Journal: iScience

Article Title: Distinct mechanisms drive sequential internalization and degradation of GABA A Rs during global ischemia and reperfusion injury

doi: 10.1016/j.isci.2023.108061

Figure Lengend Snippet:

Article Snippet: GABA A R-β3 phospho-Ser408/409 , Rockland , Cat. #612-401-D51; RRID: AB_11183444.

Techniques: Imaging, Recombinant, Reverse Transcription, SYBR Green Assay, Software

Journal: Neurobiology of disease

Article Title: Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain.

doi: 10.1016/j.nbd.2023.106248

Figure Lengend Snippet: Fig. 2. Synaptic and extrasynaptic GABAA receptor and gephyrin redistribution with seven-day DZP treatment in vivo. (A-F) Mice were treated i.p. once daily for seven days with Veh or DZP. Western blot analysis of collected cortical tissue was performed to assess protein levels of GABAAR subunits and gephyrin from total (A, B), synaptic (C, D), and extrasynaptic (E, F) fractions. Representative blots show five mice from each treatment group. Proteins quantified by western blot were normalized to GAPDH or KIR3.2 loading control. (B) Measurements of total protein levels revealed a significant increase in α1 (p = 0.0018), β3 (p = 0.0005), and γ2 (p = 0.0002) subunits. The amount of synaptic α1 (p = 0.0019), α4 (p = 0.0028), and γ2 (p = 0.0007) subunits also increased (D), while extrasynaptic α1 (p = 0.0034) and α4 (p = 0.0010) subunits were decreased and gephyrin (p = 0.0040) was increased (F) (*p ≤0.05, **p < 0.01, ***p < 0.001, Student’s t-test; n = 5–10 mice per treatment; error bars ± S.E.M.).

Article Snippet: Primary antibodies: GAPDH (RRID: AB_561053, #2118, Cell Signaling); NMDAR Receptor 1 (GluN1) (RRID: AB_1904067, #5704, Cell Signaling); NMDA NR2B subunit (GluN2B) (RRID: AB_397797, #610417, BD Biosciences); NMDA NR2A subunit (GluN2A) (RRID: AB_2492170, #1500-NR2A, PhosphoSolutions); GABAA Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABAA Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABAA Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABAA Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABAA Receptor γ2 (RRID: AB_2263066, #224003, Synaptic Systems; RRID: AB _10594245, #224004, Synaptic Systems); gephyrin (RRID: AB_640963, #sc-14003, Santa Cruz Biotechnology); Kir3.2 (RRID: AB_2040115, #APC-006, Alomone Labs).

Techniques: In Vivo, Western Blot, Control

Journal: Neurobiology of disease

Article Title: Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain.

doi: 10.1016/j.nbd.2023.106248

Figure Lengend Snippet: Fig. 3. γ2 containing GABAA receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABAAR from seven-day Veh- or DZP-treated mouse cortex was analyzed by DIA mass spectrometry to assess changes in receptor subunit composition (n = 4 mice per treatment group). The intensity of α1–5 subunit-specific peptides are shown. Inset: Relative abundance (%) of α and β subunits associated with γ2 after seven-day DZP treatment. (B,C) (B) Left: representative traces with mIPSCs from seven-day DZP-treated animals before (dark red) and after (gray) 300 nM Ro 15–4513 application. Right: averaged mIPSCs before and after Ro 15–4513. (C) Quantification shows inverse agonist activity of Ro 15–4513, consistent with predominant receptors composed of γ2 with α1, α2, α3, α5-GABAAR subunits (n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABAAR-mediated tonic current was measured in acute cortical slices from mice treated i.p. once daily for seven days with Veh or DZP. Picrotoxin-sensitive changes in holding current (Vhold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABAAR-mediated tonic current was significantly reduced (p = 0.0084) in DZP-treated mice (n = 8) relative to Veh-treated mice (n = 6). (E: **p ≤0.01, Student’s t-test; C: *p ≤ 0.05, **p ≤0.01, paired t-test; error bars ± S.E.M.).

Article Snippet: Primary antibodies: GAPDH (RRID: AB_561053, #2118, Cell Signaling); NMDAR Receptor 1 (GluN1) (RRID: AB_1904067, #5704, Cell Signaling); NMDA NR2B subunit (GluN2B) (RRID: AB_397797, #610417, BD Biosciences); NMDA NR2A subunit (GluN2A) (RRID: AB_2492170, #1500-NR2A, PhosphoSolutions); GABAA Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABAA Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABAA Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABAA Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABAA Receptor γ2 (RRID: AB_2263066, #224003, Synaptic Systems; RRID: AB _10594245, #224004, Synaptic Systems); gephyrin (RRID: AB_640963, #sc-14003, Santa Cruz Biotechnology); Kir3.2 (RRID: AB_2040115, #APC-006, Alomone Labs).

Techniques: Inhibition, Immunoprecipitation, Mass Spectrometry, Activity Assay