Adult Suprachiasmatic Nucleus Research Articles

Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures

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.

Calcium excitability and oscillations in suprachiasmatic nucleus neurons and glia in vitro

Converging lines of evidence suggest that the hypothalamic suprachiasmatic nucleus (SCN) is the site of the endogenous biological clock controlling mammalian circadian rhythms. To study the calcium responses of the cellular components that make up the clock, computer-controlled digital video and confocal scanning laser microscopy were used with the Ca2+ indicator dye fluo-3 to examine dispersed SCN cells and SCN explants with repeated sampling over time. Ca2+ plays an important second messenger role in a wide variety of cellular mechanisms from gene regulation to electrical activity and neurotransmitter release, and may play a role in clock function and entrainment. SCN neurons and astrocytes showed an intracellular Ca2+ increase in response to glutamate and 5-HT, two major neurotransmitters in afferents to the SCN. Astrocytes showed a marked heterogeneity in their response to the serial perfusion of different transmitters; some responded to both 5-HT and glutamate, some to neither, and others to only one or the other. Under constant conditions, most neurons showed irregular temporal patterns of Ca2+ transients. Expression of regular neuronal oscillations could be blocked by the inhibitory transmitter GABA. Astrocytes, on the other hand, showed very regular rhythms of cytoplasmic Ca2+ concentrations with periods ranging from 7 to 20 sec. This periodic oscillation could be initiated by in vitro application of glutamate, the putative neurotransmitter conveying visual input to the SCN critical for clock entrainment. Long-distance communication between glial cells, seen as waves of fluorescence moving from cell to cell, probably through gap junctions, was induced by glutamate, 5-HT, and ATP. These waves increased the period length of cellular Ca2+ rises to 45-70 sec. Spontaneously oscillating cells were common in culture medium, serum, or rat cerebrospinal fluid, but rare in HEPES buffer. One source for cytoplasmic Ca2+ increases was an influx of extracellular Ca2+, as seen under depolarizing conditions in about 75% of the astroglia studied. All neurotransmitter-induced Ca2+ fluxes were not dependent on voltage changes, as Ca2+ oscillations could be initiated under both normal and depolarizing conditions. Since neurotransmitters could induce a Ca2+ rise in the absence of extracellular Ca2+, the mechanisms of ultradian oscillations appear to depend on cycles of intracellular Ca2+ fluxes from Ca(2+)-sequestering organelles into the cytoplasm, followed by a subsequent Ca2+ reduction. In the adult SCN, fewer astrocytes are found than neurons, yet astrocytes frequently surround glutamate-immunoreactive axons in synaptic contact with SCN dendrites, isolating neurons from each other while maintaining contact with other astrocytes by gap junctions.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin I immunoreactivity

Synaptogenesis was studied in the rat suprachiasmatic nucleus (SCN) using quantitative ultrastructural analysis and synapsin I immunohistochemistry. SCN neurons are formed between embryonic days 13 and 17 (E13-E17), and the SCN is a distinct hypothalamic nucleus by E18. At E19 the nucleus is very immature and there are few synapses. Synaptogenesis proceeds slowly until P4 but increases rapidly between P4 and P10. At P10 the number of synapses per unit area is the same as in the adult SCN and all synaptic types present in the adult are evident. However, the SCN continues to increase in volume to the adult with approximately 30% of total synaptic number added between P10 and a young adult age. The appearance of synapsin I immunoreactivity correlates very precisely with the development of synapses in the SCN as shown by ultrastructural analysis between E19 and P6. The pattern of appearance of synapsin I immunoreactivity demonstrates that synaptogenesis in the SCN is significantly delayed in comparison to adjacent hypothalamic nuclei. Synapsin I immunohistochemistry is a reliable marker of synapse formation in the developing SCN. A correlation of these anatomical data with prior functional studies suggests that SCN neurons are born as individual circadian oscillators that undergo a rapid development in the first 10 days after birth to form a functional neural network subserving circadian rhythm generation and regulation.

Anatomic and functional development of the suprachiasmatic nuclei in the gray short-tailed opossum

The anatomic and functional development of the suprachiasmatic nuclei (SCN) was studied in the gray short-tailed opossum, Monodelphis domestica. To provide a background for developmental studies, daily patterns of wheel-running behavior and SCN metabolic activity were studied in adult animals. Adult opossums manifested robust circadian rhythms in locomotor activity that were entrained by the daily light-dark cycle. The temporal course of SCN metabolic activity, studied by the 14C-labeled deoxyglucose autoradiographic technique, showed that the adult SCN were metabolically active throughout subjective day and relatively inactive during subjective night. SCN neurogenesis, determined using 3H-thymidine autoradiography, was active at postnatal day 3, the earliest age studied, and continued until postnatal day 7. It was not until postnatal day 16 that the SCN appeared as distinct nuclei by light microscopy. Study of the ontogeny of the daily rhythm in SCN metabolic activity showed that the nuclei were metabolically active during both day and night at postnatal day 16. On day 20, a clear day-night rhythm in SCN metabolic activity was first observed; the rhythm was even more pronounced on day 27. These results indicate that the gray short-tailed opossum has a functioning circadian timing system and that the anatomic and functional development of the SCN in this species occurs during the postnatal period.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the suprachiasmatic nucleus of the perinatal rat

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was examined in the suprachiasmatic nucleus (SCN) of the perinatal rat using the unlabeled antibody enzyme method of Sternberger. Our results showed that VIP-like immunoreactivity was present in cells and fibers of the SCN prior to birth. Immunoreactive neurons at this time appeared morphologically immature, and their distribution differed somewhat from that seen in the adult SCN.

Effects of calcium ions on glucose utilization in the rat suprachiasmatic nucleus in vitro

The role of calcium ions in maintenance of the circadian rhythm in glucose utilization in the suprachiasmatic nucleus (SCN) of the hypothalamus was investigated in vitro in a rat hypothalamic slice preparation using the 2-deoxyglucose (2-DG) method. In normal Krebs solution, 2-DG uptake of adult and embryonic day 22 rat SCN was higher in subjective day than in subjective night. In calcium-free Krebs solution, however, 2-DG uptake of adult and embryonic SCN was low in both subjective day and night periods. These results indicate that the SCN rhythm in metabolic activity is dependent on calcium ions in both adult and embryonic rats. Since immunohistochemical and ultrastructural analysis of synapse formation has shown very few synapses in the SCN of embryonic day 22 rats, it is suggested that the development and maintenance of the circadian rhythm in metabolism demonstrated by the 2-DG method depends on intracellular calcium-mediated events rather than synaptic transmission.

Development of neuronal activity in the rat suprachiasmatic nucleus

The development of function was studied in the rat suprachiasmatic nucleus (SCN) by analyzing the rate and pattern of neuronal discharge in vitro from embryonic day 22 (E22) to postnatal day 14 (P14), and comparing these with adult SCN. The firing rate of SCN neurons on E22 is low but there is a clear circadian rhythm with subjective day values (1.6 ± 0.2 impulses/s) significantly higher than those for subjective night (0.9 ± 0.2 impulses/s). At E22 most neurons show an irregular firing pattern. The firing rate of SCN neurons gradually increases from E22 to P14 with emergence of patterns of firing of individual neurons that approximate those of adult SCN. In calcium-free medium the firing rate of SCN neurons at P1 is reduced and the circadian rhythm in firing rate is abolished. These results demonstrate that SCN neurons exhibit a circadian rhythm in firing rate early in development, largely prior to the formation of synaptic contacts within the SCN, and indicate that expression of the rhythm requires the presence of calcium ions.

Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamic transplants in oculo

The capacity of the rat anterior hypothalamus, and particularly the suprachiasmatic nucleus (SCN), to develop and differentiate when removed from its normal environment was examined in this study using light and electron microscopy. The hypothalamus from fetuses ranging in age from embryonic day 12 (E 12) to E 16 was transplanted to the anterior chamber of the eye of adult rats. In initial experiments, we found that transplants from E 15 fetuses and older routinely differentiated into fields of neurons with extensive neuropil with an appearance similar to the anterior hypothalamic area. Groups of small, compactly organized neurons were observed only occasionally in this tissue. Ultrastructural analysis of these transplants typically revealed well-differentiated neuronal perikarya and neuropil with a complex synaptic organization similar in appearance to the normal rat anterior hypothalamic area. Occasionally both mature and immature tissue coexisted in some of the transplants. Tissue from young embryos (E 12–14) frequently showed development of a compact, small neuron nucleus with the cytoarchitectonic appearance of the SCN. At least 45 days were required after transplantation for the successful differentiation to occur in this situation. The SCN in these transplants displayed vasoactive intestinal polypeptide-immunoreactive cells and fibers surrounded by vasopressin-immunoreactive cells and fibers, similar to the pattern observed in the normal adult SCN. Our results indicate that the anterior hypothalamus will differentiate normally in oculo and that the phenotypic specification of the SCN occurs prior to the birthdate of its component neurons.

Postnatal development of the suprachiasmatic hypothalamic nucleus of the rat

Light and electron microscopy of newborn, four day, one, two, three and five week old rats revealed principally a progressive increase in the diversity and number of synaptic contacts in the suprachiasmatic nucleus (SCN). The major increase in synaptic diversity occurred between four days and one week of age. Correlation between this finding and the adult synaptic morphology of SCN (Gülder, 1976) on the one hand, and the ontogeny of circadian rhythms on the other were made. This suggested that the retinal afferents arriving on day four form asymmetrical contacts with dendrites. While increase in synaptic number was progressive, it was most marked between three and five weeks of age. By five weeks, most features of the adult SCN were present. No significant morphological effects were evident as a result of neonatal retinal lesions.