CHARACTERIZATION OF THE SUPRACHIASMATIC NUCLEUS IN ORGANOTYPIC SLICE EXPLANT CULTURES

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were m aintained for 18-39 days in vitro as organotypic slice explants. Neuro nal subtypes containing vasopressin (VP), vasoactive intestinal polype ptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocyto chemically identifiable in these cultures. In situ hybridization histo chemistry was compatible with these SCN slice explant cultures, and mR NA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the e xplanted tissue was derived) to levels typical of adult SCNs. In contr ast, the GRP expression remained low, characteristic of early postnata l animals and far lower than adult levels. This suggests that the deve lopmental cues or programs necessary for enhanced VP expression are ma intained in these cultures, while those affecting GRP expression are a bsent or inhibited. VIP-containing neurons were numerous in the cultur es. Culture slices appeared healthy, and similar numbers and distribut ions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis reveal ed that the SCN in vitro is composed of tightly packed neurons, proces ses, and abundant synapses containing both clear and dense core vesicl es, closely resembling the SCN in vivo. Vasopressinergic neuronal soma ta contained extensive Golgi systems and labeled secretory granules, t he latter organelle being present also within processes and synaptic t erminals. GABA-immunopositive processes and synaptic profiles were abu ndant, with labeling occurring particularly over secretory vesicles an d mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for l ong periods in vitro and should be an excellent model system for study ing the intrinsic molecular mechanisms and extrinsic cues which regula te neuronal phenotype in this circadian pacemaker.