Stimulation of the hamster ventral lateral geniculate nucleus induces Fos-like immunoreactivity in suprachiasmatic nucleus cells.

The suprachiasmatic nucleus (SCN) functions as a pacemaker for circadian rhythms in rodents. Its activity and, therefore, circadian rhythms, are synchronized by light information from both a direct retinal projection and an indirect projection from the thalamic intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (vLGN) (geniculohypothalamic tract; GHT). The GHT also appears to be important for synchronizing circadian rhythms to non-photic cues. Light can activate expression of several immediate-early genes in the SCN, including c-fos. We examined whether electrical stimulation of the IGL/vLGN region would induce Fos-like immunoreactivity (lir) in the hamster SCN. Electrical stimulation was given for one hour during the dark period of the lighting cycle. Stimulation at night of the IGL and adjacent LGN regions induced Fos-lir in cells in the SCN. Labeled cells were found in the dorsolateral part of the caudal SCN, and not in rostral or central regions of the SCN, nor in the ventral part of the caudal SCN. These results indicate that activation of cells contributing to the hamster GHT can induce Fos-lir in a restricted region of the caudal SCN.     

Bifurcating axons of retinal ganglion cells terminate in the hypothalamic suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus

At least some retinal axons afferent to the hypothalamic suprachiasmatic nucleus (SCN; a circadian oscillator) bifurcate in the optic chiasm (O.E. Millhouse, Brain Res., 137 (1977) 351-355). The termination site(s) of the axonal branch that continues in the optic tract is unknown. Injection of the fluorescent tracer, True Blue, into the SCN and the fluorescent dye, Nuclear Yellow, into the lateral geniculate complex resulted in the labeling of individual retinal ganglion cells with both tracers. However, only Nuclear Yellow injections which included the intergeniculate leaflet (IGL) resulted in double-labeled ganglion cells in the retinae. These results indicate that individual retinal ganglion cells innervate both the hypothalamic SCN and the IGL of the thalamus by means of divergent axonal collaterals. Moreover, neurons of the IGL are afferent to the SCN, thereby forming a complex circuit within which photic information from the same retinal ganglion cell may influence the SCN both directly and after thalamic processing.     

Rhythmic expression of Fos-related proteins within the rat suprachiasmatic nucleus during constant retinal illumination.

Within the retinorecipient or ventrolateral subfield of suprachiasmatic nuclei (SCN) in rodents, expression of the protein product of the c-fos proto-oncogene, Fos, is regulated by light. In the present study, the expression of Fos and Fos-related proteins within the SCN was examined immunocytochemically for evidence of rhythmic variation in rats sacrificed at different circadian times during exposure to constant retinal illumination (LL). In all animals, nuclear Fos immunoreactivity was mainly confined to an area of the SCN that was coextensive with neuropeptide Y-immunopositive fibers distinguishing the ventrolateral subfield of the nucleus. Moreover, Fos-immunostaining within the ventrolateral SCN of rats exposed to LL fluctuated over the course of the circadian cycle, such that the density of immunopositive cells within this subfield was 2 times greater during the subjective night than during the subjective day. Since Fos expression within the SCN oscillates in the absence of photoperiodic time cues and since the peak of this oscillation coincides with the circadian times when light modulates the periodicity of the SCN pacemaker, these data provide further evidence that expression of the c-fos gene may be a molecular signal in the circadian timekeeping mechanism in the SCN and its regulation by photic stimuli.     

Gastrin releasing peptide and neuropeptide Y exert opposing actions on circadian phase

Microinjection of gastrin releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other's effects on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other's effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells.     

光照对哺乳类动物生物钟的调节机制

人体结构不仅存在于空间,而且存在于时间中。由于地球每24h自转一周,因此生物体内的各种功能都有明显的24h昼夜节律。在长期生物进化过程中,生物机体内发育分化出一个特殊的器官——生物钟来协调各种不同组织与器官的昼夜节律。人体的生物钟位于下丘脑的视交叉上核。由于体内生物钟是在地球昼夜环境周期性的变化中进化形成的,因此光照是影响生物钟节律最重要的因素。外界环境的光照信息是由一条独特的神经通路。从视网膜直接投射到视交叉上核,称为视网膜．下丘脑束。这条神经通路不同于经典的视觉成像通路,它不参与视觉成像功能。视锥细胞与视杆细胞全都退化的盲人或动物毫无光感,但他们的生物节律仍然受光照调节。这一现象有很重要的临床意义。本文主要讨论光照对哺乳类动物生物钟调节的神经生物学机制。     

Attenuation of circadian light induced phase advances and delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist

Circadian rhythms can be synchronised to photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus in mammals, can be phase shifted by light during the night. Non-photic stimuli reset the circadian rhythm during the day. Photic and non-photic stimuli have been shown to interact during the day and night. Precise mechanisms for these complex interactions are unknown. A possible pathway for non-photic resetting of the clock is thought to generate from the intergeniculate leaflet, which conveys information to the suprachiasmatic nucleus (SCN) through the geniculohypothalamic tract and utilises neuropeptide Y (NPY) as its primary neurotransmitter.Interactions between light and NPY were investigated during the early (2 h after activity onset) and late (6 h after activity onset) night in male Syrian hamsters. NPY microinjections into the region of the SCN significantly attenuated light-induced phase delay, during the early subjective night. Phase advances to light were completely inhibited by the administration of NPY during the late night.The precise mechanism by which NPY attenuates or blocks photic phase shifts is unclear, but the NPY Y5 receptor has been implicated in the mediation of this inhibitory effect. The NPY Y1/Y5 receptor agonist, [Leu(31),Pro(34)]NPY, was administered via cannula microinjections following light exposure during the early and late night. [Leu(31),Pro(34)]NPY significantly attenuated phase delays to light during the early night and blocked phase advances during the late night, in a manner similar to NPY.These results show the ability of NPY to attenuate phase shifts to light during the early night and block light-induced phase advances during the late night. Furthermore, this is the first in vivo study implicating the involvement of the NPY Y1/Y5 receptors in the complex interaction of photic and non-photic stimuli during the night. The alteration of photic phase shifts by NPY may influence photic entrainment within the circadian system.     

Light response of the neuronal firing activity in the suprachiasmatic nucleus of mice

To investigate the neural mechanisms underlying the mammalian photic entrainment of circadian rhythms, the response of neuronal extracellular firing activity to retinal light stimulation was investigated in the suprachiasmatic nucleus (SCN) of anesthetized mice during nighttime and daytime. In nighttime, most recorded SCN cells (83%) increased their firing frequency in response to retinal illumination. Some SCN cells (11%) responded by decreasing their firing rate. In daytime, the retinal illumination increased the firing rate in only 26% of the SCN cells, and no response was observed in the remaining cells. The light intensity threshold for the activation of SCN cells at zeitgeber time (ZT) 16 was approximately 3 x 10(11) photons cm(-2)s(-1) and the maximum response was observed at approximately 1 x 10(14) photons cm(-2)s(-1). Therefore, photic response in the firing of mouse SCN cells may be phase-dependent and have a higher threshold, which corresponds to properties of the photic entrainment in locomotor activity of mice.     

Overlap in the distribution of TrkB immunoreactivity and retinohypothalamic tract innervation of the rat suprachiasmatic nucleus

Brain-derived neurotrophic factor (BDNF) may regulate the circadian sensitivity of the clock in the hypothalamic suprachiasmatic nucleus (SCN) to light, possibly by modulating retinohypothalamic tract (RHT) input. In the present study, the anatomical distribution of the cognate receptor for BDNF, the TrkB tyrosine kinase, in RHT fibers and the SCN of rats was analyzed using combined immunohistochemical and anterograde tracing methods. Fluorescent immunostaining for the TrkB receptor was evident in fibers and cell bodies throughout the SCN. Dual labeling analysis revealed that there was substantial overlap in the distribution of TrkB immunostaining and cholera toxin subunit B (CTB)-labeling within RHT terminals and fibers projecting from the optic chiasm to the ventrolateral SCN. The present results suggest that RHT fibers may express TrkB receptors and thus provide a paracrine target for BDNF-mediated regulation of photic input to the SCN.     

Neuropeptide Y,GABA and circadian phase shifts to photic stimuli

Circadian rhythms can be phase shifted by photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus (SCN), can be phase delayed by light during the early subjective night and phase advanced during the late subjective night. Non-photic stimuli reset the clock when presented during the subjective day. A possible pathway for the non-photic resetting of the clock is thought to originate from the intergeniculate leaflet, which conveys information to the SCN through the geniculohypothalamic tract and utilizes among others neuropeptide Y (NPY) and GABA as neurotransmitters. Photic and non-photic stimuli have been shown to interact during the early and late subjective night. Microinjections of NPY or muscimol, a GABA_A receptor agonist, into the region of the SCN can attenuate light-induced phase shifts during the early and late subjective night. The precise mechanism for these interactions is unknown.

In the current study we investigate the involvement of a GABAergic mechanism in the interaction between NPY and light during the early and late subjective night. Microinjections of NPY significantly attenuated light-induced phase delays and inhibited phase advances (P<0.05). The administration of bicuculline during light exposure, before NPY microinjection did not alter the ability of NPY to attenuate light-induced phase delays and block photic phase advances.

These results indicate that NPY attenuates photic phase shifts via a mechanism independent of GABA_A receptor activation. Furthermore it is evident that NPY influences circadian clock function via differing cellular pathways over the course of a circadian cycle.     

Optic enucleation eliminates circadian rhythm shifts induced by stimulating the intergeniculate leaflet in Syrian hamsters

The intergeniculate leaflet (IGL) is a region of the lateral geniculate complex that is part of the circadian system. It receives direct innervation by specialized retinal ganglion cells involved in circadian rhythm entrainment and is also reciprocally connected to the suprachiasmatic nucleus (SCN), which is the principal circadian pacemaker. Electrical stimulation in the IGL results in shifts of circadian rhythms with a pattern of phase dependence that resembles that elicited by periods of darkness. IGL stimulation also increases levels of c-Fos in the dorsolateral part of the caudal SCN. A previous study showed that optic enucleation prevents increases in c-Fos in the SCN, suggesting the hypothesis that this increase is related to antidromic activation of retinal ganglion cells which bifurcate and project to both SCN and IGL. We tested whether phase shifts induced by IGL stimulation are also dependent on intact retinal innervation. Electrical stimulation of the IGL for 60 min at circadian time (CT)9 (with CT12 defined as activity onset) induced phase advances in nine hamsters with electrodes in the IGL, while other placements did not evoke shifts. After optic enucleation, six of these hamsters received an identical second stimulation; none showed substantial phase shifts. These results are consistent with the hypothesis that phase shifts induced by IGL stimulation depend on antidromic activation of retinal ganglion cells.     

Calbindin-D28k immunoreactivity in the suprachiasmatic nucleus and the circadian response to constant light in the rat

Recent studies in the hamster have led to the discovery that the expression of the calcium binding protein, calbindin-D28k, is a defining feature of neurons in the suprachiasmatic nucleus involved in the regulation of circadian rhythms by environmental light.(2,18, 19,32) To study further the involvement of calbindin-D28k, we examined the effect of exposure to constant light on calbindin-D28k immunoreactivity in the suprachiasmatic nucleus of intact rats and of rats treated neonatally with the retinal neurotoxin, monosodium glutamate. Exposure to constant light is known to disrupt circadian rhythms in rodents and we found previously that treatment with monosodium glutamate selectively prevents the disruptive effect of constant light on circadian rhythms in rats.(7,9) In the present study we found that exposure to light suppresses calbindin-D28k expression in the ventrolateral retinorecipient region of the suprachiasmatic nucleus of rats and that neonatal treatment with monosodium glutamate blocks the suppressive effect of constant light on calbindin-D28k expression. These findings are consistent with the proposed role of calbindin-D28k in photic signaling in the suprachiasmatic nucleus,(32) and point to the possibility that suppression of calbindin-D28k expression is linked to the mechanism by which constant light disrupts circadian rhythms.     

Functional retinal input stimulates expression of astroglial elements in the suprachiasmatic nucleus of postnatal developing rat

Astrocytes are abundant in the hypothalamic suprachiasmatic nucleus (SCN), particularly in the retinorecipient region. Using glial fibrillary acidic protein (GFAP) immunocytochemistry, we investigated the effect of light on the development of astrocytes in the SCN housing under light-dark (LD) or constant dark (DD) conditions after birth. GFAP immunoreactivity in the DD group showed lower levels than those in the LD group at P50. However, there was no difference in density of retinohypothalamic tract (RHT) terminals in the SCN between the DD and LD groups. After the adult pattern of GFAP immunoreactivity was established at P30, transferring rats to different LD conditions produced changes in GFAP immunoreactivity evident when rats were sacrificed at P50. We next examined, using a primary culture of hypothalamic astrocytes, whether neurotransmitters of RHT such as glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) can stimulate GFAP expression directly. PACAP-38 increased the length and number of astrocytic processes but glutamate did not. These findings indicate that the functional aspects of RHT such as the light stimulated release of neurotransmitters is important for the development of astrocytes in rat SCN. Dynamic plasticity of astroglial elements in the SCN occurs even after GFAP shows an adult pattern.     

Glutamate phase shifts circadian activity rhythms in hamsters

The suprachiasmatic nuclei (SCN) have been identified as a pacemaker for many circadian rhythms in mammals. Photic entrainment of this pacemaker can be accomplished via the direct retino-hypothalamic tract (RHT). Glutamate is a putative transmitter of the RHT. In the present study it is demonstrated that glutamate injections in the SCN cause phase shifts of the circadian activity rhythm of the hamster. In contrast, glutamate injections outside the SCN or vehicle injections inside the SCN did not affect the circadian phase. These data suggest that glutamate could be involved in photic entrainment of the circadian pacemaker.     

Double-stranded RNA-mediated suppression of Period2 expression in the suprachiasmatic nucleus disrupts circadian locomotor activity in rats

Circadian behavioral rhythms in mammals are controlled by a central clock located in the suprachiasmatic nucleus (SCN). PER2, the protein product of the clock gene, Period 2 (Per2), is expressed rhythmically in the SCN [Beaule C, Houle LM, Amir S (2003) Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus: Lack of effect of photic entrainment and disruption by constant light. J Mol Neurosci 21:133-148] and has been implicated in the control of circadian behavioral rhythms based on the evidence that genetic mutations in Per2 abolish free running locomotor activity rhythms in mice [Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, Eichele G, Lee CC, Bradley A (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169-173; Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:525-536]. Such mutations eradicate PER2 expression in the SCN and disrupt the SCN molecular clockwork, however, they also affect PER2 in the rest of the brain and body leaving open the possibility that the changes in behavioral rhythms might be influenced, at least in part, by disruptions in PER2 functioning outside the SCN. We used RNA interference-mediated transient knockdown of Per2 to study the effect of selective suppression of PER2 expression in the SCN, per se, on behavioral circadian rhythms. We found that transient suppression of PER2 in the SCN disrupted free running locomotor activity rhythms for up to 10 days in rats. Infusions of control dsRNA into the SCN or infusions of dsRNA to Per2 immediately dorsal to the SCN had no effect. These results constitute evidence for a direct link between PER2 expression in the SCN and the expression of behavioral circadian rhythms in mammals.     

Efferent projections of the suprachiasmatic nucleus based on the distribution of vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) immunoreactive fibers in the hypothalamus of Sapajus apella

The suprachiasmatic nucleus (SCN), which is considered to be the master circadian clock in mammals, establishes biological rhythms of approximately 24 h that several organs exhibit. One aspect relevant to the study of the neurofunctional features of biological rhythmicity is the identification of communication pathways between the SCN and other brain areas. As a result, SCN efferent projections have been investigated in several species, including rodents and a few primates. The fibers originating from the two main intrinsic fiber subpopulations, one producing vasoactive intestinal peptide (VIP) and the other producing arginine vasopressin (AVP), exhibit morphological traits that distinguish them from fibers that originate from other brain areas. This distinction provides a parameter to study SCN efferent projections. In this study, we mapped VIP (VIP-ir) and AVP (AVP-ir) immunoreactive (ir) fibers and endings in the hypothalamus of the primate Sapajus apella via immunohistochemical and morphologic study. Regarding the fiber distribution pattern, AVP-ir and VIP-ir fibers were identified in regions of the tuberal hypothalamic area, retrochiasmatic area, lateral hypothalamic area, and anterior hypothalamic area. VIP-ir and AVP-ir fibers coexisted in several hypothalamic areas; however, AVP-ir fibers were predominant over VIP-ir fibers in the posterior hypothalamus and medial periventricular area. This distribution pattern and the receiving hypothalamic areas of the VIP-ir and AVP-ir fibers, which shared similar morphological features with those found in SCN, were similar to the patterns observed in diurnal and nocturnal animals. This finding supports the conservative nature of this feature among different species. Morphometric analysis of SCN intrinsic neurons indicated homogeneity in the size of VIP-ir neurons in the SCN ventral portion and heterogeneity in the size of two subpopulations of AVP-ir neurons in the SCN dorsal portion. The distribution of fibers and morphometric features of these neuronal populations are described and compared with those of other species in the present study.     

Polysialylated neural cell adhesion molecule modulates photic signaling in the mouse suprachiasmatic nucleus

Polysialic acid (PSA), a sialic acid polymer that regulates plasticity and cell-cell interactions in neural tissues, is expressed in the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). In vivo enzymatic removal of PSA from the mouse SCN significantly impaired both the photic induction of Fos protein in SCN cells and light-induced phase-resetting of the circadian locomotor activity rhythm. Genetic deletion of PSA and it's neural cell adhesion molecule (NCAM) carrier correspondingly attenuated light-induced circadian phase-shifting. Comparison of PSA levels between young and old mice revealed a large aging-related reduction in SCN PSA content that accompanies the diminished capacity for circadian photic response reported in old rodents. Collectively these data support the contention that PSA modulates photic signaling in the SCN, and that normal reductions in the cell surface molecule contribute to aging-related deficits in SCN circadian clock function.     

Hippocampal atrophy rates in Alzheimer's disease: automated segmentation variability analysis

Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothalamic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT(1B) subtype, supposedly modulating the retinal input in a presynaptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT(1B) receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT-immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization of the circadian rhythms in recent primates.