Age-related changes in neurochemical components and retinal projections of rat intergeniculate leaflet.

Aging leads to several anatomical and functional deficits in circadian timing system. In previous works, we observed morphological alterations with age in hypothalamic suprachiasmatic nuclei, one central component of this system. However, there are few data regarding aging effects on other central components of this system, such as thalamic intergeniculate leaflet (IGL). In this context, we studied possible age-related alterations in neurochemical components and retinal projections of rat IGL. For this goal, young (3 months), adult (13 months), and aged (23 months) Wistar rats were submitted to an intraocular injection of neural tracer, cholera toxin subunit b (CTb), 5 days before a tissue fixation process by paraformaldehyde perfusion. Optical density measurements and cell count were performed at digital pictures of brain tissue slices processed by immunostaining for glutamic acid decarboxylase (GAD), enkephalin (ENK), neuropeptide Y (NPY) and CTb, characteristic markers of IGL and its retinal terminals. We found a significant age-related loss in NPY immunoreactive neurons, but not in immunoreactivity to GAD and ENK. We also found a decline of retinal projections to IGL with age. We conclude aging impairs both a photic environmental clue afferent to IGL and a neurochemical expression which has an important modulatory circadian function, providing strong anatomical correlates to functional deficits of the aged biological clock.

From blue light to clock genes in zebrafish ZEM-2S cells.

Melanopsin has been implicated in the mammalian photoentrainment by blue light. This photopigment, which maximally absorbs light at wavelengths between 470 and 480 nm depending on the species, is found in the retina of all classes of vertebrates so far studied. In mammals, melanopsin activation triggers a signaling pathway which resets the circadian clock in the suprachiasmatic nucleus (SCN). Unlike mammals, Drosophila melanogaster and Danio rerio do not rely only on their eyes to perceive light, in fact their whole body may be capable of detecting light and entraining their circadian clock. Melanopsin, teleost multiple tissue (tmt) opsin and others such as neuropsin and va-opsin, are found in the peripheral tissues of Danio rerio, however, there are limited data concerning the photopigment/s or the signaling pathway/s directly involved in light detection. Here, we demonstrate that melanopsin is a strong candidate to mediate synchronization of zebrafish cells. The deduced amino acid sequence of melanopsin, although being a vertebrate opsin, is more similar to invertebrate than vertebrate photopigments, and melanopsin photostimulation triggers the phosphoinositide pathway through activation of a G(q/11)-type G protein. We stimulated cultured ZEM-2S cells with blue light at wavelengths consistent with melanopsin maximal absorption, and evaluated the time course expression of per1b, cry1b, per2 and cry1a. Using quantitative PCR, we showed that blue light is capable of slightly modulating per1b and cry1b genes, and drastically increasing per2 and cry1a expression. Pharmacological assays indicated that per2 and cry1a responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with nitric oxide (NO) and mitogen activated protein MAP kinase (MAPK) to activate the clock genes. Our results suggest that melanopsin may be important in mediating the photoresponse in Danio rerio ZEM-2S cells, and provide new insights about the modulation of clock genes in peripheral clocks.

Morphological changes in the suprachiasmatic nucleus of aging female marmosets (Callithrix jacchus).

The suprachiasmatic nuclei (SCN) are pointed to as the mammals central circadian pacemaker. Aged animals show internal time disruption possibly caused by morphological and neurochemical changes in SCN components. Some studies reported changes of neuronal cells and neuroglia in the SCN of rats and nonhuman primates during aging. The effects of senescence on morphological aspects in SCN are important for understanding some alterations in biological rhythms expression. Therefore, our aim was to perform a comparative study of the morphological aspects of SCN in adult and aged female marmoset. Morphometric analysis of SCN was performed using Nissl staining, NeuN-IR, GFAP-IR, and CB-IR. A significant decrease in the SCN cells staining with Nissl, NeuN, and CB were observed in aged female marmosets compared to adults, while a significant increase in glial cells was found in aged marmosets, thus suggesting compensatory process due to neuronal loss evoked by aging.

Reciprocal interaction between the suprachiasmatic nucleus and the immune system tunes down the inflammatory response to lipopolysaccharide.

Several studies have shown circadian variations in the response of the immune system suggesting a role of the suprachiasmatic nucleus (SCN). Here we show that lipopolysaccharide (LPS) administration in the beginning of the active period induced more severe responses in temperature and cytokines than LPS given in the rest period. Moreover night administered LPS increased SCN basal neuronal activity indicating a direct influence of inflammation on the SCN. Bilateral lesions of the SCN resulted in an increased inflammatory response to LPS demonstrating that an interaction between the SCN and the immune system modulates the intensity of the inflammatory response.

Suprachiasmatic astrocytes modulate the circadian clock in response to TNF-α.

The immune and the circadian systems interact in a bidirectional fashion. The master circadian oscillator, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, responds to peripheral and local immune stimuli, such as proinflammatory cytokines and bacterial endotoxin. Astrocytes exert several immune functions in the CNS, and there is growing evidence that points toward a role of these cells in the regulation of circadian rhythms. The aim of this work was to assess the response of SCN astrocytes to immune stimuli, particularly to the proinflammatory cytokine TNF-α. TNF-α applied to cultures of SCN astrocytes from Per2(luc) knockin mice altered both the phase and amplitude of PER2 expression rhythms, in a phase-dependent manner. Furthermore, conditioned media from SCN astrocyte cultures transiently challenged with TNF-α induced an increase in Per1 expression in NIH 3T3 cells, which was blocked by TNF-α antagonism. In addition, these conditioned media could induce phase shifts in SCN PER2 rhythms and, when administered intracerebroventricularly, induced phase delays in behavioral circadian rhythms and SCN activation in control mice, but not in TNFR-1 mutants. In summary, our results show that TNF-α modulates the molecular clock of SCN astrocytes in vitro, and also that, in response to this molecule, SCN astrocytes can modulate clock gene expression in other cells and tissues, and induce phase shifts in a circadian behavioral output in vivo. These findings suggest a role for astroglial cells in the alteration of circadian timing by immune activation.

The retinohypothalamic tract: comparison of axonal projection patterns from four major targets.

The retinohypothalamic tract is one component of the optic nerve that transmits information about environmental luminance levels through medial and lateral branches to four major terminal fields in the hypothalamus. The spatial distribution and organization of axonal projections from each of these four terminal fields were analyzed and compared systematically with the anterograde pathway tracer PHAL in rats where the terminal fields had been labeled with intravitreal injections of a different anterograde pathway tracer, CTb. First, the well-known projections of two medial retinohypothalamic tract targets (the ventrolateral suprachiasmatic nucleus and perisuprachiasmatic region) were confirmed and extended. They share qualitatively similar projections to a well-known set of brain regions thought to control circadian rhythms. Second, the projections of a third medial tract target, the ventromedial part of the anterior hypothalamic nucleus, were analyzed for the first time and shown to resemble qualitatively those from the suprachiasmatic nucleus and perisuprachiasmatic region. And third, projections from the major lateral retinohypothalamic tract target were analyzed for the first time and shown to be quite different from those associated with medial tract targets. This target is a distinct core part of the ventral zone of the anterior group of the lateral hypothalamic area that lies just dorsal to the caudal two-thirds of the supraoptic nucleus. Its axonal projections are to neural networks that control a range of specific goal-oriented behaviors (especially drinking, reproductive, and defensive) along with adaptively appropriate and complementary visceral responses and adjustments to behavioral state.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals).

Ryanodine-sensitive intracellular Ca2+ channels in rat suprachiasmatic nuclei are required for circadian clock control of behavior.

Electrophysiological and calcium mobilization experiments have suggested that the intracellular calcium release channel ryanodine receptors (RyRs) are involved in the circadian rhythmicity of the suprachiasmatic nucleus (SCN). In the present report the authors provide behavioral evidence that RyRs play a specific and major role in the output of the molecular circadian clock in SCN neurons. They measured the circadian rhythm of drinking and locomotor behaviors in dim red light before, during, and after administration of an activator (ryanodine 0.1 microM) or an inhibitor (ryanodine 100 microM) of the RyRs. Drugs were delivered directly into the SCN by cannulas connected to osmotic minipumps. Control treatments included administration of artificial cerebrospinal fluid, KCl (20 mM), tetrodotoxin (1 microM), and anysomicin (5 microg/microl). Activation of RyRs induced a significant shortening of the endogenous period, whereas inhibition of these Ca2+ release channels disrupted the circadian rhythmicity. After the pharmacological treatments the period of rhythmicity returned to basal values and the phase of activity onset was predicted from a line projected from the activity onset of basal recordings. These results indicate that changes in overt rhythms induced by both doses of ryanodine did not involve an alteration in the clock mechanism. The authors conclude that circadian modulation of RyRs is a key element of the output pathway from the molecular circadian clock in SCN neurons in rats.

Retinal projections to the thalamic paraventricular nucleus in the rock cavy (Kerodon rupestris).

The thalamic paraventricular nucleus (PVT) receives afferents from numerous brain areas, including the hypothalamic suprachiasmatic nucleus (SCN), considered to be the major circadian pacemaker. The PVT also sends projections to the SCN, limbic system centers and some nuclei involved in the control of the Sleep-Wake cycle. In this study, we report the identification of a hitherto not reported direct retinal projection to the PVT of the rock cavy, a typical rodent species of the northeast region of Brazil. After unilateral intravitreal injections of cholera toxin subunit B (CTb), anterogradely transported CTb-immunoreactive fibers and presumptive terminals were seen in the PVT. Some possible functional correlates of the present data are briefly discussed, including the role of the PVT in the modulation of the circadian rhythms by considering the reciprocal connections between the PVT and the SCN. The present work is the first to show a direct retinal projection to the PVT of a rodent and may contribute to elucidate the anatomical substrate of the functionally demonstrated involvement of this midline thalamic nucleus in the modulation of the circadian timing system.

Internal desynchronization in a model of night-work by forced activity in rats.

Individuals engaged in shift- or night-work show disturbed diurnal rhythms, out of phase with temporal signals associated to the light/dark (LD) cycle, resulting in internal desynchronization. The mechanisms underlying internal desynchrony have been mainly investigated in experimental animals with protocols that induce phase shifts of the LD cycle and thus modify the activity of the suprachiasmatic nucleus (SCN). In this study we developed an animal model of night-work in which the light-day cycle remained stable and rats were required to be active in a rotating wheel for 8 h daily during their sleeping phase (W-SP). This group was compared with rats that were working in the wheel during their activity phase (W-AP) and with undisturbed rats (C). We provide evidence that forced activity during the sleeping phase (W-SP group) alters not only activity, but also the temporal pattern of food intake. In consequence W-SP rats showed a loss of glucose rhythmicity and a reversed rhythm of triacylglycerols. In contrast W-AP rats did not show such changes and exhibited metabolic rhythms similar to those of the controls. The three groups exhibited the nocturnal corticosterone increase, in addition the W-SP and W-AP groups showed increase of plasma corticosterone associated with the start of the working session. Forced activity during the sleep phase did not modify SCN activity characterized by the temporal patterns of PER1 and PER2 proteins, which remained in phase with the LD cycle. These observations indicate that a working regimen during the sleeping period elicits internal desynchronization in which activity combined with feeding uncouples metabolic functions from the biological clock which remains fixed to the LD cycle. The present data suggest that in the night worker the combination of work and eating during working hours may be the cause of internal desynchronization.

Changes in neuropeptide Y immunoreactivity and transcript levels in circadian system structures of the diurnal rodent, the thirteen-lined ground squirrel.

The intergeniculate leaflet (IGL) and its neuropeptide Y (NPY) projection to the main circadian clock, the suprachiasmatic nucleus (SCN), have been the focus of extensive research conducted, for the most part, on nocturnal rodent species. However, a variety of anatomical and physiological differences between the circadian system of diurnal and nocturnal species have been reported. These differences led us to question whether the role of NPY in the circadian system of the diurnal ground squirrel differs from that in nocturnal rodents. We used semi-quantitative immunohistochemistry to analyze NPY content in SCN terminals of squirrels sacrificed at specific times of the day and compared the data to previous published results from the rat. Additionally, IGL NPY mRNA was quantified using real-time PCR to determine if varying NPY immunoreactivity (-ir) levels could be the result of changes in peptide transcription. Our results demonstrate that NPY-ir levels in the ground squirrel SCN peak during the middle of the night unlike what is observed in the rat. Cell counts of NPY-ir neurons in the IGL revealed a pattern of variation 6 h out of phase compared to what was observed in the SCN. NPY mRNA levels showed only one sharp increase in the middle of the night, coinciding with increases in NPY-ir levels observed in the SCN. Differences in the pattern of fluctuation of NPY in the SCN between the rat and squirrel suggest that this peptide may serve distinct roles in the circadian system of diurnal and nocturnal species. Our data provide the first evidence of the relationship between transcript and peptide levels in the circadian system of a diurnal species.

Suprachiasmatic astrocytes as an interface for immune-circadian signalling.

The hypothalamic suprachiasmatic nuclei (SCN), the site of a mammalian circadian clock, exhibit a dense immunoreactivity for glial fibrillary acidic protein (GFAP), a specific marker for astrocytes. Although there is evidence of a circadian variation in GFAP-IR in the hamster SCN and of the participation of glial cells in input and output mechanisms of the clock, the role of these cells within the circadian system is not clearly understood. The fact that astroglia can express and respond to cytokines suggests that they could work as mediators of immune signals to the circadian system. In the present study, we have found a daily variation of GFAP-IR in the mouse SCN, peaking during the light phase. In addition, we have identified GFAP and nuclear factor-kappaB (NF-kappaB) in glial cells within the SCN and in primary cultures of the mouse SCN. Moreover, SCN glia cultures were transfected with an NF-kappaB/luc construct whose transcriptional activity was increased with lipopolysaccharide 2 mug/ml, tumor necrosis factor-alpha 20 ng/ml, or interleukin-1alpha 100 ng/ml, after 12 hr of stimulation. These results suggest that the glial cells of the SCN can mediate input signals to the mouse circadian system coming from the immune system via NF-kappaB signaling.

GFAP expression in astrocytes of suprachiasmatic nucleus and medial preoptic area are differentially affected by malnutrition during rat brain development.

The aim of the present study was investigate, in young rats, the effects of malnutrition on astrocyte distribution of two hypothalamic regions, the circadian pacemaker suprachiasmatic nucleus (SCN) and the medial preoptic area (MPA). Control rats were born from mothers fed on commercial diet since gestation and malnourished rats from mothers fed on multideficient diet, from the beginning of gestation (GLA group) or from the onset of lactation (LA group). After weaning, pups received ad libitum the same diet as their mothers, and were maintained under a 12/12 h light/dark cycle. The animals were analyzed either at 30-33, or 60-63 days of life. Brain coronal sections (50 microm) were processed to visualize glial fibrillary acidic protein (GFAP) immunoreactivity. Compared to control rats, both malnourished groups of 30 and 60 days exhibited a reduced number of GFAP-immunoreactive astrocytes in the SCN. The total GFAP-immunoreactive area in the SCN of the GLA group differed from the control group at both age ranges analyzed. The GFAP expression as measured by the relative optical density (ROD) exhibited a 50-60% reduction in the MPA in both malnourished groups, compared to controls. The results suggest that malnutrition early in life leads to alterations in gliogenesis or glial cell proliferation in both nuclei, being these alterations greater in the MPA. Compensatory plasticity mechanisms in the GFAP-expression seem to be developed in the astrocyte differentiation process in the SCN, especially when the malnutrition is installed from the lactation.

Auditory deprivation modifies biological rhythms in the golden hamster.

To assess to what extent auditory sensory deprivation affects biological rhythmicity, sleep/wakefulness cycle and 24 h rhythm in locomotor activity were examined in golden hamsters after bilateral cochlear lesion. An increase in total sleep time as well as a decrease in wakefulness (W) were associated to an augmented number of W episodes, as well as of slow wave sleep (SWS) and paradoxical sleep (PS) episodes in deaf hamsters. The number of episodes of the three behavioural states and the percent duration of W and SWS increased significantly during the light phase of daily photoperiod only. Lower amplitudes of locomotor activity rhythm and a different phase angle as far as light off were found in deaf hamsters kept either under light-dark photoperiod or in constant darkness. Period of locomotor activity remained unchanged after cochlear lesions. The results indicate that auditory deprivation disturbs photic synchronization of rhythms with little effect on the clock timing mechanism itself.

Retinohypothalamic projections in the common marmoset (Callithrix jacchus): A study using cholera toxin subunit B.

Retinal projections in vertebrates reach the primary visual, accessory optic, and circadian timing structures. The central feature of the circadian timing system is the principal circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. The direct projections from the retina to the SCN are considered the entrainment pathway of the circadian timing system. In this study, unilateral intravitreal injections of cholera toxin subunit B were used to trace the retinal projections to the marmoset hypothalamus. The retinohypothalamic tract reaches the ventral suprachiasmatic nucleus bilaterally, as anticipated from previous studies. However, labeled fibers were found in several other hypothalamic regions, such as the medial and lateral preoptic areas, supraoptic nucleus, anterior and lateral hypothalamic areas, retrochiasmatic area, and subparaventricular zone. These results reveal new aspects of retinohypothalamic projection in primates and are discussed in terms of their implications for circadian as well as noncircadian control systems.

Postnatal development of neuron number and connections in the suprachiasmatic nucleus of the hamster.

We had previously found a ca. 30% cell death during the prenatal ontogeny of the suprachiasmatic nucleus (SCN) of lambs. The period of neuron death was preceded by the establishment of the retinohypothalamic connections, a major input to this nucleus that allows the entrainment to light of the circadian rhythms generated by the SCN. The present study determined the temporal relationship between the period of ontogenetic neuron death and the establishment of the principal afferent and efferent connections of the SCN in hamsters. We found that during the first 3 postnatal days the SCN volume increases mainly by the addition of cells. After a peak 6140 neurons on each side during the third postnatal day, the SCN underwent an acute decrease of about 40% in neuron number, which led to the final adult complement of neurons, estimated in 3400 neurons per nucleus. The connections of the SCN were studied by placing DiI crystals either into the optic nerve, or into the SCN of brains fixed at different ages. We found, in agreement with previous studies, that retinal axons can be detected after the fifth postnatal day, that is, after the large decrease in neuron number. As for the SCN efferents, they began to invade the appropriate targets during the second postnatal day, followed by a large increase in the density of these efferent projection in the subsequent days. In conclusion, the massive neuronal death in the SCN was preceded by the formation of efferent connections, and followed by the formation of the retinohypothalamic tract.

Neuronal subpopulations in the suprachiasmatic nuclei based on their response to retinal and intergeniculate leaflet stimulation.

In order to characterize how suprachiasmatic nuclei (SCN) neurons integrate its visual inputs, extracellular responses from SCN and adjacent hypothalamic neurons were recorded after stimulation of either the retina, the intergeniculate leaflet (IGL) or both simultaneously. Individual stimulation of either structure elicited excitatory or inhibitory responses in 36% of SCN and 20% of non-SCN neurons. Three subpopulations of SCN neurons were found, the first two responding exclusively either to the retina or the IGL, and the third responding to both the retina and the IGL. Simultaneous stimulation of the retina and the IGL induced a change in the firing pattern of some SCN neurons, which suggests modulatory regulation of SCN neuronal activity by synaptic interactions between its visual inputs.

A putative retinohypothalamic projection containing substance P in the human.

The retinohypothalamic tract (RHT) is the principal pathway mediating the entraining effects of light on the circadian pacemaker, the suprachiasmatic nucleus (SCN). In the rat, the RHT has two components, one which projects to the SCN and the intergeniculate leaflet of the thalamus and has no known peptide content and one which projects to the SCN and, perhaps, to the olivary pretectal nucleus and contains substance P (SP). Both terminate predominantly in a zone of the SCN that contains vasoactive intestinal polypeptide (VIP)-producing neurons. In the human, there is a similar dense axonal plexus of SP-immunoreactive axons in the SCN located largely in the area occupied by VIP-immunoreactive neurons and distinct from other SP-immunoreactive axons in the area. We propose that this SP plexus represents a component of the RHT in the human brain.

Effects of central and peripheral inputs on single pineal cell activity in the rat.

The influences exerted by central and peripheral afferents to the pineal gland have been studied in rats anesthetized with urethane (1.2 g/kg, i.p.). Spontaneous action potentials arising from the pinealocytes were recorded by means of glass micropipettes filled with 3 M NaCl containing a dye. The electrical stimulation of suprachiasmatic nucleus, superior cervical ganglia, sciatic nerve and retina evoked discharge changes in a significant number of pineal cells. However, a relatively higher proportion of pinealocytes failed to respond to these afferents. Three types of responses could be observed. Inhibitions were the predominant response patterns to suprachiasmatic nucleus, superior cervical ganglia and sciatic nerve, while excitations were mainly elicited following photic stimulation, whereas the remaining evoked activity was biphasic responses, which were observed in a small number of cells after stimulation of suprachiasmatic nucleus, superior cervical ganglia and retina. These data confirm some previous neural inputs to the pineal and demonstrate the existence of a modulatory effect of the suprachiasmatic nucleus on pinealocyte discharges as well as somatosensory afferents to the gland by way of the sciatic nerve.