SNr astrocytes AMPA, mGlu and GABA_A receptors are involved in calcium spontaneous activity.

<p>(<b>A</b>) Example of two typical fluorescence variation profiles recorded in SNr astrocytes when 100 µM glutamate was perfused in the bath. (<b>B</b>) Histogram of the percentage of loaded SNr astrocytes responding to the application of 100 µM glutamate (<i>n</i> = 15) and the effect of 500 nM TTX (<i>n</i> = 12; <i>p</i> = 0.317), 10 µM CNQX (<i>n</i> = 8; <i>p</i><0.001), 100 µM LAP-3 (<i>n</i> = 11; <i>p</i> = 0.005), 50 µM AP-5 (<i>n</i> = 4; <i>p</i> = 0.453) or a cocktail containing 10 µM CNQX+100 µM LAP-3+50 µM AP-5 (<i>n</i> = 4; <i>p</i> = 0.001) on this glutamate-induced effect. (<b>C</b>) Typical fluorescence variations recorded in a SNr astrocyte when 20 µM GABA was perfused in the bath. (<b>D</b>) Histogram of the percentage of loaded SNr astrocytes responding to the application of 20 µM GABA (<i>n</i> = 18) and the effect of 500 nM TTX (<i>n</i> = 7; <i>p</i> = 0.215), 20 µM BMI (<i>n</i> = 7; <i>p</i> = 0.02) or 100 µM saclofen (<i>n</i> = 8; <i>p</i> = 0.948) on this GABA-induced effect. (<b>E</b>) Typical fluorescence variations recorded in two SNr astrocytes before and after incubation with a cocktail containing 10 µM CNQX+100 µM LAP-3+50 µM AP-5. (<b>F</b>) Histogram showing the effect of a cocktail containing 10 µM CNQX+100 µM LAP-3+50 µM AP-5 (<i>n</i> = 10; <i>p</i><0.001) or 20 µM BMI (<i>n</i> = 7; <i>p</i> = 0.003) on the spontaneous calcium activity of astrocytes in the SNr. The inhibitory effect is normalized with respect to control residual activity. *, <i>p</i><0.05; **, <i>p</i><0.01 and ***, <i>p</i><0.001.</p

SNr astrocytes display spontaneous calcium activity that is partly dependent on neuronal activity.

<p>(<b>A</b>) Fluo-4 loaded cells in the SNr area of a rat sagittal acute slice. Only small cells (astrocytes, less than 10 µm in diameter) were loaded. GABAergic and dopaminergic neurons (with cell bodies from 20 to 40 µm in diameter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041793#pone.0041793-Mailly1" target="_blank">[70]</a>) were not labelled in most cases. (<b>B</b>) Distribution of active (red frames) and non active (white frames) ROIs within the slice shown in A. (<b>C</b>) Example of typical fluorescence variations recorded in four SNr astrocytes. (<b>D</b>) Raster plot of fluorescence peaks detected in active ROIs described in B. (<b>E</b>) Example of the cumulative progress of the proportion of active astrocytes during recording over 5, 10, 15, 20, 25 and 30 minutes. (<b>F</b>) Effect of 500 nM TTX (<i>n</i> = 13 slices; <i>p</i><0.001) or 2 µM thapsigargin (Thapsi, <i>n</i> = 7 slices; <i>p</i><0.001) on the spontaneous calcium activity of astrocytes in the SNr.</p

Characterization of SNr cell populations and Fluo-4 loaded cells.

<p>(<b>A</b>) NeuN staining (green) labels few cells within SNr. Nuclei are identified by TO-PRO staining (blue). Merged image showing the proportion of neurons within the SNr (right panel). (<b>B</b>) Two-photon multistack (40 slices at 2 µm spacing) mosaic reconstruction of SNr astrocytes and vessels in an acute brain slice 2 h after sulforhodamine 101 (10 mg/ml) intravenous injection (100 µl/50 g body weight). Antero-posterior (AP) and medio-lateral (ML) orientations are shown in the upper right part of the figure. (<b>C</b>) Bi-photon imaging of sulforhodamine 101 (red) and Hoescht 33342 (blue) in the SNr. Merged image showing the proportion of astrocytes within the SNr (right panel). (<b>D</b>) Representative confocal images of SR101-labeled (red) and Fluo-4-loaded (green) cells in the SNr of a sagittal acute slice of rat brain. Merged image showing sulforhodamine 101 and Fluo-4 staining within the SNr, confirming that most of the loaded cells are astrocytes.</p