Effect of bilateral lesions of the suprachiasmatic nucleus on hyperglycemia caused by 2-deoxy-D-glucose and vasoactive intestinal peptide in rats

A disturbed calcium homeostasis characterizes diabetic pregnancy. This study documents changes in bone mineral composition in diabetic pregnant rats and examines the effect of insulin replacement. Control pregnant (CP), diabetic pregnant (DP) and insulin-treated DP (DPi) rats were assessed for femoral calcium and magnesium content, bone mineral density (BMD) and the ratio of hypertrophic to maturing and proliferative cells in the femoral growth plate. DP rats showed a significantly (P < 0.01) lower body weight, femoral weight and length than CP rats. Femoral calcium and magnesium content was also significantly (P < 0.05) lower in DP rats, as was ash weight. When calcium and magnesium were normalized for ash weight no significant differences were apparent. A significantly (P < 0.05) lower total BMD at the distal femur was seen in DP rats. This comprised a significantly (P < 0.01) lower trabecular BMD with no significant change in cortical BMD. A significantly (P < 0.05) higher ratio of hypertrophic to maturing and proliferative cells of the femoral growth plate was evident in DP animals. DPi rats showed normal blood glucose concentrations and femoral growth plate histology. DPi rats also showed normal femoral weight and length but only partially restored femoral ash weight and mineral content. Insulin failed to normalize total or trabecular BMD. Diabetes mellitus clearly has a marked effect on bone growth and mineral content in pregnancy which may be relevant to overall calcium homeostasis. The lower bone growth, bone calcium content and trabecular BMD may be unfortunate consequences of the marked hypercalciuria reported elsewhere in diabetes and may serve to maintain normocalcaemia in the disease.     

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.     

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.     

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.     

Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus

Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium. Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation. The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h. The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms. Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide. Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed. The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31. Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide. Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.     

Neurons containing gastrin-releasing peptide and vasoactive intestinal polypeptide are involved in the reception of the photic signal in the suprachiasmatic nucleus of the Syrian hamster: an immunocytochemical ultrastructural study

In mammals, the suprachiasmatic nuclei are involved in the generation of biological rhythms and are synchronized by light input coming from the retina. The targets of retinal afferents and the involvement of neurons containing gastrin-releasing and vasoactive intestinal peptides in photic reception were investigated in the suprachiasmatic nuclei of the Syrian hamster by using light- and electron-microscopic immunocytochemistry. Cholera toxin was used to trace retinal fibers and Fos immunoreactivity to visualize cellular response to light stimulation. Ultrastructural observations were made in the intermediate third of the nuclei, the area of highest overlap for the immunoreactivities investigated. Gastrin-releasing peptide and vasoactive intestinal peptide cell bodies were localized in the ventral part of the nuclei; their dense immunoreactive fiber network often displayed synaptic contacts. Both neuropeptides were colocalized in elongated cells observed near the optic chiasm. Following a light pulse in the middle of the subjective night, Fos protein was expressed in most gastrin-releasing peptide perikarya and in some vasoactive intestinal peptide cells. Retinal terminals mostly occurred in the midline zone between the suprachiasmatic nuclei. Symmetrical or asymmetrical retinal synapses were observed on gastrin-releasing peptide-immunoreactive dendrites and somata, but never on vasoactive intestinal peptide neurons. These results are discussed in relation to the photic entrainment of the circadian clock.     

Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei

The timing of the preovulatory surge of LH in female rodents is tightly coupled to the environmental light/dark cycle. This coupling is mediated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). Studies indicate that vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), which are synthesized in the SCN, transmit circadian information from the SCN to GnRH neurons, thereby regulating the timing of the LH surge. However, to date, the rhythmic expression of these two peptides in the SCN has only been examined in males. The pattern of VIP expression in males is difficult to reconcile with its role in the LH surge. The purpose of the present study was to assess the rhythm of VIP messenger RNA (mRNA) levels in the SCN of female rats under several endocrine conditions. We compared this rhythm to that in males and to AVP mRNA rhythms in all experimental groups. In all groups of females, VIP mRNA levels were rhythmic, with peak expression occurring during the light phase and a nadir occurring during the dark phase. The rhythm was approximately 12 h out of phase compared with that in males. The rhythmic expression of AVP mRNA in the SCN was virtually identical in all groups of animals. Based on these results, we conclude that 1) the rhythm of VIP seen in the SCN of females during the day may serve as a facilitory signal from the SCN to GnRH neurons; 2) the sex-specific pattern of VIP mRNA does not depend on estradiol; and 3) AVP gene expression within the SCN is not sexually differentiated or altered by estradiol.     

Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18-39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP-containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA-immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker.     

Role of the suprachiasmatic nucleus in the pathogenesis of migraine attacks

Neuroanatomic, morphometric, immunocytochemical, neurobiochemical and clinical data support the hypothesis that the suprachiasmatic nucleus of the hypothalamus might be the initial site of migraine attacks. The prodromal phase of a migraine attack could be considered a syndrome of functional suprachiasmatic nucleus insufficiency, and other phases a reactive denervation hypersensitivity with the affection of the visual, nociceptive, antinociceptive and cranial vasomotor system.     

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.     

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.     

Immunohistochemical demonstration of the intracellular localization of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat testis using the stamp preparation

Radioimmunoassay has revealed an abundance of PACAP in the rat testis. In the present study, a novel stamp preparation together with light microscopic immunohistochemistry were used to investigate in detail the intracellular localization of PACAP-like immunoreactivity (PACAP-LI) in rat germ cells. Samples were obtained by pressing the freshly cut surfaces of the testes against glass slides. PACAP-LI was clearly identified in developing acrosomes using five antisera which recognize PACAP 38, or both PACAP 27 and PACAP 38. Immunoreactivity with the antisera specific to PACAP38 was strictly localized in the developing acrosomes of the spermatids but vanished in the mature spermatids. Using an antiserum which detects PACAP 27 specifically, little staining was observed. Based on the specificities of antisera used, it was suggested that the PACAP-LI in the acrosomes represents mainly PACAP 38-LI. In addition, the present results supported the usefulness of the stamp preparation for immunohistochemical study of testicular tissues.     

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.     

Development of parvalbumin immunoreactivity in the chick Edinger Westphal nucleus

To determine when the calcium-binding protein parvalbumin appears during development, neurons in the chick Edinger Westphal nucleus were examined for parvalbumin immunoreactivity at a variety of embryonic stages. Parvalbumin immunoreactivity appeared on embryonic day 14 (E14, Hamburger and Hamilton stage 40) in predominantly lateral Edinger Westphal neurons. Cytochrome oxidase activity within the nucleus was examined throughout development, as an indicator of physiological activity, and expression of cytochrome oxidase was compared with that of parvalbumin. Cytochrome oxidase activity was found to be uniformly high in all parts of the Edinger Westphal nucleus throughout development. Either the Edinger Westphal nucleus in physiologically active quite early in its development or other energy demands mask the correlation of cytochrome oxidase with electrical activity. Cytochrome oxidase was expressed well before parvalbumin immunoreactivity appeared. Voltage-activated calcium currents were characterized in E12 Edinger Westphal neurons. In both amplitude and composition, E12 calcium currents resemble those of E16 neurons, excluding the possibility that calcium currents appear de novo during or just prior to the appearance of parvalbumin. Both cytochrome oxidase activity and calcium currents are observed in Edinger Westphal neurons well before the appearance of parvalbumin during development. These findings do not exclude the possibility that physiological activity affects the expression of parvalbumin since other factors such as changing patterns of synaptic activity or the appearance of calcium conducting NMDA receptors have yet to be examined. However, they raise the possibility that additional factors such as an intrinsic developmental program or a change in the neuron's basal intracellular calcium requirements may also be involved.     

Lack of effect of three putative vasoactive intestinal peptide receptor antagonists on vasoactive intestinal peptide-induced secretory responses in rat colon

A simple, rapid and efficient method for the preparation of a potential brain blood-flow agent, N-[11C-methyl]-chlorphentermine ([11C]NMCP), is described. Optimization of the radiochemical yield of [11C]NMCP was accomplished by a Gabriel-like reaction which permits the transformation of a primary amine to a secondary amine through a sequence of acylation, deprotonation, monomethylation and saponification. This method precludes the formation of polymethylated by-products which can reduce radiochemical yields, particularly with low specific activity 11CO2.     

Diurnal variations in the urinary excretion of calcium and phosphate in hyperparathyroidism

The urinary excretion of calcium and phosphate during the day and night was studied in 20 patients with primary hyperparathyroidism and in the same number of controls with normal function of the parathyroids. A significant difference in TRP between day and night was found in the controls but not in the HPT group. In other respects there were no substantial differences between day and night. The higher excretion of calcium observed in the HPT group was largely attributable to the patients with remal calculi. The simplified sampling procedure when only night urine is analysed has no disadvantages-it is more likely to improve the diagnostic reliability as it reduces the influence of meals, for example.     

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.     

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.