Sylvie Boisseau

Specific In Vivo Staining of Astrocytes in the Whole Brain after Intravenous Injection of Sulforhodamine Dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.

Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis

Spontaneous calcium activity can be detected in embryonic mouse cortical slices as fluorescence intensity variations, in the presence of a fluorescent calcium indicator. Current methods to detect and quantify these variations depend heavily on experimenters whose judgement may interfere with measurement. In the present work, we developed new software called CalSignal for automatic detection and tracking of cellular bodies and quantification of spontaneous calcium activity on time‐series of confocal fluorescence images. Analysis of 28 neocortical slices revealed that 21.0% of detected cells displayed peaks of fluorescence corresponding to spontaneous activity, with a mean frequency of one peak per 4 min. This activity was blocked in the absence of extracellular calcium but was not modified after depletion of calcium stores with thapsigargin or blockade of voltage‐gated calcium channels with Ni2+. Further, statistical analysis of calcium activity revealed concomitant activation of distant cells in 24 slices, and the existence of a significant network of synchrony based on such coactivations in 17 slices out of 28. These networks enclosed 84.3% of active cells, scattered throughout the neocortical wall (mean distance between cellular bodies, 111.7 µm). Finally, it was possible to identify specific cells which were synchronously active with more neighbouring cells than others. The identity of these nodal cells remains to be investigated to fully comprehend the role of spontaneous calcium activity, before synaptogenesis, in shaping cortical neurogenesis.

Heterogeneous distribution of TRPC proteins in the embryonic cortex

The present study was initiated to gain some information about the tissue distribution of transient receptor potential proteins of C-type (TRPC), a family of voltage-independent cation channels, at the beginning of neurogenesis in the telencephalon of embryonic mice. The mRNAs of all known TRPCs (TRPC1–TRPC7) could be found in the cortex at E13. TRPC1, TRPC3 and TRPC5 were the main isoforms, whereas the mRNAs for TRPC2, TRPC4, TRPC6 and TRPC7 were less abundant. The distribution throughout the cortical wall of TRPC1, TRPC3 and TRPC6 was studied by means of immuno-histochemistry. The data collected pointed to a heterogeneous expression of the channels. Three groups were identified. The first one comprises TRPC1, specifically found in the preplate but only in some post-mitotic neurons. It was mainly observed in a subset of cells distinct from the Cajal-Retzius cells. The second group is composed of TRPC3. It was found in non-neuronal cells and also in dividing (5-bromo-2′-deoxyuridine-positive) cells, indicating that TRPC3 is present in precursor cells. The third group contains TRPC6 detected in neuronal and in dividing non-neuronal cells. Double immunostaining experiments showed that TRPC3-positive cells also express TRPC6. Collectively, this report highlights a specific TRPC expression pattern in the immature cortical wall.

Disruption of dopaminergic transmission remodels tripartite synapse morphology and astrocytic calcium activity within substantia nigra pars reticulata

The substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia circuitry particularly sensitive to pathological dopamine depletion. Indeed, hyperactivity of SNr neurons is known to be responsible for some motor disorders characteristic of Parkinsons disease. The neuronal processing of basal ganglia dysfunction is well understood but, paradoxically, the role of astrocytes in the regulation of SNr activity has rarely been considered. We thus investigated the influence of the disruption of dopaminergic transmission on plastic changes at tripartite glutamatergic synapses in the rat SNr and on astrocyte calcium activity. In 6‐hydroxydopamine‐lesioned rats, we observed structural plastic changes of tripartite glutamatergic synapses and perisynaptic astrocytic processes. These findings suggest that subthalamonigral synapses undergo morphological changes that accompany the pathophysiological processes of Parkinsons disease. The pharmacological blockade of dopaminergic transmission (with sulpiride and SCH‐23390) increased astrocyte calcium excitability, synchrony and gap junction coupling within the SNr, suggesting a functional adaptation of astrocytes to dopamine transmission disruption in this output nucleus. This hyperactivity is partly reversed by subthalamic nucleus high‐frequency stimulation which has emerged as an efficient symptomatic treatment for Parkinsons disease. Therefore, our results demonstrate structural and functional reshaping of neuronal and glial elements highlighting a functional plasticity of neuroglial interactions when dopamine transmission is disrupted. GLIA 2015;63:673–683

Differential down-regulation of voltage-gated calcium channel currents by glutamate and BDNF in embryonic cortical neurons

In the embryonic brain, post‐mitotic cortical neurons migrate from their place of origin to their final location. Various external factors such as hormones, neurotransmitters or peptides regulate their migration. To date, however, only a few studies have investigated the effects of these external factors on the electrical properties of the newly formed embryonic cortical neurons. The aim of the present study was to determine whether glutamate and brain‐derived neurotrophic factor (BDNF), known to regulate neuronal cell migration, could modulate currents through voltage‐gated calcium channels (ICa) in cortical neurons isolated from embryonic day 13 (E13) mouse foetuses. Whole cell recordings of ICa showed that E13 cortical cells kept 1 day in vitro expressed functional low‐ and high‐voltage activated (LVA and HVA) Ca2+ channels of T‐, L‐ and N‐types. A 1‐day glutamate treatment non‐specifically inhibited LVA and HVA ICa whereas BDNF down‐regulated HVA with N‐type ICa being more depressed than L‐type ICa. The glutamate‐induced ICa inhibition was mimicked by NMDA. BDNF exerted its action by recruiting trkB receptors and SKF‐96365‐sensitive channels. BAPTA prevented the glutamate‐ and the BDNF‐dependent inhibition of Ica, indicating a Ca2+‐dependent mechanism of action. It is proposed that an influx of Ca2+ through NMDA receptors depresses the expression of LVA and HVA Ca2+ channels whereas a Ca2+ influx through SKF‐96365‐sensitive TRPC (transient receptor potential protein of C subtype) channels preferentially inhibits the expression of HVA Ca2+ channels. Glutamate and BDNF appear as potent modulators of the electrical properties of early post‐mitotic neurons. By down‐regulating ICa they could exert a neuroprotective action on embryonic cortical neurons.

Subthalamic Nucleus Electrical Stimulation Modulates Calcium Activity of Nigral Astrocytes

Background The substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia, delivering inhibitory efferents to the relay nuclei of the thalamus. Pathological hyperactivity of SNr neurons is known to be responsible for some motor disorders e.g. in Parkinsons disease. One way to restore this pathological activity is to electrically stimulate one of the SNr input, the excitatory subthalamic nucleus (STN), which has emerged as an effective treatment for parkinsonian patients. The neuronal network and signal processing of the basal ganglia are well known but, paradoxically, the role of astrocytes in the regulation of SNr activity has never been studied. Principal Findings In this work, we developed a rat brain slice model to study the influence of spontaneous and induced excitability of afferent nuclei on SNr astrocytes calcium activity. Astrocytes represent the main cellular population in the SNr and display spontaneous calcium activities in basal conditions. Half of this activity is autonomous (i.e. independent of synaptic activity) while the other half is dependent on spontaneous glutamate and GABA release, probably controlled by the pace-maker activity of the pallido-nigral and subthalamo-nigral loops. Modification of the activity of the loops by STN electrical stimulation disrupted this astrocytic calcium excitability through an increase of glutamate and GABA releases. Astrocytic AMPA, mGlu and GABAA receptors were involved in this effect. Significance Astrocytes are now viewed as active components of neural networks but their role depends on the brain structure concerned. In the SNr, evoked activity prevails and autonomous calcium activity is lower than in the cortex or hippocampus. Our data therefore reflect a specific role of SNr astrocytes in sensing the STN-GPe-SNr loops activity and suggest that SNr astrocytes could potentially feedback on SNr neuronal activity. These findings have major implications given the position of SNr in the basal ganglia network.