Radioautographic studies on the neurohypophysial projections of the supraoptic and paraventricular nuclei in the rat

Summary The distribution of labelled axonal pathways was studied after unilateral stereotaxic injection of ³H-leucine into either supraoptic (SON) or paraventricular nuclei (PVN). In addition to extrahypothalamic projections of both nuclei, the main efferents appeared to run towards the neurohypophysis, yet with a strikingly different pattern. At the neurohypophysial level, the SO-neurohypophysial tract crossed the inner layers of the median eminence (ME) before scattering in the neural lobe. The PV-neurohypophysial pathway, by contrast, provided an exclusive innervation to the external layer of the whole neurohypophysial organ, including the median eminence, infundibular stalk and neural lobe. The functional correlates of the clear-cut anatomical distinctness between the two magnocellular neurosecretory systems are discussed.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: III. Ontogeny of catecholamine varicosities and neurophysin neurons in the rat supraoptic and paraventricular nuclei

The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 17 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21–28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appeared to make contact with NP neurons beginning at 21–22 days postcoitus. An apparent increase in varicosity-perikaryal contacts with age was observed in both nuclei; by 14–21 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.     

P2X receptors are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei of rat hypothalamus

In the present study, the distribution of P2X receptor protein and colocalization of P2X receptors with vasopressin and oxytocin in the supraoptic and paraventricular nuclei of rat hypothalamus was studied using double-labeling fluorescence immunohistochemistry. The results showed that vasopressin-containing neurons expressed P2X₂, P2X₄, P2X₅ and P2X₆ receptor and oxytocin-containing neurons expressed P2X₂, P2X₄ and P2X₅ receptors in the supraoptic nucleus. In the paraventricular nucleus, vasopressin-containing neurons expressed P2X₄, P2X₅ and P2X₆ receptors, while oxytocin-containing neurons expressed P2X₄ receptors. This study provides the first evidence that P2X receptor subunits are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei, and hence, provides a substantial neuroanatomical basis for possible functional interactions between the purinergic and vasopressinergic systems, and the purinergic and oxytocinergic systems in the rat hypothalamus. Wei Guo and Jihu Sun contributed equally to this work.     

Changes in nucleolar dry mass of neurones of the paraventricular and supraoptic nuclei in the rat during pregnancy and lactation

Summary Interference microscopy was used to measure the dry mass of nucleoli in unfixed nuclei isolated from neurones of the paraventricular (PV) and supraoptic (SO) nuclei of female rats. Changes in nucleolar dry mass during pregnancy and lactation have been interpreted as reflecting changes in rates of synthesis of ribosomes and protein in these neurones. Measurements were made on a total of 6580 nucleoli from 135 rats. At the end of pregnancy nucleolar dry mass of both PV and SO neurones was increased compared with virgin female rats. Nucleolar dry mass of PV neurones but not SO neurones increased further during lactation. This change was biphasic, with a nadir at 2 weeks post partum. After day 5 post partum, nucleolar dry mass of PV and SO neurones was increased only in rats suckling pups. Adjustment of litter size to 10 or 22 to 24 pups on the first day post partum did not affect nucleolar changes in PV and SO neurones. Nucleolar changes were less when only one pup was nursed. The results are discussed in relation to oxytocin secretion induced by the suckling stimulus and the synthetic response of PV and SO neurones to increased secretion.     

Immunoelectronmicroscopic localization of vasopressin in the rat suprachiasmatic nucleus

Summary The classical areas for arginine-vasopressin (AVP) synthesis are the magnocellular supraoptic (SON) and paraventricular nuclei. More recently AVP was also demonstrated in neurons of the parvocellular suprachiasmatic nucleus (SCN) of the rat. This result was substantiated in the present study by means of immunoelectron microscopy, by subjecting sections to antivasopressin plasma. Conventional electron microscopy revealed dense-core vesicles in the SCN cell bodies and fibres (mean diameter 94.7±0.9 nm and 84.0±1.1 nm respectively). These vesicles were infrequent within the cell bodies and could not be accumulated by ethanol administration. Immunoelectron microscopy showed a positive reaction in the cell bodies and fibres within vesicles of 93.7±1.1 nm and 98.5±1.2 nm respectively. By comparison, the cell bodies and fibres of the SON showed immunoreactive granules of 143.0±1.8 and 147.3±1.8 nm respectively. The presence in the SCN of AVP in vesicles of different size than those in the SON suggests that synthesis of this substance is indeed occurring within the SCN cells.Supported by The Foundation for Medical Research FUNGOThe authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group Neuroendocrinology for their suggestions, Mr. P.S. Wolters and Miss A. van der Veiden for their skilled assistance     

Different patterns of circadian oscillation in the suprachiasmatic nucleus of hamster,mouse, and rat

Although spontaneous neural firing in the mammalian suprachiasmatic nucleus is accepted to peak once during mid-subjective day, dual activity peaks have been reported in horizontal brain slices taken from hamsters. These two peaks were interpreted as new evidence for the theory of dual circadian oscillators and raised the expectation that such activity would be found in other circadian model systems. We examined hamster, mouse, and rat slices in both coronal and horizontal planes and found a second peak of activity only in hamster horizontal preparations. This raises interesting questions about the relative circadian physiology of these important experimental animals.Abbreviations CT circadian time - SCN suprachiasmatic nucleus P.W. Burgoon and P.T. Lindberg contributed equally to this work.     

Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat

Summary Fine structural localization of an ACTH-like substance was obtained in neurons of the rat arcuate nucleus using immuno-electron microscopy, whereas it could not be confirmed that ACTH-containing cell bodies are present in the supraoptic nucleus. The immunoreactive cells of the arcuate nucleus appeared to be more numerous than the unreactive neurons. Immunostaining was carried out before embedding in resin. Empty vesicles of irregular shape were found in dendrites of immunoreactive arcuate neurons, but their significance and nature remain enigmatic. The reaction product was distributed uniformly throughout the cytoplasm of the ACTH-positive cells, except that the mitochondria, rough endoplasmic reticulum and Golgi vesicles and cisternae were devoid of PAP molecules. This distribution differed from the localization reported in ACTH-secreting cells of the rat anterior pituitary, where the reaction product was found in the rough endoplasmic reticulum and Golgi complex as well as in secretory granules.     

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca²⁺-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.     

Multiple oscillators in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371-387, 2001)     

Light-induced tyrosine phosphorylation of BIT in the rat suprachiasmatic nucleus

Circadian changes of protein tyrosine phosphorylation in the hypothalamic suprachiasmatic nucleus have been studied using rats maintained under 12-h light/ 12-h dark cycles as well as constant dark conditions. We found that tyrosine phosphorylation of BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs), a transmembrane glycoprotein of 90-95 kDa, was higher in the light period than in the dark period and was increased after light exposure in the dark period. Similar changes in tyrosine phosphorylation were observed under constant dark conditions, but its amplitude was weaker than that in 12-h light/12-h dark cycles. As the tyrosine-phosphorylated form of BIT is able to bind to the Src homology 2 domain of a protein tyrosine phosphatase, SHP-2, we examined association of these proteins in suprachiasmatic nucleus extracts and found that SHP-2 was coprecipitated with BIT in parallel with its tyrosine phosphorylation. These results suggest that tyrosine phosphorylation of BIT might be involved in light-induced entrainment of the circadian clock.     

Effects of suprachiasmatic nuclei lesions on circadian and ultradian rhythms in body temperature in ocular enucleated rats

Abstract

To test the hypothesis that an oscillator located outside the suprachiasmatic nuclei (SCN) controls the circadian rhythm of body temperature, we conducted a study with 14 blinded rats, 10 of which receiving a SCN lesion. Body temperature was automatically and continuously recorded for about one month by intraperitoneal radio transmitters. Food intake, drinking and locomotor activity were also recorded. Periodograms revealed that 3 rats with histologically verified total bilateral SCN lesions did not exhibit any circadian rhythmicity. The 7 other rats appeared to have partial lesions. They showed shortening of period and severe amplitude reduction in all functions. Thus, no support was found for the hypothesis of a separate circadian ‘temperature oscillator’ located outside the SCN. Nevertheless, after large partial lesions body temperature showed more persistency than some of the other behavioral rhythms.

Ultradian rhythms in temperature persisted after partial and total lesions. Other functions showed parallel ultradian rhythms. In intact rats the ultradian peaks were restricted predominantly to the subjective night. After total lesions these peaks became more or less homogeneously distributed in time but more heterogeneously after partial lesions. So the SCN plays a role in the temporal structure of ultradian rhythms but does not generate them. Non‐24‐hour actograms showed instabilities of period and phase of ultradian rhythms. Intact and lesioned rats were similar with respect to the mean (about 3.5 hrs) and standard deviation (about 1.5 hrs) of ultradian periods in temperature. These features indicate that a mechanism outside the SCN is underlying ultradian rhythmicity, capable of generating short‐term oscillations. Two approaches, homeostatic sleep‐wake relaxation oscillations and multiple circadian oscillators, are discussed.     

Ultrastructural aspects of the paraventricular nuclei in the rat

Summary The ultrastructural aspects of the paraventricular nucleus and its neuropil are described in the normal rat.Two types of neurons can be distinguished morphologically. The first type contains numerous dense-core vesicles (mean diameter: 140 nm). The cisternae of the endoplasmic reticulum are arranged parallely at the periphery of the cell body.The second type of neuron contains a few dense-core vesicles (mean diameter: 75 nm) and the endoplasmic reticulum is randomly distributed in the cytoplasm. In the neuropil, two types of dense-core vesicles are observed in separated axons. The histogram of the distribution of their mean diameter clearly indicates a double population of vesicles.The signification of the second type of neuron in the paraventricular nucleus is discussed and its possible relation to TRF synthesis is evoked.This work was supported by a grant from the Belgian National Fund for Scientific Research.The author wish to thank Mrs. Hunninck-Couck for her devoted and skillful technical assistance.     

哺乳动物昼夜节律组构中的下丘脑视交叉上核和松果腺

哺乳动物下丘脑视交叉上核（SCN）是昼夜节律最主要的起搏器,控制着机体的生理和行为的节律。它具有自身内在的节律性,同时也受光照周期信号和一些内源性化学物质的调节。检查腺分泌裉黑素（MEL）受SCN的调控,MEL通过作用于SCN上高亲和性MEL受体,启动第二、第三信使系统,调整SCN的昼夜节律活动。这种调整具有时间敏感性。     

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk,Eutamias asiaticus

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.     

Periodically fluctuating protein kinases phosphorylate CLOCK, the putative target in the suprachiasmatic nucleus

We studied nuclear protein phosphorylation in the rat suprachiasmatic nucleus (SCN) and found that a nuclear fraction of the SCN contained histone H1 kinase activity that periodically fluctuated with a diurnal rhythm, reaching a maximum at the midpoint of the light phase and a minimum at the midpoint of the dark phase. A p13suc1-bound fraction from the SCN nuclear fraction also exhibited diurnally fluctuating histone H1 kinase activity. Using in situ kinase assay, three histone H1 kinases, p45PFK, p100PFK, and p200PFK (termed periodically fluctuating protein kinases, or PFKs) were found in the p13suc1-bound fractions. p45PFK exhibited the highest level of light/dark cycle phosphorylation activity fluctuation. p45PFK highly phosphorylated the Ser-Pro-rich region of CLOCK, the putative physiological target. These results suggest that PFKs, especially p45PFK, are involved in circadian clock-related signal transduction and gene expression, through the phosphorylation of target proteins such as CLOCK.     

Spatial and temporal regulation of mitogen-activated protein kinase phosphorylation in the mouse suprachiasmatic nucleus

Circadian and photic regulation of mitogen-activated protein kinase (MAPK) has been shown to associate closely with the function of the circadian clock in vertebrate clock tissues such as the mouse suprachiasmatic nucleus (SCN). Here we show that, in the central region of the mouse SCN, MAPK exhibited circadian and daily rhythms in phosphorylation with a peak at (subjective) night, and this activation was sustained for at least 8 h. In contrast, in the dorsomedial region of the SCN, MAPK showed an overt rhythm in phosphorylation with a transient peak at early subjective day, which was antiphase to that in the central region. Noticeably, the phospho-MAPK-immunoreactive cells observed in the dorsomedial region were distributed from the rostral to the caudal end of the SCN, whereas those observed in the central region were localized within the middle SCN along the rostral-caudal axis. Furthermore, a 15-min light pulse given at subjective night transiently evoked MAPK phosphorylation throughout the ventrolateral region of the SCN peaking within 15 min after the light onset, whereas nighttime-phosphorylated MAPK signals in the central-middle SCN become undetectable within 60 min after the light onset. Thus, the mode of circadian and photic regulation of MAPK phosphorylation varies remarkably among the three subregions within the SCN, suggesting divergent and cell type-specific roles of MAPK in the clock system of the mouse SCN.     

Circadian organization in the ruin lizard Podarcis sicula: the role of the suprachiasmatic nuclei of the hypothalamus

The effects of electrolytic lesions to the suprachiasmatic nuclei of the hypothalamus (SCN) on circadian rhythms of locomotor activity were examined in ruin lizards Podarcis sicula maintained in constant darkness and constant temperature (29°C). All lizards (N=15) in which the lesion damaged 80% or more of the SCN became behaviorally arrhythmic. On the contrary, locomotor rhythms persisted in all cases (N=11) when the SCN remained intact and lesions were confined to neighbouring regions of the preoptic area. Taken together with previous work which demonstrates that the pineal and the retinae are not essential for the persistence of circadian locomotor rhythmicity in Podarcis sicula and with recent evidence showing the homology between the SCN of lizards and those of mammals the present results strongly support the view that the SCN of Podarcis sicula contain the primary pacemaker(s) for locomotor rhythms.Abbreviations DD constant darkness - LL constant light - SCN suprachiasmatic nuclei of the hypothalamus - PH nucleus periventricularis hypothalami - OC optic chiasm - te length of circadian activity - freerunning circadian period     

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Subsurface cisterns in paraventricular nuclei of the hypothalamus of the rat

Summary Structures identified as subsurface cisterns (SSC's) were found in neurons of the paraventricular nuclei of the rat hypothalamus. They appeared as cytoplasmic organelles consisting most often of stacks of parallel cisterns apposed to the neuronal plasmalemma. These SSC's were located in the interneurons of the parvocellular system, but not in neurosecretory cells and glial cells. SSC's were seen at zones of cytoplasm apposed to neuronal or glial cell processes, showing in some instances specific relationships with synaptic areas.The morphological features of these SSC's are described, and their possible functional significance is briefly discussed.     

Phase advances of circadian rhythms in somatostatin depleted rats: effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus

Somatostatin is synthesized in the suprachiasmatic nucleus (SCN), a circadian pacemaker in mammals. To explore the functional significance of somatostatin in the circadian system, we examined rhythms of rat locomotor activity and electrical firing rate of SCN neurons in the brain slice after temporal depletion of somatostatin levels in the SCN. Intraperitoneal administration of cysteamine (200 mg/kg), a somatostatin depletor, significantly reduced somatostatin level in the in vivo SCN 5 min after injection and kept low level as long as 3 to 4 days. This administration, on the other hand, induced significant phase advances of about 51 min in the subsequent free-running rhythm of locomotor activity of the rat. A marked phase advance in the circadian rhythm of firing rate in the SCN was also observed after administration of cysteamine in coronal hypothalamic slices. These persistent phase shifts after administration of a somatostatin depletor may suggest that the change of somatostatin level in the SCN have a feedback influence on the circadian pacemaker.Abbreviations SCN suprachiasmatic nucleus - AVP arginine-vasopressin - VIP vasoactive intestinal polypeptide - CT circadian time - ZT zeitgeber time - i.p. intraperitoneally - 12L:12D 12 h light and 12 h dark - ANOVA analysis of variance