Nicole Ropert

Molecular and Physiological Diversity of Cortical Nonpyramidal Cells

The physiological and molecular features of nonpyramidal cells were investigated in acute slices of sensory-motor cortex using whole-cell recordings combined with single-cell RT-PCR to detect simultaneously the mRNAs of three calcium binding proteins (calbindin D28k, parvalbumin, and calretinin) and four neuropeptides (neuropeptide Y, vasoactive intestinal polypeptide, somatostatin, and cholecystokinin). In the 97 neurons analyzed, all expressed mRNAs of at least one calcium binding protein, and the majority (n = 73) contained mRNAs of at least one neuropeptide. Three groups of nonpyramidal cells were defined according to their firing pattern. (1) Fast spiking cells (n = 34) displayed tonic discharges of fast action potentials with no accommodation. They expressed parvalbumin (n = 30) and/or calbindin (n = 19) mRNAs, and half of them also contained transcripts of at least one of the four neuropeptides. (2) Regular spiking nonpyramidal cells (n = 48) displayed a firing behavior characterized by a marked accommodation and presented a large diversity of expression patterns of the seven biochemical markers. (3) Finally, a small population of vertically oriented bipolar cells, termed irregular spiking cells (n = 15), fired bursts of action potentials at an irregular frequency. They consistently co-expressed calretinin and vasoactive intestinal polypeptide. Additional investigations of these cells showed that they also co-expressed glutamic acid decarboxylase and choline acetyl transferase. Our results indicate that neocortical nonpyramidal neurons display a large diversity in their firing properties and biochemical patterns of co-expression and that both characteristics could be correlated to define discrete subpopulations.

Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes

Although Ca2+-dependent exocytosis is considered to be a pathway for gliotransmitter release from astrocytes, the structural and functional bases of this process remain controversial. We studied the relationship between near-membrane Ca2+ elevations and the dynamics of single astroglial vesicles with styryl (FM) dyes. We show that cultured astrocytes, unlike neurons, spontaneously internalize FM dyes, resulting in the labeling of the entire acidic vesicle population within minutes. Interestingly, metabotropic glutamate receptor activation did not affect the FM labeling. Most FM-stained vesicles expressed sialin, CD63/LAMP3, and VAMP7, three markers for lysosomes and late endosomes. A subset of lysosomes underwent asynchronous exocytosis that required both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane. Lysosomal fusion occurred within seconds and was complete with no evidence for kiss and run. Our experiments suggest that astroglial Ca2+-regulated exocytosis is carried by lysosomes and operates on a timescale orders of magnitude slower than synaptic transmission.

Neurotransmitter release at the thalamocortical synapse instructs barrel formation but not axon patterning in the somatosensory cortex.

To assess the impact of synaptic neurotransmitter release on neural circuit development, we analyzed barrel cortex formation after thalamic or cortical ablation of RIM1 and RIM2 proteins, which control synaptic vesicle fusion. Thalamus-specific deletion of RIMs reduced neurotransmission efficacy by 67%. A barrelless phenotype was found with a dissociation of effects on the presynaptic and postsynaptic cellular elements of the barrel. Presynaptically, thalamocortical axons formed a normal whisker map, whereas postsynaptically the cytoarchitecture of layer IV neurons was altered as spiny stellate neurons were evenly distributed and their dendritic trees were symmetric. Strikingly, cortex-specific deletion of the RIM genes did not modify barrel development. Adult mice with thalamic-specific RIM deletion showed a lack of activity-triggered immediate early gene expression and altered sensory-related behaviors. Thus, efficient synaptic release is required at thalamocortical but not at corticocortical synapses for building the whisker to barrel map and for efficient sensory function.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

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, VGLUT1Venus 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 Pearsons 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

Non‐technical summary  Ca2+ increases in astrocytes have been suggested to trigger the release of neuroactive compounds called gliotransmitters. To study the mechanism of gliotransmitter release, it is desirable to have a method able to selectively and reproducibly evoke Ca2+ increases in astrocytes. Here, we show that the optogenetic tool the light‐gated glutamate receptor 6 (LiGluR) reproducibly evokes Ca2+ rises in cultured astrocytes. Combining LiGluR photoactivation and evanescent‐field Ca2+ imaging, we explored the cellular mechanism of gliotransmitter release from astrocytes in an all‐optical manner. The results suggest that high Ca2+ in astrocytes triggers the release of glutamate via anion channels rather than vesicular exocytosis.

Altering the concentration of GABA in the synaptic cleft potentiates miniature IPSCs in rat occipital cortex.

We have tested the effect of dextran (40 kDa, 5%) on miniature IPSCs (mIPSCs) recorded in layer V cortical pyramidal cells. This compound increases the amplitude of mIPSCs at room and physiological temperatures by 15%, leaving their duration unaffected at room temperature and slightly increased at physiological temperature. The amplitude increase is attributable to an increase in the number of receptors bound by GABA during synaptic transmission, as shown by the occlusion between the effects of dextran and zolpidem on mIPSC amplitude at room temperature. As dextran presumably enhances the concentration and dwell time of GABA in the synaptic cleft, these results demonstrate that the postsynaptic GABAA receptors are not saturated at room and physiological temperatures.

New tools for investigating astrocyte-to-neuron communication

Gray matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated. Studying astrocytes in their natural environment is challenging because: (i) astrocytes are electrically silent; (ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; (iii) the size of astrocyte processes in contact with synapses are below the resolution of confocal and two-photon microscopes (iv) bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity. In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: (i) the complexity of astrocyte Ca2+ signaling revealed by GECIs; (ii) new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signaling pathways in astrocytes; (iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; (iv) possible strategies to express specifically reporter genes in 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 (Ca2+) 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 Ca2+ homeostasis; (iii) partitioning of FM4-64 into the outer leaflet of the plasma membrane results in a facilitation of store-operated Ca2+ entry (SOCE) channel gating; (iv) FM4-64 permeates and competes with Ca2+ for entry through a SOCE channel; (v) intracellular FM4-64 mobilizes Ca2+ 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.

Expression of GABAA Receptor Subunit mRNAs by Layer V Pyramidal Cells of the Rat Primary Visual Cortex

The expression of the GABAA receptor subunit mRNAs by layer V pyramidal neurons of the primary visual cortex and cerebellar Purkinje cells was analysed by single‐cell reverse transcription of the mRNAs and amplification of the resulting cDNAs by the polymerase chain reaction. Neurons were identified by infrared Videomicroscopy, and GABAA‐mediated miniature inhibitory postsynaptic currents were recorded. In Purkinje cells, α1, β2, β3, γ2s and γ2L subunit mRNAs were detected within a single cell. In layer V pyramidal cells, a total of ten GABAA receptor subunit mRNAs could be detected, with a mean of seven subunit mRNAs per cell, suggesting GABAA receptor heterogeneity within a single pyramidal cell.