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gaba receptor β3  (PhosphoSolutions)


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    PhosphoSolutions gaba receptor β3
    Synaptic and extrasynaptic <t>GABA</t> <t>A</t> <t>receptor</t> 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 GABA A R 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.).
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    Images

    1) Product Images from "Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain"

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

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2023.106248

    Synaptic and extrasynaptic GABA A 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 GABA A R 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.).
    Figure Legend Snippet: Synaptic and extrasynaptic GABA A 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 GABA A R 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.).

    Techniques Used: In Vivo, Western Blot

    γ2 containing GABA A receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABA A R 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-GABA A R subunits ( n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABA A R-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 (V hold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABA A R-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.).
    Figure Legend Snippet: γ2 containing GABA A receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABA A R 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-GABA A R subunits ( n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABA A R-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 (V hold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABA A R-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.).

    Techniques Used: Inhibition, Immunoprecipitation, Mass Spectrometry, Activity Assay

    Synaptic and extrasynaptic GABA A 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 GABA A R 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.).
    Figure Legend Snippet: Synaptic and extrasynaptic GABA A 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 GABA A R 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.).

    Techniques Used: In Vivo, Western Blot



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    mouse anti-gaba a receptor-β3  (NeuroMab)
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    NeuroMab mouse anti-gaba a receptor-β3
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    Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection heightens visceral white adipose tissue (WAT) metabolism in obese dysmetabolic rodents. (A) Panel shows a schematic illustration of the protocol used to evaluate mice WAT oxygen consumption rate (OCR). From (B) to (H) is described the effect of high fat (HF) diet and of CSN resection on the: (B) OCR per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in mice (3 pieces of tissue from 4–6 animals); (C) average basal OCR in mice (15–27 pieces of tissue from 4–6 animals) before the stimulation with norepinephrine and dopamine; (D) average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] (3 pieces of tissue from 4–6 animals); (E) Illustration of the molecular markers involved in brown adipocytes differentiation as well as the stimuli involved in the beiging of WAT; (F) average expression of PGC1α (92 kDa) ( n = 3–5); (G) average expression of PPARγ (53‐57 kDa) on visceral WAT of rats and mice ( n = 4–5)—representative western blots are shown on the top of the graphs; and (H) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in the perienteric depot ( n = 4–5) in rats and mice; right panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells. Gray and blue colors represent, respectively, normal chow (NC) and HF rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups, # p < 0.05, ## p < 0.01, ### p < 0.001 and ### p < 0.0001 comparing values with and without CSN resection.

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

    doi: 10.1111/apha.70074

    Figure Lengend Snippet: Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection heightens visceral white adipose tissue (WAT) metabolism in obese dysmetabolic rodents. (A) Panel shows a schematic illustration of the protocol used to evaluate mice WAT oxygen consumption rate (OCR). From (B) to (H) is described the effect of high fat (HF) diet and of CSN resection on the: (B) OCR per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in mice (3 pieces of tissue from 4–6 animals); (C) average basal OCR in mice (15–27 pieces of tissue from 4–6 animals) before the stimulation with norepinephrine and dopamine; (D) average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] (3 pieces of tissue from 4–6 animals); (E) Illustration of the molecular markers involved in brown adipocytes differentiation as well as the stimuli involved in the beiging of WAT; (F) average expression of PGC1α (92 kDa) ( n = 3–5); (G) average expression of PPARγ (53‐57 kDa) on visceral WAT of rats and mice ( n = 4–5)—representative western blots are shown on the top of the graphs; and (H) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in the perienteric depot ( n = 4–5) in rats and mice; right panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells. Gray and blue colors represent, respectively, normal chow (NC) and HF rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups, # p < 0.05, ## p < 0.01, ### p < 0.001 and ### p < 0.0001 comparing values with and without CSN resection.

    Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

    Techniques: Activity Assay, Expressing, Western Blot, Labeling

    Carotid body (CB) modulates lipid fluxes in white adipose tissue (WAT) in rodents. (A) Illustration of the molecular markers involved in adipose tissue energy expenditure. From (B) to (F) is described the effect of high fat (HF) diet and of CSN resection on: (B) Rg′ values, reflecting glucose uptake on WAT depots in rats ( n = 4–7); (C) WAT average expression of HSL (83 kDa) on WAT of rats ( n = 5–8); (D) WAT average expression of phosphorylated ATGL (pATGL) (55 kDa) in rats ( n = 5–7)—representative western blots are shown on the top of the graphs; (E) glycerol levels, as an index of lipolysis ( n = 6–8) in WAT in rats; (F) WAT average expression of phosphorylated AMPK (pAMPK) (60 kDa) in rats and mice ( n = 3–6)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. ** p < 0.01 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01 and ### p < 0.001 comparing values with and without CSN resection.

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

    doi: 10.1111/apha.70074

    Figure Lengend Snippet: Carotid body (CB) modulates lipid fluxes in white adipose tissue (WAT) in rodents. (A) Illustration of the molecular markers involved in adipose tissue energy expenditure. From (B) to (F) is described the effect of high fat (HF) diet and of CSN resection on: (B) Rg′ values, reflecting glucose uptake on WAT depots in rats ( n = 4–7); (C) WAT average expression of HSL (83 kDa) on WAT of rats ( n = 5–8); (D) WAT average expression of phosphorylated ATGL (pATGL) (55 kDa) in rats ( n = 5–7)—representative western blots are shown on the top of the graphs; (E) glycerol levels, as an index of lipolysis ( n = 6–8) in WAT in rats; (F) WAT average expression of phosphorylated AMPK (pAMPK) (60 kDa) in rats and mice ( n = 3–6)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. ** p < 0.01 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01 and ### p < 0.001 comparing values with and without CSN resection.

    Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

    Techniques: Expressing, Western Blot

    Carotid sinus nerve (CSN) resection improves brown adipose tissue (BAT) metabolism in rodents. Effect of high fat (HF) diet and of CSN resection on: (A) Curves of oxygen consumption rate (OCR) per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in the BAT of mice ( n = 18–27 pieces of tissue from 6–8 animals). (B) Average basal OCR in the BAT in mice ( n = 18–27 pieces of tissue from 6–8 animals) before the stimulation with norepinephrine and dopamine; (C) Average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] ( n = 5–8 animals); (D) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in BAT ( n = 4–5) in rats and mice; bottom panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells; (E) Rg′ values, reflecting glucose uptake on BAT depots in rats ( n = 5–7); (F) BAT average expression of pATGL (55 kDa) in rats ( n = 4–5); (G) average expression of pAMPK (60 kDa) on BAT of rats and mice ( n = 3–8)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

    doi: 10.1111/apha.70074

    Figure Lengend Snippet: Carotid sinus nerve (CSN) resection improves brown adipose tissue (BAT) metabolism in rodents. Effect of high fat (HF) diet and of CSN resection on: (A) Curves of oxygen consumption rate (OCR) per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in the BAT of mice ( n = 18–27 pieces of tissue from 6–8 animals). (B) Average basal OCR in the BAT in mice ( n = 18–27 pieces of tissue from 6–8 animals) before the stimulation with norepinephrine and dopamine; (C) Average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] ( n = 5–8 animals); (D) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in BAT ( n = 4–5) in rats and mice; bottom panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells; (E) Rg′ values, reflecting glucose uptake on BAT depots in rats ( n = 5–7); (F) BAT average expression of pATGL (55 kDa) in rats ( n = 4–5); (G) average expression of pAMPK (60 kDa) on BAT of rats and mice ( n = 3–8)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

    Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

    Techniques: Labeling, Expressing, Western Blot

    Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection restores sympathetic inputs in visceral white adipose tissue (WAT). (A) schematic representation of the catecholamine biosynthesis pathways; (B) effect of HF diet and CSN resection on the levels of dopamine + DOPAC, norepinephrine and epinephrine in the WAT. From (C) to (E)—Sympathetic innervation in the WAT presented by: (C) WAT average tyrosine hydroxylase (TH) levels (60 kDa) in rats and mice ( n = 5–7); (D) WAT average dopamine β hydroxylase (DβH) levels (75 kDa) in rats; (E) from the left to the right: Intensity and fibers volume of TH immunolabeling ( n = 4–5) in WAT of rats—representative images are shown at the right panel, for animated gif of the images consult the videos White adipose tissue (WAT ); (F) average β2 receptors (β2R, 47 kDa) in rats (left panel) and β3 receptors (β3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); and (G) average dopamine type 1 receptors (D1R, 48 kDa) in rats (left panel) and dopamine type 2 receptors (D2R, 49 kDa) in rats and mice (left panel) ( n = 4–6). Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05; ## p < 0.01; ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

    doi: 10.1111/apha.70074

    Figure Lengend Snippet: Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection restores sympathetic inputs in visceral white adipose tissue (WAT). (A) schematic representation of the catecholamine biosynthesis pathways; (B) effect of HF diet and CSN resection on the levels of dopamine + DOPAC, norepinephrine and epinephrine in the WAT. From (C) to (E)—Sympathetic innervation in the WAT presented by: (C) WAT average tyrosine hydroxylase (TH) levels (60 kDa) in rats and mice ( n = 5–7); (D) WAT average dopamine β hydroxylase (DβH) levels (75 kDa) in rats; (E) from the left to the right: Intensity and fibers volume of TH immunolabeling ( n = 4–5) in WAT of rats—representative images are shown at the right panel, for animated gif of the images consult the videos White adipose tissue (WAT ); (F) average β2 receptors (β2R, 47 kDa) in rats (left panel) and β3 receptors (β3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); and (G) average dopamine type 1 receptors (D1R, 48 kDa) in rats (left panel) and dopamine type 2 receptors (D2R, 49 kDa) in rats and mice (left panel) ( n = 4–6). Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05; ## p < 0.01; ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

    Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

    Techniques: Activity Assay, Immunolabeling, Western Blot

    Carotid body (CB) regulation of norepinephrine (NE) action on β3 adrenergic receptor is key to brown adipose tissue (BAT) metabolic function. (A) effect of high fat (HF) diet and carotid sinus nerve (CSN) resection on the levels of dopamine + DOPAC, NE and epinephrine in the BAT. From (B) to (D): Sympathetic innervation to BAT assessed as: (B) BAT average TH expression (60 kDa) in rats and mice ( n = 4–6); (C) BAT average D𝛃H levels (60 kDa) in rats and mice ( n = 4–6); (D) BAT average 𝛃2 receptors (𝛃2R, 47 kDa) in rats (left panel) and 𝛃3 receptors (𝛃3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05; ** p < 0.01 and *** p < 0.001 comparing NC vs. HF groups; # p < 0.05 and ## p < 0.01 comparing values with and without CSN resection.

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

    doi: 10.1111/apha.70074

    Figure Lengend Snippet: Carotid body (CB) regulation of norepinephrine (NE) action on β3 adrenergic receptor is key to brown adipose tissue (BAT) metabolic function. (A) effect of high fat (HF) diet and carotid sinus nerve (CSN) resection on the levels of dopamine + DOPAC, NE and epinephrine in the BAT. From (B) to (D): Sympathetic innervation to BAT assessed as: (B) BAT average TH expression (60 kDa) in rats and mice ( n = 4–6); (C) BAT average D𝛃H levels (60 kDa) in rats and mice ( n = 4–6); (D) BAT average 𝛃2 receptors (𝛃2R, 47 kDa) in rats (left panel) and 𝛃3 receptors (𝛃3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05; ** p < 0.01 and *** p < 0.001 comparing NC vs. HF groups; # p < 0.05 and ## p < 0.01 comparing values with and without CSN resection.

    Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

    Techniques: Expressing, Western Blot

    Synaptic and extrasynaptic GABA A 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 GABA A R 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.).

    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: Synaptic and extrasynaptic GABA A 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 GABA A R 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); GABA A Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABA A Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABA A Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABA A Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABA A 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

    γ2 containing GABA A receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABA A R 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-GABA A R subunits ( n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABA A R-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 (V hold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABA A R-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.).

    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: γ2 containing GABA A receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABA A R 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-GABA A R subunits ( n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABA A R-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 (V hold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABA A R-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); GABA A Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABA A Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABA A Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABA A Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABA A 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

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Cerebellum-Specific Deletion of the GABA A Receptor δ Subunit Leads to Sex-Specific Disruption of Behavior

    doi: 10.1016/j.celrep.2020.108338

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: mouse anti-GABA A receptor-β3 , Neuromab , Cat# 75–149; RRID: AB_2109585.

    Techniques: Recombinant, RNAscope, Knock-Out, Software