Improved potency of pyridin-2(1H)one derivatives for the treatment of mechanical allodynia.

Mechanical allodynia, a painful sensation caused by innocuous touch, is a major chronic pain symptom, which often remains without an effective treatment. There is thus a need for new anti-allodynic treatments based on new drug classes. We recently synthetized new 3,5-disubstituted pyridin-2(1H)-one derivatives. By substituting the pyridinone at the 3-position by various aryl/heteroaryl moieties and at the 5-position by a phenylamino group, we discovered that some derivatives exhibited a strong anti-allodynic potency in rats. Here, we report that varying the substitution of the pyridinone 5-position, the 3-position being substituted by an indol-4-yl moiety, further improves such anti-allodynic potency. Compared with 2, one of the two most active compounds of the first series, eleven out of nineteen newly synthetized compounds showed higher anti-allodynic potency, with two of them completely preventing mechanical allodynia. In the first series, hit compounds 1 and 2 appeared to be inhibitors of p38α MAPK, a protein kinase known to underlie pain hypersensitivity in animal models. Depending on the substitution at the 5-position, some newly synthetized compounds were also stronger p38α MAPK inhibitors. Surprisingly, though, anti-allodynic effects and p38α MAPK inhibitory potencies were not correlated, suggesting that other biological target(s) is/are involved in the analgesic activity in this series. Altogether, these results confirm that 3,5-disubstituted pyridine-2(1H)-one derivatives are of high interest for the development of new treatment of mechanical allodynia.

Astrocytes respond to a neurotoxic Aß fragment with state-dependent Ca2+ alteration and multiphasic transmitter release.

Excessive amounts of amyloid ß (Aß) peptide have been suggested to dysregulate synaptic transmission in Alzheimer's disease (AD). As a major type of glial cell in the mammalian brain, astrocytes regulate neuronal function and undergo activity alterations upon Aß exposure. Yet the mechanistic steps underlying astrocytic responses to Aß peptide remain to be elucidated. Here by fluorescence imaging of signaling pathways, we dissected astrocytic responses to Aß25-35 peptide, a neurotoxic Aß fragment present in AD patients. In native health astrocytes, Aß25-35 evoked Ca2+ elevations via purinergic receptors, being also dependent on the opening of connexin (CX) hemichannels. Aß25-35, however, induced a Ca2+ diminution in Aß-preconditioned astrocytes as a result of the potentiation of the plasma membrane Ca2+ ATPase (PMCA). The PMCA and CX protein expression was observed with immunostaining in the brain tissue of hAPPJ20 AD mouse model. We also observed both Ca2+-independent and Ca2+-dependent glutamate release upon astrocytic Aß exposure, with the former mediated by CX hemichannel and the latter by both anion channels and lysosome exocytosis. Our results suggest that Aß peptide causes state-dependent responses in astrocytes, in association with a multiphasic release of signaling molecules. This study therefore helps to understand astrocyte engagement in AD-related amyloidopathy.

Astrocyte VAMP3 vesicles undergo Ca2+ -independent cycling and modulate glutamate transporter trafficking.

KEY POINTS: Mouse cortical astrocytes express VAMP3 but not VAMP2. VAMP3 vesicles undergo Ca(2+) -independent exo- and endocytotic cycling at the plasma membrane. VAMP3 vesicle traffic regulates the recycling of plasma membrane glutamate transporters. cAMP modulates VAMP3 vesicle cycling and glutamate uptake. ABSTRACT: Previous studies suggest that small synaptic-like vesicles in astrocytes carry vesicle-associated vSNARE proteins, VAMP3 (cellubrevin) and VAMP2 (synaptobrevin 2), both contributing to the Ca(2+) -regulated exocytosis of gliotransmitters, thereby modulating brain information processing. Here, using cortical astrocytes taken from VAMP2 and VAMP3 knock-out mice, we find that astrocytes express only VAMP3. The morphology and function of VAMP3 vesicles were studied in cultured astrocytes at single vesicle level with stimulated emission depletion (STED) and total internal reflection fluorescence (TIRF) microscopies. We show that VAMP3 antibodies label small diameter (∼80 nm) vesicles and that VAMP3 vesicles undergo Ca(2+) -independent exo-endocytosis. We also show that this pathway modulates the surface expression of plasma membrane glutamate transporters and the glutamate uptake by astrocytes. Finally, using pharmacological and optogenetic tools, we provide evidence suggesting that the cytosolic cAMP level influences astrocytic VAMP3 vesicle trafficking and glutamate transport. Our results suggest a new role for VAMP3 vesicles in astrocytes.

Eliminating unwanted far-field excitation in objective-type TIRF. Part I. identifying sources of nonevanescent excitation light.

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).

Eliminating unwanted far-field excitation in objective-type TIRF. Part II. combined evanescent-wave excitation and supercritical-angle fluorescence detection improves optical sectioning.

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.

Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks.

Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. In this study, we designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination pat-terns of arbitrary orientation and period, limited only by the illumination aperture (NA = 1.45), the response time of a fast, piezo-driven tip-tilt mirror (10 ms) and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrate the performance of our instrument by imaging mitochondrial dynamics in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope provides a versatile, compact and cost-effective arrangement for super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles--important questions in both cell biology and neurophysiology.

Lack of evidence for vesicular glutamate transporter expression in mouse astrocytes.

The concept of a tripartite synapse including a presynaptic terminal, a postsynaptic spine, and an astrocytic process that responds to neuronal activity by fast gliotransmitter release, confers to the electrically silent astrocytes an active role in information processing. However, the mechanisms of gliotransmitter release are still highly controversial. The reported expression of all three vesicular glutamate transporters (VGLUT1-3) by astrocytes suggests that astrocytes, like neurons, may release glutamate by exocytosis. However, the demonstration of astrocytic VGLUT expression is largely based on immunostaining, and the possibility of nonspecific labeling needs to be systematically addressed. We therefore examined the expression of VGLUT1-3 in astrocytes, both in culture and in situ. We used Western blots and single-vesicle imaging by total internal reflection fluorescence microscopy in live cultured astrocytes, and confocal microscopy, at the cellular level in cortical, hippocampal, and cerebellar brain slices, combined with quantitative image analysis. Control experiments were systematically performed in cultured astrocytes using wild-type, VGLUT1-3 knock-out, VGLUT1(Venus) knock-in, and VGLUT2-EGFP transgenic mice. In fixed brain slices, we quantified the degree of overlap between VGLUT1-3 and neuronal or astrocytic markers, both in an object-based manner using fluorescence line profiles, and in a pixel-based manner using dual-color scatter plots followed by the calculation of Pearson's correlation coefficient over all pixels with intensities significantly different from background. Our data provide no evidence in favor of the expression of any of the three VGLUTs by gray matter protoplasmic astrocytes of the primary somatosensory cortex, the thalamic ventrobasal nucleus, the hippocampus, and the cerebellum.

Optogenetic activation of LiGluR-expressing astrocytes evokes anion channel-mediated glutamate release.

Increases in astrocyte Ca(2+) have been suggested to evoke gliotransmitter release, however, the mechanism of release, the identity of such transmitter(s), and even whether and when such release occurs, are controversial, largely due to the lack of a method for selective and reproducible stimulation of electrically silent astrocytes. Here we show that photoactivation of the light-gated Ca(2+)-permeable ionotropic GluR6 glutamate receptor (LiGluR), and to a lesser extent the new Ca(2+)-translocating channelrhodopsin CatCh, evokes more reliable Ca(2+) elevation than the mutant channelrhodopsin 2, ChR2(H134R) in cultured cortical astrocytes. We used evanescent-field excitation for near-membrane Ca(2+) imaging, and epifluorescence to activate and inactivate LiGluR. By alternating activation and inactivation light pulses, the LiGluR-evoked Ca(2+) rises could be graded in amplitude and duration. The optical stimulation of LiGluR-expressing astrocytes evoked probabilistic glutamate-mediated signalling to adjacent LiGluR-non-expressing astrocytes. This astrocyte-to-astrocyte signalling was insensitive to the inactivation of vesicular release, hemichannels and glutamate-transporters, and sensitive to anion channel blockers. Our results show that LiGluR is a powerful tool to selectively and reproducibly activate astrocytes.

FM dyes enter via a store-operated calcium channel and modify calcium signaling of cultured astrocytes.

The amphiphilic fluorescent styryl pyridinium dyes FM1-43 and FM4-64 are used to probe activity-dependent synaptic vesicle cycling in neurons. Cultured astrocytes can internalize FM1-43 and FM4-64 inside vesicles but their uptake is insensitive to the elevation of cytosolic calcium (Ca(2+)) concentration and the underlying mechanism remains unclear. Here we used total internal reflection fluorescence microscopy and pharmacological tools to study the mechanisms of FM4-64 uptake into cultured astrocytes from mouse neocortex. Our data show that: (i) endocytosis is not a major route for FM4-64 uptake into astrocytes; (ii) FM4-64 enters astrocytes through an aqueous pore and strongly affects Ca(2+) homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca(2+) entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca(2+) for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca(2+) from the endoplasmic reticulum stores, conveying a positive feedback to activate SOCE and to sustain dye uptake into astrocytes. Our study demonstrates that FM dyes are not markers of cycling vesicles in astrocytes and calls for a careful interpretation of FM fluorescence.