rabbit anti girk2  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti girk2
    VM-patterned hPSC differentiation generates functionally mature dopaminergic (DA) neurons. ( A ) Schematic overview of the experimental design. ( B – D ) Representative bright-field images of ventral midbrain (VM) differentiation cultures at different time points (16, 30, and 60 days). Scale bars, 100 µm. ( E – G ) Immunofluorescence staining of tyrosine hydroxylase (TH), MAP2, and Ki67 at days 16, 30, and 60. Scale bars, 100 µm. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). ( H ) Immunofluorescence staining of DA markers <t>TH/GIRK2.</t> ( I ) TH/calbindin (CALB) at day 60. Scale bars, 25 µm. Nuclei were stained with DAPI. ( J – L ) Electrophysiological assessment of DA neuron-rich cultures using patch-clamp analysis. ( J ) Cells analyzed at day 60 displayed induced action potentials. ( K ) Induced action potentials upon brief depolarization. ( L ) Spontaneous firing characteristic of DA neurons.
    Rabbit Anti Girk2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 29 article reviews
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    rabbit anti girk2 - by Bioz Stars, 2022-11
    93/100 stars

    Images

    1) Product Images from "Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation"

    Article Title: Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation

    Journal: Cells

    doi: 10.3390/cells10010137

    VM-patterned hPSC differentiation generates functionally mature dopaminergic (DA) neurons. ( A ) Schematic overview of the experimental design. ( B – D ) Representative bright-field images of ventral midbrain (VM) differentiation cultures at different time points (16, 30, and 60 days). Scale bars, 100 µm. ( E – G ) Immunofluorescence staining of tyrosine hydroxylase (TH), MAP2, and Ki67 at days 16, 30, and 60. Scale bars, 100 µm. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). ( H ) Immunofluorescence staining of DA markers TH/GIRK2. ( I ) TH/calbindin (CALB) at day 60. Scale bars, 25 µm. Nuclei were stained with DAPI. ( J – L ) Electrophysiological assessment of DA neuron-rich cultures using patch-clamp analysis. ( J ) Cells analyzed at day 60 displayed induced action potentials. ( K ) Induced action potentials upon brief depolarization. ( L ) Spontaneous firing characteristic of DA neurons.
    Figure Legend Snippet: VM-patterned hPSC differentiation generates functionally mature dopaminergic (DA) neurons. ( A ) Schematic overview of the experimental design. ( B – D ) Representative bright-field images of ventral midbrain (VM) differentiation cultures at different time points (16, 30, and 60 days). Scale bars, 100 µm. ( E – G ) Immunofluorescence staining of tyrosine hydroxylase (TH), MAP2, and Ki67 at days 16, 30, and 60. Scale bars, 100 µm. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). ( H ) Immunofluorescence staining of DA markers TH/GIRK2. ( I ) TH/calbindin (CALB) at day 60. Scale bars, 25 µm. Nuclei were stained with DAPI. ( J – L ) Electrophysiological assessment of DA neuron-rich cultures using patch-clamp analysis. ( J ) Cells analyzed at day 60 displayed induced action potentials. ( K ) Induced action potentials upon brief depolarization. ( L ) Spontaneous firing characteristic of DA neurons.

    Techniques Used: Immunofluorescence, Staining, Patch Clamp

    2) Product Images from "Exercise Promotes Neurite Extensions from Grafted Dopaminergic Neurons in the Direction of the Dorsolateral Striatum in Parkinson’s Disease Model Rats"

    Article Title: Exercise Promotes Neurite Extensions from Grafted Dopaminergic Neurons in the Direction of the Dorsolateral Striatum in Parkinson’s Disease Model Rats

    Journal: Journal of Parkinson's Disease

    doi: 10.3233/JPD-191755

    Exercise enhanced neurite outgrowth in the direction of the dorsolateral striatum. (A, B) To estimate the direction of neurite outgrowth, the striatum was divided into 4 regions around the grafts. Division lines (blue) in the striatum are indicated with representative images of Girk2 staining from the Tx group (A) and Tx+TMT group (B). Girk2-positive neurites from the graft in each area were traced manually (green lines), and their total length was measured. Scale bars: 1 mm. (C) Quantification of Girk2-positive total neurite length in the dorsolateral striatum. * p = 0.016 by Mann-Whitney test. (D) Quantification of the adjusted Girk2-positive neurite length per Girk2-positive unit cell number in the dorsolateral striatum. * p = 0.016 by Mann-Whitney test. (E) Estimation of the reaching distance of Girk2-positive neurites from the graft edge in the dorsolateral striatum. Contour lines were drawn from the graft edge to 500 μm in 100 μm increments, and the number of neurites passing through those contours were counted. Scale bar: 1 mm. These lines were used for the bar graphs in (F) and (G). (F) Number of Girk2-positive neurites from the graft edge passing through each contour. 200 μm: ** p = 0.008, 400 μm: * p = 0.032, 500 μm: * p = 0.024 by Mann-Whitney test. (G) Number of Girk2-positive neurites per unit cell number from the graft edge passing through each contour. 300 μm: * p = 0.049, 400 μm: * p = 0.036 by Unpaired t test. (H–J) Girk2-positive neurite length per unit cell number in each area. Ventrolateral striatum: P = 0.1077 (H), dorsomedial striatum: p = 0.2498 (I), and ventromedial striatum: p = 0.1905 (J). Values are expressed as the mean±SD (C, D, F–J). Tx group, transplantation group; Tx+TMT group, transplantation with treadmill training group.
    Figure Legend Snippet: Exercise enhanced neurite outgrowth in the direction of the dorsolateral striatum. (A, B) To estimate the direction of neurite outgrowth, the striatum was divided into 4 regions around the grafts. Division lines (blue) in the striatum are indicated with representative images of Girk2 staining from the Tx group (A) and Tx+TMT group (B). Girk2-positive neurites from the graft in each area were traced manually (green lines), and their total length was measured. Scale bars: 1 mm. (C) Quantification of Girk2-positive total neurite length in the dorsolateral striatum. * p = 0.016 by Mann-Whitney test. (D) Quantification of the adjusted Girk2-positive neurite length per Girk2-positive unit cell number in the dorsolateral striatum. * p = 0.016 by Mann-Whitney test. (E) Estimation of the reaching distance of Girk2-positive neurites from the graft edge in the dorsolateral striatum. Contour lines were drawn from the graft edge to 500 μm in 100 μm increments, and the number of neurites passing through those contours were counted. Scale bar: 1 mm. These lines were used for the bar graphs in (F) and (G). (F) Number of Girk2-positive neurites from the graft edge passing through each contour. 200 μm: ** p = 0.008, 400 μm: * p = 0.032, 500 μm: * p = 0.024 by Mann-Whitney test. (G) Number of Girk2-positive neurites per unit cell number from the graft edge passing through each contour. 300 μm: * p = 0.049, 400 μm: * p = 0.036 by Unpaired t test. (H–J) Girk2-positive neurite length per unit cell number in each area. Ventrolateral striatum: P = 0.1077 (H), dorsomedial striatum: p = 0.2498 (I), and ventromedial striatum: p = 0.1905 (J). Values are expressed as the mean±SD (C, D, F–J). Tx group, transplantation group; Tx+TMT group, transplantation with treadmill training group.

    Techniques Used: Staining, MANN-WHITNEY, Transplantation Assay

    Exercise increased the number of dopamine neurons in the graft. (A) Serial sections of representative grafts at 6 weeks after transplantation in Tx and Tx+TMT rats. The grafts were identified with the expression of GFP. (B) Quantification of the graft volume from the Tx group (N = 4) and Tx+TMT group (N = 5). (C) Immunofluorescence images of the grafts. (D, E and F) Quantification of TH+ (D) and Girk2+ (E, F) cells in the grafts. The number of TH-positive cells was significantly larger in the Tx+TMT group (D). More Girk2-positive cells survived in the Tx+TMT group, both in total number (E) and in number adjusted by the surface area of the graft (cells/cm 2 , F). * p = 0.016 by Mann-Whitney test, ** p = 0.005 by Unpaired t test. All values are expressed as the mean±SD (B, D, E and F). Scale bars: 1 mm (A), 200 μm (C).
    Figure Legend Snippet: Exercise increased the number of dopamine neurons in the graft. (A) Serial sections of representative grafts at 6 weeks after transplantation in Tx and Tx+TMT rats. The grafts were identified with the expression of GFP. (B) Quantification of the graft volume from the Tx group (N = 4) and Tx+TMT group (N = 5). (C) Immunofluorescence images of the grafts. (D, E and F) Quantification of TH+ (D) and Girk2+ (E, F) cells in the grafts. The number of TH-positive cells was significantly larger in the Tx+TMT group (D). More Girk2-positive cells survived in the Tx+TMT group, both in total number (E) and in number adjusted by the surface area of the graft (cells/cm 2 , F). * p = 0.016 by Mann-Whitney test, ** p = 0.005 by Unpaired t test. All values are expressed as the mean±SD (B, D, E and F). Scale bars: 1 mm (A), 200 μm (C).

    Techniques Used: Transplantation Assay, Expressing, Immunofluorescence, MANN-WHITNEY

    Exercise promoted neurite outgrowth from the graft. (A–C) Representative immunofluorescence images of Girk2-positive neurites extended from the graft in Tx (A) and Tx+TMT (B, C) groups. All Girk2-positive neurites expressed GFP (C). Scale bars: 200 μm. (D, E) Quantification of Girk2-positive neurites extended from the graft. Girk2-positive neurites were traced manually and quantified into total neurite length (D). * p = 0.041 by Mann-Whitney test. (E) Adjusted length of Girk2-positive neurites by the number of Girk2-positive cells. p = 0.1864 by Unpaired t test.
    Figure Legend Snippet: Exercise promoted neurite outgrowth from the graft. (A–C) Representative immunofluorescence images of Girk2-positive neurites extended from the graft in Tx (A) and Tx+TMT (B, C) groups. All Girk2-positive neurites expressed GFP (C). Scale bars: 200 μm. (D, E) Quantification of Girk2-positive neurites extended from the graft. Girk2-positive neurites were traced manually and quantified into total neurite length (D). * p = 0.041 by Mann-Whitney test. (E) Adjusted length of Girk2-positive neurites by the number of Girk2-positive cells. p = 0.1864 by Unpaired t test.

    Techniques Used: Immunofluorescence, MANN-WHITNEY

    3) Product Images from "Hippocampal long‐term synaptic depression and memory deficits induced in early amyloidopathy are prevented by enhancing G‐protein‐gated inwardly rectifying potassium channel activity, et al. Hippocampal long‐term synaptic depression and memory deficits induced in early amyloidopathy are prevented by enhancing G‐protein‐gated inwardly rectifying potassium channel activity"

    Article Title: Hippocampal long‐term synaptic depression and memory deficits induced in early amyloidopathy are prevented by enhancing G‐protein‐gated inwardly rectifying potassium channel activity, et al. Hippocampal long‐term synaptic depression and memory deficits induced in early amyloidopathy are prevented by enhancing G‐protein‐gated inwardly rectifying potassium channel activity

    Journal: Journal of Neurochemistry

    doi: 10.1111/jnc.14946

    Effects of Aβ 1–42 on hippocampal G‐protein‐gated inwardly rectifying potassium channels (GirK) protein expression pattern in vitro. Western‐blot analysis of the (a) GIRK1 and (b) GIRK2 protein levels in hippocampus slices treated with Aβ 1–42 (0.5 μM) or vehicle (control group: vehicle, veh.) for 30 and 120 min. Results are expressed with the standard deviation; ( n = 21–28 slices per experimental group, from seven mice). Aβ, amyloid‐β; β‐tub, β‐tubulin
    Figure Legend Snippet: Effects of Aβ 1–42 on hippocampal G‐protein‐gated inwardly rectifying potassium channels (GirK) protein expression pattern in vitro. Western‐blot analysis of the (a) GIRK1 and (b) GIRK2 protein levels in hippocampus slices treated with Aβ 1–42 (0.5 μM) or vehicle (control group: vehicle, veh.) for 30 and 120 min. Results are expressed with the standard deviation; ( n = 21–28 slices per experimental group, from seven mice). Aβ, amyloid‐β; β‐tub, β‐tubulin

    Techniques Used: Expressing, In Vitro, Western Blot, Standard Deviation, Mouse Assay

    4) Product Images from "BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells"

    Article Title: BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1540-17.2018

    Timed delivery of BMP5/7 during in vitro DA differentiation causes an increase in the yield of mDA neurons. Schematic representation of the protocol used to differentiate iPSC and iNSC to DA neurons ( A ). Double Immunocytochemistry against TH and TUJ1 in iPSCs and iNSCs either in control or after 30 d of in vitro DA differentiation treated with BMP5/7 during the maturation phase ( B – E′ ). Double immunocytochemistry against TH and the DA progenitor marker LMX1A ( F – G′ ), the DA markers GIRK2 ( H ) and CALB ( I ), as well as the mature DA marker DAT ( J ) are shown. The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) and one iNSC line is twofold to threefold increased in cultures treated with BMP5/7 compared with untreated controls ( K ). Double immunocytochemistry against TH and TUJ1 in 2 iPSC lines either in control or in cultures treated with Noggin during the maturation phase is shown ( L , M ). The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) is 50% decreased after blocking the BMP pathway with Noggin compared with untreated controls ( N ). The percentage of TH + LMX1A + in iPSCs is increased in the treated BMP cultures compared with untreated controls ( O ). All staining and counting were analyzed after 30 d of DA in vitro differentiation. Two tailed unpaired Student, t test * p
    Figure Legend Snippet: Timed delivery of BMP5/7 during in vitro DA differentiation causes an increase in the yield of mDA neurons. Schematic representation of the protocol used to differentiate iPSC and iNSC to DA neurons ( A ). Double Immunocytochemistry against TH and TUJ1 in iPSCs and iNSCs either in control or after 30 d of in vitro DA differentiation treated with BMP5/7 during the maturation phase ( B – E′ ). Double immunocytochemistry against TH and the DA progenitor marker LMX1A ( F – G′ ), the DA markers GIRK2 ( H ) and CALB ( I ), as well as the mature DA marker DAT ( J ) are shown. The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) and one iNSC line is twofold to threefold increased in cultures treated with BMP5/7 compared with untreated controls ( K ). Double immunocytochemistry against TH and TUJ1 in 2 iPSC lines either in control or in cultures treated with Noggin during the maturation phase is shown ( L , M ). The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) is 50% decreased after blocking the BMP pathway with Noggin compared with untreated controls ( N ). The percentage of TH + LMX1A + in iPSCs is increased in the treated BMP cultures compared with untreated controls ( O ). All staining and counting were analyzed after 30 d of DA in vitro differentiation. Two tailed unpaired Student, t test * p

    Techniques Used: In Vitro, Multiple Displacement Amplification, Immunocytochemistry, Marker, Blocking Assay, Staining, Two Tailed Test

    In postnatal P0 Smad1 Nes mutants TH + SOX6 + and TH + GIRK2 + SN neurons are more affected than TH + CALB + VTA neurons. Coronal sections of WT and Smad1 Nes midbrains at P0 and P7 are represented. The number of TH + cells is significantly reduced in Smad1 Nes mutants ( A–A
    Figure Legend Snippet: In postnatal P0 Smad1 Nes mutants TH + SOX6 + and TH + GIRK2 + SN neurons are more affected than TH + CALB + VTA neurons. Coronal sections of WT and Smad1 Nes midbrains at P0 and P7 are represented. The number of TH + cells is significantly reduced in Smad1 Nes mutants ( A–A" ). The red nucleus visualized by POU4F1 expression does not show differences beetwen the genotypes ( B–B" ). The numbers of TH + LMX1A + ( C–C" ), TH + NURR1 + ( D–D" ), TH + EN1 + ( E–E" ) and TH + PITX3 + ( F–F" ) neurons were significantly reduced in Smad1 Nes mutants. The percentages of TH + SOX6 + ( G–G" ) and TH + GIRK2 + ( H–H" ) SN neurons of the total midbrain TH + neurons were reduced in Smad1 Nes mutants. Conversely, the TH + CALB + portion of the total TH + neurons did not show difference beetwen the genotypes ( I–I" ). Additionaly, TH + neurons ( J–J" ) as well as TH + LMX1A + ( J, J′, J′" ) and TH + NURR1 + ( K–K" ) neurons showed significant reduction in Smad1 Nes mutants at P7. Two tailed unpaired Student, t test * p

    Techniques Used: Expressing, Two Tailed Test

    5) Product Images from "BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells"

    Article Title: BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1540-17.2018

    Timed delivery of BMP5/7 during in vitro DA differentiation causes an increase in the yield of mDA neurons. Schematic representation of the protocol used to differentiate iPSC and iNSC to DA neurons ( A ). Double Immunocytochemistry against TH and TUJ1 in iPSCs and iNSCs either in control or after 30 d of in vitro DA differentiation treated with BMP5/7 during the maturation phase ( B – E′ ). Double immunocytochemistry against TH and the DA progenitor marker LMX1A ( F – G′ ), the DA markers GIRK2 ( H ) and CALB ( I ), as well as the mature DA marker DAT ( J ) are shown. The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) and one iNSC line is twofold to threefold increased in cultures treated with BMP5/7 compared with untreated controls ( K ). Double immunocytochemistry against TH and TUJ1 in 2 iPSC lines either in control or in cultures treated with Noggin during the maturation phase is shown ( L , M ). The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) is 50% decreased after blocking the BMP pathway with Noggin compared with untreated controls ( N ). The percentage of TH + LMX1A + in iPSCs is increased in the treated BMP cultures compared with untreated controls ( O ). All staining and counting were analyzed after 30 d of DA in vitro differentiation. Two tailed unpaired Student, t test * p
    Figure Legend Snippet: Timed delivery of BMP5/7 during in vitro DA differentiation causes an increase in the yield of mDA neurons. Schematic representation of the protocol used to differentiate iPSC and iNSC to DA neurons ( A ). Double Immunocytochemistry against TH and TUJ1 in iPSCs and iNSCs either in control or after 30 d of in vitro DA differentiation treated with BMP5/7 during the maturation phase ( B – E′ ). Double immunocytochemistry against TH and the DA progenitor marker LMX1A ( F – G′ ), the DA markers GIRK2 ( H ) and CALB ( I ), as well as the mature DA marker DAT ( J ) are shown. The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) and one iNSC line is twofold to threefold increased in cultures treated with BMP5/7 compared with untreated controls ( K ). Double immunocytochemistry against TH and TUJ1 in 2 iPSC lines either in control or in cultures treated with Noggin during the maturation phase is shown ( L , M ). The percentage of TH + TUJ1 + in two iPSC lines (iPSC #1 and #2) is 50% decreased after blocking the BMP pathway with Noggin compared with untreated controls ( N ). The percentage of TH + LMX1A + in iPSCs is increased in the treated BMP cultures compared with untreated controls ( O ). All staining and counting were analyzed after 30 d of DA in vitro differentiation. Two tailed unpaired Student, t test * p

    Techniques Used: In Vitro, Multiple Displacement Amplification, Immunocytochemistry, Marker, Blocking Assay, Staining, Two Tailed Test

    In postnatal P0 Smad1 Nes mutants TH + SOX6 + and TH + GIRK2 + SN neurons are more affected than TH + CALB + VTA neurons. Coronal sections of WT and Smad1 Nes midbrains at P0 and P7 are represented. The number of TH + cells is significantly reduced in Smad1 Nes mutants ( A–A
    Figure Legend Snippet: In postnatal P0 Smad1 Nes mutants TH + SOX6 + and TH + GIRK2 + SN neurons are more affected than TH + CALB + VTA neurons. Coronal sections of WT and Smad1 Nes midbrains at P0 and P7 are represented. The number of TH + cells is significantly reduced in Smad1 Nes mutants ( A–A" ). The red nucleus visualized by POU4F1 expression does not show differences beetwen the genotypes ( B–B" ). The numbers of TH + LMX1A + ( C–C" ), TH + NURR1 + ( D–D" ), TH + EN1 + ( E–E" ) and TH + PITX3 + ( F–F" ) neurons were significantly reduced in Smad1 Nes mutants. The percentages of TH + SOX6 + ( G–G" ) and TH + GIRK2 + ( H–H" ) SN neurons of the total midbrain TH + neurons were reduced in Smad1 Nes mutants. Conversely, the TH + CALB + portion of the total TH + neurons did not show difference beetwen the genotypes ( I–I" ). Additionaly, TH + neurons ( J–J" ) as well as TH + LMX1A + ( J, J′, J′" ) and TH + NURR1 + ( K–K" ) neurons showed significant reduction in Smad1 Nes mutants at P7. Two tailed unpaired Student, t test * p

    Techniques Used: Expressing, Two Tailed Test

    6) Product Images from "Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats"

    Article Title: Transplantation site influences the phenotypic differentiation of dopamine neurons in ventral mesencephalic grafts in Parkinsonian rats

    Journal: Experimental Neurology

    doi: 10.1016/j.expneurol.2017.01.010

    Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Figure Legend Snippet: Transplantation site influences distribution of A9- and A10-like dopamine neurons within VM grafts. Percentages of Girk2-ir/TH-ir and Calbindin-ir/TH-ir neurons in the periphery and in the centre of E12 (A) and E14 grafts (B) at 6 weeks post-transplantation. Note the decrease in the percentage of Girk2-ir/TH-ir neurons in the periphery of grafts in HPC compared to grafts in other brain regions. The presence of A10 dopamine innervation of the N.Acc significantly increased the percentage of A10-like neurons in the periphery of the graft compared to the graft core in E14 group. Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Techniques Used: Transplantation Assay

    Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p
    Figure Legend Snippet: Transplantation site influences A9-like dopamine neuron specification in VM grafts. Coronal sections through E12 VM grafts illustrating TH-ir (green) neurons co-expressing Girk2 (red: A–D) or Calbindin (red: E–H) in grafts in the dSTR (A, E), N.Acc (B, F), PFC (C, G) and HPC (D, H). Note the increase in the Girk2-ir/TH-ir neuron population in grafts in the dSTR compared to other grafts. (A′ and F′) High magnification images from (A and F), illustrating the co-localisation of Girk2 (A′) and Calbindin (F′) with TH and morphology of double labelled neurons within the transplant. (I) Total number of Girk2-ir/TH-ir neurons and (J) Calbindin-ir/TH-ir neurons within the grafts at each donor age and transplantation site. (K) Quantification of the proportion of Girk2-ir/TH-ir neurons and (L) Calbindin-ir/TH-ir neurons out of total TH-ir neurons within the grafts. The presence of targeted midbrain innervation of the transplantation site significantly increased the number and proportion of A9-like neurons in the grafts. Scale bars: 100 μm (A–H) and 25 μm (A′ and F′). Columns depict group means; error bars illustrate ± SEM; significance levels: *p

    Techniques Used: Transplantation Assay, Expressing

    7) Product Images from "Contribution of the respiratory network to rhythm and motor output revealed by modulation of GIRK channels, somatostatin and neurokinin-1 receptors"

    Article Title: Contribution of the respiratory network to rhythm and motor output revealed by modulation of GIRK channels, somatostatin and neurokinin-1 receptors

    Journal: Scientific Reports

    doi: 10.1038/srep32707

    SST decreased rhythmic breathing and genioglossus muscle amplitude in the wild-type and GIRK2 −/− mice. Microperfusion of SST (200 μM) into the preBötC ( a ) significantly increased breathing rate in wild-type mice ( b ) and in GIRK2 −/− mice. Mean data showed that breathing rate was decreased wild-type (n = 4) and GIRK2 −/− mice (n = 4, c ). Diaphragm muscle amplitude was not changed by SST either in wild-type nor in GIRK2 −/− mice ( d ). Genioglossus muscle amplitude was decreased by SST in wild-type, but not in GIRK2 −/− mice ( e ). *Indicate mean values significantly different from aCSF/baseline with P
    Figure Legend Snippet: SST decreased rhythmic breathing and genioglossus muscle amplitude in the wild-type and GIRK2 −/− mice. Microperfusion of SST (200 μM) into the preBötC ( a ) significantly increased breathing rate in wild-type mice ( b ) and in GIRK2 −/− mice. Mean data showed that breathing rate was decreased wild-type (n = 4) and GIRK2 −/− mice (n = 4, c ). Diaphragm muscle amplitude was not changed by SST either in wild-type nor in GIRK2 −/− mice ( d ). Genioglossus muscle amplitude was decreased by SST in wild-type, but not in GIRK2 −/− mice ( e ). *Indicate mean values significantly different from aCSF/baseline with P

    Techniques Used: Mouse Assay

    Activation of NK-1Rs increased rhythmic breathing and genioglossus muscle amplitude in the wild-type but not in the GIRK2 −/− mice. Microperfusion of the NK-1R agonist GR73632 (50 μM) into the preBötC ( a ) significantly increased breathing rate in wild-type mice ( b ), but not in GIRK2 −/− mice. Dots indicate breathing rate for each breath. Mean data showed that breathing rate was increased in wild-type mice (n = 4), but not GIRK2 −/− mice (n = 5, c ). Diaphragm muscle amplitude was not changed by GR73632 either in wild-type nor in GIRK2 −/− mice ( d ). Genioglossus muscle amplitude was increased by GR73632 in wild-type, but not in GIRK2 −/− mice ( e ). *Indicate mean values significantly different from aCSF/baseline with P
    Figure Legend Snippet: Activation of NK-1Rs increased rhythmic breathing and genioglossus muscle amplitude in the wild-type but not in the GIRK2 −/− mice. Microperfusion of the NK-1R agonist GR73632 (50 μM) into the preBötC ( a ) significantly increased breathing rate in wild-type mice ( b ), but not in GIRK2 −/− mice. Dots indicate breathing rate for each breath. Mean data showed that breathing rate was increased in wild-type mice (n = 4), but not GIRK2 −/− mice (n = 5, c ). Diaphragm muscle amplitude was not changed by GR73632 either in wild-type nor in GIRK2 −/− mice ( d ). Genioglossus muscle amplitude was increased by GR73632 in wild-type, but not in GIRK2 −/− mice ( e ). *Indicate mean values significantly different from aCSF/baseline with P

    Techniques Used: Activation Assay, Mouse Assay

    Expression of GIRK2 subunits and neurokinin-1 receptors in preBötC neurons of adult rats. In the region of the preBötC ventral to the nucleus ambiguus ( a ), GIRK2 subunits were expressed in somas (GIRK2 green, DAPI, blue , b ). Neurokinin-1 receptors were expressed in the preBötC region ( red , c ). All cells expressing NK-1Rs also expressed GIRK2 subunits (white arrow, d ). Magnification showed perinuclear expression of GIRK2 subunits in cells of the preBötC region ( e , f ). NK-1Rs were found in somas and co-expressed with GIRK2 subunits. Results are representative of three biological replicates. Scale bars 100, 50, and 10 μm for upper, middle and lower panels respectively. na, nucleus ambiguus. io, inferior olive.
    Figure Legend Snippet: Expression of GIRK2 subunits and neurokinin-1 receptors in preBötC neurons of adult rats. In the region of the preBötC ventral to the nucleus ambiguus ( a ), GIRK2 subunits were expressed in somas (GIRK2 green, DAPI, blue , b ). Neurokinin-1 receptors were expressed in the preBötC region ( red , c ). All cells expressing NK-1Rs also expressed GIRK2 subunits (white arrow, d ). Magnification showed perinuclear expression of GIRK2 subunits in cells of the preBötC region ( e , f ). NK-1Rs were found in somas and co-expressed with GIRK2 subunits. Results are representative of three biological replicates. Scale bars 100, 50, and 10 μm for upper, middle and lower panels respectively. na, nucleus ambiguus. io, inferior olive.

    Techniques Used: Expressing

    8) Product Images from "Foxa1 and Foxa2 Are Required for the Maintenance of Dopaminergic Properties in Ventral Midbrain Neurons at Late Embryonic Stages"

    Article Title: Foxa1 and Foxa2 Are Required for the Maintenance of Dopaminergic Properties in Ventral Midbrain Neurons at Late Embryonic Stages

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4774-12.2013

    SN mDA neurons are more affected than the VTA in the DAT cre/+ ;Foxa1/2;R26R YFP/+ cko mice. A , B , Markers specific to the SN, such as Girk2 were dramatically reduced in the mDA neurons of the DAT cre /+ ;Foxa1/2;R26R YFP /+ cko mice. C , D , Even the dopamine transporter (DAT) was almost lost in the absence of Foxa1/2. E–O , Markers of the VTA subregion, such as OTX2 ( E–H ), calbindin ( I–L ), and calretinin ( M–O ) exhibited only a small reduction in their expression in the DAT cre /+ ;Foxa1/2;R26R YFP /+ cko mice (MUT). Arrows in G and K highlight YFP+/TH− cells still expressing OTX2 and calbindin, respectively. * p
    Figure Legend Snippet: SN mDA neurons are more affected than the VTA in the DAT cre/+ ;Foxa1/2;R26R YFP/+ cko mice. A , B , Markers specific to the SN, such as Girk2 were dramatically reduced in the mDA neurons of the DAT cre /+ ;Foxa1/2;R26R YFP /+ cko mice. C , D , Even the dopamine transporter (DAT) was almost lost in the absence of Foxa1/2. E–O , Markers of the VTA subregion, such as OTX2 ( E–H ), calbindin ( I–L ), and calretinin ( M–O ) exhibited only a small reduction in their expression in the DAT cre /+ ;Foxa1/2;R26R YFP /+ cko mice (MUT). Arrows in G and K highlight YFP+/TH− cells still expressing OTX2 and calbindin, respectively. * p

    Techniques Used: Multiple Displacement Amplification, Mouse Assay, Expressing

    9) Product Images from "Development of histocompatible primate induced pluripotent stem cells for neural transplantation"

    Article Title: Development of histocompatible primate induced pluripotent stem cells for neural transplantation

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.662

    Analysis of neuronal grafts Confocal analysis of CM-iPSC-derived grafts, 16 weeks post-transplantation. (A) Staining for the microglial marker Iba1 showed the absence of activated microglial cells around the grafts. The insert shows high magnification of microglial cells with a resting phenotype. (B) Staining for the astrocytic marker GFAP revealed the absence of astrogliosis around the grafts. (C) Staining for the proliferation marker Ki-67 showing absence of proliferating cells 16 weeks after transplantation. (D–F) Confocal analysis of iPSC grafts showed that most grafts contained midbrain-like DA neurons. The grafted TH+ cells (red) were colabeled with antibodies against human NCAM (blue) (D–F), FOXA2 (green) (D–E), GIRK2 (blue) (E), Pitx3 (green) (F). (G) Confocal images showing TH+ neurons (red) in a representative graft co-expressing the calcium-binding protein calbindin (red). (H) Confocal images showing the localization of human syntaxin within the graft and in the host striatum. (I) Correlation between number of TH+ neurons and number of rotations (n=9; simple regression, r=0.885, r 2 =0.784, P=0.01). Scale bar: 100 µm (A–D), 50 µm (A–E), 20 µm (F–I).
    Figure Legend Snippet: Analysis of neuronal grafts Confocal analysis of CM-iPSC-derived grafts, 16 weeks post-transplantation. (A) Staining for the microglial marker Iba1 showed the absence of activated microglial cells around the grafts. The insert shows high magnification of microglial cells with a resting phenotype. (B) Staining for the astrocytic marker GFAP revealed the absence of astrogliosis around the grafts. (C) Staining for the proliferation marker Ki-67 showing absence of proliferating cells 16 weeks after transplantation. (D–F) Confocal analysis of iPSC grafts showed that most grafts contained midbrain-like DA neurons. The grafted TH+ cells (red) were colabeled with antibodies against human NCAM (blue) (D–F), FOXA2 (green) (D–E), GIRK2 (blue) (E), Pitx3 (green) (F). (G) Confocal images showing TH+ neurons (red) in a representative graft co-expressing the calcium-binding protein calbindin (red). (H) Confocal images showing the localization of human syntaxin within the graft and in the host striatum. (I) Correlation between number of TH+ neurons and number of rotations (n=9; simple regression, r=0.885, r 2 =0.784, P=0.01). Scale bar: 100 µm (A–D), 50 µm (A–E), 20 µm (F–I).

    Techniques Used: Derivative Assay, Transplantation Assay, Staining, Marker, Expressing, Binding Assay

    10) Product Images from "Morphine- and CaMKII dependent enhancement of GIRK channel signaling in hippocampal neurons"

    Article Title: Morphine- and CaMKII dependent enhancement of GIRK channel signaling in hippocampal neurons

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.2966-10.2010

    Morphine treatment increases 5-HT induced GIRK currents and Ba 2+ -sensitive basal but reduces GABA B receptor-activated GIRK currents Immunostaining of GIRK2 and PSD95 in untreated hippocampal neurons (10–14 DIV) or after ~20 h morphine treatment (100 µm). A–C ) Colocalization between GIRK2 and PSD95 increased with morphine, demonstrating a response in 11–14 DIV that was similar to 21 DIV neurons. C ) Zoom of dendrites from control and morphine-treated neurons. Scale bars: 10 and 2 µm. D–F ) Whole-cell patch-clamp recordings show changes in GIRK currents with morphine treatment. The 5-HT induced GIRK currents and the Ba 2+ -sensitive basal currents increased following 20 h morphine treatment. The GABA B -activated GIRK current decreased in morphine-treated neurons. Representative current-voltage plots show the macroscopic currents recorded in 20K, 20K + 1 mM Ba 2+ , 20K + 1 µm 5-HT, or 20K + 100 µm baclofen for control ( D ) and morphine ( E ) treated neurons. Arrow marks the equilibrium potential for potassium (E K ). F ) Bar graphs show mean current density (± SEM) for baclofen, 5-HT and Ba 2+ sensitive basal K + currents. *Student’s t-test for significance (P
    Figure Legend Snippet: Morphine treatment increases 5-HT induced GIRK currents and Ba 2+ -sensitive basal but reduces GABA B receptor-activated GIRK currents Immunostaining of GIRK2 and PSD95 in untreated hippocampal neurons (10–14 DIV) or after ~20 h morphine treatment (100 µm). A–C ) Colocalization between GIRK2 and PSD95 increased with morphine, demonstrating a response in 11–14 DIV that was similar to 21 DIV neurons. C ) Zoom of dendrites from control and morphine-treated neurons. Scale bars: 10 and 2 µm. D–F ) Whole-cell patch-clamp recordings show changes in GIRK currents with morphine treatment. The 5-HT induced GIRK currents and the Ba 2+ -sensitive basal currents increased following 20 h morphine treatment. The GABA B -activated GIRK current decreased in morphine-treated neurons. Representative current-voltage plots show the macroscopic currents recorded in 20K, 20K + 1 mM Ba 2+ , 20K + 1 µm 5-HT, or 20K + 100 µm baclofen for control ( D ) and morphine ( E ) treated neurons. Arrow marks the equilibrium potential for potassium (E K ). F ) Bar graphs show mean current density (± SEM) for baclofen, 5-HT and Ba 2+ sensitive basal K + currents. *Student’s t-test for significance (P

    Techniques Used: Immunostaining, Patch Clamp

    Proposed pathway for morphine-induced enhancement of GIRK signaling in hippocampal neurons A ) Inhibition of intracellular Ca 2+ with 50 µm BAPTA-AM prevents the morphine-induced colocalization of GIRK2 and PSD95 in 20 DIV hippocampal neurons (left graph). Exposure to the metabotropic glutamate receptor agonist ACPD (100 µm) for 20 h increases the colocalization ratio for GIRK2 and PSD95 in 20 DIV hippocampal neurons (right graph). B ) Schematic shows proposed signaling pathways for morphine and ACPD-dependent upregulation of GIRK2 in hippocampal neurons.
    Figure Legend Snippet: Proposed pathway for morphine-induced enhancement of GIRK signaling in hippocampal neurons A ) Inhibition of intracellular Ca 2+ with 50 µm BAPTA-AM prevents the morphine-induced colocalization of GIRK2 and PSD95 in 20 DIV hippocampal neurons (left graph). Exposure to the metabotropic glutamate receptor agonist ACPD (100 µm) for 20 h increases the colocalization ratio for GIRK2 and PSD95 in 20 DIV hippocampal neurons (right graph). B ) Schematic shows proposed signaling pathways for morphine and ACPD-dependent upregulation of GIRK2 in hippocampal neurons.

    Techniques Used: Inhibition

    Constitutively activate CaMKII mimics effects of morphine on GIRK signaling 20 DIV hippocampal neurons were infected with virus expressing constitutively active CaMKII (ΔCaMKII-GFP). A,B ) Representative images of dendrites 1-day after infection. Low power images show location of infected and uninfected dendrites immunostained for GIRK2 and PSD95 ( A ). Gray scale image shows GFP-channel. B ) Zoom of dendrites immunostained for GIRK2 and PSD95 from infected and uninfected neurons. Scale bars: 10 and 2 µm. C ) Whole-cell patch-clamp recording from representative neuron infected with ΔCaMKII-GFP (11–14 DIV). The response to baclofen and Ba 2+ are shown. Arrow indicates E K . D ) Bar graphs show mean (± SEM) amplitude for baclofen-induced ( left ) and Ba 2+ -sensitive basal K + currents ( right ) for uninfected and infected neurons. *Student’s t-test for significance (P
    Figure Legend Snippet: Constitutively activate CaMKII mimics effects of morphine on GIRK signaling 20 DIV hippocampal neurons were infected with virus expressing constitutively active CaMKII (ΔCaMKII-GFP). A,B ) Representative images of dendrites 1-day after infection. Low power images show location of infected and uninfected dendrites immunostained for GIRK2 and PSD95 ( A ). Gray scale image shows GFP-channel. B ) Zoom of dendrites immunostained for GIRK2 and PSD95 from infected and uninfected neurons. Scale bars: 10 and 2 µm. C ) Whole-cell patch-clamp recording from representative neuron infected with ΔCaMKII-GFP (11–14 DIV). The response to baclofen and Ba 2+ are shown. Arrow indicates E K . D ) Bar graphs show mean (± SEM) amplitude for baclofen-induced ( left ) and Ba 2+ -sensitive basal K + currents ( right ) for uninfected and infected neurons. *Student’s t-test for significance (P

    Techniques Used: Infection, Expressing, Patch Clamp

    Morphine increases colocalization of GIRK2 but not PSD95 in actin-filled dendritic spines Dissociated hippocampal cultures were infected with Sindbis actin-YFP virus at 20 DIV and were either untreated (control) or exposed to morphine (100 µm) for ~20 h. A,B ) Images show examples of actin-YFP infected dendrites immunostained for GIRK2 or PSD95 in control and morphine-treated cultures at 21DIV. Morphine significantly increased colocalization of GIRK2 with actin-YFP but did not appear to alter colocalization of PSD95 with actin-YFP. Scale bar: 5 µm. C ) Bar graphs show average colocalization for GIRK2/actin-YFP and PSD95/actin-YFP in untreated (control) or morphine-treated neurons. *Student’s t-test for significance (P
    Figure Legend Snippet: Morphine increases colocalization of GIRK2 but not PSD95 in actin-filled dendritic spines Dissociated hippocampal cultures were infected with Sindbis actin-YFP virus at 20 DIV and were either untreated (control) or exposed to morphine (100 µm) for ~20 h. A,B ) Images show examples of actin-YFP infected dendrites immunostained for GIRK2 or PSD95 in control and morphine-treated cultures at 21DIV. Morphine significantly increased colocalization of GIRK2 with actin-YFP but did not appear to alter colocalization of PSD95 with actin-YFP. Scale bar: 5 µm. C ) Bar graphs show average colocalization for GIRK2/actin-YFP and PSD95/actin-YFP in untreated (control) or morphine-treated neurons. *Student’s t-test for significance (P

    Techniques Used: Infection

    Ultrastructural analyses reveal morphine increases expression of GIRK2 in dendritic spines of hippocampal neurons The effect of morphine on the expression of GIRK2 was studied at the electron microscopic (EM) level using pre-embedding immunogold method. EM micrographs show immunoreactivity for GIRK2 in control cultured hippocampal neurons ( A ) and after 20h treatment with morphine ( B ). In control neurons ( A ), immunogold particles for GIRK2 were detected in dendritic shafts (Den) of cultured cells, both along the plasma membrane (blue arrows) and at intracellular sites (crossed arrows), and especially along the plasma membrane of dendritic spines (s) (blue arrows) establishing asymmetrical synapses with axon terminal boutons (b). After 20 h treatment with morphine ( b ), immunoreactivity for GIRK2 increased along the plasma membrane of dendritic shafts (Den) (blue arrows), dendritic spines (s) (blue arrows) and endoplasmic reticulum in soma, as well as at intracellular sites (crossed arrows). Scale bars: 0.5 µm. C ) Bar graph shows the mean relative percentage of GIRK2 immunoreactivity detected in the soma, shaft and spines of intracellular and plasma membrane compartments for control and morphine (n=4 cover slips, Student’s t-test , P
    Figure Legend Snippet: Ultrastructural analyses reveal morphine increases expression of GIRK2 in dendritic spines of hippocampal neurons The effect of morphine on the expression of GIRK2 was studied at the electron microscopic (EM) level using pre-embedding immunogold method. EM micrographs show immunoreactivity for GIRK2 in control cultured hippocampal neurons ( A ) and after 20h treatment with morphine ( B ). In control neurons ( A ), immunogold particles for GIRK2 were detected in dendritic shafts (Den) of cultured cells, both along the plasma membrane (blue arrows) and at intracellular sites (crossed arrows), and especially along the plasma membrane of dendritic spines (s) (blue arrows) establishing asymmetrical synapses with axon terminal boutons (b). After 20 h treatment with morphine ( b ), immunoreactivity for GIRK2 increased along the plasma membrane of dendritic shafts (Den) (blue arrows), dendritic spines (s) (blue arrows) and endoplasmic reticulum in soma, as well as at intracellular sites (crossed arrows). Scale bars: 0.5 µm. C ) Bar graph shows the mean relative percentage of GIRK2 immunoreactivity detected in the soma, shaft and spines of intracellular and plasma membrane compartments for control and morphine (n=4 cover slips, Student’s t-test , P

    Techniques Used: Expressing, Cell Culture

    Morphine-dependent increase in colocalization of GIRK2 and PSD95 requires activated CaMKII 20 DIV hippocampal neurons were exposed to 100 µm morphine for ~18 h and during the last 2 h were either treated with the selective CaMKII inhibitor KN-93 (10 µm) and morphine, or with the inactive peptide KN-92 (10 µm) and morphine. A–C ) Representative images show immunostaining for GIRK2 and PSD95. KN-93 but not KN-92 or the vehicle DMSO prevents the morphine-dependent increase in GIRK2 colocalization with PSD95. Scale bar: 10 µm. D ) Bar graph shows the mean colocalization ratio (± SEM) with the N indicating the number of dendritic regions analyzed. *One-way ANOVA followed by Bonferroni post hoc test for significance (P
    Figure Legend Snippet: Morphine-dependent increase in colocalization of GIRK2 and PSD95 requires activated CaMKII 20 DIV hippocampal neurons were exposed to 100 µm morphine for ~18 h and during the last 2 h were either treated with the selective CaMKII inhibitor KN-93 (10 µm) and morphine, or with the inactive peptide KN-92 (10 µm) and morphine. A–C ) Representative images show immunostaining for GIRK2 and PSD95. KN-93 but not KN-92 or the vehicle DMSO prevents the morphine-dependent increase in GIRK2 colocalization with PSD95. Scale bar: 10 µm. D ) Bar graph shows the mean colocalization ratio (± SEM) with the N indicating the number of dendritic regions analyzed. *One-way ANOVA followed by Bonferroni post hoc test for significance (P

    Techniques Used: Immunostaining

    GIRK2 is predominantly expressed in the dendritic shaft of mature cultured hippocampal neurons and overlaps little with dendritic spine markers (PSD95 and NMDA receptors) 21 DIV hippocampal neurons were immunostained for GIRK2 and a spine marker, NMDA receptors ( A ) or PSD95 ( B ). Little colocalization of GIRK2 with the dendritic spine markers was observed, suggesting GIRK channels are expressed mainly on the dendritic shaft. C ) Comparison of PSD95 and NMDA receptors. As expected, PSD95 and NMDA exhibited high degree of colocalization (yellow in merged image). The extent of colocalization was determined by deriving the Pearson’s correlation coefficient for red-green images. The Pearson’s coefficient is shown for each image. The zooms show magnification of a single dendritic branch. D ) Bar graph shows the average (± SEM) Pearson’s coefficient for GIRK2/NMDA, GIRK2/PSD95 and NMDA/PSD95 (with the number of dendritic fields indicated).
    Figure Legend Snippet: GIRK2 is predominantly expressed in the dendritic shaft of mature cultured hippocampal neurons and overlaps little with dendritic spine markers (PSD95 and NMDA receptors) 21 DIV hippocampal neurons were immunostained for GIRK2 and a spine marker, NMDA receptors ( A ) or PSD95 ( B ). Little colocalization of GIRK2 with the dendritic spine markers was observed, suggesting GIRK channels are expressed mainly on the dendritic shaft. C ) Comparison of PSD95 and NMDA receptors. As expected, PSD95 and NMDA exhibited high degree of colocalization (yellow in merged image). The extent of colocalization was determined by deriving the Pearson’s correlation coefficient for red-green images. The Pearson’s coefficient is shown for each image. The zooms show magnification of a single dendritic branch. D ) Bar graph shows the average (± SEM) Pearson’s coefficient for GIRK2/NMDA, GIRK2/PSD95 and NMDA/PSD95 (with the number of dendritic fields indicated).

    Techniques Used: Cell Culture, Marker

    11) Product Images from "The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease"

    Article Title: The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease

    Journal: Brain

    doi: 10.1093/brain/awp328

    Subtype analysis of dopamine neurons in Pitx3 WT/GFP and Pitx3 GFP/GFP grafts. Immunohistochemistry for GFP (green), GIRK2 (red) and Calbindin (blue) in coronal sections through the striatum of representative animals from the Pitx3 WT/GFP ( A ) and Pitx3 GFP/GFP ( B ) groups, 12 weeks after transplantation. The boxed areas in the main panels are shown in greater detail as individual colour channels on the left. The Pitx3 WT/GFP grafts contained a mix of GFP + midbrain dopamine subtypes including GIRK2 + /Calbindin − (arrows), GIRK2 − /Calbindin + (filled arrowheads) and GIRK2 + /Calbindin + (empty arrowheads) neurons. The Pitx3 GFP/GFP grafts were dominated by the GIRK2 − /Calbindin + cell type (filled arrowheads) and contained few GIRK2 + cells (not shown). Quantification of GIRK2 + and Calbindin + GFP expressing neurons in all grafted animals confirmed that there was a substantial difference in the midbrain dopamine subtype composition between the two graft types, with Pitx3 WT/GFP grafts ( n = 8; open bars) containing predominately the GIRK2 + /Calbindin − subtype and Pitx3 GFP/GFP ( n = 7; filled bars) grafts containing mainly GIRK2 − /Calbindin + cells. Scale: 200 µm.
    Figure Legend Snippet: Subtype analysis of dopamine neurons in Pitx3 WT/GFP and Pitx3 GFP/GFP grafts. Immunohistochemistry for GFP (green), GIRK2 (red) and Calbindin (blue) in coronal sections through the striatum of representative animals from the Pitx3 WT/GFP ( A ) and Pitx3 GFP/GFP ( B ) groups, 12 weeks after transplantation. The boxed areas in the main panels are shown in greater detail as individual colour channels on the left. The Pitx3 WT/GFP grafts contained a mix of GFP + midbrain dopamine subtypes including GIRK2 + /Calbindin − (arrows), GIRK2 − /Calbindin + (filled arrowheads) and GIRK2 + /Calbindin + (empty arrowheads) neurons. The Pitx3 GFP/GFP grafts were dominated by the GIRK2 − /Calbindin + cell type (filled arrowheads) and contained few GIRK2 + cells (not shown). Quantification of GIRK2 + and Calbindin + GFP expressing neurons in all grafted animals confirmed that there was a substantial difference in the midbrain dopamine subtype composition between the two graft types, with Pitx3 WT/GFP grafts ( n = 8; open bars) containing predominately the GIRK2 + /Calbindin − subtype and Pitx3 GFP/GFP ( n = 7; filled bars) grafts containing mainly GIRK2 − /Calbindin + cells. Scale: 200 µm.

    Techniques Used: Immunohistochemistry, Transplantation Assay, Expressing

    Subtype-specific pattern of dopamine neuronal cell loss in Pitx3 knockouts. Immunohistochemistry for GFP (green; A , B , G , H ), GIRK2 (red; C , D , G , H ) and Calbindin (blue; E , F , G , H ) in the adult Pitx3 WT/GFP ( A , C , E , G ) and Pitx3 GFP/GFP ( B , D , F , H ) brain. In the Pitx3 GFP/GFP midbrain there was a substantial loss of the GIRK2 + /GFP + midbrain dopamine neurons throughout the substantia nigra pars compacta (arrows in C and D indicate the substantia nigra pars compacta). A population of GIRK2 + midbrain dopamine neurons residing in the dorsolateral part of the ventral tegmental area (arrowhead in C , D ) appeared to be less affected in the Pitx3 knockout animals. The Calbindin + /GFP + midbrain dopamine population was also left relatively intact in the Pitx3 GFP/GFP midbrain ( E , F ). Scale: 200 µm.
    Figure Legend Snippet: Subtype-specific pattern of dopamine neuronal cell loss in Pitx3 knockouts. Immunohistochemistry for GFP (green; A , B , G , H ), GIRK2 (red; C , D , G , H ) and Calbindin (blue; E , F , G , H ) in the adult Pitx3 WT/GFP ( A , C , E , G ) and Pitx3 GFP/GFP ( B , D , F , H ) brain. In the Pitx3 GFP/GFP midbrain there was a substantial loss of the GIRK2 + /GFP + midbrain dopamine neurons throughout the substantia nigra pars compacta (arrows in C and D indicate the substantia nigra pars compacta). A population of GIRK2 + midbrain dopamine neurons residing in the dorsolateral part of the ventral tegmental area (arrowhead in C , D ) appeared to be less affected in the Pitx3 knockout animals. The Calbindin + /GFP + midbrain dopamine population was also left relatively intact in the Pitx3 GFP/GFP midbrain ( E , F ). Scale: 200 µm.

    Techniques Used: Immunohistochemistry, Knock-Out

    12) Product Images from "Spinal μ-Opioid Receptor-Expressing Dorsal Horn Neurons: Role in Nociception and Morphine Antinociception"

    Article Title: Spinal μ-Opioid Receptor-Expressing Dorsal Horn Neurons: Role in Nociception and Morphine Antinociception

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4452-07.2008

    Anatomic effects of intrathecal Derm-sap as illustrated by representative photomicrographs of lumbar enlargement dorsal horn sections. A , B , Sections were stained for MOR1 30 d after lumbar intrathecal PBS ( A ) or 500 ng of Derm-sap ( B ) in Long–Evans female rats. Note the decreased intensity of staining in lamina II after Derm-sap. D , E , Unlike MOR1, no changes in MOR1C staining were observed after intrathecal PBS ( D ) or Derm-sap ( E ) in the same rats. G , H , Derm-sap ( H ) shows decreased GIRK2 staining compared with PBS ( G ), most striking medially, in the same rats. C , F , I , Bar graphs representing densitometry of L4 dorsal horn staining for MOR1 ( C ; * p
    Figure Legend Snippet: Anatomic effects of intrathecal Derm-sap as illustrated by representative photomicrographs of lumbar enlargement dorsal horn sections. A , B , Sections were stained for MOR1 30 d after lumbar intrathecal PBS ( A ) or 500 ng of Derm-sap ( B ) in Long–Evans female rats. Note the decreased intensity of staining in lamina II after Derm-sap. D , E , Unlike MOR1, no changes in MOR1C staining were observed after intrathecal PBS ( D ) or Derm-sap ( E ) in the same rats. G , H , Derm-sap ( H ) shows decreased GIRK2 staining compared with PBS ( G ), most striking medially, in the same rats. C , F , I , Bar graphs representing densitometry of L4 dorsal horn staining for MOR1 ( C ; * p

    Techniques Used: Staining

    13) Product Images from "Identification of Dopaminergic Neurons of Nigral and Ventral Tegmental Area Subtypes in Grafts of Fetal Ventral Mesencephalon Based on Cell Morphology, Protein Expression, and Efferent Projections"

    Article Title: Identification of Dopaminergic Neurons of Nigral and Ventral Tegmental Area Subtypes in Grafts of Fetal Ventral Mesencephalon Based on Cell Morphology, Protein Expression, and Efferent Projections

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1676-05.2005

    Calbindin and Girk2 expression in dopaminergic neurons in the adult mouse mesencephalon. Confocal images show the distribution of calbindin ( A-C ) and Girk2 ( D-E ) expressing cells within dopaminergic neurons of the substantia nigra from an adult mouse. Note that the calbindin ( B ) and Girk2 ( C ) expression domains appear distinctly nonoverlapping: calbindin is expressed predominately in the VTA and also in SN pars lateralis ( C ), whereas Girk2 expression is primarily restricted to the SNpc ( F ). i-v , High-magnification images showing the expression of calbindin and Girk2 within TH + neurons of the VTA ( i , ii ), SNpc ( iii , iv ), and SN pars lateralis ( v ). The positions from which these images were taken are indicated by boxed areas in C and F . The dashed-boxed areas in A and B denote a small group of calbindin + /TH + cells consistently seen in a dorsal region of the SNpc. Calbindin and Girk2 expression are not restricted to dopaminergic neurons and are found in many TH - cells throughout the brain: note, for example, the prominent Girk2 expression in the red nucleus (RN). Scale bars: A-F , 500 μm; i-v , 50 μm.
    Figure Legend Snippet: Calbindin and Girk2 expression in dopaminergic neurons in the adult mouse mesencephalon. Confocal images show the distribution of calbindin ( A-C ) and Girk2 ( D-E ) expressing cells within dopaminergic neurons of the substantia nigra from an adult mouse. Note that the calbindin ( B ) and Girk2 ( C ) expression domains appear distinctly nonoverlapping: calbindin is expressed predominately in the VTA and also in SN pars lateralis ( C ), whereas Girk2 expression is primarily restricted to the SNpc ( F ). i-v , High-magnification images showing the expression of calbindin and Girk2 within TH + neurons of the VTA ( i , ii ), SNpc ( iii , iv ), and SN pars lateralis ( v ). The positions from which these images were taken are indicated by boxed areas in C and F . The dashed-boxed areas in A and B denote a small group of calbindin + /TH + cells consistently seen in a dorsal region of the SNpc. Calbindin and Girk2 expression are not restricted to dopaminergic neurons and are found in many TH - cells throughout the brain: note, for example, the prominent Girk2 expression in the red nucleus (RN). Scale bars: A-F , 500 μm; i-v , 50 μm.

    Techniques Used: Expressing

    Calbindin and Girk2 expression in dopaminergic cells of VM grafts. Confocal immunohistochemistry for GFP ( A ), Girk2 ( B ), and calbindin ( C ). Six weeks after transplantation into intact or 6-OHDA-lesioned adult rats, all VM grafts contained GFP + neurons expressing Girk2 or calbindin ( D ). The example shown here is from an intact adult recipient. Girk2/GFP coexpressing cells were positioned predominately in the periphery of the graft, whereas the calbindin/GFP coexpressing cells were located mainly in the center. The boxed areas in A-D incorporate a peripheral and more central aspect of the graft and are shown at higher magnification ( F-I ). As is the case in the adult SN, the grafts contained also many nondopaminergic (GFP - ) calbindin- and Girk2-expressing cells ( E , from dashed box in D ). Quantitative analysis ( J ) showed that the peripheral regions of the graft contained significantly more Girk2 + /GFP + cells and the central regions significantly more calbindin + /GFP + cells. Scale bars: A-D , 200 μm; F-I , 50 μm. Student's t test, * p
    Figure Legend Snippet: Calbindin and Girk2 expression in dopaminergic cells of VM grafts. Confocal immunohistochemistry for GFP ( A ), Girk2 ( B ), and calbindin ( C ). Six weeks after transplantation into intact or 6-OHDA-lesioned adult rats, all VM grafts contained GFP + neurons expressing Girk2 or calbindin ( D ). The example shown here is from an intact adult recipient. Girk2/GFP coexpressing cells were positioned predominately in the periphery of the graft, whereas the calbindin/GFP coexpressing cells were located mainly in the center. The boxed areas in A-D incorporate a peripheral and more central aspect of the graft and are shown at higher magnification ( F-I ). As is the case in the adult SN, the grafts contained also many nondopaminergic (GFP - ) calbindin- and Girk2-expressing cells ( E , from dashed box in D ). Quantitative analysis ( J ) showed that the peripheral regions of the graft contained significantly more Girk2 + /GFP + cells and the central regions significantly more calbindin + /GFP + cells. Scale bars: A-D , 200 μm; F-I , 50 μm. Student's t test, * p

    Techniques Used: Expressing, Immunohistochemistry, Transplantation Assay

    Retrograde tracing from dorsolateral striatum in VM grafted neonates. Six weeks after transplantation of wild-type VM tissue in 6-OHDA-lesioned neonates, CTB prelabeled with an Alexa-555 fluorophore was injected into the dorsolateral striatum. Immunohistochemistry for TH ( A ) and Girk2 ( C ) shows that many retrogradely labeled CTB + cells ( B ) in the VM grafts expressed both of these proteins ( D ). Arrowheads in A-D identify the dopaminergic (TH + ) cells that had incorporated the CTB tracer, located in the periphery of the graft. The boxed area is shown at higher magnification ( E-H ). In addition to TH + cells (arrowhead), the CTB tracer was taken up by a number of TH - cells (arrow) lying within the VM grafts ( E-H ). CTB staining resulting from passive diffusion of the tracer is illustrated at the injection site in the dorsolateral striatum ( I ) and at the level of the graft ( J ). g, Graft; v, ventricle. Scale bars: A-D , 200 μm; E-H , 50 μm; I , J , 500 μm.
    Figure Legend Snippet: Retrograde tracing from dorsolateral striatum in VM grafted neonates. Six weeks after transplantation of wild-type VM tissue in 6-OHDA-lesioned neonates, CTB prelabeled with an Alexa-555 fluorophore was injected into the dorsolateral striatum. Immunohistochemistry for TH ( A ) and Girk2 ( C ) shows that many retrogradely labeled CTB + cells ( B ) in the VM grafts expressed both of these proteins ( D ). Arrowheads in A-D identify the dopaminergic (TH + ) cells that had incorporated the CTB tracer, located in the periphery of the graft. The boxed area is shown at higher magnification ( E-H ). In addition to TH + cells (arrowhead), the CTB tracer was taken up by a number of TH - cells (arrow) lying within the VM grafts ( E-H ). CTB staining resulting from passive diffusion of the tracer is illustrated at the injection site in the dorsolateral striatum ( I ) and at the level of the graft ( J ). g, Graft; v, ventricle. Scale bars: A-D , 200 μm; E-H , 50 μm; I , J , 500 μm.

    Techniques Used: Retrograde Tracing, Transplantation Assay, CtB Assay, Injection, Immunohistochemistry, Labeling, Staining, Diffusion-based Assay

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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