Journal: bioRxiv

Article Title: Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum

doi: 10.1101/2021.05.28.446253

Figure Lengend Snippet: ( A ) Top: patch RT-PCR strategy. Bottom: representative voltage responses to current steps in CA1 pyramidal neuron (far left, black), and current responses to voltage steps in astrocytes in stratum radiatum (SR, green), stratum oriens (SO, yellow), and stratum lacunosum moleculare (SLM, blue). Scale bars: neuron, 40 mV, 500 ms; astrocytes, 6 nA, 200 ms. ( B ) Summary of I-V curves of astrocytes obtained before collecting RNA. N = 39 cells, 36 slices, 9 mice for each layer. ( C ) Summary of RT-PCR analysis for mRNA encoding indicated NMDAR subunits: GRIN1 (1), GRIN2A (2A), GRIN2B (2B), and GRIN2C (2C). Control samples (no patch) were obtained by inserting the electrode into the slice without patching cells. GRIN2D levels were undetectable in all cells examined (data not shown). ( D ) Western blots of co-immunoprecipitations performed using adult mouse hippocampal extracts with the indicated antibodies where anti-Cre antibody is used as a negative control. The blots are probed for GluN2A/2B (top row), GluN2C (middle row) or GluN1 (bottom row). ( E ) Representative brain sections of GRIN2C -Cre mice crossed to a tdTomato reporter line (Ai9), showing hippocampal area CA1 immunolabelled for GFAP, tdTomato and NeuN; scale bars, 160 μm (top), 25 μm (bottom). ( F ) Quantification of % cells that are positive for tdTomato amongst NeuN-labelled cells in stratum pyramidale (left) and SR (middle) and GFAP-labelled cells (right).

Article Snippet: Antibodies used were: mouse anti-NR1 (Synaptic Systems #114011), rabbit anti-GluN2A/2B (Synaptic Systems #244003), rabbit anti-GluN2C (generously provided by Dr. Masahiko Watanabe or purchased from Frontier Institute #GLURE3C-RB-AF270), mouse anti-Cre (Merck Millipore clone 2D8) and mouse anti-BirA (Novus biologicals #NBP2-59939).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Journal of Biomedical Science

Article Title: Generational synaptic functions of GABA A receptor β3 subunit deteriorations in an animal model of social deficit

doi: 10.1186/s12929-022-00835-w

Figure Lengend Snippet: Increased glutamatergic modifications across generations of the VPA-induced offspring. a–d Representative western blot profile and the protein levels of GluN2A ( a ), GluN2B ( b ), GluA1 ( c ), and GluA2 ( d ) in synaptoneurosomes from the amygdala. n = 5–6 rats from three or four litters for each condition. e , f Top, Representative EPSC traces at different stimuli. Scale bars, 100 pA, 50 ms. Bottom, input–output curves of NMDAR-EPSC ( e ) and AMPAR-EPSC ( f ) in response to a series of increasing stimulus intensities in the BLA pyramidal neurons. n = 6–7 cells from 4–5 rats from three or four litters for each condition. g Representative traces and bar graph show the AMPAR- to NMDAR-EPSC ratio measured from each group. Scale bars, 100 pA, 50 ms. n = 8–11 cells from 4–5 rats from three or four litters for each condition. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01 vs. indicated control; one-way ANOVA or two-way rmANOVA with Bonferroni post-hoc

Article Snippet: For GABA A R insertion, N -methyl- d -aspartate (NMDA) was purchased from Tocris (Minneapolis, MN), which was dissolved in artificial cerebrospinal fluid (aCSF) to obtain a concentration of 20 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\upmu }$$\end{document} μ M. For western blotting, the following antibodies of GluN2A (1:5000; Genetex; GTX63442), GluN2B (1:5000; Abcam; ab65783), GluA1 (1:5000; Abcam; ab109450), GluA2 (1:5000; Millipore; MAB397), gephyrin (1:5000; Alomone; AIP-005), GABA A R β3 subunit (1:5000; Abcam; ab98968), and anti- \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\upbeta }$$\end{document} β -actin antibody (1:10,000; Abcam; ab6276) were used.

Techniques: Western Blot, Control