anti girk2 kir3 2 antibody  (Alomone Labs)


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    Alomone Labs anti girk2 kir3 2 antibody
    Validation of <t>GIRK2</t> 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 <t>antibody</t> (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
    Anti Girk2 Kir3 2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons"

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

    Journal: bioRxiv

    doi: 10.1101/2022.05.24.493086

    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
    Figure Legend 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

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: Over Expression

    2) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    3) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    4) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    5) 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

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

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

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2018.01.002

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

    Techniques Used: Expressing, Western Blot, Quantitation Assay

    7) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    8) 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

    9) 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

    10) Product Images from "Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells"

    Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

    Journal: bioRxiv

    doi: 10.1101/2021.09.28.462222

    Generation of functional ventral midbrain DA neurons in vitro. a , Schematic diagram of the long-term (62 DIV) neuronal differentiation protocol. b , Representative immunohistochemical images of TH/FOXA2, TH/GIRK2, and TH/CALB1 costaining in H9 and 4X cells treated with 1 µM GSK3i on 83 DIV. Scale bars, 50 µm. c , Representative immunohistochemical analysis of COL3A1 and COL1A1 expression. Many H9 cells were double positive for COL3A1 and COL1A1, but no 4X cells were positive for COL3A1 or COL1A1. DAPI was used as a nuclear stain. Scale bars, 20 µm. d , Phase contrast image of a patched 4X neuron during whole-cell recording. Scale bar, 10 µm. e , Representative response (top trace) to a depolarizing current injection (bottom trace) showing firing of repetitive action potentials. f , Example of spontaneous firing at a resting membrane potential of -45 mV showing burst-like events. Overshooting spikes occurred in groups interspersed by periods of subthreshold membrane oscillation. g , Frequency distri bution of spontaneous cell firing showing firing frequencies ranging between 1 and 5 Hz (n = 16 cells). h , Dopamine content (normalized to the protein concentration) in 4X and H9 cells at 79 DIV, as measured by HPLC. The data are presented as the mean ± SD; n= 3. An unpaired t-test was used to compare groups. **P
    Figure Legend Snippet: Generation of functional ventral midbrain DA neurons in vitro. a , Schematic diagram of the long-term (62 DIV) neuronal differentiation protocol. b , Representative immunohistochemical images of TH/FOXA2, TH/GIRK2, and TH/CALB1 costaining in H9 and 4X cells treated with 1 µM GSK3i on 83 DIV. Scale bars, 50 µm. c , Representative immunohistochemical analysis of COL3A1 and COL1A1 expression. Many H9 cells were double positive for COL3A1 and COL1A1, but no 4X cells were positive for COL3A1 or COL1A1. DAPI was used as a nuclear stain. Scale bars, 20 µm. d , Phase contrast image of a patched 4X neuron during whole-cell recording. Scale bar, 10 µm. e , Representative response (top trace) to a depolarizing current injection (bottom trace) showing firing of repetitive action potentials. f , Example of spontaneous firing at a resting membrane potential of -45 mV showing burst-like events. Overshooting spikes occurred in groups interspersed by periods of subthreshold membrane oscillation. g , Frequency distri bution of spontaneous cell firing showing firing frequencies ranging between 1 and 5 Hz (n = 16 cells). h , Dopamine content (normalized to the protein concentration) in 4X and H9 cells at 79 DIV, as measured by HPLC. The data are presented as the mean ± SD; n= 3. An unpaired t-test was used to compare groups. **P

    Techniques Used: Functional Assay, In Vitro, Immunohistochemistry, Expressing, Staining, Injection, Protein Concentration, High Performance Liquid Chromatography

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

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

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2017.00455

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

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

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

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

    12) 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

    13) 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

    14) Product Images from "GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner"

    Article Title: GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5051-14.2015

    Constitutive Girk subunit ablation and morphine-induced motor activity. The total distance traveled by adult male wild-type (white; n = 26/dose), Girk1 −/− (gray; n = 18/dose), and Girk2 −/− (black; n = 7/dose) mice during a 60 min test following systemic administration of saline (0) or morphine (3, 10, and 30 mg/kg, i.p.) is shown. Main effects of genotype ( F (2,48) = 12.8, p
    Figure Legend Snippet: Constitutive Girk subunit ablation and morphine-induced motor activity. The total distance traveled by adult male wild-type (white; n = 26/dose), Girk1 −/− (gray; n = 18/dose), and Girk2 −/− (black; n = 7/dose) mice during a 60 min test following systemic administration of saline (0) or morphine (3, 10, and 30 mg/kg, i.p.) is shown. Main effects of genotype ( F (2,48) = 12.8, p

    Techniques Used: Activity Assay, Mouse Assay

    Generation of Girk2 flox/flox mice. A , Depiction of the mouse Girk2/Kcnj6 ). Although several alternative splice variants have been identified, using two distinct translation initiation codons (ATG) and four distinct translation stop codons (TGA), all known variants contain exon 4. Shaded regions of exons denote protein-coding sequences. Some exons (1, 4, and 6) contain internal splice acceptor sites, denoted by vertical lines. B ). The region encoded by exon 4, which includes most of the N terminus (NT), both membrane-spanning domains and extracellular loops, the pore domain, and much of the C terminus (CT), is denoted by arrows. C , The Girk2 targeting strategy included engineering a loxP site just upstream (214 bp) of exon 4 and incorporating a NEO resistance cassette containing Flp recognition target (FRT) sites and a second loxP site; this cassette was positioned 795 bp downstream from exon 4. In total, the target region spanned 1.93 Kbp and was flanked by a 5′ homology arm (extending ∼5.4 Kbp upstream from exon 4) and a 3′ homology arm (an ∼7 Kbp fragment found immediately downstream from the target region). The NEO cassette was removed by crossing selected chimeras with C57BL/6N Flp deleter mice, yielding the Girk2 flox allele. D , Tissue from the cortex ( n = 9–10/genotype; t (17) = 0.09, p = 0.9), hippocampus ( n = 6–7/genotype; t 11 = 0.4, p = 0.7), and cerebellum ( n = 7–8/genotype; t (13) = 1.3, p = 0.2) of wild-type and Girk2 flox/flox (f/f) mice was evaluated for GIRK2 mRNA levels using quantitative RT-PCR. E , GIRK2 protein immunoreactivity (ir) relative to β-actin in samples from the cortex ( n = 6–7/genotype; t (11) = 0.4, p = 0.7), hippocampus ( n = 5–7/genotype; t (10) = 0.05, p = 1.0), and cerebellum ( n = 4–7/genotype; t (9) = 0.6, p = 0.6) of wild-type and Girk2 flox/flox (f/f) mice was assessed using quantitative immunoblotting. The level of GIRK2, which is seen as a doublet (top), was compared to the level of β-actin control (bottom) in each sample. Gel images of GIRK2 and β-actin were cropped and aligned with a molecular weight scale showing the markers for 52 and 38 kDA.
    Figure Legend Snippet: Generation of Girk2 flox/flox mice. A , Depiction of the mouse Girk2/Kcnj6 ). Although several alternative splice variants have been identified, using two distinct translation initiation codons (ATG) and four distinct translation stop codons (TGA), all known variants contain exon 4. Shaded regions of exons denote protein-coding sequences. Some exons (1, 4, and 6) contain internal splice acceptor sites, denoted by vertical lines. B ). The region encoded by exon 4, which includes most of the N terminus (NT), both membrane-spanning domains and extracellular loops, the pore domain, and much of the C terminus (CT), is denoted by arrows. C , The Girk2 targeting strategy included engineering a loxP site just upstream (214 bp) of exon 4 and incorporating a NEO resistance cassette containing Flp recognition target (FRT) sites and a second loxP site; this cassette was positioned 795 bp downstream from exon 4. In total, the target region spanned 1.93 Kbp and was flanked by a 5′ homology arm (extending ∼5.4 Kbp upstream from exon 4) and a 3′ homology arm (an ∼7 Kbp fragment found immediately downstream from the target region). The NEO cassette was removed by crossing selected chimeras with C57BL/6N Flp deleter mice, yielding the Girk2 flox allele. D , Tissue from the cortex ( n = 9–10/genotype; t (17) = 0.09, p = 0.9), hippocampus ( n = 6–7/genotype; t 11 = 0.4, p = 0.7), and cerebellum ( n = 7–8/genotype; t (13) = 1.3, p = 0.2) of wild-type and Girk2 flox/flox (f/f) mice was evaluated for GIRK2 mRNA levels using quantitative RT-PCR. E , GIRK2 protein immunoreactivity (ir) relative to β-actin in samples from the cortex ( n = 6–7/genotype; t (11) = 0.4, p = 0.7), hippocampus ( n = 5–7/genotype; t (10) = 0.05, p = 1.0), and cerebellum ( n = 4–7/genotype; t (9) = 0.6, p = 0.6) of wild-type and Girk2 flox/flox (f/f) mice was assessed using quantitative immunoblotting. The level of GIRK2, which is seen as a doublet (top), was compared to the level of β-actin control (bottom) in each sample. Gel images of GIRK2 and β-actin were cropped and aligned with a molecular weight scale showing the markers for 52 and 38 kDA.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Molecular Weight

    15) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

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

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

    Journal: eNeuro

    doi: 10.1523/ENEURO.0036-19.2019

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

    Techniques Used: Expressing, Staining

    17) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    18) 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

    19) Product Images from "Regulator of G Protein Signaling 6 (RGS6) Protein Ensures Coordination of Motor Movement by Modulating GABAB Receptor Signaling"

    Article Title: Regulator of G Protein Signaling 6 (RGS6) Protein Ensures Coordination of Motor Movement by Modulating GABAB Receptor Signaling

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.297218

    Localization of RGS6 and different components of the GABA B R-GIRK signaling axis in mouse cerebellum. A , genetic ablation of RGS6 does not change expression of GABA B R2 or Gα i3 in cerebellum or cerebrum by Western blot. Actin serves as a loading control. B , co-immunostaining of RGS6 with Gβ 5 , GABA B R2, GIRK1, GIRK2, and in cerebellar sections (5 μm) from WT mice reveals expression of all proteins in cerebellar granule layer ( scale bar = 100 μm; white boxes , regions shown in enlarged merged images ; white arrows , regions of granule cell specific protein enrichment).
    Figure Legend Snippet: Localization of RGS6 and different components of the GABA B R-GIRK signaling axis in mouse cerebellum. A , genetic ablation of RGS6 does not change expression of GABA B R2 or Gα i3 in cerebellum or cerebrum by Western blot. Actin serves as a loading control. B , co-immunostaining of RGS6 with Gβ 5 , GABA B R2, GIRK1, GIRK2, and in cerebellar sections (5 μm) from WT mice reveals expression of all proteins in cerebellar granule layer ( scale bar = 100 μm; white boxes , regions shown in enlarged merged images ; white arrows , regions of granule cell specific protein enrichment).

    Techniques Used: Expressing, Western Blot, Immunostaining, Mouse Assay, Protein Enrichment

    Native expression of RGS6, Gβ 5 , GABA B R2, GIRK1, and GIRK2 in isolated CGNs. A , RGS6 immunostaining is localized to the soma and neurites of CGNs from WT mice and absent in RGS6 −/− mice. B , co-immunostaining of RGS6 with Gβ 5 , GABA B R2, GIRK1, and GIRK2 in CGNs from WT mice ( scale bar = 100 μm; white arrows , regions of overlapping expression of RGS6 with indicated protein).
    Figure Legend Snippet: Native expression of RGS6, Gβ 5 , GABA B R2, GIRK1, and GIRK2 in isolated CGNs. A , RGS6 immunostaining is localized to the soma and neurites of CGNs from WT mice and absent in RGS6 −/− mice. B , co-immunostaining of RGS6 with Gβ 5 , GABA B R2, GIRK1, and GIRK2 in CGNs from WT mice ( scale bar = 100 μm; white arrows , regions of overlapping expression of RGS6 with indicated protein).

    Techniques Used: Expressing, Isolation, Immunostaining, Mouse Assay

    RGS6 complex formation in cerebellum and cerebrum. A , RGS6 co-immunoprecipitates with Gβ 5 and R7BP but not GIRK1 and GIRK2 in cerebellar and cerebral lysates. Actin serves as a loading control for Western blots. Input represents total tissue lysates used for subsequent immunoprecipitation. B , immunoprecipitation of GIRK1 from hippocampus, cerebrum, and cerebellum fails to reveal complex formation between GIRK1 and RGS7, Gβ 5 , or RGS6. Input represents blots from isolated membrane fractions used for subsequent immunoprecipitation and Gα i3 served as a loading control for Western blots. NS = nonspecific immunoreactive band, IgG = immunoglobulin heavy chain.
    Figure Legend Snippet: RGS6 complex formation in cerebellum and cerebrum. A , RGS6 co-immunoprecipitates with Gβ 5 and R7BP but not GIRK1 and GIRK2 in cerebellar and cerebral lysates. Actin serves as a loading control for Western blots. Input represents total tissue lysates used for subsequent immunoprecipitation. B , immunoprecipitation of GIRK1 from hippocampus, cerebrum, and cerebellum fails to reveal complex formation between GIRK1 and RGS7, Gβ 5 , or RGS6. Input represents blots from isolated membrane fractions used for subsequent immunoprecipitation and Gα i3 served as a loading control for Western blots. NS = nonspecific immunoreactive band, IgG = immunoglobulin heavy chain.

    Techniques Used: Western Blot, Immunoprecipitation, Isolation

    20) Product Images from "The K+ channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia"

    Article Title: The K+ channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201980

    Absence of GIRK channels in mouse DRG neurons Traces of currents in mouse DRG neurons stimulated with hyperpolarising voltage ramps from −120 to −40 mV and treated with 10 µM DAMGO followed by washout. No differences in peak currents at −120 mV in mouse nociceptors ( n = 28) and mechanoreceptors ( n = 19) after 10 µM DAMGO application. Representative action potential shapes of nociceptors and mechanoreceptors (lower panel) were used to distinguish between DRG neuron subtypes ( p = 0.237; Mann–Whitney Rank Sum test). Representative current traces of mouse DRG neurons at −80 mV after application of 10 µM DAMGO, 3 mM barium or 20 µM naloxone recorded in high potassium solution. Peak current amplitude after agonist and antagonist application ( p = 0.066; one way repeated measures ANOVA, n = 8). Quantification of GIRK channel mRNA in mouse cerebellum, heart and DRG neurons from naïve animals and from animals with CFA-induced unilateral hindpaw inflammation (inflamed: CFA ipsi; noninflamed: CFA contra). (GIRK1 and GIRK2: n = 7; GIRK3 and GIRK4: n = 6; Kruskal–Wallis ANOVA on Ranks, Dunn's method compared to cerebellum or heart * p
    Figure Legend Snippet: Absence of GIRK channels in mouse DRG neurons Traces of currents in mouse DRG neurons stimulated with hyperpolarising voltage ramps from −120 to −40 mV and treated with 10 µM DAMGO followed by washout. No differences in peak currents at −120 mV in mouse nociceptors ( n = 28) and mechanoreceptors ( n = 19) after 10 µM DAMGO application. Representative action potential shapes of nociceptors and mechanoreceptors (lower panel) were used to distinguish between DRG neuron subtypes ( p = 0.237; Mann–Whitney Rank Sum test). Representative current traces of mouse DRG neurons at −80 mV after application of 10 µM DAMGO, 3 mM barium or 20 µM naloxone recorded in high potassium solution. Peak current amplitude after agonist and antagonist application ( p = 0.066; one way repeated measures ANOVA, n = 8). Quantification of GIRK channel mRNA in mouse cerebellum, heart and DRG neurons from naïve animals and from animals with CFA-induced unilateral hindpaw inflammation (inflamed: CFA ipsi; noninflamed: CFA contra). (GIRK1 and GIRK2: n = 7; GIRK3 and GIRK4: n = 6; Kruskal–Wallis ANOVA on Ranks, Dunn's method compared to cerebellum or heart * p

    Techniques Used: MANN-WHITNEY

    Expression of functional GIRK2 channels in DRG neurons of Nav1.8-GIRK2 transgenic mice GIRK2 mRNA expression in cerebellum, spinal cord and DRG neurons of Nav1.8-GIRK2 and wildtype mice (* p = 0.05, One-way ANOVA, Holm–Sidak method, n = 3). Skin sections from Nav1.8-GIRK2 mice labeled with antibodies specific for GIRK2 and IB4. Immunoreactivity for FLAG and GIRK2 demonstrating the expression of exogenous Flag-GIRK2 in sciatic nerve (uppermost panel) and DRG neurons (lower panels) in Nav1.8-GIRK2 mice. Sections of DRGs from Nav1.8-GIRK2 mice immunostained with anti-FLAG and co-stained with IB4, anti-NF200 and anti-CGRP. All scale bars are 50 µm. Representative current traces of Nav1.8-GIRK2 DRG neurons recorded in high potassium solution. Neurons voltage-clamped at −80 mV showing large inward currents evoked by 10 µM DAMGO and inhibited by barium (3 mM) and naloxone (20 µM). Peak current amplitude after application of agonists and antagonists in wildtype and Nav1.8-GIRK2 DRG neurons (*** p = 0.001, two way ANOVA, Holm–Sidak method, n = 9–12 cells per group). All scale bars are 50 µm (B–D).
    Figure Legend Snippet: Expression of functional GIRK2 channels in DRG neurons of Nav1.8-GIRK2 transgenic mice GIRK2 mRNA expression in cerebellum, spinal cord and DRG neurons of Nav1.8-GIRK2 and wildtype mice (* p = 0.05, One-way ANOVA, Holm–Sidak method, n = 3). Skin sections from Nav1.8-GIRK2 mice labeled with antibodies specific for GIRK2 and IB4. Immunoreactivity for FLAG and GIRK2 demonstrating the expression of exogenous Flag-GIRK2 in sciatic nerve (uppermost panel) and DRG neurons (lower panels) in Nav1.8-GIRK2 mice. Sections of DRGs from Nav1.8-GIRK2 mice immunostained with anti-FLAG and co-stained with IB4, anti-NF200 and anti-CGRP. All scale bars are 50 µm. Representative current traces of Nav1.8-GIRK2 DRG neurons recorded in high potassium solution. Neurons voltage-clamped at −80 mV showing large inward currents evoked by 10 µM DAMGO and inhibited by barium (3 mM) and naloxone (20 µM). Peak current amplitude after application of agonists and antagonists in wildtype and Nav1.8-GIRK2 DRG neurons (*** p = 0.001, two way ANOVA, Holm–Sidak method, n = 9–12 cells per group). All scale bars are 50 µm (B–D).

    Techniques Used: Expressing, Functional Assay, Transgenic Assay, Mouse Assay, Labeling, Staining

    Expression of GIRK channels in rat and human sensory neurons Current traces of rat DRG neurons voltage-clamped at −80 mV. Inward currents were recorded in high potassium solution. Currents were evoked by 10 µM DAMGO and suppressed by GIRK channel blocker barium (3 mM) and opioid receptor antagonist naloxone (20 µM). Peak currents after agonist and antagonist application (* p = 0.037; one way repeated measures ANOVA, Holm–Sidak method, n = 7). Quantification of GIRK1- and -2 mRNA in rat and (postmortem) human cerebellum and naïve DRG, and in rat DRG neurons innervating the inflamed (CFA ipsi) or the non-inflamed (CFA contra) paw ( n ≥ 3). Immunoreactivity for GIRK1 and GIRK2 in rat DRG sections. GIRK channels are detectable in nonpeptidergic IB4 positive nociceptors but not in myelinated neurons expressing NF200. Rat skin cryosections (E) and human skin paraffin sections (F) stained with antibodies specific for GIRK1, GIRK2, IB4 and PGP9.5. All scale bars are 50 µm.
    Figure Legend Snippet: Expression of GIRK channels in rat and human sensory neurons Current traces of rat DRG neurons voltage-clamped at −80 mV. Inward currents were recorded in high potassium solution. Currents were evoked by 10 µM DAMGO and suppressed by GIRK channel blocker barium (3 mM) and opioid receptor antagonist naloxone (20 µM). Peak currents after agonist and antagonist application (* p = 0.037; one way repeated measures ANOVA, Holm–Sidak method, n = 7). Quantification of GIRK1- and -2 mRNA in rat and (postmortem) human cerebellum and naïve DRG, and in rat DRG neurons innervating the inflamed (CFA ipsi) or the non-inflamed (CFA contra) paw ( n ≥ 3). Immunoreactivity for GIRK1 and GIRK2 in rat DRG sections. GIRK channels are detectable in nonpeptidergic IB4 positive nociceptors but not in myelinated neurons expressing NF200. Rat skin cryosections (E) and human skin paraffin sections (F) stained with antibodies specific for GIRK1, GIRK2, IB4 and PGP9.5. All scale bars are 50 µm.

    Techniques Used: Expressing, Staining

    Identification of a regulatory sequence in the rat Kcnj6 gene that drives expression in peripheral sensory neurons Sequence alignment of rat and mouse Kcnj6 upstream of the transcription start-site. Dotted black line indicates sequence that was absent from public databases. Dotted orange lines designate regions that were deleted from the R-1195 reporter construct. eGFP fluorescence in mouse DRG cultures transfected with R-1195-eGFP, R-1195Δ1195–1142-eGFP and R-1195Δ1141–1043-eGFP reporter constructs. Insets show phase contrast images. Co-staining of R-1195-eGFP transfected sensory neurons with anti-CGRP. Co-staining of R-1195-eGFP with IB4. All scale bars are 100 μm.
    Figure Legend Snippet: Identification of a regulatory sequence in the rat Kcnj6 gene that drives expression in peripheral sensory neurons Sequence alignment of rat and mouse Kcnj6 upstream of the transcription start-site. Dotted black line indicates sequence that was absent from public databases. Dotted orange lines designate regions that were deleted from the R-1195 reporter construct. eGFP fluorescence in mouse DRG cultures transfected with R-1195-eGFP, R-1195Δ1195–1142-eGFP and R-1195Δ1141–1043-eGFP reporter constructs. Insets show phase contrast images. Co-staining of R-1195-eGFP transfected sensory neurons with anti-CGRP. Co-staining of R-1195-eGFP with IB4. All scale bars are 100 μm.

    Techniques Used: Sequencing, Expressing, Construct, Fluorescence, Transfection, Staining

    Antinociceptive effect of peripherally applied DAMGO in Nav1.8-GIRK2 mice Changes in paw withdrawal latency in response to radiant heat in the inflamed (ipsilateral) and noninflamed (contralateral) paws of Nav1.8-GIRK2 and wildtype mice. Dose-dependent inhibition of thermal hyperalgesia 5 min after DAMGO injection into the inflamed paw of Nav1.8-GIRK2 mice (unadjusted p -values: *** p = 0.000, ** p = 0.004; two way ANOVA, Holm–Sidak method, n = 7–8). DAMGO-induced antinociception is reversed by co-injection of naloxone-methiodide (NLXM, 5µg) in the inflamed paw of Nav1.8-GIRK2 mice (*** p
    Figure Legend Snippet: Antinociceptive effect of peripherally applied DAMGO in Nav1.8-GIRK2 mice Changes in paw withdrawal latency in response to radiant heat in the inflamed (ipsilateral) and noninflamed (contralateral) paws of Nav1.8-GIRK2 and wildtype mice. Dose-dependent inhibition of thermal hyperalgesia 5 min after DAMGO injection into the inflamed paw of Nav1.8-GIRK2 mice (unadjusted p -values: *** p = 0.000, ** p = 0.004; two way ANOVA, Holm–Sidak method, n = 7–8). DAMGO-induced antinociception is reversed by co-injection of naloxone-methiodide (NLXM, 5µg) in the inflamed paw of Nav1.8-GIRK2 mice (*** p

    Techniques Used: Mouse Assay, Inhibition, Injection

    21) Product Images from "GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner"

    Article Title: GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5051-14.2015

    Constitutive Girk subunit ablation and morphine-induced motor activity. The total distance traveled by adult male wild-type (white; n = 26/dose), Girk1 −/− (gray; n = 18/dose), and Girk2 −/− (black; n = 7/dose) mice during a 60 min test following systemic administration of saline (0) or morphine (3, 10, and 30 mg/kg, i.p.) is shown. Main effects of genotype ( F (2,48) = 12.8, p
    Figure Legend Snippet: Constitutive Girk subunit ablation and morphine-induced motor activity. The total distance traveled by adult male wild-type (white; n = 26/dose), Girk1 −/− (gray; n = 18/dose), and Girk2 −/− (black; n = 7/dose) mice during a 60 min test following systemic administration of saline (0) or morphine (3, 10, and 30 mg/kg, i.p.) is shown. Main effects of genotype ( F (2,48) = 12.8, p

    Techniques Used: Activity Assay, Mouse Assay

    Generation of Girk2 flox/flox mice. A , Depiction of the mouse Girk2/Kcnj6 ). Although several alternative splice variants have been identified, using two distinct translation initiation codons (ATG) and four distinct translation stop codons (TGA), all known variants contain exon 4. Shaded regions of exons denote protein-coding sequences. Some exons (1, 4, and 6) contain internal splice acceptor sites, denoted by vertical lines. B ). The region encoded by exon 4, which includes most of the N terminus (NT), both membrane-spanning domains and extracellular loops, the pore domain, and much of the C terminus (CT), is denoted by arrows. C , The Girk2 targeting strategy included engineering a loxP site just upstream (214 bp) of exon 4 and incorporating a NEO resistance cassette containing Flp recognition target (FRT) sites and a second loxP site; this cassette was positioned 795 bp downstream from exon 4. In total, the target region spanned 1.93 Kbp and was flanked by a 5′ homology arm (extending ∼5.4 Kbp upstream from exon 4) and a 3′ homology arm (an ∼7 Kbp fragment found immediately downstream from the target region). The NEO cassette was removed by crossing selected chimeras with C57BL/6N Flp deleter mice, yielding the Girk2 flox allele. D , Tissue from the cortex ( n = 9–10/genotype; t (17) = 0.09, p = 0.9), hippocampus ( n = 6–7/genotype; t 11 = 0.4, p = 0.7), and cerebellum ( n = 7–8/genotype; t (13) = 1.3, p = 0.2) of wild-type and Girk2 flox/flox (f/f) mice was evaluated for GIRK2 mRNA levels using quantitative RT-PCR. E , GIRK2 protein immunoreactivity (ir) relative to β-actin in samples from the cortex ( n = 6–7/genotype; t (11) = 0.4, p = 0.7), hippocampus ( n = 5–7/genotype; t (10) = 0.05, p = 1.0), and cerebellum ( n = 4–7/genotype; t (9) = 0.6, p = 0.6) of wild-type and Girk2 flox/flox (f/f) mice was assessed using quantitative immunoblotting. The level of GIRK2, which is seen as a doublet (top), was compared to the level of β-actin control (bottom) in each sample. Gel images of GIRK2 and β-actin were cropped and aligned with a molecular weight scale showing the markers for 52 and 38 kDA.
    Figure Legend Snippet: Generation of Girk2 flox/flox mice. A , Depiction of the mouse Girk2/Kcnj6 ). Although several alternative splice variants have been identified, using two distinct translation initiation codons (ATG) and four distinct translation stop codons (TGA), all known variants contain exon 4. Shaded regions of exons denote protein-coding sequences. Some exons (1, 4, and 6) contain internal splice acceptor sites, denoted by vertical lines. B ). The region encoded by exon 4, which includes most of the N terminus (NT), both membrane-spanning domains and extracellular loops, the pore domain, and much of the C terminus (CT), is denoted by arrows. C , The Girk2 targeting strategy included engineering a loxP site just upstream (214 bp) of exon 4 and incorporating a NEO resistance cassette containing Flp recognition target (FRT) sites and a second loxP site; this cassette was positioned 795 bp downstream from exon 4. In total, the target region spanned 1.93 Kbp and was flanked by a 5′ homology arm (extending ∼5.4 Kbp upstream from exon 4) and a 3′ homology arm (an ∼7 Kbp fragment found immediately downstream from the target region). The NEO cassette was removed by crossing selected chimeras with C57BL/6N Flp deleter mice, yielding the Girk2 flox allele. D , Tissue from the cortex ( n = 9–10/genotype; t (17) = 0.09, p = 0.9), hippocampus ( n = 6–7/genotype; t 11 = 0.4, p = 0.7), and cerebellum ( n = 7–8/genotype; t (13) = 1.3, p = 0.2) of wild-type and Girk2 flox/flox (f/f) mice was evaluated for GIRK2 mRNA levels using quantitative RT-PCR. E , GIRK2 protein immunoreactivity (ir) relative to β-actin in samples from the cortex ( n = 6–7/genotype; t (11) = 0.4, p = 0.7), hippocampus ( n = 5–7/genotype; t (10) = 0.05, p = 1.0), and cerebellum ( n = 4–7/genotype; t (9) = 0.6, p = 0.6) of wild-type and Girk2 flox/flox (f/f) mice was assessed using quantitative immunoblotting. The level of GIRK2, which is seen as a doublet (top), was compared to the level of β-actin control (bottom) in each sample. Gel images of GIRK2 and β-actin were cropped and aligned with a molecular weight scale showing the markers for 52 and 38 kDA.

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Molecular Weight

    22) 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

    23) Product Images from "Functional Recovery from Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts is Dependent on Neurite Outgrowth"

    Article Title: Functional Recovery from Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts is Dependent on Neurite Outgrowth

    Journal: bioRxiv

    doi: 10.1101/2022.04.19.488213

    Immunohistochemical analysis of d18 and d25 iPSC-derived grafts. Based on histological analysis of the grafts from d18 and d25 preparations, we present the total mean HuNu+cells (A), graft volume (B), the density of TH+ cells per mm 3 (C), total TH+ neurons (D), percentage of TH+ cells out of total HuNu+ cells (E), the total GIRK2+ cells (G) and the percentage of GIRK2+ cells out of TH+ cells (H). The total AADC+ cell data are depicted in (J) and the percentage of AADC+ cells relative to HuNu+ cells is in (K). Representative immunohistochemistry is presented for TH (F), GIRK2 (blue) and HuNu (brown) in (I) and AADC (brown) and HuNu (blue) in (L). Main effects of cell line or days in vitro (DIV) are stated, with p*≤0.05, p**≤0.001, error bars=±SEM.
    Figure Legend Snippet: Immunohistochemical analysis of d18 and d25 iPSC-derived grafts. Based on histological analysis of the grafts from d18 and d25 preparations, we present the total mean HuNu+cells (A), graft volume (B), the density of TH+ cells per mm 3 (C), total TH+ neurons (D), percentage of TH+ cells out of total HuNu+ cells (E), the total GIRK2+ cells (G) and the percentage of GIRK2+ cells out of TH+ cells (H). The total AADC+ cell data are depicted in (J) and the percentage of AADC+ cells relative to HuNu+ cells is in (K). Representative immunohistochemistry is presented for TH (F), GIRK2 (blue) and HuNu (brown) in (I) and AADC (brown) and HuNu (blue) in (L). Main effects of cell line or days in vitro (DIV) are stated, with p*≤0.05, p**≤0.001, error bars=±SEM.

    Techniques Used: Immunohistochemistry, Derivative Assay, In Vitro

    24) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    25) Product Images from "Sex differences in GABABR-GIRK signaling in layer 5/6 pyramidal neurons of the mouse prelimbic cortex"

    Article Title: Sex differences in GABABR-GIRK signaling in layer 5/6 pyramidal neurons of the mouse prelimbic cortex

    Journal: Neuropharmacology

    doi: 10.1016/j.neuropharm.2015.03.029

    Subcellular localization of GIRK2 and GABA B R1 in layer 5/6 pyramidal neurons of male and female mice
    Figure Legend Snippet: Subcellular localization of GIRK2 and GABA B R1 in layer 5/6 pyramidal neurons of male and female mice

    Techniques Used: Mouse Assay

    26) 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

    27) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    28) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    29) Product Images from "Analgesic α-conotoxins modulate GIRK1/2 channels via GABAB receptor activation and reduce neuroexcitability"

    Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABAB receptor activation and reduce neuroexcitability

    Journal: bioRxiv

    doi: 10.1101/2020.12.02.407627

    Fluorescence micrographs representing immunolabeling of GIRK 1, GIRK 2 and GABA B R2 alone and co-localization when over-expressed recombinantly in HEK293 cells. A-C GIRK1 (red), GIRK2 (green) and GABA B R2 (red) exhibited immunoreactivity in HEK293T cells and DAPI (blue) staining denotes the nucleus of the cells. D-E DAPI (blue), GIRK1 (red), GIRK2 (green) and GABA B R2 (red) immunoreactivity in HEK293T cells. The merged/overlay images depict co-localization of GIRK1 and GIRK2 or GIRK2 and GABA B R2 upon transfection. Scale bars: 100 μm.
    Figure Legend Snippet: Fluorescence micrographs representing immunolabeling of GIRK 1, GIRK 2 and GABA B R2 alone and co-localization when over-expressed recombinantly in HEK293 cells. A-C GIRK1 (red), GIRK2 (green) and GABA B R2 (red) exhibited immunoreactivity in HEK293T cells and DAPI (blue) staining denotes the nucleus of the cells. D-E DAPI (blue), GIRK1 (red), GIRK2 (green) and GABA B R2 (red) immunoreactivity in HEK293T cells. The merged/overlay images depict co-localization of GIRK1 and GIRK2 or GIRK2 and GABA B R2 upon transfection. Scale bars: 100 μm.

    Techniques Used: Fluorescence, Immunolabeling, Staining, Transfection

    Expression of GIRK1, GIRK2 and GABA B2 subunit of GABA B R and α-conotoxin Vc1.1 potentiation of inward rectifier K + currents in adult mouse DRG neurons. A Immunolabeling of mouse DRG neurons and visual inspection by confocal microscopy revealed both GIRK1 and GIRK2 channels express and show co-localization with each other using antibodies directed against GIRK1 and GIRK2 channels. B DRG neurons showing expression of GABA B2 subunit and GIRK2 channel and both GIRK2 and GABA B2 co-localize in mouse DRG neurons. Co-expression of GIRK 1 and GIRK 2 or GIRK2 and GABA B R immunoreactivity in mouse DRG neurons was independent of cell diameter. Scale bars: 100 μm. DAPI (blue) staining marks nucleus. C Diary plot of peak inward K + currents recorded at −100 mV in small to medium diameter (
    Figure Legend Snippet: Expression of GIRK1, GIRK2 and GABA B2 subunit of GABA B R and α-conotoxin Vc1.1 potentiation of inward rectifier K + currents in adult mouse DRG neurons. A Immunolabeling of mouse DRG neurons and visual inspection by confocal microscopy revealed both GIRK1 and GIRK2 channels express and show co-localization with each other using antibodies directed against GIRK1 and GIRK2 channels. B DRG neurons showing expression of GABA B2 subunit and GIRK2 channel and both GIRK2 and GABA B2 co-localize in mouse DRG neurons. Co-expression of GIRK 1 and GIRK 2 or GIRK2 and GABA B R immunoreactivity in mouse DRG neurons was independent of cell diameter. Scale bars: 100 μm. DAPI (blue) staining marks nucleus. C Diary plot of peak inward K + currents recorded at −100 mV in small to medium diameter (

    Techniques Used: Expressing, Immunolabeling, Confocal Microscopy, Staining

    α-Conotoxin Vc1.1 modulation of heteromeric GIRK1,2 and homomeric GIRK2 channels require GABA B R. A, C Representative K + currents recorded from GIRK1/2 channels co-expressed either with GABA B R ( A ) or alone ( C ) in response to a 50 ms voltage ramp protocol (−100 to +40 mV; see inset) applied at 0.1 Hz from a holding potential of −40 mV in the absence (control) and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). B, D Corresponding diary plots of K + current amplitude at −100 mV as a function of time in cells expressing GIRK1/2 channels and GABA B R (B) and GIRK1/2 channels alone D Responses to sequential bath application of Vc1.1 (blue), baclofen (red), and Ba 2+ (grey) are indicated by the bars above. E Vc1.1 potentiates also homomeric GIRK2 channel when co-expressed with GABA B R. Representative human GIRK2-mediated K + currents obtained in the absence (Control, black), and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). F Corresponding diary plot to (E) showing peak K + current amplitude at −100 mV as a function of time in response to bath application of Vc1.1 (blue), baclofen (red) or Ba 2+ (grey). G α-Conotoxin Vc1.1 concentration-response relationship obtained for potentiation of GIRK1/2 co-expressed with GABA B R in HEK293T cells. ΔI Kir represents total I Kir (basal + potentiation) minus basal I Kir . H Bar graph of ΔI Kir density (pA/pF) at −100 mV in response to 100 μM baclofen (red), 100 μM GABA (orange), 1 μM Vc1.1 (blue), 1 μM RgIA (purple) and 1 μM PeIA (yellow) recorded from cells expressing either heteromeric GIRK1/2 (solid) or homomeric GIRK2 (hashed) co-expressed with GABA B R. Neither Vc1.1 nor baclofen potentiates K + currents in cells expressing GIRK1/2 alone. The vertical dotted line (40 pA/pF) is for reference. Data represent mean ± SEM and the number of experiments is given in parentheses.
    Figure Legend Snippet: α-Conotoxin Vc1.1 modulation of heteromeric GIRK1,2 and homomeric GIRK2 channels require GABA B R. A, C Representative K + currents recorded from GIRK1/2 channels co-expressed either with GABA B R ( A ) or alone ( C ) in response to a 50 ms voltage ramp protocol (−100 to +40 mV; see inset) applied at 0.1 Hz from a holding potential of −40 mV in the absence (control) and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). B, D Corresponding diary plots of K + current amplitude at −100 mV as a function of time in cells expressing GIRK1/2 channels and GABA B R (B) and GIRK1/2 channels alone D Responses to sequential bath application of Vc1.1 (blue), baclofen (red), and Ba 2+ (grey) are indicated by the bars above. E Vc1.1 potentiates also homomeric GIRK2 channel when co-expressed with GABA B R. Representative human GIRK2-mediated K + currents obtained in the absence (Control, black), and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). F Corresponding diary plot to (E) showing peak K + current amplitude at −100 mV as a function of time in response to bath application of Vc1.1 (blue), baclofen (red) or Ba 2+ (grey). G α-Conotoxin Vc1.1 concentration-response relationship obtained for potentiation of GIRK1/2 co-expressed with GABA B R in HEK293T cells. ΔI Kir represents total I Kir (basal + potentiation) minus basal I Kir . H Bar graph of ΔI Kir density (pA/pF) at −100 mV in response to 100 μM baclofen (red), 100 μM GABA (orange), 1 μM Vc1.1 (blue), 1 μM RgIA (purple) and 1 μM PeIA (yellow) recorded from cells expressing either heteromeric GIRK1/2 (solid) or homomeric GIRK2 (hashed) co-expressed with GABA B R. Neither Vc1.1 nor baclofen potentiates K + currents in cells expressing GIRK1/2 alone. The vertical dotted line (40 pA/pF) is for reference. Data represent mean ± SEM and the number of experiments is given in parentheses.

    Techniques Used: Expressing, Concentration Assay

    30) Product Images from "p75 Neurotrophin Receptor Mediates Neuronal Cell Death by Activating GIRK Channels through Phosphatidylinositol 4,5-Bisphosphate"

    Article Title: p75 Neurotrophin Receptor Mediates Neuronal Cell Death by Activating GIRK Channels through Phosphatidylinositol 4,5-Bisphosphate

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2699-07.2008

    Model of the proposed p75 NTR - and GIRK channel-mediated cell death signaling pathway. Death signaling is activated by metalloprotease cleavage of p75 NTR after stimulation by mature or proneurotrophins (NGF). The C-terminal fragment of p75 NTR activates Rac, which in turn activates PIPKI, with signal transduction assisted by colocalization of signaling molecules within lipid raft-like membrane domains. The locally generated PtdIns(4,5)P 2 (PIP2) then binds to the C-terminal domain of neighboring GIRK2 subunits, activating GIRK1/2 heterotrimeric channels. The resulting efflux of potassium through the GIRK channels lowers the intracellular potassium concentration, releasing inhibition of the apoptosome, and resulting in capsase activation and ultimately cell death.
    Figure Legend Snippet: Model of the proposed p75 NTR - and GIRK channel-mediated cell death signaling pathway. Death signaling is activated by metalloprotease cleavage of p75 NTR after stimulation by mature or proneurotrophins (NGF). The C-terminal fragment of p75 NTR activates Rac, which in turn activates PIPKI, with signal transduction assisted by colocalization of signaling molecules within lipid raft-like membrane domains. The locally generated PtdIns(4,5)P 2 (PIP2) then binds to the C-terminal domain of neighboring GIRK2 subunits, activating GIRK1/2 heterotrimeric channels. The resulting efflux of potassium through the GIRK channels lowers the intracellular potassium concentration, releasing inhibition of the apoptosome, and resulting in capsase activation and ultimately cell death.

    Techniques Used: Transduction, Generated, Concentration Assay, Inhibition, Activation Assay

    31) Product Images from "Distinct Populations of Spinal Cord Lamina II Interneurons Expressing G-Protein-Gated Potassium Channels"

    Article Title: Distinct Populations of Spinal Cord Lamina II Interneurons Expressing G-Protein-Gated Potassium Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3693-06.2006

    Expression of GIRK subunits in excitatory interneurons of lamina II. A – C , Low-magnification image of a transverse section (15 μm) from the lumbar enlargement of a wild-type adult mouse spinal cord colabeled with antibodies directed against GIRK2 and the excitatory interneuron marker GluR2/3. Note that GIRK2 labeling is most prominent in a relatively thin band of the SDH (lamina IIo). Specific GluR2/3 labeling is also seen in this region, but immunoreactivity for this marker is more prominent in inner lamina of the mouse SDH. D – F , High-magnification confocal image of GluR2/3 and GIRK2 subunit colabeling in lamina II. Arrows point to four cells that showed labeling for both GluR2/3 and GIRK2. Determination of cellular overlap was complicated by the overlapping but distinct subcellular distributions of GIRK subunits and GluR2/3; assignment of overlap in each instance was based on the judgment of two individuals after evaluation of a complete z -series of images spanning each neuron evaluated. Data presented are representative of experiments involving tissue from three different panels of adult wild-type mice. Similar data were obtained in GIRK1 colabeling studies. Scale bars: (in A ) A – C , 75 μm; (in D ) D – F , 30 μm.
    Figure Legend Snippet: Expression of GIRK subunits in excitatory interneurons of lamina II. A – C , Low-magnification image of a transverse section (15 μm) from the lumbar enlargement of a wild-type adult mouse spinal cord colabeled with antibodies directed against GIRK2 and the excitatory interneuron marker GluR2/3. Note that GIRK2 labeling is most prominent in a relatively thin band of the SDH (lamina IIo). Specific GluR2/3 labeling is also seen in this region, but immunoreactivity for this marker is more prominent in inner lamina of the mouse SDH. D – F , High-magnification confocal image of GluR2/3 and GIRK2 subunit colabeling in lamina II. Arrows point to four cells that showed labeling for both GluR2/3 and GIRK2. Determination of cellular overlap was complicated by the overlapping but distinct subcellular distributions of GIRK subunits and GluR2/3; assignment of overlap in each instance was based on the judgment of two individuals after evaluation of a complete z -series of images spanning each neuron evaluated. Data presented are representative of experiments involving tissue from three different panels of adult wild-type mice. Similar data were obtained in GIRK1 colabeling studies. Scale bars: (in A ) A – C , 75 μm; (in D ) D – F , 30 μm.

    Techniques Used: Expressing, Marker, Labeling, Mouse Assay

    Correlations between agonist response profiles and properties of mouse lamina II neurons. A , Membrane potentials of lamina II neurons plotted as a function of agonist response profile and genotype. DAMGO-responsive wild-type and GIRK3 knock-out lamina II neurons were relatively hyperpolarized compared with neurons of the same genotype with different agonist response profiles. Furthermore, DAMGO-responsive and baclofen-responsive GIRK1 knock-out and GIRK2 knock-out neurons were significantly depolarized compared with wild-type and GIRK3 knock-out groups. B , The apparent capacitance values of lamina II neurons plotted as a function of agonist response profile and genotype. The DAMGO-responsive lamina II neurons in all genotypes exhibited significantly greater apparent capacitance values than the baclofen-responsive neurons. * p
    Figure Legend Snippet: Correlations between agonist response profiles and properties of mouse lamina II neurons. A , Membrane potentials of lamina II neurons plotted as a function of agonist response profile and genotype. DAMGO-responsive wild-type and GIRK3 knock-out lamina II neurons were relatively hyperpolarized compared with neurons of the same genotype with different agonist response profiles. Furthermore, DAMGO-responsive and baclofen-responsive GIRK1 knock-out and GIRK2 knock-out neurons were significantly depolarized compared with wild-type and GIRK3 knock-out groups. B , The apparent capacitance values of lamina II neurons plotted as a function of agonist response profile and genotype. The DAMGO-responsive lamina II neurons in all genotypes exhibited significantly greater apparent capacitance values than the baclofen-responsive neurons. * p

    Techniques Used: Knock-Out

    ). Data presented are representative of three different immunoprecipitation experiments involving three different wild-type and GIRK2 knock-out mouse panels. WT, Wild type.
    Figure Legend Snippet: ). Data presented are representative of three different immunoprecipitation experiments involving three different wild-type and GIRK2 knock-out mouse panels. WT, Wild type.

    Techniques Used: Immunoprecipitation, Knock-Out

    Overlap between GIRK subunits, MOR, and the GABA B receptor in lamina II. A – C , Low-magnification image of a transverse section (15 μm) of the lumbar enlargement from a wild-type adult mouse spinal cord colabeled with antibodies directed against GIRK2 and the GABA B(1a/b) subunit. The GABA B(1a/b) antibody shows intense labeling in the superficial layers of the dorsal horn, encompassing the region of prominent GIRK subunit immunoreactivity. D – F , Low-magnification image of MOR and GABA B(1a/b) colabeling in lamina II. Both receptors show prominent expression in the superficial layers of the dorsal horn, with less intense labeling seen in inner layers. G – I , High-magnification confocal image of MOR and GABA B(1a/b) subunit colabeling in lamina II. Arrows point to three cells that exhibited labeling for both receptor types. Data presented are representative of experiments involving tissue from three different adult wild-type mice. Scale bars: (in A ) A – C , (in D ) D – F , 75 μm; (in G ) G – I , 30 μm.
    Figure Legend Snippet: Overlap between GIRK subunits, MOR, and the GABA B receptor in lamina II. A – C , Low-magnification image of a transverse section (15 μm) of the lumbar enlargement from a wild-type adult mouse spinal cord colabeled with antibodies directed against GIRK2 and the GABA B(1a/b) subunit. The GABA B(1a/b) antibody shows intense labeling in the superficial layers of the dorsal horn, encompassing the region of prominent GIRK subunit immunoreactivity. D – F , Low-magnification image of MOR and GABA B(1a/b) colabeling in lamina II. Both receptors show prominent expression in the superficial layers of the dorsal horn, with less intense labeling seen in inner layers. G – I , High-magnification confocal image of MOR and GABA B(1a/b) subunit colabeling in lamina II. Arrows point to three cells that exhibited labeling for both receptor types. Data presented are representative of experiments involving tissue from three different adult wild-type mice. Scale bars: (in A ) A – C , (in D ) D – F , 75 μm; (in G ) G – I , 30 μm.

    Techniques Used: Labeling, Expressing, Mouse Assay

    32) 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

    33) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    34) Product Images from "Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels"

    Article Title: Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3484-05.2005

    GIRK subunit proteins are found in the mouse hippocampus. A , Representative immunoblots of hippocampal membrane protein samples from WT, GIRK1 KO (1), GIRK2 (2), GIRK3 (3), and GIRK2/GIRK3 (2/3) KO mice. Blots were probed with antibodies (Ab) for GIRK1, GIRK2, GIRK3, and GABA B(1a/b) . GABA B ), GIRK1 immunoreactivity was visualized as three bands, the lower molecular weight versions thought to represent core (c) and core-glycosylated (g) species. Reductions in the level of the heavily glycosylated (h) GIRK1 species were correlated with the absence of GIRK2 and GIRK3, respectively. The levels of GIRK2 and GIRK3 were also lower in samples from KO mice. B , Densitometric analysis of the impact of GIRK subunit ablation on residual GIRK protein levels in the hippocampus. Hippocampal protein samples were obtained from three separate and complete panels of WT and GIRK KO mice, and the levels of GIRK1 (heavily glycosylated form, h-GIRK1), GIRK2, GIRK3, and GABA B(1) (both isoforms) were determined. There was no effect of GIRK subunit ablation GABA B(1) receptor levels ( F (4,10) = 1.764; p = 0.213) (data not shown). * p
    Figure Legend Snippet: GIRK subunit proteins are found in the mouse hippocampus. A , Representative immunoblots of hippocampal membrane protein samples from WT, GIRK1 KO (1), GIRK2 (2), GIRK3 (3), and GIRK2/GIRK3 (2/3) KO mice. Blots were probed with antibodies (Ab) for GIRK1, GIRK2, GIRK3, and GABA B(1a/b) . GABA B ), GIRK1 immunoreactivity was visualized as three bands, the lower molecular weight versions thought to represent core (c) and core-glycosylated (g) species. Reductions in the level of the heavily glycosylated (h) GIRK1 species were correlated with the absence of GIRK2 and GIRK3, respectively. The levels of GIRK2 and GIRK3 were also lower in samples from KO mice. B , Densitometric analysis of the impact of GIRK subunit ablation on residual GIRK protein levels in the hippocampus. Hippocampal protein samples were obtained from three separate and complete panels of WT and GIRK KO mice, and the levels of GIRK1 (heavily glycosylated form, h-GIRK1), GIRK2, GIRK3, and GABA B(1) (both isoforms) were determined. There was no effect of GIRK subunit ablation GABA B(1) receptor levels ( F (4,10) = 1.764; p = 0.213) (data not shown). * p

    Techniques Used: Western Blot, Mouse Assay, Molecular Weight

    Subcellular distributions of GIRK subunits in the hippocampus. Electron micrographs show immunolabeling for GIRK1 and GIRK2 in the stratum radiatum of the CA1 area of WT and GIRK KO mice. den, Dendritic shaft; b, bouton; s, dendritic spine. A , B , GIRK1 immunoparticles were observed primarily along the extrasynaptic plasma membrane (arrows) of dendritic spines of CA1 neurons from WT mice, although perisynaptic labeling (crossed arrow) was also frequently observed. GIRK1 labeling was also found associated with ER cisterna of dendritic shafts and with the spine apparatus (double arrowheads) of spines. C , GIRK1 labeling was never detected within the postsynaptic specialization, as demonstrated with postembedding techniques. D , E , GIRK1 immunoparticles at presynaptic sites were localized to the extrasynaptic plasma membrane (arrowheads) and occasionally to the presynaptic membrane specialization of axon terminals establishing putative excitatory synapses on spines, as confirmed in double-labeling experiments with VGluT1. F-H , GIRK2 immunoparticles were found along the extrasynaptic plasma membrane (arrows) of dendritic shafts of CA1 cells from WT mice, mainly associated with dendritic spines. Both perisynaptic ( G , crossed arrows) and synaptic ( H , double arrow) labeling at asymmetrical synapses was detected, the latter confirmed using postembedding techniques ( I ). J , K , GIRK2 immunoparticles were detected at presynaptic sites (arrowheads), mainly in the extra synaptic plasma membrane, and occasionally in the presynaptic membrane specialization of axon terminals establishing putative excitatory synapses on spines, as confirmed using double-labeling experiments with VGluT1. Note also the GIRK2 immunoreactivity associated with ER cisterna of dendritic shafts and the spine apparatus of dendritic spines ( J , double arrowheads). L , Distribution of GIRK1 and GIRK2 on dendritic spines of CA1 neurons. Data are displayed as percentage frequency of particles in 60-nm-wide bins, starting at the edge of the postsynaptic specialization. M-T , Electron micrographs showing GIRK1 immunoreactivity in the stratum radiatum of the CA1 area in sections taken from GIRK2 KO ( M-P ), GIRK3 KO ( Q , R ), GIRK2/GIRK3 KO ( S ), and GIRK1 KO ( T ) mice. Note that GIRK1 immunoreactivity was still observed along the extrasynaptic plasma membrane (arrows) of dendritic spines and shafts of CA1 neurons from GIRK2 KO and GIRK3 KO mice. Immunoparticles for GIRK1 were also observed at perisynaptic (data not shown) positions and at presynaptic sites along the extrasynaptic plasma membrane (arrowheads) of axon terminals establishing putative excitatory synapses on dendritic spines. A higher proportion of immunoparticles for GIRK1 was detected in the ER cisterna of dendritic shafts and the spine apparatus (double arrowhead). Immunoreactivity for GIRK1 was primarily restricted to the ER of CA1 cells in the hippocampus of GIRK2/GIRK3 KO mice and was absent in sections from a GIRK1 KO mouse. Scale bars, 0.2 μm.
    Figure Legend Snippet: Subcellular distributions of GIRK subunits in the hippocampus. Electron micrographs show immunolabeling for GIRK1 and GIRK2 in the stratum radiatum of the CA1 area of WT and GIRK KO mice. den, Dendritic shaft; b, bouton; s, dendritic spine. A , B , GIRK1 immunoparticles were observed primarily along the extrasynaptic plasma membrane (arrows) of dendritic spines of CA1 neurons from WT mice, although perisynaptic labeling (crossed arrow) was also frequently observed. GIRK1 labeling was also found associated with ER cisterna of dendritic shafts and with the spine apparatus (double arrowheads) of spines. C , GIRK1 labeling was never detected within the postsynaptic specialization, as demonstrated with postembedding techniques. D , E , GIRK1 immunoparticles at presynaptic sites were localized to the extrasynaptic plasma membrane (arrowheads) and occasionally to the presynaptic membrane specialization of axon terminals establishing putative excitatory synapses on spines, as confirmed in double-labeling experiments with VGluT1. F-H , GIRK2 immunoparticles were found along the extrasynaptic plasma membrane (arrows) of dendritic shafts of CA1 cells from WT mice, mainly associated with dendritic spines. Both perisynaptic ( G , crossed arrows) and synaptic ( H , double arrow) labeling at asymmetrical synapses was detected, the latter confirmed using postembedding techniques ( I ). J , K , GIRK2 immunoparticles were detected at presynaptic sites (arrowheads), mainly in the extra synaptic plasma membrane, and occasionally in the presynaptic membrane specialization of axon terminals establishing putative excitatory synapses on spines, as confirmed using double-labeling experiments with VGluT1. Note also the GIRK2 immunoreactivity associated with ER cisterna of dendritic shafts and the spine apparatus of dendritic spines ( J , double arrowheads). L , Distribution of GIRK1 and GIRK2 on dendritic spines of CA1 neurons. Data are displayed as percentage frequency of particles in 60-nm-wide bins, starting at the edge of the postsynaptic specialization. M-T , Electron micrographs showing GIRK1 immunoreactivity in the stratum radiatum of the CA1 area in sections taken from GIRK2 KO ( M-P ), GIRK3 KO ( Q , R ), GIRK2/GIRK3 KO ( S ), and GIRK1 KO ( T ) mice. Note that GIRK1 immunoreactivity was still observed along the extrasynaptic plasma membrane (arrows) of dendritic spines and shafts of CA1 neurons from GIRK2 KO and GIRK3 KO mice. Immunoparticles for GIRK1 were also observed at perisynaptic (data not shown) positions and at presynaptic sites along the extrasynaptic plasma membrane (arrowheads) of axon terminals establishing putative excitatory synapses on dendritic spines. A higher proportion of immunoparticles for GIRK1 was detected in the ER cisterna of dendritic shafts and the spine apparatus (double arrowhead). Immunoreactivity for GIRK1 was primarily restricted to the ER of CA1 cells in the hippocampus of GIRK2/GIRK3 KO mice and was absent in sections from a GIRK1 KO mouse. Scale bars, 0.2 μm.

    Techniques Used: Immunolabeling, Mouse Assay, Labeling

    Distribution of GIRK2 in the SNc. A , In WT sections containing the SN, GIRK2 immunoreactivity was most prominent in SNc neuron dendrites found in the SNr. B , No GIRK2 staining was observed in sections from GIRK2 KO mice. C , Colocalization of the GIRK2 subunit and TH in the SNc of WT mice, as revealed using preembedding methods. The peroxidase reaction product (TH immunoreactivity) filled somata and dendritic shafts, whereas immunoparticles (GIRK2 immunoreactivity) were located along the extrasynaptic plasma membrane (arrows). D , Immunoparticles for GIRK2 (arrowheads) were also detected along the extrasynaptic plasma membrane of dendritic shafts immunonegative for TH, likely belonging to GABAergic neurons. E , F , Electron micrographs showing colocalization of the GIRK2 and GABA B(1) in the SNc of WT mice. A peroxidase reaction product (GABA B(1) immunoreactivity) filled dendritic shafts establishing asymmetrical synapses with axon terminals, whereas immunoparticles (GIRK2 immunoreactivity) were located along the extrasynaptic plasma membrane (arrows). b, Bouton; den, dendritic shaft; nuc, nucleus. Scale bars: A , B , 200 μm; C-F , 0.2 μm.
    Figure Legend Snippet: Distribution of GIRK2 in the SNc. A , In WT sections containing the SN, GIRK2 immunoreactivity was most prominent in SNc neuron dendrites found in the SNr. B , No GIRK2 staining was observed in sections from GIRK2 KO mice. C , Colocalization of the GIRK2 subunit and TH in the SNc of WT mice, as revealed using preembedding methods. The peroxidase reaction product (TH immunoreactivity) filled somata and dendritic shafts, whereas immunoparticles (GIRK2 immunoreactivity) were located along the extrasynaptic plasma membrane (arrows). D , Immunoparticles for GIRK2 (arrowheads) were also detected along the extrasynaptic plasma membrane of dendritic shafts immunonegative for TH, likely belonging to GABAergic neurons. E , F , Electron micrographs showing colocalization of the GIRK2 and GABA B(1) in the SNc of WT mice. A peroxidase reaction product (GABA B(1) immunoreactivity) filled dendritic shafts establishing asymmetrical synapses with axon terminals, whereas immunoparticles (GIRK2 immunoreactivity) were located along the extrasynaptic plasma membrane (arrows). b, Bouton; den, dendritic shaft; nuc, nucleus. Scale bars: A , B , 200 μm; C-F , 0.2 μm.

    Techniques Used: Staining, Mouse Assay

    GIRK subunit mRNA distribution in the hippocampus and substantia nigra. GIRK mRNA distributions were evaluated by in situ hybridization in sections from WT and GIRKKO mice. In sections from WT mice, mRNAs for GIRK1 ( A ), GIRK2 ( B ), and GIRK3 ( C ) were clearly evident in CA1 and CA3 pyramidal neurons, as well as granule cells of the dentate gyrus (DG). There was no specific staining for GIRK mRNAs in sections from the appropriate GIRK KO mouse (data not shown). Images are representative of data obtained from three different WT and GIRK KO panels. The WT mRNA distributions of GIRK1 ( D ), GIRK2 ( E ), and GIRK3 ( F ) were also examined in the SN. Scale bars: A-C , 500 μm; D-F , 100 μm.
    Figure Legend Snippet: GIRK subunit mRNA distribution in the hippocampus and substantia nigra. GIRK mRNA distributions were evaluated by in situ hybridization in sections from WT and GIRKKO mice. In sections from WT mice, mRNAs for GIRK1 ( A ), GIRK2 ( B ), and GIRK3 ( C ) were clearly evident in CA1 and CA3 pyramidal neurons, as well as granule cells of the dentate gyrus (DG). There was no specific staining for GIRK mRNAs in sections from the appropriate GIRK KO mouse (data not shown). Images are representative of data obtained from three different WT and GIRK KO panels. The WT mRNA distributions of GIRK1 ( D ), GIRK2 ( E ), and GIRK3 ( F ) were also examined in the SN. Scale bars: A-C , 500 μm; D-F , 100 μm.

    Techniques Used: In Situ Hybridization, Mouse Assay, Staining

    35) Product Images from "Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells"

    Article Title: Enhanced Production of Mesencephalic Dopaminergic Neurons from Lineage-Restricted Human Undifferentiated Stem Cells

    Journal: bioRxiv

    doi: 10.1101/2021.09.28.462222

    Generation of functional ventral midbrain DA neurons in vitro. a , Schematic diagram of the long-term (62 DIV) neuronal differentiation protocol. b , Representative immunohistochemical images of TH/FOXA2, TH/GIRK2, and TH/CALB1 costaining in H9 and 4X cells treated with 1 µM GSK3i on 83 DIV. Scale bars, 50 µm. c , Representative immunohistochemical analysis of COL3A1 and COL1A1 expression. Many H9 cells were double positive for COL3A1 and COL1A1, but no 4X cells were positive for COL3A1 or COL1A1. DAPI was used as a nuclear stain. Scale bars, 20 µm. d , Phase contrast image of a patched 4X neuron during whole-cell recording. Scale bar, 10 µm. e , Representative response (top trace) to a depolarizing current injection (bottom trace) showing firing of repetitive action potentials. f , Example of spontaneous firing at a resting membrane potential of -45 mV showing burst-like events. Overshooting spikes occurred in groups interspersed by periods of subthreshold membrane oscillation. g , Frequency distri bution of spontaneous cell firing showing firing frequencies ranging between 1 and 5 Hz (n = 16 cells). h , Dopamine content (normalized to the protein concentration) in 4X and H9 cells at 79 DIV, as measured by HPLC. The data are presented as the mean ± SD; n= 3. An unpaired t-test was used to compare groups. **P
    Figure Legend Snippet: Generation of functional ventral midbrain DA neurons in vitro. a , Schematic diagram of the long-term (62 DIV) neuronal differentiation protocol. b , Representative immunohistochemical images of TH/FOXA2, TH/GIRK2, and TH/CALB1 costaining in H9 and 4X cells treated with 1 µM GSK3i on 83 DIV. Scale bars, 50 µm. c , Representative immunohistochemical analysis of COL3A1 and COL1A1 expression. Many H9 cells were double positive for COL3A1 and COL1A1, but no 4X cells were positive for COL3A1 or COL1A1. DAPI was used as a nuclear stain. Scale bars, 20 µm. d , Phase contrast image of a patched 4X neuron during whole-cell recording. Scale bar, 10 µm. e , Representative response (top trace) to a depolarizing current injection (bottom trace) showing firing of repetitive action potentials. f , Example of spontaneous firing at a resting membrane potential of -45 mV showing burst-like events. Overshooting spikes occurred in groups interspersed by periods of subthreshold membrane oscillation. g , Frequency distri bution of spontaneous cell firing showing firing frequencies ranging between 1 and 5 Hz (n = 16 cells). h , Dopamine content (normalized to the protein concentration) in 4X and H9 cells at 79 DIV, as measured by HPLC. The data are presented as the mean ± SD; n= 3. An unpaired t-test was used to compare groups. **P

    Techniques Used: Functional Assay, In Vitro, Immunohistochemistry, Expressing, Staining, Injection, Protein Concentration, High Performance Liquid Chromatography

    36) Product Images from "G-Protein-Gated Potassium Channels Containing Kir3.2 and Kir3.3 Subunits Mediate the Acute Inhibitory Effects of Opioids on Locus Ceruleus Neurons"

    Article Title: G-Protein-Gated Potassium Channels Containing Kir3.2 and Kir3.3 Subunits Mediate the Acute Inhibitory Effects of Opioids on Locus Ceruleus Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-11-04328.2002

    Kir3 protein levels in wild-type and knock-out mouse brains. Membrane proteins from wild-type ( WT ), Kir3.2 knock-out ( 2 ko ), Kir3.3 knock-out ( 3 ko ), and Kir3.2/3.3 double knock-out ( 2/3 ko ) mouse brains were electrophoresed and probed using anti-Kir3 antibodies. Loading consistency was evaluated using an anti-NMDA receptor subunit ( NR1 ). Note that the heavily glycosylated form of Kir3.1 is most affected by the ablation of Kir3.2 and/or Kir3.3. Ab , Primary antibody.
    Figure Legend Snippet: Kir3 protein levels in wild-type and knock-out mouse brains. Membrane proteins from wild-type ( WT ), Kir3.2 knock-out ( 2 ko ), Kir3.3 knock-out ( 3 ko ), and Kir3.2/3.3 double knock-out ( 2/3 ko ) mouse brains were electrophoresed and probed using anti-Kir3 antibodies. Loading consistency was evaluated using an anti-NMDA receptor subunit ( NR1 ). Note that the heavily glycosylated form of Kir3.1 is most affected by the ablation of Kir3.2 and/or Kir3.3. Ab , Primary antibody.

    Techniques Used: Knock-Out

    Average resting membrane potentials of LC neurons from wild-type (−60.2 ± 1.0 mV), Kir3.3 knock-out ( 3 ko ) (−36.9 + 4.6 mV), Kir3.2 heterozygous ( 2 het ) (−45.2 ± 2.7 mV), Kir3.2 heterozygous/Kir3.3 knock-out ( 2 het/3 ko ) (−40.4 ± 2.0 mV), Kir3.2 knock-out ( 2 ko ) (−45.5 ± 3.2 mV), and Kir3.2/3.3 double knock-out ( 2/3 ko ) (−38.8 ± 2.9 mV) mice. The number of independent experiments for each group is shown at the top of each column; values shown are means ± SEM. * p
    Figure Legend Snippet: Average resting membrane potentials of LC neurons from wild-type (−60.2 ± 1.0 mV), Kir3.3 knock-out ( 3 ko ) (−36.9 + 4.6 mV), Kir3.2 heterozygous ( 2 het ) (−45.2 ± 2.7 mV), Kir3.2 heterozygous/Kir3.3 knock-out ( 2 het/3 ko ) (−40.4 ± 2.0 mV), Kir3.2 knock-out ( 2 ko ) (−45.5 ± 3.2 mV), and Kir3.2/3.3 double knock-out ( 2/3 ko ) (−38.8 ± 2.9 mV) mice. The number of independent experiments for each group is shown at the top of each column; values shown are means ± SEM. * p

    Techniques Used: Knock-Out, Mouse Assay

    The opioid-induced inhibition of voltage-gated calcium channels is normal in LC neurons from Kir3 knock-out mice. A , Example of the inward calcium/barium current elicited by a voltage step from −60 to −20 mV in an LC neuron from a Kir3.2/Kir3.3 double knock-out mouse ( control ), and its inhibition during ME perfusion. B , Summary of the calcium/barium current inhibition by ME observed in wild-type and Kir3 knock-out mice. Values shown are means ± SEM, and the number of cells tested in each group shown at the top of each column. 3 ko , Kir3.3 knock-out; 2 het , Kir3.2 heterozygous; 2 het/3 ko , Kir3.2 heterozygous/Kir3.3 knock-out; 2 ko , Kir3.2 knock-out; 2/3 ko , Kir3.2/3.3 double knock-out.
    Figure Legend Snippet: The opioid-induced inhibition of voltage-gated calcium channels is normal in LC neurons from Kir3 knock-out mice. A , Example of the inward calcium/barium current elicited by a voltage step from −60 to −20 mV in an LC neuron from a Kir3.2/Kir3.3 double knock-out mouse ( control ), and its inhibition during ME perfusion. B , Summary of the calcium/barium current inhibition by ME observed in wild-type and Kir3 knock-out mice. Values shown are means ± SEM, and the number of cells tested in each group shown at the top of each column. 3 ko , Kir3.3 knock-out; 2 het , Kir3.2 heterozygous; 2 het/3 ko , Kir3.2 heterozygous/Kir3.3 knock-out; 2 ko , Kir3.2 knock-out; 2/3 ko , Kir3.2/3.3 double knock-out.

    Techniques Used: Inhibition, Knock-Out, Mouse Assay

    37) Product Images from "Amelioration of non-motor dysfunctions after transplantation of human dopamine neurons in a model of Parkinson's disease"

    Article Title: Amelioration of non-motor dysfunctions after transplantation of human dopamine neurons in a model of Parkinson's disease

    Journal: Experimental Neurology

    doi: 10.1016/j.expneurol.2016.02.003

    Immunohistological analysis of hVM tissue at 20 weeks post-graft. Immunohistochemistry of TH + ve neurons (brown) and HuNu (blue) in the hVM graft (top panel). From left to right, images depict representative tissue from a Control rat (A), Lesion rat (B) and a large hVM graft (C). The central panel depicts A9 TH + ve neurons (green) co-labelled with Girk2 (red, D); A10 TH + ve neurons (green) co-labelled with Calbindin (red, E); × 10 magnification of hVM cells with TH + ve neurons stained in brown and HuNu + ve cells in blue (F). The bottom panel depicts the number of TH + ve cells per individual graft (G) and as a group mean (H), as well as the proportion of girk2 + ve (I) and calbindin + ve (J) cells out of the total number of TH + ve neurons. Scale bar = 1000 μm. Error bars = ± standard error of mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Immunohistological analysis of hVM tissue at 20 weeks post-graft. Immunohistochemistry of TH + ve neurons (brown) and HuNu (blue) in the hVM graft (top panel). From left to right, images depict representative tissue from a Control rat (A), Lesion rat (B) and a large hVM graft (C). The central panel depicts A9 TH + ve neurons (green) co-labelled with Girk2 (red, D); A10 TH + ve neurons (green) co-labelled with Calbindin (red, E); × 10 magnification of hVM cells with TH + ve neurons stained in brown and HuNu + ve cells in blue (F). The bottom panel depicts the number of TH + ve cells per individual graft (G) and as a group mean (H), as well as the proportion of girk2 + ve (I) and calbindin + ve (J) cells out of the total number of TH + ve neurons. Scale bar = 1000 μm. Error bars = ± standard error of mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Immunohistochemistry, Staining

    38) Product Images from "Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??"

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    Journal:

    doi: 10.1113/jphysiol.2009.173229

    GIRK2 is not regulated by Gα i3 in whole oocytes
    Figure Legend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Techniques Used:

    Functional differences between homomeric GIRK1 and GIRK2
    Figure Legend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Techniques Used: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches
    Figure Legend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Techniques Used:

    Biochemical and functional differences between GIRK1 and GIRK2
    Figure Legend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Techniques Used: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2
    Figure Legend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Techniques Used: Activity Assay

    39) Product Images from "The K+ channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia"

    Article Title: The K+ channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201201980

    Absence of GIRK channels in mouse DRG neurons Traces of currents in mouse DRG neurons stimulated with hyperpolarising voltage ramps from −120 to −40 mV and treated with 10 µM DAMGO followed by washout. No differences in peak currents at −120 mV in mouse nociceptors ( n = 28) and mechanoreceptors ( n = 19) after 10 µM DAMGO application. Representative action potential shapes of nociceptors and mechanoreceptors (lower panel) were used to distinguish between DRG neuron subtypes ( p = 0.237; Mann–Whitney Rank Sum test). Representative current traces of mouse DRG neurons at −80 mV after application of 10 µM DAMGO, 3 mM barium or 20 µM naloxone recorded in high potassium solution. Peak current amplitude after agonist and antagonist application ( p = 0.066; one way repeated measures ANOVA, n = 8). Quantification of GIRK channel mRNA in mouse cerebellum, heart and DRG neurons from naïve animals and from animals with CFA-induced unilateral hindpaw inflammation (inflamed: CFA ipsi; noninflamed: CFA contra). (GIRK1 and GIRK2: n = 7; GIRK3 and GIRK4: n = 6; Kruskal–Wallis ANOVA on Ranks, Dunn's method compared to cerebellum or heart * p
    Figure Legend Snippet: Absence of GIRK channels in mouse DRG neurons Traces of currents in mouse DRG neurons stimulated with hyperpolarising voltage ramps from −120 to −40 mV and treated with 10 µM DAMGO followed by washout. No differences in peak currents at −120 mV in mouse nociceptors ( n = 28) and mechanoreceptors ( n = 19) after 10 µM DAMGO application. Representative action potential shapes of nociceptors and mechanoreceptors (lower panel) were used to distinguish between DRG neuron subtypes ( p = 0.237; Mann–Whitney Rank Sum test). Representative current traces of mouse DRG neurons at −80 mV after application of 10 µM DAMGO, 3 mM barium or 20 µM naloxone recorded in high potassium solution. Peak current amplitude after agonist and antagonist application ( p = 0.066; one way repeated measures ANOVA, n = 8). Quantification of GIRK channel mRNA in mouse cerebellum, heart and DRG neurons from naïve animals and from animals with CFA-induced unilateral hindpaw inflammation (inflamed: CFA ipsi; noninflamed: CFA contra). (GIRK1 and GIRK2: n = 7; GIRK3 and GIRK4: n = 6; Kruskal–Wallis ANOVA on Ranks, Dunn's method compared to cerebellum or heart * p

    Techniques Used: MANN-WHITNEY

    Expression of functional GIRK2 channels in DRG neurons of Nav1.8-GIRK2 transgenic mice GIRK2 mRNA expression in cerebellum, spinal cord and DRG neurons of Nav1.8-GIRK2 and wildtype mice (* p = 0.05, One-way ANOVA, Holm–Sidak method, n = 3). Skin sections from Nav1.8-GIRK2 mice labeled with antibodies specific for GIRK2 and IB4. Immunoreactivity for FLAG and GIRK2 demonstrating the expression of exogenous Flag-GIRK2 in sciatic nerve (uppermost panel) and DRG neurons (lower panels) in Nav1.8-GIRK2 mice. Sections of DRGs from Nav1.8-GIRK2 mice immunostained with anti-FLAG and co-stained with IB4, anti-NF200 and anti-CGRP. All scale bars are 50 µm. Representative current traces of Nav1.8-GIRK2 DRG neurons recorded in high potassium solution. Neurons voltage-clamped at −80 mV showing large inward currents evoked by 10 µM DAMGO and inhibited by barium (3 mM) and naloxone (20 µM). Peak current amplitude after application of agonists and antagonists in wildtype and Nav1.8-GIRK2 DRG neurons (*** p = 0.001, two way ANOVA, Holm–Sidak method, n = 9–12 cells per group). All scale bars are 50 µm (B–D).
    Figure Legend Snippet: Expression of functional GIRK2 channels in DRG neurons of Nav1.8-GIRK2 transgenic mice GIRK2 mRNA expression in cerebellum, spinal cord and DRG neurons of Nav1.8-GIRK2 and wildtype mice (* p = 0.05, One-way ANOVA, Holm–Sidak method, n = 3). Skin sections from Nav1.8-GIRK2 mice labeled with antibodies specific for GIRK2 and IB4. Immunoreactivity for FLAG and GIRK2 demonstrating the expression of exogenous Flag-GIRK2 in sciatic nerve (uppermost panel) and DRG neurons (lower panels) in Nav1.8-GIRK2 mice. Sections of DRGs from Nav1.8-GIRK2 mice immunostained with anti-FLAG and co-stained with IB4, anti-NF200 and anti-CGRP. All scale bars are 50 µm. Representative current traces of Nav1.8-GIRK2 DRG neurons recorded in high potassium solution. Neurons voltage-clamped at −80 mV showing large inward currents evoked by 10 µM DAMGO and inhibited by barium (3 mM) and naloxone (20 µM). Peak current amplitude after application of agonists and antagonists in wildtype and Nav1.8-GIRK2 DRG neurons (*** p = 0.001, two way ANOVA, Holm–Sidak method, n = 9–12 cells per group). All scale bars are 50 µm (B–D).

    Techniques Used: Expressing, Functional Assay, Transgenic Assay, Mouse Assay, Labeling, Staining

    Expression of GIRK channels in rat and human sensory neurons Current traces of rat DRG neurons voltage-clamped at −80 mV. Inward currents were recorded in high potassium solution. Currents were evoked by 10 µM DAMGO and suppressed by GIRK channel blocker barium (3 mM) and opioid receptor antagonist naloxone (20 µM). Peak currents after agonist and antagonist application (* p = 0.037; one way repeated measures ANOVA, Holm–Sidak method, n = 7). Quantification of GIRK1- and -2 mRNA in rat and (postmortem) human cerebellum and naïve DRG, and in rat DRG neurons innervating the inflamed (CFA ipsi) or the non-inflamed (CFA contra) paw ( n ≥ 3). Immunoreactivity for GIRK1 and GIRK2 in rat DRG sections. GIRK channels are detectable in nonpeptidergic IB4 positive nociceptors but not in myelinated neurons expressing NF200. Rat skin cryosections (E) and human skin paraffin sections (F) stained with antibodies specific for GIRK1, GIRK2, IB4 and PGP9.5. All scale bars are 50 µm.
    Figure Legend Snippet: Expression of GIRK channels in rat and human sensory neurons Current traces of rat DRG neurons voltage-clamped at −80 mV. Inward currents were recorded in high potassium solution. Currents were evoked by 10 µM DAMGO and suppressed by GIRK channel blocker barium (3 mM) and opioid receptor antagonist naloxone (20 µM). Peak currents after agonist and antagonist application (* p = 0.037; one way repeated measures ANOVA, Holm–Sidak method, n = 7). Quantification of GIRK1- and -2 mRNA in rat and (postmortem) human cerebellum and naïve DRG, and in rat DRG neurons innervating the inflamed (CFA ipsi) or the non-inflamed (CFA contra) paw ( n ≥ 3). Immunoreactivity for GIRK1 and GIRK2 in rat DRG sections. GIRK channels are detectable in nonpeptidergic IB4 positive nociceptors but not in myelinated neurons expressing NF200. Rat skin cryosections (E) and human skin paraffin sections (F) stained with antibodies specific for GIRK1, GIRK2, IB4 and PGP9.5. All scale bars are 50 µm.

    Techniques Used: Expressing, Staining

    Identification of a regulatory sequence in the rat Kcnj6 gene that drives expression in peripheral sensory neurons Sequence alignment of rat and mouse Kcnj6 upstream of the transcription start-site. Dotted black line indicates sequence that was absent from public databases. Dotted orange lines designate regions that were deleted from the R-1195 reporter construct. eGFP fluorescence in mouse DRG cultures transfected with R-1195-eGFP, R-1195Δ1195–1142-eGFP and R-1195Δ1141–1043-eGFP reporter constructs. Insets show phase contrast images. Co-staining of R-1195-eGFP transfected sensory neurons with anti-CGRP. Co-staining of R-1195-eGFP with IB4. All scale bars are 100 μm.
    Figure Legend Snippet: Identification of a regulatory sequence in the rat Kcnj6 gene that drives expression in peripheral sensory neurons Sequence alignment of rat and mouse Kcnj6 upstream of the transcription start-site. Dotted black line indicates sequence that was absent from public databases. Dotted orange lines designate regions that were deleted from the R-1195 reporter construct. eGFP fluorescence in mouse DRG cultures transfected with R-1195-eGFP, R-1195Δ1195–1142-eGFP and R-1195Δ1141–1043-eGFP reporter constructs. Insets show phase contrast images. Co-staining of R-1195-eGFP transfected sensory neurons with anti-CGRP. Co-staining of R-1195-eGFP with IB4. All scale bars are 100 μm.

    Techniques Used: Sequencing, Expressing, Construct, Fluorescence, Transfection, Staining

    Antinociceptive effect of peripherally applied DAMGO in Nav1.8-GIRK2 mice Changes in paw withdrawal latency in response to radiant heat in the inflamed (ipsilateral) and noninflamed (contralateral) paws of Nav1.8-GIRK2 and wildtype mice. Dose-dependent inhibition of thermal hyperalgesia 5 min after DAMGO injection into the inflamed paw of Nav1.8-GIRK2 mice (unadjusted p -values: *** p = 0.000, ** p = 0.004; two way ANOVA, Holm–Sidak method, n = 7–8). DAMGO-induced antinociception is reversed by co-injection of naloxone-methiodide (NLXM, 5µg) in the inflamed paw of Nav1.8-GIRK2 mice (*** p
    Figure Legend Snippet: Antinociceptive effect of peripherally applied DAMGO in Nav1.8-GIRK2 mice Changes in paw withdrawal latency in response to radiant heat in the inflamed (ipsilateral) and noninflamed (contralateral) paws of Nav1.8-GIRK2 and wildtype mice. Dose-dependent inhibition of thermal hyperalgesia 5 min after DAMGO injection into the inflamed paw of Nav1.8-GIRK2 mice (unadjusted p -values: *** p = 0.000, ** p = 0.004; two way ANOVA, Holm–Sidak method, n = 7–8). DAMGO-induced antinociception is reversed by co-injection of naloxone-methiodide (NLXM, 5µg) in the inflamed paw of Nav1.8-GIRK2 mice (*** p

    Techniques Used: Mouse Assay, Inhibition, Injection

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    Alomone Labs anti girk2 kir3 2 antibody
    Validation of <t>GIRK2</t> 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 <t>antibody</t> (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
    Anti Girk2 Kir3 2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    GIRK2 is not regulated by Gα i3 in whole oocytes

    Journal:

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    doi: 10.1113/jphysiol.2009.173229

    Figure Lengend Snippet: GIRK2 is not regulated by Gα i3 in whole oocytes

    Article Snippet: The asymmetric interaction of GIRK1 and GIRK2 with Gαi3 βγ suggested a different regulation of function of each subunit within the heteromeric channel.

    Techniques:

    Functional differences between homomeric GIRK1 and GIRK2

    Journal:

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    doi: 10.1113/jphysiol.2009.173229

    Figure Lengend Snippet: Functional differences between homomeric GIRK1 and GIRK2

    Article Snippet: The asymmetric interaction of GIRK1 and GIRK2 with Gαi3 βγ suggested a different regulation of function of each subunit within the heteromeric channel.

    Techniques: Functional Assay

    Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Journal:

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    doi: 10.1113/jphysiol.2009.173229

    Figure Lengend Snippet: Gα i3 GA does not regulate GIRK2 in excised plasma membrane patches

    Article Snippet: The asymmetric interaction of GIRK1 and GIRK2 with Gαi3 βγ suggested a different regulation of function of each subunit within the heteromeric channel.

    Techniques:

    Biochemical and functional differences between GIRK1 and GIRK2

    Journal:

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    doi: 10.1113/jphysiol.2009.173229

    Figure Lengend Snippet: Biochemical and functional differences between GIRK1 and GIRK2

    Article Snippet: The asymmetric interaction of GIRK1 and GIRK2 with Gαi3 βγ suggested a different regulation of function of each subunit within the heteromeric channel.

    Techniques: Functional Assay

    The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Journal:

    Article Title: Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K+ channel by G?iGDP and G??

    doi: 10.1113/jphysiol.2009.173229

    Figure Lengend Snippet: The basal activity is Gβγ dependent in GIRK1*, but Gβγ independent in GIRK2

    Article Snippet: The asymmetric interaction of GIRK1 and GIRK2 with Gαi3 βγ suggested a different regulation of function of each subunit within the heteromeric channel.

    Techniques: Activity Assay