girk2  (Alomone Labs)


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    Structured Review

    Alomone Labs girk2
    Expression of <t>GIRK2</t> subunit is unchanged post-SE. A , B , D , E , All representative images follow the same format. Upper left, Transverse slice from dorsal hippocampus with the nuclear stain, Hoechst 33342, from control group. Upper right, Representative hippocampal staining of GIRK2. The blue box shows the portion of CA1 expanded below. The yellow shaded region shows the region selected for quantification from the alveus to the fissure in both channels. Bottom, GIRK2 staining in CA1, where the lighter shade of gray reflects more immunoreactivity for GIRK2 protein. Staining is evident in the somatic layer (S.P.) and dendritic layers. Scale bars = 500 µm. A , Representative section from the dorsal hippocampus with GIRK2 staining from a control rat. B , Representative section from the dorsal hippocampus with GIRK2 staining from a post-SE rat. C , Quantification of average grayscale pixel intensity along the length of the somatodendritc axis on dorsal CA1. Since the radial length can differ between sections, the lengths were normalized and binned into 20 segments. Dotted lines reflect transitions between layers abbreviated S.O. (stratum oriens), S.P. (stratum pyramidale), S.R. (stratum radiatum), and S.L.M. (stratum lacunosum moleculare). Comparisons between equivalent radial locations were tested between control and post-SE group data. D , GIRK2 staining in the ventral hippocampus of control rat. E , GIRK2 staining in the ventral hippocampus of a post-SE rat. F , Quantification along the normalized length of the somatodendritic/radial axis in ventral CA1. Equivalent radial locations were compared between control and post-SE group data.
    Girk2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Epilepsy-Induced Reduction in HCN Channel Expression Contributes to an Increased Excitability in Dorsal, But Not Ventral, Hippocampal CA1 Neurons"

    Article Title: Epilepsy-Induced Reduction in HCN Channel Expression Contributes to an Increased Excitability in Dorsal, But Not Ventral, Hippocampal CA1 Neurons

    Journal: eNeuro

    doi: 10.1523/ENEURO.0036-19.2019

    Expression of GIRK2 subunit is unchanged post-SE. A , B , D , E , All representative images follow the same format. Upper left, Transverse slice from dorsal hippocampus with the nuclear stain, Hoechst 33342, from control group. Upper right, Representative hippocampal staining of GIRK2. The blue box shows the portion of CA1 expanded below. The yellow shaded region shows the region selected for quantification from the alveus to the fissure in both channels. Bottom, GIRK2 staining in CA1, where the lighter shade of gray reflects more immunoreactivity for GIRK2 protein. Staining is evident in the somatic layer (S.P.) and dendritic layers. Scale bars = 500 µm. A , Representative section from the dorsal hippocampus with GIRK2 staining from a control rat. B , Representative section from the dorsal hippocampus with GIRK2 staining from a post-SE rat. C , Quantification of average grayscale pixel intensity along the length of the somatodendritc axis on dorsal CA1. Since the radial length can differ between sections, the lengths were normalized and binned into 20 segments. Dotted lines reflect transitions between layers abbreviated S.O. (stratum oriens), S.P. (stratum pyramidale), S.R. (stratum radiatum), and S.L.M. (stratum lacunosum moleculare). Comparisons between equivalent radial locations were tested between control and post-SE group data. D , GIRK2 staining in the ventral hippocampus of control rat. E , GIRK2 staining in the ventral hippocampus of a post-SE rat. F , Quantification along the normalized length of the somatodendritic/radial axis in ventral CA1. Equivalent radial locations were compared between control and post-SE group data.
    Figure Legend Snippet: Expression of GIRK2 subunit is unchanged post-SE. A , B , D , E , All representative images follow the same format. Upper left, Transverse slice from dorsal hippocampus with the nuclear stain, Hoechst 33342, from control group. Upper right, Representative hippocampal staining of GIRK2. The blue box shows the portion of CA1 expanded below. The yellow shaded region shows the region selected for quantification from the alveus to the fissure in both channels. Bottom, GIRK2 staining in CA1, where the lighter shade of gray reflects more immunoreactivity for GIRK2 protein. Staining is evident in the somatic layer (S.P.) and dendritic layers. Scale bars = 500 µm. A , Representative section from the dorsal hippocampus with GIRK2 staining from a control rat. B , Representative section from the dorsal hippocampus with GIRK2 staining from a post-SE rat. C , Quantification of average grayscale pixel intensity along the length of the somatodendritc axis on dorsal CA1. Since the radial length can differ between sections, the lengths were normalized and binned into 20 segments. Dotted lines reflect transitions between layers abbreviated S.O. (stratum oriens), S.P. (stratum pyramidale), S.R. (stratum radiatum), and S.L.M. (stratum lacunosum moleculare). Comparisons between equivalent radial locations were tested between control and post-SE group data. D , GIRK2 staining in the ventral hippocampus of control rat. E , GIRK2 staining in the ventral hippocampus of a post-SE rat. F , Quantification along the normalized length of the somatodendritic/radial axis in ventral CA1. Equivalent radial locations were compared between control and post-SE group data.

    Techniques Used: Expressing, Staining

    2) Product Images from "Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice"

    Article Title: Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2018.01.002

    Tau deficiency did not impact the expression of DAT and Girk2. (A, B) Western blot showing the expression level of DAT in the midbrain and striatum (A), and Girk2 in the midbrain (B) of tau +/+ , tau +/ − , and tau − / − at 18 months of age. (C) Quantitation of DAT expression level by Western blot analyses, normalized with β-actin ( n = 5 per genotype, P > 0.05). (D) Quantitation of Girk2 expression level by Western blot analyses, normalized with β-actin ( n = 5 per genotype, P > 0.05). All data were presented as mean ± SEM.
    Figure Legend Snippet: Tau deficiency did not impact the expression of DAT and Girk2. (A, B) Western blot showing the expression level of DAT in the midbrain and striatum (A), and Girk2 in the midbrain (B) of tau +/+ , tau +/ − , and tau − / − at 18 months of age. (C) Quantitation of DAT expression level by Western blot analyses, normalized with β-actin ( n = 5 per genotype, P > 0.05). (D) Quantitation of Girk2 expression level by Western blot analyses, normalized with β-actin ( n = 5 per genotype, P > 0.05). All data were presented as mean ± SEM.

    Techniques Used: Expressing, Western Blot, Quantitation Assay

    3) Product Images from "A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells"

    Article Title: A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2017.08.002

    DA Neuronal-Subtype-Specific Innervation of Host Targets Small deposits of Fluorogold (FG) were injected into the dorsolateral striatum (A) or frontal cortex (C) 27 weeks after grafting. The deposits were verified as being located remotely to the graft core for each animal as shown in photomontages of fluorescent detection of EGFP (grayscale; note EGFP + fiber network can be seen in the striatum) and FG (red) in representative coronal sections (A and B). Detection of FG (grayscale), EGFP (green), GIRK2 (red), and CALBINDIN (blue) in animals receiving FG in either the dorsolateral striatum (B) or frontal cortex (D) highlight representative examples of DA neuronal phenotypes projecting to these regions. A total of 79 FG + grafted cells were detected in animals injected with FG in the dorsolateral striatum, of which 54 were EGFP + DA neurons and a total of 105 were detected in animals injected in the frontal cortex, of which only 12 were EGFP + DA neurons (E). For each FG + cell the relative location on the axis between the center of the graft and the graft-host border is indicated as well as the soma size (maximum diameter) and the GIRK2/CALBINDIN expression profile. Dopamine neurons (EGFP + ) retrogradely labeled (FG + ) from the dorsolateral striatum are plotted on the left of the graph and EGFP + /FG + neurons retrogradely labeled from frontal cortex are plotted on the right. A number of examples of GABA-containing neurons retrogradely labeled from cortex were also observed in the grafts (F). Scale bars, 1 mm (A and C); 50 μm (B, D, and F). The schematic in (A) is modified from an original created by Bengt Mattsson (Lund University, Sweden).
    Figure Legend Snippet: DA Neuronal-Subtype-Specific Innervation of Host Targets Small deposits of Fluorogold (FG) were injected into the dorsolateral striatum (A) or frontal cortex (C) 27 weeks after grafting. The deposits were verified as being located remotely to the graft core for each animal as shown in photomontages of fluorescent detection of EGFP (grayscale; note EGFP + fiber network can be seen in the striatum) and FG (red) in representative coronal sections (A and B). Detection of FG (grayscale), EGFP (green), GIRK2 (red), and CALBINDIN (blue) in animals receiving FG in either the dorsolateral striatum (B) or frontal cortex (D) highlight representative examples of DA neuronal phenotypes projecting to these regions. A total of 79 FG + grafted cells were detected in animals injected with FG in the dorsolateral striatum, of which 54 were EGFP + DA neurons and a total of 105 were detected in animals injected in the frontal cortex, of which only 12 were EGFP + DA neurons (E). For each FG + cell the relative location on the axis between the center of the graft and the graft-host border is indicated as well as the soma size (maximum diameter) and the GIRK2/CALBINDIN expression profile. Dopamine neurons (EGFP + ) retrogradely labeled (FG + ) from the dorsolateral striatum are plotted on the left of the graph and EGFP + /FG + neurons retrogradely labeled from frontal cortex are plotted on the right. A number of examples of GABA-containing neurons retrogradely labeled from cortex were also observed in the grafts (F). Scale bars, 1 mm (A and C); 50 μm (B, D, and F). The schematic in (A) is modified from an original created by Bengt Mattsson (Lund University, Sweden).

    Techniques Used: Injection, Expressing, Labeling, Modification

    Immunohistochemical Identification of DA Neuronal Subtypes in Grafts at 28 Weeks (A) The vast majority of EGFP + cells were TH + with typical midbrain dopamine neuron morphology. (B–G) Most EGFP + DA neurons also expressed VMAT and DAT with a punctate pattern typical for these proteins (closed arrowheads). Cytoplasmic distribution of EGFP showed a mix of neuronal morphologies including smaller spherical neurons typical for A10 identity (C), as well as large, angular cell soma typical for A9 neurons (D). The GFP + neurons were also mixed based on GIRK2 and CALBINDIN expression and included EGFP + /GIRK2 + /CALBINDIN − (E–G; arrows, particularly at the periphery of the grafts), EGFP + /GIRK2 + /CALBINDIN + (E–G; open arrowheads), and a smaller contribution of EGFP + /GIRK2 − /CALBINDIN + neurons (E–G; closed arrowheads). (H) Comparison of the mean diameter (horizontal lines) of EGFP + cells in the periphery of the graft (n = 100; sampled across 3 grafts) showed these cells were significantly larger than those located more centrally (n = 100; sampled across 3 grafts, Student's t test: ∗∗∗∗ p
    Figure Legend Snippet: Immunohistochemical Identification of DA Neuronal Subtypes in Grafts at 28 Weeks (A) The vast majority of EGFP + cells were TH + with typical midbrain dopamine neuron morphology. (B–G) Most EGFP + DA neurons also expressed VMAT and DAT with a punctate pattern typical for these proteins (closed arrowheads). Cytoplasmic distribution of EGFP showed a mix of neuronal morphologies including smaller spherical neurons typical for A10 identity (C), as well as large, angular cell soma typical for A9 neurons (D). The GFP + neurons were also mixed based on GIRK2 and CALBINDIN expression and included EGFP + /GIRK2 + /CALBINDIN − (E–G; arrows, particularly at the periphery of the grafts), EGFP + /GIRK2 + /CALBINDIN + (E–G; open arrowheads), and a smaller contribution of EGFP + /GIRK2 − /CALBINDIN + neurons (E–G; closed arrowheads). (H) Comparison of the mean diameter (horizontal lines) of EGFP + cells in the periphery of the graft (n = 100; sampled across 3 grafts) showed these cells were significantly larger than those located more centrally (n = 100; sampled across 3 grafts, Student's t test: ∗∗∗∗ p

    Techniques Used: Immunohistochemistry, Expressing

    4) Product Images from "G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channel Activation by the p75 Neurotrophin Receptor Is Required for Amyloid β Toxicity"

    Article Title: G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channel Activation by the p75 Neurotrophin Receptor Is Required for Amyloid β Toxicity

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2017.00455

    GIRK channel down-regulation inhibits Aβ 42 -induced neuronal degeneration. Western blots (A) and quantification (B) of total and surface GIRK1 and GIRK2 protein levels in mature hippocampal neurons treated for 2 h with either control Aβ 16 or oligomeric Aβ 42 and baclofen ( N = 4 experiments). Baclofen treatment reversed the Aβ-induced upregulation of surface GIRK channel subunits. (C) Potassium loss from cultured neurons treated with Aβ 42 was reduced by co-treatment with baclofen ( n = 443 neurons). (D) Representative relief contrast and fluorescence (Asante Potassium Green-2) photographs of neuronal cultures taken immediately and 160 min after Aβ and baclofen treatment. (E) Photomicrographs of hippocampal cultures immunostained for β-III tubulin 20 h after treatment with Aβ peptides, baclofen (Bac) and/or the GABA B receptor antagonist CGP55845 (CPGt). (F) Quantification of neurite integrity of the treated cultures ( N = 3 experiments). (G) Percentage survival of neurons cultured in the presence of Aβ and baclofen for 20 h. Down-regulation of GIRK channels by chronic baclofen treatment inhibited cell death, but the neurotoxicity of Aβ 42 was restored when neurons were co-cultured with the GABA B receptor antagonist CGP55845 (CGP; N = 5 experiments). * p
    Figure Legend Snippet: GIRK channel down-regulation inhibits Aβ 42 -induced neuronal degeneration. Western blots (A) and quantification (B) of total and surface GIRK1 and GIRK2 protein levels in mature hippocampal neurons treated for 2 h with either control Aβ 16 or oligomeric Aβ 42 and baclofen ( N = 4 experiments). Baclofen treatment reversed the Aβ-induced upregulation of surface GIRK channel subunits. (C) Potassium loss from cultured neurons treated with Aβ 42 was reduced by co-treatment with baclofen ( n = 443 neurons). (D) Representative relief contrast and fluorescence (Asante Potassium Green-2) photographs of neuronal cultures taken immediately and 160 min after Aβ and baclofen treatment. (E) Photomicrographs of hippocampal cultures immunostained for β-III tubulin 20 h after treatment with Aβ peptides, baclofen (Bac) and/or the GABA B receptor antagonist CGP55845 (CPGt). (F) Quantification of neurite integrity of the treated cultures ( N = 3 experiments). (G) Percentage survival of neurons cultured in the presence of Aβ and baclofen for 20 h. Down-regulation of GIRK channels by chronic baclofen treatment inhibited cell death, but the neurotoxicity of Aβ 42 was restored when neurons were co-cultured with the GABA B receptor antagonist CGP55845 (CGP; N = 5 experiments). * p

    Techniques Used: Western Blot, Cell Culture, Fluorescence, BAC Assay

    Aβ 42 -induced potassium efflux and apoptosis are mediated by p75 NTR . (A) Western blot of p75 NTR cleavage in the presence of Aβ and TAPI or the cleavage stimulator PMA (positive control) for 3 h and quantification of the C-terminal fragment (CTF) band intensity (FL, full length; ICD, intracellular domain fragment; N = 2 experiments). (B) Percentage survival of neurons cultured in the presence of Aβ and treated with the metalloprotease inhibitor TAPI for 20 h, which significantly inhibited Aβ-induced cell death. Western blots (C) and quantification (D) of total and surface GIRK1 and GIRK2 protein levels in hippocampal neurons treated for 2 h with either control Aβ 16 or oligomeric Aβ 42 and c29 or scrambled (SC) peptides ( N = 8 replicates). Neither peptide treatment altered the levels of Aβ-induced upregulation of surface GIRK channel subunits. (E) Average decrease in potassium concentration of individual neurons in cultures treated for 160 min with Aβ 42 and the dominant-negative p75 NTR peptide c29 ( n = 458 neurons). (F) Representative relief contrast and fluorescence (Asante Potassium Green-2) photographs of neuronal cultures taken immediately and 160 min after Aβ and c29 treatment. (G) Percentage survival of neurons cultured in the presence of Aβ and control or c29 peptides over 20 h. c29 but not a scrambled peptide inhibited Aβ 42 -initiated death ( N = 3 experiments). * p
    Figure Legend Snippet: Aβ 42 -induced potassium efflux and apoptosis are mediated by p75 NTR . (A) Western blot of p75 NTR cleavage in the presence of Aβ and TAPI or the cleavage stimulator PMA (positive control) for 3 h and quantification of the C-terminal fragment (CTF) band intensity (FL, full length; ICD, intracellular domain fragment; N = 2 experiments). (B) Percentage survival of neurons cultured in the presence of Aβ and treated with the metalloprotease inhibitor TAPI for 20 h, which significantly inhibited Aβ-induced cell death. Western blots (C) and quantification (D) of total and surface GIRK1 and GIRK2 protein levels in hippocampal neurons treated for 2 h with either control Aβ 16 or oligomeric Aβ 42 and c29 or scrambled (SC) peptides ( N = 8 replicates). Neither peptide treatment altered the levels of Aβ-induced upregulation of surface GIRK channel subunits. (E) Average decrease in potassium concentration of individual neurons in cultures treated for 160 min with Aβ 42 and the dominant-negative p75 NTR peptide c29 ( n = 458 neurons). (F) Representative relief contrast and fluorescence (Asante Potassium Green-2) photographs of neuronal cultures taken immediately and 160 min after Aβ and c29 treatment. (G) Percentage survival of neurons cultured in the presence of Aβ and control or c29 peptides over 20 h. c29 but not a scrambled peptide inhibited Aβ 42 -initiated death ( N = 3 experiments). * p

    Techniques Used: Western Blot, Positive Control, Cell Culture, Concentration Assay, Dominant Negative Mutation, Fluorescence

    5) Product Images from "GIRK2 splice variants and neuronal G protein-gated K+ channels: implications for channel function and behavior"

    Article Title: GIRK2 splice variants and neuronal G protein-gated K+ channels: implications for channel function and behavior

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01820-2

    Overlap of GIRK2a and GIRK2c with PSD-95 in pyramidal neurons. ( A,B ) Representative images showing GIRK2 (red) and PSD-95 (green) immunolabeling, and their overlay, in a Girk2 −/− hippocampal pyramidal neuron expressing GIRK2a ( A ) or GIRK2c ( B ). Scale bars: 50 microns. The inset (Bi) highlights the limited overlap between PSD-95 and GIRK2, as demonstrated with GIRK2c. Scale bars: 20 microns. ( C ) Quantification of overlap between PSD-95 and GIRK2a or GIRK2c, under control conditions and following morphine treatment (n = 8 per isoform and treatment condition). Main effects of isoform (F 1,28 = 9.2, P
    Figure Legend Snippet: Overlap of GIRK2a and GIRK2c with PSD-95 in pyramidal neurons. ( A,B ) Representative images showing GIRK2 (red) and PSD-95 (green) immunolabeling, and their overlay, in a Girk2 −/− hippocampal pyramidal neuron expressing GIRK2a ( A ) or GIRK2c ( B ). Scale bars: 50 microns. The inset (Bi) highlights the limited overlap between PSD-95 and GIRK2, as demonstrated with GIRK2c. Scale bars: 20 microns. ( C ) Quantification of overlap between PSD-95 and GIRK2a or GIRK2c, under control conditions and following morphine treatment (n = 8 per isoform and treatment condition). Main effects of isoform (F 1,28 = 9.2, P

    Techniques Used: Immunolabeling, Expressing

    GIRK2 expression in the mouse hippocampus. ( A ) Girk2a and Girk2c mRNA levels as assessed by RNA-Seq in CA1 cell body and neuropil samples, taken from 3 adult mice. Main effects of isoform (F 1,8 = 14.3, P
    Figure Legend Snippet: GIRK2 expression in the mouse hippocampus. ( A ) Girk2a and Girk2c mRNA levels as assessed by RNA-Seq in CA1 cell body and neuropil samples, taken from 3 adult mice. Main effects of isoform (F 1,8 = 14.3, P

    Techniques Used: Expressing, RNA Sequencing Assay, Mouse Assay

    Fear learning in CaMKIICre(+): Girk2 fl/fl mice expressing GIRK2a or GIRK2c. ( A ) EGFP expression in the dorsal CA1 of a CaMKIICre(+): Girk2 fl/fl mouse, 2 wk after infusion of the AAV8-CaMKIIα-DIO-GIRK2a-IRES-EGFP virus. Scale bar: 500 microns. ( B ) Representative somato-dendritic currents (V hold = −60 mV) evoked by baclofen (200 μM) and reversed by the GABA B R antagonist CGP54626 (2 μM) in dorsal CA1 pyramidal neurons from CaMKIICre(+): Girk2 fl/fl mice, 2 wk after infusion of Cre-dependent control (con, mCherry), GIRK2a, or GIRK2c virus. Scale: 100 pA/100 s. ( C ) Summary of baclofen-induced currents in dorsal CA1 pyramidal neurons from CaMKIICre(+): Girk2 fl/fl mice, 2 wk after infusion of Cre-dependent control (con, mCherry), GIRK2a, or GIRK2c virus (F 2,11 = 24.3, P
    Figure Legend Snippet: Fear learning in CaMKIICre(+): Girk2 fl/fl mice expressing GIRK2a or GIRK2c. ( A ) EGFP expression in the dorsal CA1 of a CaMKIICre(+): Girk2 fl/fl mouse, 2 wk after infusion of the AAV8-CaMKIIα-DIO-GIRK2a-IRES-EGFP virus. Scale bar: 500 microns. ( B ) Representative somato-dendritic currents (V hold = −60 mV) evoked by baclofen (200 μM) and reversed by the GABA B R antagonist CGP54626 (2 μM) in dorsal CA1 pyramidal neurons from CaMKIICre(+): Girk2 fl/fl mice, 2 wk after infusion of Cre-dependent control (con, mCherry), GIRK2a, or GIRK2c virus. Scale: 100 pA/100 s. ( C ) Summary of baclofen-induced currents in dorsal CA1 pyramidal neurons from CaMKIICre(+): Girk2 fl/fl mice, 2 wk after infusion of Cre-dependent control (con, mCherry), GIRK2a, or GIRK2c virus (F 2,11 = 24.3, P

    Techniques Used: Mouse Assay, Expressing

    Subcellular distribution of GIRK2a and GIRK2c in Girk2 −/− pyramidal neurons. ( A,B ) Representative images showing GIRK2 (red) and MAP2 (green) immunolabeling, and their overlay, in Girk2 −/− hippocampal pyramidal neurons expressing either GIRK2a ( A ) or GIRK2c ( B ). Scale bars: 50 microns. The insets highlight different densities of GIRK2a and GIRK2c puncta along proximal/primary (Ai,Bi) and distal/secondary (Aii,Bii) dendritic segments. Scale bars: 5 microns. ( C ) Quantification of GIRK2a and GIRK2c labeling in dendrites from infected Girk2 −/− pyramidal neurons. GIRK2 fluorescence intensity was measured in 2–3 primary ( t 27 = 1.4, P = 0.17), secondary ( t 29 = 2.1, * P
    Figure Legend Snippet: Subcellular distribution of GIRK2a and GIRK2c in Girk2 −/− pyramidal neurons. ( A,B ) Representative images showing GIRK2 (red) and MAP2 (green) immunolabeling, and their overlay, in Girk2 −/− hippocampal pyramidal neurons expressing either GIRK2a ( A ) or GIRK2c ( B ). Scale bars: 50 microns. The insets highlight different densities of GIRK2a and GIRK2c puncta along proximal/primary (Ai,Bi) and distal/secondary (Aii,Bii) dendritic segments. Scale bars: 5 microns. ( C ) Quantification of GIRK2a and GIRK2c labeling in dendrites from infected Girk2 −/− pyramidal neurons. GIRK2 fluorescence intensity was measured in 2–3 primary ( t 27 = 1.4, P = 0.17), secondary ( t 29 = 2.1, * P

    Techniques Used: Immunolabeling, Expressing, Labeling, Infection, Fluorescence

    GPCR-GIRK currents in Girk2 −/− pyramidal neurons expressing GIRK2a or GIRK2c. ( A ) Whole-cell currents (V hold = −70 mV) evoked by baclofen (100 μM) in a wild-type pyramidal neuron expressing EGFP (WT, black), as well as Girk2 −/− pyramidal neurons expressing EGFP ( Girk2 −/− , gray), GIRK2a (red), or GIRK2c (blue). Scale: 1 nA/5 s. ( B ) Summary of baclofen-induced, steady-state current densities (pA/pF) in wild-type control and Girk2 −/− pyramidal neurons expressing EGFP, GIRK2a, or GIRK2c (F 3,53 = 44.8, P
    Figure Legend Snippet: GPCR-GIRK currents in Girk2 −/− pyramidal neurons expressing GIRK2a or GIRK2c. ( A ) Whole-cell currents (V hold = −70 mV) evoked by baclofen (100 μM) in a wild-type pyramidal neuron expressing EGFP (WT, black), as well as Girk2 −/− pyramidal neurons expressing EGFP ( Girk2 −/− , gray), GIRK2a (red), or GIRK2c (blue). Scale: 1 nA/5 s. ( B ) Summary of baclofen-induced, steady-state current densities (pA/pF) in wild-type control and Girk2 −/− pyramidal neurons expressing EGFP, GIRK2a, or GIRK2c (F 3,53 = 44.8, P

    Techniques Used: Expressing

    Synaptic GIRK currents in Girk2 −/− neurons expressing GIRK2a and GIRK2c. ( A ) Image showing EGFP expression in the CA1 region of an organotypic hippocampal slice, taken 7 d after infection with AAV8-hSyn-GIRK2a-IRES-EGFP. The dotted white line highlights key features of slice morphology. Scale bar: 500 microns (inset: 50 microns). ( B ) Summary of peak baclofen-induced somatodendritic current amplitudes (I baclofen ) in dorsal CA1 pyramidal neurons, measured 7 d after CA1 infusion of control virus (con) to wild-type (WT) organotypic slices, or GIRK2 expression viruses (2a, 2c) to Girk2 −/− organotypic slices (F 2,17 = 1.2, P
    Figure Legend Snippet: Synaptic GIRK currents in Girk2 −/− neurons expressing GIRK2a and GIRK2c. ( A ) Image showing EGFP expression in the CA1 region of an organotypic hippocampal slice, taken 7 d after infection with AAV8-hSyn-GIRK2a-IRES-EGFP. The dotted white line highlights key features of slice morphology. Scale bar: 500 microns (inset: 50 microns). ( B ) Summary of peak baclofen-induced somatodendritic current amplitudes (I baclofen ) in dorsal CA1 pyramidal neurons, measured 7 d after CA1 infusion of control virus (con) to wild-type (WT) organotypic slices, or GIRK2 expression viruses (2a, 2c) to Girk2 −/− organotypic slices (F 2,17 = 1.2, P

    Techniques Used: Expressing, Infection

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    Alomone Labs anti girk2 kir3 2 antibody
    Ethanol treatment reduced KCNJ6 haplotype differences in iN excitability and <t>GIRK2</t> expression. A Morphological analysis of IEE iNs generated from affected and unaffected individuals, showing no difference in: (a) neuronal soma size ( p = 0.32); (b) soma circularity ( p = 0.54); (c) soma solidity ( p = 0.92); and (d) total neurite area ( p = 0.98). B There was no difference in GIRK2 expression in the IEE AF group iNs compared with UN by (a) puncta counts ( p = 0.46), (b) puncta size ( p = 0.36), (c) puncta circularity ( p = 0.052), or (d) solidity ( p = 0.47). C Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) for each cell line. D Electrophysiological analysis of passive neuronal properties in IEE iNs, showing (a) a small decrease in AF membrane capacitance ( p = 9.6 × 10 –9 ), but no difference in (b) membrane resistance ( p = 0.63), (c) spontaneous EPSCs frequency ( p = 0.32), or (d) spontaneous EPSCs amplitude ( p = 0.19). (e) Representative sEPSCs traces for each cell line. (f) The AF group exhibited no change in resting membrane potential after IEE ( p = 0.79). E Electrophysiological analysis of active neuronal properties found no difference in IEE iNs for (a) current required to shift resting membrane potential to −65 mV ( p = 0.32), (b) maximum number of action potentials (APs) induced with the “step” protocol ( p = 0.48), with (c) representative traces of APs induced with the “step” protocol, (d) number of action potentials (APs) induced with the “ramp” protocol ( p = 0.95), with (e) representative traces of APs induced with the “ramp” protocol. F Representative images from individual lines of iNs, exposed to 7d of IEE, marked with arrows pointing to individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray). G Summarized results from all lines showing differences in GIRK2 expression levels before and after 7 days of 20 mM IEE with ethanol (EtOH; p = 1.0 × 10 −20 ). H Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) prior and following 7 days 20 mM IEE with ethanol. Sample images are from line 246. I Representative images of FISH detection of KCNJ6 mRNA for each cell line. J Quantification of FISH. (a) The number of KCNJ6 puncta normalized to the number of cells in an image shows decreased expression in control AF compared with UN ( p = 8.2 × 10 −3 ) and increased expression following IEE ( p = 8.2 × 10 −3 ; using Tukey’s pairwise comparisons). (b) The percentage of KCNJ6 -expressing MAP2 + cells substantially increase in the (c) AF group but not in the (b) UN group. Numbers of KCNJ6 puncta were analyzed by expression levels per cell, as recommended by the FISH manufacturer in (d) UN or (e) AF cells. (f) KCNJ6 puncta within the neuronal soma show increases following IEE in both UN and AF groups ( p = 0.006 for genotype, Tukey’s post-hoc for UN, p = 0.01, for AF, p = 0.01). (g) A similar analysis of non-somatic puncta, presumably within neurites, showed no differences following IEE between genotypes ( p = 0.23).
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    Ethanol treatment reduced KCNJ6 haplotype differences in iN excitability and GIRK2 expression. A Morphological analysis of IEE iNs generated from affected and unaffected individuals, showing no difference in: (a) neuronal soma size ( p = 0.32); (b) soma circularity ( p = 0.54); (c) soma solidity ( p = 0.92); and (d) total neurite area ( p = 0.98). B There was no difference in GIRK2 expression in the IEE AF group iNs compared with UN by (a) puncta counts ( p = 0.46), (b) puncta size ( p = 0.36), (c) puncta circularity ( p = 0.052), or (d) solidity ( p = 0.47). C Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) for each cell line. D Electrophysiological analysis of passive neuronal properties in IEE iNs, showing (a) a small decrease in AF membrane capacitance ( p = 9.6 × 10 –9 ), but no difference in (b) membrane resistance ( p = 0.63), (c) spontaneous EPSCs frequency ( p = 0.32), or (d) spontaneous EPSCs amplitude ( p = 0.19). (e) Representative sEPSCs traces for each cell line. (f) The AF group exhibited no change in resting membrane potential after IEE ( p = 0.79). E Electrophysiological analysis of active neuronal properties found no difference in IEE iNs for (a) current required to shift resting membrane potential to −65 mV ( p = 0.32), (b) maximum number of action potentials (APs) induced with the “step” protocol ( p = 0.48), with (c) representative traces of APs induced with the “step” protocol, (d) number of action potentials (APs) induced with the “ramp” protocol ( p = 0.95), with (e) representative traces of APs induced with the “ramp” protocol. F Representative images from individual lines of iNs, exposed to 7d of IEE, marked with arrows pointing to individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray). G Summarized results from all lines showing differences in GIRK2 expression levels before and after 7 days of 20 mM IEE with ethanol (EtOH; p = 1.0 × 10 −20 ). H Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) prior and following 7 days 20 mM IEE with ethanol. Sample images are from line 246. I Representative images of FISH detection of KCNJ6 mRNA for each cell line. J Quantification of FISH. (a) The number of KCNJ6 puncta normalized to the number of cells in an image shows decreased expression in control AF compared with UN ( p = 8.2 × 10 −3 ) and increased expression following IEE ( p = 8.2 × 10 −3 ; using Tukey’s pairwise comparisons). (b) The percentage of KCNJ6 -expressing MAP2 + cells substantially increase in the (c) AF group but not in the (b) UN group. Numbers of KCNJ6 puncta were analyzed by expression levels per cell, as recommended by the FISH manufacturer in (d) UN or (e) AF cells. (f) KCNJ6 puncta within the neuronal soma show increases following IEE in both UN and AF groups ( p = 0.006 for genotype, Tukey’s post-hoc for UN, p = 0.01, for AF, p = 0.01). (g) A similar analysis of non-somatic puncta, presumably within neurites, showed no differences following IEE between genotypes ( p = 0.23).

    Journal: Molecular Psychiatry

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1038/s41380-022-01818-x

    Figure Lengend Snippet: Ethanol treatment reduced KCNJ6 haplotype differences in iN excitability and GIRK2 expression. A Morphological analysis of IEE iNs generated from affected and unaffected individuals, showing no difference in: (a) neuronal soma size ( p = 0.32); (b) soma circularity ( p = 0.54); (c) soma solidity ( p = 0.92); and (d) total neurite area ( p = 0.98). B There was no difference in GIRK2 expression in the IEE AF group iNs compared with UN by (a) puncta counts ( p = 0.46), (b) puncta size ( p = 0.36), (c) puncta circularity ( p = 0.052), or (d) solidity ( p = 0.47). C Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) for each cell line. D Electrophysiological analysis of passive neuronal properties in IEE iNs, showing (a) a small decrease in AF membrane capacitance ( p = 9.6 × 10 –9 ), but no difference in (b) membrane resistance ( p = 0.63), (c) spontaneous EPSCs frequency ( p = 0.32), or (d) spontaneous EPSCs amplitude ( p = 0.19). (e) Representative sEPSCs traces for each cell line. (f) The AF group exhibited no change in resting membrane potential after IEE ( p = 0.79). E Electrophysiological analysis of active neuronal properties found no difference in IEE iNs for (a) current required to shift resting membrane potential to −65 mV ( p = 0.32), (b) maximum number of action potentials (APs) induced with the “step” protocol ( p = 0.48), with (c) representative traces of APs induced with the “step” protocol, (d) number of action potentials (APs) induced with the “ramp” protocol ( p = 0.95), with (e) representative traces of APs induced with the “ramp” protocol. F Representative images from individual lines of iNs, exposed to 7d of IEE, marked with arrows pointing to individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray). G Summarized results from all lines showing differences in GIRK2 expression levels before and after 7 days of 20 mM IEE with ethanol (EtOH; p = 1.0 × 10 −20 ). H Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin-positive processes (gray) prior and following 7 days 20 mM IEE with ethanol. Sample images are from line 246. I Representative images of FISH detection of KCNJ6 mRNA for each cell line. J Quantification of FISH. (a) The number of KCNJ6 puncta normalized to the number of cells in an image shows decreased expression in control AF compared with UN ( p = 8.2 × 10 −3 ) and increased expression following IEE ( p = 8.2 × 10 −3 ; using Tukey’s pairwise comparisons). (b) The percentage of KCNJ6 -expressing MAP2 + cells substantially increase in the (c) AF group but not in the (b) UN group. Numbers of KCNJ6 puncta were analyzed by expression levels per cell, as recommended by the FISH manufacturer in (d) UN or (e) AF cells. (f) KCNJ6 puncta within the neuronal soma show increases following IEE in both UN and AF groups ( p = 0.006 for genotype, Tukey’s post-hoc for UN, p = 0.01, for AF, p = 0.01). (g) A similar analysis of non-somatic puncta, presumably within neurites, showed no differences following IEE between genotypes ( p = 0.23).

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (BioLegend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Generated, Fluorescence In Situ Hybridization

    GIRK2 overexpression mimics ethanol response. A Current required to shift resting membrane potential to −65 mV, in untreated (control, p = 5.9 × 10 −4 ), lentiviral KCNJ6 overexpression (over., p = 0.23), or 1 d 20 mM EtOH ( p = 0.75) cultures. B Representative traces of APs induced with the “ramp” protocol. C Quantification of maximum number of action potentials (APs) induced with “ramp” protocol, control ( p = 6.8 × 10 −6 ), overexpression ( p = 0.014), or 1 d 20 mM EtOH ( p = 0.10). D quantification of GIRK2 puncta after overexpression (line 376, one-tailed Student’s t test p = 0.04).

    Journal: Molecular Psychiatry

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1038/s41380-022-01818-x

    Figure Lengend Snippet: GIRK2 overexpression mimics ethanol response. A Current required to shift resting membrane potential to −65 mV, in untreated (control, p = 5.9 × 10 −4 ), lentiviral KCNJ6 overexpression (over., p = 0.23), or 1 d 20 mM EtOH ( p = 0.75) cultures. B Representative traces of APs induced with the “ramp” protocol. C Quantification of maximum number of action potentials (APs) induced with “ramp” protocol, control ( p = 6.8 × 10 −6 ), overexpression ( p = 0.014), or 1 d 20 mM EtOH ( p = 0.10). D quantification of GIRK2 puncta after overexpression (line 376, one-tailed Student’s t test p = 0.04).

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (BioLegend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Over Expression, One-tailed Test

    Experimental design and gene expression analysis. A Diagram outlining experimental design. Lymphocytes from subjects with or without AUD diagnosis and KCNJ6 haplotype variants were selected, reprogrammed into iPSC, induced into excitatory iNs, and analyzed by morphometry, immunocytochemistry, gene expression, and electrophysiology. B Sequencing alignment and depth analysis of bulk RNA sequencing confirmed expression of KCNJ6 mRNA in iN cultures, specifically the ENST00000609713 isoform, containing an 18.1 kilobase 3′UTR region. KCNJ6 exons are mapped to chromosome locations (marked in mb, megabases) and the position of the gene is indicated by the red box on the chromosome 21 pictogram, top, with transcription direction on the minus strand indicated by the broken arrow. Variant analysis of RNA sequences predicts a region of linkage disequilibrium of 22 SNPs, including the 3 SNPs used to select subjects (red, Table 1 ), and 19 additional SNPs (blue, Supplementary Table 1 ). Depth: number of sequencing reads per base aligned by position. Frequency: thickness of curved lines represents the relative frequency of splice site utilization between exons. C Single-cell RNAseq identifies a cluster of induced neurons (lower left), expressing markers consistent with neuronal function including SYP , SLC17A6 , GRIN2B , SCN3A , KCNJ3 , and KCNJ6 ; distinct from “transition neurons” that either do not express these markers or express sporadically. Additional markers are plotted in Supplementary Fig. 2E . Isolating KCNJ6 mRNA expression, aggregated by subject and treatment, AF neurons expressed a trend towards lower levels than UN neurons ( p = 0.0508; Wald test), but treatment of 7d with IEE at 20 mM peak concentration increased AF expression above untreated ( p = 0.0225) to levels similar to UN control ( p = 0.322, not denoted on figure). D Volcano plot for untreated UN vs. AF neurons, highlighting genes significantly different (FDR > 0.05) and at least 1.5-fold changed (red dots). Genes below the fold-change cut-off are marked in blue, and those not significantly different are marked in green. Significantly different genes are listed in Supplementary Table 3 . E Gene ontology (GO) enrichment of top 10 biological process (BP) terms for up- or downregulated genes. Plots indicate the number of regulated genes from the term and the color indicates the adjusted p value (q-value; key). All enriched terms are listed in Supplementary Tables 4 , 5 .

    Journal: Molecular Psychiatry

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1038/s41380-022-01818-x

    Figure Lengend Snippet: Experimental design and gene expression analysis. A Diagram outlining experimental design. Lymphocytes from subjects with or without AUD diagnosis and KCNJ6 haplotype variants were selected, reprogrammed into iPSC, induced into excitatory iNs, and analyzed by morphometry, immunocytochemistry, gene expression, and electrophysiology. B Sequencing alignment and depth analysis of bulk RNA sequencing confirmed expression of KCNJ6 mRNA in iN cultures, specifically the ENST00000609713 isoform, containing an 18.1 kilobase 3′UTR region. KCNJ6 exons are mapped to chromosome locations (marked in mb, megabases) and the position of the gene is indicated by the red box on the chromosome 21 pictogram, top, with transcription direction on the minus strand indicated by the broken arrow. Variant analysis of RNA sequences predicts a region of linkage disequilibrium of 22 SNPs, including the 3 SNPs used to select subjects (red, Table 1 ), and 19 additional SNPs (blue, Supplementary Table 1 ). Depth: number of sequencing reads per base aligned by position. Frequency: thickness of curved lines represents the relative frequency of splice site utilization between exons. C Single-cell RNAseq identifies a cluster of induced neurons (lower left), expressing markers consistent with neuronal function including SYP , SLC17A6 , GRIN2B , SCN3A , KCNJ3 , and KCNJ6 ; distinct from “transition neurons” that either do not express these markers or express sporadically. Additional markers are plotted in Supplementary Fig. 2E . Isolating KCNJ6 mRNA expression, aggregated by subject and treatment, AF neurons expressed a trend towards lower levels than UN neurons ( p = 0.0508; Wald test), but treatment of 7d with IEE at 20 mM peak concentration increased AF expression above untreated ( p = 0.0225) to levels similar to UN control ( p = 0.322, not denoted on figure). D Volcano plot for untreated UN vs. AF neurons, highlighting genes significantly different (FDR > 0.05) and at least 1.5-fold changed (red dots). Genes below the fold-change cut-off are marked in blue, and those not significantly different are marked in green. Significantly different genes are listed in Supplementary Table 3 . E Gene ontology (GO) enrichment of top 10 biological process (BP) terms for up- or downregulated genes. Plots indicate the number of regulated genes from the term and the color indicates the adjusted p value (q-value; key). All enriched terms are listed in Supplementary Tables 4 , 5 .

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (BioLegend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Immunocytochemistry, Sequencing, RNA Sequencing Assay, Variant Assay, Concentration Assay

    Validation of GIRK2 expression and function in human induced neurons. A Representative confocal images of GIRK2 immunoreactivity in mouse cortical neurons. Arrowheads indicate locations of GIRK2-staining puncta, with an example punctum enlarged in the inset, overlapping or adjacent to βIII-tubulin-positive processes. We observed two cellular expression patterns—one where the entire neuron is decorated with GIRK2 antibody (Supplementary Fig. 4 ), or another where GIRK2 expression is relatively faint and observed mostly on neuronal processes, shown here. B GIRK2 expression patterns in human induced neurons (iNs), showing representative confocal images from line 420. Inset shows two adjacent puncta. GIRK2 immunoreactivity matched a pattern of process-selective expression in mouse ( A and Supplementary Fig. 4A ), where GIRK2 was detected as relatively small (~0.5 µm diameter) puncta scattered primarily along the processes. Localization of GIRK2 immunoreactivity in human iN did not directly colocalize with synaptic vesicle marker VGLUT2 or synaptic marker Syn1 (Supplementary Fig. 5 ), but instead was found most frequently adjacent to synapses but overlapping the shafts of the βIII tubulin-positive processes, and less so on MAP2 positive processes (Figs. 2D.a, 3C, E ). Cultured neurons express βIII tubulin throughout the cell, but not as strongly in axonal processes [ 69 ]. We previously found that processes in human iN cells stained for ankyrin G, identifying the axonal initial segment, which similarly lacked βIII-tubulin [ 70 ]. Detection of GIRK2 primarily on βIII-tubulin + /MAP - processes, therefore, suggests pre-axonal, and likely presynaptic, localization. C Following infection of iN cultures with lentivirus expressing both KCNJ6 and mCherry, large numbers of GIRK2 + puncta are seen in representative images (line 420). D Evaluation of GIRK2 function in iNs, (a) quantification of GIRK2 expression on MAP2 + vs. βIII-tubulin + neuronal processes. GIRK2 is more abundant on βIII-tubulin processes ( p = 0.0006, one-tailed Student’s t test, n = 15 cells per group, cell line 420). (b) Basal levels (upper pie plot) of the GIRK current in iNs as percent of neurons responding with hyperpolarization to the selective GIRK activator (160 nM ML297); compared with responding percentage when GIRK2 is overexpressed (lower pie chart). (c) Representative image of iN overexpressing GIRK2, as confirmed with mCherry fluorescence. (d) Representative traces of induced action potential firing before and after GIRK activation, demonstrating the contribution of GIRK function to cell excitability. (e) Representative trace of spontaneous postsynaptic current (sEPSCs) recordings during ML297 (160 nM) GIRK activator wash-in, demonstrating a shift of 7 mV holding current (amplifier-dependent compensation of GIRK-mediated membrane hyperpolarization). (f) Quantification of neuronal excitability at baseline and following GIRK activation with 160 nM ML297 ( p = 0.015, paired Student’s t test, n = 9 cells before/after ML297, cell line 376).

    Journal: Molecular Psychiatry

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1038/s41380-022-01818-x

    Figure Lengend Snippet: Validation of GIRK2 expression and function in human induced neurons. A Representative confocal images of GIRK2 immunoreactivity in mouse cortical neurons. Arrowheads indicate locations of GIRK2-staining puncta, with an example punctum enlarged in the inset, overlapping or adjacent to βIII-tubulin-positive processes. We observed two cellular expression patterns—one where the entire neuron is decorated with GIRK2 antibody (Supplementary Fig. 4 ), or another where GIRK2 expression is relatively faint and observed mostly on neuronal processes, shown here. B GIRK2 expression patterns in human induced neurons (iNs), showing representative confocal images from line 420. Inset shows two adjacent puncta. GIRK2 immunoreactivity matched a pattern of process-selective expression in mouse ( A and Supplementary Fig. 4A ), where GIRK2 was detected as relatively small (~0.5 µm diameter) puncta scattered primarily along the processes. Localization of GIRK2 immunoreactivity in human iN did not directly colocalize with synaptic vesicle marker VGLUT2 or synaptic marker Syn1 (Supplementary Fig. 5 ), but instead was found most frequently adjacent to synapses but overlapping the shafts of the βIII tubulin-positive processes, and less so on MAP2 positive processes (Figs. 2D.a, 3C, E ). Cultured neurons express βIII tubulin throughout the cell, but not as strongly in axonal processes [ 69 ]. We previously found that processes in human iN cells stained for ankyrin G, identifying the axonal initial segment, which similarly lacked βIII-tubulin [ 70 ]. Detection of GIRK2 primarily on βIII-tubulin + /MAP - processes, therefore, suggests pre-axonal, and likely presynaptic, localization. C Following infection of iN cultures with lentivirus expressing both KCNJ6 and mCherry, large numbers of GIRK2 + puncta are seen in representative images (line 420). D Evaluation of GIRK2 function in iNs, (a) quantification of GIRK2 expression on MAP2 + vs. βIII-tubulin + neuronal processes. GIRK2 is more abundant on βIII-tubulin processes ( p = 0.0006, one-tailed Student’s t test, n = 15 cells per group, cell line 420). (b) Basal levels (upper pie plot) of the GIRK current in iNs as percent of neurons responding with hyperpolarization to the selective GIRK activator (160 nM ML297); compared with responding percentage when GIRK2 is overexpressed (lower pie chart). (c) Representative image of iN overexpressing GIRK2, as confirmed with mCherry fluorescence. (d) Representative traces of induced action potential firing before and after GIRK activation, demonstrating the contribution of GIRK function to cell excitability. (e) Representative trace of spontaneous postsynaptic current (sEPSCs) recordings during ML297 (160 nM) GIRK activator wash-in, demonstrating a shift of 7 mV holding current (amplifier-dependent compensation of GIRK-mediated membrane hyperpolarization). (f) Quantification of neuronal excitability at baseline and following GIRK activation with 160 nM ML297 ( p = 0.015, paired Student’s t test, n = 9 cells before/after ML297, cell line 376).

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (BioLegend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Staining, Marker, Cell Culture, Infection, One-tailed Test, Fluorescence, Activation Assay

    Impact of AUD-associated KCNJ6 haplotype on neuronal properties. A Principles of morphological analysis of induced neurons: (a) Neurite area was the total TuJ1 + (βIII-tubulin + ) staining area outside the cell soma. (b) Solidity is the area of the soma divided by its convex hull area. (c) Soma size was the area of the MAP2 + cell body. (d) Circularity compared the perimeter to the area. B Morphometry of iNs from KCNJ6 haplotype variant and affected ( AF , cyan) or unaffected ( UN , gray) individuals. Results are summed by group (left) or plotted individually by cell line (right), with subjects identified by line number (see Table 1 —females identified with gray numbers). Individual cells are plotted as dots with the bar showing the mean, with error bars indicating the standard error of the mean (SEM). No significant differences were found in (a) soma size, (b) circularity, or (c) soma solidity, but (d) total neurite area was increased in the AF group ( p = 0.018). C Representative images of iNs from individual lines, with arrows identifying individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). D GIRK2 expression was decreased in the AF as measured by puncta counts (a, p = 0.0012), circularity (c, p = 0.007), or solidity (d, p = 0.037), while there was no difference in puncta size (b). E Representative images of individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). F Electrophysiological analysis of passive neuronal properties, showing no difference in (a) membrane capacitance (b) membrane resistance, or (c) spontaneous EPSCs frequency. (d) Representative sEPSCs traces for each line. (e) Spontaneous EPSCs amplitude. G Electrophysiological analysis of induced neuronal properties. (a) Quantification of current required to shift resting membrane potential to −65 mV in pA: difference by group p = 1.2 × 10 –5 ; (b) quantification of maximum number of action potentials (APs) induced with the “step” protocol, p = 0.086; (c) representative traces of APs induced with the “step” protocol; (d) quantification of number of action potentials (APs) induced with the “ramp” protocol, p = 2.0 × 10 –9 ; (e) representative traces of APs induced with the “ramp” protocol. A generalized linear model was used to evaluate group differences for morphometry and GIRK2 expression, and generalized estimation equations was used for electrophysiology results. Numbers of cell lines and replicates for each experiment are shown in Supplementary Table 6 .

    Journal: Molecular Psychiatry

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1038/s41380-022-01818-x

    Figure Lengend Snippet: Impact of AUD-associated KCNJ6 haplotype on neuronal properties. A Principles of morphological analysis of induced neurons: (a) Neurite area was the total TuJ1 + (βIII-tubulin + ) staining area outside the cell soma. (b) Solidity is the area of the soma divided by its convex hull area. (c) Soma size was the area of the MAP2 + cell body. (d) Circularity compared the perimeter to the area. B Morphometry of iNs from KCNJ6 haplotype variant and affected ( AF , cyan) or unaffected ( UN , gray) individuals. Results are summed by group (left) or plotted individually by cell line (right), with subjects identified by line number (see Table 1 —females identified with gray numbers). Individual cells are plotted as dots with the bar showing the mean, with error bars indicating the standard error of the mean (SEM). No significant differences were found in (a) soma size, (b) circularity, or (c) soma solidity, but (d) total neurite area was increased in the AF group ( p = 0.018). C Representative images of iNs from individual lines, with arrows identifying individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). D GIRK2 expression was decreased in the AF as measured by puncta counts (a, p = 0.0012), circularity (c, p = 0.007), or solidity (d, p = 0.037), while there was no difference in puncta size (b). E Representative images of individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). F Electrophysiological analysis of passive neuronal properties, showing no difference in (a) membrane capacitance (b) membrane resistance, or (c) spontaneous EPSCs frequency. (d) Representative sEPSCs traces for each line. (e) Spontaneous EPSCs amplitude. G Electrophysiological analysis of induced neuronal properties. (a) Quantification of current required to shift resting membrane potential to −65 mV in pA: difference by group p = 1.2 × 10 –5 ; (b) quantification of maximum number of action potentials (APs) induced with the “step” protocol, p = 0.086; (c) representative traces of APs induced with the “step” protocol; (d) quantification of number of action potentials (APs) induced with the “ramp” protocol, p = 2.0 × 10 –9 ; (e) representative traces of APs induced with the “ramp” protocol. A generalized linear model was used to evaluate group differences for morphometry and GIRK2 expression, and generalized estimation equations was used for electrophysiology results. Numbers of cell lines and replicates for each experiment are shown in Supplementary Table 6 .

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (BioLegend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Staining, Variant Assay, Expressing

    Validation of GIRK2 expression and function in human induced neurons. A . Representative confocal images of GIRK2 immunoreactivity in mouse cortical neurons. Arrowheads indicate locations of GIRK2-staining puncta, with an example punctum enlarged in the inset, overlapping or adjacent to βIII-tubulin-positive processes. We observed two cellular expression patterns – one where the entire neuron is decorated with GIRK2 antibody (Supplemental Fig. 5), or another where GIRK2 expression is relatively faint and observed mostly on neuronal processes, shown here. B . GIRK2 expression patterns in human induced neurons (iNs), showing representative confocal images from line 420. Inset shows two adjacent puncta. C . Following infection of iN cultures with lentivirus expressing KCNJ6 and mCherry, large numbers of GIRK2 + puncta are seen in representative images (line 420). D . Evaluation of GIRK2 function in iNs, (a) quantification of GIRK2 expression on MAP2 + vs. βIII-tubulin + neuronal processes. GIRK2 is more abundant on βIII-tubulin processes (p=0.014). (b) Basal levels (upper pie plot) of the GIRK current in iNs as percent of neurons responding with hyperpolarization to the selective GIRK activator (160 nM ML297); compared with responding percentage when GIRK2 is overexpressed (lower pie chart). (c) Representative image of iN overexpressing GIRK2, as confirmed with mCherry fluorescence. (d) Representative traces of induced action potential firing before and after GIRK activation, demonstrating the contribution of GIRK function to cell excitability. (e) Representative trace of spontaneous postsynaptic potential (sEPSCs) recordings during ML297 (160 nM) GIRK activator wash-in, demonstrating a shift of 7mV holding current (amplifier-dependent compensation of GIRK-mediated membrane hyperpolarization). (f) Quantification of neuronal excitability at baseline and following GIRK activation with ML297 (160 nM). Student’s t-test was used to evaluate differences (*p

    Journal: bioRxiv

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1101/2022.05.24.493086

    Figure Lengend Snippet: Validation of GIRK2 expression and function in human induced neurons. A . Representative confocal images of GIRK2 immunoreactivity in mouse cortical neurons. Arrowheads indicate locations of GIRK2-staining puncta, with an example punctum enlarged in the inset, overlapping or adjacent to βIII-tubulin-positive processes. We observed two cellular expression patterns – one where the entire neuron is decorated with GIRK2 antibody (Supplemental Fig. 5), or another where GIRK2 expression is relatively faint and observed mostly on neuronal processes, shown here. B . GIRK2 expression patterns in human induced neurons (iNs), showing representative confocal images from line 420. Inset shows two adjacent puncta. C . Following infection of iN cultures with lentivirus expressing KCNJ6 and mCherry, large numbers of GIRK2 + puncta are seen in representative images (line 420). D . Evaluation of GIRK2 function in iNs, (a) quantification of GIRK2 expression on MAP2 + vs. βIII-tubulin + neuronal processes. GIRK2 is more abundant on βIII-tubulin processes (p=0.014). (b) Basal levels (upper pie plot) of the GIRK current in iNs as percent of neurons responding with hyperpolarization to the selective GIRK activator (160 nM ML297); compared with responding percentage when GIRK2 is overexpressed (lower pie chart). (c) Representative image of iN overexpressing GIRK2, as confirmed with mCherry fluorescence. (d) Representative traces of induced action potential firing before and after GIRK activation, demonstrating the contribution of GIRK function to cell excitability. (e) Representative trace of spontaneous postsynaptic potential (sEPSCs) recordings during ML297 (160 nM) GIRK activator wash-in, demonstrating a shift of 7mV holding current (amplifier-dependent compensation of GIRK-mediated membrane hyperpolarization). (f) Quantification of neuronal excitability at baseline and following GIRK activation with ML297 (160 nM). Student’s t-test was used to evaluate differences (*p

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (Bio legend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Staining, Infection, Fluorescence, Activation Assay

    Ethanol treatment reduced KCNJ6 haplotype differences in iN excitability and GIRK2 expression. A . Morphological analysis of IEE iNs generated from affected and unaffected individuals, showing no difference in: (a) neuronal soma size (p = 0.38); (b) soma circularity (p = 0.47); (c) soma solidity (p = 0.87); and (d) total neurite area (p = 0.98). B . There was no difference in GIRK2 expression in the IEE AF group iNs compared with UN by (a) puncta counts (p = 0.28), (b) puncta size (p = 0.34), or (d) solidity (p = 0.43), but there was a slight increase in (c) puncta circularity (p = 0.046). C . Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray) for each cell line. D . Electrophysiological analysis of passive neuronal properties in IEE iNs, showing (a) a small decrease in AF membrane capacitance (p = 9.6 × 10 −9 ), but no difference in (b) membrane resistance (p = 0.63), (c) spontaneous EPSCs frequency (p = 0.32), or (d) spontaneous EPSCs amplitude (p = 0.19). (d) Representative sEPSCs traces for each cell line. (f) The AF group exhibited no change in resting membrane potential after IEE (p = 0.79). E . Electrophysiological analysis of active neuronal properties found no difference in IEE iNs for (a) current required to shift resting membrane potential to -65mV (p = 0.32), (b) maximum number of action potentials (APs) induced with the “step” protocol (p = 0.48), with (c) representative traces of APs induced with the “step” protocol, (d) number of action potentials (APs) induced with the “ramp” protocol (p = 0.95), with (e) representative traces of APs induced with the “ramp” protocol. F . Representative images from individual lines of iNs, marked with arrows pointing to individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray). G . Summarized results from all lines showing differences in GIRK2 expression levels before and after 7 days of 20 mM IEE with ethanol (EtOH). H . Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray) prior and following 7 days 20 mM IEE with ethanol. I . Representative images of FISH detection of KCNJ6 mRNA for each cell line. J . Quantification of FISH. (a) The number of KCNJ6 puncta normalized to the number of cells in an image shows decreased expression in control AF compared with UN (p = 1.6 × 10 −3 ), increased expression following IEE (p = 1.1 × 10 −3 ; Tukey’s pairwise comparisons for UN p = 9.0 × 10 −3 , for AF p = 5.5 × 10 −3 ). (b) The percentage of KCNJ6 -expressing MAP2 + cells substantially increase in the (c) AF group but not in the (b) UN group. Numbers of KCNJ6 puncta were analyzed by expression levels per cell, as recommended by the FISH manufacturer in (d) UN or (e) AF cells. (f) KCNJ6 puncta within the neuronal soma show increases following IEE in both UN and AF groups (p = 4.1 × 10 −4 , Tukey’s post-hoc for UN, p = 1.1 × 10 −3 , for AF, p = 1.1 × 10 −3 ). (g) A similar analysis of non-somatic puncta, presumably within neurites, showed no differences following IEE (p=0.24).

    Journal: bioRxiv

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1101/2022.05.24.493086

    Figure Lengend Snippet: Ethanol treatment reduced KCNJ6 haplotype differences in iN excitability and GIRK2 expression. A . Morphological analysis of IEE iNs generated from affected and unaffected individuals, showing no difference in: (a) neuronal soma size (p = 0.38); (b) soma circularity (p = 0.47); (c) soma solidity (p = 0.87); and (d) total neurite area (p = 0.98). B . There was no difference in GIRK2 expression in the IEE AF group iNs compared with UN by (a) puncta counts (p = 0.28), (b) puncta size (p = 0.34), or (d) solidity (p = 0.43), but there was a slight increase in (c) puncta circularity (p = 0.046). C . Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray) for each cell line. D . Electrophysiological analysis of passive neuronal properties in IEE iNs, showing (a) a small decrease in AF membrane capacitance (p = 9.6 × 10 −9 ), but no difference in (b) membrane resistance (p = 0.63), (c) spontaneous EPSCs frequency (p = 0.32), or (d) spontaneous EPSCs amplitude (p = 0.19). (d) Representative sEPSCs traces for each cell line. (f) The AF group exhibited no change in resting membrane potential after IEE (p = 0.79). E . Electrophysiological analysis of active neuronal properties found no difference in IEE iNs for (a) current required to shift resting membrane potential to -65mV (p = 0.32), (b) maximum number of action potentials (APs) induced with the “step” protocol (p = 0.48), with (c) representative traces of APs induced with the “step” protocol, (d) number of action potentials (APs) induced with the “ramp” protocol (p = 0.95), with (e) representative traces of APs induced with the “ramp” protocol. F . Representative images from individual lines of iNs, marked with arrows pointing to individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray). G . Summarized results from all lines showing differences in GIRK2 expression levels before and after 7 days of 20 mM IEE with ethanol (EtOH). H . Representative images of individual GIRK2 puncta (red) localized on βIII-Tubulin positive processes (gray) prior and following 7 days 20 mM IEE with ethanol. I . Representative images of FISH detection of KCNJ6 mRNA for each cell line. J . Quantification of FISH. (a) The number of KCNJ6 puncta normalized to the number of cells in an image shows decreased expression in control AF compared with UN (p = 1.6 × 10 −3 ), increased expression following IEE (p = 1.1 × 10 −3 ; Tukey’s pairwise comparisons for UN p = 9.0 × 10 −3 , for AF p = 5.5 × 10 −3 ). (b) The percentage of KCNJ6 -expressing MAP2 + cells substantially increase in the (c) AF group but not in the (b) UN group. Numbers of KCNJ6 puncta were analyzed by expression levels per cell, as recommended by the FISH manufacturer in (d) UN or (e) AF cells. (f) KCNJ6 puncta within the neuronal soma show increases following IEE in both UN and AF groups (p = 4.1 × 10 −4 , Tukey’s post-hoc for UN, p = 1.1 × 10 −3 , for AF, p = 1.1 × 10 −3 ). (g) A similar analysis of non-somatic puncta, presumably within neurites, showed no differences following IEE (p=0.24).

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (Bio legend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Generated, Fluorescence In Situ Hybridization

    Impact of AUD-associated KCNJ6 haplotype on neuronal properties. A . Principles of morphological analysis of induced neurons: (a) Neurite area was the total TuJ1 + (βIII-tubulin) + staining area outside the cell soma. (b) Solidity is the area of the soma divided by its convex hull area. (c) Soma size was the area of the MAP2 + cell body. (d) Circularity compared the perimeter to the area. B . Morphometry of iNs from KCNJ6 haplotype variant and affected ( AF , cyan) or unaffected ( UN , grey) individuals. Results are summed by group (left) or plotted individually by cell line (right), with subjects identified by line number (see Table 1 --females identified with grey numbers). Individual cells are plotted as dots with the bar showing the mean, with bars indicating the standard error of the mean (SEM). No significant differences were found in (a) soma size, (b) circularity, or (c) soma solidity, but total neurite area was increased in the AF group (p = 0.0007). C . Representative images of iNs from individual lines, with arrows identifying individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). D . GIRK2 expression was decreased in the AF as measured by puncta counts (a, p = 0.043), while there was no difference in puncta size (b), circularity (c), or solidity (d). E . Representative images of individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). F . Electrophysiological analysis of passive neuronal properties, showing no difference in (a) membrane capacitance (b) membrane resistance, or (c) spontaneous EPSCs frequency. (d) Representative sEPSCs traces for each line. (e) Spontaneous EPSCs amplitude. G . Electrophysiological analysis of active neuronal properties. (a) Quantification of current required to shift resting membrane potential to -65mV in pA: difference by group p = 0.048; (b) quantification of maximum number of action potentials (APs) induced with the “step” protocol, p = 0.014; (c) representative traces of APs induced with the “step” protocol; (d) quantification of number of action potentials (APs) induced with the “ramp” protocol, p = 0.036; (e) representative traces of APs induced with the “ramp” protocol. A generalized linear model was used to evaluate group differences for morphometry and GIRK2 expression, and generalized estimation equations was used for electrophysiology results (*p

    Journal: bioRxiv

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1101/2022.05.24.493086

    Figure Lengend Snippet: Impact of AUD-associated KCNJ6 haplotype on neuronal properties. A . Principles of morphological analysis of induced neurons: (a) Neurite area was the total TuJ1 + (βIII-tubulin) + staining area outside the cell soma. (b) Solidity is the area of the soma divided by its convex hull area. (c) Soma size was the area of the MAP2 + cell body. (d) Circularity compared the perimeter to the area. B . Morphometry of iNs from KCNJ6 haplotype variant and affected ( AF , cyan) or unaffected ( UN , grey) individuals. Results are summed by group (left) or plotted individually by cell line (right), with subjects identified by line number (see Table 1 --females identified with grey numbers). Individual cells are plotted as dots with the bar showing the mean, with bars indicating the standard error of the mean (SEM). No significant differences were found in (a) soma size, (b) circularity, or (c) soma solidity, but total neurite area was increased in the AF group (p = 0.0007). C . Representative images of iNs from individual lines, with arrows identifying individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). D . GIRK2 expression was decreased in the AF as measured by puncta counts (a, p = 0.043), while there was no difference in puncta size (b), circularity (c), or solidity (d). E . Representative images of individual GIRK2 puncta (red) localized on βIII-tubulin + processes (gray). F . Electrophysiological analysis of passive neuronal properties, showing no difference in (a) membrane capacitance (b) membrane resistance, or (c) spontaneous EPSCs frequency. (d) Representative sEPSCs traces for each line. (e) Spontaneous EPSCs amplitude. G . Electrophysiological analysis of active neuronal properties. (a) Quantification of current required to shift resting membrane potential to -65mV in pA: difference by group p = 0.048; (b) quantification of maximum number of action potentials (APs) induced with the “step” protocol, p = 0.014; (c) representative traces of APs induced with the “step” protocol; (d) quantification of number of action potentials (APs) induced with the “ramp” protocol, p = 0.036; (e) representative traces of APs induced with the “ramp” protocol. A generalized linear model was used to evaluate group differences for morphometry and GIRK2 expression, and generalized estimation equations was used for electrophysiology results (*p

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (Bio legend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Staining, Variant Assay, Expressing

    Experimental design and gene expression analysis A . Diagram outlining experimental design. Lymphocytes from subjects with or without AUD diagnosis and KCNJ6 haplotype variants were selected, reprogrammed into iPSC, induced into excitatory iNs, and analyzed by morphometry, immunocytochemistry, gene expression, and electrophysiology. B . Sequencing alignment and depth analysis of bulk RNA sequencing confirmed expression of KCNJ6 mRNA in iN cultures, specifically the ENST00000609713 isoform, containing an 18.1 kilobase 3’UTR region. Depth: number of sequencing reads per base aligned by position. Frequency: thickness of curved lines represents the relative frequency of splice site utilization between exons. Variant analysis of RNA sequences predicts a region of linkage disequilibrium of 22 SNPs, including the 3 SNPs used to select subjects (red, Table 1 ), and 19 additional SNPs (blue, Supplemental Table 1). C . Single-cell RNAseq identifies a cluster of induced neurons (upper right), expressing markers consistent with neuronal function including SYP, SCN3A, SLC17A6, GRIN2B, KCNJ3 , and KCNJ6 ; distinct from “transition neurons” that either do not express these markers or express sporadically. Isolating KCNJ6 mRNA expression, aggregated by subject and treatment, AF neurons expressed a trend towards lower levels than UN neurons (p = 0.0508; Wald test), but treatment of 7d with IEE at 20 mM peak concentration increased AF expression above untreated (p = 0.0225) to levels similar to UN control (p = 0.322, not denoted on figure). D . Volcano plot for untreated UN vs AF neurons, highlighting genes significantly different (FDR > 0.05) and at least 1.5-fold changed (red dots). Genes belopuncta circularity w the fold-change cut-off are marked in green, and those not significantly different are marked in blue. Significantly different genes are listed in Supplemental Table 2. F . Gene ontology (GO) enrichment of top 10 biological process (BP) terms for up-or down-regulated genes. Plots indicate the number of regulated genes from the term and the color indicates the adjusted p-value (q-value; key). All enriched terms are listed in Supplemental Table 3.

    Journal: bioRxiv

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1101/2022.05.24.493086

    Figure Lengend Snippet: Experimental design and gene expression analysis A . Diagram outlining experimental design. Lymphocytes from subjects with or without AUD diagnosis and KCNJ6 haplotype variants were selected, reprogrammed into iPSC, induced into excitatory iNs, and analyzed by morphometry, immunocytochemistry, gene expression, and electrophysiology. B . Sequencing alignment and depth analysis of bulk RNA sequencing confirmed expression of KCNJ6 mRNA in iN cultures, specifically the ENST00000609713 isoform, containing an 18.1 kilobase 3’UTR region. Depth: number of sequencing reads per base aligned by position. Frequency: thickness of curved lines represents the relative frequency of splice site utilization between exons. Variant analysis of RNA sequences predicts a region of linkage disequilibrium of 22 SNPs, including the 3 SNPs used to select subjects (red, Table 1 ), and 19 additional SNPs (blue, Supplemental Table 1). C . Single-cell RNAseq identifies a cluster of induced neurons (upper right), expressing markers consistent with neuronal function including SYP, SCN3A, SLC17A6, GRIN2B, KCNJ3 , and KCNJ6 ; distinct from “transition neurons” that either do not express these markers or express sporadically. Isolating KCNJ6 mRNA expression, aggregated by subject and treatment, AF neurons expressed a trend towards lower levels than UN neurons (p = 0.0508; Wald test), but treatment of 7d with IEE at 20 mM peak concentration increased AF expression above untreated (p = 0.0225) to levels similar to UN control (p = 0.322, not denoted on figure). D . Volcano plot for untreated UN vs AF neurons, highlighting genes significantly different (FDR > 0.05) and at least 1.5-fold changed (red dots). Genes belopuncta circularity w the fold-change cut-off are marked in green, and those not significantly different are marked in blue. Significantly different genes are listed in Supplemental Table 2. F . Gene ontology (GO) enrichment of top 10 biological process (BP) terms for up-or down-regulated genes. Plots indicate the number of regulated genes from the term and the color indicates the adjusted p-value (q-value; key). All enriched terms are listed in Supplemental Table 3.

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (Bio legend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Expressing, Immunocytochemistry, Sequencing, RNA Sequencing Assay, Variant Assay, Concentration Assay

    GIRK2 overexpression mimics ethanol response A . Current required to shift resting membrane potential to -65mV, in untreated (control, p = 5.9 × 10 −4 ), lentiviral KCNJ6 overexpression (over., p = 0.23), or 1 d 20 mM IEE (EtOH, p = 0.75) cultures. B . Representative traces of APs induced with the “ramp” protocol. C . Quantification of maximum number of action potentials (APs) induced with “ramp” protocol, control (p = 6.8 × 10 −6 ), overexpression (p = 0.014), or 1 d 20 mM IEE (p = 0.10). D . quantification of GIRK2 puncta after overexpression (line 376, one-sided t-test p = 0.04).

    Journal: bioRxiv

    Article Title: Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

    doi: 10.1101/2022.05.24.493086

    Figure Lengend Snippet: GIRK2 overexpression mimics ethanol response A . Current required to shift resting membrane potential to -65mV, in untreated (control, p = 5.9 × 10 −4 ), lentiviral KCNJ6 overexpression (over., p = 0.23), or 1 d 20 mM IEE (EtOH, p = 0.75) cultures. B . Representative traces of APs induced with the “ramp” protocol. C . Quantification of maximum number of action potentials (APs) induced with “ramp” protocol, control (p = 6.8 × 10 −6 ), overexpression (p = 0.014), or 1 d 20 mM IEE (p = 0.10). D . quantification of GIRK2 puncta after overexpression (line 376, one-sided t-test p = 0.04).

    Article Snippet: Primary antibodies used: rabbit anti-GIRK2 (Alomone labs, APC-006, 1:400), mouse anti-βIII-tub (Bio legend, MMS-435P,1:1000), chicken anti-MAP2 (Millipore AB5543, 1:1000), mouse anti-Syn1 (SYSY,106-011, 1:200), mouse anti-PSD 95 (SYSY, 124-011, 1:2000), mouse anti-mCherry (Thermofisher Scientific, M11217, 1:100).

    Techniques: Over Expression