Fig 2: P2X7-EGFP expression in retina, sciatic nerves, spinal cord, and at the neuromuscular synapse.(A) EGFP exclusively co-localizes with microglia and endothelial cells in the adult mouse retina. Upper panel: Middle, retinal slice labeled for GFP (600 101 215, Rockland), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Müller glia). Left and right, retinal flat mounts scanned at the plane of the ganlion cell layer (GCL) and outer plexiform layer (OPL), respectively, to delineate microglia residing in these retinal layers. Astrocytes in the GCL were labeled with GFAP (G6171, Sigma-Aldrich). IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Lower panel: Co-staining of EGFP with neuronal marker PKCa (left) and glutamine synthetase (two right panels) at higher contrast and resolution to show absence of neuronal P2X7-EGFP. Cell nuclei were counterstained with Hoechst 33342 (blue) Scale bars: 20 µm. n = 2 individual line 61 in FVB/C57b/6 hybrid mice (B) Confocal images of GFP (ab6556, Abcam; A10262 or Thermo Fisher Scientific) co-immunostaining with antibodies against the indicated marker proteins in transgenic mice line 17 spinal cord slices (GFAP (MAB360, Millipore), S100ß (S2532, Sigma Aldrich)). Representative images were taken from the areas shown in the schematic overview. Arrows indicate co-staining for S100ß and GFP. Scale bar: 40 µm. Cell nuclei were counterstained with DAPI (blue). Representative images from n = 3 animals are shown. (C) Comparison of transgenic P2X7-EGFP fluorescence and endogenous P2X7 immunofluorescence (P2X7 antibody, Synaptic Systems) in teased sciatic nerve fibers of line 61 and wt mice, respectively. Representative images from at least 3 animals are shown. (D) Co-staining of P2X7-EGFP (A11122, Thermo Fischer Scientific, dilution 1:1000) in teased sciatic nerve fibers of line 46 with antibodies against axonal marker proteins demonstrates localization of the transgene at perinodal regions of Schwann cells. Scale bars: 50 µm. (E) Reconstructed 3-D images of the neuromuscular junction showing co-staining of P2X7-EGFP (ab6556, Abcam; or A10262, Thermo Fisher Scientific) with perisynaptic Schwann cells (S100ß (S2532, Sigma Aldrich)) as well as postsynaptic (a-Bungarotoxin, a-Bgt) and presynaptic (synaptophysin, Syn) marker proteins. The side view in the right panel shows no overlap between GFP and synaptophysin staining. Scale bars: 10 µm and 20 µm, respectively. Representative images from n = 3 animals are shown. For antibodies not specified in the legend see Key resources table.

Fig 3: Identity and quantity of P2X7-EGFP expressing cell types in the CA1 region and comparison with P2X7 expression in wt mice.(A–F) Co-labeling of tg line 17 brain slices with anti-GFP antibody (ab6556, Abcam; A10262, Thermo Fisher Scientific) and antibodies for the indicated marker proteins (GFAP (MAB360, Millipore), S100ß (S2532, Sigma Aldrich)). Hippocampal CA1 regions are shown. Arrows indicate co-staining for S100ß and GFP. Cell nuclei were counterstained with DAPI (blue). PL, pyramidal cell layer; SR, stratum radiatum. Scale bar: 50 µm (G) Quantitative analysis of 10 ‘counting boxes’ (as shown in C–F) from five sections/mouse in each experiment. Bars represent mean ±SEM of three independent experiments/animals (total cell numbers in transgenic versus wt animals were: 14.4% vs. 12.2% Iba1 +cells, 10.4% vs. 11.0% Olig2 +cells, 7.3% vs. 8.4% NG2 +cells, 16.1 vs. 14.1% S100ß + cells). (H) Quantitative analysis of P2X7 protein reduction in conditional P2X7-/- mice (CNP-cre, Cx3cr1-cre). 75 µg cerebrum extracts (1% NP40) were analyzed by western blotting and infrared imaging with antibodies against P2X7 (Synaptic Systems) and fluorescent secondary antibodies (LI-COR 680RD dk anti-rb; LI-COR 800CW gt anti-ms). Data were normalized to P2X7 protein in wt animals. Bars represent mean ± SEM from 6 to 9 animals analyzed in three independent experiments. Significance between means was analyzed using two-tailed unpaired Student’s t-test and indicated as ****p<0.0001 compared to P2rx7fl/fl. For antibodies not specified in the legend see Key resources table.

Fig 4: Proper functioning of Cre system in the MR. VGLUT3-Cre mouse acquired from the Jacksons Laboratory (USA; stock No.: #028534) and bred locally at the Institute of Experimental Medicine was injected with AAV containing the sequence of EYFP in a DIO construct (Addgene, catalog No.: #20298-AAV5) (see Figure 2B). Brain slices of 30 µm thickness were stained with a-VGLUT3 (rabbit, Synaptic Systems, stock No.: #135203) and a-EYFP (chicken, Life Technologies, stock No.: #A10262), then with Alexa-647 conjugated a-rabbit (donkey host, Jackson, stock No.: #711-605-152) and Alexa-488 conjugated a-chicken (goat host, Life Technologies, stock No.: #A11039) antibodies, respectively. EYFP-positive (green) cells represent Cre activity. (A) Viral staining in the whole MR. (B) Example of a proper Cre system functioning: VGLUT3+ (purple) neurons were able to inverse and express the gene sequence of EYFP that was coded in the injected AAV in the MR. We found no ectopic (that is, EYFP in non-VGLUT3+ cells) expression. (C,D) Double immunofluorescent positive neurons in the MR. Double fluorescent immunohistochemistry was done by László Bíró, and the pictures were taken by him at the Nikon Centre at the Institute of Experimental Medicine with C2 confocal laser-scanning microscope at 4×, 20×, and 60× magnification. AAV: adeno-associated viral vectors; EYFP: enhanced yellow fluorescent protein; DIO: double-floxed inverted open reading frame; MR: median raphe; VGLUT3: vesicular glutamate transporter type 3.

Fig 5: Distribution pattern of transgenic P2X7-EGFP.(A) DAB staining with an antibody against GFP (A11122, Thermo Fisher Scientific). Scale bars: 200 µm and 50 µm in hippocampus and cerebellum, respectively. A representative result from at least three animals is shown (B) Ratios of transgenic (line 17) and endogenous P2X7 protein in different brain regions. Protein extracts (1% NP40) were prepared and 75 µg per lane separated by SDS-PAGE. Bands were quantified upon western blotting by infrared imaging with antibodies against P2X7 (Synaptic Systems) and fluorescent secondary antibodies (LI-COR 680RD dk anti rb). Data are presented as means from three animals. (C) Co-labeling of line 17 P7 cerebellum with antibodies against GFP (A10262, Thermo Fisher Scientific) and S100ß (S2532, Sigma Aldrich). A typical staining pattern for radial glia is seen. The close up of a representative area in the Purkinje cell layer (right) shows punctate P2X7 staining on cells with Bergmann glia morphology. Cell nuclei were counterstained with DAPI (blue). Scale bars represent 100 µm and 10 µm, respectively. CA1/3, cornu ammonis regions 1/3; DG, dentate gyrus; ML, molecular layer; GL, granular layer; WM, white matter; EGL, external granular layer; PCL, Purkinje cell layer. (D) Co-labeling of wt and tg line 17 cerebellar slices from P7 pubs with an anti-GFP antibody (A10262, Thermo Fischer Sci.) and the novel P2X7-specific nanobody-rbIgG fusion construct 7E2-rbIgG (Danquah et al., 2016) confirms the endogenous expression pattern and the specificity of the P2X7-EGFP signal. Representative results from n = 3 (Tg) and n = 2 (Wt) pubs are shown. Scale bar: 50 µm, DAPI staining in blue. PCL, Purkinje cell layer; GL, granular layer; ML, molecular layer; DG, dentate gyrus; CA3, cornu ammonis region 3; EGL, external granular layer. (E) Comparison of DAB staining in transgenic P2X7-EGFP mice, wt, and P2X7-/- mice with 7E2-rbIgG (Danquah et al., 2016). Scale bar: 100 µm. Representative results from three animals per line (line 17 and wt) are shown. For antibodies not specified in the legend see Key resources table.