Distribution Of calbindin-D28k Research Articles

Calbindin Immunoreactivity in Rat Cerebral Cortex and Cerebellum Neurons

Objective. To carry out an immunohistochemical assessment of the distribution of calbindin D28K in cerebral cortex (parietal and frontal) and cerebellum neurons in rats.Materials and methods. Experiments were performed on 18 mongrel white male rats weighing 200–250 g. Fragments of the frontal and parietal cortex and cerebellar cortex were collected. Paraffi n sections were processed using primary polyclonal antibodies to detect calbindin D28K immunoreactivity.Results. In layer II of the frontal and parietal cortex, most neurons were moderately immunopositive, while layers III, V, and VI contained occasional neurons with high calbindin immunoreactivity. Individual nerve fibers, mainly the dendrites of pyramidal neurons, were clearly seen. Some of these showed a distinctive staining pattern: immunopositive parts alternated with immunonegative parts. In the cerebellar cortex, the bodies and dendritic branches of Purkinje cells were well stained. A significant proportion of neurons in the granular layer were moderately immunopositive, and included some afferent nerve fibers running from the white matter.Conclusions. Calbindin immunoreactivity in cerebral cortex structures in normal rats varied significantly and could be related both to the type of neuron and their functional state.

The distribution of calbindinD-28k and parvalbumin immunoreactive neurons in the somatosensory area of the pigeon pallium.

GABAergic interneurons regulate the degree of glutamatergic excitation and output of projection neurons. In this study, we investigated the distribution of calbindinD-28k (CB) and parvalbumin (PV) in the somatosensory area of the pigeon pallium using immunohistochemical method. Our results show that anatomical structures of the somatosensory area of the pigeon pallium consisted of several subdivisions including the hyperpallium, intercalated hyperpallium, mesopallium, nidopallium and basorostralis. Neuronal density was significantly higher in the intercalated hyperpallium and basorostralis than that in the other subdivisions. The density of the CB immunoreactive neurons was generally similar in all the subdivisions; however, the density of PV immunoreactive neurons was particularly prominent in the basorostralis compared with that in the other subdivisions. In addition, the mean proportion of PV immunoreactive neurons to total neurons was higher than that in the CB immunoreactive neurons in all the subdivisions. In brief, our present study shows that PV immunoreactive neurons in the somatosensory area of the pigeon pallium were significantly abundant compared with CB immunoreactive neurons. This finding needs more studies regarding CB- and PV-related functions in the somatosensory area of the avian pallium.

Calbindin‐D28K Immunoreactivity in the Human Cerebellar Cortex

Calbindin-D28k (CB) is a calcium-binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer-specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed.

Expression of the calcium-binding proteins in the central, medial and cortical nuclei of the rabbit amygdaloid complex during postnatal development.

Calbindin-D28k (CB), parvalbumin (PV) and calretinin (CR) are calcium-binding proteins (CaBPs) considered to be markers for certain subpopulations of neurons in the central nervous system. The aim of this study was to describe the pattern of distribution of CB-, PV- and CR-immunoreactive elements in the rabbit corticomedial amygdaloid complex during the postnatal period. The time course of changes in CaBPs expression during maturation of the selected nuclei indicates their diversity. During the first month after birth, CaBPs expression stabilizes earliest in the anterior cortical and then in the medial nuclei. Later, during the second month of postnatal life, the posteromedial and posterolateral cortical nuclei maturate. The central nucleus requires a considerably longer time to reach maturity - about three months are needed to stabilize CaBPs expression in all its subdivisions. This nucleus also shows the most differentiated, time-dependent distribution of CaBPs immunoreactivity (especially CB), distinct in its divisions. The differences in the CaBPs immunoreactivity confirm previous reports concerning dissimilar origin and development, and also reflect the diversity of connectivity of the amygdaloid body - the collection of nuclei, considered as one functional integrity.

Regulation of Calbindin-D28k Expression by Msx2 in the Dental Epithelium

Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.

Changes in the distribution of calbindin D28-k, parvalbumin, and calretinin in the hippocampus of the circling mouse

The circling (cir) mouse strain, a murine model of deafness caused by a spontaneous mutation, exhibits characteristic behaviors of circling and hyperactivity. In an induced-noise paradigm, cir mice display a significant loss in their spatial orientation abilities, and this has been suggested to be due at least in part to changes in calcium homeostasis. Auditory information is transferred from the cochlear nucleus to the hippocampus, where it is processed to modulate motor and sensory activity. Such a pathway could be affected at the cellular level by alterations in neurotransmission, including alterations that involve Ca2+. However, there have been no studies in a hearing deficit model examining the concomitant molecular alterations in the hippocampus. Thus, in the present study we used immunohistochemistry to compare the distribution of the calcium-binding proteins (CaBPs) calbindin D-28k, parvalbumin, and calretinin in the hippocampi of heterozygous (+/cir), homozygous (cir/cir), and wild-type (+/+) mice. The expression of the CaBPs in various hippocampal subfields appeared to be significantly lower in cir mice (+/− and −/−) than in +/+ mice. Such a decrease in CaBP expression in cir/cir mice would alter calcium homeostasis, which in turn could affect the connection of the tri-synaptic circuit of the hippocampus as well as the cortical region. A decrease in CaBPs and the probable resultant glutamate-mediated excitability could contribute to the functional changes that lead to the characteristic behavioral features of cir mice.

Calcium-binding proteins and GFAP immunoreactivity alterations in murine hippocampus after 1 month of exposure to 835 MHz radiofrequency at SAR values of 1.6 and 4.0 W/kg

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

Immunohistochemical localization of calbindin D28k and calretinin in the retina of two lungfishes, Protopterus dolloi and Neoceratodus forsteri: Colocalization with choline acetyltransferase and tyrosine hydroxylase

There are no previous studies of the distribution of calbindin-D28k (CB) and calretinin (CR) in retinal neurons in lungfishes. The present study aimed to characterize the distribution of these neurons in a lepidosirenid lungfish ( Protopterus dolloi) and a ceratodontid lungfish ( Neoceratodus forsteri) using the same immunohistochemical protocol, with specific poly- and monoclonal antibodies, used in a previous comparative study of amphibians. The pattern for both proteins was similar in the two lungfishes studied, but some differences were also found. CB was found in photoreceptors, in subpopulations of bipolar and amacrine cells, and in horizontal and ganglion cells. Photoreceptors were negative for CR, whereas subpopulations of horizontal, bipolar, and amacrine cells contained CR, as did cells in the ganglion cell layer. Colocalization of CB/CR occurred in some cells of the inner nuclear layer. Tyrosine hydroxylase (TH) immunoreactivity colocalized with CB in some amacrine cells and, less frequently, with CR, but colocalization of TH with CB or CR in cholinergic cells was never found. Our data suggest a specific pattern for CB and CR distribution in the retinal neurons of lungfishes, a pattern that is highly comparable to that seen in amphibians, with unique features not observed in other vertebrates.

Calcium-binding protein immunoreactivity characterizes the auditory system of Gekko gecko.

Geckos use vocalizations for intraspecific communication, but little is known about the organization of their central auditory system. We therefore used antibodies against the calcium-binding proteins calretinin (CR), parvalbumin (PV), and calbindin-D28k (CB) to characterize the gecko auditory system. We also examined expression of both glutamic acid decarboxlase (GAD) and synaptic vesicle protein (SV2). Western blots showed that these antibodies are specific to gecko brain. All three calcium-binding proteins were expressed in the auditory nerve, and CR immunoreactivity labeled the first-order nuclei and delineated the terminal fields associated with the ascending projections from the first-order auditory nuclei. PV expression characterized the superior olivary nuclei, whereas GAD immunoreactivity characterized many neurons in the nucleus of the lateral lemniscus and some neurons in the torus semicircularis. In the auditory midbrain, the distribution of CR, PV, and CB characterized divisions within the central nucleus of the torus semicircularis. All three calcium-binding proteins were expressed in nucleus medialis of the thalamus. These expression patterns are similar to those described for other vertebrates.

Immunohistochemical localization of calbindin-D28k and calretinin in the brainstem of anuran and urodele amphibians

Calbindin-D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB- and CR-immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident. J. Comp. Neurol. 515:503–537, 2009. © 2009 Wiley-Liss, Inc.

Immunohistochemical localization of calbindin‐D28k and calretinin in the brainstem of anuran and urodele amphibians

Calbindin-D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB- and CR-immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident.

The Effect of 835 MHz Radiofrequency Radiation Exposure on the Immunohistochemical Distribution of Calbindin D28k and Calretinin in the Mouse Cerebellar Cortex

Widespread use of mobile phones and subsequent electromagnetic field (EMF) exposure have raised crucial question of their possible biological effects on the nervous system. The study on the effect of radiofrequency (RF) radiation on the nervous system, however, did not precede enough to determine the biological hazard to brain. Until now, several studies have reported decreases in neuron number and neuronal damage in the cortex, hippocampus, and basal ganglia in the brains of animals exposed to RF radiation. However, there were few reports about the cerebellum, the main voluntary motor control center. In this regard, by using immunohistochemisty, current study intended to investigate the changes in the calbindin D28k (CB) and calretinin (CR)-immunoreactivity (IR) in the mouse cerebellar cortex after EMF exposure at 835 ㎒ for different exposure times and absorption rates, 1h/day for 5 days at 1.6 W /㎏, 1 h/day for 5 days at 4.0 W /㎏, 5 h/day for 1 day at 1.6 W /㎏, 5 h/day for 1 day at 4.0 W /㎏, daily exposure for one month at 1.6 W /㎏. Among groups, most prominent CB IR was observed in the Purkinje cell layer followed by molecular and granular layer. The highest CB IR was noted in 5 h/day for 1 day at 1.6 W /㎏ in the entire three layers while the lowest was noted in one month at 1.6 W /㎏. Similarly CR IR was maximum in one month at 1.6 W /㎏ whilst the lowest was observed in 1 h/day for 5 days at 4.0W/㎏. EMF exposure for 5 days at 1.6 W/㎏ reduced CB-IR. The CR-IR was mainly localized in small cells in the granular layer, with maximum IR observed after one month exposure. Therefore, the present study suggest the possibility of alterations of calcium ion concentration, which playa role in maintaining metabolic homeostasis, in the cerebellum after long-term exposure to 835 ㎒ of RF radiation, which might lead to the disruption of normal trait.

Comparative analysis of calbindin D-28K and calretinin in the retina of anuran and urodele amphibians: Colocalization with choline acetyltransferase and tyrosine hydroxylase

Previous studies in amphibians yielded contradictory results about the distribution of calbindin-D28k (CB) and calretinin (CR) in retinal neurons, most likely due to the different antibodies used. The present comparative study aimed to characterize the distribution of CB and CR in relation to retinal neurons in six species of anuran and urodele amphibians by using the same immunohistochemical protocol with specific poly- and monoclonal antibodies. CB was specifically found in cones, in subpopulations of bipolar and amacrine cells and in sparse neurons in the ganglion cell layer. All photoreceptors were negative for CR, whereas subpopulations of horizontal, bipolar and amacrine cells as well as cells in the ganglion cell layer contained CR. CB/CR colocalization occurred in some amacrine cells and in cells of the ganglion cell layer, with variable proportions among species. Most of the ganglion cells identified by retrograde labeling from the optic nerve contained CB and/or CR. Cholinergic cells, visualized by choline acetyltransferase (ChAT) immunoreactivity, constituted a subpopulation of the CR-positive amacrine cells in anurans and a high percentage (40-90%) of cholinergic cells were CR immunoreactive in urodeles. CB/ChAT colocalization was between 10 and 30% in anurans and lower in urodeles (7-10%). Finally, CB colocalized in 6-8% of the tyrosine hydroxylase (TH) amacrine cells only in anurans, whereas CR and TH colocalized in 5% of TH cells in the urodele retina. Our data suggest a specific pattern for CB and CR distribution in the retinal neurons of amphibians comparable to amniotes in some cell types showing, however, peculiar features not observed previously in other vertebrates.

Distribution of calbindin-D28k, neuronal nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the lateral nucleus of the sheep amygdaloid complex

This study describes calbindin-D28k (CB), neuronal nitric oxide synthase (nNOS), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) expression in the lateral nucleus of the sheep amygdaloid complex. Double immunofluorescence protocol was used in order to determine whether there is colocalization of CB and nNOS. The CB-immunoreactive (IR) neuronal population was composed especially of non-pyramidal neurons, but a few pyramidal cells were also present. The non-pyramidal neurons showed a multipolar and, occasionally, a fusiform morphology. The comparison between single-labeled CB-IR non-pyramidal neurons and cells belonging to CB-IR neuronal population showed they were identical for morphology, mean size, and distribution. The single-labeled CB-IR non-pyramidal neurons were only the 17.8% of the total non-pyramidal neurons counted. The nNOS-IR neuronal population was represented by non-pyramidal multipolar and fusiform neurons. Single-labeled nNOS-IR non-pyramidal neurons had the same morphology, mean area, and distribution as cells belonging to nNOS-IR neuronal population. Single-labeled nNOS-IR non-pyramidal neurons were more numerous than single-labeled CB-IR, and represented the 73.7% of total non-pyramidal neurons counted. NADPH-d-positive cells had the same morphology and distribution as the nNOS-IR neurons. Double immunolabeling (CB/nNOS) was found mostly in non-pyramidal multipolar neurons and only in a few non-pyramidal fusiform cells. These neurons had a mean perikaryal area significantly higher and significantly smaller than that of single-labeled nNOS and single-labeled CB-IR non-pyramidal neurons, respectively. CB and nNOS coexist only in a minority of non-pyramidal neurons (8.5%). The 32.4% of all CB-IR non-pyramidal neurons were nNOS-positive; only 10.4% of nNOS-IR non-pyramidal neurons were CB-positive. These results indicate that CB and nNOS are expressed by selective neurons and that the majority of nNOS-IR non-pyramidal neurons are lacking in CB.

Distribution of Calbindin D-28k in Odontoblasts after Cavity Preparation in Human Teeth

Distribution of Calbindin D-28k in Odontoblasts after Cavity Preparation in Human Teeth

Calbindin D-28k distribution in odontoblasts underneath tertiary dentine in human carious teeth

Objective: The aim of this study was to examine the calbindin D-28k immunoreactivity in carious teeth to know whether this protein may have a function in tertiary dentine formation. Methods: Human extracted teeth with or without carious lesions were immersion-fixed with Zamboni fixative, demineralized in 4.13% EDTA solution (pH 7.4), frozen-sectioned, and processed for calbindin immunoreactivity and hematoxylin-eosin stain. The intensity of the immunostaining was evaluated by quantitative densitometry. Results: In intact teeth, numerous odontoblasts were aligned underneath the secondary dentine and their cell bodies showed the immunoreactivity. In carious teeth, tertiary dentine had poor- or rich tubular patterns under the carious lesion. Underneath the tubule-poor tertiary dentine, distinct odontoblasts could not be seen at the central site. However, some cells with a flat appearance were located at this site and were immunonegative for calbindin D-28k. On the other hand, columnar odontoblasts were seen at the peripheral site, and their cell bodies and processes showed strong immunoreactivity. Underneath the tubule-rich tertiary dentine, columnar odontoblasts were abundantly distributed, and the strong immunoreactivity was observed in their cell bodies and processes. The immunoreactivity in odontoblasts underneath the tertiary dentine with poor or rich tubular pattern was more intense than that for the secondary dentine in intact teeth ( P<0.05). On the other hand, the intensity of the immunoreactivity in odontoblasts was similar underneath the secondary dentine in intact and carious teeth. Conclusions: The present study demonstrated that calbidin D-28k was actively synthesised by odontoblasts under the carious lesion. These findings may suggest that this protein plays an important role in the tertiary dentine formation.

Differential distribution of calbindin D28k and parvalbumin among functionally distinctive sets of structures in the macaque brainstem.

In a study of brainstem in the cynomolgus monkey, we found that the distribution of calbindin D28K (CB) and parvalbumin (PV) is nonoverlapping among functionally distinct sets of brainstem structures. Nuclei involved in representation and regulation of the organism's internal state contain CB, whereas those involved in the representation of the external environment and the representation or execution of externally directed actions contain only PV. Moreover, our findings indicate that different nuclei known as components of the ascending reticular activating system (ARAS) contain either CB or PV or both, suggesting that this system in primates operates with both CB and PV. In line with previously reported findings, we also found that unmyelinated pathways contain only CB, whereas myelinated pathways contain PV. Distribution of CB and PV in the macaque brainstem follows a pattern comparable to, but in some instances significantly different than, the pattern previously reported in the rat. We argue that the nonoverlapping distribution of CB and PV among different structures of the brainstem might reflect underlying differences in the physiological, anatomic, and perhaps phylogenetic properties of these structures. Considering our recent findings of selective vulnerability of brainstem structures to Alzheimer's disease, the present data suggest that the majority of macaque brainstem nuclei that contain CB are vulnerable to neurofibrillary tangles in humans. By contrast, only few nuclei that contain PV exhibit pathologic changes. Some of these nuclei are affected with a high number of neuritic plaques without ever developing neurofibrillary tangles.

Calcification processes in the chick otoconia and calcium binding proteins: patterns of tetracycline incorporation and calbindin-D28K distribution

In order to clarify the otoconia formation and turnover, tetracycline, an antibiotic that precipitates at calcifying fronts and serves as a fluorescent marker, was injected into eggs at different stages of chick embryonic development, as well as into postnatal chicken and into adult animals. The changes in the intensity, location patterns and time course of fluorescent labelling in each examined stage in the otolithic organs was studied. The presence and distribution of calbindin (CB)-D28K, one of the calcium-binding proteins constantly found in the mammalian and chicken cochlea and also in otolithic membrane of some adult mammals, was studied. Results in embryonal stages, postnatal and adult animals allow us to postulate that otoliths are mainly produced during the embryonal phase, but they may also be produced throughout the whole life span. Results also indicate that otoconia are dynamic structures which undergo turnover. The correspondence between the patterns of CB-D28K immunoreactivity and tetracycline fluorescence may indicate that CB-D28K participates in the formation of otoconia.

Hypoxia-Induced Subcellular Distribution of Calbindin-D28k in the Cerebellum of the Term Guinea Pig Fetus

Hypoxia-Induced Subcellular Distribution of Calbindin-D 28k in the Cerebellum of the Term Guinea Pig Fetus

Molecular aspects of tibial dyschondoplasia in the chicken: I. Expression of calbindin-D 28k gene

Avian tibial dyschondroplasia (TD) is a skeletal disorder characterized by a cartilage plug in the proximal metaphysis of the tibiotarsus. Experiments were carried out to investigate whether the expression of calbindin-D 28k gene is altered in this disease or in the presence of vitamin D metabolites. The intestinal mRNA levels for calbindin-D 28k in normal and TD chicks were determined by northern blot analysis, and the distribution of calbindin-D 28k in the normal growth plate (LTD) and tibial dyschondroplastic cartilage of chicks were examined immunocytochemically. There were no differences in the intestinal calbindin-D 28k mRNA levels between LTD and TD chicks fed diets sufficient in vitamin D 3. However, reduced calbindin-D 28k mRNA levels were found in TD chicks fed a diet supplemented with 10 μg/kg 1,25 (OH) 2D 3 as compared to LTD chicks. Calbindin-D 28k was distributed throughout the growth plate, but concentrated in the hypertrophic zones. Depletion of vitamin D resulted in the absence of calbindin-D 28k in rachitic growth plates. These findings suggest that calbindin-D 28k is vitamin D dependent in this tissue. This protein was also found to be present in the TD- associated epiphyseal growth plate, but appeared to be less intensely concentrated than that in LTD growth plate. The proximal part of the dyschondroplastic cartilage was lightly stained, and no evidence of calbindin was found in the middle and distal parts of the lesion. These results suggest that decreased calbindin-D 28k gene expression is observed in the TD lesion.