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Synapses of double bouquet cells in monkey cerebral cortex visualized by calbindin immunoreactivity.

In the monkey neocortex, immunoreactivity for the 28-kDa vitamin D-dependent calcium binding protein (Calbindin) is contained in a set of GABAergic intrinsic neurons whose small size and laminar locations render them very distinct from a second set of GABAergic intrinsic neurons that show immunoreactivity for another calcium-binding protein, parvalbumin. A conspicuous feature of many calbindin-immunoreactive cells is their possession of long, vertically oriented bundles of immunoreactive processes that descend or ascend vertically through several cortical layers. These are components of the radial fasciculi of the cortex and are here shown by correlative electron microscopic immunocytochemistry to consist of both immunoreactive dendrites and unmyelinated axons. The morphology of the bundles and the parent cells indicates that the cells are classical double bouquet cells. The calbindin-positive axons in the radial fasciculi in the present study formed symmetric synapses on unlabeled dendritic shafts (62%) and spines (38%). Despite the close-packed nature of the immunoreactive axons, relatively few terminals of the same axon converged on a single postsynaptic profile. The postsynaptic profiles were identified in certain cases as side branches of pyramidal cell apical and basal dendrites. Mainstem apical dendrites generally did not receive synapses derived from the calbindin-positive axons. These results indicate that double bouquet cells can be distinguished both by their GABAergic character and by their possession of calbindin immunoreactivity. They are probably major contributors to the vertical flow of inhibitory influences across laminae of the cerebral cortex.

Animals↗

Calbindin D-28k and choline acetyltransferase are expressed by different neuronal populations in pedunculopontine nucleus but not in nucleus basalis in squirrel monkeys.

Single- and double-immunostaining procedures were used to study the distribution of the acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) and the calcium binding protein calbindin D-28k in the nucleus basalis of Meynert (nbM) and in the pedunculopontine nucleus (PPN) of the squirrel monkey (Saimiri sciureus). As expected from previous studies in other primates, including humans, the nbM in the squirrel monkey is enriched with large ChAT-immunoreactive neurons that form clusters in the substantia innominata. Some ChAT-positive neurons are also scattered more dorsally within the internal and external medullary laminae of the pallidal complex. A smaller number of calbindin-immunoreactive cells occur in the same locations and their mean cross-sectional somatic area (424 microns 2) is not significantly different from that of the ChAT-immunoreactive cells (450 microns 2). Furthermore, 60% of the ChAT-immunopositive cells in the nbM display calbindin immunoreactivity. Most of these double-immunoreactive neurons occur in the typical clusters of the nbM, whereas the large neurons scattered in between the clusters display ChAT immunoreactivity only. In the PPN, ChAT-positive neurons are scattered around and partly within the superior cerebellar peduncle and also form a dense cluster in the lateral portion of the mesopontine tegmentum. Calbindin-immunoreactive cells also abound around the superior cerebellar peduncle, but they are more sparsely distributed and cover a larger sector of the tegmentum than the ChAT-positive neurons. These calbindin-immunoreactive cells are significantly smaller (200 microns 2) than the ChAT-immunoreactive cells (471 microns 2) and no double-immunostained neurons are present in the PPN.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Comparative distribution of calbindin and Met-enkephalin immunoreactivities in the guinea-pig lateral septum, with reference to electrophysiologically characterized neurons in the mediolateral part.

The distribution both of Met-enkephalinergic nerve terminals and of calbindin-containing neurons was investigated in the guinea-pig lateral septum, using a double-immunostaining technique. The findings show that the two immunoreactivities overlapped for neurons located in areas of the dorsal part and of the mediolateral part of the lateral septum. Nine cells of the mediolateral part which were electrophysiologically characterized and intracellularly labelled were subjected to the double-immunostaining protocol. All these cells displayed characteristic discharges due to the activation of high-threshold Ca2+ conductances. Two of them contained calbindin and were the target of enkephalinergic inputs; they possessed somatic spines. These data demonstrate: (1) that the guinea-pig lateral septum contains subpopulations of calbindin neurons which are postsynaptic to enkephalinergic inputs; (2) that Ca2+ conductances are not related to the presence of calbindin; (3) that somatospiny neurons, which are involved in the regulation of the hippocampo-septo-hypothalamic complex, contain calbindin and are the target of enkephalinergic endings.

Animals↗

Calbindin D-28k immunoreactivity in the rat accessory olfactory bulb.

The distribution pattern and the morphology of calbindin D-28k-immunoreactive neurons were studied in the accessory olfactory bulb of the rat using a monoclonal antibody and the avidin-biotin-immunoperoxidase method. Positive neurons were observed in all layers but the vomeronasal nerve layer. Scarce mono-dendritic periglomerular neurons were calbindin D-28k-immunoreactive. Different morphological types of short-axon cells were calbindin D-28k-immunostained, with different degrees of intensity, in the boundary between the internal and external plexiform layer. In addition, deep short-axon cells located in the granule cell layer were calbindin D-28k-immunopositive. By contrast, previous studies described all cells in the rat accessory olfactory bulb as calbindin D-28k-immunonegative. The staining pattern in the rat accessory olfactory bulb showed both similarities and differences with the distribution pattern of the same calcium-binding protein in the main olfactory bulb.

Animals↗

Responsiveness to ATP with an increase in intracellular free Ca2+ is not a distinctive feature of calbindin-D28 immunoreactive neurons in myenteric ganglia.

The aim of this study was to test the hypothesis that ATP elevates cytosolic free Ca2+ levels ([Ca2+]i) in myenteric neurons expressing the Ca2+ binding protein, calbindin-D28. A laser microbeam marked the location of cultured neurons on coverslips and provided unequivocal relocation of ATP-responsive neurons after immunocytochemistry. All myenteric multipolar neurons displayed ATP Ca2+ transients, and 42% also expressed calbindin-D28 reactivity. Statistical analysis of the kinetics and shape of ATP Ca2+ transients revealed no differences between calbindin and non-calbindin neurons. The identity of other responsive neurons is unknown. Less than 8% of ganglion cells with ATP Ca2+ transients were immunopositive for the glial protein S-100. We conclude that one of the actions of ATP in myenteric ganglia is to increase [Ca2+]i which may activate gKCa leading to membrane hyperpolarization in AH, Dogiel Type II neurons expressing calbindin-D28. An efficient buffering mechanism for handling large purinergic Ca2+ loads is a common feature of all types of myenteric ganglion cells.

Adenosine Triphosphate↗

Appearance and distribution of neuron-specific enolase and calbindin (CaBP 28 kDa) in the developing human inner ear.

The onset and development of neuron-specific enolase (NSE) and calbindin immunoreactivities were studied in the inner ear of human fetuses aged from 6-7 to 14 weeks of gestation. NSE occurred very early in ganglion neurons. Its appearance in vestibular sensory cells at 8 weeks coincided with the formation of the first afferent synapses, and showed an apex/base gradient in the cristae. Calbindin was found in vestibular ganglion neurons at 6-7 weeks and in the cochlear ganglion neurons at 8-9 weeks. Vestibular sensory cells and the whole ventral wall of the cochlear duct were stained from 8-9 weeks. At 14 weeks, calbindin staining occurred only in the sensory cells of the cochlear neuroepithelium. Non-neuronal secretory structures, i.e. Kölliker's organ and some cells of the transitional zone of the utricle, were also reactive. Staining appeared in Kölliker's organ with a base to apex gradient and disappeared from it with an internal to external gradient. Calbindin appeared in vestibular sensory cells later than NSE staining, synapse formation and sensory hair bundle differentiation. By contrast in the cochlea, calbindin staining appeared in the neuroepithelium before sensory cell differentiation, but remained only in the hair cells after they had differentiated and been contacted by the afferent fibers.

Antibodies↗

Transient expression of calbindin-D28k immunoreactivity in layer V pyramidal neurons during postnatal development of kitten cortical areas.

Calbindin-D28k is a 28 kDa calcium binding protein that has been shown to colocalize with a specific subpopulation of gamma-aminobutyric acid inhibitory interneurons in mammalian neocortex. We have examined the ontogeny of calbindin in neonatal kitten cortex in areas 17,18,19,7, medial and lateral suprasylvian visual areas, splenial visual area and cingulate cortex from the day of birth (P0) through maturation of the brain (P101). Transient staining of immature layer V pyramidal cells was seen in kittens six weeks old and younger. This transient staining of pyramidal cells was most intense and the stained neurons were most numerous in cingulate cortex. Apical dendrites of pyramidal cells in cingulate cortex were prominently stained and could be followed to layer I, where they were seen to branch extensively. There were very few calbindin immunoreactive pyramidal cells in primary cortical areas postnatally. Transient staining in extrastriate visual cortical areas disappeared first from the lateral suprasylvian areas, and persisted longest in area 7. Pyramidal neurons in the cingulate gyrus expressed calbindin longest, but calbindin expression by pyramidal neurons ceased by the sixth postnatal week in all areas of the brain.

Aging↗

Relationships between calbindin (Mr 28,000) and calcium transport by the eggshell gland.

1. Eggshell density (mg/cm2) and eggshell gland calbindin decreased in the aged hens. 2. Aged hens which laid eggs with a low shell weight and shell density had significantly lower intestinal and eggshell gland calbindin as compared with those which laid eggs with a high shell weight and shell density. 3. Significant correlations were found in aged hens between duodenal or eggshell calbindin and shell weight or shell density. 4. The results suggest that: (a) aged hens forming light shells absorbed calcium with a lower efficiency than those forming heavy shells or than young hens; (b) the decline in shell density in the aged hens is caused by a physiological calcium deficiency or by a defect in the hens' ability to alter calbindin synthesis in response to calcium needs; (c) in the aged hens, the deposition of calcium into the eggshell is dependent on, or at least associated with, eggshell gland calbindin.

Aging↗

Calbindin D-28K and parvalbumin immunoreactivity is confined to two separate neuronal subpopulations in the cat visual cortex, whereas partial coexistence is shown in the dorsal lateral geniculate nucleus.

Calbindin D-28K-immunoreactive cells were localized in the supragranular layers of the striate cortex of the cat, while parvalbumin-stained cells occurred from the bottom half of layer II through layer VI, making the two distributions almost complementary. Calbindin- and parvalbumin-positive cells occurred throughout the 3 layers of the dorsal lateral geniculate nucleus (dLGN), but calbindin-immunoreactive cells outnumbered parvalbumin-positive cells. Double labeling on single sections was performed in order to determine the possible coexistence of calbindin and parvalbumin in single cells of cat visual cortex and dLGN. Calbindin and parvalbumin immunoreactivity was found in two separate neuronal populations in the visual cortex, while in the dLGN about 50% of the cells were doubly stained.

Animals↗

Calbindin immunoreactivity in sensory and autonomic ganglia in the guinea pig.

Immunoreactivity (IR) for the calcium binding protein, calbindin, was localized in sensory ganglia (nodose, trigeminal and dorsal root), in parasympathetic ganglia (otic and sphenopalatine) in sympathetic chain ganglia and in sympathetic pre-vertebral ganglia of guinea pig. In sensory ganglia, fine nerve fibres with calbindin-IR surrounded the majority of cell bodies, a low proportion of which were themselves reactive. In cranial parasympathetic and in sympathetic chain ganglia, a small proportion of nerve cells was surrounded with baskets of calbindin-IR nerve fibres, but very few cell bodies were reactive. In prevertebral sympathetic ganglia, dense networks of terminals surrounded many cell bodies, but few somata were themselves reactive. In the coeliac and inferior mesenteric ganglia, the calbindin-IR nerve fibres surrounded somatostatin-IR cell bodies, but not those with neuropeptide Y-IR. It is concluded that specific subgroups of peripheral autonomic and sensory neurones have calbindin-IR.

Animals↗

Calbindin D-28k immunoreactivity in the rat cerebellar cortex: age-related changes.

The present study was designed to analyze age-dependent changes in the expression of calbindin D-28k immunoreactivity in the cerebellar cortex of male Wistar rats aged 3 months (young), 12 months (adult) and 24 months (old). Calbindin D-28k immunoreactivity was localized primarily in the cytoplasm of Purkinje neurons and in the basal portion of their dendritic processes entering in the molecular layer. The expression of calbindin D-28k immunoreactivity was highest in Purkinje neurons of adult rats and lowest in Purkinje neurons of old rats. Moreover, the number of Purkinje neurons displaying calbindin D-28k immunoreactivity was decreased in aged rats. These results demonstrate the occurrence of age-related-changes in the expression of immunoreactivity of the Ca2+ binding protein calbindin D-28k in the rat cerebellar cortex. The possibility that impairment in the expression of Ca2+ binding proteins may be involved in changes of intracellular Ca2+ homeostasis reported in aging and in some neuro-degenerative disorders is discussed.

Aging↗

Compartmentalization of calbindin and parvalbumin in different parts of rat rubrospinal neurons.

The distribution of calbindin-immunoreactive neurons in the red nucleus and the subcellular distribution of the calbindin and parvalbumin in tracer-identified rubrospinal neurons of the rat were studied. Only a fraction of the retrogradely labelled rubrospinal neurons was found to contain calbindin. These neurons filled the caudal part of the red nucleus and also appeared sporadically along the ventromedial border of the middle segment of the red nucleus. In addition to the somata, calbindin was found in the dendritic arbors of tracer-identified rubrospinal neurons, revealed by injecting the fluorescent dye Lucifer Yellow into their cell bodies. The axons of rubrospinal neurons located in the caudal red nucleus were marked by performing anterograde tracing with fluorescent dextran tracer in freshly prepared brainstem slices. Parvalbumin was found to locate in swellings along these tracer-identified axons as well as at their cut ends. The results indicate that calbindin and parvalbumin are segregated to the somadendritic and axonal compartments of the rat rubrospinal neurons, respectively. This anatomical segregation suggests that they may have different functions in neurons.

Animals↗

Glial cell line-derived neurotrophic factor protects striatal calbindin-immunoreactive neurons from excitotoxic damage.

The neostriatum is one of the areas with relatively high levels of glial cell line-derived neurotrophic factor (GDNF) messenger RNA expression in the developing and adult brain. GDNF expression in the neostriatum has been suggested to be involved in promoting the survival of nigral dopaminergic neurons, acting as a target-derived neurotrophic factor. However, GDNF messenger RNA expression in the striatum starts several days before dopaminergic and other afferent neurons reach the striatum, suggesting additional trophic effects of this factor on striatal neurons. In the present report, we have examined whether GDNF is able to prevent the degeneration of striatal calbindin- and parvalbumin-immunoreactive neurons in a lesion model of Huntington's disease. Fischer 344 rat 3T3 fibroblast cell line expressing high levels of GDNF (F3A-GDNF) was used to assess the protective effect of this factor, on striatal neurons, against excitotoxicity. Quinolinate (34 nmol) was injected at two different coordinates, and calbindin, parvalbumin and tyrosine hydroxylase immunoreactivity were examined seven days after lesion. Dopaminergic afferents were spared after quinolinate injection, but the number of calbindin- and parvalbumin-immunoreactive neurons was decreased. Interestingly, implantation of F3A-GDNF cells increased the density of tyrosine hydroxylase staining in the intact and also in the quinolinate-lesioned striatum. Furthermore, GDNF partially protected calbindin- but not parvalbumin-immunoreactive neurons from quinolinate excitotoxicity. Instead, mock-transfected fibroblasts did not affect any of these parameters. Our results show that GDNF specifically protects a subpopulation of striatal calbindin-immunoreactive neurons against quinolinate lesion, suggesting that GDNF administration may have a potential therapeutic application in the prevention and treatment of striatonigral degenerative disorders.

3T3 Cells↗

Changes in structure and stability of calbindin-D(28K) upon calcium binding.

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.

Animals↗

Modulating calbindin and parvalbumin immunoreactivity in the cochlear nucleus by moderate noise exposure in mice. . A quantitative study on the dorsal and posteroventral cochlear nucleus.

The number of calbindin D-28k and parvalbumin immunoreactive (IR) neurons were characterized on sections from the cochlear nucleus, dorsal cochlear nucleus (DCN) and posteroventral cochlear nucleus (PVCN) using two-dimensional quantification. After noise exposure (6-12 kHz, 2 h, at either 80 dB SPL or 103 dB SPL), the number of calbindin and parvalbumin immunoreactive neurons increased in CBA/CBA mice. Quantitative analysis of calbindin-IR in the PVCN did not show a statistically significant difference between any of the groups, whereas statistically significant differences in calbindin-IR were found in the DCN for the 103 dB and 80 dB group compared to the control group, and 103 dB compared to the 80 dB group, respectively. A statistically significant increase in the number of parvalbumin-IR neurons in the PVCN and the DCN was evident in the 103 dB and 80 dB group compared to the control group, and in the 103 dB compared to the 80 dB group. The data indicate that increasing sound stimulation causes a graded increase in the expression of calcium-binding protein immunoreactivity in the DCN and PVCN neurons and neuropil. This increase of protein expression is due to increased positive immunoreactivity in 'silent' neurons. These findings implicate that these neurons have the possibility to react against trauma and display calbindin or parvalbumin as a rescue event. The ability to map sound-induced calcium-binding protein changes in auditory neurons may be useful in future studies designed for detecting early patterns of neurodegeneration and neuroprotection in the central auditory pathway.

Acoustic Stimulation↗

Association of spinothalamic lamina I neurons and their ascending axons with calbindin-immunoreactivity in monkey and human.

The calbindin-immunoreactivity of spinothalamic (STT) lamina I neurons and their ascending axons was examined in two experiments. In the first experiment, lamina I STT neurons in macaque monkeys were double-labeled for calbindin and for retrogradely transported WGA*HRP following large (n=2) or small (n=1) injections that included the posterior thalamus. Most, but not all (78%) of the contralateral retrogradely labeled lamina I STT cells were positive for calbindin. Calbindin-immunoreactivity was not selectively associated with any particular anatomical type of lamina I STT cell; 82% of the fusiform cells, 78% of the pyramidal cells and 67% of the multipolar cells were double-labeled. In the second experiment, oblique transverse sections from upper cervical spinal segments of three macaque monkeys, one squirrel monkey and five humans were stained for calbindin-immunoreactivity. In each case, a distinct bundle of fibers was densely stained in the middle of the lateral funiculus. This matches the location of anterogradely labeled ascending lamina I axons observed in prior work in cats and monkeys, and it matches the location of the classically described 'lateral spinothalamic tract' in humans. This bundle had variable shape across cases, an observation that might have clinical significance. These findings support the view that lamina I STT neurons are involved in spinal cordotomies that reduce pain, temperature and itch sensations.

Animals↗

Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in areas of the brain, including the septal nucleus. Certain growth factors may promote the long-term survival of this subpopulation of neurons at risk. This study was undertaken to characterize growth factors' long-term effects on survival and development of neurons expressing the calcium-binding protein calbindin. In order to accomplish this, embryonic day 16 rat septal neurons were grown in bilaminar culture with astrocytes and in the absence of serum. These cultures were chronically treated with estrogen (Es), insulin-like growth factors I/II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Insulin-like growth factor II significantly increased the number of neurons immunoreactive for calbindin by 155%, suggesting either an increase in the survival of this subpopulation or an increase in the percentage of cells expressing calbindin. Chronic treatment with NGF, IGF-II, and Es significantly increased the number of primary neuritic processes on calbindin-positive neurons, whereas NGF and Es caused significant increases in the number of secondary processes and in the total lengths of the neuritic processes. Thus, effects of IGF-II, estrogen, and NGF on survival and maintenance of this neuronal subpopulation may be dependent on alterations in neurons which are immunopositive for calbindin.

Animals↗

Postnatal development of parvalbumin and calbindin D-28k immunoreactivities in the canine hippocampus.

The calcium-binding proteins, parvalbumin and calbindin D-28k, are markers of different classes of GABAergic interneurons and display different functions. The present study was attempted to determine immunoreactivities and colocalization of the parvalbumin and calbindin D-28k in the developing canine hippocampus by immunohistochemistry. The calcium-binding protein-containing neurons showed different developmental patterns. The first appearance of parvalbumin immunoreactive nonpyramidal cells was observed at P7. Parvalbumin immunoreactivity was elicited by the sequence from CA3 to CA1 to reach an adult-like distribution pattern, which was reached at P60, while calbindin D-28k immunoreactivity appeared from P0, including pyramidal and nonpyramidal cells. The characteristic distribution of calbindin D-28k immunoreactive pyramidal cells was clarified by P28, and an adult-like distribution pattern was reached by the end of the second postnatal month. Double-labeled nonpyramidal cells were frequently seen in the subareas, CA3 of P14/CA1-CA2 of P28, where parvalbumin immunoreactive nonpyramidal cells were emerging. These data suggest that the colocalization of the two calcium-binding proteins during development is related closely to the area-specific maturation of parvalbumin expression, although either prenatal expression of calbindin D-28k or parvalbumin was not determined.

Animals↗