Immunohistochemical detection of tyrosine hydroxylase and concentrations of monoamines in the substantia nigra and hypothalamus of hereditary microphthalmic rats.

We compared tyrosine hydroxylase immunoreactivity in the substantia nigra and hypothalamus of hereditary microphthalmic rats with that of normal rats. A considerable number of neuronal cell bodies expressing tyrosine hydroxylase were present in the substantia nigra of the microphthalmic mutant as well as normal rats. Neuronal cells positive for tyrosine hydroxylase in the hypothalamus were fewer than in the substantia nigra in both rats. The concentrations of monoamines (dopamine, noradrenaline, adrenaline, and serotonin) in the substantia nigra and hypothalamus in the microphthalmic mutant were approximately the same as those of normal rats, although the diurnal fluctuation of a few monoamines was observed in normal rats. These results suggest that the metabolic aspects of catecholamine in the substantia nigra and hypothalamus of the microphthalmic mutant rat do not markedly differ from those of normal rats.     

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75^NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.     

Properties of the serotonergic cerebral ganglion neurons of the gastropod mollusc, Philine aperta

The characteristics of a pair of identified neurons found in the cerebral ganglia of the gastropod mollusc Philine aperta have been examined. Because they appear to contain serotonin, and since they probably also use serotonin as a neurotransmitter, these neurons were named the serotonergic cerebral neurons (SCNs). Each SCN sent an axon out of the ipsilateral cerebro-buccal connective to the buccal ganglia. The SCNs also had extensive projections to all the ipsilateral, and most of the contralateral, buccal nerve trunks. Stimulating the SCNs produced the hyperpolarization of a pair of identified buccal ganglion mechano-sensory neurons (the S-cells), and had an excitatory action on the electrical activity of acinar cells of the salivary glands. A comparison of the properties of the Philine SCNs with those of similar serotonin-containing cerebral ganglion neurons in other gastropod molluscs provides evidence of homology with these neurons.     

Little or no induction of hyperglycemia by 2-deoxy-D-glucose in hereditary blind microphthalmic rats

Studies were made on whether hereditary microphthalmic rats (1), which are congenitally blind, showed a hyperglycemic response to intracerebroventricular injection of 2-deoxy-D-glucose (2DG) in their subjective light period. In contrast to previous findings in normal rats in which 2DG injection caused light-cycle dependent hyperglycemia (2) and bilateral lesion of the suprachiasmatic nucleus (SCN) completely abolished this hyperglycemia (3), 2DG injection caused no and only slight hyperglycemia in male and female rats with hereditary microphthalmia, respectively. Gross and histological examinations indicated that these rats had no optic nerve or retinohypothalamic tract and that their SCN had an abnormal structure. Locomotive activity recordings showed that all the blind rats had a free-running circadian activity rhythm. These findings suggest that the projection sites of the retinohypothalamic tract to the SCN are involved in the mechanism of the hyperglycemic response to 2DG, but that neural cells, which may be responsible for the generation of circadian rhythms, are not. We have reported that when adult rats were blinded by orbital enucleation, their hyperglycemic response to 2DG was suppressed temporarily 3-5 weeks after the operation, but that their plasma insulin level was basically higher and increased further after 2DG injection during this period (4). In congenitally blind rats, however, the basal plasma insulin level was not higher and the level did not change after 2DG treatment. This difference is discussed from the view point of the role of the premature SCN in regulation of the plasma insulin concentration.     

Chronic 2G exposure affects c-Fos reactivity to a light pulse within the rat suprachiasmatic nucleus

This study examined the effect of the hyperdynamic environment on the function of the retinohypothalamic tract. Rats were exposed to either 2 days or 21 days of 2G via centrifugation. During the last hour of 2G exposure, one series of rats was exposed to a 1 hour phase-shifting light pulse while the second series of rats did not receive a light pulse. In addition a groups of 1G controls was exposed to the same 1 hour lighting paradigm. All animals were processed for c-Fos within the SCN. The 1G controls showed the normal response to light in which significantly greater numbers of c-Fos positive neurons were found in the SCN of the light pulsed rats relative to that of the nonlight pulsed rats. However, rats exposed to 2 days of 2G did not show the same response to light. Light pulsed rats and nonlight pulsed rats exhibited few c-Fos positive neurons within the SCN. A recovery in the effect of light to induce c-Fos reactivity within SCN neurons occurred in the rats exposed to 21 days of 2G. These results suggest that exposure to 2G can temporarily suppress the responsiveness of the SCN to the phase-shifting effects of light mediated by the retinohypothalamic tract.     

Fractal Patterns of Neural Activity Exist within the Suprachiasmatic Nucleus and Require Extrinsic Network Interactions

The mammalian central circadian pacemaker (the suprachiasmatic nucleus, SCN) contains thousands of neurons that are coupled through a complex network of interactions. In addition to the established role of the SCN in generating rhythms of ∼24 hours in many physiological functions, the SCN was recently shown to be necessary for normal self-similar/fractal organization of motor activity and heart rate over a wide range of time scales—from minutes to 24 hours. To test whether the neural network within the SCN is sufficient to generate such fractal patterns, we studied multi-unit neural activity of in vivo and in vitro SCNs in rodents. In vivo SCN-neural activity exhibited fractal patterns that are virtually identical in mice and rats and are similar to those in motor activity at time scales from minutes up to 10 hours. In addition, these patterns remained unchanged when the main afferent signal to the SCN, namely light, was removed. However, the fractal patterns of SCN-neural activity are not autonomous within the SCN as these patterns completely broke down in the isolated in vitro SCN despite persistence of circadian rhythmicity. Thus, SCN-neural activity is fractal in the intact organism and these fractal patterns require network interactions between the SCN and extra-SCN nodes. Such a fractal control network could underlie the fractal regulation observed in many physiological functions that involve the SCN, including motor control and heart rate regulation.     

Fos expression within vasopressin-containing neurons in the suprachiasmatic nucleus of diurnal rodents compared to nocturnal rodents

The underlying neural causes of the differences between nocturnal and diurnal animals with respect to their patterns of rhythmicity have not yet been identified. These differences could be due to differences in some subpopulation of neurons within the suprachiasmatic nucleus (SCN) or to differences in responsiveness to signals emanating from the SCN. The experiments described in this article were designed to address the former hypothesis by examining Fos expression within vasopressin (VP) neurons in the SCN of nocturnal and diurnal rodents. Earlier work has shown that within the SCN of the diurnal rodent Arvicanthis niloticus, approximately 30% of VP-immunoreactive (IR) neurons express Fos during the day, whereas Fos rarely is expressed in VP-IR neurons in the SCN of nocturnal rats. However, in earlier studies, rats were housed in constant darkness and pulsed with light, whereas Arvicanthis were housed in a light:dark (LD) cycle. To provide data from rats that would permit comparisons with A. niloticus, the first experiment examined VP/Fos double labeling in the SCN of rats housed in a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase. Fos was significantly elevated in the SCN of animals sacrificed during the light compared to the dark phase, but virtually no Fos at either time was found in VP-IR neurons, confirming that the SCN of rats and diurnal Arvicanthis are significantly different in this regard. The authors also evaluated the relationship between this aspect of SCN function and diurnality by examining Fos-IR and VP-IR in diurnal and nocturnal forms of Arvicanthis. In this species, most individuals exhibit diurnal wheel-running rhythms, but some exhibit a distinctly different and relatively nocturnal pattern. The authors have bred their laboratory colony for this trait and used animals with both patterns in this experiment. They examined Fos expression within VP-IR neurons in the SCN of both nocturnal and diurnal A. niloticus kept on a 12:12 LD cycle and perfused 4 h into the light phase or 4 h into the dark phase, and brains were processed for immunohistochemical identification of Fos and VP. Both the total number of Fos-IR cells and the proportion of VP-IR neurons containing Fos (20%) were higher during the day than during the night. Neither of these parameters differed between nocturnal and diurnal animals. The implications of these findings are discussed.     

Cellular Requirements of Suprachiasmatic Nucleus Transplants for Restoration of Orcadian Rhythm

Fetal neurografts containing the suprachiasmatic nucleus (SCN) can restore the circadian locomotor and drinking rhythm of SCN-lesioned (SCNX) rat and hamster. This functional outcome finally proves that the endogenous biological clock autonomously resides in the SCN. Observations on the cellular requirements of the “new” SCN for restoration of the arrhythmic SCNX animals have led to some new insights and confirmed findings from other studies. A critical mass of SCN neurons appeared necessary for functional effects, whereas the temporal profile of reinstatement of rhythm correlated with the delayed maturation of the grafted SCN. Cytoarchitectoni-cally, the grafted SCN does not seem to develop normally for all anatomical aspects. Complementary clusters of vasoactive intestinal polypeptide(VIP)-and vasopressin(VP)ergic neurons are formed, but somatostatin(SOM)ergic neurons do not always “join” this group, as is normally seen in situ. Nevertheless, these new SCNs can restore the ablated functions. As the period length of restored rhythms tends to vary, it might be that the grafted SCN underwent an altered or impaired maturation that resulted in a different setting of its clock mechanism. A prominent role of VIPergic neurons seems indicated by their presence in all functional grafts, but, although they may be required, these cells do not appear to be a sufficient condition for restoration of rhythm. Many grafts exhibit the presence of VIPergic cells without counteracting the arrhythmia, whereas VP- and SOMergic SCN neurons are usually present as well. Findings with VP-deficient Brattleboro rat grafts indicated that VP is not the primary obligatory signal of circadian activity. It is argued that perhaps the role of SOMergic neurons in the clock function of the (grafted) SCN has been insufficiently considered. However, one should keep in mind that the peptides of the various types of SCN neurons may function only as cofactors, mutually modulating molecular or bioelectrical cellular activities within the nucleus or the message of the main transmitter γ-aminobutyric acid.     

Cytological features of serotonin-containing neurons and their processes in the retina of the carp (Cyprinus carpio). An immunohistochemical study using flat-mount preparations

The morphological features of serotonin-containing neurons (SCNs) and their processes in the retina of the carp (Cyprinus carpio) were immunohistochemically studied by applying a modified peroxidase-antiperoxidase technique using flat-mount preparations. The somata of immunoreactive SCNs were mostly located in the innermost part of the inner nuclear layer (INL). These cells were distributed at a density of 64.8 cells/mm2, this being similar to the density of dopamine-containing interplexiform cells. The processes of the SCNs ramified successively into two finer branches, eventually forming a broad, extensive network in the thin layer just subjacent to the plane of the somata of the SCNs. Processes originating from neighboring SCNs exhibited cytoplasmatic continuity with each other at the lightmicroscope level. Due to their location and cytological features, these SCNs appeared to correspond to amacrine cells.     

Knockdown of clock genes in the suprachiasmatic nucleus blocks prolactin surges and alters FRA expression in the locus coeruleus of female rats

The nature of the circadian signal from the suprachiasmatic nucleus (SCN) required for prolactin (PRL) surges is unknown. Because the SCN neuronal circadian rhythm is determined by a feedback loop of Period (Per) 1, Per2, and circadian locomotor output cycles kaput (Clock) gene expressions, we investigated the effect of SCN rhythmicity on PRL surges by disrupting this loop. Because lesion of the locus coeruleus (LC) abolishes PRL surges and these neurons receive SCN projections, we investigated the role of SCN rhythmicity in the LC neuronal circadian rhythm as a possible component of the circadian mechanism regulating PRL surges. Cycling rats on proestrous day and estradiol-treated ovariectomized rats received injections of antisense or random-sequence deoxyoligonucleotide cocktails for clock genes (Per1, Per2, and Clock) in the SCN, and blood samples were taken for PRL measurements. The percentage of tyrosine hydroxylase-positive neurons immunoreactive to Fos-related antigen (FRA) was determined in ovariectomized rats submitted to the cocktail injections and in a 12:12-h light:dark (LD) or constant dark (DD) environment. The antisense cocktail abolished both the proestrous and the estradiol-induced PRL surges observed in the afternoon and the increase of FRA expression in the LC neurons at Zeitgeber time 14 in LD and at circadian time 14 in DD. Because SCN afferents and efferents were probably preserved, the SCN rhythmicity is essential for the magnitude of daily PRL surges in female rats as well as for LC neuronal circadian rhythm. SCN neurons therefore determine PRL secretory surges, possibly by modulating LC circadian neuronal activity.     

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.     

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     

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     

Reengineering the Order Fulfillment Process in Supply Chain Networks

An order fulfillment process (OFP) starts with receiving orders from customers and ends with delivering the finished goods. The order fulfillment process is complex because it is composed of several activities, executed by different functional entities, and heavily interdependent among the tasks, resources, and agents involved in the process. A supply chain network (SCN) is a network of autonomous or semi-autonomous business entities involved, through upstream and downstream links, in the different processes and activities that produce goods or services to customers. As manufacturing practice shifts toward the outsourcing paradigm, the OFP is more likely to be executed throughout SCNs. It becomes imperative to integrate the OFP into SCNs to improve the OFP. Generalizing from the variety and complexity of SCNs, this study identifies several main types of SCN structures and addresses OFP issues based on them. The objective of reengineering the OFP is to achieve agility in the process in terms of efficiency, flexibility, robustness, and adaptability. We propose a multiagent information system (MAIS) approach for reengineering the OFP in SCNs. The MAIS models the OFP in SCNs and evaluates OFP performance by applying the proposed strategies. A multiagent simulation platform, called Swarm, is enhanced and applied for modeling the MAIS; and experiments are conducted to simulate the OFP and then evaluate the potential improvement strategies to identify useful strategies for improving the OFP. The strategies we evaluated include (1) coordinating demand management policies, (2) information sharing strategies, (3) synchronizing material and capacity availability, (4) dynamic resource allocation, and (5) the combination of various strategies. The results shed light on identifying the main effects of various strategies on OFP performance. The insights from utilizing various strategies in different SCNs help reengineer the OFP in SCNs.     

Rearrangement of serotonin-immunoreactive fibers in the denervated rat suprachiasmatic nucleus after transplantation of fetal raphe tissue

Summary Pieces of fetal midbrain raphe tissue were transplanted into the third ventricle or the ventral hypothalamic region near the suprachiasmatic nucleus (SCN) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. The ability of grafted serotonin neurons to reinnervate the SCN in the host rats was studied by means of immunohistochemistry 1 and 3 months after transplantation. In both the intraventricular and intraparenchymal transplant experiments, reinnervation by outgrowing serotonin fibers was observed in the hypothalamus of host rats at 1 and 3 months after surgery. At both survival periods, there was no abundant arborization of serotonin fibers in the SCN, while the preoptic and periventricular areas of the host rats displayed a pattern of serotonergic innervation resembling that in normal (untreated) rats. It is suggested that within the SCN the regenerating serotonin fibers may be exposed to an inhibitory environment.     

Cytological features of serotonin-containing neurons and their processes in the retina of the carp (Cyprinus carpio)

Summary The morphological features of serotonin-containing neurons (SCNs) and their processes in the retina of the carp (Cyprinus carpio) were immunohistochemically studied by applying a modified peroxidase-antiperoxidase technique using flat-mount preparations. The somata of immunoreactive SCNs were mostly located in the innermost part of the inner nuclear layer (INL). These cells were distributed at a density of 64.8 cells/mm², this being similar to the density of dopamine-containing interplexiform cells. The processes of the SCNs ramified successively into two finer branches, eventually, forming a broad, extensive network in the thin layer just subjacent to the plane of the somata of the SCNs. Processes originating from neighboring SCNs exhibited cytoplasmatic continuity with each other at the lightmicroscope level. Due to their location and cytological features these SCNs appeared to correspond to amacrine cells.In honour of Prof. P. van Duijn     

Novel phase-shifting effects of GABAA receptor activation in the suprachiasmatic nucleus of a diurnal rodent

The vast majority of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals, contain the inhibitory neurotransmitter GABA. Most studies investigating the role of GABA in the SCN have been performed using nocturnal rodents. Activation of GABA(A) receptors by microinjection of muscimol into the SCN phase advances the circadian activity rhythm of nocturnal rodents, but only during the subjective day. Nonphotic stimuli that reset the circadian pacemaker of nocturnal rodents also produce phase advances during the subjective day. The role of GABA in the SCN of diurnal animals and how it may differ from nocturnal animals is not known. In the studies described here, the GABA(A) agonist muscimol was microinjected directly into the SCN region of diurnal unstriped Nile grass rats (Arvicanthis niloticus) at various times in their circadian cycle. The results demonstrate that GABA(A) receptor activation produces large phase delays during the subjective day in grass rats. Treatment with TTX did not affect the ability of muscimol to induce phase delays, suggesting that muscimol acts directly on pacemaker cells within the SCN. These data suggest that the circadian pacemakers of nocturnal and diurnal animals respond to the most abundant neurochemical signal found in SCN neurons in opposite ways. These findings are the first to demonstrate a fundamental difference in the functioning of circadian pacemaker cells in diurnal and nocturnal animals.     

Modeling the spontaneous activity in suprachiasmatic nucleus neurons: Role of cation single channels

A population of interconnected neurons of the mammalian suprachiasmatic nuclei (SCN) controls circadian rhythms in physiological functions. In turn, a circadian rhythm of individual neurons is driven by intracellular processes, which via activation of specific membrane channels, produce circadian modulation of electrical firing rate. Yet the membrane target(s) of the cellular clock have remained enigmatic. Previously, subthreshold voltage-dependent cation (SVC) channels have been proposed as the membrane target of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons. We tested this hypothesis with computational modeling based on experimental results from on-cell recording of SVC channel openings in acutely isolated SCN neurons and long-term continuous recording of activity from dispersed SCN neurons in a multielectrode array dish (MED). The model reproduced the circadian behavior if the number of SVC channels or their kinetics were modulated in accordance with protein concentration in a model of the intracellular clock (Scheper et al., 1999. J. Neurosci. 19, 40-47). Such modulation changed the average firing rate of the model neuron from zero (“subjective-night” silence) up to 18 Hz (“subjective-day” peak). Furthermore, the variability of interspike intervals (ISI) and the circadian pattern of firing rate (i.e. silence-to-activity ratio and shape of circadian peaks) are in reasonable agreement with experimental data obtained in dispersed SCN neurons in MED. These results suggest that the variability of ISI in intact SCN neurons is mostly due to stochastic single-channel openings, and that the circadian pattern of the firing rate is specified by threshold properties of dependence of the spontaneous firing rate on the number of single channels (R-N relationship). This plausible mathematical modeling supports the hypothesis that SVC channels could be a critical element in circadian modulation of firing rate in SCN neurons.