Journal: bioRxiv

Article Title: Perineuronal Net and Inhibitory Synapse Remodeling on Striatal Fast-spiking Interneurons by Chronic Alcohol Exposure

doi: 10.1101/2025.07.08.663744

Figure Lengend Snippet: A ) Schematic of viral injection of virus expressing a cre-dependent Gephyrin-GFP intrabody into parvalbumin (PV)-cre mice. B ) Representative image of an FSI expressing gephyrin-GFP intrabody (green) colocalized to an immunofluorescent marker for vesicular GABA transporter (VGAT) puncta. VGAT positive puncta were masked onto the GFP signal and colocalized signals (white dots) were labeled with a dot function model to quantify GABAergic synapses (scale bar 10µm). (right) Representative fluorescent image of proximal neuronal branch from control and CIE treated mice depicting colocalized presynaptic VGAT (purple) puncta onto gephyrin (green)-positive postsynaptic elements (scale bar 1µm). CIE dramatically reduced the density of colocalized VGAT puncta onto somata ( C ), proximal dendrites ( D ), and distal dendrites ( E ) (N=8 mice per group, 4M 4F). F ) Schematic of whole-cell patch clamp recording from an FSI during photo-uncaging of RuBi-GABA. G ) Representative traces of photo-uncaged GABA currents from FSIs of control (left) and CIE (right) treated mice at increasing stimulus intensities (scale bar = 200pA, 100ms). H ) CIE did not impact photo-uncaged IPSCs compared to control treated mice (n=8 mice per group, 4M 4F). All data represented as mean + SEM. *P < .05, **p<.01. Portions of this figure were created with BioRender.com

Article Snippet: Presynaptic GABAergic terminals were fluorescently labeled with an anti-vesicular GABA transporter (VGAT) antibody (rabbit; 1:1,000; Synaptic Systems # 131002).

Techniques: Injection, Virus, Expressing, Marker, Labeling, Control, Patch Clamp

Journal: bioRxiv

Article Title: Perineuronal Net and Inhibitory Synapse Remodeling on Striatal Fast-spiking Interneurons by Chronic Alcohol Exposure

doi: 10.1101/2025.07.08.663744

Figure Lengend Snippet: A ) schematic of whole-cell patch recording from PNN enriched (PNN+) or PNN poor (PNN-) FSI identified by WFA stain after recordings. B ) PNN+ FSIs exhibited larger eIPSC amplitudes compared to undetected PNN-FSIs (N=11 PNN+ cells, 5M 6F; 6 PNN-cells, 3M 3F). Inset: representative traces from PNN+ (pink) and PNN+ (black) FSIs (scale bar 1nA, 100ms). C ) The presence of PNNs did not impact GABA release probability onto FSIs. D-F ) Spontaneous IPSC events (sIPCSs) recorded from FSIs (N=11 PNN+ cells, 5M 6F; 6 PNN-cells, 3M 3F). Inset: representative traces from PNN+(pink) and PNN-(black) FSIs (scale bar 100pA, 1s). D,E ) PNN+ FSIs exhibited a greater frequency of spontaneous IPSC events but ( F ) no change in IPSC amplitude compared to PNN-FSIs. G ) Schematic of unilateral microinjection of chondroitinase ABC (ChABC) into the dorsolateral striatum to enzymatically degrade PNNs. H ) Degrading PNNs with ChABC reduced eIPSC event amplitude onto FSIs but ( I ) did not impact GABA release probability (N=10 mice, 5M 5F; scale bar 1nA, 100ms). J,K ) Degrading PNNs with ChABC reduced the frequency of sIPSC events onto FSIs but ( L ) did not impact event amplitude (N=8 mice, 4M 4F; scale bar 100pA, 1s). M ) Schematic of viral injection of cre-dependent Gephyrin-GFP intrabody into PV-cre mice followed by ChABC. N) Degrading PNNs with ChABC did not reduce inhibitory synapses onto somata but ( O ) did reduce inhibitory synapse number on proximal dendrites (N= 13 mice, 6M 7F). P ) Schematic of whole-cell patch clamp recording during photo-uncaging of RuBi-GABA onto FSIs treated with ChABC. Q ) Representative traces of photo-uncaged GABA postsynaptic currents on control (left) and ChABC (right) treated FSIs at increasing stimulus intensity (scale bar = 200pA, 100ms). G ) There was no change in photo-uncaged IPSC event amplitude between control (black) and ChABC (pink) FSIs (N=8 mice per group, 4M 4F). All data represented as mean + SEM. *P < 0.05. ** P < 0.01. Portions of this figure were created with BioRender.com

Article Snippet: Presynaptic GABAergic terminals were fluorescently labeled with an anti-vesicular GABA transporter (VGAT) antibody (rabbit; 1:1,000; Synaptic Systems # 131002).

Techniques: Staining, Microinjection, Injection, Patch Clamp, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: GABAergic synaptic components are largely preserved across human and mouse neuronal models

doi: 10.3389/fncel.2025.1588894

Figure Lengend Snippet: Morphological characterization of GABAergic neurons. (A–D) Scatter-plot showing mean soma areas (A) , dendritic thickness (B) , dendritic lengths (C) , and VGAT synapse puncta (D) . (E) Representative images of neuronal morphology showing immunostaining for MAP2 (color coded) and VGAT (black). Scale bars, 40 μm. Data shown as mean ± SEM. Kruskal-Wallis test: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Specifically, primary antibodies dilutions were as follows: chicken anti-microtubule-associated protein 2 (MAP2) (1:2000, Millipore), rabbit anti-vesicular GABA transporter (VGAT) (1:1000, Synaptic Systems).

Techniques: Immunostaining

Journal: Current Research in Neurobiology

Article Title: Regulation of perisomatic synapses from cholecystokinin basket interneurons through NrCAM and Ankyrin B

doi: 10.1016/j.crneur.2025.100150

Figure Lengend Snippet: CCK-basket cell synaptic puncta on soma of cortical pyramidal neurons (PNs) are decreased in mPFC of NrCAM-null mutant mice. A. PNs in mPFC layer 2/3 of WT and NrCAM-null mice (adult P60) were immunostained for somal marker MATH2 (blue). CCK-BC presynaptic puncta on PN soma were immunostained for VGLUT3 (red, arrowheads). Confocal images were captured with a 40X objective in a field of view of 1878× 1878 pixels, or 160 μm × 160 μm. The mean numbers of soma per image were: WT 26 ± 11, NrCAM-null 22 ± 7. Representative regions of confocal images were obtained from maximum intensity projections of 5 optical sections (∼0.3 μm each). Bars = 20 μm. B. Quantification of VGLUT3+ synaptic puncta per MATH2+ PN soma in WT and NrCAM-null mPFC. Data points represent the number of puncta per soma per image. The mean number of puncta per soma across all images was significantly different between genotypes (two-tailed t -test: p∗ = 0.01). C. PNs were immunostained for MATH2 (blue) and perisomatic puncta from CCK-BCs and PV-BCs were immunostained for VGAT (red, arrowheads) in WT and NrCAM-null mice. Confocal mages were captured with a 63X objective in a field of view of 2048× 2048 pixels, or 102 μm × 102 μm. The mean numbers of soma per image were: WT 6 ± 2, NrCAM-null 6 ± 2. Representative images as described in A are shown. Bars = 20 μm. D. Quantification of VGAT + synaptic puncta per MATH2+ PN soma in WT and NrCAM-null mPFC. Data points represent the number of puncta per soma per image. The mean number of puncta per soma were significantly different between genotypes (two-tailed t -test: ∗p = 0.02). E. Immunostaining of CCK-BC marker NECAB1 (white, arrowheads) in WT and NrCAM-null mPFC. Non-confocal images were obtained by EVOS microscopy. Images were captured with a 10X objective in a field of view of 2048× 1536 pixels, or 632 μm × 474 μm. The mean numbers of soma per image were: WT 11 ± 4, NrCAM-null 10 ± 4. Representative regions of images are shown. Bar = 100 μm. F. Quantification of the mean number of NECAB1+ soma per mm 2 in EVOS images. Data points represent the number of puncta per soma per image. Differences between WT and NrCAM-null genotypes were not significantly different (ns, two-tailed t -test: p = 0.99).

Article Snippet: Mouse monoclonal antibody to vesicular GABA transporter (VGAT) was from Synaptic Systems (131 011.)

Techniques: Mutagenesis, Marker, Two Tailed Test, Immunostaining, Microscopy

Journal: Current Research in Neurobiology

Article Title: Regulation of perisomatic synapses from cholecystokinin basket interneurons through NrCAM and Ankyrin B

doi: 10.1016/j.crneur.2025.100150

Figure Lengend Snippet: Perisomatic synaptic puncta from CCK-BCs onto PN soma are decreased in mPFC of Nex1Cre-ERT2: Ank2+/+ (WT) and Ank2 F/F mPFC. A. Immunofluorescent staining of VGLUT3+ synaptic puncta (red, arrowheads) on EGFP + PN soma (blue) in Ank2 +/+ (WT) and Ank2 F/F mPFC layer 2/3 (∼P60). Confocal images were captured with 40X objective and a field of view of 1878× 1878 pixels, or 160 μm × 160 μm. The mean numbers of soma per image were: WT 18 ± 3, Ank2 F/F 8 ± 1. Representative regions of confocal images were obtained from average intensity projections of 5 optical sections (∼0.3 μm each). Bars = 20 μm. B. Quantitation of VGLUT3+ synaptic puncta on EGFP + soma. Data points represent the number of puncta per soma for each image. The mean number of VGLUT3+ puncta per EGFP + soma was significantly different for genotypes Ank2 +/+ and Ank2 F/F (two-tailed t -test, p < 0.0001∗). C. Immunofluorescent staining of NECAB1+ soma (arrowheads) on EGFP + PN soma in Ank2+/+ and Ank2 F/F mice. Non-confocal images were obtained by EVOS microscopy. Images were captured with a 10X objective in a field of view of 2048× 1536 pixels, or 1264 μm × 948 μm. The mean numbers of soma per image were: Ank2 +/+ 12 ± 3, Ank2 F/F 15 ± 4. Representative regions of images are shown. Bars = 10 μm. D. Quantitation of NECAB1+ EGFP + soma/mm 2 (x10 −1 ). Data points represent the number of puncta per soma for each image. Mean differences between Ank2+/+ and Ank2 F/F genotypes were not significant (ns) (two-tailed t -test, p = 0.25.) E. Immunofluorescent staining of Syt2b + synaptic puncta (red, white arrowheads) on EGFP + PN soma (blue) in Ank2 +/+ and Ank2 F/F mPFC. Confocal images were captured with a 40X objective in a field of view of 1878× 1878 pixels, or 160 μm × 160 μm. The mean numbers of soma per image were: Ank2 +/+ 7 ± 3, Ank2 F/F 4 ± 1. Representative regions of images were obtained from maximum intensity projections of 5 optical sections (∼0.3 μm each). Bars = 20 μm. F. Quantitation of Syt2b + puncta per EGFP + soma (x10 −1 ). Data points represent the number of puncta per soma in each image. Mean differences in Syt2b + puncta per EGFP + soma were not significant (ns) between Ank2 +/+ and Ank2 F/F genotypes (two-tailed t -test, p = 0.99.) Bars = 20 μm. G. Immunofluorescent staining of VGLUT3 (magenta) and VGAT (green) in Nex1Cre-ERT2: Ank2+/+ mPFC layer 2/3 (P63) (EGFP, blue) showed double staining of perisomatic puncta on EGFP + PNs (white arrowheads), in confocal images, as seen in 2 representative single optical sections (0.3 μm). Quantitation indicated that ∼88 % of VGLUT3+ puncta co-expressed VGAT (38 double labeled puncta scored on 43 EGFP + soma in 8 confocal z-stacks). Control staining with secondary antibodies alone was negative. Confocal images were captured with a 63X objective in a field of view of 512× 512 pixels, or 160 μm × 160 μm. Bars = 10 μm.

Article Snippet: Mouse monoclonal antibody to vesicular GABA transporter (VGAT) was from Synaptic Systems (131 011.)

Techniques: Staining, Quantitation Assay, Two Tailed Test, Microscopy, Double Staining, Labeling, Control