Evoked responses to single pulse electrical stimulation reveal impaired striatal excitability in a rat model of Parkinson's disease.

BACKGROUND: Sensorimotor beta oscillations are increased in Parkinson's disease (PD) due to the alteration of dopaminergic transmission. This electrophysiological read-out is reported both in patients and in animal models such as the 6-OHDA rat model obtained with unilateral nigral injection of 6-hydroxydopamine (6-OHDA). Current treatments, based on dopaminergic replacement, transiently normalize this pathological beta activity and improve patients' quality of life. OBJECTIVES: We wanted to assess in vivo whether the abnormal beta oscillations can be correlated with impaired striatal or cortical excitability of the sensorimotor system and modulated by the pharmacological manipulation of the dopaminergic system. METHODS: In the unilateral 6-OHDA rat model and control animals, we used intra-striatal and intra-cortical single-pulse electrical stimulation (SPES) and concurrent local field potentials (LFP) recordings. In the two groups, we quantified basal cortico-striatal excitability from time-resolved spectral analyses of LFP evoked responses induced remotely by intracerebral stimulations. The temporal dependance of cortico-striatal excitability to dopaminergic transmission was further tested using electrophysiological recordings combined with levodopa injection. RESULTS: LFP evoked responses after striatal stimulation showed a transient reduction of power in a large time-frequency domain in the 6-OHDA group compared to the sham group. This result was specific to the striatum, as no significant difference was observed in cortical LFP evoked responses between the two groups. This impaired striatal excitability in the 6-OHDA group was observed in the striatum at least during the first 3 months after the initial lesion. In addition, the striatum responses to SPES during a levodopa challenge showed a transient potentiation of the decrease of responsiveness in frequencies below 40 Hz. CONCLUSION: The spectral properties of striatal responses to SPES show high sensitivity to dopaminergic transmission in the unilateral 6-OHDA rat model. We thus propose that this approach could be used in preclinical models as a time-resolved biomarker of impaired dopaminergic transmission capable of monitoring progressive neurodegeneration and/or challenges to drug intake.

Differential Effects of Antiepileptic Drugs on Focal Seizures in the Intrahippocampal Kainate Mouse Model of Mesial Temporal Lobe Epilepsy.

AIMS: Mesial temporal lobe epilepsy (MTLE) is the most common form of drug-refractory epilepsy. Most of the morphological and electrophysiological features of human MTLE can be reproduced in a mouse by a unilateral intrahippocampal injection of kainate (MTLE mouse model). The effects of antiepileptic drugs (AEDs) on the occurrence of recurrent focal hippocampal seizures in this model remain to be specified. Here, we addressed the pharmacological reactivity of this model to the most commonly used AEDs. METHODS: Using depth electroencephalographical (EEG) recordings, we tested the dose-response effects of acute injection of nine AEDs on the occurrence of hippocampal paroxysmal discharges (HPDs) as well as on ictal and interictal power spectra in the MTLE mouse model. RESULTS: Valproate, carbamazepine, and lamotrigine dose dependently suppressed HPDs and modified the general behavior and/or EEG activity. Levetiracetam and pregabalin suppressed HPDs at high doses but without any behavioral nor interictal EEG changes. Finally, phenobarbital, tiagabine, vigabatrin, and diazepam suppressed HPDs in a dose-dependent manner at doses devoid of obvious behavioral effects. CONCLUSION: The MTLE mouse model displays a differential sensitivity to AEDs with a greater efficacy of drug that facilitates GABAergic transmission. This model provides an efficient tool to identify new treatment for drug-resistant forms of focal epilepsies.

Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.

Advanced fluorescence technologies help to resolve long-standing questions about microbial vitality.

Advances in fundamental physical and optical principles applied to novel fluorescence methods are currently resulting in rapid progress in cell biology and physiology. Instrumentation devised in pioneering laboratories is becoming commercially available, and study findings are now becoming accessible. The first results have concerned mainly higher eukaryotic cells but many more developments can be expected, especially in microbiology. Until now, some important problems of cell physiology have been difficult to investigate due to interactions between probes and cells, excretion of probes from cells and the inability to make in situ observations deep within the cell, within tissues and structures. These technologies will enable microbiologists to address these topics. This Review aims at introducing the limits of current physiology evaluation techniques, the principles of new fluorescence technologies and examples of their use in this field of research for evaluating the physiological state of cells in model media, biofilms or tissue environments. Perspectives on new imaging technologies, such as super-resolution imaging and non-linear highly sensitive Raman microscopy, are also discussed. This review also serves as a reference to those wishing to explore how fluorescence technologies can be used to understand basic cell physiology in microbial systems.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes.

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30 degrees C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-beta-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.

Chronic administration of atypical antipsychotics improves behavioral and synaptic defects of STOP null mice.

INTRODUCTION: Recent studies have suggested that schizophrenia is associated with alterations in the synaptic connectivity involving cytoskeletal proteins. The microtubule-associated protein stable tubule only polypeptide (STOP) plays a key role in neuronal architecture and synaptic plasticity, and it has been demonstrated that STOP gene deletion in mice leads to a phenotype mimicking aspects of positive and negative symptoms and cognitive deficits classically observed in schizophrenic patients. In STOP null mice, behavioral defects are associated with synaptic plasticity abnormalities including defects in long-term potentiation. In these mice, long-term administration of typical antipsychotics has been shown to partially alleviate behavioral defects but, as in humans, such a treatment was poorly active on deficits related to negative symptoms and cognitive impairments. Here, we assessed the effects of risperidone and clozapine, two atypical antipsychotics, on STOP null mice behavior and synaptic plasticity. RESULTS: Long-term administration of either drug results in alleviation of behavioral alterations mimicking some negative symptoms and partial amelioration of some cognitive defects in STOP null mice. Interestingly, clozapine treatment also improves synaptic plasticity of the STOP null animals by restoring long-term potentiation in the hippocampus. DISCUSSION: All together, the pharmacological reactivity of STOP null mice to antipsychotics evokes the pharmacological response of humans to such drugs. Totally, our study suggests that STOP null mice may provide a useful preclinical model to evaluate pharmacological properties of antipsychotic drugs.

High heterogeneity of plasma membrane microfluidity in multidrug-resistant cancer cells.

Diffusion-time distribution analysis (DDA) has been used to explore the plasma membrane fluidity of multidrug-resistant cancer cells (LR73 carcinoma cells) and also to characterize the influence of various membrane agents present in the extracellular medium. DDA is a recent single-molecule technique, based on fluorescence correlation spectroscopy (FCS), well suited to retrieve local organization of cell membrane. The method was conducted on a large number of living cells, which enabled us to get a detailed overview of plasma membrane microviscosity, and plasma membrane micro-organization, between the cells of the same line. Thus, we clearly reveal the higher heterogeneity of plasma membrane in multidrug-resistant cancer cells in comparison with the nonresistant ones (denoted sensitive cells). We also display distinct modifications related to a membrane fluidity modulator, benzyl alcohol, and two revertants of multidrug resistance, verapamil and cyclosporin-A. A relation between the distribution of the diffusion-time values and the modification of membrane lateral heterogeneities is proposed.

Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts.

Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY-2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl-beta-cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl-conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid-phase heterogeneity in correlation with the disruption of phytosterol-rich domains. Methyl-beta-cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains.

Influence of the chain length of ubiquinones on their interaction with DPPC in mixed monolayers.

The thermodynamic behavior of representative short (UQ2), middle (UQ4 and UQ6) and long-chain (UQ10) ubiquinones (UQ) mixed with dipalmitoyl-phosphatidylcholine (DPPC) was studied in monolayers at the air-water interface. The influence of isoprenoid chain-length of UQ on miscibility of both lipids was investigated by analysis of surface pressure-area isotherms and using fluorescence microscopy. Analysis of excess areas (A(ex)) and free energies of mixing (DeltaGm), calculated from compression isotherms in the full range of ubiquinones concentrations, has given evidences for UQ-rich constant-size (UQ6, UQ10) or less growth limited (UQ2, UQ4) microdomains formation within mixed films. Fluorescence microscopy observation revealed that ubiquinones are preferentially soluble in the expanded phase. When lateral pressure increased, concomitant evolutions of A(ex) and DeltaGm parameters, and composition dependence of collapse surface pressures, argue for an evolution towards a total segregation, never reached due to expulsion of ubiquinones from the film. The possible significance of these observations is discussed in relation to ubiquinones organization and similar chain length effects in membranes.

Fibrinogen mediates bladder cancer cell migration in an ICAM-1-dependent pathway.

Fibrinogen (fg), present in tumor matrices, has been demonstrated to be determinant in metastatic potential. We have recently shown that fg/ICAM-1 interactions are involved in leukocyte migration across endothelial cell monolayers. Using bladder transitional cell carcinoma as a model, we will show in this study that bladder high grade tumor cell lines express ICAM-1, and that this expression induces an fg-mediated migration. This phenomenon was dependent on ICAM-1/fg interaction as well as RhoA activity. ICAM-1 was concentrated in focal adhesion plaques when tumor cells were allowed to adhere on immobilized fg, suggesting a role in cell migration. The addition of fg induced a 3- to 6-fold enhancement of bladder tumor cell migration through HUVEC monolayers. This process was inhibited by an anti-ICAM-1 antibody blocking fg binding, demonstrating that ICAM-1/fg interaction was involved in the extravasation process. Finally, immunohistological studies revealed that the expression of ICAM-1 was closely associated with an infiltrative histological phenotype.