PubMed Health⌕ Search

Biomedical subjects

M Robello

Publications and source records attributed to M Robello.

At least 19 recordsLinked to original sources
PubMed2005

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABA(A) receptor function.

Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present. The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA(A) receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA(A) receptors.

Animals↗
PubMed2003

Immunocytochemical study by two photon fluorescence microscopy of the distribution of GABA(A) receptor subunits in rat cerebellar granule cells in culture.

An immunocytochemical investigation of the expression of alpha(1), alpha(6), beta(2/3), gamma(2) and delta subunits was performed on rat cerebellum granule cells in culture by the two photon microscopy technique. The first four subunits appear to be expressed abundantly in these cells, whereas the delta one seems to be expressed at a lower level. Another major difference in the distribution of these subunits is that whereas alpha(6), beta(2/3) and gamma(2) appear only on plasma membranes alpha(1) and delta are present mainly in the cell bodies cytoplasm. Still another difference was found in that the presence of gamma(2) on neurites is "polarized", preferentially labelling neurites with the appearance of dendrites. The subunits alpha(6) and beta(2/3) appear to label all types of neurites, with beta(2/3) being by far the most heavily expressed subunit type. A final distinct characteristic is that alpha(6) and, even more, gamma(2) appear to accumulate in the cytoplasmic domains immediately below the cone of emergence of neurites. This suggests a conspicuous transport of such subunits from the site of synthesis in the cell body to the site of final expression in the neurites (dendrites and axon terminals).

Animals↗
PubMed2002

Modulation by Zn(2+) and Cd(2+) of GABA(A) receptors of rat cerebellum granule cells in culture.

This study aims to characterize more closely the different populations of GABA(A) receptors present on the cerebellar granule cells of the rat. The effects of two divalent cations, Zn(2+) and Cd(2+), on GABA-activated chloride currents were studied using the whole-cell patch-clamp technique. Zinc cations inhibit differently the peak and the steady-state current elicited by 10 micro M GABA. In fact, Zn(2+) appears to be more potent in inhibiting the steady-state component, with a lower IC(50). The inhibition of the peak component is of the competitive type, whereas the inhibition of the steady-state one is mixed, being partly competitive and partly allosteric. In addition, Cd(2+) has an inhibitory effect on GABA-activated chloride currents. In terms of the peak component, its effect is limited in extent with a maximal inhibition of only 26%, but with a high affinity (IC(50) as low as 0.03 micro M). The steady-state component is inhibited by 20% independently from the Cd(2+) concentration, in the 10(-2)-10(2) micro M range. In this case, the inhibitory mechanism appears to be of the competitive type for the peak component and of the allosteric type for the steady-state one. We suggest these data are a further confirmation that the rapidly and slowly desensitizing components of the GABA-activated chloride currents, corresponding respectively to the peak and the steady-state components, are made up of two different receptor populations.

Animals↗
PubMed2002

Three-dimensional mapping of cholinergic molecules by confocal laser scanning microscopy in sea urchin larvae.

Confocal laser scanning microscopy (CLSM) was used to examine molecules related to the cholinergic neurotransmission system and detected at all the larval stages of Paracentrotus lividus, by histochemical and immunohistochemical methods. CLSM, providing spatial resolution of the cells located both at the larval surface and at depth, allows a complete mapping in a three-dimensional volumetric frame. At early larval stages acetylcholinesterase- as well as choline acetyltransferase-like molecules were found mainly in the gut wall cells, and along the ciliary bands of the arms, together with muscarinic acetylcholine receptors. At perimetamorphic stages, cholinergic molecules were present in the ciliate strands along the arms, in the larval body and in the rudiment. At metamorphosis, positivity to cholinergic molecules translocated to the juvenile, where a high frequency of mAChR- and ChAT-like positive cells was found.

Animals↗
PubMed2001

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity.

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.

Alkaline Phosphatase↗
PubMed2001

Modulation by lanthanum ions of gamma-aminobutyric acid(A) receptors of rat cerebellum granule cells in culture: clues on their subunit composition.

Gamma-aminobutyric acid (GABA) activated chloride currents were studied in rat cerebellum granule cells in culture by the whole cell patch-clamp technique. Both the peak and steady state currents were inhibited by 100 microM lanthanum. In the first case, inhibition is due to an increase of the EC50 for GABA. The inhibitory effect of lanthanum on the peak current at 3 microM GABA increased with the cation concentration. A tendency towards the same behavior was found also for the inhibition of the steady state current, at 3 microM GABA, as a function of lanthanum concentration, although inhibition in this case was lower. The comparison of the results with published data about the effects of lanthanum on recombinant GABA(A) receptors likely to occur in granule cells allows suggestions about the receptor types giving, respectively, the peak and the steady state component.

Animals↗
PubMed2001

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy.

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.

Animals↗
PubMed2001

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells.

GABAA receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABAA receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABAA receptors mediating the tonic form of inhibition in cerebellar granules.

Animals↗
PubMed2001

Two-photon microscopy and spectroscopy based on a compact confocal scanning head.

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Animals↗
PubMed2000

Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration.

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.

Algorithms↗
PubMed2000

Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106-126.

Prion diseases are neurodegenerative pathologies characterized by the accumulation, in the brain, of altered forms of the prion protein (PrP), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) was reported to maintain the neurodegenerative characteristics of PrP(Sc). We investigated the intracellular mechanisms involved in PrP106-126-dependent degeneration of primary cultures of cerebellar granule neurons. Prolonged exposure of such neurons to PrP106-126 induced apoptotic cell death. The L-type voltage-sensitive calcium channel blocker nicardipine reproduced this effect, suggesting that blockade of Ca(2+) entry through this class of calcium channels may be responsible for the granule cell degeneration. Microfluorometric analysis showed that PrP106-126 caused a reduction in cytosolic calcium levels, elicited by depolarizing K(+) concentrations in these neurons. Electrophysiological studies demonstrated that PrP106-126 and nicardipine selectively reduce the L-type calcium channel current. These data demonstrate that PrP106-126 alters the activity of L-type voltage-sensitive calcium channels in rat cerebellar granule cells and suggest that this phenomenon is related to the cell death induced by the peptide.

Animals↗
PubMed2000

Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures.

Techniques based on two-photon excitation (TPE) allow three-dimensional (3D) imaging in highly localized volumes, of the order of magnitude of a fraction of a femtolitre up to single-molecule detection. In TPE microscopy a fundamental advantage over conventional widefield or confocal 3D fluorescence microscopy is given by the use of infrared (IR) instead of ultraviolet (UV) radiation to excite those fluorophores requiring UV excitation, hence causing little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sample compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realized a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture is a mode-locked Ti:sapphire infrared pulsed laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse width from 65 to 95 fs as a function of wavelength (690-830 nm). The scanning head allows conventional and two-photon confocal imaging. Point spread function measurements are reported with examples of applications to the study of biological systems.

Animals↗
PubMed2000

Changes in the endoplasmic reticulum structure of Paramecium primaurelia in relation to different cellular physiological states.

The fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], a vital dye utilized to stain the endoplasmic reticulum (ER) of animal and plant cells, has been used to visualize the ER-type structures of Paramecium primaurelia under confocal laser scanning microscopy (CLSM). The morphology of the ER has been studied in paramecia in different physiological conditions. Cells are analysed in early and late logarithmic growth phases, in stationary and in death phases, during shift-up by refeeding after starvation and shift-down by using a starvation medium. In log-phase growing paramecia, the ER constitutes an anastomosing membrane system consisting of short tubules and flattened sacs forming a peripheral network, which is abundant in the cortical region around the trichocysts and the ciliary basal bodies. The tubular network and cytoplasmic membranes are reduced in stationary-phase cells; the original conditions are restored in starved cells after refeeding. The analysis of serial optical sections collected by CLSM at 0.5 microm intervals and three-dimensional reconstruction from these sections allow us to visualize differences between differently growing cells.

Animals↗
PubMed2000

GABA(A) receptor modulation in rat cerebellum granule cells.

The inhibitory GABA(A) receptor is a key element in determining the pattern of nerve cell electrical activity. Thus, modulation of its function is of paramount impact in shaping neuronal functional activity under physiological and pathological conditions. This applies to cerebellar granule neurons as to all the other neurons in the brain. The culture of cerebellar granules from newborn rats is a convenient means by which to approach these cells for electrophysiological studies provided that they maintain, as far as GABA(A) receptors are concerned, the same characteristics as in situ. Thus, the regulation of GABA(A) receptor activity in these neurons has been studied by the patch-clamp technique, both in the whole-cell and outside-out configuration. An obvious first level of control of such receptors' activity is their desensitization under continued agonist application, with biphasic kinetics. The data do not allow one to conclude whether one is dealing with two different populations of receptors or with a single population with two desensitization phases; although the presence of two GABA(A) receptor populations is suggested by a host of observations. The granule cell GABA(A) receptors are modulated by changes in extracellular pH with lower pH resulting in an enhanced receptor activity. They display, under the conditions of whole-cell recording, a run-down phenomenon which is most probably due to a tyrosine phosphatase activity which is in turn under control by a protein serine kinase. Thus, in situ tyrosine phosphorylation is a key element in determining the efficiency of GABA mediated inhibition. Activation of protein kinase A or protein kinase G (PKG) down-regulates GABA(A) receptors' activity. This last event is involved in the depression of those receptors' activity by L-arginine via the production of nitric oxide. In addition, the activity of calmodulin-activated adenylate cyclase I is controlled by GABA(B) receptors. Dendritic GABA(A) receptor activity is partially blocked by previous activation of N-methyl-D-aspartate (NMDA) receptors via calcineurin mediated dephosphorylation/activation of protein tyrosine phosphatase and concomitant production of nitric oxide and PKG activation. The site phosphorylated by PKG is evidently not available for calcineurin-mediated serine dephosphorylation, due to calcineurin-specific membrane localization in respect of the GABA(A) receptor. Overall, a complex network of biochemical signals appear to keep granule cells GABA(A) receptors under a fine balance between up- and down-regulatory mechanisms. The overall data appear also to indicate the presence of two GABA(A) receptor populations: a dendritic one which can be modulated by Ca++ entering via NMDA receptors and a cell body one. The two populations are probably different in terms of desensitization kinetics and benzodiazepine sensitivity.

Animals↗
PubMed1999

Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization.

GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors.

Animals↗
PubMed1999

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Animals↗
PubMed1999

GABA(B) receptor activation protects GABA(A) receptor from cyclic AMP-dependent down-regulation in rat cerebellar granule cells.

Interaction between GABAA and GABA(B) receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA(B) agonist (-)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (-)baclofen dose-dependently prevents the reduction of GABA(A) receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA(B) receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA(A) receptor function. On the whole, the data indicate the presence of complex modulation of GABA(A) receptors by GABA(B) receptor types in cerebellum granule cells.

Adenylate Cyclase Toxin↗