Calbindin-D(28K) cells selectively contact intra-SCN neurons

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.

Fig. 1

Schematic representing the average distribution of cells and fibers of the peptides studied in the rostral, mid-anterior, mid-posterior and caudal aspects of the SCN. Each panel shows the fiber distribution between CalB and VIP, AVP, CCK, GRP, NPY or 5-HT. The left columns in the first four panels represent the distribution of the different peptidergic cells and fibers (gray) in relation to the CalB cells (black), the right columns represent the distribution of CalB cells and fibers (gray) in relation to the other peptidergic cells (black). The last two columns represent the distribution of afferent NPY and 5-HT fibers (gray) in relation to the CalB cells (black).

Fig. 2

Photomicrographs of double-labeled coronal brain sections through the SCN. The top row are light microscopy photomicrographs showing the distribution of CalB- and of VIP-, AVP (VP)-, CCK- and GRP-ir in 50 μm thick sections of the mid-posterior SCN. The CalB cells form a dense subnucleus in the ventrolateral region, some sparse CalB cells are also seen in the dorsal SCN. The second and third rows are1-μm confocal sections. The second row shows the presence (or absence) of VIP-, AVP-, CCK- and GRP-like immunoreactivity (left to right, respectively) within the CalB subregion. VIP makes numerous appositions with the CalB cells. AVP and CCK make few appositions. GRP cells are distributed among the CalB cells and GRP fibers contact CalB cells. The third row shows the presence (or absence) of CalB fibers within the region of VIP- (ventral SCN), AVP- (dorsomedial SCN), CCK- (medial SCN) and GRP-like (ventromedial SCN) immunoreactivity (left to right, respectively). Arrows indicate some of the appositions. Some CalB fibers make appositions with VIP cells. In the second column, no CalB-ir fibers are seen in this section of the dorsomedial AVP region. CalB-ir fibers are seen among the CCK-ir cells and some contact the cells. CalB-ir fibers make appositions with GRP-ir cells, and some cells contain both CalB and GRP.

Fig. 3

Photomicrographs of coronal brain sections double-labeled for NPY and CalB (left) and 5-HT and CalB (right). The top panels shows the distribution of the NPY and 5-HT fibers within the mid-posterior SCN at the level of the CalB sub-nucleus. The bottom panels are 1-μm confocal sections showing the NPY fibers surrounding and making appositions onto the CalB cells and the sparse 5-HT fibers contacting the CalB cells. Although both NPY and 5-HT fibers contact all the CalB cells, the contacts on each cell were more numerous for NPY than for 5-HT.

Fig. 4

Light microscopy photomicrographs of 25-μm sections showing the injection sites of BDA in the first column, stained with the FITC fluorophore. The size of the injection is represented by dashed lines drawn on alternate sections stained with SG chromogen. BDA-ir efferent projections are shown in the second column. The same section as in the second column double-labeled for CalB is shown in the last column. First row: BDA was injected in the ventral SCN. BDA-ir fibers innervate the whole SCN. Second row: An injection in the medial SCN results in fibers that densely innervate the whole SCN except the CalB subregion which receives only sparse fibers.

Fig. 5

Confocal microscopy photomicrographs of 1-μm optical sections of the CalB subnucleus stained for CalB (first column) and for SP-, GRP-and VIP-like immunoreactivity (second column), and the overlay of the two laser wavelengths (third column). The arrowheads indicate some of the double-labeled cells. For VIP, the captured images are from the ventral part of the CalB subnucleus. The proportions of double-labeled cells are shown in Fig. 6.

Fig. 6

Bar graphs showing the proportion of cells double-labeled within the SCN. For SP, the numbers refer to cells within the SP subnucleus only. For VIP, the numbers refer to cells within the ventral CalB subnucleus only. (A) Percentage (mean ± S.E.M.) of CalB cells that also colocalize each peptide. The counts were done on 74 CalB cells in three animals for SP, 154 CalB cells in eight animals for GRP and 40 CalB cells in three animals for VIP. (B) Percentage (mean ± S.E.M.) of peptidergic cells that colocalize CalB. The counts were done on 48 SP cells in three animals, 122 GRP cells in eight animals and 25 VIP cells in three animals. In four animals, 40 AVP (VP)-ir cells and 26 CalB-ir cells were sampled in the rostral and dorsomedial SCN, and no cell colocalized AVP and CalB.

Fig. 7

Schematic representing the connections between the CalB cells and CCK, AVP, GRP, NPY, VIP and the retina. The arrow thickness and the percentages are the proportion of cells receiving three or more appositions (Table 1). For retinal afferents, the thickness of the arrow is derived from previous data (Bryant et al., 2000; Silver et al., 1996).