LOCALIZATION AND CHARACTERIZATION OF NITRIC-OXIDE SYNTHASE IN THE RATSUPRACHIASMATIC NUCLEUS - EVIDENCE FOR A NITRERGIC PLEXUS IN THE BIOLOGICAL CLOCK

Behavioral and electrophysiological evidence indicates that the biolog ical clock in the hypothalamic suprachiasmatic nuclei (SCN) can be res et at night through release of glutamate from the retinohypothalamic t ract and subsequent activation of nitric oxide synthase (NOS). However , previous studies using NADPH-diaphorase staining or immunocytochemis try to localize NOS found either no or only a few positive cells in th e SCN. By monitoring conversion of L-[H-3]arginine to L-[H-3]citrullin e, this study demonstrates that extracts of SCN tissue exhibit NOS spe cific activity comparable to that of rat cerebellum. The enzymatic rea ction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endotheli al isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN coll ected in day versus night, either by western blot or by enzyme activit y measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate tha t neuronal fibers within the rat SCN express abundant nNOS and that th e level of the enzyme does not vary temporally. The distribution and q uantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.