Taurine treatment inhibits CaMKII activity and modulates the presence of calbindin D28k,calretinin, and parvalbumin in the brain

Taurine, 2‐aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems and has several biological functions. In addition, it has been related to a neuroprotective role against several diseases such as epilepsy. In the present work, we treated mice with taurine and examined its effects on the expression of proteins in the hippocampus associated with calcium regulation. Taurine treatment alters the presence of calbindin‐D28k, calretinin, and parvalbumin in the brain, mainly in the hippocampus. It also reduced CaMKII activity, indicating that taurine could alter calcium signaling pathways. However, the activity of calpain, a protease related to apoptosis induced by calcium signalling, did not change. The concentration of taurine in the hippocampus was also unaffected by the treatment. These results provide new insight into the role of taurine in calcium homeostasis. © 2009 Wiley‐Liss, Inc.     

The role of parvalbumin and calbindin D28k in experimental scrapie

Aims: Prion diseases are generally characterized by pronounced neuronal loss. In particular, a subpopulation of inhibitory neurones, characterized by the expression of the calcium-binding protein parvalbumin (PV), is selectively destroyed early in the course of human and experimental prion diseases. By contrast, nerve cells expressing calbindin D28k (CB), another calcium-binding protein, as well as PV/CB coexpressing Purkinje cells, are well preserved. Methods: To evaluate, if PV and CB may directly contribute to neuronal vulnerability or resistance against nerve cell death, respectively, we inoculated PV- and CB-deficient mice, and corresponding controls, with 139A scrapie and compared them with regard to incubation times and histological lesion profiles. Results: While survival times were slightly but significantly diminished in CB–/–, but not PV–/– mice, scrapie lesion profiles did not differ between knockout mice and controls. There was a highly significant and selective loss of isolectin B₄-decorated perineuronal nets (which specifically demarcate the extracellular matrix surrounding the 'PV-expressing' subpopulation of cortical interneurones) in scrapie inoculated PV+/+, as well as PV–/– mice. Purkinje cell numbers were not different in CB+/+ and CB–/– mice. Conclusions: Our results suggest that PV expression is a surrogate marker for neurones highly vulnerable in prion diseases, but that the death of these neurones is unrelated to PV expression and thus based on a still unknown pathomechanism. Further studies including the inoculation of mice ectopically (over)expressing CB are necessary to determine whether the shortened survival of CB–/– mice is indeed due to a neuroprotective effect of this molecule.     

Localization of parvalbumin,calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15–20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions. J. Comp. Neurol. 390:377–391, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Deafferentation removes calretinin immunopositive terminals,but does not induce degeneration of calbindin D-28k and parvalbumin expressing neurons in the hippocampus of adult rats

Unilateral combined transections of the fimbriafornix and angular bundle in adult Fischer 344 rats were used to study the effects of deafferentation on hippocampal expression of calretinin, calbindin D-28k, and parvalbumin. Reflecting the widespread degeneration of synaptic contacts, immunostaining for glial fibrillary acidic protein 6 days after the lesions was increased in lacunosum-molecular and oriens layers of CA1, 2, and 3 in ipsi- and contralateral hippocampus and in the ipsilateral dentate gyrus outer molecular layer. At 21 days the immunoreactivity had decreased to control levels except for a still slightly increased signal in the oriens layer of CA1-3. At 6 and 21 days after the combined lesions the numbers of hippocampal neurons containing calretinin, parvalbumin, and calbindin D-28k was unaltered. The combined lesions abolished calretinin containing terminals in the dentate gyrus inner molecular layer on the deafferentated side. This could be reproduced by single unilateral fimbria-fornix transections, suggesting that the axons of these calretinin positive terminals project to the hippocampus through the fimbria-fornix. The most likely origin of the calretinin positive terminals are neurons in the supramammillary hypothalamic nucleus. Our findings demonstrate that the extensive lesion-induced synaptic rearrangements in the adult hippocampus do not induce degeneration of hippocampal neurons expressing calretinin, calbindin D-28k, and parvalbumin, but do remove calretinin containing terminals which reach their targets in the hippocampus through the fimbria-fornix. © 1994 Wiley-Liss, Inc.     

Local circuit neurons immunoreactive for calretinin,calbindin D-28k or parvalbumin in monkey prefronatal cortex: Distribution and morphology

In the cerebral cortex, local circuit neurons provide critical inhibitory control over the activity of pyramidal neurons, the major class of excitatory efferent cortical cells. The calciumbinding proteins, calretinin, calbindin, and parvalbumin, are expressed in a variety of cortical local circuit neurons. However, in the primate prefrontal cortex, relatively little is known, especially with regard to calretinin, about the specific classes or distribution of local circuit neurons that contain these calcium-binding proteins. In this study, we used immunohistochemical techniques to characterize and compare the morphological features and distribution in macaque monkey prefrontal cortex of local circuit neurons that contain each of these calcium-binding proteins. On the basis of the axonal features of the labeled neurons, and correlations with previous Golgi studies, calretinin appeared to be present in double-bouquet neurons, calbindin in neurogliaform neurons and Martinotti cells, and parvalbumin in chandelier and wide arbor (basket) neurons. Calretinin was also found in other cell populations, such as a distinctive group of large neurons in the infragranular layers, but it was not possible to assign these neurons to a known cell class. In addition, although the animals studied were adults, immunoreactivity for both calretinin and calbindin was found in Cajal-Retzius neurons of layer I. Dual labeling studies confirmed that with the exception of the Cajal-Retzius neurons, each calcium-binding protein was expressed in separate populations of prefrontal cortical neurons. Comparisons of the laminar distributions of the labeled neurons also indicated that these calcium-binding proteins were segregated into discrete neuronal populations. Calretinin-positive neurons were present in greatest density in deep layer I and layer II, calbindin-immuno-reactive cells were most dense in layers II-superficial III, and parvalbumin-containing neurons were present in greatest density in the middle cortical layers. In addition, the relative density of calretinin-labeled neurons was approximately twice that of the calbindin- and parvalbumin-positive neurons. However, within each group of labeled neurons, their laminar distribution and relative density did not differ substantially across regions of the prefrontal cortex. These findings demonstrate that calretinin, calbindin, and parvalbumin are markers of separate populations of local circuit neurons in monkey prefrontal cortex, and that they may be useful tools in unraveling the intrinsic inhibitory circuitry of the primate prefrontal cortex in both normal and disease states.     

Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus.

The possible coexistence of calbindin D28k with parvalbumin and of calbindin D28k with cholecystokinin was studied in nonpyramidal cells of the rat dorsal hippocampal formation. Neighbouring Vibratome sections were immunostained either for calbindin D28k and parvalbumin or for calbindin D28k and cholecystokinin. The cells, halved during sectioning, were identified in both sections immunostained for different antigens. The coexistence of calbindin D28k and parvalbumin in the same neuron was rare throughout the hippocampal formation with the exception of stratum oriens of the CA1 region, where 9.6% of the parvalbumin-immunoreactive cells also contained calbindin D28k. In stratum radiatum of the CA3 region, calbindin D28k and cholecystokinin coexisted in 12.5% and 21.2% of the calbindin D28k and cholecystokinin-immunoreactive cells, respectively. In other regions of the hippocampal formation, the two markers coexisted in less than 5% of the cells of either type. The present results demonstrate that calbindin D28k-, parvalbumin- and cholecystokinin-containing nonpyramidal cells represent largely nonoverlapping cell populations and may thus be involved in different inhibitory circuits.     

Parvalbumin,calbindin D-28k,and calretinin immunoreactivity in the ascending auditory pathway of horseshoe bats

In the subcortical auditory system of Rhinolophus rouxi, antibodies directed against the calcium-binding proteins parvalbumin, calbindin D-28k, and calretinin yield partly overlapping and partly complementary labeling patterns which are described in detail for each nucleus. The most general features of the labeling patterns are that: (1) Parvalbumin is a potent marker for large and heterogenous populations of cells and puncta (presumed axon terminals) throughout the auditory pathway. (2) Immunostaining with the monoclonal calbindin-antiserum was typically absent or sparse in most auditory brainstem centers, but prominent in auditory nerve fibers and in cells of the medial geniculate body (MGB). (3) Calretinin label is abundant but more restricted to subsets of auditory nuclei or subpopulations of cells than parvalbumin. (4) Calcium-binding proteins are useful markers to define particular subregions or cell types in auditory nuclei: for example, (i) different labeling patterns are obtained within the nuclei of the lateral lemniscus and adjacent tegmental zones; (ii) in the inferior colliculus both calbindin- and calretinin-antisera yield similar regional specific staining patterns, but label different cell types; (iii) subregions of the medial geniculate body have characteristic profiles of calcium-binding proteins; and (iv) analyses of different nuclei showed that there is no simple common denominator for cells characterized by the expression of particular calcium-binding proteins, nor does labeling correspond in a straightforward way with specific functional systems. (5) there are profound differences between the calbindin labeling patterns seen in Rhinolophus and those in other mammals.     

Distribution of GABAergic interneurons immunoreactive for calretinin, calbindin D28K, and parvalbumin in the cerebral cortex of the lizard Podarcis hispanica.

The types and distribution of cells containing three calcium-binding proteins, calretinin, calbindin D28K, and parvalbumin, have been studied by immunocytochemistry in different areas of the cerebral cortex of lizards. Cross-reactivity of the antisera has been excluded by demonstrating the existence of several cell groups immunoreactive for one but not the other two calcium-binding proteins. In the dorsal and dorsomedial cortices all three proteins coexist in a single subpopulation of gamma-aminobutyric acid (GABA)ergic neurons, the terminals of which form pericellular baskets around cell bodies of bipyramidal neurons. The somata of these neurons are largely restricted to the cellular and inner plexiform layers, but the dendrites usually penetrate all layers, allowing the neurons to sample input from all possible sources. A small number of parvalbumin-containing neurons in the outer plexiform layer do not contain the other two proteins. The medial cortex, which is likely to be homologous to the mammalian dentate gyrus, only contains parvalbumin-immunoreactive neurons. The dendritic trees of these cells appear to avoid the Timm-positive fields receiving input from zinc-rich fiber collaterals, originating from principal cells. The lateral cortex contains calbindin D28K-immunoreactive GABAergic neurons, which lack the other two calcium-binding proteins. These neurons have horizontally running dendrites in the outer plexiform layer, but their axon terminals could not be visualized. The present study uncovered important similarities and differences between the lizard and the mammalian archicortex in the types of neurons containing calcium-binding proteins. As in mammals, different cell types evolved in the lizard to inhibit the perisomatic versus the distal dendritic region of principal cells, the calcium-binding protein-containing neurons being responsible for the former, and neuropeptide-containing neurons for the latter. The results also suggest that further neurochemical diversion of GABAergic interneurons coupled to a functional specialization took place during phylogenetic development from reptiles to mammals.     

Alterations in Purkinje cell spines of calbindin D-28 k and parvalbumin knock-out mice

The second messenger Ca2+ is known to act in a broad spectrum of fundamental cell processes, including modifications of cell shape and motility, through the intermediary of intracellular calcium-binding proteins. The possible impact of the lack of the intracellular soluble Ca2+-binding proteins parvalbumin (PV) and calbindin D-28 k (CB) was tested on spine morphology and topology in Purkinje cell dendrites of genetically modified mice. Three different genotypes were studied, i.e. PV or CB single knock-out (PV-/-, CB-/-) and PV and CB double knock-out mice (PV-/-CB-/-). Purkinje cells were microinjected with Lucifer Yellow and terminal dendrites scanned at high resolution with a confocal laser microscope followed by three-dimensional (3-D) reconstruction. The absence of PV had no significant effect on spine morphology, whereas the absence of CB resulted in a slight increase of various spine parameters, most notably spine length. In double knock-out mice, the absence of both PV and CB entailed a doubling of spine length, an increase in spine volume and spine surface, a higher spine density along the dendrites, as well as a more clustered spine distribution. In all three genotypes, a reduction in the number of stubby spines was observed compared with wild-type animals. These results suggest a morphological compensation for the lack of the soluble calcium buffers in the cytoplasm of Purkinje cell dendritic spines. The increase in various spine parameters, particularly volume, may counteract the lack of the calcium buffers, such as to adjust Ca2+-transients at the transitional zone between spines and dendrites.     

Colocalization of calbindin D-28k,calretinin, and GABA immunoreactivities in neurons of the human temporal cortex

The calcium-binding proteins calbindin D-28k (CalB) and calretinin (CalR) have been shown to be useful markers of neuronal subpopulations located mainly in layers II–III of the neocortex of a variety of species, including human. Double labeling immunocytochemical studies of CalB, CalR, and GABA in experimental animals have shown that CalB and CalR are present in separate subpopulations of neurons. However, there are no studies of colocalization of these calcium-binding proteins and GABA in the human neocortex. The principal goal of the present work was to investigate the degree of colocalization of these substances in layers II–III of the human temporal neocortex, using a postembedding immunocytochemical method. The patterns of staining for CalB, CalR, and GABA in the human cortex were similar to those found in monkey neocortex. However, the degree of colocalization for certain combinations was different from that reported in the monkey and other experimental animals. A relatively large proportion of CalB- and CalR-immunoreactive cells (approximately 71% and 74%, respectively) were found to be immunoreactive for GABA. However, the degree of colocalization of CalB with CalR was low (between 4% and 6%). Thus, our quantitative and qualitative data suggest that these calcium-binding proteins are present in similar cortical circuits in all primates, but that in the human neocortex, there might be additional GABAergic and perhaps also non-GABAergic interneurons with unique chemical characteristics. © 1996 Wiley-Liss, Inc.     

'New' functions for 'old' proteins: the role of the calcium-binding proteins calbindin D-28k,calretinin and parvalbumin,in cerebellar physiology. Studies with knockout mice

Calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) belong to the large family of EF-hand calcium-binding proteins, which comprises more than 200 members in man. Structurally these proteins are characterized by the presence of a variable number of evolutionary well-conserved helix-loop-helix motives, which bind Ca2+ ions with high affinity. Functionally, they fall into two groups: by interaction with target proteins, calcium sensors translate calcium concentrations into signaling cascades, whereas calcium buffers are thought to modify the spatiotemporal aspects of calcium transients. Although CR, CB and PV are currently being considered calcium buffers, this may change as we learn more about their biology. Remarkable differences in their biophysical properties have led to the distinction of fast and slow buffers and suggested functional specificity of individual calcium buffers. Evaluation of the physiological roles of CR, CB and PV has been facilitated by the recent generation of mouse strains deficient in these proteins. Here, we review the biology of these calcium-binding proteins with distinct reference to the cerebellum, since they are particularly enriched in specific cerebellar neurons. CR is principally expressed in granule cells and their parallel fibres, while PV and CB are present throughout the axon, soma, dendrites and spines of Purkinje cells. PV is additionally found in a subpopulation of inhibitory interneurons, the stellate and basket cells. Studies on deficient mice together with in vitro work and their unique cell type-specific distribution in the cerebellum suggest that these calcium-binding proteins have evolved as functionally distinct, physiologically relevant modulators of intracellular calcium transients. Analysis of different brain regions suggests that these proteins are involved in regulating calcium pools critical for synaptic plasticity. Surprisingly, a major role of any of these three calcium-binding proteins as an endogenous neuroprotectant is not generally supported.     

Embryonic and postnatal development of GABA, calbindin, calretinin, and parvalbumin in the mouse claustral complex

We analyzed the development of immunoreactive expression patterns for the neurotransmitter gamma-aminobutyric acid (GABA) and the calcium-binding proteins calbindin, calretinin, and parvalbumin in the embryonic and postnatal mouse claustral complex. Each calcium-binding protein shows a different temporal and spatial pattern of development. Calbindin-positive cells start to be seen very early during embryogenesis and increase dramatically until birth, thus becoming the most abundant cell type during embryonic development, especially in the ventral pallial part of the claustrum. The distribution of calbindin neurons throughout the claustrum during embryonic development partly parallels that of GABA neurons, suggesting that at least part of the calbindin neurons of the claustral complex are GABAergic and originate in the subpallium. Parvalbumin cells, on the other hand, start to be seen only postnatally, and their number then increases while the density of calbindin neurons decreases. Based on calretinin expression in axons, the core/shell compartments of the dorsal claustrum start to be clearly seen at embryonic day 18.5 and may be related to the development of the thalamoclaustral input. Comparison with the expression of Cadherin 8, a marker of the developing dorsolateral claustrum, indicates that the core includes a central part of the dorsolateral claustrum, whereas the shell includes a peripheral area of the dorsolateral claustrum, plus the adjacent ventromedial claustrum. The present data on the spatiotemporal developmental patterns of several subtypes of GABAergic neurons in the claustral complex may help for future studies on temporal lobe epilepsies, which have been related to an alteration of the GABAergic activity.     

Mono- and dual-frequency fast cerebellar oscillation in mice lacking parvalbumin and/or calbindin D-28k

Calbindin is a fast Ca2+-binding protein expressed by Purkinje cells and involved in their firing regulation. Its deletion produced approximately 160-Hz oscillation sustained by synchronous, rhythmic Purkinje cells in the cerebellar cortex of mice. Parvalbumin is a slow-onset Ca2+-binding protein expressed in Purkinje cells and interneurons. In order to assess its function in Purkinje cell firing regulation, we studied the firing behavior of Purkinje cells in alert mice lacking parvalbumin (PV-/-), calbindin (CB-/-) or both (PV-/- CB-/-) and in wild-type controls. The absence of either protein resulted in Purkinje cell firing alterations (decreased complex spike duration and pause, increased simple spike firing rate) that were more pronounced in CB-/- than in PV-/- mice. Cumulative effects were found in complex spike alterations in PV-/- CB-/- mice. PV-/- and CB-/- mice manifested approximately 160-Hz oscillation that was sustained by Purkinje cells firing rhythmically and synchronously along the parallel fiber axis. This oscillation was dependent on GABA(A), N-methyl-D-aspartate and gap junction transmission. PV-/- CB-/- mice exhibited a dual-frequency (110 and 240 Hz) oscillation. The instantaneous spectral densities of both components were inversely correlated. Simple and complex spikes of Purkinje cells were phase-locked to one of the two oscillation frequencies. Mono- and dual-frequency oscillations presented similar pharmacological properties. These results demonstrate that the absence of the Ca2+ buffers parvalbumin and calbindin disrupts the regulation of Purkinje cell firing rate and rhythmicity in vivo and suggest that precise Ca2+ transient control is required to maintain the normal spontaneous arrhythmic and asynchronous firing pattern of the Purkinje cells.     

Calbindin-D28K, parvalbumin and calretinin in primate lower motor neurons

It has been suggested that lower motor neurons containing calcium-binding proteins (CBP) may be resistant to degeneration in motor neuron disease. The testing of this hypothesis is hampered by lack of comprehensive information regarding the presence of CBPs in motor neurons. To address this shortcoming, we investigated the distribution of the CBPs calbindin-D28K (CB), parvalbumin (PV) and calretinin (CRT) in lower motor neurons in the normal human and two non-human primates (rhesus monkey and common marmoset) using immunohistochemistry. A variable proportion of motor neurons in cranial nerve motor nuclei contained immunoreactivity for one or more CBPs. A subpopulation of spinal cord alpha-motor neurons was also CBP-positive. Comparison of staining for choline acetyltransferase (ChAT) and CBPs in the human spinal cord demonstrated that approximately 63% of ventral horn motor neurons contained PV, 53% contained CRT and 56% contained CB. CBP immunoreactivity within motor neurons was of variable staining intensity. It remains to be established whether the presence of these CBPs confers protection against the pathogenic mechanisms of motor neuron disease.     

Differences in locomotor behavior revealed in mice deficient for the calcium-binding proteins parvalbumin, calbindin D-28k or both

We investigated the role of the two calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) in the locomotor activity and motor coordination using null-mutant mice for PV (PV-/-), CB (CB-/-) or both proteins (PV-/-CB-/-). These proteins are expressed in distinct, mainly non-overlapping populations of neurons of the central and peripheral nervous system and PV additionally in fast-twitch muscles. In a test measuring repeated locomotor activity during 18-20 days, the analysis revealed a slightly increased activity in mice lacking either protein, while the lack of both decreased the number of beams crossed during active periods. An increase in the characteristic speed during the first 8 days could be attributed to PV-deficiency, while the elimination of CB in CB-/- and double-KO mice decreased the percentage of fast movements at all time points. In the latter, additionally a reduction of the fastest speed was observed. The alterations in locomotor activity (fast movements, fastest speed) strongly correlate with the impairment in locomotor coordination in mice deficient for CB evidenced in the runway assay and the rotarod assay. The graded locomotor phenotype (CB>PV) is qualitatively correlated with alterations in Purkinje cell firing reported previously in these mice. The presence or absence of either protein did not affect the spontaneous locomotor activity when animals were placed in a novel environment and tested only once for 30 min. In summary, the lack of these calcium-binding proteins yields characteristic, yet distinct phenotypes with respect to locomotor activity and coordination.     

Distribution of parvalbumin, calretinin, and calbindin-D(28k) immunoreactivity in the rat amygdaloid complex and colocalization with gamma-aminobutyric acid

To understand the organization of inhibitory circuitries in the rat amygdala, the distribution of parvalbumin, calretinin, and calbindin immunoreactivity was investigated in the rat amygdaloid complex. Colocalization of various calcium-binding proteins with the inhibitory transmitter gamma-aminobutyric acid (GABA) was studied by using the mirror technique. Parvalbumin-immunoreactive (-ir) elements were located mostly in the deep amygdaloid nuclei, whereas the calretinin-ir and calbindin-ir staining were most intense in the cortical nuclei as well as in the central nucleus and the amygdalohippocampal area. Second, the distribution of immunopositive neurons largely parallelled the distribution of terminal and neuropil labeling. Third, immunostained neurons could be divided into four major morphologic types (types 1-4) based on the characteristics of the somata and the dendritic trees. The fourth lightly stained neuronal type that had a pyramidal GABA-negative soma was observed only in calretinin and calbindin preparations. Fourth, parvalbumin-ir terminals formed basket-like plexus and cartridges, which suggests that parvalbumin labels GABAergic inhibitory basket cells and axo-axonic chandelier cells, respectively. Colocalization studies indicated that 521 of 553 (94%) of parvalbumin-ir, 419 of 557 (75%) of calbindin-ir, and 158 of 657 (24%) of calretinin-ir neurons were GABA-positive in the deep amygdaloid nuclei. A high density of large GABA-negative calbindin-ir neurons was observed caudally in the medial division of the lateral nucleus and GABA-negative calretinin-ir neurons were observed in the magnocellular division of the accessory basal nucleus as well as in the intermediate and parvicellular divisions of the basal nucleus. These data suggest that in various amygdaloid areas, neuronal excitability is controlled by GABAergic neurons that contain different calcium-binding proteins. The appearance of basket-like plexus and cartridges in the parvalbumin preparations, but not in calretinin preparations, suggests that like in the hippocampus, the distribution of inhibitory terminals in the dendritic and perisomatic regions of postsynaptic neurons in the rat amygdala is organized in a topographic manner.     

Developmental changes in calbindin-D28k and calretinin expression in the mouse suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus, the primary circadian pacemaker in mammals, and the retinohypothalamic tract, the retinal afferent fibres to the suprachiasmatic nucleus, both mature during early postnatal life. The establishment of circadian rhythms is thought to depend on input from the retina, but the mechanism remains unknown. Here we examined developmental changes in the expression of the Ca2+-binding proteins calbindin-D28k and calretinin in the mouse hypothalamus. Robust calbindin-D28k immunoreactivity was observed in the dorsomedial suprachiasmatic nucleus and the supraoptic nucleus in neonatal mice (postnatal day 3). The calbindin-D28k immunoreactivity decreased significantly in the suprachiasmatic nucleus but not in the supraoptic nucleus during postnatal days 9-15, when retinohypothalamic tract projections to the suprachiasmatic nucleus are completed. Calretinin immunoreactivity was low in the neonatal suprachiasmatic nucleus and increased with development in the ventrolateral suprachiasmatic nucleus, in parallel with the developmental reduction of calbindin-D28k immunoreactivity observed in the dorsomedial suprachiasmatic nucleus. Developmentally stable calretinin immunoreactivity was also observed in retinohypothalamic tract fibres. Organotypic slice cultures of the suprachiasmatic nucleus were prepared from postnatal day 3 mice to examine the effect of the absence of retinohypothalamic tract inputs on developmental changes in calbindin-D28k and calretinin expression. After 12 days in vitro, the cultured suprachiasmatic nucleus slices exhibited dense calbindin-D28k immunoreactivity similar to neonatal mice, and calretinin immunoreactivity in the ventrolateral suprachiasmatic nucleus similar to young adult mice. These results demonstrate a developmental reduction in calbindin-D28k expression that paralleled retinohypothalamic tract formation and a developmental increase in calretinin expression that is independent of retinohypothalamic tract connections to suprachiasmatic nucleus neurons.     

Expression of calbindin D-28k and parvalbumin in cerebral cortical dysgenesis induced by administration of ethylnitrosourea to rats at the stage of neurogenesis

It has been reported that transplacental administration of ethylnitrosourea (ENU), which is cytotoxic immediately after administration, to rat fetuses at the neurogenesis stage induces dysgenesis of the cerebral cortex, characterized by neuronal sparseness and architectural irregularity. In the present study, we examined the topographic distribution of neurons containing 5-bromo-2-deoxyuridine (BrdU), and those containing calbindin D-28k (CaBP) and parvalbumin (PV), most of latter two are considered to be interneurons located in particular layers of the normal cerebral cortex in rats with experimentally induced cerebral cortical dysgenesis. Pregnant Wistar albino rats were given a single transplacental administration of ENU on embryonic day 16, followed 4, 8, 16, 24, 36, or 48 h later by a single intraperitoneal injection of BrdU. The pups were killed 10 weeks after birth. In the normal cerebral cortex, BrdU-immunopositive neurons showed an inside-out pattern according to the time of BrdU injection, whereas in ENU-treated rats the topographic localization of the BrdU-immunopositive neurons was irregular and the inside-out pattern was disrupted. Although the number of CaBP- and PV-immunopositive neurons was lower in ENU-treated animals, no topographic difference was evident between the normal and the dysgenetic cerebral cortices. These findings indicate that the expression of CaBP and PV in the neurons of the rat cerebral cortex is extrinsic, and depends on the position of the neurons rather than on the time of their formation or on genetic control. This suggests the existence of re-regulation of the expression of CaBP and PV in the developing brain, which may be one of the effective mechanisms by which the cerebral cortex can maintain its normal function in spite of cytoarchitectural abnormality.     

Postnatal development of parvalbumin and calbindin D-28k immunoreactivities in the canine anterior cingulate cortex: transient expression in layer V pyramidal cells

We have examined the ontogeny of parvalbumin (PV) and calbindin D-28k (CB) immunoreactivities in the canine anterior cingulate cortex (ACC) from the day of birth (P0) through P180. At P7, PV immunoreactivity first appeared in layer VI multipolar cells. The PV immunoreactivity in GABAergic nonpyramidal cells appeared to follow an inside-out gradient of radial emergence. Although immunoreaction was limited mainly to the developing nonpyramidal cells, pyramid-like PV immunoreactive cells were transitorily observed in layer V from P14 to P90. The developmental pattern of CB immunoreactivity differed from that of PV immunoreactivity. CB immunoreactivity first developed in layer V pyramidal cells from P0, which continued through P90. CB immunoreactive nonpyramidal cells were located in the infragranular layers and white matter at P0 and maturated in both the supragranular and infragranular layers without clear inside-out gradient.This developmental study revealed the comparable belated expression of PV immunoreactivity and the transient expression of both calcium-binding proteins in layer V pyramidal cells. These results suggest that the transient expression of calcium-binding proteins in layer V pyramidal cells might be related to the critical period of early postnatal development.