The intestine is the only gate for the entry of Ca to the body in humans and mammals. The entrance of Ca occurs via paracellular and intracellular pathways. All steps of the latter pathway are regulated by calcitriol and by other hormones. Dietary and pharmacological compounds also modulate the intestinal Ca absorption process. Among them, dietary Ca and P are known to alter the lipid and protein composition of the brush-border and basolateral membranes and, consequently, Ca transport. Ca intakes are below the requirements recommended by health professionals in most countries, triggering important health problems. Chronic low Ca intake has been related to illness conditions such as osteoporosis, hypertension, renal lithiasis and incidences of human cancer. Carbohydrates, mainly lactose, and prebiotics have been described as positive modulators of intestinal Ca absorption. Apparently, high meat proteins increase intestinal Ca absorption while the effect of dietary lipids remains unclear. Pharmacological compounds such as menadione, dl-butionine-S,R-sulfoximine and ursodeoxycholic acid also modify intestinal Ca absorption as a consequence of altering the redox state of the epithelial cells. The paracellular pathway of intestinal Ca absorption is poorly known and is under present study in some laboratories. Another field that needs to be explored more intensively is the influence of the gene × diet interaction on intestinal Ca absorption. Health professionals should be aware of this knowledge in order to develop nutritional or medical strategies to stimulate the efficiency of intestinal Ca absorption and to prevent diseases.

Immunocytochemical techniques were used to determine the distribution of the calcium-binding protein calretinin in the cat retina. Comparisons were made with parvalbumin and calbindin as well as with the inhibitory neurotransmitters GABA and glycine. Calretinin immunoreactivity was seen in horizontal cells and multiple subpopulations of amacrine and ganglion cells. Cone outer segments were also stained. Calbindin immunoreactivity was present in cone photoreceptors, horizontal cells, at least two subtypes of cone bipolar cell, numerous amacrine cells, and cells residing in the ganglion cell layer. Heavy staining for parvalbumin was found in both A- and B-type horizontal cells, distinct subpopulations of amacrine and ganglion cells, and a small population of cone photoreceptor cells. To confirm the identity of cone photoreceptors, comparisons were made with retinas stained for opsins specific for red/green or blue cones (Szé1 et al., 1986). The localization of parvalbumin corresponded with that of blue-type cones only whereas calretinin and calbindin staining showed the same distribution as both red/green and blue cones. Double-label immunofluorescence studies revealed colocalization of all three of the calcium-binding proteins in a number of neurons including horizontal cells and AII amacrine cells. To assess a possible transmitter-specific relationship for calretinin, double-label studies were carried out with GABA and glycine. However, the staining patterns for each of these inhibitory amino acids differed substantially from that of calretinin. The possibility remains that calretinin and other calcium-binding proteins may play a role in neurotransmission through interactions with receptors or second-messenger agents.

Architectonic subdivisions of the inferior pulvinar (PI) complex were delineated in New World owl and squirrel monkeys and Old World macaque monkeys. Brain sections were processed for Nissl substance, myelin, cytochrome oxidase (CO), acetylcholinesterase (AChE), calbindin-D28K (Cb), or with the monoclonal antibody Cat-301. In all three primates, we identified the posterior nucleus (PIp) and the medial nucleus (PIm) of previous reports, and divided the previously recognized central nucleus (PIc) into two subdivisions, medial (PIcm) and lateral (PIcl). Each nucleus had several features that allowed it to be readily distinguished. (1) PIp was dark in Cb, and moderately dark in AChE and CO preparations. (2) PIm was Cb light, and AChE and CO dark. (3) PIcm was Cb dark, and AChE and CO light. (4) PIcl was Cb moderate with a scattering of dark neurons, and moderately dark for AChE and CO. (5) In sections processed for Cat-301, PIm in macaque monkeys and PIcm and PIp in squirrel monkeys stained darkly, while little staining was apparent in owl monkeys. The results allowed subdivisions of the inferior pulvinar to be more clearly defined, homologized, and compared across taxa. All monkeys appear to have the same four subdivisions of the PI, although properties vary.

The morphology and distribution of normally placed and displaced A horizontal cells were studied in the retina of a diurnal hystricomorph rodent, the agouti Dasyprocta aguti. Cells were labeled with anti-calbindin immunocytochemistry. Dendritic-field size reaches a minimum in the visual streak, of about 9000 μm2, and increases toward the retinal periphery both in the dorsal and ventral regions. There is a dorsoventral asymmetry, with dorsal cells being larger than ventral cells at equal distances from the streak. The peak value for cell density of 281 ± 28 cells/mm2 occurs in the center of the visual streak, decreasing toward the dorsal and ventral retinal periphery, paralleling the increase in dendritic-field size. Along the visual streak, the decline in cell density is less pronounced, remaining between 100–200 cells/mm2 in the temporal and nasal periphery. Displaced horizontal cells are rare and occur in the retinal periphery. They tend to be smaller than normally placed horizontal cells in the ventral region, whilst no systematic difference was observed between the two cell groups in the dorsal region. Mosaic regularity was studied using nearest-neighbor analysis and the Ripley function. When mosaic regularity was determined removing the displaced horizontal cells, there was a slight increase in the conformity ratio, but the bivariate Ripley function indicated some repulsive dependence between the two mosaics. Both results were near the level of significance. A similar analysis performed in the capybara retina, a closely related hystricomorph rodent bearing a higher density of displaced horizontal cells than found in the agouti, suggested spatial independence between the two mosaics, normally placed versus displaced horizontal cells.

As more human retinas affected with genetic or immune-based diseases become available for morphological analysis, it is important to identify immunocytochemical markers for specific subtypes of retinal neurons. In this study, we have focused on bipolar cell markers in central retina. We have done single and double labeling using several antisera previously utilized in macaque monkey or human retinal studies and two new antisera (1) to correlate combinations of antisera labeling with morphological types of bipolar cells in human retina, and (2) to compare human labeling patterns with those in monkey retina. Human bipolar cells showed a wide range of labeling patterns with at least ten different bipolar cell types identified from their anatomy and marker content. Many bipolar cell bodies in the outer part of the inner nuclear layer contained combinations of protein kinase C alpha (PKCα), Islet-1, glycine, and Goα. Bipolar cells labeled with these markers had axons terminating in the inner half of the inner plexiform layer (IPL), consistent with ON bipolar cells. Bipolar cell bodies adjacent to the amacrine cells and with axons in the outer half of the IPL contained combinations of recoverin, glutamate transporter-1, and PKCβ, or CD15 and calbindin. Bipolar cells labeled with these markers were presumed OFF bipolar cells. Calcium-binding protein 5 (CaB5) labeled both putative ON and OFF bipolar cells. Using this cell labeling as a criteria, most cell bodies close to the horizontal cells were ON bipolar cells and almost all bipolar cells adjacent to the amacrine cells were OFF with a band in the middle 2–3 cell bodies thick containing intermixed ON and OFF bipolar cells. Differences were found between human and monkey bipolar cell types labeled by calbindin, CaB5, and CD15. Two new types were identified. One was morphologically similar to the DB3, but labeled for CD15 and CaB5. The other had a calbindin-labeled cell body adjacent to the horizontal cell bodies, but did not contain any accepted ON markers. These results support the use of macaque monkey retina as a model for human, but caution against the assumption that all labeling patterns are identical in the two primates.

Three series of experiments were conducted with fast-growing chickens in order: to evaluate the effects of dietary Ca and P on cholecalciferol metabolism and expression; to determine dietary Ca requirements; to determine dietary P requirements. The results of the first series confirmed previous results on the effects of dietary Ca and P on some variables of vitamin D metabolism and expression, Ca homeostasis and P metabolism in the young chicken (1- to 21-d-old), and extended them to older birds (22- to 43-d-old). The bone formation rate and the duodenal calbindin content were maintained at high levels until the age of 43 d. Dietary Ca or P restriction increased duodenal calbindin and decreased bone ash in both 22- and 43-d-old chickens, but the effect on bone ash was less pronounced in the 43-d-old birds than in the younger ones. These results suggest that: (a) the capabilities for adaptation to dietary Ca and P restriction remain high during the whole growing period; (b) the growing broilers express a high adaptive capability even when the diet contains the recommended Ca and P contents. The results of the second and third series of experiments suggest that: (c) unlike the Ca requirements of the 1- to 22-d-old chick, P requirements for growth and bone ash are similar, and are as high in the older chicks as in the younger ones (7·4–8·3 g P/kg or 4·8–5·7 g non-phytate P/kg diet); (d) although growth and bone ash in the 29- to 43-d-old chickens appear to be less sensitive to dietary Ca content, within a range close to the calculated P requirement, 10 g Ca/kg diet appears to be required for best tibia mineralization, and to a lesser extent for better growth at this age.

We previously reported that two types of visual thalamic interneurons (class III and class V) could be distinguished by the presence or absence of the enzyme brain nitric oxide synthase (bNOS; Bickford et al., 1999). In the present study, we found that further immunocytochemical characterization can be used to isolate class V and class III neurons: class V neurons express GABA and bNOS but not the calcium binding protein calbindin, whereas class III neurons express GABA and calbindin, but not bNOS. By pairing staining for bNOS or calbindin with staining for GABA, we were able to distinguish class III and class V neurons, thereby allowing us to characterize the synaptic inputs to these two interneuron populations within the cat pulvinar nucleus. We found class III and class V interneurons participate in distinctly different circuitries. Class III cells receive the majority of their input from large terminals with round vesicles (RL profiles, that presumably originate from cortical layer V) while class V cells receive all of their input from small terminals with round vesicles (RS profiles, that presumably originate from cortical layer VI). These two types of interneurons also target different regions of the dendritic arbors of thalamocortical cells; class III cells contact large caliber (presumably proximal) thalamocortical dendrites within glomeruli, while class V cells contact the small caliber (presumably distal) thalamocortical cell dendrites within the extraglomerular neuropil. Thus, a dual inhibitory system exists within the visual thalamus that integrates different types of corticothalamic input and targets distinct regions of thalamocortical cell dendritic arbors.

Bipolar cells are not only important for visual processing but input from these cells may underlie the reorganization of ganglion cell dendrites in the inner plexiform layer (IPL) during development. Because little is known about the development of bipolar cells, here we have used immunocytochemical markers and dye labeling to identify and follow their differentiation in the neonatal ferret retina. Putative cone bipolar cells were immunoreacted for calbindin and recoverin, and rod bipolar cells were immunostained for protein kinase C (PKC). Our results show that calbindin-immunoreactive cone bipolar cells appear at postnatal day 15 (P15), at which time their axonal terminals are already localized to the inner half of the IPL. By contrast, recoverin-immunoreactive cells with terminals in the IPL are present at birth, but many of these cells may be immature photoreceptors. By the second postnatal week, recoverin-positive cells resembling cone bipolar cells were clearly present, and with increasing age, two distinct strata of immunolabeled processes occupied the IPL. PKC-containing rod bipolar cells emerged by the fourth postnatal week and at this age have stratified arbors in the inner IPL. The early bias of bipolar axonal arbors in terminating in the inner or outer half of the IPL is confirmed by dye labeling of cells with somata in the inner nuclear layer. At P10, several days before ribbon synapses have been previously observed in the ferret IPL, the axon terminals of all dye-labeled bipolar cells were clearly stratified. The results suggest that bipolar cells could provide spatially localized interactions that are suitable for guiding dendritic lamination in the inner retina.

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.

Two types of cone bipolar cells, the blue cone bipolar cell and the diffuse bipolar cell (DB3), were labelled immunohistochemically and investigated in the retina of a New World monkey, the marmoset. Blue cone bipolar cells were labelled with an antiserum against cholecystokinin. Short-wavelength-sensitive (SWS) cones were labelled with an antiserum against the SWS cone opsin. The DB3 cells were labelled with antibodies to calbindin. Blue cone bipolar cells in marmoset do not form a regular mosaic but instead follow the random distribution of the SWS cones. Nevertheless, the SWS cone to blue cone bipolar cell connectivity in marmoset is very similar to that previously described for macaque. In contrast to the blue cone bipolar cells, the DB3 cells form a regular mosaic. The synaptic connectivity of DB3 cells in the inner plexiform layer was analyzed. They make output synapses onto ganglion cells and amacrine cells, and gap junctions with each other. Our results provide further evidence for the existence of parallel bipolar cell pathways in the primate retina and support the view that the retinae of Old World and New World primates have common neuronal connectivity. The random distribution of SWS cones and blue cone bipolar cells is an exception to the general rule of a regular mosaic distribution of cell populations in the retina.

Antibodies directed against the calcium-binding proteins, parvalbumin and calbindin, can be used to label distinct neuronal subgroups in the primate visual pathway. We analyzed parvalbumin immunoreactivity (P-IR) and calbindin immunoreactivity (C-IR) in the lateral geniculate nucleus (LGN) and visual cortex of the marmoset, Callithrix jacchus. We compared marmosets which were identified as having dichromatic or trichromatic color vision. Within the LGN, the density of P-IR neurones is highest in the parvocellular and magnocellular laminae, but C-IR neurones are found mainly in the koniocellular division of the LGN, that is, the interlaminar zones and S laminae. Not all interlaminar zone cells are C-IR. In the visual cortex, P-IR neurones are present in all laminae except lamina 1, in areas V1 and V2. Neurones which are strongly C-IR are mainly located in laminae 2 and 3 in V1 and V2. Lightly C-IR neurones are concentrated in lamina 4, and are more numerous in V1 than in V2. Quantitative analysis showed no differences in the density or distribution of IR neurones in either LGN or visual cortex when dichromat and trichromat animals were compared. We conclude that this functional difference is not associated with differences in the neurochemistry of calcium-binding proteins in the primary visual pathways.