Age-dependent effects of conditioning on cholinergic and vasopressin systems in the rat suprachiasmatic nucleus

Active shock avoidance was used to explore the impact of behavioural stimulation on the neurochemistry of the suprachiasmatic nucleus. We have found previously that the expression of muscarinic acetylcholine receptors in the suprachiasmatic nucleus of young rats was significantly enhanced 24 hours after fear conditioning. Here, we investigated whether this observation is age-dependent. We used 26 month-old Wistar rats with a deteriorated circadian system, and compared them with young rats (4 months of age) with an intact circadian system. Vasopressin, representing a major output system of the suprachiasmatic nucleus, was studied in addition to muscarinic receptors. Young rats showed a significant increase in immunostaining for muscarinic acetylcholine receptors 24 h after training, corroborating earlier observations. Aged rats did not show such an increase. In contrast, aged rats did show an increase in vasopressin immunoreactivity 24 h after fear conditioning, both at the level of content and cell number, while young rats did not reveal a significant rise. Thus, it seems that these two neurochemical systems in the suprachiasmatic nucleus are regulated independently. The results further demonstrate that the circadian pacemaker is influenced by fear conditioning in an age-dependent manner.     

Retinohypothalamic projection in the mouse: Electron microscopic and iontophoretic investigations of hypothalamic and optic centers

The problem of the direct retinohypothalamic projection in mammals (Moore, 1973) was reinvestigated in the laboratory mouse by electron microscopy and cobalt chloride-iontophoresis. The time-course of the axonal degeneration in the suprachiasmatic nucleus was studied 3, 6 and 12 h, 1, 2, 4, 6, 9 and 12 days after unilateral retinectomy. Specificity of the degenerative changes was controlled by investigation of the superficial layers of the superior colliculus. The ratio of crossed to uncrossed optic fibers could could be determined by counting degenerating structures (axons and terminals) in the optic chiasma and the ipsilateral and contralateral areas of the optic tract, the suprachiasmatic nucleus, and the superior colliculus. The number of degenerating axons in the suprachiasmatic nucleus showed a maximum one day after unilateral retinectomy and was, at all stages studied, two to three times higher in the contralateral than in the ipsilateral nuclear area. In the optic tract and in the superior colliculus the number of degenerating profiles was three times higher in the contralateral than in the ipsilateral area. Retinohypothalamic connections and crossing pattern of retinal fibers were studied light microscopically using impregnation with cobalt sulfide in whole mounts of brains. Most of the optic fibers in the laboratory mouse are crossed crossed (70-80%). A bundle of predominantly crossed optic fibers runs to the suprachiasmatic nucleus.     

Long-Lasting Effects of Sepsis on Circadian Rhythms in the Mouse

Daily patterns of activity and physiology are termed circadian rhythms and are driven primarily by an endogenous biological timekeeping system, with the master clock located in the suprachiasmatic nucleus. Previous studies have indicated reciprocal relationships between the circadian and the immune systems, although to date there have been only limited explorations of the long-term modulation of the circadian system by immune challenge, and it is to this question that we addressed ourselves in the current study. Sepsis was induced by peripheral treatment with lipopolysaccharide (5 mg/kg) and circadian rhythms were monitored following recovery. The basic parameters of circadian rhythmicity (free-running period and rhythm amplitude, entrainment to a light/dark cycle) were unaltered in post-septic animals compared to controls. Animals previously treated with LPS showed accelerated re-entrainment to a 6 hour advance of the light/dark cycle, and showed larger phase advances induced by photic stimulation in the late night phase. Photic induction of the immediate early genes c-FOS, EGR-1 and ARC was not altered, and neither was phase-shifting in response to treatment with the 5-HT-1a/7 agonist 8-OH-DPAT. Circadian expression of the clock gene product PER2 was altered in the suprachiasmatic nucleus of post-septic animals, and PER1 and PER2 expression patterns were altered also in the hippocampus. Examination of the suprachiasmatic nucleus 3 months after treatment with LPS showed persistent upregulation of the microglial markers CD-11b and F4/80, but no changes in the expression of various neuropeptides, cytokines, and intracellular signallers. The effects of sepsis on circadian rhythms does not seem to be driven by cell death, as 24 hours after LPS treatment there was no evidence for apoptosis in the suprachiasmatic nucleus as judged by TUNEL and cleaved-caspase 3 staining. Overall these data provide novel insight into how septic shock exerts chronic effects on the mammalian circadian system.     

c-fos expression in the putative avian suprachiasmatic nucleus

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups. Accepted: 15 November 1996     

The suprachiasmatic nucleus of the mink (Mustela vison): apparent absence of vasopressin-immunoreactive neurons

The hypothalamic suprachiasmatic nucleus is centrally involved in generation of several circadian rhythms. Neurons of the mammalian suprachiasmatic nucleus express a number of neuropeptides including vasopressin. The suprachiasmatic nucleus of the mink (Mustela vison) is easily distinguished from neighbouring hypothalamic areas and the underlying optic chiasm as a small nucleus containing densely packed parvocellular neurons. A dorsal and ventral subdivision were clearly recognized within the midportion and caudal part of the nuclcus. Using immunohistochemistry, we have identified vasopressin-, neurophysin-, and vasoactive intestinal peptide-immunoreactive neuronal elements in the hypothalamus of the mink. Vasoactive intestinal peptide-immunoreactive neurons can be observed in the ventral aspect of the suprachiasmatic nucleus, but to our surprise, no vasopressin immunoreactive perikarya are found within the suprachiasmatic nucleus, this absence being independent of the experienced annual cycle. The hypothalamic paraventricular and supraoptic nuclei contain large numbers of vasopressin-, neurophysin-and vasoactive intestinal peptide-immunoreactive magnocellular neurons with extensive projections towards the infundibulum and neurohypophysis. A comparative analysis of the distribution of vasopressin-immunoreactive elements in a number of conventional laboratory animals has demonstrated that, in contrast to the rat, golden hamster and Mongolian gerbil, neither vasopressin-containing perikarya in the suprachiasmatic nucleus nor fine calibered immunoreactive fibres entering the adjacent subparaventricular zone are present in the mink. The mink is a photodependent seasonal breeder, and thus vasopressin-immunoreactive neurons in the suprachiasmatic nuclei may not be essential for the photoperiodic regulation of reproduction and seasonal events experienced by this species.     

Relationship between androgenization and differentiation of hypothalamic centers

Dependence of the sterilizing action of androgen on differentiation of the hypothalamic centers was studied during the postnatal development of female rats. Asynhronous differentiation in the development of the arcuate nucleus (tonic centre) and suprachiasmatic nucleus (cyclic centre) was observed. Neurocytes of the arcuate nucleus started to produce secretory granules in 20-day embryos, while in the suprachiasmatic nucleus the neurocytes with secretory granules appeared in 5--7-day rats. Injection of testosterone propionate induced an anovulatory cycle in females the first 7 days after birth; this is associated with the inhibition of development of the hypothalamic cyclic centre.     

罗汉果双受精过程的细胞学观察

罗汉果（Ｓｉｒａｉｔｉａｇｒｏｓｖｅｎｏｒｉ（Ｓｗｉｎｇｌｅ）Ｃ．Ｊｅｍｅｙ）双受精过程属有丝分裂前配子融合类型,授粉后２４～４８ｈ,花粉管进入胚囊,穿过一个助细胞,放出两个精子。雌雄核融合和雄核与次生核融合同时发生在授粉后６２～７２,雄核与次生核融合速度快于配子融合,７２ｈ后即可见到初生胚乳核分裂。合子中的雌雄核仁在授粉后第５～６ｄ融合,授粉后８～９ｄ合成分裂形成二细胞胚。在双受精过程中,多次观察到有多条花粉管进入胚囊和多精入极核现象。原胚期有附加花粉管从珠孔进入。     

Effects of partial destruction of the suprachiasmatic nuclei on two circadian parameters: wheel-running activity and short-day induced testicular regression

Summary Many circadian rhythms in mammals are regulated by the suprachiasmatic nuclei located in the anterior hypothalamus. The suprachiasmatic nuclei are a heterogeneous population of neurons loosely segregated into regions. In an effort to determine if a regional specificity of control of different circadian rhythms exists within the SCN, the effect of small electrolytic lesions of the suprachiasmatic nuclei was examined on two parameters which are known to depend on the circadian system for their normal expression: wheel-running activity and short-day induced testicular regression. While some SCN lesions altered both the circadian rhythm of locomotor activity and the normal temporal pattern of gonadal regression on short-days, other partial lesions of the suprachiasmatic nuclei were found to effect one parameter without effecting the other. Detailed histological analysis of the neural damage sustained by the suprachiasmatic nuclei did not indicate an obvious regional specificity of function within the nuclei. However, the results do suggest that functionally specific neural pathways emerging from the suprachiasmatic nuclei carry circadian information to independent neural circuits responsible for locomotor activity and neuroendocrine-gonadal function.Abbreviations SCN suprachiasmatic nucleus - SCG superior cervical ganglion - NAT N-acetyltransferase - LD light/dark cycle - DD constant darkness - circadian period - phase angle of entrainment - HRP horseradish peroxidase - SEM standard error of the mean - vPVN hypothalamic paraventricular nucleus - DMH hypothalamic dorsomedial nucleus - PVT thalamic paraventricular nucleus - IMLN intermediolateral nucleus     

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel,Spermophilus richardsonii

Summary In the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Microinjections of growth hormone-releasing factor into the medial preoptic area/suprachiasmatic nucleus region of the hypothalamus stimulate food intake in rats

The role of the suprachiasmatic nucleus/medial preoptic area region of the hypothalamus in the expression of rat hypothalamic growth hormone-releasing factor-induced feeding in the rat was examined. Rats were tested for their 90-min food intake following microinjections of growth hormone-releasing factor (0.0, 0.01, 0.1 or 1.0 pmol) aimed at the suprachiasmatic nucleus/medial preoptic area region. It was found that growth hormone-releasing factor dose-dependently stimulated food intake with the 1.0 pmol dose being the most effective, increasing food intake by approximately 200%. Injections outside the suprachiasmatic nucleus/medial preoptic area region were ineffective. These data are taken to suggest that the suprachiasmatic nucleus/medial preoptic area region of the hypothalamus is important for the central stimulatory effects of growth hormone-releasing factor on feeding.     

Retinohypothalamic projections and immunocytochemical analysis of the suprachiasmatic region of the desert iguana Dipsosaurus dorsalis

Two separate and distinct retinal projections to the hypothalamus in the iguanid lizard Dipsosaurus dorsalis were described using horseradish peroxidase and cobalt-filling techniques. Both of the projections were unilateral and completely crossed; one terminated in the supraoptic nucleus and the other in the suprachiasmatic nucleus. Immunocytochemical analysis showed that the supraoptic nucleus contained cell bodies and fibers that cross-react with antibodies raised against arginine vasopressin, while the suprachiasmatic nucleus contained arginine vasopressin-like immunoreactive fibers emanating from cells in the nearby paraventricular nucleus. The suprachasmatic nucleus contained a dense plexus of fibers that cross-reacted with neuropeptide-Y antibody. Antiserum against vasoactive intestinal polypeptide showed no reactivity in any part of the forebrain, while antiserum against serotonin showed sparse and uniform reactivity throughout the forebrain, including the suprachiasmatic nucleus. These results, together with other data, indicate that the suprachiasmatic nucleus of D. dorsalis is homologous to the suprachiasmatic nuclei of rodents, structures known to contain circadian pacemakers. We suggest that the suprachiasmatic nucleus may play a similar role in the circadian system of D. dorsalis.     

The source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during the postnatal period in rats

Suprachiasmatic nucleus in the rats during early postnatal development is transitorily innervated by tyrosine hydroxylase-immunoreactive fibers that are neither catecholamine- nor serotoninergic. The goal of this immunocytochemical investigation was to find out if tyrosine hydroxylase-immunoreactive neurons of anterior hypothalamus could be the source of this innervation. According to the obtained immunocytochemical data, multiple multipolar tyrosine hydroxylase-immunoreactive neurons are localized around the suprachiasmatic nucleus in the rats at days 2 and 10 of postnatal development. Most of them were observed ventrally and laterally to the nucleus. The axons of the neurons are oriented towards the suprachiasmatic nucleus. Further investigation demonstrated considerably decreased number of tyrosine hydroxylase-immunoreactive neurons surrounding the suprachiasmatic nucleus in the adult animals as compared to early postnatal period, which correlates to the number of tyrosine hydroxylase-immunoreactive fibers in this nucleus. Hence, tyrosine hydroxylase-immunoreactive neurons in the ventral region of anterior hypothalamus can be considered as a potential source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during early postnatal development.     

The neurofilament architecture of the rat suprachiasmatic nucleus

The neurofilament architecture within the suprachiasmatic nucleus of the rat was analyzed immunocytochemically using neurofilament monoclonal antibodies. The topographic distribution of neurofilament containing structures was restricted mainly to the ventral and caudal part of the suprachiasmatic nucleus, coinciding with the entrance area of the retino-suprachiasmatic fibres of this nucleus. Within the nucleus itself an axonal organization was present. The axons were grouped, forming clusters. These clusters existed of a core of myelinated axons surrounded by unmyelinated axons. The myelinated/unmyelinated axon ratio could reach 1:25. Within the nucleus the myelinated axons extended upwards to the middle part of the suprachiasmatic nucleus, where the fibers of the axon clusters fanned out.     

The Source of Transitory Innervation of Suprachiasmatic Nucleus by Tyrosine Hydroxylase-Immunoreactive Fibers during Postnatal Period in Rats

Suprachiasmatic nucleus in the rats during early postnatal development is transitorily innervated by tyrosine hydroxylase-immunoreactive fibers that are neither catecholamine- nor serotoninergic. The goal of this immunocytochemical investigation was to find out if tyrosine hydroxylase-immunoreactive neurons of anterior hypothalamus could be the source of this innervation. According to the obtained immunocytochemical data, multiple multipolar tyrosine hydroxylase-immunoreactive neurons are localized around the suprachiasmatic nucleus in the rats at days 2 and 10 of postnatal development. Most of them were observed ventrally and laterally to the nucleus. The axons of the neurons are oriented towards the suprachiasmatic nucleus. Further investigation demonstrated considerably decreased number of tyrosine hydroxylase-immunoreactive neurons surrounding the suprachiasmatic nucleus in the adult animals as compared to early postnatal period, which correlates to the number of tyrosine hydroxylase-immunoreactive fibers in this nucleus. Hence, tyrosine hydroxylase-immunoreactive neurons in the ventral region of anterior hypothalamus can be considered as a potential source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during early postnatal development.     

5HT7 receptors in the rodent suprachiasmatic nucleus

Serotonergic modulation of circadian rhythms in rodent model preparations has received considerable attention over the past decade. Investigators have also been trying to determine which of the many serotonin receptor subtypes may be mediating the effects of serotonin in the suprachiasmatic nucleus, the location of the biological clock that generates the circadian rhythms. A single study in 1993 using the in vitro rat hypothalamic slice preparation suggested that serotonergic modulation of circadian rhythms at the level of the suprachiasmatic nucleus was acting via the newly discovered 5HT7 receptor subtype. Since that initial claim, serotonin modulation of circadian rhythms at the level of the suprachiasmatic nucleus has generally been attributed to 5HT7 receptor activation. However, when trying to cite relevant literature in support of 5HT7 involvement, it becomes evident that attributing rhythm-related serotonin activity in the suprachiasmatic nucleus to 5HT7 receptors may be somewhat premature. There are issues related to pharmacological specificity, species-specific results, and significant knowledge gaps that necessitate a careful review of the literature to make a judgment as to whether 5HT7 receptors are responsible for serotonergic activity in the rodent suprachiasmatic nucleus. In addition, there is sufficient data available at present to make an initial determination as to the degree of 5HT7 receptor involvement at any level in the generation or modulation of circadian rhythms in rodent species.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

Distribution of vasoactive intestinal peptide-like and neurophysin-like immunoreactive neurons and acetylcholinesterase staining in the ring dove hypothalamus with emphasis on the question of an avian suprachiasmatic nucleus

Summary Two nuclei, termed here the medial hypothalamic nucleus and the lateral hypothalamic retinorecipient nucleus, are possible homologs of the mammalian suprachiasmatic nucleus. As the mammalian suprachiasmatic nucleus is characterized by a dense concentration of vasoactive intestinal peptide (VIP)-and neurophysin (NP)-immunoreactive neurons and an absence of acetylcholinesterase (AChE) staining, we decided to examine these factors in the ring dove hypothalamus. Neither the medial hypothalamic nucleus nor the lateral hypothalamic retinorecipient nucleus contained either VIP-or NP-like immunoreactive neurons. The lateral hypothalamic retinorecipient nucleus stained darkly for AChE. Although there was some overlap in the distribution of VIP-and NP-like immunoreactive neurons, a clustering of both types into a well defined nucleus was not observed. Therefore, an avian homolog to the mammalian suprachiasmatic nucleus must differ in its chemoarchitecture from that of mammalian species described to date.     

Light-Induced Variations in AP-1 Binding Activity and Composition in the Rat Suprachiasmatic Nucleus

Abstract : 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 Modification of Uterine Responses by Day-Night Dexamethasone Treatment during Decidualization in Rats

The effect of treatment (1 mg/rat for 5 days, post-implantation) with the synthetic glucocorticoid, dexamethasone (Dex) on the biochemical components in uterine proliferation during decidualization in rats was assessed. Dex was administered subcutaneously either at only one time (two hours after lights off) of the scotophase, or again at one time (two hours after lights on) during the photophase of a fixed 24 hours (12L: 12D) photoperiod. The uterine parameters included the wet weight, protein and DNA concentrations, nuclear estrogen receptors, enzymatic activities of alkaline phosphatase, isocitrate dehydrogenase and the matrix metalloproteinases, and serum levels of estradiol and progesterone. Certainly the rat circadian system, under the influence of the hypothalamic suprachiasmatic nucleus is fully entrained by the alternation of the experiment. Hence, the results suggest that between the two injections times, there may be a circadian difference in the uterine responses to Dex. The findings have meaningful implications for Dex and the optimal effectiveness of its application times for implantation problems.     

Circadian Modification of Uterine Responses by Day-Night Dexamethasone Treatment during Decidualization in Rats

The effect of treatment (1 mg/rat for 5 days, post-implantation) with the synthetic glucocorticoid, dexamethasone (Dex) on the biochemical components in uterine proliferation during decidualization in rats was assessed. Dex was administered subcutaneously either at only one time (two hours after lights off) of the scotophase, or again at one time (two hours after lights on) during the photophase of a fixed 24 hours (12L: 12D) photoperiod. The uterine parameters included the wet weight, protein and DNA concentrations, nuclear estrogen receptors, enzymatic activities of alkaline phosphatase, isocitrate dehydrogenase and the matrix metalloproteinases, and serum levels of estradiol and progesterone. Certainly the rat circadian system, under the influence of the hypothalamic suprachiasmatic nucleus is fully entrained by the alternation of the experiment. Hence, the results suggest that between the two injections times, there may be a circadian difference in the uterine responses to Dex. The findings have meaningful implications for Dex and the optimal effectiveness of its application times for implantation problems.