Calbindin-D28K cells selectively contact intra-SCN neurons

doi:10.1016/S0306-4522(01)00604-2

Neuroscience

Volume 111, Issue 3, 30 May 2002, Pages 575-585

https://doi.org/10.1016/S0306-4522(01)00604-2 Get rights and content

Abstract

Calbindin-D_28K-immunoreactive cells are tightly packed within a discrete region of the caudal aspect of the suprachiasmatic nuclei of hamsters. These cells receive direct retinal input and are Fos-positive in response to a light pulse. Knowledge of their afferent and efferent connections is necessary to understand suprachiasmatic nucleus organization. The first aim of the present study is to identify interconnections between calbindin and other peptidergic cells of the suprachiasmatic nuclei, using epi- and confocal microscopy and intra-suprachiasmatic nucleus tract tracing. The results indicate that essentially all calbindin cells receive numerous appositions from vasoactive intestinal polypeptide (VIP), neuropeptide Y and serotonin fibers and that most receive appositions from gastrin releasing peptide (GRP) and cholecystokinin (CCK) fibers. Reciprocal connections are seen from VIP, GRP and CCK cells but surprisingly, not from dorsomedial vasopressin cells. Injection of biotinylated dextran amine into the suprachiasmatic nucleus indicates that the ventrolateral suprachiasmatic nucleus projects to the entire nucleus, while the dorsal and medial regions of the suprachiasmatic nucleus project densely to most of the nucleus, except to the calbindin region. Analysis of colocalization of the peptides in the calbindin cell region shows that 91% of the substance P cells, 42% of the GRP cells and 60% of the VIP cells in the calbindin subnucleus coexpress calbindin-D_28K.

Our results reveal a highly specialized topographical organization of connections among suprachiasmatic nucleus cells.

Section snippets

Animals and housing

Subjects were LVG hamsters (Mesocricetus auratus) obtained from Charles River Laboratories. Animals were housed in translucent propylene cages (48×27×20 cm) and provided with ad libitum access to food and water. They were kept in a 14:10 light:dark cycle, with lights on at 08.00 h. The room was kept at 21±1°C. All handling of animals was done in accordance with Institutional Animal Care and Use Committee guidelines of Columbia University.

Perfusion

Hamsters were heavily anesthetized with sodium

Intra-suprachiasmatic CalB connections

CalB cells make selective intra-SCN connections. Figure 1 is a schematic representing the distribution of cells and fibers and the intra-SCN interconnections between the CalB cells and the VIP, AVP, CCK or GRP cells. The SCN is represented at four different levels from rostral to caudal.

Figure 2 shows photomicrographs of representative parts of the SCN, double-labeled for CalB and VIP, AVP, CCK or GRP. The top row shows light microscopic images of 50 μm thick sections through the CalB subregion

Connections of CalB-immunoreactive cells within the SCN

The molecular machinery that generates rhythmicity at the cellular level is being elucidated (Dunlap, 1999, King and Takahashi, 2000, Reppert and Weaver, 2001); however, the mechanisms of entrainment, coupling between entraining cues and oscillators and output are poorly understood. Knowledge of SCN organization is necessary to understand the generation and control of circadian rhythmicity in mammals.

This study focuses on the connections of the CalB cells of the hamster which has several

Acknowledgements

Supported by NIH Grants NS 37919 to R.S. and DK 07328 to L.J.K.

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In rat, it is partly colocalized with VIP in the most ventral aspect of the SCN where it would define a functionally distinct group within the VIP population (Albers et al., 1991; Romijn et al., 1997; Kawamoto et al., 2003; Guillaumond et al., 2007). In hamster, it is coexpressed in some of the CalB cells (LeSauter et al., 2002). Astrocytes are also important cellular components of the SCN.
The circadian timing system orchestrates daily variations in physiology and behavior through coordination of multioscillatory cell networks that are highly plastic in responding to environmental changes. Over the last decade, it has become clear that this plasticity involves structural changes and that the changes may be observed not only in central brain regions where the master clock cells reside but also in clock-controlled structures. This review considers experimental data in invertebrate and vertebrate model systems, mainly flies and mammals, illustrating various forms of structural circadian plasticity from cellular to circuit-based levels. It highlights the importance of these plastic events in the functional adaptation of the clock to the changing environment.
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The CALB cells in the hamster SCNce contact neurons identifiably VIP-, GRP- or CCK-IR and each of those cell types project back onto the CALB-IR cells. In contrast, VP-IR neurons appear to have no connections with CALB-IR cells (LeSauter et al., 2002). A confocal laser scanning microscopic study (Romijn et al., 1997) analyzed connections between cells representing the major phenotypes in rat SCN, including VP-, VIP-, GRP- and SS-IR neurons.
The suprachiasmatic nucleus (SCN), site of the primary clock in the circadian rhythm system, has three major afferent connections. The most important consists of a retinohypothalamic projection through which photic information, received by classical rod/cone photoreceptors and intrinsically photoreceptive retinal ganglion cells, gains access to the clock. This information influences phase and period of circadian rhythms. The two other robust afferent projections are the median raphe serotonergic pathway and the geniculohypothalamic (GHT), NPY-containing pathway from the thalamic intergeniculate leaflet (IGL). Beyond this simple framework, the number of anatomical routes that could theoretically be involved in rhythm regulation is enormous, with the SCN projecting to 15 regions and being directly innervated by about 35. If multisynaptic afferents to the SCN are included, the number expands to approximately brain 85 areas providing input to the SCN. The IGL, a known contributor to circadian rhythm regulation, has a still greater level of complexity. This nucleus connects abundantly throughout the brain (to approximately 100 regions) by pathways that are largely bilateral and reciprocal. Few of these sites have been evaluated for their contributions to circadian rhythm regulation, although most have a theoretical possibility of doing so via the GHT. The anatomy of IGL connections suggests that one of its functions may be regulation of eye movements during sleep. Together, neural circuits of the SCN and IGL are complex and interconnected. As yet, few have been tested with respect to their involvement in rhythm regulation.
Neurons identified by NeuN/Fox-3 immunoreactivity have a novel distribution in the hamster and mouse suprachiasmatic nucleus
2011, Brain Research
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VP-IR cells also tend to be scattered elsewhere in the SCN, particularly in the mouse. CCK-IR cells have a distribution similar to that for VP-IR neurons, but are also found ventrolaterally (LeSauter et al., 2002; Silver et al., 1999). CCK has been less widely studied than VP and the extent to which it is present in the SCN of diverse species is not known.
The suprachiasmatic nucleus (SCN) has several structural characteristics and cell phenotypes shared across species. Here, we describe a novel feature of SCN anatomy that is seen in both hamster and mouse. Frozen sections through the SCN were obtained from fixed brains and stained for the presence of immunoreactivity to neuronal nuclear protein (NeuN-IR) using a mouse monoclonal antibody which is known to exclusively identify neurons. NeuN-IR did not identify all SCN neurons as medial NeuN-IR neurons were generally not present. In the hamster, NeuN-IR cells are present rostrally, scattered in the dorsal half of the nucleus. More caudally, the NeuN-IR cells are largely, but not exclusively, scattered inside the lateral and dorsolateral border. At mid- to mid-caudal SCN levels, a dense group of NeuN-IR cells extends from the dorsolateral border ventromedially to encompass the central subnucleus of the SCN (SCNce). The pattern is similar in the mouse SCN. NeuN-IR does not co-localize with either cholecystokinin- or vasoactive intestinal polypeptide, but does with vasopressin-IR in the caudal SCN. In the hamster SCNce, numerous cells contain both calbindin- and NeuN-IR. The distribution of NeuN-IR cells in the SCN is unique, especially with regard to its generally lateral location through the length of the nucleus. The distribution of NeuN-IR cells is not consistent with most schemas representing SCN organization or with terminology referring to its widely accepted subdivisions. NeuN has recently been identified as Fox-3 protein. Its function in the SCN is not known, nor is it known why a large proportion of SCN cells do not contain NeuN-IR.
Investigating the role of substance P in photic responses of the circadian system: Individual and combined actions with gastrin-releasing peptide
2010, Neuropharmacology
The suprachiasmatic nucleus (SCN) contains the master mammalian circadian pacemaker. It is comprised of several phenotypically distinct cell groups, some of which are situated in the weakly rhythmic retinoresponsive ventrolateral region while others are found in the rhythmic, non-retinoresponsive dorsomedial region. The mechanism by which retinorecipient cells convey photic information to the dorsomedial clock cells is unclear. The ventrolateral SCN core contains a variety of cell phenotypes. Two neuropeptides, namely substance P (SP) and gastrin-releasing peptide (GRP) extensively colocalize with calbindin D28K, a marker for SCN cells that are strongly light-responsive. Previous studies have implicated these neuropeptides in photic phase shifting of the circadian system. The present study examines how these peptides interact to regulate photic responses of the circadian system. It was observed that 55.5 ± 9.1% of SP cells colocalized GRP. SP did not enhance GRP-induced phase shifts in the early-subjective night, while it significantly attenuated GRP-induced phase shifts during the late-subjective night. SP induced significant phase shifts that did not resemble light in the early-subjective night, but was not necessary for light-induced phase shifts and Fos expression at this time. SP induced significant Fos expression only in the late subjective night. SP may not be a necessary component in the pathway(s) involved in photic phase shifting during the early-subjective night, but may modulate phase shifts during the late-subjective night. Distinct biochemical mechanisms that underlie behavioral phase shifts may account for the differences observed in the early- vs. late-subjective night.
Calcium-binding proteins in the circadian centers of the common marmoset (Callithrix jacchus) and the rock cavy (Kerodon rupestris) brains
2008, Brain Research Bulletin
Citation Excerpt :
For example, it was noticed that the rupture of the circadian rhythm of locomotor activity following exposure to prolonged constant light parallels the suppression of CB expression in the retinorecipient area of the rat SCN [3]. Current statements point to the fact that CB present in the SCN cells are part of a potential intercellular pathway of the SCN population, sustaining local neurons that drive photic information between areas of the nucleus [17,21]. Also, CB expression is negatively correlated to the day length, i.e., the number of CB-positive cells in the SCN increases in hamsters exposed to short photoperiods when compared with hamsters exposed to long photoperiods, supporting the idea that CB-IR neurons in the SCN are involved in the encoding of seasonal information by the SCN [26].
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.
Enhancement of photic shifts with the 5-HT<inf>1A</inf> mixed agonist/antagonist NAN-190: Intra-suprachiasmatic nucleus pathway
2008, Neuroscience
Chronic desynchronization between the mammalian circadian pacemaker and its external environment, such as that observed from shift work or jet lag, can lead to various long-term health consequences. The circadian clock can be reset by exposure to light, although the magnitude of such adjustments is modest. 5-HT modulates the effects of light, and 5-HT_1A mixed agonist/antagonists, such as NAN-190, have been found to potentiate the phase resetting ability of light. The mechanism for this potentiation has yet to be uncovered, although it has been proposed that these drugs inhibit raphe output while simultaneously blocking post-synaptic 5-HT_1A receptors. The current study takes advantage of the heterogeneous network organization of the circadian clock to identify where in the circadian system NAN-190 exerts its influence. Retinorecipient cells in the ventrolateral suprachiasmatic nucleus (SCN) are activated by glutamate and release either gastrin-releasing peptide (GRP) or vasoactive intestinal polypeptide. Application of the glutamate agonist N-methyl-d-aspartic acid (NMDA) or either of these neuropeptides to the SCN mimics the effects of light. We hypothesized that NAN-190 would modify responses to treatments that activate the circadian system upstream, but not downstream, of where NAN-190 is acting. Hamsters were pretreated with NAN-190 or vehicle, followed by one of the neurochemicals 45 min later, during the early- and/or late-subjective night. NAN-190 potentiated NMDA-induced phase advances and delays as well as GRP-induced advances, but attenuated GRP-induced delays. NAN-190 did not potentiate NMDA-induced Fos expression, however greater GRP-induced Fos expression was found within the dorsolateral region of the SCN. These data suggest that NAN-190 acts, in part, by modifying the responsiveness of retinorecipient cells in the circadian clock. An understanding of the neural events that underlie the potentiation of photic phase shifts by NAN-190 could guide the development of novel chronobiotics which could be used to treat a variety of sleep and circadian disorders.

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Calbindin-D28K cells selectively contact intra-SCN neurons

Abstract

Section snippets

Animals and housing

Perfusion

Intra-suprachiasmatic CalB connections

Connections of CalB-immunoreactive cells within the SCN

Acknowledgements

Brain Res.

Neuroscience

Cell

Neuroscience

Brain Res.

Prog. Brain Res.

Prog. Brain Res.

Neuroscience

Neurosci. Lett.

Neuron

Brain Res.

Neuroscience

Dev. Brain Res.

Brain Res.

Neuroscience

Neurons containing gastrin-releasing peptide and vasoactive intestinal polypeptide are involved in the reception of the photic signal in the suprachiasmatic nucleus of the Syrian hamster: an immunocytochemical ultrastructural study

Cell Tissue Res.

Interaction of colocalized neuropeptides: functional significance in the circadian timing system

J. Neurosci.

Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei

Proc. Natl. Acad. Sci. USA

Retinal innervation of calbindin-D28K cells in the hamster suprachiasmatic nucleus: ultrastructural characterization

J. Biol. Rhythms

Calbindin-D_28K cells selectively contact intra-SCN neurons