Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 1. Representative confocal immunofluorescence images demonstrating expression of GABAAR α1 in the stargazer primary SoCx. 10× (Objective: 10× Plan) magnification from control (NE) littermate and epileptic (E) stargazer are shown here. (A) Pseudo-green channel shows diffuse labelling for GABAAR α1 subunit across the cortical layers with higher intensity in layer IV. (B) Pseudo-red channel shows labelling for PV+ neurons. The soma of PV+ neurons are present in all layers except layer I. (C) Pseudo-blue channel shows VGlut2 which assists in identification of the cortical layers. Intense labelling can be seen, predominantly, in layer IV due to the presence of thalamocortical excitatory nerve terminals.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Expressing, Control

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 2. Merged confocal immunofluorescence images demonstrating the co-labelling for GABAAR α1 and PV in the primary SoCx of control (NE) littermate and epileptic (E) stargazer. (A) 10× magnification of GABAAR α1/PV merged image shows co-localization of GABAAR α1 with PV+ somas and processes. (B) 60× (Objective: 60× PlanApo oil) magnification of GABAAR α1/PV merged image clearly shows GABAAR α1 present throughout the cortex as well on PV somas.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Control

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 3. WB analysis for GABAAR α1 and α3 subunits in the primary SoCx. Data compare expression between control (NE) littermates and epileptic (E) stargazers. (A) Representative blots display GABAAR α1 in the primary SoCx with β-actin as the loading control. Bar graphs represent the relative expression levels of GABAAR α1 (mean ± SEM). WB analysis revealed a statistically significant reduction in whole-tissue expression levels of GABAAR α1 in the primary SoCx of the stargazers (NE: 0.960 ± 0.066 [n = 18], E: 0.78 ± 0.046 [n = 14], p = 0.041); (B) Representative blots display GABAAR α3 in the primary SoCx with β-actin as the loading control. Bar graphs represent the relative expression levels of GABAAR α3. There was no statistically significant change in whole-tissue expression levels of GABAAR α3 in the primary SoCx of the stargazers (NE: 1.000 ± 0.036 [n = 17], E: 0.935 ± 0.049 [n = 15], p = 0.519). The significance threshold was set at 0.05 with * indicating p < 0.05. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Expressing, Control

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 4. WB analysis for GABAAR β2, β3, and γ2 subunits in the primary SoCx. The data compare expression between control (NE) littermates and epileptic (E) stargazers. (A–C) Representative blots display tissue expression of GABAAR β2, β3, and γ2 subunits in the primary SoCx with β-actin as the loading control. Bar graphs represent the relative expression levels of GABAAR β2, β3, and γ2 subunits, respectively. Bar graphs show a lack of statistically significant change in whole-tissue primary SoCx expression levels for β2 (NE: 0.917 ± 0.091 [n = 12], E: 0.678 ± 0.050 [n = 10]; p = 0.093), β3 (NE: 1.000 ± 0.067 [n = 12], E: 1.070 ± 0.109 [n = 10]; p = 0.859), and γ2 (NE: 1.000 ± 0.159 [n = 9], E: 0.946 ± 0.079 [n = 7]; p = 0.900). The significance threshold was set at 0.05. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Expressing, Control

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 5. WB analysis for the principal tonic GABAAR α4, α5, and δ subunits. Analysis compares the primary SoCx of control (NE) littermates and the epileptic (E) stargazers. (A–C) Representative blots display tissue expression of GABAAR α4, α5, and δ subunits in the primary SoCx with β-actin as the loading control. Bar graphs represent the relative expression levels of GABAAR α4, α5 and δ subunits, respectively. Bar graphs reveal a lack of statistically significant change in whole-tissue primary SoCx expression levels for GABAAR α4 (NE: 1.000 ± 0.063 [n = 18], E: 1.117 ± 0.095 [n = 15]; p = 0.325), α5 (NE: 1.000 ± 0.048 [n = 23], E: 0.937 ± 0.083 [n = 13]; p = 0.672), and δ (NE: 1.000 ± 0.041 [n = 27], E: 0.899 ± 0.069 [n = 23]; p = 0.215). The significance threshold was set at 0.05. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Control, Expressing

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 6. Pilot WB run for biochemically isolated primary SoCx subcellular fractions (total lysate, cytosol, extra-synaptic and synaptic). This run confirmed the successful isolation of the synaptic fractions given the intense labelling for both PSD95 and GABAAR α1. Extra-synaptic fraction, on the other hand, showed no bands for PSD95, but low intensity bands were seen for GABAAR α1 and β-actin. PanC showed good expression in all subcellular fractions.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Isolation, Expressing

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 7. WB analyses of biochemically isolated fractions from the primary SoCx for GABAAR α1 and α3 subunits. Isolated subcellular fractions were compared between control (NE) litter- mates and epileptic (E) stargazers. (A–D) Biochemical fractionation analysis revealed a 12.2% reduction in the synaptic expression of phasic GABAAR α1 in the primary SoCx of the stargaz- ers compared to their control littermates (NE: 1.000 ± 0.146 [n = 10], E: 0.878 ± 0.037 [n = 10]; p = 0.002); no significant difference was revealed from the other subcellular components: total lysate (NE: 1.000 ± 0.043 [n = 7], E: 0.961 ± 0.052 [n = 6]; p = 0.531), cytosol (NE: 1.000 ± 0.034 [n = 7], E: 1.100 ± 0.098 [n = 6]; p = 0.443), and extra-synaptic (NE: 1.000 ± 0.074 [n = 9], E: 0.912 ± 0.094 [n = 9]; p = 0.385). (E–H) Biochemical fractionation analysis revealed no significant change in GABAAR α3 in all subcellular components from the primary SoCx of stargazers compared to their control littermates: Total lysate (NE: 1.000 ± 0.026 [n = 12], E: 1.035 ± 0.060 [n = 10]; p = 0.381), cytosol (NE: 1.000 ± 0.037 [n = 11], E: 1.181 ± 0.118 [n = 10]; p = 0.349), extra-synaptic (NE: 1.000 ± 0.031 [n = 11], E: 1.093 ± 0.125 [n = 11]; p = 0.133), and synaptic (NE: 0.967 ± 0.034 [n = 11], E: 1.205 ± 0.153 [n = 9], p = 0.359). The sig- nificance threshold was set at 0.05 with ** indicating p < 0.01. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Isolation, Control, Fractionation, Expressing

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 8. WB analyses of biochemically isolated fractions from primary SoCx for GABAAR α4, α5, and δ subunits. Subcellular fractions were isolated from the control (NE) littermates and epileptic epileptic (E) stargazers. (A–D) Biochemical fractionation analysis revealed no signif- icant change in GABAAR α4 in all subcellular components from the primary SoCx of stargaz- ers compared to their NE littermates: total lysate (NE: 1.000 ± 0.063 [n = 9], E: 1.353 ± 0.227 [n = 9]; p = 0.257), cytosol (NE: 1.000 ± 0.088 [n = 14], E: 1.308 ± 0.223 [n = 10]; p = 0.172), extra-synaptic (NE: 1.000 ± 0.051 [n = 14], E: 0.789 ± 0.183 [n = 9]; p = 0.516), and synaptic (NE: 1.000 ± 0.047 [n = 14], E: 0.955 ± 0.122 [n = 9]; p = 0.369). (E–H) GABAAR α5 anal- ysis revealed no significant change in all subcellular components from the primary SoCx of stargazers compared to their control littermates: total lysate (NE: 1.000 ± 0.094 [n = 10], E: 0.919 ± 0.102 [n = 10]; p = 0.566), cytosol (NE: 1.000 ± 0.113 [n = 14], E: 1.295 ± 0.362 [n = 8]; p = 0.920), extra-synaptic (NE: 1.000 ± 0.068 [n = 13], E: 1.324 ± 0.214 [n = 9]; p = 0.431) and, synaptic (NE: 1.000 ± 0.075 [n = 13], E: 1.190 ± 0.195 [n = 9]; p = 0.744). (I–L) No statistically significant change was also seen for GABAAR δ subunit in all subcellular compo- nents from the primary SoCx of stargazers compared to their control littermates: total lysate (NE:1.000 ± 0.071 [n = 15], E: 1.088 ± 0.201 [n = 9]; p = 0.815), cytosol (NE: 1.000 ± 0.083 [n = 15], E: 0.831 ± 0.138 [n = 9]; p = 0.379), extra-synaptic (NE: 1.000 ± 0.078 [n = 15], E: 1.419 ± 0.241 [n = 10]; p = 0.191), and synaptic (NE: 1.000 ± 0.053 [n = 15], E: 1.092 ± 0.094 [n = 9]; p = 0.263). The significance threshold was set at 0.05. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Isolation, Control, Fractionation

Journal: International journal of molecular sciences

Article Title: Altered GABA A Receptor Expression in the Primary Somatosensory Cortex of a Mouse Model of Genetic Absence Epilepsy.

doi: 10.3390/ijms232415685

Figure Lengend Snippet: Figure 9. ICC-EM analysis for GABAAR α1 at PV+ and non-PV inhibitory synapses in the primary SoCx compared and analyzed between control (NE) littermates and epileptic (E) stargazers. From top to bottom: Representative EM micrographs demonstrate PV labelling in the presynaptic terminals (pre), which appear darker stained, in stargazer and NE littermate primary SoCx. Arrow represents 20 nm immunogold particle labelling PV while arrowhead represents 10 nm immunogold labelling GABAAR α1. (A) For analysis for GABAAR α1 at inhibitory synapses from non-PV terminals onto non-PV profiles, as indicated by lack of 20 nm gold particles, 490 synapses in stargazers and 490 in control littermates primary SoCx were analyzed from seven pairs distributed equally. No significant difference was seen in the seven pairs (NE: 10.65 ± 0.918 [n = 7], E: 10.72 ± 0.751 [n = 7]; p = 0.804). (B) Analysis for GABAAR α1 at inhibitory synapses with PV (20 nm gold) in the presynaptic terminals revealed no significant difference between the stargazers (63 synapses analyzed) and control littermates (63 synapses analyzed) (NE: 11.82 ± 1.325 [n = 7], E: 11.15 ± 1.007 [n = 7], p > 0.999). The significance threshold was set at 0.05. ‘ns’ indicates no significant change found from analyses.

Article Snippet: Membranes were probed using GABAAR α1 (1:500; AGA-001, Alomone, Jerusalem, Israel), GABAAR α3 (1:500; AGA-003, Alomone, Jerusalem, Israel), GABAAR α4 (1:200; AGA-008, Alomone, Jerusalem, Israel), GABAAR α5 (1:1000; AB9678, Sigma-Aldrich, St. Louis, MO, USA), GABAAR β2 (1:1000; ab8340, Abcam, Cambridge, UK), GABAAR β3 (1:1000; ab4046, Abcam, Cambridge, UK), GABAAR γ2 (1:200; AGA-005, Alomone, Jerusalem, Israel), and GABAAR δ (1:200; AGA014, Alomone, Jerusalem, Israel) with β-actin (1:1000; ab8226, Abcam, Cambridge UK) used Int.

Techniques: Control, Staining