Sexual dimorphism among calbindin-D28K immunoreactive cells in the rat pineal body

Calbindin antibodies have been used in neuroanatomical studies to give excellent cytoarchitecural staining and visualization of a Golgi-like cellular morphology. Calbindin-D28K immunoreactivity used in rat pineal gland as a marker detected two classes of pineal cells. One class of small cells representing exclusively glial cells was strongly immunoreactive, and presented a large variety of individual shapes. The majority were a pyramidal shape with one or more processes while others displayed a cytoplasmic lipid droplet. Some small cells occurred around pericapillary spaces. The second class of calbindin-D28K positive cells corresponding to type II pinealocytes were characterized by their large size and less intensive labelling. Type II pinealocytes were round or rectangular; the nucleus was infolded and large with a prominent nucleolus. These large cells were preferentially distributed in the vicinity of vessels and assembled in a cluster of more than ten cells. The lack of S-100 and myeloperoxidase immunoreactivities in large calbindin-D28K cells excluded their possible characterization as glial cells and mononuclear phagocytes, while their size (>15 m) excluded microglial cells. A sex difference was detected between large calbindin-D28K positive cells. The mean calculated number of large positive cells for males was 6361±1504 (n=8) compared to 2162±1235 (n=7) for females. No significative difference was detected between males and females for small calbindin-D28K positive cells.     

Hamster pulmonary endocrine cells with positive immunostaining for calbindin-D28K

 We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D_28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells. Accepted: 17 June 1997     

Catecholaminergic innervation of the suprachiasmatic nucleus in the adult rat: ultrastructural relationships with neurons containing vasoactive intestinal peptide or vasopressin

Catecholaminergic fibers in the suprachiasmatic nucleus of adult rats were investigated by use of light- and electron-microscopic immunocytochemistry. The suprachiasmatic nucleus receives a modest density of tyrosine hydroxylase-containing axons, homogeneously distributed in the nucleus and forming varicosities throughout its entire rostro-caudal extension. Immunolabeling with antibodies against dopamine showed that this catecholamine input comprises a dopaminergic component. Many tyrosine hydroxylase-positive cells were localized at the immediate periphery of the suprachiasmatic nucleus. With electron-microscopic examination, dendrites of these neurons were found within the limits of the nucleus as well as at a border zone between the suprachiasmatic nucleus proper and the optic tract where they received unlabeled synapses, providing a morphological support for a possible role of dopaminergic neurons in the integration and/or transfer of light-related signals. More than 91% of catecholaminergic axonal varicosities were found to establish morphologically defined synapses with dendrites. To investigate whether these synapses might be shared with neurons of one or both of the two main peptidergic populations of the nucleus, namely vasoactive intestinal peptide- and vasopressin-containing neurons, we carried out doublelabeling experments combining immunoperoxidase and immunogold-silver labeling. Results showed only a few cases of direct association of the catecholaminergic terminals with these peptidergic categories. In both types of dually stained sections, catecholaminergic synapses were preferentially made with unlabeled dendrites. The homogeneous distribution of tyrosine hydroxylase-immunoreactive fibers in the suprachiasmatic nucleus could therefore reflect a lack of significant catecholaminergic innervation of both vasoactive intestinal peptide- and vasopressin-synthesizing neurons.     

Dual role of calbindin-D28K in vesicular catecholamine release from mouse chromaffin cells

Calbindin-D(28K) is suggested to play a postsynaptic role in neurotransmission and in the regulation of the intracellular Ca(2+) concentration. However, it is still unclear whether calbindin-D(28K) has a role in the regulation of exocytosis, either as Ca(2+) buffer or as Ca(2+) sensor. Amperometric recordings of catecholamine exocytosis from wild-type and calbindin-D(28K) knockout mouse chromaffin cells reveal a strong reduction in the number of released vesicles, as well as in the amount of neurotransmitter released per fusion event in knockout cells. However, Ca(2+) current recordings and Ca(2+) imaging experiments, including video-rate confocal laser scanning microscopy, revealed that the intracellular Ca(2+) dynamics are remarkably similar in wild-type and knockout cells. The combined results demonstrate that calbindin-D(28K) plays an important and dual role in exocytosis, affecting both release frequency and quantal size, apparently without strong effects on intracellular Ca(2+) dynamics. Consequently, the possibility that calbindin-D(28K) functions not only as a Ca(2+) buffer but also as a modulator of vesicular catecholamine release is discussed.     

Development of hamster circadian rhythms: Role of the maternal suprachiasmatic nucleus

Summary During development, the circadian rhythms of rodents become entrained to rhythmicity of the mother. Rhythms in behavior and in neuroendocrine function are regulated by a circadian pacemaker thought to be located within the suprachiasmatic nucleus (SCN) of the hypothalamus. Evidence indicates that this pacemaker begins to function and to be entrained by maternal rhythms before birth. Although the maternal rhythms which mediate prenatal entrainment of the fetal circadian pacemaker have not been identified, it is likely that they are regulated by the maternal SCN.The role of the maternal SCN in entrainment of the offspring was examined in Syrian hamsters (Mesocricetus auratus) by measuring the activity/rest rhythms of pups. Using the synchrony among the rhythms of pups within a litter as an indication that the pups had been entrained, the effect on entrainment of ablating the maternal SCN was determined. Lesions of the maternal SCN which were performed early in gestation (day 7) and which destroyed at least 75% of the SCN were found to disrupt the normal within litter synchrony among pups, indicating interference with the normal mechanism of entrainment.The effect of lesions on day 7 of gestation could mean that the maternal SCN is important for entrainment of the pups before birth, after birth, or during both of these times. To determine if the maternal SCN is specifically important for prenatal entrainment, lesions were performed two days before birth on day 14 of gestation. Lesions of the maternal SCN on day 14 were not as disruptive as were lesions on day 7. This suggests that the maternal SCN is important between days 7 and 14 of gestation and that the synchrony normally observed at weaning is already established, in part, on or before day 14 of gestation. This further suggests that an entrainable circadian pacemaker is present in the fetus only two weeks after fertilization.Abbreviations SCN suprachiasmatic nucleus - L:D light:dark - LL constant light - r mean vector length - 2DG 2-deoxyglucose - NAT N-acetyltransferase     

Post-coital ultrastructural changes in neurons of the suprachiasmatic nucleus of the rabbit

Summary The effects of coitus on the ultrastructure of neurons in the suprachiasmatic nucleus of the rabbit were studied. Changes first became apparent 1/2 h after coitum in neurons located near capillaries. More pronounced ultrastructural changes were observed in large neurons removed at 1 h post-coitus. These changes, characterized by well developed Golgi systems and rough endoplasmic reticulum, the presence of large dense-core vesicles and a significant increase in both neuronal and nuclear size, were also evident in neurons observed at 2 to 10 h post coitum. Similar ultrastructural features were not observed in the neurons of the control animals. The post-coital ultrastructural changes observed within these neurons suggest high synthetic activity which may concern the production sites of the neurohormone LH-RF. Two populations of dense-core vesicles were observed: a) those with a mean diameter of 849 Å, and b) those with a mean diameter of 1542 Å. The small dense-core vesicle is probably monoamine in nature; the larger vesicle may contain the neurohormone LH-RF. A third vesicle type with a mean diameter of 1836 Å and characterized by a granular content of low electron density was also observed. That this vesicle represents the immature form of the large (1542 Å) dense-core vesicle is suggested; however, morphological evidence supporting this hypothesis is inconclusive. There is also no evidence for the storage of secreted materials within the soma of these neurons. Immediate transport toward the median eminence is suggested.This investigation was supported by grants to the two senior authors from the Medical Research Council of Canada.     

Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells

Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i.     

Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta.

The appearance of the calcium-binding proteins (CaBP-D28K and CaBP-D9K) in embryonic mice tissues was determined using a sensitive immunohistochemical assay. CaBP-D28K first appears in myenteric nerve plexuses of the duodenum on day E15, in duodenal villus cells on day E16, in Purkinje cells of the cerebellum on day E19, in cells of the mesonephric duct on day E11 and in the metanephric duct on day E12. CaBP-D9K first appears in enterocytes of the duodenum on day E18, in trophoblastic giant cells (TGC) of the placenta on day E10, and in the metanephric duct on day E15. A differential time of appearance and colocalization of the two CaBPs is demonstrated in the embryonic mouse kidney, suggesting either that vitamin D does not control both CaBPs in the foetus or that the vitamin D control is unequal. The early appearance and location of CaBP-D9K in TGCs may suggest that these cells play an important role in transplacental transfer of calcium.     

Ontogeny of calbindin-D28K and calretinin in developing chick kidney

The ontogeny of two calcium-binding proteins (calbindin-D_28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.     

Immunohistochemical demonstration of calbindin-D 28K (CABP28K) in the spinal cord motoneurons of teleost fish

Summary The distribution and localization of the calciumbinding protein, calbindin-D 28K (CaBP28K), in the spinal cord motoneurons of larvae of the teleost fish, Apteronotus leptorhynchus (Gymnotidae) and Pollimyrus isidori (Mormyridae), and in the adult goldfish, Carassius auratus (Cyprinidae), were determined by means of immunohistochemistry. Sections of whole larvae and goldfish spinal cord were reacted with a polyclonal antibody to rat renal CaBP28K. CaBP28K was located by the PAP technique (Sternberger). It was found in the soma, dendrites, axons and axon terminals of spinal motoneurons but not in those of electromotoneurons of Apteronotus leptorhynchus, whereas it occurred in both motoneurons and electromotoneurons of the larval electric organ of Pollimyrus isidori. In these species CaBP28K was also present in the electromotoneuron axon terminals that make synaptic contacts with the pedicles of the electrocytes. In adult Carassius auratus, CaBP28K was found in the soma, dendrites and axons of certain spinal motoneurons. The results indicate that, in teleosts, the motoneurons containing CaBP28K may represent a well-defined population within the spinal cord; the role of this protein in these cells remains to be determined.     

Rhythmic coupling among cells in the suprachiasmatic nucleus

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells. There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. One mechanism that mediates coordinated electrical activity is direct electrical connections between cells formed by gap junctions. In the present study, we used a brain slice preparation to show that developing SCN cells are dye coupled. Dye coupling was observed in both the ventrolateral and dorsomedial subdivisions of the SCN and was blocked by application of a gap junction inhibitor, halothane. Dye coupling in the SCN appears to be regulated by activity-dependent mechanisms as both tetrodotoxin and the GABA(A) agonist muscimol inhibited the extent of coupling. Furthermore, acute hyperpolarization of the membrane potential of the original biocytin-filled neuron decreased the extent of coupling. SCN cells were extensively dye coupled during the day when the cells exhibit synchronous neural activity but were minimally dye coupled during the night when the cells are electrically silent. Immunocytochemical analysis provides evidence that a gap-junction-forming protein, connexin32, is expressed in the SCN of postnatal animals. Together the results are consistent with a model in which gap junctions provide a means to couple SCN neurons on a circadian basis.     

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light-entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co-extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT_1A or 5ht₇ receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT_1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.     

Presynaptic adenosine A1 receptors regulate retinohypothalamic neurotransmission in the hamster suprachiasmatic nucleus

Adenosine has been implicated as a modulator of retinohypothalamic neurotransmission in the suprachiasmatic nucleus (SCN), the seat of the light-entrainable circadian clock in mammals. Intracellular recordings were made from SCN neurons in slices of hamster hypothalamus using the in situ whole-cell patch clamp method. A monosynaptic, glutamatergic, excitatory postsynaptic current (EPSC) was evoked by stimulation of the optic nerve. The EPSC was blocked by bath application of the adenosine A(1) receptor agonist cyclohexyladenosine (CHA) in a dose-dependent manner with a half-maximal concentration of 1.7 microM. The block of EPSC amplitude by CHA was antagonized by concurrent application of the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist CGS21680 was ineffective in attenuating the EPSC at concentrations up to 50 microM. Trains of four consecutive stimuli at 25 ms intervals usually depressed the EPSC amplitude. However, after application of CHA, consecutive responses displayed facilitation of EPSC amplitude. The induction of facilitation by CHA suggested a presynaptic mechanism of action. After application of CHA, the frequency of spontaneous EPSCs declined substantially, while their amplitude distribution was unchanged or slightly reduced, again suggesting a mainly presynaptic site of action for CHA. Application of glutamate by brief pressure ejection evoked a long-lasting inward current that was unaffected by CHA at concentrations sufficient to reduce the evoked EPSC amplitude substantially (1 to 5 microM), suggesting that postsynaptic glutamate receptor-gated currents were unaffected by the drug. Taken together, these observations indicate that CHA inhibits optic nerve-evoked EPSCs in SCN neurons by a predominantly presynaptic mechanism.     

Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod

The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro.     

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

In mammals, the suprachiasmatic nuclei are involved in the generation of biological rhythms and are synchronized by light input coming from the retina. The targets of retinal afferents and the involvement of neurons containing gastrin-releasing and vasoactive intestinal peptides in photic reception were investigated in the suprachiasmatic nuclei of the Syrian hamster by using light- and electron-microscopic immunocytochemistry. Cholera toxin was used to trace retinal fibers and Fos immunoreactivity to visualize cellular response to light stimulation. Ultrastructural observations were made in the intermediate third of the nuclei, the area of highest overlap for the immunoreactivities investigated. Gastrin-releasing peptide and vasoactive intestinal peptide cell bodies were localized in the ventral part of the nuclei; their dense immunoreactive fiber network often displayed synaptic contacts. Both neuropeptides were colocalized in elongated cells observed near the optic chiasm. Following a light pulse in the middle of the subjective night, Fos protein was expressed in most gastrin-releasing peptide perikarya and in some vasoactive intestinal peptide cells. Retinal terminals mostly occurred in the midline zone between the suprachiasmatic nuclei. Symmetrical or asymmetrical retinal synapses were observed on gastrin-releasing peptide-immunoreactive dendrites and somata, but never on vasoactive intestinal peptide neurons. These results are discussed in relation to the photic entrainment of the circadian clock.     

Ultrastructural identification of calbindin-D28k-immunoreactive cells in the rat superficial pineal body

The vitamin-D-dependent calcium-binding protein, calbindin-D28k, was detected immunocytochemically in the majority of pericapillary pinealocytes of the rat superficial pineal body. The interstitial cells were calbindin-negative. The significance of these findings is discussed.     

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca²⁺-dependent fashion. Molecular docking and conformational sampling studies of the Ca²⁺-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.

Structured summary of protein interactions

Calbindin-D28K and Caspase-3bind by isothermal titration calorimetry(View interaction)