The existence of an endogenous pacemaker has been strengthened by more recent experiments conducted on humans and animals in free-running conditions. One study protocol showed that the main circadian rhythms (temperature, sleep, and hormone secretion) also persist in experimental isolation from all the exogenous entrainments, including light (18). Under constant external environmental conditions, the persistent biological rhythm of the majority of living beings has a period not exactly of 24 hours, but of around 25 hours (about a day). In the early 1970s, the finding that a bilateral lesion of a small part of the hypothalamus of rodents led to a disruption of the circadian biological fluctuations represented the first step in identifying the location of the endogenous pacemaker in the SCN (19,20). When the SCN cells are isolated in vitro or in vivo they maintain a rhythm of firing and secretion of approximately 24 hours; this demonstrates that these cells owe the intrinsic fundamental capacity of autodepolarization to a neu-rophysiologic mechanism that should reside, and can be generated, within the same neurons (21). Transplantation of SCN tissue into the diencephalon of SCN-lesioned animals restores circadian oscillations, with a rhythm that reflects the specific characteristics of the donor and not of the recipient (22). The physiology of SCN can be simplified into three main functions: autodepolarization, a response to external input stimuli as light, and a faculty to influence circadian rhythms by output signals (23,24). Even though most of the underlying cellular mechanisms supporting the SCN autodepolarization are still unsolved, the discovery of clock (cir-cadian locomotor output cycles kaput) genes represented an important step toward understanding the problem. The term clock genes identifies a family of loci involved in circadian physiology, which exhibit a clear circadian rhythm of transcription, or a transcriptional response, to entrainment inputs (24). Period (Per), Cryptochrome (Cry), and Rev-Erba are typical examples of clock genes.
Mammals' SCN consists in a bilateral symmetric group of about 20,000 neurons, located in the anterior part of the hypothalamus (25). Cells of the SCN have differing phenotype, innervation, functions, and gene expression. In rodents, the dorsal medial shell part of SCN produces vasopressin (VP), and receives little afferent neurons from the retina. The ventrolateral part of the SCN receives more consistent innervations from the retina, and is composed of three different kinds of cells containing: vasoactive intestinal peptide (VIP), calbinding and substance P (SP), and gastrin-releasing peptide (GRP). The shell part of the SCN has a self-sustained rhythm ascribed to a circadian autoregulatory transcriptional-translation loop involving clock gene expression (26-29). In these cells, the dimerization of two nuclear proteins CLOCK and BMAL allows their binding to the promoter region of Per, Cry, and Rev-Erba genes, activating their transcription. As the respective Per and Cry proteins accumulate in the cytoplasm, they dimerize and move into the nucleus to exert an inhibitory effect on the CLOCK and BMAL, resulting in an inhibition of their own transcription and closing of a negative feedback cycle. The SCN shell neurons converse with the targets by y-amino butyric acid (GABA) and VP secretion (24).
Although the core part of the SCN does not express an autonomous circadian rhythm, it shows a strong expression of Per genes in response to light stimulus, and it seems to be essential for an harmonic synchronization of the different parts of the SCN (30). Light stimulus, through the retina and RHT, induces glutamate (GLU) and SP release in the SCN core (31,32). GLU binding with NMDA is followed by an increase of influx calcium, which activates protein kinases that phosphorylate the c-AMP-response-element-binding protein (CREB) (33). Phosphorylated CREB binds the promoter regions of Per-1 and Per-2, inducing their transcription. By VIP, GRP, and SP, the SCN core neurons communicate to the SCN shell cells where Per transcription occurs with a constant delay with respect to the external stimulus and the Per transcription in the SCN core (34).
While the two main RHT and GHT afferent nervous pathways to the SCN are well-defined, the efferent projections from SCN are less well-known, especially in humans. Efferent neurons to the paraventricular nucleus (PVN), ventromedialis nucleus, subparaventricular zone (SPZ) of the hypothalamus, and SCN itself have been demonstrated (35). The SPZ could be differentiated into a ventral and in a dorsal part. Lesional experiments showed that the ventral SPZ is a relay region mainly involved in the circadian control of sleep-wake and locomotor activity, while the dorsal SPZ principally regulates temperature body rhythm. The SCN could also drive circadian rhythm via diffusible substance, in agreement with the evidence that driving function is preserved in SCN cell transplantation (35,36). Therefore, local nuclei could be affected without direct traditional axonal connections. Because in vivo monitoring of the SCN is almost impossible, body core temperature and MLT secretion are generally used as phase markers of SCN activity, given that their oscillation periods closely resemble that of the hypothalamic pacemaker.
When free-running experiments are extended for a long time, some vegetative functions, such as body temperature, cortisol secretion, and REM sleep, desynchronized from the behavioral functions such as motor activity or food and drink intake, which continue to follow the SCN rhythm. This phenomenon, described as ''internal desynchronization,'' suggested the existence of other different endogenous clocks, and a strong dependence of behavioral input to the SCN rhythm. The existence of these different endogenous pacemakers and this possible hierarchical relationship with the SCN is still debated (37).
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