A new method for manufacturing carbon-fibre microelectrodes.

A new, fast and low-cost method using a carbon-fibre microelectrode is proposed. The microelectrode is constructed by glueing one or more carbon fibres (5 mm in diameter) with a silver resin on a silver wire or stainless steel tube. The characteristics of that microelectrode have been measured and compared with those of commercially available glass carbon-fibre microelectrodes. According to our measures, the impedance, capacity and current noise were lowered which permits these electrodes to be used in voltammetry and in detection of very low currents generated during formation of antigen-antibody complex.

Antibody-coated electrodes for detecting somatic exocytosis of somatostatin-like material in Helix neurones.

Immunoelectrodes have been developed which can be used to detect minute amounts of somatostatin. They were made with electrochemically treated glassy carbon fibres coated with anti-somatostatin antibodies. Calibration and various controls were carried out to ensure that the immunoelectrodes responded specifically to the presence of femtomolar somatostatin. Electrophysiological experiments were performed on anti-somatostatin immunoreactive neurones in the snails Helix aspersa and H. pomatia. Somatostatin-like material was released in response to sustained firing. The release was measured at the soma, which means that it occurred in the extrasynaptic area. The finding that the fluorescent dye FM 1-43 was incorporated into somatic vesicles confirmed that exocytosis actually occurred during sustained neuronal firing.

Odorant-evoked potassium changes in the frog olfactory epithelium.

Electroolfactogram (EOG) and extracellular potassium activity (aK) measurements were carried out in frog olfactory epithelia in vivo. Odorant-evoked changes in aK were characterized on the basis of depth profile analysis. Following an olfactory stimulation with butanol vapours, an increase in aK was measured in the mucus and the proximal part of the epithelium; this response started after the beginning of the EOG and was proportional to the amplitude of the latter. In the deeper part of the epithelium, the aK response had complex waveforms showing an initial K decrease which was suppressed by local application of ouabain, suggesting the existence of a pumping mechanism at this level. The results are discussed in terms of extracellular accumulation of K ions following neuroreceptor activation with respect to EOG generation theories.

Influence of GABA, baclofen and bicuculline on the extracellular K+ activity in the olfactory bulb of the developing rat.

gamma-Aminobutyric acid (GABA) and baclofen were iontophoresed into the glomerular and plexiform layers of the olfactory bulb of rats at different ages. Subsequent changes in K+ activity and mitral cell firing rates were monitored to specify GABA receptor activation. Only bicuculline-insensitive receptors are shown to be already present in the glomerular layer of the newborn rat. The possible distribution and maturation of GABA receptors on the olfactory nerve endings or on the dendrites of the mitral or periglomerular cells is discussed.

Electrical stimulation of primary olfactory nerve induces two types of variations in the extracellular potassium activity within the glomerulus of the rat olfactory bulb in vivo.

Primary olfactory nerve stimulation induces a two-step increase in extracellular potassium, as recorded by K+-sensitive microelectrodes inserted into single glomeruli of the rat olfactory bulb. The second rise in K+ (K-peak) occurs in a graded, activity-dependent manner at the basal pole of the glomerular neuropile. This phenomenon appears to be directly related to the rate of extracellular K+ accumulation and is accompanied by a substantial depression of the mitral cell firing rate.

Na+,K(+)-ATPase in the rat olfactory bulb: evidence for a higher enzymatic activity in the glomerular layer.

Protein content and ATPase activities have been determined in the superficial and deep layers of the rat olfactory bulb. Protein levels, Mg2(+)-ATPase and Na+,K(+)-ATPase activities were significantly higher in the whole homogenate for the superficial layers. These differences were amplified when activity was expressed on a wet weight basis in a crude microsomal preparation isolated by differential centrifugation. Specific activities, however, showed similar values in the microsomal fractions from superficial and deep layers. The results are discussed in terms of differences in the density of neuronal processes and efficiency of K+ reuptake mechanisms.

Evidence for an absence of K+ spread in the glomerular layer of the rat olfactory bulb.

Using K+-sensitive microelectrodes inserted into the olfactory bulb, the effects of distant K+ ejection on the extracellular K+ activity (aK), were monitored in the glomerular and plexiform layers. A ouabain-sensitive mechanism, which appeared to be markedly more efficient in the glomerular layer, prevents spread of distal ejected K+. The results are discussed on the basis of Na+,K+-pump activation in both glomerular neuronal networks and the glial capsule enclosing each glomerulus.

Organotypic culture of neuroepithelium attached to olfactory bulb from adult mouse as a tool to study neuronal regeneration after ZnSO4 neuroepithelial trauma.

Chemical destruction of the olfactory mucosa leads to a neuronal regeneration. A new organotypic culture model is perfected to improve the regenerating processes study. Explants of neuroepithelium attached to olfactory bulbs were removed from adult mice and cultured, 12 h after ZnSO4 intranasal application. After 3 days in culture, explants showed a necrosis in the olfactory epithelium and a thinning of the olfactory bulb nervous layer. From the fifth day of culture, and mostly the tenth, new cells showed positive immunoreactivity with the olfactory marker protein (OMP), meaning they were regenerating olfactory neurons. Simultaneously, OMP immunoreactivity increased in the nervous and glomerular layers of the olfactory bulb, indicating epithelio-bulbar reconnection. This organotypic culture model could allow further investigations on the regenerating process kinetic.

Functional alterations in the olfactory bulb of the staggerer mutant mouse.

Putative alterations of the functional activity in the staggerer mutant mouse olfactory bulb neuronal network have been studied by recording odor induced evoked field potentials (EFP) in the mitral cells layer. In standard conditions, the main feature observed in mutants was a significant increase in latency preceding the functional response of the mitral cells to the odorant. In these animals, all parameters of the average EFP were widely modified when compared with those recorded in wild mice. Amplitudes and most of the duration of the EFP phases were significantly decreased. Functional alterations were discussed according to the structural disorganization previously described in staggerer mutant mouse olfactory bulb.

[The sense of smell: analysis of odors and olfactory memory]. / L'odorat: analyse des odeurs et mémoire olfactive.

Olfactory transduction is mediated by neuroepithelial cells localized on turbinal crests in naris. Odorant molecules are uptaked by the mucosa which flows on epithelial surface and some of the molecules enter in contact with olfactory cells tentacles. Odorant molecules interact with molecular receptors bound to cell membrane and induced successive reactions leading to ion channels opening and then to receptor potential appearance. The receptor cells degenerate when molecular receptors are saturated and, further regenerate, according a genetic program and olfactory learning action potentials which appear at the basal pole of the receptor cell are propagated along the small olfactory nerves to the olfactory bulb glomerulus. Several hundred axons enter in one glomerulus among others and make synaptic contacts with one dendritic tree of a mitral cell. The selection of the numerous signals which converge in the glomerulus, the codage in intensity, duration and contrast are made at this level. One odorant molecule activates some glomeruli (rarely one); a complex odor activates a great number of glomeruli according a complex spatial distribution. Following odor stimulation, the electrical signals are analysed in the olfactory bulb which delivers to the olfactory cortex various informations about the odor components that are to be selected and compared to anterior olfactory learning. Animal experimentation and clinical data show that memory storage is made using short, medium and long term memorization. This memory is integrated in the limbic system at the interface of neural structures which regulate the main behaviors and physiological functions. This explains the modulatory role of smell in some behaviors but complicate our understanding of access to olfactory memory.

Postnatal variations of extracellular free calcium levels in the rat. Influence of undernutrition.

Ontogenetic changes in calcium activity were directly measured using an ion-selective micropipette in rat blood plasma and olfactory bulb extracellular fluid. Significant differences were observed according to the age and the nutritional state of the animal.

Intercorrelations among tests of lateralisation in the BALB/c mouse.

In order to examine the reliability of lateralised behaviours, BALB/c mice were tested in three different situations: the Collins paw preference test (PPT), the rotatory swimming test (RST), and the T maze test (TMT). The results showed a significant correlation between the scores of lateralisation in the PPT and the RST, but a lack of lateralisation in the TMT. Considering the tasks involved in these tests, these results appear to support the hypothesis of close links between lateralised behaviours, emotional processes, and neural pathways.

[Modifications of the olfactory bulb electrocorticogram and trans-glomerular evoked potentials in staggerer mutant mice]. / Modifications de l'électrocorticogramme du bulbe olfactif et des potentiels évoqués transglomérulaires chez la souris mutante cérébelleuse staggerer.

Electrophysiological observations have been made on normal C57-BL/6J and staggerer mutant mice. Morphological observations have given evidence it existed various neurones modifications which affected the olfactory bulb in the mutant mice. Olfactory bulb electrocorticograms (ECoG) of mutant have shown rare bursts of potentials of longer time duration than in normal mice. These bursts were less affected by odor stimulations (ammonia and urine of opposite sexes) than in the normal mice and never varied under urine odor influence in female mutant. Evoked potential induced by the odors had long latency and long duration (up to 50 ms vs 30 ms). In a large amount of them, the late phases and the late oscillatory potentials, which generally followed the evoked potential, were absent. All these results improved the idea that staggerer mutation, which mainly affected the N-CAM gene, not only induced cerebellar diseases, but also functionally affects the olfactory bulb.

Immunoelectrodes in protein detection: comparison between glassy carbon and a semimetallic Ni/P thin film as binding support. Biological applications.

Though immunoelectrodes can allow direct detection of very low protein amounts (about 0.1 pmol) in vitro and in vivo, they are not yet widely used because they need quality improvement. Based on a few works devoted to the basic electrochemical phenomenon occurring when antibodies are linked onto a solid support and during antigen/antibody complex formation, we have coated two different supports with antibodies: the classical glassy carbon fiber or an epoxy plate covered with an amorphous semimetallic (nickel/phosphorus) thin film obtained by means of an electrochemical deposit. The antibody/antigen complex formation induces direct and/or indirect ionic movements and a current flow through the conductive support toward a very low-noise and high-sensitivity preamplifier stage in an I/V configuration. The proposed electrochemical treatment (hydrophilization), applied to both carbon and Ni/P electrodes, improves antibody binding and reliability of the response to antigens. The Ni/P probes present several advantages when compared to carbon fiber: better conductivity, possibility of surface quality control, and semimetallic nature, making them unbreakable. Several applications were proposed: somatostatin-14 detection with both carbon fiber and Ni/P plate electrodes, and histamine detection in simple and complex fluid media. Dose-response curves and analysis of the results lead us to conclude that the obtained currents are directly related to the quantity of antigen.

Structural and immunohistological modifications in olfactory bulb of the staggerer mutant mouse.

In the present study, we describe the structural and cytological changes observed in staggerer mutant olfactory bulbs, as compared to normal mice. On the basis of photonic and ultrastructural observations we tried to define the alterations induced by the mutation: i.e. a reduction of bulb size, a reduction in the volume of three out of the six architectonic layers (glomerular, external and internal plexiform), a reduction of glomeruli size, a loss of half the mitral cells and a slight decrease in juxtaglomerular interneuron number. In staggerer, an hypertrophy of glial ensheathing cell processes was especially evident at the level of each glomerulus, whereas the density of the astrocyte network was weaker in the granular layer and the nerve layer not apparently impaired. An immunofluorescent labelling study combined with confocal scanning microscopy was performed in order to identify the cellular type and the differentiation degree of the various elements. Antibodies anti-GFAP, a protein present in both ensheathing cells and astrocytes, and anti-OMP, the specific maturation protein of the nerve layer, were used for that purpose. Data confirmed the reality of the gliosis and the persistence of the sensory component in the mutant. All the structural alterations described in staggerer olfactory bulb were in close agreement with the functional troubles previously recorded. Our results are discussed in connection with the present knowledge on embryonal origin, fetal development and adult cellular renewal of the olfactory bulb.

Effects of amphetamine and phenylethylamine on catecholamine release in the glomerular layer of the rat olfactory bulb.

In the present work, we have shown electrochemically that in the rat olfactory bulb (OB), extracellular dopamine (DA) was highest in the glomerular layer (GL), whereas extracellular noradrenaline (NA) appeared to be more uniformly distributed across layers. The GL catecholamine (CA) responses to amphetamine (AMPH) and phenylethylamine (PEA) were also characterized electrochemically using an in vivo model. Results of this investigation show that at a lower dose (1 mg/kg), PEA had no effect on CA release. In contrast, at a higher dose (10 mg/kg), it produced similar increases in either extracellular DA (17.5 +/- 7%) or extracellular NA (14 +/- 3%), and DA exhibited dose-independent increases to AMPH (93 +/- 8%: 1 mg/kg vs. 97 +/- 6%: 10 mg/kg) whereas NA exhibited dose-dependent increases to AMPH (24.5 +/- 6%: 1 mg/kg vs. 39 +/- 7%: 10 mg/kg). These data indicate that (i) PEA may increase CA release but less efficiently than AMPH. (ii) AMPH is more efficient on the DAergic than on the NAergic system since AMPH-induced DA release exceeded 2-4 times the AMPH-induced NA release.