Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.     

Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.     

尾核P物质对胃运动的抑制效应系通过黑质,迷走背核经迷走神经所介导

本工作观察损毁下丘脑外侧区,黑质,迷走背核及其传出神经对尾核微量注射Ｐ物质引起的胃肌电快波和胃运动抑制效应的影响。实验结果：该抑制效应不依赖于下丘脑外侧区的完整但可被损毁黑质,迷走背核或迷走上所消除。用利血平耗竭交感神经递质则不影响该效应。这些结果表明：尾核ＳＰ的抑胃效应系通过黑质、迷走背核经迷走神经所传出。     

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.     

Immunocytochemical localization of calcium-binding proteins, calbindin D28K-, calretinin-, and parvalbumin-containing neurons in the dog visual cortex

Although the dog is widely used to analyze the function of the brain, it is not known whether the distribution of calcium-binding proteins reflects a specific pattern in the visual cortex. The distribution of neurons containing calcium-binding proteins, calbindin D28K, calretinin, and parvalbumin in adult dog visual cortex were studied using immunocytochemistry. We also compared this labeling to that of gamma-aminobutyric acid (GABA). Calbindin D28K-immunoreactive (IR) neurons were predominantly located in layer II/III. Calretinin- and parvalbumin-IR neurons were located throughout the layers with the highest density in layers II/III and IV. The large majority of calbindin D28K-IR neurons were multipolar stellate cells. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. And the large majority of parvalbumin-IR neurons were multipolar stellate and round/oval cells. More than 90% of the calretinin- and parvalbumin-IR neurons were double-labeled with GABA, while approximately 66% of the calbindin D28K-IR neurons contained GABA. This study elucidates the neurochemical structure of calcium-binding proteins. These data will be informative in appreciating the functional significance of different laminar distributions of calcium-binding proteins between species and the differential vulnerability of calcium-binding proteins-containing neurons, with regard to calcium-dependent excitotoxic procedures.     

Calcium-binding proteins in the retina of a calbindin-null mutant mouse

Calcium-binding proteins are abundantly expressed in many neurons of mammalian retinae. Their physiological roles are, however, largely unknown. This is particularly true for calcium-modulating proteins (“calcium buffers”) such as calbindin D28k. Here, we have studied retinae of wildtype (+/+) and calbindin-null mutant (–/–) mice by using immunocytochemical methods. Although calbindin immunoreactivity was completely absent in the calbindin (–/–) retinae, those cells that express the protein in wildtype retinae, such as horizontal cells, were still present and appeared normal. This was verified by immunostaining horizontal cells for various neurofilament proteins. In order to assess whether other calcium-binding proteins are upregulated in the mutant mouse and may thus compensate for the loss of calbindin, mouse retinae were also immunolabeled for parvalbumin, calretinin, and a calmodulin-like protein (CALP). In no instance could a change in the expression pattern of these proteins be detected by immunocytochemical methods. Thus, our results show that calbindin is not required for the maintenance of the light-microscopic structure of the differentiated retina and suggest roles for this protein in retinal function.     

Antibody Recognition of Calcium-Binding Proteins Depends on Their Calcium-Binding Status

Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.     

大白鼠脚内核的传入性联系

众所周知,肉食动物和大白鼠的脚内核,相当于灵长类的内侧苍白球(Nagy et al.1978;Fox and Schmitz 1944);它们的细胞形态、传入及传出均相同。早期以及近年来的一些研究工作者,虽然在研究其他核团的投射时,联系到一些本核团的传入,但是尚缺乏对本核团传人的系统研究。本实验即是应用辣根过氧化物酶的逆行传递法来研究大白鼠脚内核的传入性联系。     

Preproenkephalin-like immunoreactive and calcium-binding proteins-like immunoreactive double-labelled neurons in the spinal trigeminal nucleus caudalis of the rat

Immunofluorescence histochemical double-staining for preproenkephalin (PPE) and calbindin-D28k (CB), calretinin (CR) or parvalbumin (PV) were performed in the spinal trigeminal nucleus caudalis (Vc) of the rat. Neuronal cell bodies exhibiting PPE-like immunoreactivity were present in all laminae of the Vc, with a higher concentration in lamina II. Most of the CB-, CR- and PV-like immunoreactive neurons were located in lamina II, and some of them were also found in laminae I and III of the Vc. Some PPE-like immunoreactive neurons also showed CB-, CR-, or PV-like immunoreactivities. CB/PPE, CR/PPE and PV/PPE double-labelled neurons were mainly observed in lamina II. The percentages of CB/PPE double-labelled neurons in the total numbers of the CB- and PPE-like immunoreactive neurons were 3.5–1.5% and 3.3–15.7%, respectively. Of all CR- and PPE-like immunoreactive neurons, 4.7–13.5% and 3.7–14.2% showed both CR- and PPE-like immunoreactivities. The ratios of PV/PPE double-labelled neurons in all PV- and PPE-like immunoreactive neurons were 9.7–28.1% and 2.1–8.7%, respectively. The present results indicate that some enkephalinergic neurons in the Vc of the rat also contain calcium-binding proteins.     

Co-existence of protein kinase C gamma and calcium-binding proteins in neurons of the medullary dorsal horn of the rat

Protein kinase C gamma isoform (PKCgamma) is present at high levels in the spinal and medullary dorsal horns and is thought to play a role in the sensitization of dorsal horn neurons in certain pain states. Calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) are the most commonly expressed calcium-binding proteins and are located abundantly in the medullary dorsal horn (also called the caudal subnucleus of the spinal trigeminal nucleus). In the present study, immunofluorescence histochemical double staining for PKCgamma and CB, CR or PV was performed in the rat medullary dorsal horn. Most of the PKCgamma-, CB-, CR- and PV-immunoreactive neurons were observed in lamina II; some were also encountered in lamina I and lamina III of the medullary dorsal horn. Neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were also mainly found in lamina II, while in lamina I and lamina III, only a few neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were encountered. The percentages of neurons co-expressing CB/PKCgamma in the total numbers of CB- and PKCgamma-immunoreactive neurons were 6.7 and 5.9%, respectively. Of the total numbers of CR- and PKCgamma-immunoreactive neurons, 5.0 and 5.6%, respectively, showed both CR and PKCgamma immunoreactivities. The percentages of neurons co-expressing PV/PKCgamma in the total numbers of PV- and PKCgamma-immunoreactive neurons were 25.7 and 4.1%, respectively. Most of these neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were small (/=36 microm) multipolar neurons were infrequently seen. The present results indicate that there are some neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma in the medullary dorsal horn. These neurons might play important roles in the nociceptive modulation from the oro-facial region.     

Differential effect of psychotropic drugs on dihydroxyphenylacetic acid (DOPAC) in the rat substantia nigra and caudate nucleus

The effect of different psychotropic drugs on DOPAC levels in the caudate nucleus and in the substantia nigra was compared. While pargyline, haloperidol and damphetamine caused parallel changes in both areas, apomorphine and reserpine influenced DOPAC levels in the caudate nucleus and in the substantia nigra differently. In both areas pargyline caused a rapid fall in DOPAC concentrations, while haloperidol caused a marked increase. D-amphetamine was slightly more potent in decreasing DOPAC level in the substantia nigra than in the caudate nucleus. On the other hand, apomorphine was very potent in decreasing DOPAC level in the caudate nucleus but failed to influence it in the substantia nigra. Finally, reserpine, which depleted dopamine stores in both areas, increased DOPAC in the caudate nucleus but decreased it in the substantia nigra.     

Calcium-binding Proteins Calbindin D28K, Calretinin, and Parvalbumin Immunoreactivity in the Rabbit Visual Cortex

The distribution and morphology of neurons containing three calcium-binding proteins, calbindin D28K, calretinin, and parvalbumin in the adult rabbit visual cortex were studied. The calcium-binding proteins were identified using antibody immunocytochemistry. Calbindin D28K-immunoreactive (IR) neurons were located throughout the cortical layers with the highest density in layer V. However, calbindin D28K-IR neurons were rarely encountered in layer I. Calretinin-IR neurons were mainly located in layers II and III. Considerably lower densities of calretinin-IR neurons were observed in the other layers. Parvalbumin-IR neurons were predominantly located in layers III, IV, V, and VI. In layers I and II, parvalbumin-IR neurons were only rarely seen. The majority of the calbindin D28K-IR neurons were stellate, round or oval cells with multipolar dendrites. The majority of calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicularly to the pial surface. The morphology of the majority of parvalbumin-IR neurons was similar to that of calbindin D28K: stellate, round or oval with multipolar dendrites. These results indicate that these three different calcium-binding proteins are contained in specific layers and cells in the rabbit visual cortex.     

Distribution of metabolic activity (cytochrome oxidase) and immunoreactivity to calcium-binding proteins in turtle brainstem auditory nuclei

Using histochemical and immunohistochemical techniques, distribution of activity of oxidative mitochondrial enzyme cytochrome oxidase (CO) and of immunoreactivity to calcium-binding proteins has been studied in spiral ganglion and auditory nuclei of brainstem in two turtle species. It has been shown that immunoreactivity to calbindin, parvalbumin, and calretinin in neurons and neuropil of nuclei of cochlear and superior olivary complexes, in nucleus of lateral lemniscus, and in spiral ganglion neurons coincides topographically with the high CO activity. The similarity of the studied metabolic and neurochemical characteristics of these auditory centers in reptiles, birds, and mammals indicates the existence of some common principles of their organization in amniotes in spite of phylogenetic differences and peculiarities of specialization of the auditory system in different species.     

Topical organization of the neuronal projections of the substantia nigra and ventral field of the tectum mesencephali onto the head of the caudate nucleus in the cat brain

The investigation performed on cats by means of retrograde axonal transport of horseradish peroxidase and luminophores has presented the data demonstrating spatial organization of separate part projections of the nigral complex and the tegmental ventral field to various segments of the caudate nucleus head. Terminal fields from neurons of various parts of the substantia nigra and the tegmental ventral field are demonstrated to overlap in segments of the caudate nucleus. Experiments with double fluorescent labelling demonstrate divergence of axons of the nigral neurons.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Calbindin D-28k immunoreactive nerve fibers in the carotid body of normoxic and chronically hypoxic rats

The distribution and ultrastructural characteristics of calbindin D-28k immunoreactive nerve fibers were examined in the carotid body of the normoxic control rats by light and electron microscopy, and the abundance of calbindin D-28k fibers in the carotid body was compared in normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). Calbindin D-28k immunoreactivity was recognized in nerve fibers within the carotid body. Calbindin D-28k immunoreactive nerve fibers appeared as thin processes with many varicosities. They were distributed around clusters of glomus cells, and around blood vessels. Immunoelectron microscopy revealed that the calbindin D-28k immunoreactive nerve terminals are in close apposition with the glomus cells, and membrane specialization is visible in some terminals. Some dense-cored vesicles in the glomus cells were aggregated in this contact region. The chronically hypoxic carotid bodies were found to be enlarged several fold, and a relative abundance of calbindin D-28k fibers was lesser than in the normoxic carotid bodies. When expressed by the density of varicosities per unit area of the parenchyma, the density of calbindin D-28k fibers associated with the glomus cells in chronically hypoxic carotid bodies was decreased by 70%. These immunohistochemical findings indicate a morphological basis for involvement of calcium binding protein in the neural pathway that modulates carotid body chemoreception.     

Structure of the human brain calcium-binding protein calretinin and its expression in bacteria

Calbindin D28k and calretinin are two closely related intracellular calcium-binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin-D-dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31,520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium-binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 x 10(-9) amino acid-1 year-1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium-binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.     

Transient co-localization of calretinin,parvalbumin, and calbindin-D28k in developing visual cortex of monkey

Brain Cell Biology - This paper reports a double-labelling immunocytochemical study of the three calcium-binding proteins calretinin, parvalbumin, and calbindin-D28k in developing and adultMacaca...     

The role of the substantia nigra in the control of amygdaloid paroxysmal activity

Both in acute and chronic cats focal paroxysmal activity evoked in the ventro-basal complex of the amygdala has been inhibited by substantia nigra conditioning stimulation, to a greater extent, than by caudate nucleus activation. Injection of kainic acid into substantia nigra resulted in the disappearance of the caudate inhibition. It is suggested that the final control, exerted by the striatum on the amygdaloid seizures, occurs by means of the substantia nigra.     

Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.