Journal: Cell reports

Article Title: Astrocyte glypican 5 regulates synapse maturation and stabilization

doi: 10.1016/j.celrep.2025.115374

Figure Lengend Snippet: (A–D) GLUA2 protein level is decreased at intracortical synapses in Gpc5 cKO mice at P28. (A) Immunostaining of intracortical presynaptic VGLUT1 and postsynaptic GLUA2 in L2/3. (B–D) Numbers of VGLUT1 (B), GLUA2 (C), and colocalized (D) puncta. N = 5 mice/condition. (E–H) GLUA1 protein level is unchanged at intracortical synapses in Gpc5 cKO mice at P28. (E) Immunostaining of presynaptic VGLUT1 and postsynaptic GLUA1 in L2/3. (F–H) Numbers of VGLUT1 (F), GLUA1 (G), and colocalized (H) puncta. N = 6 mice/condition. (I–P) Thalamocortical synapses have unaltered AMPAR levels in Gpc5 cKO mice at P28. (I) Immunostaining of thalamocortical presynaptic VGLUT2 and postsynaptic GLUA2 in L4. (J–L) Numbers of VGLUT2 (J), GLUA2 (K), and colocalized (L) puncta. N = 5 mice/condition. (M) Immunostaining of thalamocortical presynaptic VGLUT2 and postsynaptic GLUA1 in L4. (N–P) Numbers of VGLUT2 (N), GLUA1 (O), and colocalized (P) puncta. N = 5 mice/condition. (Q–S) VGLUT2 puncta volume is decreased in P28 Gpc5 cKO mice. (Q) Immunostaining of VGLUT2 puncta in L1 VC. (R and S) VGLUT2 puncta volume in L1 and L4. N = 6 mice/condition L1, N = 5 mice/condition L4. (T) Summary of synaptic changes in Gpc5 cKO mice at P28. Scale bars: 5 μm (A, E, I, M, and Q, main) and 1 μm (A, E, I, and M, magnified). Graphs: mean ± SEM. Individual data points represent mice. Statistics: t test. See also .

Article Snippet: Guinea pig polyclonal anti VGlut2 , Millipore , CAT# AB2251; RRID:AB_2665454.

Techniques: Immunostaining

Journal: Cell reports

Article Title: Astrocyte glypican 5 regulates synapse maturation and stabilization

doi: 10.1016/j.celrep.2025.115374

Figure Lengend Snippet: (A–H) GLUA2 puncta numbers are recovered at intracortical synapses in Gpc5 cKO mice at P120. (A and E) Immunostaining of intracortical presynaptic VGLUT1 and postsynaptic GLUA2 in L1 (A) and L2/3 (E). (B–D and F–H) VGLUT1 (B and F), GLUA2 (C and G), and colocalized (D and H) puncta in L1 and L2/3. (I–K) VGLUT2 puncta volume is recovered at P120 in Gpc5 cKO mice. (I) Immunostaining of VGLUT2 in layer 1 VC. (J and K) VGLUT2 puncta volume in L1 (J) and L4 (K). N = 5 mice/condition. Scale bars: 5 μm (A, E, and I, main) and 1 μm (A and E, magnified). Graphs: mean ± SEM. Individual points represent mice. Statistics: t test. (L) Summary of synapse findings in Gpc5 cKO mice at P120. (M and N) Absence of astrocyte GPC5 enables enhanced ocular dominance plasticity in adulthood. (M) Arc mRNA in the VC ipsilateral to the nondeprived eye 12 hours or 5 days after ME. Scale bar: 500 μm. (N) Width of the binocular zone. Graph: mean ± SEM. Individual points represent mice. N = 5 mice/condition. Statistics: two-way ANOVA. (O) Summary of phenotypes in Gpc5 astrocyte-specific cKO mice. See also .

Article Snippet: Guinea pig polyclonal anti VGlut2 , Millipore , CAT# AB2251; RRID:AB_2665454.

Techniques: Immunostaining

Journal: Cell reports

Article Title: Astrocyte glypican 5 regulates synapse maturation and stabilization

doi: 10.1016/j.celrep.2025.115374

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Guinea pig polyclonal anti VGlut2 , Millipore , CAT# AB2251; RRID:AB_2665454.

Techniques: Recombinant, Modification, Saline, RNAscope, Multiplex Assay, Electron Microscopy, Negative Control, Software, Cell Culture, Fluorescence, Microscopy, Hybridization

Journal: bioRxiv

Article Title: Parvalbumin expression identifies subicular principal cells with high projection specificity

doi: 10.1101/2024.08.13.606964

Figure Lengend Snippet: A) Example image of a double fluorescent in situ hybridization on a coronal brain section showing co-localization of VGLUT2 (white) and PV (red) mRNA in the subiculum. Nuclei are stained with DAPI (blue). Scale bar 100 µm. i, Magnification of the boxed area in A. The white arrow indicates a cell co-expressing PV and VGLUT2 mRNA. Scale bar 5 µm. B) A small percentage of subicular PV+ neurons co-expressed VGLUT2 (n=747 cells in 3 mice) or VGLUT1 (n=569 cells in 1 mouse) mRNA. C) To topographically describe the occurrence of VGLUT1/2 expressing PV+ neurons, we divided the long and short axes of the subiculum into 4 and 3 equal parts, respectively, to obtain 12 sub-fields along the proximal/distal axis and superficial/deep layers (upper panel). The relative occurrence of double labelled neurons is displayed in a heat map (double labelled cells per subfield/total double labelled cells, lower panel). CA1 cornus ammonis, DG dentate gyrus, S, subiculum; alv, alveus; RSG, retrosplenial cortex granular. D) Cartoon illustrating the electrophysiological recording of postsynaptic responses in pyramidal cells (PC, white) during optogenetic stimulation of PV+ neurons (green) in the subiculum of PV-Cre mice. E) 46% of recorded neurons showed exclusively inhibitory responses, 27% of cells showed both excitatory and inhibitory responses, 27% of recorded cells were unresponsive (n=30 cells in 5 mice). Example traces are displayed adjacent to the respective groups in the pie chart. Inhibitory responses could be blocked with gabazine (GBZ), excitatory responses could be blocked with NBQX and APV. Scale bar 2 mV, 50 ms. F) Cartoon depicting the location of the pyramidal cells receiving inhibitory input (gray), excitatory and inhibitory input (black) or no input (white) in the dorsal subiculum (cells are reported on a sagittal atlas plate 0.96 mm lateral (Paxinos and Franklin 2012). G, H) Current injections in CHR2-YFP-expressing PV+ neurons in distal subiculum revealed two different electrophysiological phenotypes: fast spiking, non-accommodating cells (G, left panel) and cells with slow and accommodating firing (H, left panel). Scale 20 mV, 200 ms. Both types of neurons showed high fidelity in their firing response following short pulses of light (right panels in G and H). Pulse duration 1 ms at 10% LED power; scale 10 mV, 50 ms at 10 Hz.

Article Snippet: Primary antibodies used were: rabbit polyclonal anti-GFP (1:1000, A6455, Invitrogen, California, USA), mouse monoclonal anti GFP (1:1000, A11120, Invitrogen, California, USA) rabbit polyclonal anti-PV (1:1000 to 1:2000, PV25, Swant, Switzerland), mouse monoclonal anti-PV (1:5000, PV235, Swant, Switzerland), rabbit polyclonal anti-RFP (1:2000, 600-401-379, Rockland, Pennsylvania, USA), mouse monoclonal anti-RFP (1:2000, 200-301-379, Rockland, Pennsylvania, USA), rabbit polyclonal anti-VGAT (1:1000, 131003, Synaptic Systems, Germany), guinea pig polyclonal anti-VGLUT2 (1:5000, AB2251, Millipore, Germany), guinea pig polyclonal anti-VGLUT2 (1:500, 135-404, Synaptic Systems, Germany).

Techniques: In Situ Hybridization, Staining, Expressing

Journal: bioRxiv

Article Title: Parvalbumin expression identifies subicular principal cells with high projection specificity

doi: 10.1101/2024.08.13.606964

Figure Lengend Snippet: A) Coronal brain sections in rostro-caudal sequence depicting the pathway of PV+ axonal projections in a PV-Cre mouse injected with mCherry-expressing Cre-dependent rAAVs (AAV-FLEX-mCherry) in the dorsal subiculum. Insets i, ii and iii show magnifications of the boxed areas demonstrating the absence of fibers from the internal capsule (ic) and the reticular thalamus (RT) but their presence in the dorsal fornix (df) and fornix (f), respectively. iv, close up of the boxed area in the preceding image (bregma level -1.03) showing the high density of labeled fibers in the antero-ventral thalamus (AV). v, magnification of the boxed area in the preceding image (bregma -2.80), showing some mCherry-positive fibers also in the medial mammillary nucleus (mMB). Scale bar 500 µm in all overview images, i -iii scale bar 50 µm, iv and v scale bar 500 µm. B,C) Close up of the boxed areas in iv,v, respectively, showing the dense network of mCherry-positive fibers in the AV and sparse positive fibers in the mMB. Scale bar 10 µm. Note the injection site in the Subiculum (S) is visible at bregma level -2.60. D,E) Images from the AV (D) and the mMB (E) of a PV-Cre mouse injected with a GFP-expressing Cre-dependent rAAV in the dorsal subiculum, stained for GFP (green) as well as the presynaptic markers VGLUT2 (purple) and VGAT (white). Images on the right show magnifications of the boxed areas on the left. Circles indicate boutons immunoreactive for GFP, VGLUT2 and VGAT. Image stacks are 2.5 µm in D and 0.6 µm in E. Scale bar 5µm in the overview, 1 µm in the magnifications. AD, antero-dorsal nucleus of the thalamus; AV, antero-ventral nucleus of the thalamus; DG, dentate gyrus; df, dorsal fornix; f, fornix; Hb, habenula; ic, internal capsule; LD, lateral dorsal nucleus of the thalamus; MD, mediodorsal nucleus of the thalamus; mMB, medial mammillary nucleus; RT, reticular thalamus; sm, stria medullaris of the thalamus; S, Subiculum; SuM, Supramammillary nucleus.

Article Snippet: Primary antibodies used were: rabbit polyclonal anti-GFP (1:1000, A6455, Invitrogen, California, USA), mouse monoclonal anti GFP (1:1000, A11120, Invitrogen, California, USA) rabbit polyclonal anti-PV (1:1000 to 1:2000, PV25, Swant, Switzerland), mouse monoclonal anti-PV (1:5000, PV235, Swant, Switzerland), rabbit polyclonal anti-RFP (1:2000, 600-401-379, Rockland, Pennsylvania, USA), mouse monoclonal anti-RFP (1:2000, 200-301-379, Rockland, Pennsylvania, USA), rabbit polyclonal anti-VGAT (1:1000, 131003, Synaptic Systems, Germany), guinea pig polyclonal anti-VGLUT2 (1:5000, AB2251, Millipore, Germany), guinea pig polyclonal anti-VGLUT2 (1:500, 135-404, Synaptic Systems, Germany).

Techniques: Sequencing, Injection, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: Parvalbumin expression identifies subicular principal cells with high projection specificity

doi: 10.1101/2024.08.13.606964

Figure Lengend Snippet: A) Illustration of the electrophysiological recording set up. B) Paired pulse ratios (PPRs) of EPSCs at different interstimulus intervals indicated that PV+ projections in the AV display synaptic depression (n= at least 4, for 100 ms n=18, 150 ms n=10, 200 ms n=8). Data are shown as mean ± sem. C) A train of stimuli at 10Hz shows synaptic depression at different light intensities of stimulation (minimal stimulation at 8% corresponds to 1.7, maximal at 30 to 5.6 mW/mm 2 ). The top shows example traces, the bottom EPSP ratios. Scale bar 5 pA, 100 ms. D) Example traces of (top) a cell that responds with action potential firing and (bottom) a cell that does not show an action potential response. When held at -50mV and stimulated with light, 3 out of 8 cells responded with action potential firing. Scale bar 5 mV, 100 ms. E) Example voltage clamp recordings in a cell that showed light-evoked EPSCs (top, n=5) or did not (bottom, n=3) during high frequency stimulation (100 Hz, 1 ms pulse duration, 30% LED power, held at -50 mV) in the presence of ionotropic glutamate and GABA antagonists (NBQX, APV and GBZ) showed no indication of metabotropic responses. Scale bar 5 pA, 1 s. F) Left, 3D isometric view of a thalamic neuron filled with biocytin (blue) and presynaptic VGLUT2-(purple) and GFP-(green) immunoreactive buttons of subicular projections. Right, 3D-surface reconstruction of the neuron on the left at higher magnification. Only VGLUT2-and GFP-positive surfaces with a distance of 0.5 µm from the filled neuron are depicted. Colocalization of VGLUT2 and GFP is shown in yellow (arrowheads). G) Putative synapses were found more frequently on proximal dendrites and somata (Pr) than on distal dendrites (Di). p=0.0111, paired t-test. H) The size of GFP+ presynaptic terminals showed considerable variation. I) Electron micrographs showing synaptic contacts (arrow head and arrows) between GFP-positive (peroxidase reaction product) and VGLUT2-positive (immunoparticles) boutons (b, b1, b2) and large calibre dendritic shafts (d) of thalamic neurons. S, dendritic spine; b3 and b4, GFP-positive but VGLUT2-negative axon terminals. Scale bar 1 µm.

Article Snippet: Primary antibodies used were: rabbit polyclonal anti-GFP (1:1000, A6455, Invitrogen, California, USA), mouse monoclonal anti GFP (1:1000, A11120, Invitrogen, California, USA) rabbit polyclonal anti-PV (1:1000 to 1:2000, PV25, Swant, Switzerland), mouse monoclonal anti-PV (1:5000, PV235, Swant, Switzerland), rabbit polyclonal anti-RFP (1:2000, 600-401-379, Rockland, Pennsylvania, USA), mouse monoclonal anti-RFP (1:2000, 200-301-379, Rockland, Pennsylvania, USA), rabbit polyclonal anti-VGAT (1:1000, 131003, Synaptic Systems, Germany), guinea pig polyclonal anti-VGLUT2 (1:5000, AB2251, Millipore, Germany), guinea pig polyclonal anti-VGLUT2 (1:500, 135-404, Synaptic Systems, Germany).

Techniques: