Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

褪黑素对大鼠下丘脑薄片视交叉上核神经元放电昼夜节律性的调制

先用持续光照和松果腺切除预处理大鼠,然后制成下丘脑薄片,记录其视交叉上核（ＳＣＮ）神经元自发放电,观察其昼夜变化和褪黑素（ＭＥＬ）对它的影响。实验结果表明：⑴在正常光照（光照：黑暗＝１２：１２）条件下,ＳＣＮ神经元自发放电频率呈现昼夜低的节律性。在昼夜时间（ＣＴ）６－８出现放电高峰,频率约为８．３Ｈｚ;在ＣＴ１８－２０出现低谷,频率约为３．８Ｈｚ。松果腺切除后,ＳＣＮ神经元自发放电的昼夜节律性基本     

Temporary Suppression of the Hyperglycemic Response to 2-Deoxy-d-Glucose by Blinding

Studies were made on whether there is a time-dependency in the hyperglycemic response to intracranial injection of 2-deoxy-d-glucose (2DG) in blind rats. 2DG was given to blind rats in their subjective light and dark periods to see if the response free-runs like their circadian locomotor and feeding rhythms. The following results were obtained: (1) In control period and week 3 after blinding, 2DG caused greater hyperglycemia in the subjective light period than in the subjective dark period; (2) In weeks 4 and 6, however, 2DG caused only slight hyperglycemia, while it caused considerable hyperinsulinemia in both the subjective light and dark periods with no time-dependency. (3) In week 8, the hyperglycemic response to 2DG was completely restored while the hyperinsulinemic response was lost. These findings indicate that the subjective time-dependency in the hyperglycemic response to intracranial injection of 2DG exists until week 3 and after week 8 after blinding, however, in week 4 and 6 after blinding the subjective time-dependency appeared to disappear and the hyperglycemic response is largely suppressed in association with hyperinsulinemia. Together with a previous finding that bilateral lesions of the SCN completely abolished the response to 2DG and the fact that blind rats showed circadian rhythms of feeding and locomotive activity even in weeks 4 and 6 after blinding, these findings present the possibility that the site responding to 2DG is in the vicinity of the SCN, but is indifferent neuronal cells from those of the circadian oscillator. However, it is also possible that the blinding elicits the suppression of hyperglycemia due to 2DG through disturbing neural pathway outside the SCN which control blood glucose concentration.     

Functional changes of the SCN in spontaneous hypertension but not after the induction of hypertension

ABSTRACT

The present study investigates the circadian behavior of spontaneously hypertensive rats (SHRs) during the pre-hypertensive and hypertensive stage, with the aim to gain insight into whether observed changes in the functionality of suprachiasmatic nucleus (SCN) in the hypertensive state are cause or consequence of hypertension. Four types of animals were used in this study: (1) SHRs which develop hypertension genetically; (2) their normotensive controls, Wistar Kyoto rats (WKYs); (3) Wistar rats whereby hypertension was surgically induced (2 Kidney 1 Clamp (2K1C) method); and (4) sham-operated control Wistar rats. Period length and activity levels and amplitude changes of locomotor and wheel running activity were determined, in constant conditions, as a measure of the functionality of the SCN. Hereto two conditions were used, constant darkness (0 lux) and constant dim (5 lux) light. SHRs showed a shortened period of their locomotor and running wheel activity rhythms in constant darkness during both pre-hypertensive and hypertensive stages and exhibited period lengthening in constant dim light conditions, only during hypertensive stages. Total amount as well as the amplitude of daily running wheel rhythms showed an inverse correlation with the period length, and this relation was significantly different in SHRs compared to WKYs. None of the aforementioned changes in circadian rhythms were observed after the surgical induction of hypertension. The present findings suggest early functional changes of the SCN in the etiology of spontaneous hypertension.     

Effect on the plasma renin-aldosterone system of lesions to suprachiasmatic nuclei and central serotonin depletion in rats

Renin activity, angiotensin-converting enzyme activity and aldosterone concentration were measured in the plasma of 8 experimental groups of rats: I--sham operated non-treated rats, II--suprachiasmatic nuclei (SCN) lesioned non-treated: III--sham operated + furosemide (4 mg/kg i.p.), IV--SCN lesioned + furosemide, V--shams + 24-hour water deprivation: VI--SCN + 24-hour water deprivation, VII--intact rats + saline: and VIII--intact rats + p-chlorophenylalanine (pCPA, 300 mg/kg, i.p.). No significant changes in basal levels of the three parameters were found after SCN, lesions in comparison with sham operated controls. Furosemide caused a similar increase in all three parameters of both sham and SCN lesioned rats. Similar changes were observed in SCN rats 24 hours after water deprivation and in intact rats 48 hours after serotonin depletion by pCPA: suppressed renin activity together with increased aldosterone concentration. It is concluded that the central serotonergic system and SCN play a similar role in control of the renin-aldosterone system in rats under conditions of negative water-salt balance.     

Restoration of Orcadian Behavior by Anterior Hypothalamic Grafts Containing the Suprachiasmatic Nucleus: Graft/Host Interconnections

Destruction of the hypothalamic suprachiasmatic nucleus (SCN) disrupts circadian behavior. Transplanting SCN tissue from fetal donors into SCN-lesioned recipients can restore circadian behavior to the arhythmic hosts. In the transplantation model employing fetal hamster donors and SCN-lesioned hamsters as hosts, the period of the restored circadian behavior is hamster-typical. However, when fetal rat anterior hypothalamic tissue containing the SCN is implanted into SCN-lesioned rats, the period of the restored circadian rhythm is only rarely typical of that of the intact rat. The use of an anterior hypothalamic heterograft model provides new approaches to donor specificity of restored circadian behavior and with the aid of species-specific markers, provides a means for assessing connectivity between the graft and the host. Using an antibody that stains rat and mouse neuronal tissue but not hamster neurons, it has been demonstrated that rat and mouse anterior hypothalamic heterografts containing the SCN send numerous processes into the host (hamster) neuropil surrounding the graft, consistent with graft efferents reported in other hypothalamic transplantation models in which graft and host tissue can be differentiated (i.e., Brattleboro rat and hypogonadal mouse). Moreover, SCN neurons within anterior hypothalamic grafts send an appropriately restricted set of efferent projections to the host brain which may participate in the functional recovery of circadian locomotor activity.     

Suprachiasmatic nucleus and circadian core temperature rhythm in the rat

Core temperature was telemetered from 26 singly-housed adult male inbred Fischer rats standardized in an ambient temperature of 24 ± 1°C, in light from 0600–1800 alternating with darkness (L:D 12:12), with food and water freely available. The rats were operated upon first for bilateral electrolytic lesioning of the suprachiasmatic nucleus (SCN) or by a sham-operation, which consisted of an inserted electrode which neither penetrated into the SCN area nor was activated to produce a lesion. Next, a temperature sensor was implanted intraperitoneally. The telemetered data obtained at 10-min intervals from each rat were analyzed by the least-squares fit of certain trial periods (cosinor methods). A circadian population rhythm persisted in the SCN-lesioned rats which sustained destruction of both SCN (P < 0.01). The amplitude of the circadian temperature rhythm was attenuated(P < 0.01) and the rhythm's acrophase advanced (P < 0.05) from mid-dark to a time near the transition from light to darkness. Unilateral lesions of the suprachiasmatic nuclei altered the circadain amplitude but not the phasing.     

Neuropeptide changes in the suprachiasmatic nucleus are associated with the development of hypertension

ABSTRACT

Human postmortem studies as well as experimental animal studies indicate profound changes in neuropeptide expression in the suprachiasmatic nucleus (SCN) in several pathological conditions including hypertension. In addition, animal experimental observations show that the SCN peptides, vasopressin (AVP) and vasoactive intestinal peptide (VIP) are essential for adequate rhythmicity. These data prompted us to investigate whether changes in these neuronal populations could be the cause or consequence of hypertension. Changes in blood pressure and levels of neuropeptide expression in the SCN were determined during development of hypertension in spontaneously hypertensive rats (SHR), in 2K1C reno-vascular induced hypertensive animals and their respective controls. During the pre-hypertensive stage (5 weeks of age), the VIP and AVP content was higher and the somatostatin (SOM) content was lower in the SHR SCN. At the onset of hypertension (12 weeks of age), when blood pressure levels had just reached about 140 mmHg, AVP and SOM content in the SCN was not different anymore in SHRs compared to control, but VIP was still higher. After 16 weeks, the AVP content was decreased, but SOM was increased and the overall level of VIP in the SCN was still higher in SHRs compared to controls. None of the aforementioned changes in the SCN was observed after induction of hypertension in the 2K1C model. However, while VIP was increased in the NTS projecting medial region of the SCN in SHR animals only after the establishment of hypertension, VIP was decreased in the same region in the 2K1C induced hypertensive rats. Consequently, the present findings confirm previous studies in human and rat indicating that changes in the SCN are strongly associated with the development of hypertension. In addition, the changes in peptide content in the 2K1C animals indicate that the SCN is also able to respond to increases in blood pressure.     

Morphological correlates of serotonin-neuropeptide Y interactions in the rat suprachiasmatic nucleus: Combined radioautographic and immunocytochemical data

Summary The morphological substrate of putative serotonin (5-HT)/neuropeptide Y (NPY) interactions in thé suprachiasmatic nucleus (SCN) was investigated by combined radioautography and immunocytochemistry after intraventricular administration of (³H)5-HT in the rat. In the ventral portion of the SCN, the distribution of (³H)5-HT uptake sites overlapped closely the NPY-immunoreactive terminals. Previous investigations have shown that the dense 5-HT and NPY innervations of the SCN originate in different structures, i.e., the midbrain raphe nuclei and the ventral lateral geniculate nucleus, respectively. Accordingly, in the present study, destruction of 5-HT afferents by 5,7-dihydroxytryptamine was not found to induce any modification in NPY staining and, in ultrastructural immuno-radioautographic preparations, two distinct pools of axonal varicosities could be identified. Both 5-HT and NPY terminals established morphologically defined synaptic junctions, sometimes on the same neuronal target. Some cases of direct axo-axonic appositions between the two types of terminals were also encountered. These data constitute additional criteria for characterizing the cytological basis of the multiple transmitter interactions presumably involved in the function of the SCN as a central regulator of circadian biological rhythms.     

DIURNAL,AGE, AND IMMUNE REGULATION OF INTERLEUKIN-1β AND INTERLEUKIN-1 TYPE 1 RECEPTOR IN THE MOUSE SUPRACHIASMATIC NUCLEUS

Circadian clocks serve to impose a near-24-h temporal architecture on an organism's physiology, metabolism, and behavior. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master circadian pacemaker. There is growing evidence that immunomodulators, such as cytokines, may impinge on circadian timekeeping. We examined whether there is endogenous expression of the proinflammatory cytokine interleukin-1β (IL-1β) and its signaling receptor IL-1R1 in the SCN of young and older mice across the diurnal cycle. We found expression of both IL-1β and IL-1R1 in the young SCN, although only IL-1R1 displayed temporal regulation. In the older SCN, levels of IL-1β were expressed at lower levels than in the young SCN, and IL-1R1 did not vary across the 24?h. We also report age-related day-night variation of IL-1β and IL-1R1 in the paraventricular nucleus (PVN) of the hypothalamus. Further, we examined the effect of peripheral immune challenge on IL-1β and IL-1R1 in the SCN. We found that IL-1β immunoreactivity was not altered 6 or 24?h after a septic dose of lipopolysaccharide (LPS; 5?mg/kg), whereas IL-1R1 was significantly up-regulated in the SCN both 6 and 24?h after LPS. We also demonstrate cellular activation in the SCN 24?h following LPS treatment, as evidenced by increased c-Fos and p65-NF-κB (nuclear factor kappa B) expression. Our results indicate that IL-1β and its associated signaling system may play a role in mediating the response of the circadian timing system to immune challenge as well as potentially contributing to the basal functioning of the SCN clock. (Author correspondence: andrew.coogan@nuim.ie)     

Reduced light response of neuronal firing activity in the suprachiasmatic nucleus and optic nerve of cryptochrome-deficient mice

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day-night difference was not observed in Cry-deficient mice. In addition, we observed day-night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals.     

Effect of serotonin on differentiation of neurons producing vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus

This study was aimed to evaluate the morphogenetic influence of serotonin on the differentiating vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus (SCN) in rats. The comparative morpho-functional analysis of VIP neurons was made in adult rats which developed under normal metabolism of serotonin or in its deficiency. The serotonin deficiency was induced in foetuses by injections of p-chlorophenilalanine to pregnant mothers. Control rats received the saline. According to our data, there was no difference in the VIP mRNA concentration and most probably in VIP synthesis in the SCN in adult rats following prenatal serotonin depletion compared to the control. However, the serotonin deficiency resulted in increase of the VIP concentration in cell bodies and of the VIP neurones number. The size of the VIP-neurones did not change in pharmacologically treated rats compared to the controls showing no functional hypertrophy of the neurones as a result of the activation of specific syntheses. From the above data, it follows that serotonin provides an imprinting influence differentiating the VIP neurones, contributing most probably to development of the VIP release mechanism.     

Suprachiasmatic nucleus: Numbers of synaptic appositions and various types of synapses

Summary The suprachiasmatic nucleus (SCN) of male rats was estimated to contain 16×10⁶ synaptic appositions (unilaterally) or 250×10⁶ appositions in 1 mm³ tissue of the nucleus with an average of 1404 appositions per neuron. There are significantly fewer synaptic appositions in the suprachiasmatic nucleus of female rats (15×10⁶ per SCN or 236×10⁶ in 1 mm³ tissue of SCN with 1264 appositions per neuron on an average). Additionally, numbers of various types of synapses (axo-somatic, invaginated, dendrodendritic and optic) are estimated.Supported by National Health & Medical Research Council of Australia     

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性

本研究旨在观察和比较视交叉上核（suprachiasmatic nucleus,SCN）与松果体（pineal gland,pG）中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗（constant darkness,DD）和12h光照：12h黑暗交替（12hourlight：12hour-darkcycle,LD）光制下分别被饲养8周（n=36）和4周n=36）后,在一昼夜内每隔4h采集一组SCN和PG组织（n=6）,提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点（circadian times．CT or zeitgeber times．ZT）各样品中Clock基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：（1）SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化（P〈0．05）,PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚（SCN为CTl5,PG为CT18）,谷值位于主观白天（SCN为CT3,PG为CT6）（P〉0．05）。（2）LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡（P〈0．05）,但与其DD光制下节律外观相比,呈现反时相节律变化（P〈0．05）,且其表达的振幅及峰值的mRNA水平均增加（P〈0．05）,而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反（P〈0．05）。（3）在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN（P〈0．05）,即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。     

Neural interaction of gonadotropin-regulating hormone immunoreactive neurons and the suprachiasmatic nucleus with the paraventricular organ in the Japanese grass lizard (Takydromus tachydromoides)

Our previous study demonstrated that the paraventricular organ (PVO) in the hypothalamus of the Japanese grass lizard (Takydromus tachydromoides) showed immunoreactivity against the light signal-transducing G-protein, transducin. This finding suggested that the PVO was a candidate for the deep-brain photoreceptor in this species. To understand functions of the PVO, we investigated distributions of transducin, serotonin, gonadotropin-releasing hormone (GnRH), and gonadotropin-inhibitory hormone (GnIH) in the lizard's brain. We immunohistochemically confirmed co-localization of transducin and serotonin in PVO neurons that showed structural characteristics of cerebrospinal fluid (CSF)-contacting neurons. GnRH-immunoreactive (ir) cells were localized in the posterior commissure and lateral hypothalamic area. Some of the serotonin-ir fibers extending from the PVO to the lateral hypothalamic area contacted the GnRH-ir cell bodies. GnIH-ir cells were localized in the nucleus accumbens, paraventricular nucleus, and upper medulla, and GnIH-ir fibers from the paraventricular nucleus contacted the lateral processes of serotonin-ir neurons in the PVO. In addition, we found that serotonin-ir fibers from the PVO extended to the suprachiasmatic nucleus (SCN), and the retrograde transport method confirmed the PVO projections to the SCN. These findings suggest that the PVO, by means of innervation mediated by serotonin, plays an important role in the regulation of pituitary function and the biological clock in the Japanese grass lizard.     

Effect of Continuous Infusion of Anti-L1 Antibody into the Third Cerebral Ventricle above the Suprachiasmatic Nucleus on the Circadian Rhythm of Locomotor Activity in Rats

During an investigation into the role of the neural cell adhesion molecules such as L1 and NCAM in the generation mechanism of circadian rhythms, we observed that L1-like immunoreactive substance is expressed in the hypothalamic suprachiasmatic nucleus (SCN). Therefore, we examined the effect of continuous infusion of anti-L1 antibody into the third cerebral ventricle above the SCN using an Alzet osmotic minipump, on the circadian rhythm of locomotor activity in rats under constant red dim light (less than 1 lx) condition, in order to elucidate the role of L1 in the mechanism of circadian rhythm. Continuous infusion of intact rabbit IgG into the third cerebral ventricle above the SCN, which was done as a control experiment, shifted the phase of the free-running circadian rhythm and reduced daily locomotor activity for an initial few days, however, it did not eliminate the circadian rhythm. In contrast, continuous infusion of anti-L1 antibody temporarily disrupted the circadian rhythm during the infusion period. Furthermore, the infusion of the anti-L1 antibody but not that of control IgG caused a change in the SCN conformation, from which it appeared that SCN neurons displaced in dorsal direction, 4 days after the start of the infusion. These findings suggest that the cell adhesion molecule, L1, might be involved in the generation and/or transduction of the time signal of the circadian rhythm in the SCN.     

Effect of ethanol on the concentration of serotonin in the brain of the rat and the excretion of 5-hydroxyindoleacetic acid

It is found that serotonin content in the brain areas and heart of rats with low alcohol motivation decreases after 5 months of chronic consumption of 48% ethanol solution in a dose of 4 g/kg; in animals with high alcohol motivation serotonin content decreases only in the hypothalamus. Under chronic alcoholization for 1 and 12 months no considerable changes were found in serotonin level of the studied tissues. 60 min after intraperitoneal administration of 20% ethanol solution in a dose of 3 g/kg in intact animals there occurs an increase of serotonin content in the brain hemispheres and heart and its decrease in the hypothalamus; in rat with low alcohol motivation after taking ethanol for 5 months this administration evokes a decrease of serotonin content in the hypothalamus and truncus cerebri; in rats with high alcohol motivation--its decrease in the hypothalamus. Excretion of 5-oxyindoleacetic acid with urine decreases 10 months after alcohol intoxication. When rats were not given ethanol after its chronic taking for 3 months serotonin oxidation was intensified for the first day, which was not observed after 7-month alcoholization of animals.     

Kinetic study of serotonin metabolism in the suprachiasmatic nucleus of the rat: neuroendocrine incidence (author's transl)

The suprachiasmatic nucleus (SCN), which is implicated in the regulation of several cyclic neuroendocrine rhythms, displays a conspicuous and apparently specific serotoninergic innervation. Our study was intended to establish more precise correlations between the incidence of serotonin (5 HT) metabolism in the activity of the SCN, and neuroendocrine rhythms. For this purpose, castrated female rats, having subcutaneous implants of estradiol, were studied. These animals display very marked circadian fluctuations in plasma levels of the pituitary hormones ACTH, LH and PRL; a relatively well-synchronized increment in all these hormones occurs between 11.00 and 15.00. Punches were obtained to determine the endogenous content of 5 HT, measured by a radioenzymatic technique, simultaneously in SCN and median eminence (ME). An index of SCN activity was determined from in vivo SCN 2-deoxy (1-14C) glucose (DG) uptake; the retention was estimated on radioautographs. Endogenous level of 5 HT displayed a marked circadian rhythm with a peak between 12.00 and 15.00 in the SCN; 5 HT levels were constant throughout the day in the ME. 14C-DG uptake was greater at 15.00 than at 9.00. However, after PCPA treatment or raphe lesions, the uptake of 14C-DG was the same at 9.00 and 15.00. Taken together, these results strongly suggest that in our model: (1) SCN displays a rhythm of activity in the light period; (2) SCN displays specific rhythms in the content of 5 HT; (3) the SCN rhythm of activity must be under an inhibitory control of 5 HT, since the lowest metabolic level is increased at 9.00 by inactivation of 5 HT system; (4) the close relationships between the initial release phase of several pituitary hormones, the increase of metabolic activity in the SCN and the higher level of the 5 HT in the nucleus suggest that 5 HT terminals in the SCN play an important part in the control of cyclic hormonal secretion.     

Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock

Abstract: Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of l -[³H]arginine to l -[³H]citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca²⁺/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.     

Long-term serotonin administration leads to higher bone mineral density, affects bone architecture, and leads to higher femoral bone stiffness in rats

New evidence suggests a control of bone mass by the central nervous system. We have previously shown that functional serotonin receptors are present in bone cells and that serotonin stimulates proliferation of osteoblast precursor cells in vitro. In the present study we investigated the effects of serotonin on bone tissue in vivo. Ten, 2-month-old female Sprague-Dawley rats were injected with serotonin subcutaneously (s.c.) (5 mg/kg) once daily for 3 months, controls received saline. Using microdialysis and HPLC, free circulating serotonin levels were measured. DXA scans were made after 3 months of serotonin administration. Bone architecture and mechanical properties were investigated by micro-computed tomography (microCT), histomorphometry, and mechanical testing. A long-lasting hyperserotoninemia with a >10-fold increase in serotonin appeared. Total body BMD was significantly higher (0.1976+/-0.0015 vs. 0.1913+/-0.0012 g/cm2) in rats receiving serotonin. Cortical thickness (Ct.Th) measured by microCT analysis was also higher, whereas trabecular bone volume (BV) was lower. Interestingly, the perimeter and cross-sectional moment of inertia (MOI), a proxy for geometrical bone strength, were the same in both groups. These data suggest that serotonin reduces resorption or/and increases apposition of endosteal bone. Mechanical testing showed that femoral stiffness was higher in serotonin-dosed animals. The energy absorption also seemed slightly, but not significantly higher. In conclusion, hyperserotoninemia led to a higher BMD, altered bone architecture and higher femural bone stiffness in growing rats, demonstrating that serotonin may have important effects on bone in vivo.