Arlette Brehier

Transient biochemical compartmentalization of Purkinje cells during early cerebellar development

It has recently been observed that during early cerebellar development--from embryonic Day 17 to postnatal Day 3 in the rat--only certain discrete clusters of Purkinje cells (PCs) are immunoreactive to cyclic GMP-dependent protein kinase (cGK). In contrast, at later stages and in the adult, all the PCs are immunoreactive. These results obtained with cGK suggest a transitory intrinsic heterogeneity in the immature cerebellar cortex. It seemed therefore interesting to investigate the distribution of other PC markers during early development in the rat and in other species. The results presented here were obtained with two other antibodies--against vitamin D-dependent calcium binding protein and against Purkinje cell specific glycoprotein--which, like cGK, label all adult PCs. Each antibody gave a different and reproducible mosaic of positive and negative clusters of PCs in the perinatal cerebellum, thus indicating a transient biochemical compartmentalization resulting from the differential expression of parts of the same genotype by clusters of PCs. This compartmentalization in concomitant with the ingrowing of the cerebellar afferents. Once synaptogenesis starts, the biochemical heterogeneity of PCs disappears.

Cerebellar mutations affecting the postnatal survival of Purkinje cells in the mouse disclose a longitudinal pattern of differentially sensitive cells.

The pattern of surviving Purkinje cells (PCs) was investigated in three cerebellar mutant mice with severe postnatal PC death. Two of these mutations, nervous (nr) and Purkinje cell degeneration (pcd) mutations are already well characterized. The third mutation is a new one, which appeared spontaneously in DW/J-Pas mice and was called tambaleante (tbl). PCs were identified by immunocytochemistry using an antibody against vitamin D-dependent calcium-binding protein which labels all the PCs in adult control mice. In each of the three mutations, surviving PCs are arranged according to a different and reproducible pattern which is symmetric relative to the midline. In NR and young PCD mutants, PCs are closely packed in broad sagittal bands. In TBL, they are more loosely arranged in a rather patchy pattern. In PCD and in TBL mutants the death of resistant PCs is only shortly delayed but in NR there is little change in the number of surviving PCs after 3 months. The differential sensitivity of subsets of PCs to the effect of nr, pcd, and tbl mutations is topographically determined. These results provide a new evidence of the PC heterogeneity which has been previously demonstrated by histochemical and immunohistochemical techniques. Moreover, in the anterior vermis of control mice, three thin sagittal bands of PCs are labeled by the Q113 monoclonal antibody. Similarly, in the anterior lobe of the NR cerebellum, the thin longitudinal strips of missing PCs coincide with the absence of Q113 immunoreactivity: in this region the nr mutation affects specifically the survival of Q113 positive cells. However, other clusters of Q113 immunoreactive PCs do survive in NR mice suggesting that susceptibility to the nr mutation and Q113 positivity are two independent markers of the underlying PC compartmentalization.

Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28K) in vestibular sensory hair cells and vestibular ganglion neurones of the cat.

Vestibular sensory hair cells, afferent fibres and vestibular ganglion neurones of the cat are intensely labelled by a specific antibody to rat kidney vitamin D-dependent calcium-binding protein (CaBP-28K). Type I hair cells are more weakly CaBP immunoreactive than type II hair cells. Ganglion neurones also present a differential staining. The presence of calcium-binding protein in sensory hair cells could be of interest for the understanding of transductional mechanisms.

Cholecalcin (28-kDa calcium-binding protein) in the rat hippocampus: development in normal animals and in altered thyroid states. An immunocytochemical study.

An immunocytochemical study of cholecalcin (28-kDa calcium-binding protein, CaBP, calbindin) was carried out during the development of the rat hippocampus. In normal animals, the protein appeared from Postnatal Day 3 in the granule cells of the dentate gyrus and from Day 5 in the CA1-CA2 pyramidal cells of Ammons horn. The cells of both regions thus showed positive cholecalcin labeling about 1 week after their formation. The sequence of labeling of the granule cells was a reflection of the major sequences of neurogenesis. Cholecalcin could not be detected in hippocampal cells until dendritic arborization and axon growth had occurred. There was a good correlation between the appearance of cholecalcin and the onset of synaptogenesis. In animals with an experimentally altered thyroid state, in which hippocampal development is retarded or accelerated due to abnormal cell maturation, cholecalcin appearance was similarly retarded or accelerated. Cholecalcin seems to be synthesized at the same time as the hippocampal cells become functional.

Calbindin (CaBP 28 kDa) localization in the peripheral vestibular system of various vertebrates

Previous reports on calbindin, a 28 kDa vitamin D-induced calcium-binding protein, located in the mammalian peripheral vestibular system indicated that it is specifically distributed and postulated that it could play a role in the electrophysiological functioning of the sensory cells. This immunocytochemical investigation of the distribution of calbindin in the vestibular system of various vertebrates: fishes (goldfish and sea-perch), amphibia (frog), birds (chicken) and mammals (mouse, cat and baboon), was performed to verify these observations. In the vestibular ganglion, only a few neurons were faintly immunoreactive in the fishes and the frog, while the staining was more intense but still not present in all neurons of the chicken, the mouse and the cat. All the neurons were immunoreactive in the baboon. No immunoreactivity was observed in the sensory epithelia of the fishes. All hair cells were strongly immunoreactive in the frog. In the other species, most of the hair cells in the cristae were immunostained except those situated in the peripheral areas. In the maculae, the hair cells of the striola were either the only ones stained or were more intensely stained or were more intensely stained than the others. The localization of calbindin in specific cellular types and its increasing abundance from the fishes to the mammals suggest that calbindin is associated with the capacity of sensory and nerve cells to analyze precise mechanical or biochemical stimulations.

Effect of polyisobutylcyanoacrylate nanoparticles and Lipofectin® loaded with oligonucleotides on cell viability and PKCα neosynthesis in HepG2 cells

The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.

Immuno-electronmicroscopic localization of ‘vitamin D-dependent’ calcium-binding protein (CaBP-28k) in the vestibular hair cells of the cat

The PAP immunohistochemical method was used to carry out a light- and electronmicroscopic study of the distribution of the vitamin D-dependent calcium-binding protein (CaBP-28k, calbindin, cholecalcin) in the vestibule of the young cat. It was found that the two types of hair cells, types I and II, were stained differently. Type II cells were intensely immunoreactive and their staining did not vary with the location of the cells within the crista ampullaris. Type I cells at the top of the cristae were lightly stained, or unstained, while the type I cells laterally or basally were frequently intensely stained. The nerve fibers arriving at the top of the cristae are highly immunoreactive while the fibers of the base are not stained. Immunostaining for CaBP was correlated with differences in the innervation of hair cells at the top and base of the cristae. This differential CaBP-immunostaining may reflect differences in the physiological activity of the cells. The electronmicroscopic study showed that CaBP is present throughout the cytoplasm of the hair cells but that its concentration was particularly high in the cuticular plate and stereocilia. This specific intracellular distribution of CaBP is discussed with the possible role of Ca2+ in the physiology of the vestibular hair cells.

The comparative immunocytochemical distribution of 28 kDa cholecalcin (CaBP) in the hippocampus of rat, guinea pig and hedgehog

The distribution of 28 kDa cholecalcin (calcium-binding protein, CaBP) in the hippocampal formation of the rat, guinea pig and European hedgehog was examined by immunocytochemistry. The extension of the mossy fibers (the axons of the granule cells of the dentate gyrus) was also studied using the Timms sulfide-silver method. Cholecalcin was present in all mossy fibers. In the rat, only those pyramidal cells not reached by the labeled mossy fibers displayed cholecalcin immunoreactivity. Immunocytochemical staining of the hedgehog hippocampus showed that contacts between cholecalcin-containing mossy fibers and cholecalcin-containing pyramidal cells are possible. Consequently, the protein is probably not involved in the control of mossy fiber extension. Strikingly, no guinea pig pyramidal cells showed cholecalcin immunoreactivity. The possible involvement of cholecalcin in the differential excitability of pyramidal cells in the CA3 and CA1 areas of the hippocampus could therefore be tested in a comparative study of rat, guinea pig and hedgehog.

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

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

Evidence for the presence of Calbindin-D 28K (CaBP-28K) in the tibial growth cartilages of rats.

SummaryThe distribution of the vitamin-D dependent calcium-binding protein (Calbindin-D 28K) (CaBP-28K) in the tibial growth plate cartilage of the rat has been studied immunohistochemically using an antibody raised against rat renal CaBP-28K. The protein was detected mainly in the nuclei of chondrocytes and occasionally in the juxta-nuclear cytoplasm. The distribution was not uniform throughout the growth plate, but concentrated in the proliferatively active chondrocytes of the resting and proliferative zones. These findings raise the possibility that CaBP-28K may be involved in the mitotic activity of the chondrocytes, acting as a regulator of the proliferative process, perhaps via intranuclear calcium.