus

Abstract

The suprachiasmatic nucleus (SCN) regulates a number of circadian rhythms in mammals. A neuropeptide Y (NPY)-containing pathway from the intergeniculate leaflet of the lateral geniculate to the SCN is considered to carry information of the environmental light-dark cycle. Antisera directed against NPY, Cys-NPY(32–36)amide or the C-terminal extended peptide of proNPY(68–97) (CPON) and avidin-biotin immunohistochemistry were used to define the precise distribution of NPYergic nerve fibers in the SCN, and to compare the location of the various fragments of proNPY in these nerves. Gel chromatography and specific radioimmunoassays were applied to quantify the efficiency of the amidation of NPY, and to study the size of peptides demonstrating NPY- and NPYamide-immunoreactivity in anterior hypothalamic extracts. NPY-, NPYamide-, and CPON-immunoreactive nerve fibers exhibited apparently the same distribution and morphology in the SCN. Immunoreactive fibers were preferentially located in the ventral part of the SCN, but along the rostrocaudal axis of the nucleus, the density and the precise distribution of immunoreactive elements changed. From the rostral third of the SCN to the middle third, the number of immunoreactive fibers increased and their distribution extended in a dorsal and lateral direction. In the caudal part of the SCN, the number of immunoreactive elements decreased and the innervation spread to an even more dorsolateral location. Dorsal aspects of the rostral SCN contained a moderate number of fibers, whereas the dorsomedial quadrant of the caudal 2 3 of the SCN was almost devoid of immunoreactivity. The number of NPY-, NPYamide-, and CPON-immunoreactive elements differed in the SCN: the CPON displaying the highest, and NPYamide the lowest number. Gel filtration and specific radioimmunoassays showed the existence of a single NPY-immunoreactive peptide in tissue extracts coeluting with NPY(1–36)amide. No proNPY was detected, which indicates that the proNPY molecule is cleaved to NPY and CPON, and NPY is further amidated at the C-terminal end. The localization of NPYamide-immunoreactive nerves in the SCN suggests an important functional role, acting directly on oscillator neurons. The biological function of the CPON in relation to circadian rhythm and in other systems remains to be established.