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Morphological features of neurons containing calcium-binding proteins in the human striatum.

An immunohistochemical approach was used to characterize the morphological phenotype of neurons containing the calcium-binding proteins calretinin (CR), parvalbumin (PV), or calbindin-D28k (CB) in the normal human striatum. The protein CR occurs in at least four morphologically distinct types of neurons. Apart from the numerous medium-sized aspiny interneurons and the less abundant giant aspiny interneurons, CR also labels some medium-sized spiny neurons morphologically identical to striatal projection neurons. This finding indicates that CR is not only confined to striatal interneurons but also may be involved in the function of certain projection neurons. Some small and peculiar bushy-like aspiny neurons also are enriched with CR. These neurons could correspond to the dwarf or neurogliform neurons first described by Ramón y Cajal (1911). Three types of PV-immunoreactive striatal neurons can be visualized in the human striatum: 1) the common medium-sized aspiny leptodendritic neurons, 2) some smaller and profusely arborized aspiny neurons, and 3) a few large and intensely stained neurons with conspicuously beaded and poorly branched dendrites. The protein CB labels virtually all medium-sized spiny projection neurons located in the striatal matrix but also identifies a small subset of large and more intensely immunostained aspiny neurons. The latter finding indicates that CB is not entirely confined to striatal projection neurons but also may play a role in local circuit neurons. These normative data should help our understanding of the chemical anatomy of the human striatum in both health and disease.

Adult↗

Effect of GDNF on differentiation of cultured ventral mesencephalic dopaminergic and non-dopaminergic calretinin-expressing neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for ventral mesencephalic (VM) dopaminergic neurons. Subpopulations of dopaminergic and non-dopaminergic VM neurons express the calcium-binding proteins calbindin (CB) and calretinin (CR). Characterization of the actions of GDNF on distinct subpopulations of VM cells is of great importance for its potential use as a therapeutic molecule and for understanding its role in neuronal development. The present study investigated the effects of GDNF on the survival and morphological differentiation of dopaminergic and non-dopaminergic neurons in primary cultures of embryonic day (E) 18 rat VM. As expected from our results obtained using E14 VM cells, GDNF significantly increased the morphological complexity of E18 CB-immunoreractive (CB-ir), tyrosine hydroxylase (TH)-ir, and CR-ir neurons and also the densities of CB-ir and TH-ir neurons. Interestingly, densities of E18 CR-ir neurons, contrarily to our previous observations on E14 CR-ir neurons, were significantly higher after GDNF treatment (by 1.5-fold). Colocalization analyses demonstrated that GDNF increased the densitiy of dopaminergic neurons expressing CR (TH+/CR+/CB-), while no significant effects were observed for TH-/CR+/CB- cell densities. In contrast, we found that GDNF significantly increased the total fiber length (2-fold), number of primary neurites (1.4-fold), number of branching points (2.5-fold), and the size of neurite field per neuron (1.8-fold) of the non-dopaminergic CR-expressing neurons (TH-/CR+/CB-). These cells were identified as GABA-expressing neurons. In conclusion, our findings recognize GDNF as a potent differentiation factor for the development of VM dopaminergic and non-dopaminergic CR-expressing neurons.

Animals↗

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding.

Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.

Alkylation↗

Local-circuit neurones in the medial prefrontal cortex (areas 25, 32 and 24b) in the rat: morphology and quantitative distribution.

This paper is a light microscopical study describing the detailed morphology and quantitative distribution of local circuit neurones in areas 25, 32, and 24b of the medial prefrontal cortex (mPFC) in the rat. Cortical interneurones were identified immunocytochemically by their expression of calretinin (CR), parvalbumin (PV), and calbindin D-28k (CB) immunoreactivity. Neurones immunoreactive for gamma-aminobutyric acid (GABA) were also investigated, as were interneurones containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity. Several distinct classes of CR+, PV+, and CB+ neurones were identified; the most frequent were: bipolar/bitufted CR+ cells in upper layer 3; multipolar PV+ neurones in layers 3 and 5; and bitufted/multipolar CB+ neurones in lower layer 3. CB+ neurones resembling Martinotti and neurogliaform cells were also present in layers 5/6. The morphologies and depth distributions of each cell type were consistent across the three areas of mPFC studied. Seven classes of diaphorase-reactive mPFC neurone are described; these cells were composed about 0.8% of the total neurone population and had a peak distribution located in mid- to lower layer 5 in each area. In areas 32 and 25, three defined bands of diffuse NADPH diaphorase staining were located in layer 2 and in upper and deep layer 5. Diaphorase reactivity was very infrequently colocalised with either CR, PV, or CB immunoreactivities. The numerical densities of neurones (N(V), number of cells per mm3) in each layer were calculated stereologically. The mean total neuronal N(V) estimate for areas 25, 32, and 24b was 51,603 +/- 3,324 (mean +/- S.D.; n = 8). Significant interareal differences were detected. From cortical thickness data and neuronal N(V) estimates, the absolute number of neurones under 1 mm2 of cortical surface (N(C)) have been derived. The mean N(C) value for areas 25, 32, and 24b was 57,328 +/- 7,505 neurones. In immunolabelled Nissl-stained sections, CR+ neurones constituted an overall 4.0%, PV+ cells 5.6%, and CB+ 3.4% of the total neurone populations in mPFC. GABA+ cells represented a mean of 16.2% (14.8-17.2%) of neurones in areas 25, 32 and 24b. The absolute numbers of CR+, PV+, CB+, and GABA+ neurones within individual layers in a column of cortex under 1 mm2 of cortical surface (N(L)) have also been derived, with significant interareal differences in N(L) values being detected. The data provide the structural basis for a qualitative and quantitative definition of local cortical circuits in the rat mPFC.

Animals↗

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.

Animals↗

Urinary 28-kD calbindin-D as a new marker for damage to distal renal tubules caused by cisplatin-based chemotherapy.

Calbindin-D, a vitamin D-dependent calcium-binding protein of 28 kD, is found predominantly in the distal tubules of the kidney and central nervous system tissues in humans. To evaluate damage to the renal tubules caused by cisplatin-based chemotherapy, levels of urinary and serum calbindin-D were determined in patients treated with cisplatin- or carboplatin-based chemotherapies using a highly sensitive enzyme immunoassay system developed in our laboratory. Levels of urinary 28-kD calbindin-D were also determined in patients with benign and malignant urological diseases. The mean urinary calbindin-D level was 2.44 + or - 0.31 (mean + or - SE) ng/mg creatinine in 40 healthy subjects. Urinary calbindin-D levels were elevated (>10 ng/mg creatinine) in 2 of 33 patients (6%) with benign and 1 of 50 (2%) with malignant urological diseases. Urinary calbindin-D levels were significantly increased after cisplatin-based chemotherapy in 14 patients, with peaks (71.8 + or - 13.5 ng/mg creatinine) being found 8 days after administration of cisplatin, and then a gradual return to the baseline. On the other hand, 7 patients receiving carboplatin-based chemotherapy demonstrated no significant elevation (highest level 7.7 + or - 2.5 ng/mg creatinine). In 7 patients treated with cisplatin-based chemotherapy the serum calbindin-D level was also raised after treatment, with a good correlation to urinary values. These findings suggest that urinary and serum calbindin-D may be kidney-derived and that 28-kDa calbindin-D is a useful marker for damage to the distal renal tubules associated with cisplatin-based chemotherapy.

Acetylglucosaminidase↗

Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions.

The companion paper (Gabbott and Bacon [1996] J. Comp. Neurol.) describes the morphology of calretinin (CR)-, parvalbumin (PV)-, calbindin (CB)-, and GABA-immunoreactive neurons, and NADPH diaphorase-reactive cells, in the medial prefrontal cortex (mPFC; areas 24a, 24b, 24c, 25 and 32) of the adult monkey. Since these local circuit neurons play crucial functional roles, the aim of this study was to provide supportive quantitative data defining their areal and laminar distribution in mPFC. The numerical densities of neurons (Nv, number of cells per mm3) in each area and layer were calculated stereologically. The mean total neuronal NV estimates across mPFC was 55,727 +/- 3,319 per mm3 (mean +/- S.D.; n = 3); values ranged from 50,489 +/- 8,374 per mm3 (area 24a) to 59,938 +/- 7,214 per mm3 (area 24c). Interareal differences were not significant. Cortical depth measurements and neuronal NV estimates for each area allowed the absolute number of neurons in a column of cortex under 1 mm2 of surface to be calculated; values varied between 86,457 +/- 15,063 (area 24a) and 128,464 +/- 24,050 (area 24c). Using immunolabelled Nissl-stained sections of mPFC, CR+ neurons constituted 11.2%, PV+ neurons 5.9%, and CB+ neurons 5.0% of the total neuron population. GABA+ neurons represented an overall 24.9% (23.5-27.3%) of neurons in the mPFC. Differences between areas were not significant. The cortical depth distribution histograms of CR+, PV+, CB+, and GABA+ cell populations in each area were derived and the percentage of a given cell population in each layer subsequently calculated. Peaks in the cortical depth distributions of CR+ and CB+ neurons occurred in layer 2 and upper layer 3, respectively; the peak distribution of PV+ neurons occurred between lower layer 3 and upper layer 5. The depth distribution of GABA+ cells reflected the combined distributions of CR+, PV+ and CR+ neurons. In all areas, the majority (74.4-84.0%) of the GABA cell population was located in layers 2/3. The depth distributions for each cell type were similar between areas. Diaphorase-reactive neurons accounted for 0.25% (0.2-0.32%) of all cortical neurons in mPFC and were distributed in two horizontal strata, in midlayer 3 and in mid/upper layer 6. A large population of diaphorase-reactive cells was present in the white matter. The absolute numbers of CR+, PV+, CB+ and GABA+ neurons within individual layers in a column of cortex under 1 mm2 and 50 x 50 microns of cortical surface have been derived. The data presented provide the basis for a quantitative definition of cortical circuits in monkey mPFC.

Afferent Pathways↗

AAV-mediated gene delivery of BDNF or GDNF is neuroprotective in a model of Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder that results in the progressive loss of GABAergic medium spiny projection neurons in the striatum. Neurotrophic factors have demonstrated neuroprotective actions on striatal neurons, suggesting that increased neurotrophic factor expression may prevent or reduce neuronal loss in the HD brain. We investigated whether enhanced expression of brain-derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor (GDNF), achieved by adeno-associated viral (AAV) vector-mediated gene delivery, could protect striatal neurons in the quinolinic acid (QA) rodent model of HD. Adult Wistar rats received unilateral intrastriatal injections of AAV-BDNF, AAV-GDNF, AAV-GFP, or PBS. Three weeks later, the rats were lesioned with QA, a toxin that induces striatal neuron death by an excitotoxic process. Both AAV-BDNF and AAV-GDNF significantly reduced the loss of both NeuN- and calbindin-immunopositive striatal neurons 2 weeks after lesion compared to controls. AAV-BDNF also provided significant neurotrophic support to NOS-immunopositive striatal interneurons, while AAV-GDNF-treated rats demonstrated significant protection of parvalbumin-immunopositive striatal interneurons compared to controls. These results indicate that AAV-mediated gene transfer of BDNF or GDNF into the striatum provides neuronal protection in a rodent model of HD.

Animals↗

Variable in vivo regulation of rat vitamin D-dependent genes (osteopontin, Ca,Mg-adenosine triphosphatase, and 25-hydroxyvitamin D3 24-hydroxylase): implications for differing mechanisms of regulation and involvement of multiple factors.

In these studies, the regulation of renal osteopontin (OPN), intestinal calcium pump, and renal and intestinal 25-hydroxyvitamin D3 24-hydroxylase (24OHase) was compared to the regulation of calbindin by Northern blot analysis. The time course of induction of the calbindins and calcium pump messenger RNAs (mRNAs) in vitamin D-deficient rats in response to 1,25-hydroxyvitamin D3 [1,25-(OH)2D3] was similar [first induction 3 h after 1,25-(OH)2D3 (200 ng/100 g BW), reaching a maximum at 12-24 h]. However, the maximal induction of calbindin mRNA was greater (8.2 +/- 1.2-fold) than that of intestinal calcium pump mRNA (21.3 +/- 0.5-fold). 24-Hydroxylase mRNA showed a different time course of induction by 1,25-(OH)2D3. 24-Hydroxylase mRNA was undetectable in vitamin D-deficient rat intestine and kidney. In intestine, 24OHase mRNA was maximally induced early [3-6 h after 1,25-(OH)2D3] and did not accumulate long after 1,25-(OH)2D3 administration. Unlike the other vitamin D-regulated genes, OPN was uninduced by 1,25-(OH)2D3 in the kidney of D-deficient rats. However, in vitamin D-replete rats given 1,25-(OH)2D3 for 5 days, a significant induction in renal OPN was observed (6.0-fold; P < 0.01). Developmental studies indicated that renal 24OHase and intestinal calcium pump mRNAs, similar to calbindin-D9K mRNA, were induced at 3 weeks of age, the time of weaning when active intestinal calcium absorption is induced. A comparison of the effect of calcium status showed that low dietary calcium resulted in an induction of intestinal calcium pump as well as calbindin-D9K mRNA, consistent with the requirement for an increase in intestinal calcium absorption during dietary calcium restriction. We next examined gender- and age-related changes in the expression of these genes. Our results indicate that renal OPN mRNA increases markedly in males with aging (10-fold between 3-20 months; P < 0.01), whereas OPN mRNA levels are unchanged in females. In both males and females, significant increases (2.7- to 4.5-fold) in renal 24OHase mRNA were observed at 20 months compared to 3 months of age.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption.

Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.

Absorption↗

Embryonic stem-cell derived neurones express a maturation dependent pattern of voltage-gated calcium channels and calcium-binding proteins.

There are remarkable changes of calcium binding proteins and voltage dependent Ca(2+) channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi- and multipolar neurones, expressing all Ca(2+) channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N- and L-type channel contribution in favour of P/Q- and R-type participation in bi- and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca(2+) homeostasis, and clarifies contradictory reports on the development of Ca(2+) channel expression using primary cultures of neurones already committed to certain brain compartments.

Animals↗

alpha-actinin-2 in rat striatum: localization and interaction with NMDA glutamate receptor subunits.

Alpha-actinin (alpha-actinin-2) is a protein which links the NR1 and NR2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptors to the actin cytoskeleton. Because of the importance of NMDA receptors in modulating the function of the striatum, we have examined the localization of alpha-actinin-2 protein and mRNA in striatal neurons, and its biochemical interaction with NMDA receptor subunits present in the rat striatum. Using an alpha-actinin-2-specific antibody, we found intense immunoreactivity in the striatal neuropil and within striatal neurons that also expressed parvalbumin, calretinin and calbindin. Conversely, alpha-actinin-2 immunoreactivity was not detected in neurons expressing choline acetyltransferase and neuronal nitric oxide synthase. Dual-label in situ hybridization revealed that the highest expression of alpha-actinin-2 mRNA is in substance P-containing striatal projection neurons. The alpha-actinin-2 mRNA is also present in enkephalinergic projection neurons and interneurons expressing parvalbumin, choline acetyl transferase and the 67-kDa isoform of glutamic acid decarboxylase, but was not detected in somatostatin-expressing interneurons. Immunoprecipitation of membrane protein extracts showed that alpha-actinin-2 is present in heteromeric complexes of NMDA subunits, but is not associated with AMPA receptors in the striatum. A subunit-specific anti-NR1 antibody co-precipitated major fractions of NR2A and NR2B subunits, but only a minor fraction of striatal alpha-actinin-2. Conversely, alpha-actinin-2 antibody immunoprecipitated only modest fractions of striatal NR1, NR2A and NR2B subunits. These data demonstrate that alpha-actinin-2 is a very abundant striatal protein, but exhibits cellular specificity in its expression, with very high levels in substance-P-containing projection neurons, and very low levels in somatostatin and neuronal nitric oxide synthase interneurons. Despite the high expression of this protein in the striatum, only a minority of NMDA receptors are linked to alpha-actinin-2. This interaction may identify a subset of receptors with distinct anatomical and functional properties.

Actinin↗

The role of gonadal hormones in gene expression of calbindin (Mr 28,000) in the laying hen.

Acute and chronic changes in calbindin (Mr 28,000) mRNA and calbindin concentrations were determined to assess the roles of steroid hormones in calbindin mRNA and calbindin synthesis in the eggshell gland (ESG). The results support an earlier suggestion that calbindin gene expression in the ESG is associated with Ca2+ flux through the ESG. The evidence includes wide oscillation of the mRNA during the diurnal egg cycle, in close temporal association with egg shell calcification. Progesterone (single im injection of 1 mg/kg body weight, BW) prolonged the period of egg formation and reduced the rate of Ca2+ transport and the concentration of calbindin mRNA in the ESG. Dexamethasone (single im injection of 5 mg/kg BW) prolonged the period of egg formation, increased shell Ca2+, and reduced calbindin mRNA in the ESG and intestine. Testosterone (single im injection of 2 mg/kg BW) did not affect calbindin mRNA synthesis in the ESG. The effects of estradiol on the synthesis of calbindin mRNA in the ESG of sexually immature or laying birds were minor, while it affected plasma Ca in the same birds. The antiestrogen Tamoxifen (60 mg/kg BW, given orally) reduced plasma Ca, but did not affect the synthesis of calbindin mRNA in the ESG. The antiprogesterone RU-38486 (20 mg/kg BW, orally) increased shell Ca2+ but had no effect on plasma Ca or the synthesis of calbindin mRNA. It appears that estrogens alone cannot account for the markedly elevated synthesis of calbindin mRNA in the ESG of the laying bird. The hypothesis that the regulatory mechanism for the synthesis of calbindin mRNA in the ESG may involve a stimulator(s), associated with the onset of production, and an oscillating depressor(s) is supported and both appear to be closely linked to the reproductive cycle. The specific in vivo effect of progesterone on calbindin mRNA in the ESG, together with its already known changes during the ovulatory cycle in birds, supports the idea that it is a depressor.

Animals↗

Distribution of calbindin-D28K in the brain of the fetal sheep in late gestation.

Immunocytochemical distribution of calbindin-D28K was examined in the brains of fetal sheep at 100-105 and 120-125 days of gestation (dGA) and at term in labor (term approximately 150 dGA) and compared to adult sheep. Brains were perfused, cut frozen and immunostained as freely floating sections with a polyclonal anti-calbindin-D28K antibody. Light microscopy revealed that calbindin-D28K immunogenicity in fetal cerebellum, rhombencephalon, mesencephalon, diencephalon and telencephalon was overall very similar to that seen in the brains of adult sheep. In addition even though fetal sheep peripheral plasma cortisol concentrations increase 10-20 times over the last 3-4 weeks of gestation to peak during labor and delivery, no glucocorticoid dependent increases in calbindin-D28K in the hippocampal formation could be seen in the sheep fetus as have been demonstrated for the adult rat given exogenous glucocorticoids. It is concluded that: (1) the basic pattern of calbindin-D28K distribution seen in adult sheep brains is established by at least 100 dGA in the sheep fetus; and (2) the pattern of calbindin-D28K expression in fetal sheep hippocampal formation is not related to peripheral plasma glucocorticoid concentrations.

Animals↗

Moderate elevation of extracellular potassium transiently inhibits regeneration of sensory axons in cultured adult sciatic nerves.

The adult frog dorsal root ganglia (DRG) together with the sciatic nerve (ScN) has previously been shown to survive in organ culture for several days. If a local test crush is made at the beginning of culturing, there is an initial delay of about 3 days before the sensory axons start to grow into the distal nerve stump at a rate of about 0.6-0.9 mm/day. The present results showed that axonal growth was unaffected in preparations maintained for 8 days in medium containing 10 mM K+ (5 mM is the physiological level). In contrast, the outgrowth was markedly reduced by 15 mM K+ and still more by 20 and 25 mM K+. The growth inhibition was partially counteracted by nifedipine, a Ca(2+)-channel antagonist. Other experiments clearly showed that high K+ exerted its effects during the early phase of the regeneration and lacked effects at later stages. The possibility that Ca(2+)-binding proteins, e.g. calbindin, which showed immunohistochemical reactivity in different structures, contribute to the growth adaptation to high K+ will be considered. The generality of the findings was supported by inhibition of axonal outgrowth of adult mouse sciatic sensory axons by high K+.

Animals↗

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver.

The cDNA of a Ca(2+)-binding protein regucalcin was cloned from a rat liver cDNA library which was constructed in lambda ZAPII by immunoscreening. Positive clones were obtained from which spanned the region of interest, and they gave a sequence of 1.7 kb by sequencing with the dideoxynucleotide method. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the regucalcin molecule. Regucalcin was composed of 299 amino acid residues and its molecular weight was estimated to be 33,388 Da. The hydropathy profile of regucalcin showed a highly hydrophilic character. The nucleotide and amino acid sequences of regucalcin did not have statistically significant homology, as compared with the registered sequences which are found in the EMBL and GenBank databases containing several other Ca(2+)-binding proteins (calmodulin, calbindin-D28k and S-100 beta). The regucalcin molecule did not contain the EF-hand motif as a Ca(2+)-binding domain. The present study demonstrates that regucalcin is a unique Ca(2+)-binding protein in the liver of rats.

Amino Acid Sequence↗

Differentiation of chemically defined neuronal populations in the transplanted olfactory bulb without olfactory receptor innervation.

Olfactory bulbs (OBs) from embryonic day 15 and 17 and postnatal day 1 mice were transplanted into the lateral ventricle of juvenile host mice without bulbectomy, and fine structural and chemical features of neurons and glia in the OB transplants were investigated immunocytochemically and electron microscopically. In the OB transplants there were neither clearly defined glomeruli nor layers, nor olfactory marker protein immunoreactive elements. However, chemically defined neuronal populations resembling those in the normal OBs such as those immunoreactive for gamma-aminobutyric acid (GABA), tyrosine hydroxylase and Ca(2+)-binding proteins (calbindin-D28K, calretinin, parvalbumin) were observed. Electron microscopically, dendrodendritic and somatodendritic reciprocal synapses, that is, synapses characteristic of the OB, were occasionally observed in the OB transplants. These results indicated that at least some embryonic or newborn mouse OB neurons and/or precursor cells could exhibit chemical properties and form typical synaptic contacts observed in normal OB, even when they received no inputs from olfactory receptor cells.

Animals↗

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells.

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Animals↗