Roland Pochet

Calbindin and calretinin localization in retina from different species

Calbindin-D28K and calretinin are homologous calcium-binding proteins localized in many neurons of the central nervous systems. We have compared polyclonal antibodies against calbindin and calretinin and have shown by western blots using purified calbindin and calretinin from rat that (1) anti-calretinin does not recognize calbindin and (2) anti-calbindin presents some cross-reactivity with calretinin. In this report, we have compared by immunohistochemistry the localization of both calcium-binding proteins in the retina of monkey, pig, sheep, rat, cat, pigeon, and salamander. These results are compared with previous data for chick. There are many differences between species and not within species, but some aspects of the distribution are conserved. All species, except rat and monkey, have some cones which contain calbindin only. Most species also have some bipolar cells containing calbindin only. Calretinin is rarely seen in photoreceptors or bipolar cells. All species have horizontal cells which contain calretinin or calbindin or both. All species have amacrine cells and ganglion cells containing one or other protein. In the cat ganglion cell layer, the calretinin antisera define a new, asymmetric, type of cell.

CALCIUM BINDING PROTEINS AND NEUROPEPTIDES AS MOLECULAR MARKERS OF GABAERGIC INTERNEURONS IN THE CAT VISUAL CORTEX

SummaryIn the cat visual cortex, almost all parvalbumin-positive cells are GABAergic, and about 80% of the calbindin D-28K-positive neurons are also GABA-immunoreactive. About 37% of the GABAergic neurons contain parvalbumin and a smaller fraction (about 18%) contains calbindin. Furthermore, parvalbumin and calbindin are localized in two separate neuronal populations in the cat visual cortex, suggesting that two GABAergic populations can be distinguished, one containing parvalbumin and one containing calbindin. Double staining for parvalbumin and neuropeptides (CCK, SRIF and NPY), revealed no double-labeled cells, with the exception of a few SRIF- and parvalbumin-positive neurons. These results show that cortical GABAergic cells can be differentiated on basis of their calcium binding protein and neuropeptide immunoreactivity.

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

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

Topographical organization of the projections from physiologically identified areas of the motor cortex to the striatum in the rat.

The present study was undertaken to determine in the rat the topography of the neostriatal projections originating from the motor cortex. For that purpose, anterograde tracers (Phaseolus vulgaris leucoagglutinin: PHA-L; wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) were deposited in discrete cortical sites physiologically identified by microstimulation. Five major motor areas were considered in this study: the rostral (RFL) and caudal (CFL) forelimb areas, the hindlimb (HL) area, the vibrissae motor-frontal eye field (V-FEF) region and the jaw, lips and tongue (JLT) area (according to the nomenclature of Neafsey et al.). The results indicate that functionally different regions of the motor cortex project to different sectors of the caudate putamen (CPU). All 3 distinct limb areas RFL, CFL and HL project to the dorsolateral quarter of the CPU, V-FEF area projects to the dorsomedial quarter, whereas the JLT area projects to the ventrolateral quarter. The pattern of terminal labeling is relatively consistent, whatever the cortical area in which the tracer is deposited. This pattern is characterized by the presence of two or more labeled bands which are obliquely oriented along a ventrolateral-dorsomedial axis. Control experiments were also undertaken in which a retrograde tracer (WGA-HRP) was deposited in various neostriatal loci. The results are congruent with the findings of the anterograde study and further indicate that a given neostriatal sector receives projections from cytoarchitectonically different but functionally related regions of the neocortex. The somatotopic features of both motor and somatosensory corticostriatal projections appear to be in register. In addition, the striatal distribution of motor cortical fibers was compared in 6 experimental cases to the compartmental subdivision of the striatum in patches and matrix, following immunohistochemical localization of calbindin 28 kDa. The calbindin-immunoreactivity is extremely weak in the dorsolateral sector but is higher in the central and ventrolateral parts of the CPU. In these deep striatal regions receiving fibers from V-FEF, JLT and, to a lesser extent, from the limb areas, the cortical fibers are mostly directed to the matrix. The band-like organization of the projection from the motor cortex is correlated to the patch-matrix organization. The patches correspond to the bands of low density of terminal fibers and the matrix to the bands of high terminal density. The present results provide an anatomical basis to both electrophysiological and behavioral observations suggesting that functional distinctions can be established between subregions of the striatum.

Impaired blood-brain and blood-spinal cord barriers in mutant SOD1-linked ALS rat.

Blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) impairment is an additional accident occurring during the amyotrophic lateral sclerosis (ALS) progression. In this work, we aimed to decipher if BBB/BSCB leakage appeared before critical detrimental events and could serve as a marker preceding clinical symptoms. Three different BBB leakage markers: Evans blue, IgG and hemosiderin, were used to look at the SOD1-linked ALS rat model at presymptomatic and symptomatic stages. Although IgG and hemosiderin could be detected at presymptomatic stage, Evans blue extravasation which fits best with BBB/BSCB impairment could only be seen at symptomatic stages. BBB/BSCB impairment was further substantiate by showing at symptomatic stages decreased mRNA expression of ZO-1 and occludin as well as agrin, a basal membrane constituent. Electron microscopic data substantiate a toxic environment around endothelial cell and peri-vascular swollen astrocyte end-feet showing oedema-linked BBB opening.

Supratentorial Pilocytic Astrocytomas, Astrocytomas, Anaplastic Astrocytomas and Glioblastomas are Characterized by a Differential Expression of S100 Proteins

The levels of expression of the S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B proteins were immunohistochemically assayed and quantitatively determined in a series of 95 astrocytic tumors including 26 World Health Organization (WHO) grade I (pilocytic astrocytomas), 23 WHO grade II (astrocytomas), 25 WHO grade III (anaplastic astrocytomas) and 21 WHO grade IV (glioblastomas) cases. The level of the immunohistochemical expression of the S100 proteins was quantitatively determined in the solid tumor tissue (tumor mass). In addition twenty blood vessel walls and their corresponding perivascular tumor astrocytes were also immunohistochemically assayed for 10 cases chosen at random from each of the four histopathological groups. The data showed modifications in the level of S100A3 protein expression; these modifications clearly identified the pilocytic astrocytomas from WHO grade II‐IV astrocytic tumors as a distinct biological group. Modifications in the level of S100A6 protein expression enabled a clear distinction to be made between low (WHO grade I and II) and high (WHO grade III and IV) grade astrocytic tumors. Very significant modifications occurred in the level of S100A1 protein expression (and, to a lesser extent, in their of the S100A4 and S100B proteins) in relation to the increasing levels of malignancy. While the S100A5 protein was significantly expressed in all the astrocytic tumors (but without any significant modifications in the levels of malignancy), the S100A2 protein was never expressed in these tumors. These data thus indicate that several S100 proteins play major biological roles in human astrocytic tumors.

Calbindin in vertebrate classes: immunohistochemical localization and Western blot analysis.

Calbindin immunoreactivity was investigated in various vertebrates. Positive labeling was observed in the absorptive cells of the duodenum of all birds and reptiles but not in mammals, amphibia, or fish. Staining was present in the kidney distal convoluted tubule from amphibia and higher vertebrates. Fish kidney was negative. In the central nervous system of all species investigated, cellular bodies and fibers were Calbindin positive. Their distribution was quite broad and correlates well with the previously reported mapping for chick and rat. Western blot analysis revealed two Calbindins in brain from mammals, birds, reptiles, and amphibia (27,000 and 29,000 Da). Only one band was detected in fish. We conclude that Calbindin from the evolutionary point of view is primarily a neuronal protein, with a highly conservative character.

Rat brain synthesizes two ‘vitamin D-dependent’ calcium-binding proteins

Two proteins from rat brain reacting against anti-chick intestinal vitamin D-dependent calcium-binding protein were characterized in terms of their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their molecular size. The proteins were present in the isolated cytoplasm and were produced following translation of brain mRNA in the rabbit reticulocyte lysate system. Their apparent molecular weight was 29,000 and 27,000 daltons whereas rat kidney contained only one protein cross-reacting with this antiserum and with a molecular weight of 27,000 daltons.

S100A2, a Putative Tumor Suppressor Gene, Regulates In Vitro Squamous Cell Carcinoma Migration

It has been previously shown that S100A2 is down-regulated in tumor cells and can be considered a tumor suppressor. We have recently shown that this down-regulation can be observed particularly in epithelial tissue, where S100A2 expression decreases remarkably in tumors as compared with normal specimens. In the present paper we investigate whether S100A2 could play a tumor-suppressor role in certain epithelial tissues by acting at the cell migration level. To this end, we made use of five in vitro human head and neck squamous cell carcinoma lines in which we characterized S100A2 expression at both RNA and protein level. To characterize the influence of S100A2 on cell kinetic and cell motility features, we used two complementary approaches involving specific antisense oligonucleotides and the addition of S100A2 to the culture media. The different expression analyses gave a coherent demonstration of the fact that the FADU and the RPMI-2650 cell lines exhibit high and low levels of S100A2 expression, respectively. Antisense oligonucleotides (in FADU) and extracellular treatments (in RPMI) showed that, for these two models, S100A2 had a clear inhibitory influence on cell motility while modifying the cell kinetic parameters only slightly. These effects seem to be related, at least in part, to a modification in the polymerization/depolymerization dynamics of the actin microfilamentary cytoskeleton. Furthermore, we found evidence of the presence of the receptor for advanced glycation end-products (RAGE) in RPMI cells, which may act as a receptor for extracellular S100A2. The present study therefore presents experimentally based evidence showing that S100A2 could play a tumor-suppressor role in certain epithelial tissues by restraining cell migration features, at least in the case of head and neck squamous cell carcinomas.

Re-induction of hyponatremia after rapid overcorrection of hyponatremia reduces mortality in rats.

Osmotic demyelination syndrome is a devastating neurologic disorder often seen after the rapid correction of chronic hyponatremia. The permeability of the blood-brain barrier is increased in experimental osmotic demyelination, and some have suggested that corticosteroids protect against this disorder by keeping the permeability of the blood-brain barrier low. We previously reported that re-lowering of the serum sodium after rapid correction of chronic hyponatremia was beneficial if performed early in the course (12 to 24 h). Here we compared mortality, blood-brain barrier permeability, and microglial activation in rats after the rapid correction of chronic hyponatremia. We studied three groups of rats after correction of chronic hyponatremia: and treated them with sodium chloride, with or without dexamethasone; or with sodium chloride followed by re-induction of hyponatremia. We found that treatment with dexamethasone or re-induction of hyponatremia effectively prevented the opening of the blood-brain barrier, reduced neurological manifestations, and decreased microglial activation; however, only re-induction of hyponatremia resulted in a significant decrease in mortality 5 days after the correction of chronic hyponatremia. Restoring the permeability of the blood-brain barrier to normal levels did not decrease mortality. Our results suggest that after inadvertent rapid correction of hyponatremia, treatment options should favor re-lowering serum sodium. The increased permeability of blood-brain barrier seen in osmotic demyelination syndrome may not be a primary pathophysiologic insult of this syndrome.