Journal: The Journal of Neuroscience

Article Title: Regulation of Perineuronal Nets in the Adult Cortex by the Activity of the Cortical Network

doi: 10.1523/jneurosci.0434-21.2021

Figure Lengend Snippet: Figure 1. Chemogenetic paradigm and histologic analysis of PNN density in the V1 visual cortex. A, Four weeks after hemilateral AAV injection in the V1 cortex, DREADD- or PSAM-GlyR-expressing mice received four injections of relevant ago- nist or PBS starting at P58, and brains were processed for histochemistry at P61. B, Macrotome fluorescence-negative picture of a coronal section of mouse brain at the level of the visual cortex showing PNN staining with WFA. The superimposed sec- tion of the mouse brain atlas delineates the densely stained V1 area flanked by V2L and V2ML areas exhibiting faint PNN labeling. Throughout this study, analyses of PNN density were performed in layers IV-V of the V1 area as represented by the red rectangle. C, PNN density analyses were performed around PV-immunopositive cells, or around cells showing expression of both PV and the chemogenetic tool, as exemplified here for the hM4Di-mCherry fusion protein. D, In order to quantify PNN density, widefield fluorescence pictures were acquired (left), background-subtracted using the Subtract Background func- tion of the ImageJ software (middle), and PNNs were delineated manually around the soma based on WFA staining intensity (right, red lines), to create ring-shaped ROIs using the XOR function of ImageJ. Scale bars: C, D, 20 mm.

Article Snippet: Adeno-associated pseudovirions (AAVs) encoding designer receptor exclusively activated by designer drug (DREADD) (Alexander et al., 2009) AAV2/5-hSyn-DIO-hM4Di-mCherry (titer: 5.2 1012 gc/ml) and AAV2/5-hSyn-DIO-hM3Dq-mCherry (7.8 1012 gc/ml) were produced from Addgene plasmids #44362 and #44361 at the facility of Nantes University (Unite Mixte de Recherche 1089).

Techniques: Injection, Expressing, Fluorescence, Staining, Labeling, Software

Journal: The Journal of Neuroscience

Article Title: Regulation of Perineuronal Nets in the Adult Cortex by the Activity of the Cortical Network

doi: 10.1523/jneurosci.0434-21.2021

Figure Lengend Snippet: Figure 3. Targeted inhibition of PV interneurons using DREADD hM4Di induces PNN regression. Confocal fluorescence images acquired in the V1 cortex illustrate the PNN (WFA) surrounding hM4Di-expressing PV interneurons in layers IV-V after PBS or CNO treatment of the mice. Note the low PNN density around hM4Di1 cells after CNO treatment, as exemplified in high-magnification images. Scale bars: left, 100mm; right, 10mm. Plot of PNN density around PV1 (contralateral uninjected hemicortex) and hM4Di1/ PV1 (ipsilateral injected) cells in the V1 cortex normalized for each mouse to mean density in contralateral hemicortex. Indicated in bars are the number of cells analyzed in 3 CNO-treated and 3 PBS-treated mice. *Significantly different from other conditions.

Article Snippet: Adeno-associated pseudovirions (AAVs) encoding designer receptor exclusively activated by designer drug (DREADD) (Alexander et al., 2009) AAV2/5-hSyn-DIO-hM4Di-mCherry (titer: 5.2 1012 gc/ml) and AAV2/5-hSyn-DIO-hM3Dq-mCherry (7.8 1012 gc/ml) were produced from Addgene plasmids #44362 and #44361 at the facility of Nantes University (Unite Mixte de Recherche 1089).

Techniques: Inhibition, Fluorescence, Expressing, Injection

Journal: The Journal of Neuroscience

Article Title: Regulation of Perineuronal Nets in the Adult Cortex by the Activity of the Cortical Network

doi: 10.1523/jneurosci.0434-21.2021

Figure Lengend Snippet: Figure 2. Targeted expression of DREADD hM4Di in PV interneurons. A, Stereotaxic injection of Cre-dependent AAV encoding hM4Di fused to mCherry in the visual cortex of PV-Cre mice. Macrotome fluorescence picture showing expression of hM4Di- mCherry revealed by anti-RFP immunohistochemistry 5 weeks after injection. The superimposed section of the mouse brain atlas represents the V1 area. B, Confocal fluorescence images acquired in the V1 cortex after dual immunostaining showing hM4Di-mCherry expression in PV-positive cells. Scale bar, 100 mm.

Article Snippet: Adeno-associated pseudovirions (AAVs) encoding designer receptor exclusively activated by designer drug (DREADD) (Alexander et al., 2009) AAV2/5-hSyn-DIO-hM4Di-mCherry (titer: 5.2 1012 gc/ml) and AAV2/5-hSyn-DIO-hM3Dq-mCherry (7.8 1012 gc/ml) were produced from Addgene plasmids #44362 and #44361 at the facility of Nantes University (Unite Mixte de Recherche 1089).

Techniques: Expressing, Injection, Fluorescence, Immunohistochemistry, Immunostaining

Journal: Frontiers in cellular neuroscience

Article Title: A requirement for astrocyte IP 3 R2 signaling for whisker experience-dependent depression and homeostatic upregulation in the mouse barrel cortex.

doi: 10.3389/fncel.2022.905285

Figure Lengend Snippet: FIGURE 2 Opto- and Chemogenetically activated astrocytes drive synaptic and spike activity in L2/3 neurons of the barrel cortex in vitro and in vivo. (A) Images showing Melanopsin expression in astrocytes: (i) Low magnification image displaying extent of Melanopsin staining in horizontal slices. (ii) Composite image showing co-expression (yellow) of melanopsin -CFP and S100b. (iii) image of NeuN staining (blue). (iv) Melanopsin-CFP (green). (v) S100b staining (red). Images (vi) are expanded region from (ii). (vii) The expanded region from (iv). (Viii) The expanded region from (v). (B) Images showing DREADD expression in astrocytes: (i) Low magnification image displaying extent of DREADD staining in horizontal slices. Note barrels delineated by DAPI staining. (ii) Composite image showing co-expression of DREADD and S100b (yellow). (iii) Image of NeuN staining (blue). (iv) DREADD-mCitrine (green). (v) S100b staining (red). Images below show staining in the expanded region. (vi) The expanded region from (ii). (vii) The expanded region from (iv). (viii) the expanded region from (v). (C) Image of an ex vivo slice from an animal transfected with GFAP-Melanopsin-CFP and a bulk loaded with Fluo-4AM. Fluorescence traces from circled CFP positive astrocytes are shown below, illustrating calcium elevations in response to light stimulation (blue dashes). (D) Example voltage clamp recording from an L2/3 cortical neuron in an ex vivo slice from a WT animal transfected with GFAP-melanopsin, illustrating an increase in excitatory current frequency, following 470-nm light stimulation (blue flashes), and example recording from a similar experiment in an IP3R2-/- slice. A scale bar, 500 pA, 100 s (expanded inset scale bars, WT 200 pA; 10 ms. IP3R2-/- 20 pA, 100 ms). Bar graphs on right show summary effect of 470-nm stimulation in slices from WT (white bars) and IP3R2-/- (red bars) animals expressing GFAP-Melanopsin on sEPSCs. (E) Data from in vivo recordings showing (i) example spike activity recording from a control non-melanopsin animal before and (ii) after 470-nm stimulation. An (Continued)

Article Snippet: In the case of Designer Receptor Exclusively Activated by Designer Drug (DREADD) experiments, local field potentials (LFPs) were recorded with 0.1-Hz high pass filter along with a Humbug 50-Hz noise eliminator (Digitimer).

Techniques: Activity Assay, In Vitro, In Vivo, Expressing, Staining, Ex Vivo, Transfection, Fluorescence, Control