mouse anti-vesicular glutamate transporter 1 (vglut1  (Synaptic Systems)

Journal: bioRxiv

Article Title: Gluk4-containing kainate receptors regulate synaptic communication in the motor cortex and reduce axon degeneration in adult mice

doi: 10.1101/2024.02.29.582867

Figure Lengend Snippet: a-b) Reconstructed basal dendrite segment from a control (Ctrl, a ) or Grik4 -/- ( b ) mouse, with spines classified as thin (red), stubby (yellow), or mushroom (blue) morphological subtypes. Scale bars represent 1µm. c) Number of spines per 10µm stretch of basal dendrite, average per mouse [unpaired t-test, t = 4.64, p = 0.002]. Number of thin, stubby or mushroom spines per 10µm stretch of basal dendrite, average per mouse [2-way mixed ANOVA: Spine type (within-subjects) F(2,16) = 136.1, p<0.0001; Genotype F(1,8) = 21.50, p = 0.0017; Interaction F(2,16)=35.54, p<0.0001]. d) Basal dendritic spine morphological types expressed as percentage of total spines within each dendrite, averaged per mouse [Mann-Whitney U compare mean ranks between genotypes, within each spine type, Bonferroni adjusted p-values; thin rank diff = 5, p=0.0286; stubby rank diff = -1.25, p >0.999; mushroom rank diff = -5, p = 0.029]. e-j) Thy1-YFPH + neuronal soma in layer V of the motor cortex of a WT ( e-g ) or Grik4 -/- ( h-j ) mouse, stained to detect GFP (green), vesicular GABA Transporter (VGAT; red; marking inhibitory synaptic terminals), and vesicular glutamate transporter 1 (VGlut1; blue; marking excitatory synaptic terminals). Imaris software was used to identify and mark puncta that were close to the GFP + neuronal surface (grey spots in f , g , i and j ). k) The density of VGlut1 puncta in GFP + Ctrl neurons (n=69) and Grik4 -/- neurons (n=37) [Unpaired t-test, t=3.281, P=0.0014]. l) The density of VGlut1 puncta at GFP + neuron soma, average per mouse [Unpaired t-test t=5.760, p=0.0007]. m) The density of VGAT puncta in individual GFP + neurons of Ctrl mice (n=69 cells) and Grik4 -/- mice (n=37 cells) [Unpaired t-test, t = 2.342, p = 0.021]. n) The density of VGAT puncta at GFP + neuron soma, average per mouse [Unpaired t-test t=1.196, p=0.2707]. Data presented as mean ± SD. Outcome of Šídák’s multiple comparisons tests (c), Bonferroni-corrected Mann-Whitney U tests (d) or t-tests (k-n): * p<0.05, **p<0.01, *** p<0.001 or **** p<0.0001. n = 4-6 mice in each group.

Article Snippet: Sections were incubated overnight at 4°C in PBS blocking solution [0.1% (v/v) Triton X-100 and 10% foetal calf serum in PBS] containing primary antibodies including: mouse anti-NeuN (1:200; Millipore, #MAB337); mouse anti-parvalbumin (1:1000, Swant, #235), rabbit anti-somatostatin (1:1000, Immunostar, #20067), mouse anti-SMI32 (1:1000, Covance/Biolegend, #801702), mouse anti-SMI312 (1:1000, Covance/Biolegend, #837904), rabbit anti-amyloid precursor protein (APP, C-terminal, 1:1000, Sigma, #A8717), rat anti-green fluorescent protein (GFP, 1:2000, Nacalai Tesque, #04404-84), mouse anti-vesicular glutamate transporter 1 (VGlut1, 1:200, Synaptic Systems, #135 511), and chicken anti-vesicular GABA transporter (VGAT, 1:1000, Synaptic Systems, #131 006).

Techniques: MANN-WHITNEY, Staining, Software

Journal: bioRxiv

Article Title: Imbalanced expression of clustered protocadherins in pre- and post-synaptic compartments of CA1 pyramidal cells during hippocampal development

doi: 10.1101/2023.04.15.536995

Figure Lengend Snippet: A : Images of Lucifer yellow-injected CA1 pyramidal cells. Scale bar: left, 200 μm; right, 5 μm. B : Image of pyramidal cell dendrites of P7 to 8-week old mice. Scale bar: 2 μm. C : Immunostaining for vGluT1 and magnified view (bottom) of the white square in the upper image. Scale bar: 100 μm (upper) and 20 μm (lower). D : Developmental stage-associated changes in spine density in the CA1 pyramidal cell radiatum region; mean ± SD. E : Developmental stage-associated changes in vGluT1 immunosignals in the CA1 stratum radiatum region; mean ± SD. F : Criteria for spinal morphology G : Developmental stage-associated changes in the proportion of each spine type.

Article Snippet: Similarly, slices were incubated overnight with rabbit anti-Lucifer yellow antibody and mouse anti-vesicular glutamate transporter-1 (vGluT1) antibody (0.4 μg/ml; sc-377425, Santa Cruz Biotechnology, Inc.) in the same primary solution.

Techniques: Injection, Immunostaining

Journal: bioRxiv

Article Title: Imbalanced expression of clustered protocadherins in pre- and post-synaptic compartments of CA1 pyramidal cells during hippocampal development

doi: 10.1101/2023.04.15.536995

Figure Lengend Snippet: A : cPcdhγCR immunosignals at P7. B : vGluT1 immunosignals. C : Merged image of cPcdhγ and vGluT1 expression. A’-C’ : Magnified view of the CA1 stratum radiatum regions. A”-C” : Magnified view of the upper images. D-F: Synaptic localization of cPcdhγCR immunosignals at P21. D : Green indicates dendrites and spines visualized using Lucifer yellow. cPcdhγ and vGluT1 were co-immunostained. The localization of cPcdhγ in synapses was categorized into four groups. E : Schematic representation of the immunosignal location categories. F : Proportion of cPcdhγ localization in the categories shown in E. Scale bar: 100 μm ( A-C ), 20 μm ( A’-C’ ), 1 μm ( A’’-C’’ ), 200 nm ( D ).

Article Snippet: Similarly, slices were incubated overnight with rabbit anti-Lucifer yellow antibody and mouse anti-vesicular glutamate transporter-1 (vGluT1) antibody (0.4 μg/ml; sc-377425, Santa Cruz Biotechnology, Inc.) in the same primary solution.

Techniques: Expressing

Journal: bioRxiv

Article Title: Imbalanced expression of clustered protocadherins in pre- and post-synaptic compartments of CA1 pyramidal cells during hippocampal development

doi: 10.1101/2023.04.15.536995

Figure Lengend Snippet: A-D’ : cPcdhγ (5 nm gold particles, red circles) and vGluT1 (15 nm gold particles) in the CA1 stratum radiatum. A, Pre-synaptic localization of cPcdhγ. cPcdhγ-immunoparticles were found near both the active zone ( A ) and the extra-synaptic area ( B ). C-C’ and D-D’ are images of paired replicas. C, D , Localization of cPcdhγ at postsynaptic sites. C’, D’, Localization of cPcdhγ at pre-synaptic sites. E, Proportion of cPcdhγ+ pre-synaptic sites identified using vGluT1 labelling. F, Number of cPcdhγ-immunoparticles in active zones and extra-active zones. G, Number of cPcdhγ-immunoparticles in pre- and postsynaptic faces of paired replicas. H, Proportion of cPcdhγ+ postsynapses in cPcdhγ+ pre-synapses. Scale bar: 200 nm.

Article Snippet: Similarly, slices were incubated overnight with rabbit anti-Lucifer yellow antibody and mouse anti-vesicular glutamate transporter-1 (vGluT1) antibody (0.4 μg/ml; sc-377425, Santa Cruz Biotechnology, Inc.) in the same primary solution.

Techniques:

Journal: Molecular Neurobiology

Article Title: Celsr2 Knockout Alleviates Inhibitory Synaptic Stripping and Benefits Motoneuron Survival and Axon Regeneration After Branchial Plexus Avulsion

doi: 10.1007/s12035-022-03198-3

Figure Lengend Snippet: Celsr2 inactivation slows down inhibitory synaptic stripping on injured motoneurons. C5–C7 spinal sections are for double immunostaining at day 7, 14, and 21 after BPA, and for EM studies on day 7 after BPA. A – C Anti-VGAT (green) and -ChAT (red) double immunostaining shows that the coverage of VGAT-immunoreactivity on motoneuron membranes decreases on injured sides compared to intact sides in two groups ( A ). The linear density of VGAT immunoreactivity is comparable in two groups on intact sides ( P > 0.05), but significantly increased at day 7, 14, and 21 on injured sides in the mutant compared to the control ( B ; P < 0.0001 at day 7, P < 0.05 at day 14 and 21). Normalized to intact sides, the VGAT loss is decreased in the mutant compared to the control at three timepoints after BPA ( P < 0.01 at day 7 and 14, P < 0.05 at day 21; C ). D – F Anti-vGlut1 (green) and -ChAT (red) double immunostaining shows that the coverage of vGlut1-immunoreactivity on motoneuron membranes decreases on injured sides compared to intact sides at day 7, 14, and 21 in two groups ( D ). Linear density is comparable in two groups on both sides (E, P > 0.05). Normalized to intact sides, the vGlut1 loss is similar in two groups (F, P > 0.05). G – I Ultrastructure of ventral horns under EM shows motoneuron (MN) membrane, presynaptic terminals (T), F- and S- boutons 7 days after BPA in two groups ( G ). The number of F-boutons within 100-µm motoneuron membrane is significantly increased in the mutant on injured sides ( P < 0.01), but comparable in two groups on intact sides ( P > 0.05); the density of S-Boutons shows no differences in two groups ( P > 0.05) ( H ). The bouton density ratio (injured to intact sides) shows an increase of F-boutons ( P < 0.01), but not of S-boutons ( P > 0.05), in the mutant compared to the control ( I ). Two-way ANOVA with Sidak’s multiple comparisons; immunofluorescent staining: n = 4 animals in the control and 5 animals in the mutant at day 7, n = 5 animals in the control and 6 animals in the mutant at day 14, n = 5 animals/group at day 21; EM study: n = 6 neurons from 3 animals in each group; dpi days post injury

Article Snippet: Primary antibodies were: goat anti- choline acetyltransferase (ChAT, 1:500, ab144p, Millipore), chicken anti- β -gal (1:500, ab9361, Abcam), rabbit anti-Calretinin (1:300, ab702, Abcam), mouse anti-Parvalbumin (1:1000, Mab1572, Millipore), rabbit anti-CAMKII (1:500, ab104224, Abcam), rabbit anti-vesicular GABA transporter (VGAT; 1:800, NO131013, Synaptic Systems), mouse anti-vesicular glutamate transporter 1 (vGlut1; 1:1000, Mab5502, Millipore), rat anti-major histocompatibility complex 1 (MHC1; 1:300, sc-59199, Santa Cruz), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, AB7260, Abcam), rabbit anti-Iba1(1:1000, 019–19,741, Wako), and rabbit anti-Oligo2 (1:500, ab9610, Merck Millipore).

Techniques: Stripping Membranes, Double Immunostaining, Mutagenesis, Staining

Journal: Molecular Neurobiology

Article Title: Celsr2 Knockout Alleviates Inhibitory Synaptic Stripping and Benefits Motoneuron Survival and Axon Regeneration After Branchial Plexus Avulsion

doi: 10.1007/s12035-022-03198-3

Figure Lengend Snippet: Conditional inactivation of Celsr2 in astrocytes does not affect synaptic withdrawal after BPA. A – C In spinal sections of adult Celsr2 LacZ mouse, double immunostaining shows GFAP-positive cells co-express ß -gal (arrows, A ). Conditional inactivation of Celsr2 in astrocytes and the experimental design are illustrated in the schematic ( B ). Celsr2 mRNA levels in spinal samples are significantly downregulated in Aldh1l1–CreER T2 ; Celsr2 f /− mice ( Aldh1l1 cKO) upon 3-days tamoxifen induction, but still higher than that in Celsr2 −/− mice ( C ). D – F The coverage of inhibitory synaptic vesicles on spinal motoneuron membranes is visualized by anti-VGAT and -ChAT double immunostaining ( D ). The linear densities of VGAT-positive vesicles on both sides and the percentage of VGAT-positive vesicles loss (normalized to intact sides) on injury sides show similar changes in two groups ( E , F ; P > 0.05). G – I Anti-vGlut1 and -ChAT double immunostaining shows BPA-induced excitatory synapse withdrawal on spinal motoneuron membranes in two groups ( G ). The reduction is comparable in two groups at day 7, 14, and 21 after BPA ( H , I ; P > 0.05). Two-way ANOVA with Sidak’s multiple comparisons, n = 6 mice at each timepoint in each group; dpi, days post-injury

Article Snippet: Primary antibodies were: goat anti- choline acetyltransferase (ChAT, 1:500, ab144p, Millipore), chicken anti- β -gal (1:500, ab9361, Abcam), rabbit anti-Calretinin (1:300, ab702, Abcam), mouse anti-Parvalbumin (1:1000, Mab1572, Millipore), rabbit anti-CAMKII (1:500, ab104224, Abcam), rabbit anti-vesicular GABA transporter (VGAT; 1:800, NO131013, Synaptic Systems), mouse anti-vesicular glutamate transporter 1 (vGlut1; 1:1000, Mab5502, Millipore), rat anti-major histocompatibility complex 1 (MHC1; 1:300, sc-59199, Santa Cruz), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, AB7260, Abcam), rabbit anti-Iba1(1:1000, 019–19,741, Wako), and rabbit anti-Oligo2 (1:500, ab9610, Merck Millipore).

Techniques: Double Immunostaining