Synaptology of the rat suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the rat the fine structure of the synapses and some features of their topological arrangement were studied. Five types of synapses could be distinguished with certainty: A. Two types of Gray-type-I (GTI) or asymmetrical synapses (approximately 33%). The presynaptic elements contain strikingly different types of mitochondria. Size of clear vesicles: approximately 450 A. Synapses with subjunctional bodies often occur, among these also "crest synapses". Localization: dendritic shafts and spines, rarely somata. B. Three types of Gray-type-2 (GTII) or symmetrical synapses (approximately 66%):1) Axo-dendritic and -somatic (=AD) synapses. Size of clear vesicles: approximately 500 A. 2) Invaginated axo-dendritic and -somatic (=IAD) synapses with club-like postsynaptic protrusions within the presynaptic elements (PreE1). Size of clear vesicles is very variable: approximately 400-1,000 A. 3) Dendro-dendritic, -somatic and somato-dendritic (=DD) synapses occurring at least partly in reciprocal arrangements. They represent an intrinsic system. Shape of clear vesicles: often oval; sucrose treatment partly produces flattening. Dense core-vesicles (dcv) are found in all GTII- and most of the GTI-synapses after three-dimensional reconstruction. All types of synapses (mostly GTII-synapses) can be enclosed by multilamellar astroglial formations. The synapses often occur in complex synaptic arrangements. Dendrites and somata of females show significantly more multivesiculated bodies than those of males. Further pecularities of presynaptic (PreELs) and postsynaptic elements (PostELs) within the SCN are described and discussed.     

The neurofilament architecture of the rat suprachiasmatic nucleus

The neurofilament architecture within the suprachiasmatic nucleus of the rat was analyzed immunocytochemically using neurofilament monoclonal antibodies. The topographic distribution of neurofilament containing structures was restricted mainly to the ventral and caudal part of the suprachiasmatic nucleus, coinciding with the entrance area of the retino-suprachiasmatic fibres of this nucleus. Within the nucleus itself an axonal organization was present. The axons were grouped, forming clusters. These clusters existed of a core of myelinated axons surrounded by unmyelinated axons. The myelinated/unmyelinated axon ratio could reach 1:25. Within the nucleus the myelinated axons extended upwards to the middle part of the suprachiasmatic nucleus, where the fibers of the axon clusters fanned out.     

Temperature-sensitive properties of rat suprachiasmatic nucleus neurons

The hypothalamic suprachiasmatic nucleus (SCN) contains a heterogeneous population of neurons, some of which are temperature sensitive in their firing rate activity. Neuronal thermosensitivity may provide cues that synchronize the circadian clock. In addition, through synaptic inhibition on nearby cells, thermosensitive neurons may provide temperature compensation to other SCN neurons, enabling postsynaptic neurons to maintain a constant firing rate despite changes in temperature. To identify mechanisms of neuronal thermosensitivity, whole cell patch recordings monitored resting and transient potentials of SCN neurons in rat hypothalamic tissue slices during changes in temperature. Firing rate temperature sensitivity is not due to thermally dependent changes in the resting membrane potential, action potential threshold, or amplitude of the fast afterhyperpolarizing potential (AHP). The primary mechanism of neuronal thermosensitivity resides in the depolarizing prepotential, which is the slow depolarization that occurs prior to the membrane potential reaching threshold. In thermosensitive neurons, warming increases the prepotential's rate of depolarization, such that threshold is reached sooner. This shortens the interspike interval and increases the firing rate. In some SCN neurons, the slow component of the AHP provides an additional mechanism for thermosensitivity. In these neurons, warming causes the slow AHP to begin at a more depolarized level, and this, in turn, shortens the interspike interval to increase firing rate.     

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue

Summary Pieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.     

Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat

The risk for cardiovascular incidents is highest in the early morning, which seems partially due to endogenous factors. Endogenous circadian rhythms in mammalian physiology and behavior are regulated by the suprachiasmatic nucleus (SCN). Recently, anatomical evidence has been provided that SCN functioning is disturbed in patients with essential hypertension. Here we review neural and neuroendocrine mechanisms by which the SCN regulates the cardiovascular system. First, we discuss evidence for an endogenous circadian rhythm in cardiac activity, both in humans and rats, which is abolished after SCN lesioning in rats. The immediate impact of retinal light exposure at night on SCN-output to the cardiovascular system, which signals 'day' in both diurnal (human) and nocturnal (rat) mammals with opposite effects on physiology, is discussed. Furthermore, we discuss the impact of melatonin treatment on the SCN and its potential medical relevance in patients with essential hypertension. Finally, we argue that regional differentiation of the SCN and autonomous nervous system is required to explain the multitude of circadian rhythms. Insights into the mechanisms by which the SCN affects the cardiovascular system may provide new strategies for the treatment of disease conditions known to coincide with circadian rhythm disturbances, as is presented for essential hypertension.     

Immunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus

Summary The classical areas for arginine-vasopressin (AVP) synthesis are the magnocellular supraoptic (SON) and paraventricular nuclei. More recently AVP was also demonstrated in neurons of the parvocellular suprachiasmatic nucleus (SCN) of the rat. This result was substantiated in the present study by means of immunoelectron microscopy, by subjecting sections to antivasopressin plasma. Conventional electron microscopy revealed dense-core vesicles in the SCN cell bodies and fibres (mean diameter 94.7±0.9 nm and 84.0±1.1 nm respectively). These vesicles were infrequent within the cell bodies and could not be accumulated by ethanol administration. Immunoelectron microscopy showed a positive reaction in the cell bodies and fibres within vesicles of 93.7±1.1 nm and 98.5±1.2 nm respectively. By comparison, the cell bodies and fibres of the SON showed immunoreactive granules of 143.0±1.8 and 147.3±1.8 nm respectively. The presence in the SCN of AVP in vesicles of different size than those in the SON suggests that synthesis of this substance is indeed occurring within the SCN cells.Supported by The Foundation for Medical Research FUNGOThe authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group Neuroendocrinology for their suggestions, Mr. P.S. Wolters and Miss A. van der Veiden for their skilled assistance     

Postnatal development of the suprachiasmatic hypothalamic nucleus of the rat

Summary 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 (Güldner, 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.Supported in part by grants NS-12265, NS-12267, HD04583 and HD-08658 from the National Institutes of Health, USPHS. The electron microscopic facilities of the California Regional Primate Center, supported by NIH grant RR-00169, were utilized. The technical assistance of Mrs. Viviana Wong is gratefully acknowledged. A preliminary report of a portion of this data was given at the Society for Neuroscience, November, 1974 in St. Louis, Missouri.     

Beyond the suprachiasmatic nucleus

The suprachiasmatic nucleus is the master oscillator controlling circadian rhythms in mammals. Yet extensive temporal restructuring of behavior can occur without participation of the suprachiasmatic nucleus. This raises questions about current thinking about how to cope with jet lag and shift work.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Synapses of optic nerve afferents in the rat suprachiasmatic nucleus

Summary The identification of optic synapses in the rat suprachiasmatic nucleus (Güldner, 1978) has made it possible to study them morphometrically. The measurements followed the check-list introduced by Palay and Chan-Palay (1976). There are several items which could usefully be added to this list. The structure of essential synaptic components varies considerably in what is apparently one synaptic population based on morphological criteria. The possible reasons for the variable sizes of the optic boutons containing different amounts of clear and dense core vesicles are discussed in terms of different activities or metabolic states of the individual boutons and/or different metabolic states of neuronal and glial elements in their vicinity. The active zones of optic synapses are also extremely variable. One optic bouton can form several active zones of very different sizes, or form Gray-type-I (asymmetric), Gray-type-II (symmetric) and intermediate contacts at the same time. The function and/or functional efficiency of a single optic bouton therefore could then be quite different with respect to its various postsynaptic elements. The different appearance of the active zones is discussed mainly in terms of possible regulative influences from neighboring synapses via the postsynaptic neuron.The author wishes to thank Mrs. Bassirat Pirouzmandi for her excellent technical assistance     

Light-induced tyrosine phosphorylation of BIT in the rat suprachiasmatic nucleus

Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12-h light/ 12-h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs), a transmembrane glycoprotein of 90-95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12-h light/12-h dark cycles. As the tyrosine-phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP-2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP-2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light-induced entrainment of the circadian clock.     

Forced desynchronization of dual circadian oscillators within the rat suprachiasmatic nucleus

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) contains multiple autonomous single-cell circadian oscillators and their basic intracellular oscillatory mechanism is beginning to be identified. Less well understood is how individual SCN cells create an integrated tissue pacemaker that produces a coherent read-out to the rest of the organism. Intercellular coupling mechanisms must coordinate individual cellular periods to generate the averaged, genotype-specific circadian period of whole animals. To noninvasively dissociate this circadian oscillatory network in vivo, we (T.C. and A.D.-N.) have developed an experimental paradigm that exposes animals to exotic light-dark (LD) cycles with periods close to the limits of circadian entrainment. If individual oscillators with different periods are loosely coupled within the network, perhaps some of them would be synchronized to the external cycle while others remain unentrained. In fact, rats exposed to an artificially short 22 hr LD cycle express two stable circadian motor activity rhythms with different period lengths in individual animals. Our analysis of SCN gene expression under such conditions suggests that these two motor activity rhythms reflect the separate activities of two oscillators in the anatomically defined ventrolateral and dorsomedial SCN subdivisions. Our "forced desychronization" protocol has allowed the first stable separation of these two regional oscillators in vivo, correlating their activities to distinct behavioral outputs, and providing a powerful approach for understanding SCN tissue organization and signaling mechanisms in behaving animals.     

Circadian activity rhythms and phase-shifting of cultured neurons of the rat suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1- to 3-day-old rats cultured on multi-microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine-vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase-shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase-shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase-shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase-shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase-shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase-shifts.     

Distinct pharmacological mechanisms leading to c-fos gene expression in the fetal suprachiasmatic nucleus.

Maternal treatment with cocaine or a D1-dopamine receptor agonist induces c-fos gene expression in the fetal suprachiasmatic nuclei (SCN). Other treatments that induce c-fos expression in the fetal SCN include caffeine and nicotine. In the current article, the authors assessed whether these different pharmacological treatments activate c-fos expression by a common neurochemical mechanism. The results indicate the presence of at least two distinct pharmacological pathways to c-fos expression in the fetal rat SCN. Previous studies demonstrate that prenatal activation of dopamine receptors affects the developing circadian system. The present work shows that stimulant drugs influence the fetal brain through multiple transmitter systems and further suggests that there may be multiple pathways leading to entrainment of the fetal biological clock.     

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in 'arbitrary units', is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.     

Morphometric and immunohistochemical studies of the suprachiasmatic nucleus in the hereditary microphthalmic rat]

Morphometric and immunohistochemical analyses of the suprachiasmatic nucleus (SCN) were performed on hereditary microphthalmic rats. In normal rats, the number of cells and the volume of the SCN were 11, 631 and 6.7 x 10(-2) mm3 (an average taken from 12 SCNs). However, the neuronal population and volume of the SCN in hereditary microphthalmic rats were 7,450 and 4.5 x 10(-2) mm3 (an average taken from 14 SCNs), respectively. There were no significant differences in the size of neurons between normal and microphthalmic SCN neurons. Immunohistochemical studies showed that a considerable number of antivasopressin positive neurons were present in microphthalmic rats, despite their lack of the optic nerve. However, further detailed studies revealed that the number of antivasopressin positive neurons present in microphthalmic rats was only 68% of those found in normal rats. These findings suggest that the complete development of the SCN and vasopressin neurons depends on the visual input.