Day-night changes in c-fos expression in the fetal sheep suprachiasmatic nucleus at late gestation

The suprachiasmatic nucleus (SCN) is a circadian oscillator in mammals and shows day-night changes in metabolic activity. To investigate whether the fetal sheep SCN behaves as a circadian oscillator, day-night changes in c-fos expression, a marker of neuronal activity, were measured. Eight fetal sheep were sacrificed at 135 days gestation--four at day-time (1200 hours) and four at night-time (2400 hours). Fetal brains were fixed, removed and cut in 40-microns serial coronal sections. Alternate sections were incubated with anti-Fos antibody (1:500) and Fos expression was revealed with extra-avidin-peroxidase and 3,3'-diaminobenzidine or stained with cresyl violet. The number of Fos-immunoreactive (Fos-ir) neurons per mm2 in the rostral, intermediate and caudal regions of the fetal sheep SCN was counted. Fetuses sacrificed in the day-time showed a higher number of Fos-ir neurons per mm2 (mean +/- s.e.; 516.7 +/- 60.1) in the three regions of the SCN than fetuses sacrificed at night-time (140.5 +/- 21.8). In addition, at night-time Fos-ir neurons were mainly localized to the ventrolateral area of the SCN. These findings demonstrate day-night changes in molecular activity consistent with the presence of a circadian oscillator in the fetal sheep SCN.     

Organization of neural inputs to the suprachiasmatic nucleus in the rat

The circadian timing of the suprachiasmatic nucleus (SCN) is modulated by its neural inputs. In the present study, we examine the organization of the neural inputs to the rat SCN using both retrograde and anterograde tracing methods. After Fluoro-Gold injections into the SCN, retrogradely labeled neurons are present in a number of brain areas, including the infralimbic cortex, the lateral septum, the medial preoptic area, the subfornical organ, the paraventricular thalamus, the subparaventricular zone, the ventromedial hypothalamic nucleus, the posterior hypothalamic area, the intergeniculate leaflet, the olivary pretectal nucleus, the ventral subiculum, and the median raphe nuclei. In the anterograde tracing experiments, we observe three patterns of afferent termination within the SCN that correspond to the photic/raphe, limbic/hypothalamic, and thalamic inputs. The median raphe projection to the SCN terminates densely within the ventral subdivision and sparsely within the dorsal subdivision. Similarly, areas that receive photic input, such as the retina, the intergeniculate leaflet, and the pretectal area, densely innervate the ventral SCN but provide only minor innervation of the dorsal SCN. A complementary pattern of axonal labeling, with labeled fibers concentrated in the dorsal SCN, is observed after anterograde tracer injections into the hypothalamus and into limbic areas, such as the ventral subiculum and infralimbic cortex. A third, less common pattern of labeling, exemplified by the paraventricular thalamic afferents, consists of diffuse axonal labeling throughout the SCN. Our results show that the SCN afferent connections are topographically organized. These hodological differences may reflect a functional heterogeneity within the SCN.     

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness

Synapses of optic afferents (optic synapses) in the suprachiasmatic nucleus of hooded rats were morphometrically evaluated after exposing the animals to 12 h, 14 days, 2 months, and 8 months of constant light (light rats) and darkness (dark rats). Compared with dark rats, optic synapses from light rats have larger boutons with larger mitochondria, more clear vesicles, fewer dense-core vesicles and front-line vesicles, smaller presynaptic dense projections, a smaller amount of postsynaptic density material, a smaller relative number of Gray-type I (asymmetric) junctions, a greater relative number of Gray-type II (symmetric) junctions, as well as more and larger mitochondria in the postsynaptic dendrites. Junctions of optic synapses are mostly straight, but the small number of positively curved contacts are more flattened in light rats than in dark rats. An age-related increase in the size of presynaptic dense projections was also observed. There are no changes in the sizes of clear and dense-core vesicles, in the size of synaptic junctions and their numerical density in area, and in the unspecific contact area between pre- and postsynaptic elements. The changes in optic boutons are characteristic for activated and relatively disused synapses with a slow, tonic firing rate. It appears that (1) the amount of postsynaptic density material is proportional to the strength of Gray-type I synapses, and that (2) some excitatory optic synapses become inhibitory after long-term activity, whereas some inhibitory synapses turn into excitatory contacts after long-term disuse.     

Tracer and electrical coupling of rat suprachiasmatic nucleus neurons

Whole-cell recording from single neurons of the suprachiasmatic nucleus with an electrode containing the tracer neurobiotin resulted in the staining of multiple neurons in 30% of the cases. Typically, one neuron was darkly stained with dendritic processes and an axon clearly visible while other neurons were lightly stained. The darkly-stained cells were identified as the recorded neuron and tracer-coupled to one to five lightly stained neurons. The resting membrane potential, input membrane conductance, membrane capacitance, the decay time constant and the maximum H-current amplitude of the recorded neurons with tracer-coupled cells were not significantly different from those of neurons not showing tracer coupling. Stimulation of the preoptic area activated an antidromic action potential or an all-or-none small slow inward current in some neurons when the synaptic transmission was blocked by a calcium-free/Mn2+ solution. The small slow inward current did not "collide" with an orthodromically activated action spike suggesting that the current represents the signal from an electrotonically-coupled neuron. In addition, the frequency of biphasic field currents from a neighbouring cell firing were increased by depolarization and decreased by hyperpolarization of the recorded cell. These data demonstrate a chemical and electrical low-resistance coupling of suprachiasmatic nucleus neurons, which could be important in synthesizing the suprachiasmatic nucleus circadian rhythm.     

GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus

The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABAA agonist muscimol (10 microM) potentiated SCN field potentials by 23%, while application of the GABAA antagonist bicuculline (10 microM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA, agonist baclofen (1.0 microM) inhibited SCN field potentials by 48%, while the GABAB antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABAA activity tends to increase, and GABAB activity tends to decrease the response of SCN neurons to optic nerve stimulation.     

Memory on long but not on short days is stored in the rat suprachiasmatic nucleus

Whether or not a representative sample of the nasopharyngeal microflora can be obtained by introducing a cotton swab through the nasal cavity has been evaluated. Also, a correlation between these results and those achieved from the middle ear effusions, has been searched. Ninety adenoidectomy-pending children, fifty of whom also presented otitis media with effusion, were included in the study. The research showed that there were a coincidence (p < 0.001) among the results obtained from the nasal cotton swab, those obtained from cultures of adenoid biopsies and the middle ear effusions.     

Light-induced variations in AP-1 binding activity and composition in the rat suprachiasmatic nucleus

Expression of immediate early genes, including fos-like and jun-like genes, in the suprachiasmatic nucleus is believed to be part of the mechanism for photic entrainment of circadian rhythms to the environmental light/dark cycle. However, the effects of a light stimulus on activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus remain unclear. The photic regulation of AP-1 DNA-binding activity and composition in the rat suprachiasmatic nucleus was evaluated by using an electrophoretic mobility shift assay. A light pulse given during subjective night induced an increase in AP-1 binding activity when either nuclear or whole-cell extracts from suprachiasmatic nuclei were used. Under constant dark conditions, proteins that are predominant components of AP-1 complexes are Fra-2 and Jun-D. Under light stimulation, c-Fos and Jun-B consistently increased, as expected, but this was also the case for Fra-2, Jun-D, and c-Jun, although to a lesser extent. An immunocytochemical study of the Fra-2 expression pattern demonstrated the presence of the protein in the ventrolateral as well as in the dorsomedial subdivisions of the suprachiasmatic nucleus. Light regulation of Fra-2 immunoreactivity, however, appeared to be restricted to the ventrolateral subdivision. It is concluded that light may be acting both by increasing constitutive AP-1 complexes and by inducing the expression of specific complexes.     

Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat

The retinal projections to gastrin-releasing peptide (GRP)-expressing neurons in the rat suprachiasmatic nucleus (SCN) were investigated by double immunofluorescence and immunoelectron microscopy. Optic nerve terminals labeled by cholera toxin B subunit (CTb) which was transported from the retinal ganglion cells were intermingled with GRP-immunoreactive cell bodies and processes in the ventrolateral portion of the SCN. Ultrastructural analysis revealed that CTb-immunoreactive retinal terminals made synaptic contacts with GRP-immunoreactive dendritic processes. These results demonstrated that photic information is directly input from the optic nerve to GRP neurons in the SCN and these GRP neurons may be involved in circadian entrainment by light.     

Electrophysiological effects of pressure-ejected bombesin-like peptides on hamster suprachiasmatic nucleus neurons in vitro

The suprachiasmatic nuclei (SCN) of the rodent hypothalamus function as a light entrainable circadian pacemaker. The SCN contain moderate to high concentrations of a number of neuropeptides including peptides showing structural homology with the amphibian derived tetradecapeptide, bombesin (BN), called bombesin-like peptides (BNLPs). BNLPs include the 27 amino acid peptide, gastrin releasing peptide (GRP1-27), a smaller decapeptide (GRP18-27) and another decapeptide with less structural homology, neuromedin B (NmB). Immunoreactivity for BN and receptors for BNLPs have been demonstrated in the region of the rat SCN receiving photic input. We studied the effects of local pressure ejections of BNLPs dissolved in saline/1% bovine serum albumin (BSA) vehicle on the extracellularly recorded firing rates of Syrian hamster SCM neurons in a hypothalamic slice preparation. In one study, an ejecting electrode containing BN (10(-8) to 10(-4) M) was positioned 20 to 60 microm from a recording electrode. Of 74 cells tested with BN, 50 (67.6%) showed significant increases in firing rate, while 3 of 29 cells (15.8%) tested with vehicle ejections were activated. In a second study, a single electrode was used for both recordings and pressure ejections. Of 48 cells tested, BN (10(-6) to 10(-4) M) activated 30 (62.5%) and suppressed firing in 4 (8.3%). Of 208 cells tested with GRP1-27 (10(-9) to 10(-4) M), 105 (50.5%) were activated and 2 (1.0%) were suppressed. The percentage of cells responding increased with the concentration of GRP1027 used in the electrode. No circadian variation in responsiveness to GRP1-27 was detected. GRP18-27 (5 x 10(-5) to 10(-4) M) activated 10 out of 18 cells tested (55.6%), while NmB (10(-4) M) activated 2 out of 30 cells tested (6.7%) and vehicle ejections activated 1 out of 36 cells tested (2.8%). GRP1-27, GRP18-27 and BN, the BNLPs showing the greatest degree of structural homology, activate approximately 50% of SCN cells, apparently via the BN/GRP-preferring receptor subtype, and may play a role in photic entrainment.     

Changes in vasoactive intestinal peptide mRNA levels in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment under light/dark conditions

Photic stimulus and serotonin (5-hydroxytryptamine; 5-HT) are two factors known to regulate vasoactive intestinal peptide (VIP) synthesis in the suprachiasmatic nucleus (SCN). To explore the role of 5-HT in the photic stimulus-induced change in VIP synthesis, we investigated the changes in level of VIP mRNA under a 12 h light/12 h dark cycle following depletion of 5-HT by intraperitoneal administration of p-chlorophenylalanine (PCPA) methyl ester (200 mg/kg concentration) for 3 successive days. To estimate VIP mRNA expression, we performed in situ hybridization using imaging plates combined with microcomputer-based imaging analysis. In light-phase, total signals of VIP mRNA from the PCPA-treated rats showed a significant decrease compared with those from the saline-treated control rats. However, in dark-phase, there were no significant decreases between the PCPA-treated rats and the saline-control rats. The present results strongly suggest that 5-HT neuronal inputs to the SCN interfere with the effect of photic stimulus on VIP synthesis at the mRNA level.     

Pituitary adenylate cyclase-activating polypeptide and melatonin in the suprachiasmatic nucleus: effects on the calcium signal transduction cascade

The suprachiasmatic nucleus (SCN) harbors an endogenous oscillator generating circadian rhythms that are synchronized to the external light/dark cycle by photic information transmitted via the retinohypothalamic tract (RHT). The RHT has recently been shown to contain pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitter/neuromodulator. PACAPergic effects on cAMP-mediated signaling events in the SCN are restricted to distinct time windows and sensitive to melatonin. In neurons isolated from the SCN of neonatal rats we investigated by means of the fura-2 technique whether PACAP and melatonin also influence the intracellular calcium concentration ([Ca2+]i). PACAP elicited increases of [Ca2+]i in 27% of the analyzed neurons, many of which were also responsive to the RHT neurotransmitters glutamate and/or substance P. PACAP-induced changes of [Ca2+]i were independent of cAMP, because they were not mimicked by forskolin or 8-bromo-cAMP. PACAP caused G-protein- and phospholipase C-mediated calcium release from inositol-trisphosphate-sensitive stores and subsequent protein kinase C-mediated calcium influx, demonstrated by treatment with GDP-beta-S, neomycin, U-73122, calcium-free saline, thapsigargin, bisindolylmaleimide, and chelerythrine. The calcium influx was insensitive to antagonists of voltage-gated calcium channels of the L-, N-, P-, Q- and T-type (diltiazem, nifedipine, verapamil, omega-conotoxin, omega-agatoxin, amiloride). Immunocytochemical characterization of the analyzed cells revealed that >50% of the PACAP-sensitive neurons were GABA-immunopositive. Our data demonstrate that in the SCN PACAP affects the [Ca2+]i, suggesting that different signaling pathways (calcium as well as cAMP) are involved in PACAPergic neurotransmission or neuromodulation. Melatonin did not interfere with calcium signaling, indicating that in SCN neurons the hormone primarily affects the cAMP signaling pathway.     

Sources of p75-nerve growth factor receptor-like immunoreactivity in the rat suprachiasmatic nucleus

In mammals, the suprachiasmatic nucleus is critical for the generation of circadian rhythms and their entrainment to environmental cues. In the rat, the ventrolateral aspect of the suprachiasmatic nucleus receives a robust retinal input. This region also exhibits the most intense immunolabeling for the low-affinity nerve growth factor receptor in the forebrain. Our study was aimed at identifying the sources of this low-affinity nerve growth factor receptor immunoreactivity using immunohistochemistry combined with retrograde tract-tracing, and orbital enucleation. To determine the origin of the low-affinity nerve growth factor receptor immunoreactivity from sources extrinsic to the suprachiasmatic nucleus, unilateral injections of the retrograde tracer, Fluorogold, were made into the suprachiasmatic nucleus. Retrogradely labeled neurons that were also immunopositive for the low-affinity nerve growth factor receptor were found in both the basal forebrain and the retina. In the basal forebrain, such cells were found throughout its rostrocaudal extent, with the majority also being immunoreactive for the cholinergic marker, choline acetyltransferase. In the retina, cells retrogradely labeled with Fluorogold that were immunoreactive for low-affinity nerve growth factor receptor were located in the ganglion cell layer. Orbital enucleations were performed to confirm the findings observed following retrograde labeling in the retina. Unilateral orbital enucleations resulted in a significant reduction in low-affinity nerve growth factor receptor immunoreactivity in the contralateral suprachiasmatic nucleus compared to that seen on the ipsilateral side when examined one week post-surgery. Bilateral enucleations resulted in an equal decrease on both sides of the suprachiasmatic nucleus. Similar low-affinity nerve growth factor-like immunoreactivity was seen in the suprachiasmatic nucleus even two to four weeks after bilateral enucleations. Taken together, these findings suggest that low-affinity nerve growth factor receptors in the suprachiasmatic nucleus derive from multiple sources. While some receptors may be intrinsic to suprachiasmatic nucleus neurons, most appear to be of extrinsic origin and are located on axon terminals of basal forebrain cholinergic neurons and retinal ganglion cells.     

Neuropeptidergic organization of the suprachiasmatic nucleus in the blind mole rat (Spalax ehrenbergi)

The blind mole rat, Spalax, is a subterranean rodent with atrophied, subcutaneous eyes. Whereas most of the visual system is highly degenerated, the retino-hypothalamic pathway in this species has remained intact. Although Spalax is considered to be visually blind, circadian locomotor rhythms are entrained by the light/dark cycle. In the present study we used anterograde tracing techniques to demonstrate retinal afferents to the suprachiasmatic nucleus (SCN) and immunohistochemistry to examine the distribution of neuropeptides that are known to be involved in the regulation or expression of circadian rhythmicity. Based on the localization of retinal afferents and neuropeptides, the SCN can be divided into two subdivisions. The ventral region, which receives retinal afferents, also contains vasoactive intestinal polypeptide (VIP)-containing neurons, and fibers that are immunopositive to neuropeptide Y (NPY) and serotonin (5-HT). The dorsal region contains vasopressinergic neurons, but this latter cell population is extremely sparse compared to that described in other rodents. The dorsal region is also characterized by numerous VIP-immunoreactive fibers. The presence of NPY and 5-HT fibers suggests that the SCN receives afferent projections from the intergeniculate leaflet and from the raphe nuclei, respectively. These neuroanatomical results, together with previous studies of behavior, visual tract tracing, and immediate early gene expression, confirm that an endogenous clock and the capacity for light entrainment of circadian rhythms are conserved in the blind mole rat.     

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

With the advent of combinatorial chemistry a new paradigm is evolving in the field of drug discovery. The approach is based on an integration of chemistry, high-throughput screening and automation engineering. The chemistry arm is usually based on solid-phase synthesis technology as the preferred approach to library construction. One of the most powerful of the solid-phase methods is encoded split synthesis, in which the reaction history experience by each polymeric bead is unambiguously recorded. This split-and-pool approach, employing chemically robust tags, was used to construct a 85,000-membered dihydrobenzopyran library.     

Alteration of serotonergic innervation in the suprachiasmatic nucleus of the rat following removal of input fibers from retina and lateral geniculate nucleus

We report the results of sclerotherapy in 20 patients with bleeding gastric varices due to hepatic schistosomiasis. In an endemic area, patients with hepatic schistosomiasis, and bleeding gastric varices seen on endoscopy to be inferior extension of esophageal varices, were treated with emergency endoscopic injection just proximal to the cardia. Hemostasis was achieved in 17. Obliteration of varices was achieved in all patients with sclerotherapy, combined with surgery. Thirteen patients who had not been operated on in the past and consented to surgery underwent esophagogastric devascularization with splenectomy. Surgery was carried out as an emergency in the three patients who did not respond to sclerotherapy and electively in 10 patients after control of bleeding. After surgery, sclerotherapy was required for remnant varices. One patient with Child-Pugh grade C cirrhosis died of hepatic encephalopathy after control of the bleed. During a median follow-up of 9 months (range, 1-25 months), recurrence of bleeding in one patient and recurrent varices in two others were controlled with sclerotherapy. One patient had a fatal hemorrhage at home. We conclude that sclerotherapy effectively controls acutely bleeding type 1 gastric varices. Combined with esophagogastric devascularization and splenectomy, long-term results may be encouraging in patients with hepatic schistosomiasis.     

Expression of brain-derived neurotrophic factor and its cognate receptor, TrkB, in the rat suprachiasmatic nucleus

Brainerd and Reyna (1998, this issue) have described fuzzy-trace theory as a basic-processing theory, emphasizing age differences in children's disposition to use verbatim versus gist representations. The theoretical climate of the 1980's, when fuzzy-trace theory was first formulated, is described. Fuzzy-trace theory integrated new ideas about how cognitive development was viewed into a coherent framework, which only gradually gained acceptance as critical aspects of the theory were confirmed, counterintuitive findings were predicted and demonstrated, and other researchers began applying the theory. Fuzzy-trace theory converges with other contemporary theoretical accounts in raising the general issue of the relation between two developing representational systems and is consistent with the idea that immature (a bias toward verbatim encoding) and mature (a bias toward gist encoding) have both advantages and disadvantages at different times in development. By integrating the theory with ideas from social-contextual perspectives, the theory may have a greater impact in the future for issues of social significance.     

N-methyl-D-aspartate receptors are indispensable for the formation of long-term potentiation in the rat suprachiasmatic nucleus in vitro

Optic nerve (ON) stimulation caused a postsynaptic field potential in the suprachiasmatic nucleus (SCN) of rat hypothalamic slices. The postsynaptic field potential was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, in a concentration-dependent manner, but not affected by D-amino-5-phosphonovaleric acid (APV), a competitive NMDA receptor antagonist. Tetanic stimulation to the ON induced long-term potentiation (LTP) in the SCN. Application of APV at 50 microM inhibited the induction of LTP by tetanic stimulation but CNQX at lower dose (5 microM) didn't inhibit it. These results suggest that NMDA receptors are indispensable for the induction of LTP after tetanic stimulation.     

c-fos gene expression in postnatal rat retinas with light/dark cycle

We examined the diurnal variation of c-fos gene expression during a 12:12 light/dark cycle in developing rat retinas by in situ hybridization histochemistry. c-fos Gene was not expressed before postnatal day 10 (P10) but was expressed on P15 in the outer nuclear layer throughout the dark period and in the inner nuclear layer and the ganglion cell layer during the light period. These results demonstrated that the earliest c-fos gene expression occurred between P11 and P15. The good correlation between the expression of c-fos gene and the genes coding for proteins involved in phototransduction, in terms of their diurnal variation and in their development, suggested that c-fos gene may play a role in the regulation of these genes in retinal cells during the light/dark cycle.