Concentrations and distribution of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and peptide histidine valine (PHV) in the cerebral cortex and the suprachiasmatic nucleus of the mouse

Prepro-vasoactive intestinal peptide (prepro-VIP) is processed to at least three biologically active peptides: VIP, peptide histidine isoleucine (PHI) and an extended PHI, peptide histidine valine (PHV). The aim of the present investigation was by chromatography combined with RIA and immunocytochemistry to determine which of these peptides were present in the cerebral cortex and the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. These regions were chosen since they are known to contain a high concentration of VIP but the relative concentration of PHI and PHV is not known. Tissue was extracted and subjected to gel chromatography and high-pressure liquid chromatography (HPLC). VIP and PHI immunoreactivities co-eluted with synthetic rat VIP and PHI. A minor peak of PHI and prepro-VIP(111–122) immunoreactivities eluted at the position of synthetic PHV. Surprisingly, a major peak of prepro-VIP(111–122) immunoreactivity eluted in a position not related to any other immunoreactivity indicating the presence of prepro-VIP(111–122). Measurements of these immunoreactivities in cortical and suprachiasmatic extracts revealed that VIP was found in the highest concentration whereas PHV was found in the lowest. Immunoreactivity for PHI and prepro-VIP(111–122) was found in moderate concentrations. Except for prepro-VIP(111–122) which was found to be 3×higher concentrated in the SCN than in the cerebral cortex, the other immunoreactivities were found in almost similar relative concentrations in the two tissues. Using immunocytochemistry, elongated neurons mostly of the bipolar type with prominent processes observed in the cerebral cortex reacted with all antisera tested. More PHI/PHV/prepro-VIP(111–122)- than VIP-immunoreactive (ir) nerve fibers were found in the cerebral cortex. In the SCN, the density of immunoreactivity was the same whatever antiserum used. VIP-, PHI- and prepro-VIP(111–122)/PHV-ir neurons were observed in the ventral part of the nucleus with numerous axons coursing caudodorsally into the subparaventricular area. A substantial number of terminals was detected caudal to the paraventricular nucleus. Minor projections spread to the medial part of the anterior nucleus and to the medial preoptic area hypothalamic. These data show that VIP and PHI are the major active peptides derived from prepro-VIP in the mouse cerebral cortex and SCN whereas PHV was found in minor concentrations. Prepro-VIP(111–122), which so far has been found to have no functional significance, is, therefore, most likely a vaste fragment of processing of PHI in central neurons. The presence of all these peptides in axons indicate that the neurotransmission involving VIP is more complex, due to roles of other peptides processed from the same prepro-VIP molecule.     

Ventral lateral geniculate nucleus efferents to the rat suprachiasmatic nucleus exhibit avian pancreatic polypeptide-like immunoreactivity

The distribution of avian pancreatic polypeptide-like (APP) immunoreactivity was investigated in the suprachiasmatic nucleus (SCN) of the rat hypothalamus with immunohistochemical methods. Specificity of the antisera was established by the absence of all immunoreactive staining in tissue incubated in antisera which had been preabsorbed with the pure APP antigen. In addition, the antisera exhibited no significant cross reactivity with vasoactive intestinal polypeptide, vasopressin, somatostatin, or secretin. Within the rat SCN, APP immunoreactivity is restricted to varicose axons in the ventral and lateral aspects of the nucleus; the dorsomedial component of the nucleus is totally devoid of immunoreactivity and immunoreactive perikarya are not present in any portion of the SCN. The immunoreactive axons in the ventrolateral portion of the nucleus form an extensive plexus which is distributed in a pattern closely corresponding to the distribution of retinal and ventral lateral geniculate (vLGN) efferents to the SCN. No immunoreactive perikarya are observed in the retina following immunoperoxidase staining for APP and neither unilateral nor bilateral enucleation causes an observable alteration in the pattern of APP axon distribution within the SCN, thus indicating that the fiber plexus is not of retinal origin. In contrast, APP immunoreactive neurons are present in the same area of the vLGN in which retrogradely filled neurons have been demonstrated following iontophoretic injection of HRP into the SCN. Bilateral electrolytic lesions of the vLGN result in a total loss of immunoreactive axons in both SCN. Unilateral vLGN lesions cause a loss of approximately 60 to 75% of the immunoreactive fibers in the ipsilateral SCN with a lesser contralateral loss. These observations provide further information on the organization of afferents to the rat SCN and demonstrate that the vLGN projection is chemically distinct from other SCN afferents.     

Immunocytochemical evidence for a diurnal rhythm of neurons showing colocalization of VIP with GRP in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN), which functions as a biological clock, contains several neuropeptides such as vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and gastrin-releasing peptide (GRP). Studies from several laboratories have provided evidence for the coexistence of VIP with PHI and GRP, but reliable data about the proportions of colocalization and a possible diurnal rhythmicity are lacking. In the present study, we therefore aimed at studying these aspects. To this end, rats were killed by perfusion fixation during the middle of the day (Zeitgeber time [ZT] 7) and during the second part of the night (ZT 19). Coronal Vibratome sections through the SCN were double-immunolabeled for the presence of VIP and PHI or for VIP and GRP. Analysis of the sections was done by semi-quantitative confocal laser scanning fluorescence microscopy. It turned out that, in keeping with previous literature data, VIP and PHI always coexist at the cellular level. This was seen in all possible ratios, both during the day and at night. Part of these VIP/PHI-containing neurons (21%) and part of the GRP-containing neurons (33%) showed colocalization during the middle of the day. During the second part of the night, these percentages increased significantly to 28% and 40%, respectively. This increase in percentages was due to a significant, nocturnal increase of the number of profiles showing colocalization, in contrast to the number of profiles exclusively immunoreactive for VIP or GRP. J. Comp. Neurol. 391:397–405, 1998. © 1998 Wiley-Liss, Inc.     

Calcium-binding proteins in the circadian centers of the common marmoset (Callithrix jacchus) and the rock cavy (Kerodon rupestris) brains

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic intergeniculate leaflet (IGL) are considered to be the main centers of the mammalian circadian timing system. In primates, the IGL is included as part of the pregeniculate nucleus (PGN), a cell group located mediodorsally to the dorsal lateral geniculate nucleus. This work was carried out to comparatively evaluate the immunohistochemical expression of the calcium-binding proteins calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) into the circadian brain districts of the common marmoset and the rock cavy. In both species, although no fibers, terminals or perikarya showed PV-immunoreaction (IR) into the SCN, CB-IR perikarya labeling was detected throughout the SCN rostrocaudal extent, seeming to delimit its cytoarchitectonic borders. CR-IR perikarya and neuropil were noticed into the ventral and dorsal portions of the SCN, lacking immunoreactivity in the central core of the marmoset and filling the entire nucleus in the rock cavy. The PGN of the marmoset presented a significant number of CB-, PV-, and CR-IR perikarya throughout the nucleus. The IGL of the rocky cavy exhibited a prominent CB- and CR-IR neuropil, showing similarity to the pattern found in other rodents.By comparing with literature data from other mammals, the results of the present study suggest that CB, PV, and CR are differentially distributed into the SCN and IGL among species. They may act either in concert or in a complementary manner in the SCN and IGL, so as to participate in specific aspects of the circadian regulation.     

Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamic transplants in oculo

The capacity of the rat anterior hypothalamus, and particularly the suprachiasmatic nucleus (SCN), to develop and differentiate when removed from its normal environment was examined in this study using light and electron microscopy. The hypothalamus from fetuses ranging in age from embryonic day 12 (E 12) to E 16 was transplanted to the anterior chamber of the eye of adult rats. In initial experiments, we found that transplants from E 15 fetuses and older routinely differentiated into fields of neurons with extensive neuropil with an appearance similar to the anterior hypothalamic area. Groups of small, compactly organized neurons were observed only occasionally in this tissue. Ultrastructural analysis of these transplants typically revealed well-differentiated neuronal perikarya and neuropil with a complex synaptic organization similar in appearance to the normal rat anterior hypothalamic area. Occasionally both mature and immature tissue coexisted in some of the transplants. Tissue from young embryos (E 12-14) frequently showed development of a compact, small neuron nucleus with the cytoarchitectonic appearance of the SCN. At least 45 days were required after transplantation for the successful differentiation to occur in this situation. The SCN in these transplants displayed vasoactive intestinal polypeptide-immunoreactive cells and fibers surrounded by vasopressin-immunoreactive cells and fibers, similar to the pattern observed in the normal adult SCN. Our results indicate that the anterior hypothalamus will differentiate normally in oculo and that the phenotypic specification of the SCN occurs prior to the birthdate of its component neurons.     

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.     

A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleus

A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575-11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (beta-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed beta-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN.     

Evidence from Confocal Fluorescence Microscopy for a Dense, Reciprocal Innervation Between AVP-,somatostatin-, VIP/PHI-, GRP- and VIP/PHI/GRP-immunoreactive Neurons in the Rat Suprachiasmatic Nucleus

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP ? VIP/PHI, AVP ? GRP, AVP ? somatostatin, somatostatin ? VIP/PHI, somatostatin ? GRP, VIP/PHI ? GRP, VIP/PHI/GRP ? GRP, VIP/PHI/GRP ? VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.     

Day-night variation in prepro vasoactive intestinal peptide/peptide histidine isoleucine mRNA within the rat suprachiasmatic nucleus

Neurons within the suprachiasmatic nuclei of the hypothalamus (SCN) appear to function as a circadian clock that controls the timing of many physiological systems. The SCN contain several chemically distinct neuronal subpopulations, including a large group of interneurons within the ventrolateral SCN that exhibit co-localizable immunoreactivity for both vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The purpose of the present study was to determine whether VIP/PHI neurons within the rat SCN exhibit rhythmicity in the cellular levels of the messenger RNA encoding the precursor from which both VIP and PHI are derived. Using both quantitative in situ and solution hybridization prepro-VIP/PHI mRNA levels early in the dark phase were demonstrated to be significantly higher than those 5 h after the onset of the daily light period. Since no statistically reliable (P greater than 0.05) day-night variation was observed in the levels of prepro-VIP/PHI mRNA within cortex, these data suggest that the rhythmicity in prepro-VIP/PHI mRNA is an intrinsic property of VIP/PHI-containing SCN neurons, or rhythmically driven by local synaptic events within the SCN.     

Suprachiasmatic nucleus projections to the paraventricular thalamic nucleus in nocturnal rats (Rattus norvegicus) and diurnal nile grass rats (Arviacanthis niloticus)

The circadian pacemaker of the suprachiasmatic nucleus (SCN) is likely to control the timing of the sleep-wake cycle in mammals by modulating the daily activity patterns of brain regions important in sleep and wakefulness. One such brain region is the paraventricular nucleus of the thalamus (PVT). In both nocturnal rats and the diurnal rodent Arvicanthis niltoicus (Nile grass rat), expression of Fos (the product of the immediate-early gene c-fos) in the PVT increases at times of day when the animals are most active. To compare the projections of the SCN to the PVT in these two species, the retrograde tracer cholera toxin (beta subunit; CTbeta) was microinjected into the PVT and the SCN was examined to identify labeled neurons. Further, the PVT-projecting SCN cells containing either arginine vasopressin (AVP) or gastrin releasing peptide (GRP) were also compared between species. In both nocturnal rats and diurnal Nile grass rats, the SCN sends a substantial projection to the PVT. In both species, many PVT-projecting SCN neurons contain AVP, and few contain GRP. Other work has shown that some AVP-containing neurons of the SCN function differently in rats and Nile grass rats. Projections from functionally distinct SCN neurons to the PVT may contribute to the difference in the temporal distribution of sleep and wakefulness seen between these two species.     

The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems

The suprachiasmatic nucleus (SCN) temporally organizes behavior in part by sustaining arousal during the wake period of the sleep/wake cycle to consolidate adaptive waking behavior. In this study, we demonstrate direct projections from the SCN, in both the rat and the human brains, to perikarya and proximal dendrites of two groups of posterior hypothalamic neurons with axonal projections that suggest they are important in the regulation of arousal, one producing hypocretins (HCT) and the other melanin-concentrating hormone (MCH). In addition, we demonstrate that both HCT and MCH-producing neurons are immunoreactive for glutamate (GLU). These observations support the hypothesis that direct projections from the SCN to the posterior hypothalamus mediate the arousal function of the circadian timing system.     

Cellular levels of messenger ribonucleic acids encoding vasoactive intestinal Peptide and gastrin-releasing Peptide in neurons of the suprachiasmatic nucleus exhibit distinct 24-hour rhythms

There is strong evidence supporting the view that the Suprachiasmatic nucleus (SCN) functions as a circadian clock; however, the neural and molecular events underlying SCN function remain unclear. A specific subpopulation of neurons within the ventrolateral aspect of the SCN that contains three peptides, vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP), play an important role in SCN function. VIP-containing neurons of the SCN receive synapses from photic projections, and co-injection of all three peptides mimics the phase-delaying effects of light on circadian activity rhythms. In principle, the signaling potential of a neuron containing several transmitters may be affected by the concentration ratio of co-released factors; hence, one mechanism by which VIP/PHI/GRP-containing neurons could influence SCN function is by changing the concentration ratio of these peptides throughout the light-dark cycle. The present study was performed to examine this possibility. Relative cellular levels of mRNA encoding both VIP/PHI and GRP were determined within the SCN every 4 h in rats housed in a 14 h light: 10 h dark cycle. Quantitative in situ hybridization revealed a statistically significant (P<0.005) 24-h profile of changes in VIP/PHI mRNA that peaked during the dark phase, and a significant (P<0.005) 24-h profile of changes in GRP mRNA that peaked during the light phase. These data support the interpretation that cellular levels of mRNAs encoding VIP/PHI and GRP within the SCN exhibit distinct profiles of changes throughout the light-dark cycle. Further, these findings are consistent with the hypothesis that the concentration ratio of VIP and PHI to GRP changes over the light-dark cycle, and that this may be an important mechanism by which circadian rhythms are generated or entrained.     

Suprachiasmatic nucleus projections to the paraventricular thalamic nucleus in nocturnal rats (Rattus norvegicus) and diurnal nile grass rats (Arviacanthis niloticus)

The circadian pacemaker of the suprachiasmatic nucleus (SCN) is likely to control the timing of the sleep–wake cycle in mammals by modulating the daily activity patterns of brain regions important in sleep and wakefulness. One such brain region is the paraventricular nucleus of the thalamus (PVT). In both nocturnal rats and the diurnal rodent Arvicanthis niltoicus (Nile grass rat), expression of Fos (the product of the immediate-early gene c-fos) in the PVT increases at times of day when the animals are most active. To compare the projections of the SCN to the PVT in these two species, the retrograde tracer cholera toxin (β subunit; CTβ) was microinjected into the PVT and the SCN was examined to identify labeled neurons. Further, the PVT-projecting SCN cells containing either arginine vasopressin (AVP) or gastrin releasing peptide (GRP) were also compared between species. In both nocturnal rats and diurnal Nile grass rats, the SCN sends a substantial projection to the PVT. In both species, many PVT-projecting SCN neurons contain AVP, and few contain GRP. Other work has shown that some AVP-containing neurons of the SCN function differently in rats and Nile grass rats. Projections from functionally distinct SCN neurons to the PVT may contribute to the difference in the temporal distribution of sleep and wakefulness seen between these two species.     

Effect of orbital enucleation on glucose homeostasis and morphology of the suprachiasmatic nucleus.

In rats there is a direct neural connection called the retinohypothalamic tract (RHT) from retinal ganglion cells to the ventrolateral part of the suprachiasmatic nucleus (SCN), which has neurons containing vasoactive intestinal polypeptide (VIP)-like substance. Previously, we observed that bilateral orbital enucleation (blinding) caused temporary suppression of the hyperglycemic response to intracranial injection of 2-deoxy-D-glucose (2DG) from week 4 to 6 after blinding. Moreover, bilateral lesions of the SCN had a similar effect. From these findings, we supposed that the neurons responsible for the hyperglycemic response to 2DG were present in the SCN, that after blinding these neurons temporarily lost their activity, and that this functional change was reflected in the morphology of the SCN. To investigate this possibility, we examined the morphological changes of the SCN by Nissl staining and immunohistochemical studies with anti-VIP and anti-peptide histidine isoleucine (PHI) antibodies in blinded rats, and the relationship between these morphological changes and the hyperglycemic response to 2DG. After surgical blinding, we observed following changes. (1) The optic chiasm became thinner. (2) The SCN became displaced rostrally. (3) The density of neurons in the middle to caudal part of the SCN, where the retinal ganglion cells projected, decreased markedly without change in cell number during the period when the hyperglycemic response to intracranial injection of 2DG was temporarily suppressed after blinding. The first and second changes seemed to reflect reduction of fibers and axon terminals of retinal ganglion cells and their innervation, respectively. As the third change was parallel with suppression of the hyperglycemic response to 2DG injection, it may reflect functional change of the neurons in the SCN that are responsible for the hyperglycemia due to 2DG.     

Changes in vasopressin cells of the rat suprachiasmatic nucleus with aging

The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be the endogenous clock of the mammalian brain, regulating circadian rhythmicity of a great number of physiological and behavioural parameters. Numerous studies have shown that the circadian organization in the rat is progressively disturbed in senescence. However, a recent study by Peng et al.17 using conventionally stained material, revealed no decrease in overall SCN cell number of senescent rats. Their results have now been confirmed in this study. In addition, an increase in SCN volume (P = 0.02) and nucleus diameter (P = 0.001) and an overall decrease in cell density (P = 0.006) was observed. All these parameters seem to confirm the absence of a general degeneration in the senescent SCN. However, the major aim of the present study was to determine whether a well-defined population of neurons, i.e. the vasopressinergic (AVP) cells of the SCN, shows changes with aging. Immunocytochemical staining with antivasopressin and morphometry revealed a decrease of 31% (P = 0.007) in the number of these SCN neurons, whereas the remaining vasopressin cells became larger (P = 0.001). There were no statistical significant differences between rats housed in standard cages and those housed in an enriched environment in either age group, but the groups were relative small. Changes in either the number or stainability of SCN vasopressin neurons may be a morphological correlate of changed circadian rhythms in senescence.     

Identification of a second category of α-melanocyte-stimulating hormone (α-MSH) neurons in the rathypothalamus

An immunocytochemical localization of alpha-melanocyte-stimulating hormone (alpha-MSH) as well as ACTH and a fragment (16K) of the common precursor of ACTH and beta-lipotropin (beta-LPH) was performed in rat brain. Two different groups of neuronal cell bodies showing alpha-MSH-like immunoreactivity (alpha-MSH-LI) were observed in the hypothalamus. One group of neurons located in the arcuate nucleus was shown to contain not only alpha-MSH-LI, but also ACTH and the 16K fragment. A second category of alpha-MSH-LI-containing neurons was characterized by the complete absence of staining for ACTH and 16K fragment. These neurons were mainly located in the dorsal-lateral portion of the hypothalamus. Immunoelectron microscopy showed that immunostaining for alpha-MSH was restricted to dense core vesicles in the positive perikarya. Nerve fibers staining for alpha-MSH (but not for ACTH and 16K fragment) were also observed outside the ACTH-beta-LPH pathway, especially in the cortex, caudate-putamen nucleus and hippocampus. These findings strongly suggest the presence of two different neuronal systems reacting with antibodies to alpha-MSH.     

NPY-like immunoreactivity in the brain of the teleost Tinca tinca (Cyprinidae)

The distribution of neuropeptide tyrosine (NPY) in the brain of the tench (Tinca tinca) was mapped immunohistochemically by the peroxidase-anti-peroxidase (PAP) technique. NPY-immunoreactive (NPYi) neurons were found in the nucleus entopeduncularis (ne). These perikarya were intensely immunostained and were surrounded by a large number of axons and fibre terminals. Additional immunoreactive neurons appeared in the nucleus dorsolateralis thalami (ndl) at the level of the commissura posterior (cp). Only a few scattered NPY-positive perikarya occurred in the nucleus ventromedialis (nvm) and in the nucleus posterior periventricularis (nppv). At more caudal levels immunoreactive NPY-neurons were found in the nucleus lateralis valvulae of the dorsal tegmentum mesencephali. Besides some scattered neurons in the medulla oblongata the only immunostained neurons were located in a subependymal nucleus at the lateral border of the fourth ventricle. However, these perikarya were only weakly immunostained by the NPY-antiserum. From its location this cell group is considered as nucleus motorius nervi vagi (nmX). NPYi fibres and axon terminals were found in the bulbus olfactorius, in the dorsal and lateral areas of the telencephalon, in the near surrounding of the ne, below most of the hypothalamic nuclei, as a connection between the nucleus posterior periventribularis and the nucleus recessus lateralis, in the ventral hypothalamus, in the lateral parts of the pituitary and in the caudal diencephalic inferior lobe. NPY-fibres occurred in the medial and deep layers of the tectum opticum, in the marginal areas of the tegmentum and the torus semicircularis, and as a lateral fibre tract through the medulla oblongata, connecting to the rostral parts of the spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of lateral geniculate-hypothalamic connections in the rat

The location and chemical identity of neurons interconnecting the lateral geniculate complex and the hypothalamus were analyzed in order to provide further information on the anatomical substrates for the entrainment of circadian rhythms. A particular objective of the study was to characterize the neurons projecting between the intergeniculate leaflet (IGL) of the lateral geniculate complex and the suprachiasmatic nucleus (SCN) and related anterior hypothalamic areas. The connectivity experiments employed five combinations of fluorescent tracer injection and were combined with immunohistochemical localization of either neuropeptide Y (NPY), met-enkephalin (mENK) or the vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI) group. IGL efferents. Injection of tracer into the SCN results in retrograde labeling of NPY-immunoreactive neurons in the IGL as would be expected from prior work. These neurons and their terminals also contain the C-flanking peptide of the NPY precursor molecule (CPON). In addition, there are two additional groups of neurons in the IGL that project either to the SCN or the contralateral IGL but do not exhibit NPY immunoreactivity. These include a substantial population of cells that project to the SCN and an even larger group of neurons which project to the contralateral IGL and contain mENK immunoreactivity. Hypothalamic efferents. Injection of tracer into the IGL results in retrograde labeling of scattered neurons throughout the SCN and immediately adjacent anterior hypothalamus ipsilaterally and also in labeling of a small number of neurons in the same areas on the contralateral side of the brain. In rare instances, individual SCN neurons appear to project to both IGLs. However, the retrochiasmatic area (RCA) contains the largest number of retrogradely labeled neurons following tracer injections into the IGL. These neurons are concentrated along the midsagittal plane and in the lateral RCA ipsilateral to the injected IGL. None of the labeled neurons in the SCN or adjacent anterior hypothalamus exhibit VIP or PHI immunoreactivity. These observations indicate that the anatomical relations between the geniculate complex and the anterior hypothalamus are more complex than previously shown. First, the geniculohypothalamic tract arises from two distinct groups of IGL neurons: one contains NPY/CPON immunoreactivity; the chemical content of the other is not characterized at the present time. Second, the commissural projection between the two IGLs is formed by a third group of neurons, and these cells contain mENK immunoreactivity. Finally, reciprocal projections from the hypothalamus to the IGL arise from neurons in the retrochiasmatic area, SCN, and adjacent anterior hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen-receptive neurons in the anteroventral periventricular nucleus are synaptic targets of the suprachiasmatic nucleus and peri-suprachiasmatic region

The anteroventral periventricular nucleus (AVPv) in the rat preoptic area is a key site underlying control of the steroid dependent preovulatory gonadotropin surge. Estrogen and progesterone receptor-containing neurons in the preoptic/hypothalamic continuum, particularly those in the AVPv, are believed to transduce steroidal signals and, in turn convey this information to the LHRH system, which lacks steroid receptors. In addition to the influence of the gonadal steroids, the precise timing of the preovulatory gonadotropin surge is believed to be regulated by the hypothalamic suprachiasmatic nucleus (SCN). The SCN and peri-SCN neurons send efferent projections rostrally to the anterior preoptic area suggesting that circadian signals are communicated synaptically to steroid-responsive neurons in the AVPv. To test this hypothesis, ultrastructural double label immunocytochemistry was conducted to determine whether SCN efferents contact estrogen receptor-immunoreactive neurons in the AVPv. Brain sections with SCN injections of phaseolus vulgaris leucoagglutinin (PHA-L) were immunostained for estrogen receptors and PHA-L. Light and electron microscopic data show that the anterior preoptic area received robust PHA-L-immunoreactive efferents from SCN neurons and immediately adjacent subparaventricular zone. In particular, the AVPv contained abundant labeled fibers and terminal boutons. Ultrastructurally, SCN- and subparaventricular zone-derived terminals synaptically contacted the perikaryon of many estrogen receptor-immunoreactive neurons in the AVPv. The perikarya of unlabeled neurons were also contacted, but the majority of the labeled contacts were observed upon neuronal processes. These results demonstrate that estrogen responsive AVPv neurons are regulated by SCN efferents. Furthermore, the present data provide strong support to the idea of collective control of pituitary gonadotropin release by steroid sensitive and circadian signal neural pathways.     

Nitric oxide synthase in the hypothalamic suprachiasmatic nucleus of rat: evidence from histochemistry, immunohistochemistry and Western blot; and colocalization with VIP

Nitric oxide (NO) is a neuroactive substance of high potency. Physiological results revealed the involvement of NO in circadian regulation of rats. Since neuronal structures containing NO-synthase (NOS) were previously not found in the circadian oscillator, the hypothalamic suprachiasmatic nucleus (SCN), in this species but are present in the hamster, we investigated the distribution of NO-producing structures in the rat SCN by Western blot analysis, immunohistochemistry of NOS, and by histochemistry (NADPH-diaphorase (NADPH-d) activity of NOS). Western blot analysis of SCN homogenates from rat (and, for comparison, hamster) showed a NOS-like immunoreactive (-LI) protein band of apparent molecular mass of 150 kDa, consistent with the neuronal NOS molecule. In the rat SCN, perikarya exhibiting NADPH-d staining or NOS-LI with a complete overlapping of both were found. Double-immunofluorescence experiments revealed that NOS cells are a subgroup of the neuronal SCN population that is characterized by immunoreactivity to vasoactive intestinal polypeptide. These data provide evidence for the existence of neuronal nitric oxide synthase in the rat SCN and may explain the involvement of NO in the mediation of photic information.