Cell malpositioning has been described in laminated structures of the spontaneous mutation, reeler, including the cerebellum, the hippocampus, and the neocortex. Despite the ectopic positions of different neuronal populations, the specificity of synaptic connections is maintained. The metabolic consequences of this form of neuropathology were examined in Reln(rl) mutant mice by quantitative measures of cytochrome oxidase (CO) activity, a mitochondrial enzyme essential for oxidative metabolism in neurons. Despite severe tissue disorganization but in line with the intact synaptic organization, the reeler mutation did not affect global metabolic activity of the laminated structures of the brain. CO activity, however, was altered in specific subregions of the cerebellum, hippocampus, and neocortex, as well as in septum and various brainstem (medial pontine, paramedial reticular, paragigantocellular reticular) regions anatomically related to these structures, attesting to large functional alterations in Reln(rl-orl) brain. Metabolic activity variations were also detected in the ventral tegmental area and ventral neostriatum of the mesolimbic dopaminergic pathway. The results are discussed and compared to the regional CO variations found in other ataxic mice, in regard to the structural defects, the integrity of the connections, and the mutation-specific effects. (C) 2006 Wiley-Liss, Inc.

The Relnrl-orl mutation is characterized by a marked deficit in cerebellar granule cell and Purkinje cell number as well as ectopias in cerebellum, hippocampus, and neocortex. By comparison to Balb/c controls, Relnrl-orl mutants did not alternate spontaneously in a T-maze and were deficient for visuomotor guidance in a water maze. Despite cerebellar ataxia and motor coordination impairments on stationary beam, coat-hanger, and rotorod tests, the horizontal motor activity of Relnrl-orl mutants was not reduced in an open-field. The elevated cytochrome oxidase (CO) activity in Purkinje cells and the reduced CO activity in the roof nuclei (interpositus and dentate) of the mutants were associated with poor performance on the small stationary beam. In addition, deficient CO activity of the granular layer of the motor cortex was associated with shorter latencies before falling from the larger stationary beam and a lower number of rears in the open-field. Conversely, elevated CO activity in the polymorphic layer of primary somatosensory cortex was congruent with higher latencies before falling from the same apparatus, indicating functional compensation.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

Here we examine the role of Reelin, an extracellular protein involved in neuronal migration, in the formation of hippocampal connections. Both at prenatal and postnatal stages, the general laminar and topographic distribution of entorhinal projections is preserved in the hippocampus of reeler mutant mice, in the absence of Reelin. However, developing and adult entorhinal afferents show severe alterations, including increased numbers of misrouted fibers and the formation of abnormal patches of termination from the medial and lateral entorhinal cortices. At perinatal stages, single entorhinal axons in reeler mice are grouped into thick bundles, and they have decreased axonal branching and decreased extension of axon collaterals. We also show that the number of entorhino-hippocampal synapses is lower in reeler mice than in control animals during development. Studies performed in mixed entorhino-hippocampal co-cultures combining slices from reeler and wild-type mice indicate that these abnormalities are caused by the lack of Reelin in the target hippocampus. These findings imply that Reelin fulfills a modulatory role during the formation of layer-specific and topographic connections in the hippocampus. They also suggest that Reelin promotes maturation of single fibers and synaptogenesis by entorhinal afferents.

In studies of dog atopy, we have found that the high IgE responder status is inherited in a dominant manner. However, various environmental factors are required in addition to allow full expression of the gene. Such are a very early contact with some allergen to start the IgE response, allergen administration by injection and the absence of feeding with allergen during the first months of life. These observations show that the late determination of the allergic phenotype does not enable to make correct conclusions about the atopic genotype. Various factors which drive the dogs to IgE responses and atopy appear also to play a role in man.

Allergy diagnosis in dogs is still a difficult task for the veterinary surgeon. Skin tests, which are often considered as "golden standard", are cumbersome and in part unreliable, as shown here by investigations with various commercial allergen extracts. Serological determinations of allergen-specific IgE with the help of polyclonal anti-IgE antibodies are also unreliable, because they detect IgG antibodies aside from IgE. Intensive serological and experimental research in this field in the past three years has permitted: to establish accurate, sensitive and highly specific methods for determination of total and allergen-specific IgE. This has enabled development of reliable diagnostic tests which can be performed in the veterinary practice; to obtain a clear picture on genetics of IgE regulation (segregation between allergic and non allergic dogs); to evaluate the clinical relevance of IgE with the help of skin tests, bronchial provocation and experimental induction of skin lesions very similar to the spontaneously occurring atopic dermatitis; to achieve better understanding of pathophysiology of IgE mediated reactions thanks to cellular tests. The results of four clinical studies with a total of about 500 dogs have shown that strip tests performed with monoclonal anti-IgE antibodies enable a sensitive and very specific allergy diagnosis in dogs, which is also usable in the veterinary practice. The available monoclonal anti-IgE antibodies enable determination of total and allergen-specific IgE in a simple procedure, Thanks to an improved serological diagnosis, indications and future results of specific immunotherapy can be put on a scientific basis. Besides serological determination of IgE, skin tests are a very useful diagnostic tool. There are, however, several possible errors in their performance and interpretation. Epidermal allergens and mould appear frequently to produce false positive reactions. Skin tests should no longer be considered as sole "gold standard" in diagnosis of dog allergy.

Le test Immunodot Top Screen consiste en un test sur bandelettes pour la détermination sérologique des anticorps IgE spécifiques. Il permet avec une manipulation simple d'effectuer un large dépistage des allergies IgE-dépendantes. Avec l'anamnèse, le test Immunodot Top Screen permet de poser le diagnostic d'atopie à peu de frais et avec une grande exactitude.

Top Screen is a serologic strip test for the detection of allergen-specific IgE directed against several allergen groups. It enables with a single manipulation to achieve a broad screening for IgE-mediated allergies. Together with patient's history, Top Screen permits to diagnose atopy with high reliability and with low cost. In addition, in case of a positive result, the test gives indications about the directions in which further investigations should be performed. In hands not trained for allergology practice, Top Screen enables a primary allergy diagnosis with better quality control than is possible with skin tests.

A new technique for determination of total IgE and specific IgE using immunodot nitrocellulose strips is presented. In the kit proposed for adults, the total IgE test gives reproducible results in the range of 10 to 400 IgE IU/ml while in the kit for investigation of cord blood, the range is 0,5 to 20 IgE IU/ml. The test shows excellent correlation with the Pharmacia PRIST test for the range of 10 to 400 IU/ml. In cord blood, the Immunodot test detects a larger number of sera over 1 IU/ml than the Ultra Low PRIST assay, due to the fact that the Immunodot test detects also IgE complexed with IgG anti-IgE antibodies transmitted to the newborn by the mother. The test developed for detection of specific IgE enables in a simple way to evaluate antibodies directed against various pollen allergens (e.g. grasses, rye, birch, mugwort, plantain) or indoor allergens (house dust mites, cat, dog, moulds). The results, expressed in Units (ERU/ml) or in classes show a good correlation with several other diagnostic serological methods, such as RAST, CAP, EAST and MATRIX. Although not automated, this technique enables to test easily and simultaneously 30-40 sera with minimal handling, economically and without costly investments for instruments.

Demonstration of NADPH diaphorase (NADPH-d) activity in the nervous system has recently gained considerable interest since it has been shown that this enzyme is a nitric oxide synthase (NOS). Therefore, histochemical staining of NADPH-d activity provides a specific labelling of neurons that use nitric oxide (NO). In this work, spatiotemporal distribution of NADPH-d neurons has been determined during forebrain ontogenesis. NADPH-d neurons first appeared between embryonic days 15 and 16 and were confined to the fronto-lateral aspect of the incipient corpus striatum and cortical subplate but were not present in the cortical plate. Until birth, NADPH-d neurons differentiated progressively in cortical subplate and striatum in rostro-caudal and latero-medial directions. For both regions, the adult cortical pattern was established during the first postnatal week. The pattern of genesis of NADPH-d neurons might be related to the spatiotemporal ontogenesis of catecholaminergic afferents to the forebrain described previously in the literature.

Cajal-Retzius cells, which are present transiently in the first layer of the mammalian neocortex, have been revealed in the mouse by DiI. This lipophilic fluorescent dye, locally applied over the cortex after formaldehyde fixation, allowed the global view of cortical cells. During ontogenesis, Cajal-Retzius cells retained their initial characteristic bipolar shape and orientation parallel to the meningeal surface. The bright fluorescent light emitted by this dye allowed visualization of the labelled cells by "microtomoscopy" using a confocal scanning laser microscope and analysis of the detailed aspect of these neurons and of their connections.

Cajal-Retzius cells, which are present transiently in the first layer of the mammalian neocortex, have been revealed in the mouse by DiI. This lipophilic fluorescent dye, locally applied over the cortex after formaldehyde fixation, allowed the global view of cortical cells. During ontogenesis, Cajal-Retzius cells retained their initial characteristic bipolar shape and orientation parallel to the meningeal surface. The bright fluorescent light emitted by this dye allowed visualization of the labelled cells by "microtomoscopy" using a confocal scanning laser microscope and analysis of the detailed aspect of these neurons and of their connections.

The ontogenetic development of Cajal-Retzius cells was studied in mouse by local application of horseradish peroxidase over the developing neocortex, revelation with 3,3′-diaminobenzidene and examination from horizontal thick sections. Cajal-Retzius cells were completely stained in Golgi-like fashion. The Cajal-Retzius cells were seen to be elongated spindle-shaped bipolar neurons with their main processes horizontally oriented. They were exclusively located in the first cortical layer and were connected to the cortex surface by numerous vertical appendages. Except for these appendages, the Cajal-Retzius cells were two-dimensional, with an immature structure at their tips resembling a growth cone. Cajal-Retzius cell dendrites were up to 400-μm-long and reached their maximal length prenatally. Their axon and its collaterals were very fine and sometimes measured several millimetres. It followed a random but planar trajectory confined to the first layer. Healthy Cajal-Retzius cell bearing growth cones were seen until one week after birth when signs of Cajal-Retzius cell degeneration began to occur and intensified in the days that followed. Rough endoplasmic reticulum and Golgi complex swelling along with a progressive darkening of the Cajal-Retzius cells were revealed by electron microscopy, strongly suggesting that most Cajal-Retzius cells disappear from the first cortical layer. Usually neuronal death is the result of cell deafferentation following synapse retraction; however, this effect does not seem to apply to Cajal-Retzius cells engaged in the process of death since normal synaptic junctions were seen on them. No signs of the morphological transformation of Cajal-Retzius cells into persisting horizontal first layer cells were observed.

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.