Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures

Abstract

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18-39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA 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 explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP-containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA-immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker.

The Suprachiasmatic Nucleus is a Functionally Heterogeneous Timekeeping Organ

“…For example, although AVP and VIP are present "organotypically" in cultured slices, gastrin releasing peptide (GRP; a prominent ventrolateral neurotransmitter) expression at the adult level is lost (Wray et al, 1993). Importantly, the apparent functional redundancy in cell and slice cultures contrasts with data obtained from acutely-prepared slices.…”

Morphological Survey of the Suprachiasmatic Nucleus in Slice Culture Using Roller Tube Method: 1. Architecture of the Suprachiasmatic Nucleus in vitro.

“…After 4 weeks of culture, electron microscopic analysis demonstrated many synapses in the neuronal zone, similar to the finding of Wray et al (31). At P4, the time of the extraction of the SCN, synapses were already formed, but poorly developed compared to those of the adult SCN (17).…”

“…This suggests that many of the synapses detected in this experiment developed in the present in vitro slice culture system. By using immunoelectron microscopic technique, Wray et al (31) further demonstrated that GABA-immunoreactive and neurophysin-immunoreactive terminals made synaptic contacts with nonimmunoreactive dendrites in slice explant culture.…”

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

“…Accordingly, our in vitro SCN system may provide a valuable model for investigating the multi-oscillator system. Morphological characteristics of the cultured SCN have been well described (11,23). Two weeks after culturing, a SCN slice is flattened to a few cell layers and two neuronal cell masses surrounded by a peripheral glial cell-dispersed zone in the slice are observed.…”