Journal: ACS nano

Article Title: AptBCis1, An Aptamer-Cisplatin Conjugate, Is Effective in Lung Cancer Leptomeningeal Carcinomatosis.

doi: 10.1021/acsnano.4c04680

Figure Lengend Snippet: Figure 6. AptB1 interacting with EAAT2, Nucleolin, and YB-1. (a) Results of AptB1-AP/MS study revealed three candidate AptB1- interacting proteins: EAAT2, YB-1, and Nucleolin. (b) AP-immunoblots verified the interaction between AptB1 and EAAT2, Nucleolin, as well as YB-1 in the PC9 cells and in the mouse brain. (c) The confocal microscopy images showed colocalization (yellow) of AptB1 (red) and Nucleolin (green, upper panel) or YB-1 (green, lower panel) in the PC9 cells. (d) AptB1-treated cells were fractionated into cytosol and nucleus fractions. GAPDH is a cytosolic marker, and Histone H3 is a nucleus marker. The AptB1 sequences were successfully amplified in both cellular fractions. (e) The Coomassie Blue stain and the immunoblots showed the purified GST and GST-YB-1 proteins. (f) The YB-1 exonuclease assay results supported the role of YB-1 as an exonuclease for AptB1. (g) Scheme illustrating the proposed mechanism of AptBCis1 as therapeutics for lung cancer with and without LM.

Article Snippet: Anti-luciferase antibody (sc-74548), EAAT2 siRNA (sc-35256), Nucleolin siRNA (sc-29230), YB-1 siRNA (sc38634), and GAPDH antibody (sc-32233) were purchased from Santa Cruz. γH2AX (9718), YB-1 (4202), and EAAT2 (20848) antibodies were purchased from Cell Signaling.

Techniques: Protein-Protein interactions, Western Blot, Confocal Microscopy, Marker, Amplification, Staining, Purification

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: Primer sequences for quantitative polymerase chain reaction

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX restores the expression of EAAT2 after spinal cord injury. (A) Representative western blot of EAAT2 protein level in spinal cord tissues 28 days after injury. (B) Quantification of the protein expression of EAAT2. (C) EAAT2 mRNA expression in spinal cord tissues 28 days after injury. (D) Representative images of western blotting of EAAT2 in each group at 7 dpi. (E) Quantitative assessment of EAAT2 protein expression. (F) EAAT2 mRNA expression in each group at 7 dpi. (G, H) Representative figures and quantitative assessment of immunofluorescence staining for EAAT2 (red, Alexa Fluor 594), GFAP (green, Alexa Fluor 488), and DAPI (blue) in reactive astrocytes in each group at 7 dpi. In reactive astrocytes, the fluorescence intensity of EAAT2 in the SCI + Veh group was lower compared with the Sham group, whereas EAAT2 expression was increased in the SCI + RUX group compared with the SCI + Veh group. The arrow indicates the expression of EAAT2 in the processes of astrocytes. Scale bar: 10 µm. (I) Measurement of glutamate levels in injured tissues in each group at 7 dpi ( n = 3). (J) Representative western blot of p-JAK2, JAK2, p-STAT3, and STAT3 in each group at 7 dpi. (K, L) Quantification of p-JAK2/JAK2 and p-STAT3/STAT3 levels in each group. In B, C, E, F, H, I, K, and L, data were normalized by the sham group. Data are presented as the mean ± SD. The experiments were performed in triplicate. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). AS: Scar-forming reactive astrocytes; DAPI: 4′,6-diamidino-2-phenylindole; dpi: days post-injury; EAAT2: excitatory amino acid transporter 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; JAK2: Janus kinase 2; NA: naïve astrocytes; ns: not significant; p-JAK2: phosphorylated Janus kinase 2; p-STAT3: phosphorylated signal transducer and activator of transcription 3; RA: reactive astrocytes; RUX: ruxolitinib; SCI: spinal cord injury; STAT3: signal transducer and activator of transcription 3; Veh: vehicle.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX rescues astrocyte EAAT2 loss and restores glutamate uptake in vitro. (A) The effect of RUX (0.5, 1, 1.5, 2 and 2.5 µM) on the viability of astrocytes was assessed via CCK-8 assay ( n = 3/ group). (B, C) Western blotting of EAAT2 expression in A1IM-treated astrocytes in each group pre-treated with RUX (0.2, 0.5 and 1 µM) as shown ( n = 3 per group). (D) Quantitative analysis of EAAT2 protein levels in each group ( n = 3/group). (E, F) Representative immunofluorescence staining images and quantification of EAAT2 (red, Alexa Fluor 594), GFAP (green, Alexa Fluor 488), and DAPI (blue) in primary mouse astrocytes for each group ( n = 3 per group). The A1IM group displayed lower fluorescence intensity of EAAT2 compared with the Control group, whereas the RUX group exhibited higher fluorescence intensity of EAAT2 compared with the A1IM group. Scale bar: 20 µm. (G) Relative glutamate uptake of astrocytes in each group ( n = 3). The experiments were conducted in triplicate. Data are normalized by the sham group. Data are presented as the mean ± SD. ## P < 0.01, vs . control group; ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). A1IM: A1-like astrocyte induction medium; C1q: complement component 1q; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; EAAT2: excitatory amino acid transporter 2; GFAP: glial fibrillary acidic protein; IL-1α: interleukin-1 alpha; ns: not significant; RUX: ruxolitinib; TNF-α: tumor necrosis factor-alpha.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: In Vitro, CCK-8 Assay, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01863

Figure Lengend Snippet: RUX restores EAAT2 loss in astrocytes by inhibiting the activation of STAT3 in vitro. (A) Representative western blotting of p-JAK2, JAK2, p-STAT3 and STAT3 expression of astrocytes in each group pre-treated with RUX (0.2, 0.5 and 1 µM) ( n = 3 per group). (B) Quantitative analysis of p-JAK2/JAK2 and p-STAT3/STAT3 level in each group. (C) Immunocytochemistry of GFAP (green, Alexa Fluor 488) and p-STAT3 (red, Alexa Fluor 594) in primary mouse astrocytes. DAPI (blue) was used to stain nuclei. The A1IM group exhibited higher nuclear p-STAT3 expression compared with the Control group, whereas the RUX group displayed decreased nuclear p-STAT3 expression compared with the A1IM group. Scale bar: 40 µm. (D) Quantitative results of relative p-STAT3 intensity. (E) Representative western blotting of STAT3 expression of astrocytes pre-treated with S3I-201 or vehicle. (F) Quantitative results of relative STAT3 expression level. (G) Primary mouse astrocytes were pretreated with S3I-201 (50 μM) or RUX for 1 hour and then exposed to LPS (1 µg/mL) or PBS for 5 hours. Western blotting was conducted to assess EAAT2 expression level, followed by quantitative analysis of EAAT2 protein expression (H). (I) Glutamate (100 nM) was introduced into each culture medium, and the relative glutamate uptake of astrocytes in each group was measured. Data are normalized to the control group. Data are presented as the mean ± SD ( n = 3 per group). ## P < 0.01, vs. control group; * P < 0.05, ** P < 0.01, vs. A1IM group (one-way analysis of variance followed by Tukey’s post hoc test). A1IM: A1-like astrocyte induction medium; Ctrl: control; DAPI: 4′,6-diamidino-2-phenylindole; EAAT2: excitatory amino acid transporter 2; GFAP: glial fibrillary acidic protein; JAK2: Janus kinase 2; ns: not significant; p-JAK2: phosphorylated JAK2; p-STAT3: phosphorylated STAT3; RUX: ruxolitinib; S3I-201: a STAT3 inhibitor; STAT3: signal transducer and activator of transcription 3.

Article Snippet: After blocking with Immunol Staining Blocking Buffer at room temperature for 1 hour, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-GFAP (1:500), rabbit anti-EAAT2 (1:50), rabbit anti-p-STAT3 (1:200), mouse anti-neuronal nuclei (NeuN; 1:500, Proteintech, Wuhan, China, Cat# 66836-1-Ig, RRID: AB_2882179), rabbit anti-MAP2 (1:500, Proteintech, Cat# 17490-1-AP, RRID: AB_2137880), rat anti-C3 (1:500), rabbit anti-IL-1β (1:250, Proteintech, Cat# 16806-1-AP, RRID: AB_10646432), rabbit anti-IL-6 (1:500, Proteintech, Cat# 21865-1-AP, RRID: AB_11142677), rabbit anti-TNF-α (1:400, Proteintech, Cat# 17590-1-AP, RRID: AB_2271853), rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, Cat# ab178945, RRID: AB_2861417), rabbit anti-Arginase-1 (Arg1, Proteintech, Cat# 16001-1-AP, RRID: AB_2289842), and mouse anti- ionized calcium-binding adapter molecule 1 (IBA1, Santa Cruz Biotechnology, Cat# sc-32725, RRID: AB_667733).

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Immunocytochemistry, Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: A Distinct Metabolite Profile Correlates with Neurodegenerative Conditions and the Severity of Congenital Hydrocephalus.

doi: 10.1093/jnen/nly097

Figure Lengend Snippet: FIGURE 5. Immunohistochemistry/immunofluorescence for glutamate, glutaminase, and the excitatory amino acid transporter 2 (EAAT2). Immunolabeling in vibratome sections for glutamate (Glu, fluorescence in green), kidney-type glutaminase (KGA, fluorescence in green and DAB-nickel reaction), and GFAP (fluorescence in red) in the neocortex (ncx) of normal mice (wt; A, B, F, G) and hyh mice with rapidly progressive hydrocephalus (RPH; C–E, H, I). (D0/I0) and (D00/I00) correspond to the green and red channels for immunofluorescence of (D/I) to show colocalizations (arrowheads). A confocal plane (1 mm-thick) is shown in (D). Nuclear staining in blue with DAPI. Arrowheads in (B) point to astrocytes immunolabeled for GFAP and glutamate in the gray matter and white matter (wm) of the neocortex. Arrows in (C–E) point to the periventricular astrocyte reaction in the white matter. Abbreviations: hip, hippocampus. Immunofluorescence in frozen sections (confocal planes 1 mm thick) for EAAT2

Article Snippet: Antibodies Antibody Source Reference Type Use Dilution EAAT2 Santa Cruz Biotechnology Sc-365634 Mouse monoclonal FR 1:100 GFAP Sigma G-A-5 Mouse monoclonal PF, VB, FR 1:1000 Glutamate Sigma G6642 Rabbit polyclonal VB, FR 1:4000 Glutathione peroxidase Abcam ab108427 Rabbit monoclonal PF, VB, FR 1:150 Glutamine synthetase Abcam ab49873 Rabbit polyclonal PF, VB, FR 1:5000 Glutathione synthetase GeneTex GTX63666 Rabbit monoclonal PF, VB, FR 1:100 Kidney-type glutaminase (KGA) * * Rabbit polyclonal VB, FR 1:500 NeuN Merck Millipore MAB377 Clone A60 Mouse monoclonal VB, FR 1:500 Olig2 R&D Systems AF2418 Goat polyclonal VB, FR 1:1000 Taurine Merck Millipore AB5022 Rabbit polyclonal VB, FR 1:100 Abbreviations: FR, frozen section; PF, paraffin section; VB, vibratome section.

Techniques: Immunohistochemistry, Immunofluorescence, Immunolabeling, Fluorescence, Staining

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Immunohistochemical stain of GLT1 (red), astrocytes (green), and DAPI (blue) at ~P28 in mouse visual cortex. GLT1 is expressed by astrocytes throughout cortical layers. B) High-magnification image of a single GFAP-labelled astrocyte with surrounding GLT1 expression. C) Quantification of GLT1-mRNA across developmental time points showing a significant increase from birth and peaking at P21. Levels are normalized to P28 (P0 (n=4), P7 (n=4), P14 (n=3), P21 (n=3), P28 (n=4), P42 (n=3), P60 (n=4); one-way ANOVA, F(6,18)=86.7, p=2.7×10 −12 ). D) Western blot quantification showing that transgenic mice with heterozygous expression of GLT1 (HET) have significantly less GLT1 expression compared to wildtype (WT) littermates (n=WT(11), HET(11); unpaired t-test, t=4.64, p=2.7×10 −4 ), but comparable expression of GLAST (WT (n=8), HET (n=4); unpaired t-test, t=0.62, p=0.55). E) Example images of GLT1 WT and HET astrocytes labeled using a custom GFAP-tdTomato transgenic mouse line (red). Astrocyte volume is reconstruction from imaged z-stacks (green). F) Quantification of astrocyte volume from 3D reconstructions show no difference between GLT1 WT and HET animals (WT (n=3), HET (n=13); unpaired t-test, t=0.53, p=0.60). G) Images of the lateral geniculate nucleus (LGN) after CTB-594 (red) and CTB-488 (green) injection into the contralateral and ipsilateral eyes respectively. Merged overlays from GLT1 WT and HET mice show normal retinothalamic axon segregation. H) Quantification of ipsilateral area in GLT1 WT and HETs across several binary thresholds (0, 5, 30%) showing no difference in absolute ipsilateral area (WT (n=3), HET (n=6); two-way ANOVA, genotype effect F(1,7)=1.93, p=0.21). I) Quantification of contra/ipsi projection overlap showing no difference in contra/ipsi segregation (WT (n=3), HET (n=6); two-way ANOVA, genotype effect, F(1,7)=0.43, p=0.53). *p<0.05, **p<0.01, ***p<0.005

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Transgenic Assay, Labeling, Injection

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Schematic of experimental design. Top: Visual gratings were separately presented to the contra (green) and ipsi (blue) eyes in P28 mice and neuronal responses recorded. Bottom: schematic of measures. Ocular dominance index (ODI) was calculated as (max Contra − max Ipsi ) / max Contra +max Ipsi . Orientation Selectivity Index (OSI) was calculated as described previously . Difference in preferred orientation (ΔPO) was calculated as the difference between preferred orientations of the max contralateral and ipsilateral responses. B) Example cells in GLT1 WT (top) and GLT1 HET (bottom) animals. Left: in vivo images of neuronal somas measured in binocular visual cortex using the calcium indicator, GCaMP6f. Right: Tuning curves of three cells (white circles in left) to contra (green) and ipsi (blue) stimulation. Note the matched tuning and contralateral bias in WT animals and the mismatched tuning curves and lack of contralateral bias in GLT1 HETs. C) Quantification of the average response to PO in GLT1 WT and HET mice. WT mice have a significantly higher contralateral response than ipsilateral response while HET mice have approximately equal contralateral and ipsilateral responses (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=7.72, p=0.0056, interaction F(1,674)=4.243, p=0.040). D) Quantification of ocular dominance index showing that GLT1 HET mice have significantly decreased ODI (n=4-6 animals, 23-52 cells per animal, t-test, p=0.0018). E) Quantification of OSI showing that GLT1 HET mice have a significantly decreased OSI of ipsilateral responses compared to both contra and ipsi responses in GLT1 WT animals (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=12.46, p=4.5×10 −4 ). F) Quantification of ΔPO showing an increased difference in the preferred orientations between contralateral and ipsilateral inputs to neurons in GLT1 HET animals (n=4-6 animals, 23-52 cells per animal, t-test, p=1.0×10 −4 ). *p<0.05, **p<0.01, ***p<0.005

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: In Vivo

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Left: low-magnification image of neurons in visual cortex of GFP-M transgenic mice. Right: higher-magnification image of layer 2/3 neurons (dotted box in left). B) Images of basal dendrites of layer 2/3 neurons in GLT1 WT (top) and HET (bottom) mice. WT example is from dotted box in right panel of A. C) GLT1 HET mice have increased spine density on basal dendrites of layer 2/3 neurons in visual cortex (n=4 animals, 5 slices, 10 dendrites per animal, t-test, p=0.041). D) Example traces of miniature excitatory post-synaptic currents (mEPSCs) from voltage-clamped layer 2/3 neurons in the visual cortex of GLT1 WT and HET mice. E) Quantification of mEPSC amplitude showing no difference in magnitude of mEPSCs (n=8-13 cell, t-test, p=0.49). F) Neurons from GLT1 HET mice have a trend towards increased mEPSC frequency (n=8-13 cells, t-test, p=0.052). G) Example images of parvalbumin positive (PV+, green) and somatostatin positive (SST+, magenta) interneurons in visual cortex of GLT1 WT and HET mice). H) GLT1 HET mice have a trend towards decreased PV+ neuron density (n=4 animals, 5 slices per animal, t-test, p=0.12). I) GLT1 HET mice have a significant increase in SST+ cell density (n=4 animals, 5 slices per animal, t-test, p=0.0023). J) Example images of perineuronal nets (PNNs) visualized using wisteria floribunda agglutin (WFA) staining. K) GLT1 HET mice have significantly decreased PNN density compared to WT littermates (n=9 animals, 5 slices per animal, t-test, p=0.0068). L) Model of net decrease in cortical inhibition through increased SST+ cell density inhibiting PV+ interneurons yielding increase in excitatory pyramidal neuron responses (Pyr). *p<0.05, **p<0.01,***p<0.005

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: Transgenic Assay, Staining, Inhibition

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Schematic of experimental setup for intrinsic signal optical imaging. Drifting bars are presented to each eye individually and phase maps are generated by the retinotopic activity in visual cortex. Averaged responses of multiple sweeps yield an amplitude map. Ocular Dominance Index (ODI) is calculated as the contralateral response − ipsilateral response / contralateral + ipsilateral responses. B) ODI for GLT1 WT (grays) and GLT1 HET (reds) mice that were either non-deprived (ND), or had the contralateral eye monocularly deprived for 4 days (4dMD) or 7 days (7dMD). GLT1 WT mice display a typical contralateral bias in ND conditions. After 4dMD and 7dMD the ODI significantly decreases demonstrating intact ocular dominance plasticity. GLT1 HET mice display an abnormal lack of contralateral ODI bias under ND conditions, a significant decrease in ODI at 4dMD, and a return to no bias at 7dMD (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). C) Comparison of eye-specific amplitudes for GLT1 WT and HET mice. ND GLT1 HET mice have approximately equal responses to contralateral and ipsilateral inputs. After 4dMD, GLT1 HET mice have a significant decrease in contralateral responses and at 7dMD, significant decrease in both contralateral and ipsilateral responses (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p<0.05, **p<0.01, ***p<0.005

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: Optical Imaging, Generated, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Quantification of GLT1 mRNA in WT (grays) and HET (reds) mice in ND, 4dMD, and 7dMD conditions. GLT1 HET mice have significantly less GLT1 mRNA in ND conditions, but no difference at 4dMD and 7dMD compared to WT mice (n=2-3 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). B) Example western blots for GLT1 protein in WT (top) and HET (bottom) mice in ND, 4dMD, and 7dMD conditions. C) Quantification of western blots showing significantly less GLT1 protein in HET mice in ND, but no difference in 4dMD and 7dMD compared to WT littermates (n=4-9 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p<0.05, **p<0.01, ***p<0.005

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

doi: 10.1101/2020.05.25.113613

Figure Lengend Snippet: A) Schematic showing the contralateral (contra, green) and ipsilateral (ipsi, blue) inputs to binocular visual cortex synapsing onto layer 2/3 pyramidal cells (L2/3, Pyr, gray). Astrocytes (Ast, orange) have fine processes that surround excitatory synapses. Dashed box is magnified below, with details. B) At eye-opening, GLT1 WT and HET animals have similar astrocyte volume and LGN refinement. With visual experience, GLT1-WT animals undergo activity-dependent plasticity resulting in decreased spine density, binocular matching of preferred orientation, and a contralaterally biased ocular dominance. GLT1-HET animals have comparatively decreased GLT1 protein resulting in increased dendritic spines, increased ipsi responses, reduced contra bias and orientation tuning, and decreased binocular matching of orientation preference. Following 4 days of MD in GLT1-WT mice, contralateral responses decrease and after 7 days of MD, ipsilateral responses increase. In GLT1-HET animals, after 4 days of MD contralateral responses decrease resulting in a negative ODI. However, after 7 days of MD, increased GLT1 expression also decreases ipsilateral inputs resulting in no ocular dominance bias. These results are reasonably explained by a selective influence of GLT1 on ipsilateral inputs and responses during development.

Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

Techniques: Activity Assay, Expressing