Neurogenesis of the hamster suprachiasmatic nucleus

Neurogenesis of the hypothalamic suprachiasmatic nucleus (SCN) was described in the Syrian hamster (Mesocricetus auratus) using tritiated [3H]thymidine autoradiography. Pregnant hamsters were given single intraperitoneal injections of [3H]thymidine at different times during prenatal development, and labeled cells were analyzed in the offspring of 4-5 weeks of age. Cells of the hamster SCN became postmitotic (were 'born') over two and a half days from 10.5 to 13.0 days postfertilization (dpf) with a peak around 11.5 dpf, 4 days before birth. Two gradients in SCN neurogenesis were observed. Posterior cells were produced somewhat earlier than anterior cells and ventrolateral cells were produced before dorsomedial cells. An exception to the second gradient was a small population of ventrolateral cells produced near the end of SCN neurogenesis. The pattern of SCN neurogenesis in the hamster was similar to that described in the rat, including a predominant ventrolateral to dorsomedial gradient and the presence of ventral or ventrolateral cells produced relatively late, contrary to the predominant gradient.     

Responses of the suprachiasmatic nucleus to retinohypothalamic tract volleys in a slice preparation of the mouse hypothalamus

The electrophysiological responses of the mouse suprachiasmatic nucleus (SCN) to stimulated synaptic input from the retinohypothalamic tract (RHT) were investigated using a hypothalamic slice preparation that includes the entire SCN, optic chiasm and optic nerves. Extracellular recordings of single-unit activity reveal a population of neurons in the ventrolateral SCN that are activated at a median latency of 10 ms after stimulation of the contralateral optic nerve. These neurons apparently receive direct excitatory input from RHT synapses. Other SCN neurons are activated at longer latencies, possibly through input from interneurons. The population field potentials evoked in the SCN by optic nerve volleys consist of a calcium-insensitive transient generated by optic tract axons in the chiasm, followed by calcium-sensitive waves generated by postsynaptic activity. The postsynaptic waves have the form of a field EPSP, negative in the dorsolateral SCN and positive in the ventrolateral SCN, upon which is superimposed a population spike of opposite polarity. The population spike occurs at the same latency as the monosynaptic single unit responses, which were all found near or ventral to the point of reversal of field potential. These findings suggest that neurons in the ventrolateral SCN are excited by synapses on dorsally extended dendrites. The conduction velocity of the RHT in the optic nerve was found to be 0.59 +/- 0.03 mm/ms, while that of the optic tract volley was 2.4 +/- 0.75 mm/ms. The low conduction velocity of the RHT indicates that, within the optic nerve, these axons are thin and/or unmyelinated in the optic nerve.     

The retinohypothalamic projection and oxidative metabolism in the suprachiasmatic nucleus of primates and tree shrews

This study compared the patterns of retinal projections and oxidative metabolism in the hypothalamus of squirrel monkeys, Bonnet macaques, and tree shrews. Intraocular injections of horseradish peroxidase in primates demonstrated that retinal terminals were present from the anterior to posterior poles of the suprachiasmatic nucleus (SCN). The terminals were primarily located in the ventral and ventrolateral regions of the SCN. In addition, there was a relatively even density and distribution of retinal terminals between the ipsilateral and contralateral projections. The pattern of oxidative metabolism in the hypothalamus of the primates examined demonstrated that the SCN is highly metabolic relative to the surrounding area, and distinct regions of the SCN exhibit clear differences in metabolism. These distinct metabolic regions may reflect functional subdivisions within the SCN. In addition, elevated metabolism is found along the hypothalamo-optic chiasm border. The retinal projection to the hypothalamus in tree shrews was very different from that of the primates examined. The contralateral retinal projection was very dense, but the ipsilateral retinal projection was very sparse. Retinal terminals were primarily distributed along the lateral border of the SCN. Both the SCN and the region lateral to the SCN exhibited elevated oxidative metabolism relative to the surrounding hypothalamus.     

Retinal input to the suprachiasmatic nucleus before and after puberty in Djungarian hamsters

Retinal projections to the suprachiasmatic nucleus (SCN) mediate the effect of photoperiod to entrain circadian rhythms and to control reproductive maturation in the Djungarian hamster. To determine whether the retinal innervation of the SCN had fully developed by the onset of puberty in this hamster species, prepubertal and postpubertal hamsters received an intraocular unilateral injection of horseradish peroxidase (HRP), and after 24 h, the anterograde transport of HRP to the SCN was studied. In prepubertal hamsters, the retinohypothalamic tract (RHT) was found to project to the medial and caudal SCN, principally the ventrolateral regions and, to an extent, the dorsomedial portion of the nucleus. RHT innervation was asymmetric; the SCN contralateral to the monocular injection received the dominant projection. A similar pattern of retinal projections was found postpubertally; however, the ipsilateral SCN was less extensively labelled with HRP and smaller as determined by Nissl counterstain compared to that in prepubertal hamsters. These findings indicate that modifications in the retinal innervation of the SCN occur as late as puberty, and may be part of a developmental change in the mechanism which processes photoperiodic information during sexual maturation.     

Indirect projections from the suprachiasmatic nucleus to the ventrolateral preoptic nucleus: a dual tract-tracing study in rat

The suprachiasmatic nucleus (SCN) contains a master clock for most circadian rhythms in mammals, including daily sleep-wake cycles. The ventrolateral preoptic nucleus (VLPO) plays a key role in sleep generation and, as such, might be an important target of the SCN circadian signal. However, direct SCN projections to the VLPO are limited, suggesting that most of the SCN output to the VLPO might be conveyed indirectly. We examined this possibility by microinjecting selected known major targets of SCN efferents with biotinylated dextran-amine and/or cholera toxin B subunit, followed by analyses of retrograde labelling in the SCN and anterograde labelling in the VLPO. Retrograde labelling results confirmed that the medial preoptic area, subparaventricular zone, dorsomedial hypothalamic nucleus and posterior hypothalamic area all received projections from the SCN; these projections arose predominantly from the shell, as opposed to the core, of the SCN. Anterograde labelling results indicated that these same nuclei also projected to the VLPO, mainly its medial and ventral aspects. Comparison of the results of injections of similar sizes across different target groups indicated that the rostral part of the medial preoptic area and the caudal part of the dorsomedial hypothalamic nucleus were particularly noteworthy for the abundance of both SCN source neurons and efferent fibres and terminals in the VLPO. These results suggest that the SCN might provide indirect input to the VLPO via the medial preoptic area and the dorsomedial hypothalamic nucleus, and that these indirect neuronal pathways might play a major role in circadian control of sleep-wake cycles.     

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalmus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immunoreactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness. In the absence of photic time cues, temporal variation in the pattern of Fos immunostaining in the SCN, or within specific subfields of the nucleus, was evident only within dorsomedial SCN during exposure to LL, such that the density of immunopositive cells was greater during the subjective day than during the subjective night. These data demonstrate that light stimulation causes an increase in the expression of Fos protein(s) in the retina and within the ventrolateral, but not the dorsomedial, subfield of the SCN. The inductive effect of light on Fos expression within the retina and the ventrolateral or retinorecipient subfield of the SCN suggests that Fos protein(s) may play a role in the transduction of light signals by the primary neural components governing the generation and photoentrainment of circadian rhythms in mammals.     

Influence of environmental light-dark cycle and enucleation on activity of suprachiasmatic neurons in slice preparations

The influence of environmental light-dark cycle (LD) and bilateral enucleation on single neuronal activity in the suprachiasmatic nucleus (SCN) was examined using a hypothalamic slice preparation. Firstly, we reconfirmed previous results that the discharge rate in slices taken from animals kept on normal LD was higher during the light than during the dark period. Secondly, the day time discharge rate in the ventrolateral part of the SCN was decreased by bilateral enucleation and DD housing, while in the dorsomedial part it was unaffected. Thirdly, LL housing suppressed the discharge rates in both parts during the day and night periods. The present results suggest that the dorsomedial part of the SCN is more important in regulation of the circadian rhythm of SCN neuronal activity than the ventrolateral.     

Circannual morphometric investigations of the rat suprachiasmatic nucleus after pinealectomy, ganglionectomy and thyroidectomy

We karyometrically investigated the nucleus suprachiasmaticus (SCN) which had been manipulated in several ways in order to analyze the functional importance of the pineal gland on the primary pacemaker of mammals in the course of the year. The manipulation modes were (i) pinealectomy (PX), and (ii) sympathetic denervation of the pineal by bilateral extirpation of the upper cervical ganglia (ganglion ectomy, GX). Additionally, the influence of the inactivated pineal, obtained through hypothyroidism which was realized by (iii) subtotal thyroidectomy (TX), was also investigated. With respect to annual oscillations the results of our investigations were able to illustrate the following. (1) The SCN consists of at least two parts (ventrolateral and dorsomedial) each with different functions and relationships. The nuclei of the ventrolateral cells are bigger and there are many indications both in our own research and from literature that the neurons of this part are involved in the generation of rhythms. (2) The size of the cell nuclei of the ventrolateral part shows annual patterns. In the course of the year the maxima of the nuclear volume were registered in March and September (bimodal pattern, equinox, L:D = 12:12). PX, GX or TX only negligibly changed the bimodal annual pattern. However, in comparison the smallest cell nuclei were registered in the winter (short day). The much smaller cell nuclei of the dorsomedial part likewise show bimodal patterns but only in the experimental groups. The control group of this part shows an unimodal annual curve with a minimum at long-day conditions (June. L:D = 16:8). (3) All manipulations which inactivated the pineal or reduced the content of melatonin (PX, GX, and TX) were followed by an increase (activation) of cell nuclei of the SCN. In contrast to these effects, an increase of thyroxine (by exposure to cold), has an opposite effect (not documented here). In conclusion these results indicate, without a doubt, that a negative correlation exists, functionally, between the SCN and the pineal (in the same annual experiment the nuclear size of the pinealocytes was increased, under short-day conditions in December, and decreased under long-day circumstances in June). Additionally, it could be shown that the degree of negative correlation between the pineal and the SCN was seasonally dependent. The lowest effects of PX, GX and TX were registered at short-day conditions (December, L:D = 8:16).     

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.     

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms.

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalamus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immuno-reactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin depletion decreases light induced c-fos in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is the locus of the biological clock in mammals. Daily light cycles entrain the endogenous circadian rhythms in mammals through direct and indirect neural pathways from the retinae to the suprachiasmatic nucleus. We have studied the effect of serotonin depletion on the photic induction of the early response gene c-fas in the SCN of rats. Serotonin depletion, verified by immunohistochemistry, produced a significant decrease (42%) in the number of c-FOS positive cells in the ventrolateral portion of the SCN. These results support the involvement of serotonin as a mediator of photic information to the SCN through the retinal projection to the dorsal raphe nucleus.     

The circadian rhythm of Per1 gene product in the rat suprachiasmatic nucleus and its modulation by seasonal changes in daylength

The suprachiasmatic nucleus (SCN) of rats maintained under a 12-h light, 12-h dark cycle (LD12:12) as well as of those released into darkness exhibited the rhythm of a clock gene Per1 product, PER1 protein, with the maximum late in the subjective day and early night and minimum in the morning. The rhythm was phase delayed by 6-8 h compared with the reported rhythm of Per1 mRNA in the rat SCN [L. Yan et al. Neuroscience 94 (1999) 141]. Under a long, LD16:8, artificial photoperiod, the interval of elevated PER1-immunoreactivity was at least 4 h longer than that under a short, LD 8:16 photoperiod, due mainly to an earlier PER1 day-time rise under the long photoperiod. Under a natural photoperiod, profiles of the PER1 rhythm in summer and in winter resembled those under corresponding artificial photoperiods; therefore, twilight did not affect the rhythm in a substantial way. Under all photoperiods, when PER1 immunoreactivity was elevated, immunopositive cells were localized in the dorsomedial rather than in the ventrolateral part of the SCN. As the Per1 gene is a part of a molecular clockwork and as the rhythm of its product is modulated by the photoperiod, it appears that the whole molecular clockwork in the rat SCN is photoperiod-dependent and thus shaped by the season of the year.     

A sparse projection from the suprachiasmatic nucleus to the sleep active ventrolateral preoptic area in the rat

The circadian clock of the suprachiasmatic nucleus (SCN) may control the sleep-wake cycle by modulating the activity of brain regions important in sleep onset and maintenance, such as the ventrolateral preoptic area (VLPO). The aim of this study was to determine whether the VLPO receives direct projections from the SCN. The retrograde tracer cholera toxin (beta subunit; CT beta) was injected into the VLPO of male rats and the SCN was examined for the presence of labeled, VLPO-projecting neurons. After injections restricted to the VLPO only a few labeled cells were found within the SCN, with more labeled cells located around the nucleus. Therefore, the circadian regulation of the VLPO is likely to be achieved through multisynaptic pathways or via a diffusible signal, rather than by direct axonal outputs from the SCN to the VLPO.     

Direct projections from serotonergic neurons in the dorsal and median raphe nuclei of midbrain to the suprachiasmatic nucleus in Tupaia belangeri chinensis

This study investigated the direct serotonergic projections to the suprachiasmatic nucleus (SCN) from the dorsal and median raphe nuclei (DR/MR) of the midbrain in Tupaia belangeri chinensis (TBC) by combined application of retrograde horseradish peroxidase (HRP) tract tracing, immunohistochemistry, and electron microscope techniques. The results provide evidence for the direct projections to the SCN from serotonergic neurons distributed predominantly in the MR (mainly in its lateral portion) and to a lesser degree in the DR (in its ventrolateral portion) more caudally in the midbrain, and the existence of abundant symmetrical and asymmetrical synaptic connections between the serotonergic terminals and the postsynaptic elements in the SCN TBC. The results also revealed that almost all DR neurons projecting to the SCN contained serotonin, whereas about one-half of MR neurons projecting to the SCN were immunoreactive for serotonin.     

Differential expression patterns of inositol trisphosphate receptor types 1 and 3 in the rat suprachiasmatic nucleus.

Inositol 1,4,5-trisphosphate receptor types 1 (IP(3)-R1) and 3 (IP(3)-R3) were found in the rat suprachiasmatic nucleus (SCN) and the levels of both of IP(3)-R1 and IP(3)-R3 mRNA showed a reciprocal circadian rhythm. IP(3)-R1 immunoreactive cells were localized in neuronal cells in the SCN. In contrast, IP(3)-R3 immunoreactive cells were mainly localized in the astrocytes in the ventrolateral region of the SCN. These results suggest that IP(3)-R1 and IP(3)-R3 may have differential roles in the SCN in the mammalian circadian rhythm.     

Alpha-bungarotoxin binding in relation to functional organization of the rat suprachiasmatic nucleus

[125I]alpha-Bungarotoxin (alpha-Btx) binding was quantified in autoradiographs of in vitro labeled sections through the rat suprachiasmatic nucleus (SCN). Recent evidence suggests that acetylcholine and alpha-Btx binding sites in the SCN region may mediate effects of light on circadian functions. In contrast to the ventrolateral termination of retinal axons, there was a consistent pattern of relatively high alpha-Btx binding levels in the dorsolateral SCN and low levels in the ventromedial SCN. In addition, enucleation had little or no effect. We also investigated the possibility that phase-dependent influences of light on circadian functions could reflect the presence of light-induced or daily rhythms in alpha-Btx binding. Animals were tested in a light-dark cycle or after 1-2 days in constant light. The dorsolateral SCN showed a small but significant decline of alpha-Btx binding in animals in light at night compared to darkness at night. alpha-Btx binding in the SCN showed no statistically significant daily variation in animals in the light-dark cycle.     

Voltage-gated calcium channels play crucial roles in the glutamate-induced phase shifts of the rat suprachiasmatic circadian clock

The resetting of the circadian clock based on photic cues delivered by the glutamatergic retinohypothalamic tract is an important process helping mammals to function adaptively to the daily light-dark cycle. To see if the photic resetting relies on voltage-gated Ca(2+) channels (VGCCs), we examined the effects of VGCC blockers on the glutamate-induced phase shifts of circadian firing activity rhythms of suprachiasmatic nucleus (SCN) neurons in hypothalamic slices. First, we found that a cocktail of amiloride, nimodipine and omega-conotoxin MVIIC (T-, L- and NPQ-type VGCC antagonists, respectively) completely blocked both phase delays and advances, which were, respectively, induced by glutamate application in early and late night. Next, we discovered that: (i) amiloride and another T-type VGCC antagonist, mibefradil, completely obstructed the delays without affecting the advances; (ii) nimodipine completely blocked the advances while having less impact on delays; and (iii) omega-conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole-cell recordings revealed that T-type Ca(2+) currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L-type Ca(2+) currents did not differ from early to late night in both regions. These results indicate that VGCCs play important roles in glutamate-induced phase shifts, T-type being more important for phase delays and L-type being so for phase advances. Moreover, the results point to the possibility that a nocturnal modulation of T-type Ca(2+) current in retinorecipient neurons is related to the differential involvement of T-type VGCC in phase delays and advances.     

Effects of optogenetic stimulation of vasopressinergic retinal afferents on suprachiasmatic neurones

Physiological circadian rhythms are orchestrated by the hypothalamic suprachiasmatic nucleus (SCN). The activity of SCN cells is synchronised by environmental signals, including light information from retinal ganglion cells (RGCs). We recently described a population of vasopressin‐expressing RGCs (VP‐RGC) that send axonal projections to the SCN. To determine how these VP‐RGCs influence the activity of cells in the SCN, we used optogenetic tools to specifically activate their axon terminals within the SCN. Rats were intravitreally injected with a recombinant adeno‐associated virus to express the channelrhodopsin‐2 and the red fluorescent protein mCherry under the vasopressin promoter (VP‐ChR2mCherry). In vitro recordings in acute brain slices showed that approximately 30% of ventrolateral SCN cells responded to optogenetic stimulation with an increase in firing rate that progressively increased during the first 200 seconds of stimulation and which persisted after the end of stimulation. Finally, application of a vasopressin V1A receptor antagonist dampened the response to optogenetic stimulation. Our data suggest that optogenetic stimulation of VP‐RGC axons within the SCN influences the activity of SCN cells in a vasopressin‐dependent manner.     

AMPA/kainate receptor antagonist DNQX blocks the acute increase of Per2 mRNA levels in most but not all areas of the SCN

The daily light:dark cycle synchronizes the circadian timing system by resetting the phase of the circadian pacemaker on a daily basis. Light acutely increases mRNA levels of the clock genes Per1 and Per2 in the suprachiasmatic nucleus (SCN), the site of the primary circadian pacemaker in mammals. Light is conveyed to the SCN through the retinohypothalamic tract (RHT), an efferent projection from retinal ganglion cells that releases the excitatory amino acid (EAA) neurotransmitter glutamate in the SCN. EAA receptor activation in the SCN is critical for the ability of light to phase-shift the circadian pacemaker. In a previous study, we demonstrated that EAA receptor activation is necessary and sufficient for light to acutely increase Per1 mRNA levels in the SCN. In the current study, we determined whether EAA receptor activation in the SCN is necessary for the ability of light to increase Per2 mRNA levels in the SCN in Syrian hamsters. The NMDA receptor antagonist AP5 and the AMPA/kainate receptor antagonist DNQX inhibited the ability of light and NMDA to acutely increase Per2 mRNA levels in the SCN. In hamsters injected with DNQX, Per1 and Per2 mRNA levels remained slightly elevated in the ventrolateral SCN, suggesting that AMPA/kainate receptor activation in this region is not critical for the effects of light on the circadian pacemaker.