rabbit anti trek 1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti trek 1
    MOR and <t>TREK-1</t> co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
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    Images

    1) Product Images from "Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes"

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00319

    MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
    Figure Legend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Techniques Used: Staining, Labeling

    Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P
    Figure Legend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Techniques Used: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

    MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.
    Figure Legend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Techniques Used: Mouse Assay, Staining

    2) Product Images from "Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ"

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00013

    Genetic deletion of TWIK-1 and TREK-1 genes together does not alter the electrophysiological properties of astrocytes. (A,B) Bar graph summary of the V M and R in from WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current profiles from WT and TWIK-1 −/− /TREK-1 −/− astrocytes, respectively. (D) Averaged I-V plots from these two genotypes, where the whole-cell current amplitudes in both inward and outward directions were comparable. (E) The RI values were also comparable between the two genotypes.
    Figure Legend Snippet: Genetic deletion of TWIK-1 and TREK-1 genes together does not alter the electrophysiological properties of astrocytes. (A,B) Bar graph summary of the V M and R in from WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current profiles from WT and TWIK-1 −/− /TREK-1 −/− astrocytes, respectively. (D) Averaged I-V plots from these two genotypes, where the whole-cell current amplitudes in both inward and outward directions were comparable. (E) The RI values were also comparable between the two genotypes.

    Techniques Used:

    Quinine does not reveal the functional contribution of TWIK-1 and TREK-1 in single or double gene knockout mice. (A) Representative astrocyte V M recordings first in 100 μM BaCl 2 bath application for 5 min, followed by addition of 400 μM quinine for 20 min, from a WT, TREK-1 −/− , TWIK-1 −/− and TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 400 μM quinine-induced V M depolarization (Δ V M 1) and the total V M depolarization induced by BaCl 2 plus quinine from all four genotypes. (C) Representative whole-cell current recordings first in aCSF as control, then in 100 μM BaCl 2 plus 400 μM quinine, and washout. Representative I–V relationships were shown in the right panel. (D) Summary of RI values from four genotypes obtained from astrocyte recordings in the presence of 400 μM quinine and 100 μM BaCl 2 together in bath. The RI values were comparable among the four genotypes.
    Figure Legend Snippet: Quinine does not reveal the functional contribution of TWIK-1 and TREK-1 in single or double gene knockout mice. (A) Representative astrocyte V M recordings first in 100 μM BaCl 2 bath application for 5 min, followed by addition of 400 μM quinine for 20 min, from a WT, TREK-1 −/− , TWIK-1 −/− and TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 400 μM quinine-induced V M depolarization (Δ V M 1) and the total V M depolarization induced by BaCl 2 plus quinine from all four genotypes. (C) Representative whole-cell current recordings first in aCSF as control, then in 100 μM BaCl 2 plus 400 μM quinine, and washout. Representative I–V relationships were shown in the right panel. (D) Summary of RI values from four genotypes obtained from astrocyte recordings in the presence of 400 μM quinine and 100 μM BaCl 2 together in bath. The RI values were comparable among the four genotypes.

    Techniques Used: Functional Assay, Double Knockout, Mouse Assay, In Situ

    TREK-1 gene deletion does not alter the electrophysiological properties of astrocytes. (A) Identification of astrocyte in hippocampal slices based on cellular morphology and positive SR101 staining in CA1 region. (B,C) Membrane potential ( V M ), and input resistance ( R in ) in WT and TREK-1 −/− astrocytes. (D) Representative astrocyte whole-cell passive conductance from a WT and a TREK-1 −/− astrocyte are shown separately as indicated. The voltage commands used for whole-cell current induction are displayed on the left panel. (E) I-V plots show the averaged current amplitudes from WT and TREK-1 −/− astrocytes. The equation for rectification index (RI) is also illustrated and the resulted RI values are summarized in (F) .
    Figure Legend Snippet: TREK-1 gene deletion does not alter the electrophysiological properties of astrocytes. (A) Identification of astrocyte in hippocampal slices based on cellular morphology and positive SR101 staining in CA1 region. (B,C) Membrane potential ( V M ), and input resistance ( R in ) in WT and TREK-1 −/− astrocytes. (D) Representative astrocyte whole-cell passive conductance from a WT and a TREK-1 −/− astrocyte are shown separately as indicated. The voltage commands used for whole-cell current induction are displayed on the left panel. (E) I-V plots show the averaged current amplitudes from WT and TREK-1 −/− astrocytes. The equation for rectification index (RI) is also illustrated and the resulted RI values are summarized in (F) .

    Techniques Used: Staining

    K ir4.1 inhibition does not reveal the functional contribution of TREK-1 in TREK-1 −/− astrocytes. (A) Representative V M response to K ir 4.1 inhibitor 100 μM BaCl 2 from a WT and a TREK-1 −/− astrocyte, as indicated in situ . Δ V M indicates the peak V M depolarization during a 5 min BaCl 2 bath application. (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. (D) I-V plots derived from recordings in (C) . The Ba 2+ -sensitive currents in I-V plots were obtained from sweep subtraction. The Ba 2+ -sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and TREK-1 KO, RI = 0.90, respectively. (E) Summary of RI values from WT and TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.
    Figure Legend Snippet: K ir4.1 inhibition does not reveal the functional contribution of TREK-1 in TREK-1 −/− astrocytes. (A) Representative V M response to K ir 4.1 inhibitor 100 μM BaCl 2 from a WT and a TREK-1 −/− astrocyte, as indicated in situ . Δ V M indicates the peak V M depolarization during a 5 min BaCl 2 bath application. (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. (D) I-V plots derived from recordings in (C) . The Ba 2+ -sensitive currents in I-V plots were obtained from sweep subtraction. The Ba 2+ -sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and TREK-1 KO, RI = 0.90, respectively. (E) Summary of RI values from WT and TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Techniques Used: Inhibition, Functional Assay, In Situ, Derivative Assay

    TREK-1 channels are predominantly located in cytoplasm. (A) Fractionation western blot results revealed the subcellular distribution of TREK-1 channels in cytoplasmic fraction vs . membrane fraction in two independent tests of mice hippocampal samples. Anti-glial fibrillary acidic protein (GFAP) (50 kDa) and ATP1α2 (112 kDa) were markers for cytoplasmic and membrane fractions, respectively. The blots shown in (A) were first incubated with anti-TREK-1 antibody and then re-probed with the rest of other primary antibodies sequentially after the original membranes were stripped with stripping buffer (see “Materials and Methods” Section). (B) Bar graph summary showing the relative ratio of TREK-1 proteins located in cytoplasmic vs . membrane fractions. Data are shown as mean ± SEM. Numbers indicate the times of observations. ** p
    Figure Legend Snippet: TREK-1 channels are predominantly located in cytoplasm. (A) Fractionation western blot results revealed the subcellular distribution of TREK-1 channels in cytoplasmic fraction vs . membrane fraction in two independent tests of mice hippocampal samples. Anti-glial fibrillary acidic protein (GFAP) (50 kDa) and ATP1α2 (112 kDa) were markers for cytoplasmic and membrane fractions, respectively. The blots shown in (A) were first incubated with anti-TREK-1 antibody and then re-probed with the rest of other primary antibodies sequentially after the original membranes were stripped with stripping buffer (see “Materials and Methods” Section). (B) Bar graph summary showing the relative ratio of TREK-1 proteins located in cytoplasmic vs . membrane fractions. Data are shown as mean ± SEM. Numbers indicate the times of observations. ** p

    Techniques Used: Fractionation, Western Blot, Mouse Assay, Incubation, Stripping Membranes

    K ir 4.1 inhibition does not reveal the functional contribution of TWIK-1/TREK-1 in double gene knockout mice. (A) Representative V M response to K ir 4.1 inhibitor, 100 μM BaCl 2 , from a WT and a TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. I–V relationships were shown in (D) . (D) I–V plots derived from recordings in (C) . The Ba 2+ -sensitive currents, in I-V plots were obtained from sweep subtraction. The Ba 2+ - sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and double gene knockout mice, RI = 0.90, respectively. (E) Summary of RI values from WT and TWIK-1 −/− /TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.
    Figure Legend Snippet: K ir 4.1 inhibition does not reveal the functional contribution of TWIK-1/TREK-1 in double gene knockout mice. (A) Representative V M response to K ir 4.1 inhibitor, 100 μM BaCl 2 , from a WT and a TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. I–V relationships were shown in (D) . (D) I–V plots derived from recordings in (C) . The Ba 2+ -sensitive currents, in I-V plots were obtained from sweep subtraction. The Ba 2+ - sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and double gene knockout mice, RI = 0.90, respectively. (E) Summary of RI values from WT and TWIK-1 −/− /TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Techniques Used: Inhibition, Functional Assay, Double Knockout, Mouse Assay, In Situ, Derivative Assay

    Expression of astrocyte K + channels in TREK-1, TWIK-1 single, and TWIK-1/TREK-1 double gene knockout mice. (A) Schematic illustrations of genetic deletion of four transmembrane domains, including two pore-forming regions, in TREK-1 knockout (TREK-1 −/− , upper), and TWIK-1 knockout (TWIK-1 −/− , lower) mice. (B) PCR genotyping confirmation of successful genetic knockout of the targeted genes in TWIK-1 −/− , TREK-1 −/− , and TWIK-1/TREK-1 double knockout (TWIK-1 −/− /TREK-1 −/− ) mice. (C) The relative quantity of mRNA of K + channels in freshly dissociated mature astrocytes. The results were examined from wild type (WT), TWIK-1 −/− , TREK- 1−/− , and TWIK-1 −/− /TREK-1 −/− astrocytes using qRT-PCR.
    Figure Legend Snippet: Expression of astrocyte K + channels in TREK-1, TWIK-1 single, and TWIK-1/TREK-1 double gene knockout mice. (A) Schematic illustrations of genetic deletion of four transmembrane domains, including two pore-forming regions, in TREK-1 knockout (TREK-1 −/− , upper), and TWIK-1 knockout (TWIK-1 −/− , lower) mice. (B) PCR genotyping confirmation of successful genetic knockout of the targeted genes in TWIK-1 −/− , TREK-1 −/− , and TWIK-1/TREK-1 double knockout (TWIK-1 −/− /TREK-1 −/− ) mice. (C) The relative quantity of mRNA of K + channels in freshly dissociated mature astrocytes. The results were examined from wild type (WT), TWIK-1 −/− , TREK- 1−/− , and TWIK-1 −/− /TREK-1 −/− astrocytes using qRT-PCR.

    Techniques Used: Expressing, Double Knockout, Mouse Assay, Knock-Out, Polymerase Chain Reaction, Genotyping Assay, Quantitative RT-PCR

    3) Product Images from "Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction"

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908792

    Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.
    Figure Legend Snippet: Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Techniques Used: Immunohistochemistry

    TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).
    Figure Legend Snippet: The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Techniques Used:

    4) Product Images from "Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice"

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1485-5

    Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p
    Figure Legend Snippet: Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p

    Techniques Used: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

    Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p
    Figure Legend Snippet: Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p

    Techniques Used: Expressing, Staining

    Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p
    Figure Legend Snippet: Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p

    Techniques Used: Functional Assay, Staining, Double Staining, TUNEL Assay

    Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p
    Figure Legend Snippet: Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p

    Techniques Used: Expressing, Mouse Assay, Staining, Western Blot

    TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p
    Figure Legend Snippet: TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p

    Techniques Used: Mouse Assay, Magnetic Resonance Imaging, Staining, Western Blot, Expressing

    Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p
    Figure Legend Snippet: Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p

    Techniques Used: Expressing, Staining, Western Blot

    Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p
    Figure Legend Snippet: Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p

    Techniques Used: Transmission Electron Microscopy

    5) Product Images from "Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction"

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908792

    Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.
    Figure Legend Snippet: Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Techniques Used: Immunohistochemistry

    TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).
    Figure Legend Snippet: The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Techniques Used:

    6) Product Images from "Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction"

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908792

    Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.
    Figure Legend Snippet: Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Techniques Used: Immunohistochemistry

    TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).
    Figure Legend Snippet: The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Techniques Used:

    7) Product Images from "The Knockdown of TREK-1 in Hippocampal Neurons Attenuate Lipopolysaccharide-Induced Depressive-Like Behavior in Mice"

    Article Title: The Knockdown of TREK-1 in Hippocampal Neurons Attenuate Lipopolysaccharide-Induced Depressive-Like Behavior in Mice

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20235902

    Lipopolysaccharide (LPS) increases the expression of TREK-1 in the hippocampus. ( A ) Experimental procedure for the LPS injection test schedule. Viruses were injected into the bilateral dentate gyrus, followed by a 21-day recovery (Day −21). LPS (1.2 mg/kg) or its vehicle was administered 1 time (Day 0), and subsequently, 4 h later the open field test (OFT) and 24 h later the OFT and tail suspension test were performed. ( B ) An illustration of a hippocampal slice of pSico-Red-shTREK-1 mice showing the site of AAV-hSyn-BFP (neuronal control) or AAV-hSyn-BFP-Cre (neuronal Cre, nCre) injection. ( C ) Immunohistochemical staining of the hippocampal slice with the anti-TREK-1 antibody. ( D ) Quantitative real-time polymerase chain reaction analysis of TREK-1 in the dentate gyrus. The numbers inside each bar indicate the number of sample ( E ) Protein expression of the green fluorescent protein, mCherry, and TREK-1 in the dentate gyrus. Data are presented as means ± standard error of the mean (* p
    Figure Legend Snippet: Lipopolysaccharide (LPS) increases the expression of TREK-1 in the hippocampus. ( A ) Experimental procedure for the LPS injection test schedule. Viruses were injected into the bilateral dentate gyrus, followed by a 21-day recovery (Day −21). LPS (1.2 mg/kg) or its vehicle was administered 1 time (Day 0), and subsequently, 4 h later the open field test (OFT) and 24 h later the OFT and tail suspension test were performed. ( B ) An illustration of a hippocampal slice of pSico-Red-shTREK-1 mice showing the site of AAV-hSyn-BFP (neuronal control) or AAV-hSyn-BFP-Cre (neuronal Cre, nCre) injection. ( C ) Immunohistochemical staining of the hippocampal slice with the anti-TREK-1 antibody. ( D ) Quantitative real-time polymerase chain reaction analysis of TREK-1 in the dentate gyrus. The numbers inside each bar indicate the number of sample ( E ) Protein expression of the green fluorescent protein, mCherry, and TREK-1 in the dentate gyrus. Data are presented as means ± standard error of the mean (* p

    Techniques Used: Expressing, Injection, Mouse Assay, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    Reduced neurotrophic factor expression in lipopolysaccharide (LPS)-induced depression was preserved in the hippocampal neuronal TREK-1 conditional knockdown mice. ( A ) Serum corticosterone levels in saline or LPS-injected mice. ( B ) Enzyme-linked immunosorbent assay for the vascular endothelial growth factor (VEGF) in mice hippocampal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of insulin growth factor-1 (IGF-1) ( C ) and brain-derived neurotrophic factor (BDNF) ( D ). I Western blot analysis from the hippocampal tissues infected with neuronal CTL or neuronal Cre virus ( E ). Corresponding quantified densitometry of the western blotting results, as shown in I for IGF-1 ( F ) and BDNF ( G ). qRT-PCR analysis of VEGFR receptor-1 ( H ), VEGFR receptor-2 (VEGFR-2) ( I ), IGF-1r ( J ), and Ntrk2 ( K ). ( L ) Western blot analysis of hippocampal tissues. Blots were performed using the antibodies indicated at right. Corresponding quantified densitometry of the western blotting results, as shown in ( L ) for VEGFR-2 ( M ), IGF-1r ( N ), and Ntrk2 ( O ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (* p
    Figure Legend Snippet: Reduced neurotrophic factor expression in lipopolysaccharide (LPS)-induced depression was preserved in the hippocampal neuronal TREK-1 conditional knockdown mice. ( A ) Serum corticosterone levels in saline or LPS-injected mice. ( B ) Enzyme-linked immunosorbent assay for the vascular endothelial growth factor (VEGF) in mice hippocampal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of insulin growth factor-1 (IGF-1) ( C ) and brain-derived neurotrophic factor (BDNF) ( D ). I Western blot analysis from the hippocampal tissues infected with neuronal CTL or neuronal Cre virus ( E ). Corresponding quantified densitometry of the western blotting results, as shown in I for IGF-1 ( F ) and BDNF ( G ). qRT-PCR analysis of VEGFR receptor-1 ( H ), VEGFR receptor-2 (VEGFR-2) ( I ), IGF-1r ( J ), and Ntrk2 ( K ). ( L ) Western blot analysis of hippocampal tissues. Blots were performed using the antibodies indicated at right. Corresponding quantified densitometry of the western blotting results, as shown in ( L ) for VEGFR-2 ( M ), IGF-1r ( N ), and Ntrk2 ( O ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (* p

    Techniques Used: Expressing, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Western Blot, Infection

    Validation of the hippocampal TWIK-related potassium channel-1 (TREK-1) conditional knockdown (cKD) mice. ( A ) An illustration of a hippocampal slice of TREK-1 cKD mice to show the site of adeno-associated virus (AAV)-elongation factor 1 alpha (EF1α)-mCherry (Control [CTL]) or AAV-EF1α-mCherry-IRES-Cre (Cre) injection. ( B ) Scheme representation of green fluorescent protein, mCherry, and TREK-1 under CTL or Cre virus infection. TREK-1 signals were compared by immunohistochemical staining in the dentate gyrus injected with CTL or Cre virus in pSico-Red-shTREK-1 mice. Validation of TREK-1 cKD efficiency of messenger ribonucleic acid levels by real-time polymerase chain reaction (RT-PCR) ( C – E ) and quantitative RT-PCR ©. Validation of TREK-1 cKD efficiency of protein levels by Western blotting ( F , G ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (* p
    Figure Legend Snippet: Validation of the hippocampal TWIK-related potassium channel-1 (TREK-1) conditional knockdown (cKD) mice. ( A ) An illustration of a hippocampal slice of TREK-1 cKD mice to show the site of adeno-associated virus (AAV)-elongation factor 1 alpha (EF1α)-mCherry (Control [CTL]) or AAV-EF1α-mCherry-IRES-Cre (Cre) injection. ( B ) Scheme representation of green fluorescent protein, mCherry, and TREK-1 under CTL or Cre virus infection. TREK-1 signals were compared by immunohistochemical staining in the dentate gyrus injected with CTL or Cre virus in pSico-Red-shTREK-1 mice. Validation of TREK-1 cKD efficiency of messenger ribonucleic acid levels by real-time polymerase chain reaction (RT-PCR) ( C – E ) and quantitative RT-PCR ©. Validation of TREK-1 cKD efficiency of protein levels by Western blotting ( F , G ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (* p

    Techniques Used: Mouse Assay, Injection, Infection, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    The knockdown of neuronal TREK-1 in the hippocampus did not show cytokine changes under lipopolysaccharide (LPS)-induced depression. ( A ) Multiple cytokine array dot blots in the hippocampal lysates infected with neuronal control or neuronal Cre virus. Quantitative real-time polymerase chain reaction analysis of interleukin 1 beta ( B ) and tumor necrosis factor α ( C ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (** p
    Figure Legend Snippet: The knockdown of neuronal TREK-1 in the hippocampus did not show cytokine changes under lipopolysaccharide (LPS)-induced depression. ( A ) Multiple cytokine array dot blots in the hippocampal lysates infected with neuronal control or neuronal Cre virus. Quantitative real-time polymerase chain reaction analysis of interleukin 1 beta ( B ) and tumor necrosis factor α ( C ). The numbers inside each bar indicate the number of samples. Data are presented as means ± standard error of the mean (** p

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    The knockdown of neuronal TREK-1 in the hippocampus exhibited antidepressant behavior. ( A ) Bodyweight changes in the lipopolysaccharide (LPS) or saline-injected groups (neuronal control [nCTL] saline = 8, nCTL LPS = 8, neuronal Cre [nCre] saline = 8, nCre LPS = 8). ( B ) The representative track generated from the open field test on mice injected with LPS or saline. Velocity ( C ) and total distance ( D ) moved time of mice administered with LPS and saline after 4 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). Vel©ty ( E ) and total distance ( F ) moved time of mice administered with LPS and saline after 24 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). ( G ) Immobility time and ( H ) struggling time from the tail suspension test on mice injected with LPS after 24 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). Data are presented as means ± standard error of the mean (** p
    Figure Legend Snippet: The knockdown of neuronal TREK-1 in the hippocampus exhibited antidepressant behavior. ( A ) Bodyweight changes in the lipopolysaccharide (LPS) or saline-injected groups (neuronal control [nCTL] saline = 8, nCTL LPS = 8, neuronal Cre [nCre] saline = 8, nCre LPS = 8). ( B ) The representative track generated from the open field test on mice injected with LPS or saline. Velocity ( C ) and total distance ( D ) moved time of mice administered with LPS and saline after 4 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). Vel©ty ( E ) and total distance ( F ) moved time of mice administered with LPS and saline after 24 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). ( G ) Immobility time and ( H ) struggling time from the tail suspension test on mice injected with LPS after 24 h (nCTL saline = 8, nCTL LPS = 8, nCre saline = 8, nCre LPS = 8). Data are presented as means ± standard error of the mean (** p

    Techniques Used: Injection, Generated, Mouse Assay

    Generation of transgenic mice for the Cre-dependent knockdown of TWIK-related potassium channel-1 (TREK-1). ( A ) Scheme of the mice model of TREK-1 conditional knockdown. ( B ) Genotypes of mice were identified using genomic deoxyribonucleic acid. Primers elongation factor 1 alpha pro and mCherry-N were used to distinguish the targeted allele (515 bp) from the untargeted wild-type allele. ( C ) Protein samples from mice hippocampus were tested for the protein expression of green fluorescent protein, mCherry, and TREK-1 by western blot analysis. Actin was used as a loading control.
    Figure Legend Snippet: Generation of transgenic mice for the Cre-dependent knockdown of TWIK-related potassium channel-1 (TREK-1). ( A ) Scheme of the mice model of TREK-1 conditional knockdown. ( B ) Genotypes of mice were identified using genomic deoxyribonucleic acid. Primers elongation factor 1 alpha pro and mCherry-N were used to distinguish the targeted allele (515 bp) from the untargeted wild-type allele. ( C ) Protein samples from mice hippocampus were tested for the protein expression of green fluorescent protein, mCherry, and TREK-1 by western blot analysis. Actin was used as a loading control.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Western Blot

    8) Product Images from "Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice"

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1485-5

    Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p
    Figure Legend Snippet: Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p

    Techniques Used: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

    Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p
    Figure Legend Snippet: Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p

    Techniques Used: Expressing, Staining

    Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p
    Figure Legend Snippet: Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p

    Techniques Used: Functional Assay, Staining, Double Staining, TUNEL Assay

    Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p
    Figure Legend Snippet: Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p

    Techniques Used: Expressing, Mouse Assay, Staining, Western Blot

    TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p
    Figure Legend Snippet: TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p

    Techniques Used: Mouse Assay, Magnetic Resonance Imaging, Staining, Western Blot, Expressing

    Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p
    Figure Legend Snippet: Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p

    Techniques Used: Expressing, Staining, Western Blot

    Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p
    Figure Legend Snippet: Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p

    Techniques Used: Transmission Electron Microscopy

    9) Product Images from "Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels"

    Article Title: Spadin Modulates Astrocytic Passive Conductance via Inhibition of TWIK-1/TREK-1 Heterodimeric Channels

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21249639

    Spadin inhibits TREK-1 mediated current in cultured astrocytes. ( A ) Current densities induced by voltage ramping were measured before and after application of 10 µM spadin (SP) in astrocytes transfected with scrambled control (Sc) short hairpin-forming interfering RNA (shRNA) (Sc sh) or TREK-1 shRNA. ( B ) Summary bar graph showing averaged currents with or without spadin treatment at +50 mV and −150 mV in ( A ). The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p
    Figure Legend Snippet: Spadin inhibits TREK-1 mediated current in cultured astrocytes. ( A ) Current densities induced by voltage ramping were measured before and after application of 10 µM spadin (SP) in astrocytes transfected with scrambled control (Sc) short hairpin-forming interfering RNA (shRNA) (Sc sh) or TREK-1 shRNA. ( B ) Summary bar graph showing averaged currents with or without spadin treatment at +50 mV and −150 mV in ( A ). The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p

    Techniques Used: Cell Culture, Transfection, shRNA

    Spadin does not affect astrocytic passive conductance in mice with TREK-1 or TWIK-1 knockdown (KD). ( A ) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in hippocampal astrocytes after injection with lentiviruses expressing scrambled control shRNA (ScRNA), TWIK-1 shRNA, or TREK-1 shRNA in the absence or presence of 10 µM spadin, respectively. ( B – D ) I–V curves of passive conductance in astrocytes infected with ScRNA ( B ), TWIK-1 shRNA ( C ), or TREK-1 shRNA ( D ), respectively. The curves obtained from hippocampi treated with spadin are presented in different colors (red, blue, or purple). ( E ) The bar graph shows averaged current at −160 mV and +40 mV of holding potentials. Th number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant and *** p
    Figure Legend Snippet: Spadin does not affect astrocytic passive conductance in mice with TREK-1 or TWIK-1 knockdown (KD). ( A ) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in hippocampal astrocytes after injection with lentiviruses expressing scrambled control shRNA (ScRNA), TWIK-1 shRNA, or TREK-1 shRNA in the absence or presence of 10 µM spadin, respectively. ( B – D ) I–V curves of passive conductance in astrocytes infected with ScRNA ( B ), TWIK-1 shRNA ( C ), or TREK-1 shRNA ( D ), respectively. The curves obtained from hippocampi treated with spadin are presented in different colors (red, blue, or purple). ( E ) The bar graph shows averaged current at −160 mV and +40 mV of holding potentials. Th number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant and *** p

    Techniques Used: Mouse Assay, Injection, Expressing, shRNA, Infection

    Spadin inhibits TWIK-1 mediated K + currents in a TREK-1-dependent manner in cultured astrocytes. ( A ) Schematic representation of the genomic structure of the TWIK-1 gene. Target sequences of gRNA are located within exon2 of mouse TWIK-1 gene. Examples of indel mutations are displayed. ( B ) Immunocytochemical images of control gRNA (Con gRNA) or TWIK-1 gRNA transfected cultured astrocytes stained with anti-TWIK-1 antibody. ( C ) Current densities before and after application of 10 µM spadin (SP) to control gRNA or TWIK-1 gRNA transfected astrocytes. ( D ) Summary bar graph of control gRNA or TWIK-1 gRNA currents with or without spadin (SP) plotted at +50 mV and −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p
    Figure Legend Snippet: Spadin inhibits TWIK-1 mediated K + currents in a TREK-1-dependent manner in cultured astrocytes. ( A ) Schematic representation of the genomic structure of the TWIK-1 gene. Target sequences of gRNA are located within exon2 of mouse TWIK-1 gene. Examples of indel mutations are displayed. ( B ) Immunocytochemical images of control gRNA (Con gRNA) or TWIK-1 gRNA transfected cultured astrocytes stained with anti-TWIK-1 antibody. ( C ) Current densities before and after application of 10 µM spadin (SP) to control gRNA or TWIK-1 gRNA transfected astrocytes. ( D ) Summary bar graph of control gRNA or TWIK-1 gRNA currents with or without spadin (SP) plotted at +50 mV and −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p

    Techniques Used: Cell Culture, Transfection, Staining

    Spadin efficiently controls astrocytic passive conductance in hippocampus. ( A , B ) Representative fluorescence images of dual immunostaining of TREK-1 (green) and GFAP (red), with DAPI staining of astrocyte nuclei (blue) in mouse hippocampus. The images show that TREK-1 was highly expressed in astrocytes. Scale bar, 150 μm. ( C ) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in hippocampal astrocytes treated with vehicle (black), 10 μM spadin (red) or 200 μM quinidine (blue) respectively. ( D ) Current–voltage ( I – V) curves of passive conductance in ( C ). ( E , F ) Bar graphs showing averaged currents at −160 mV and +40 mV of holding potentials ( E ) and reversal potentials (Vrev) ( F ), respectively. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. ** p
    Figure Legend Snippet: Spadin efficiently controls astrocytic passive conductance in hippocampus. ( A , B ) Representative fluorescence images of dual immunostaining of TREK-1 (green) and GFAP (red), with DAPI staining of astrocyte nuclei (blue) in mouse hippocampus. The images show that TREK-1 was highly expressed in astrocytes. Scale bar, 150 μm. ( C ) Representative traces of passive conductance induced by voltage stepping from −160 mV to +40 mV in hippocampal astrocytes treated with vehicle (black), 10 μM spadin (red) or 200 μM quinidine (blue) respectively. ( D ) Current–voltage ( I – V) curves of passive conductance in ( C ). ( E , F ) Bar graphs showing averaged currents at −160 mV and +40 mV of holding potentials ( E ) and reversal potentials (Vrev) ( F ), respectively. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. ** p

    Techniques Used: Fluorescence, Immunostaining, Staining

    Spadin regulates K + currents mediated by TWIK-1/TREK-1 heterodimeric channels. Current densities induced by voltage ramping were measured in COS-7 cells transfected with control vector (Con) or several channel expressing vectors. ( A , C , E ) Current densities induced by voltage ramping were measured in COS-7 cells transfected with TREK-1 ( A ), TWIK-1 (K274E) ( C ), and TWIK-1/TREK-1 ( E ) before and after treatment with 10 µM spadin, respectively. Summary histograms show averaged current densities recorded from cells expressing TREK-1 ( B ) and TWIK-1/TREK-1 ( F ) with or without spadin treatment at +50 mV. The number on each bar indicates n for each condition. ( D ) TWIK-1(K274E)-mediated current densities were averaged from the plots in ( C ) at −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, ** p
    Figure Legend Snippet: Spadin regulates K + currents mediated by TWIK-1/TREK-1 heterodimeric channels. Current densities induced by voltage ramping were measured in COS-7 cells transfected with control vector (Con) or several channel expressing vectors. ( A , C , E ) Current densities induced by voltage ramping were measured in COS-7 cells transfected with TREK-1 ( A ), TWIK-1 (K274E) ( C ), and TWIK-1/TREK-1 ( E ) before and after treatment with 10 µM spadin, respectively. Summary histograms show averaged current densities recorded from cells expressing TREK-1 ( B ) and TWIK-1/TREK-1 ( F ) with or without spadin treatment at +50 mV. The number on each bar indicates n for each condition. ( D ) TWIK-1(K274E)-mediated current densities were averaged from the plots in ( C ) at −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, ** p

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    Spadin’s effects on astrocytic K + current were abrogated by genetic ablation of TREK-1 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. ( A ) Schematic representation of the genomic structure of the TREK-1 gene. Target sequences of gRNA are located within exon3 of mouse TREK-1 gene. Examples of indel mutations are displayed. ( B ) Immunocytochemical images of control gRNA (Con gRNA) or TREK-1 gRNA transfected cultured astrocytes stained with anti-TREK-1 antibody. ( C ) Current densities showing before and after application of 10 µM spadin (SP) to control gRNA or TREK-1 gRNA transfected astrocytes. ( D ) Summary bar graph of control gRNA or TREK-1 gRNA currents with or without spadin (SP) plotted at +50 mV and −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p
    Figure Legend Snippet: Spadin’s effects on astrocytic K + current were abrogated by genetic ablation of TREK-1 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. ( A ) Schematic representation of the genomic structure of the TREK-1 gene. Target sequences of gRNA are located within exon3 of mouse TREK-1 gene. Examples of indel mutations are displayed. ( B ) Immunocytochemical images of control gRNA (Con gRNA) or TREK-1 gRNA transfected cultured astrocytes stained with anti-TREK-1 antibody. ( C ) Current densities showing before and after application of 10 µM spadin (SP) to control gRNA or TREK-1 gRNA transfected astrocytes. ( D ) Summary bar graph of control gRNA or TREK-1 gRNA currents with or without spadin (SP) plotted at +50 mV and −150 mV. The number on each bar indicates n for each condition. All values are mean ± s.e.m. p -values were obtained with one-way ANOVA followed by Turkey’s post hoc test. n.s: not significant, * p

    Techniques Used: CRISPR, Transfection, Cell Culture, Staining

    10) Product Images from "Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior"

    Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00863

    Sub-cellular location of the TREK-1 channel protein in the brain of Sort1 −/− and WT mice. (A) The TREK-1 expression was decreased by 36% (histogram, Left) in the plasma membrane (PM) prepared from Sort1 −/− mouse brain when compared to PM prepared from WT mouse brain as visualized by Western blots (Right). (B) The expression of TREK-1 channels remained unchanged in high and low density vesicles (H/LDM) prepared from brains of Sort1 −/− and WT mice, as well as in total brain extracts (C) . The sub-cellular compartments were identified by specific markers: NaKATPase for plasma membranes (A) , TGN38 for H/LDM (B) , and actin for total brain extracts (C) . ∗ p
    Figure Legend Snippet: Sub-cellular location of the TREK-1 channel protein in the brain of Sort1 −/− and WT mice. (A) The TREK-1 expression was decreased by 36% (histogram, Left) in the plasma membrane (PM) prepared from Sort1 −/− mouse brain when compared to PM prepared from WT mouse brain as visualized by Western blots (Right). (B) The expression of TREK-1 channels remained unchanged in high and low density vesicles (H/LDM) prepared from brains of Sort1 −/− and WT mice, as well as in total brain extracts (C) . The sub-cellular compartments were identified by specific markers: NaKATPase for plasma membranes (A) , TGN38 for H/LDM (B) , and actin for total brain extracts (C) . ∗ p

    Techniques Used: Mouse Assay, Expressing, Western Blot

    11) Product Images from "Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior"

    Article Title: Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00863

    Sub-cellular location of the TREK-1 channel protein in the brain of Sort1 −/− and WT mice. (A) The TREK-1 expression was decreased by 36% (histogram, Left) in the plasma membrane (PM) prepared from Sort1 −/− mouse brain when compared to PM prepared from WT mouse brain as visualized by Western blots (Right). (B) The expression of TREK-1 channels remained unchanged in high and low density vesicles (H/LDM) prepared from brains of Sort1 −/− and WT mice, as well as in total brain extracts (C) . The sub-cellular compartments were identified by specific markers: NaKATPase for plasma membranes (A) , TGN38 for H/LDM (B) , and actin for total brain extracts (C) . ∗ p
    Figure Legend Snippet: Sub-cellular location of the TREK-1 channel protein in the brain of Sort1 −/− and WT mice. (A) The TREK-1 expression was decreased by 36% (histogram, Left) in the plasma membrane (PM) prepared from Sort1 −/− mouse brain when compared to PM prepared from WT mouse brain as visualized by Western blots (Right). (B) The expression of TREK-1 channels remained unchanged in high and low density vesicles (H/LDM) prepared from brains of Sort1 −/− and WT mice, as well as in total brain extracts (C) . The sub-cellular compartments were identified by specific markers: NaKATPase for plasma membranes (A) , TGN38 for H/LDM (B) , and actin for total brain extracts (C) . ∗ p

    Techniques Used: Mouse Assay, Expressing, Western Blot

    12) Product Images from "Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes"

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00319

    MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
    Figure Legend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Techniques Used: Staining, Labeling

    Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P
    Figure Legend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Techniques Used: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

    MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.
    Figure Legend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Techniques Used: Mouse Assay, Staining

    13) Product Images from "Involvement of TREK-1 Channel in Cell Viability of H9c2 Rat Cardiomyoblasts Affected by Bupivacaine and Lipid Emulsion"

    Article Title: Involvement of TREK-1 Channel in Cell Viability of H9c2 Rat Cardiomyoblasts Affected by Bupivacaine and Lipid Emulsion

    Journal: Cells

    doi: 10.3390/cells8050454

    Effect of TREK-1 channel modulators on cell viability. ( a ) Increase in cell viability by TREK-1 channel activators (100 μM carbamazepine, 1 mM LiCl 2 , 5 μM linoleic acid, and 5 μM arachidonic acid). † p
    Figure Legend Snippet: Effect of TREK-1 channel modulators on cell viability. ( a ) Increase in cell viability by TREK-1 channel activators (100 μM carbamazepine, 1 mM LiCl 2 , 5 μM linoleic acid, and 5 μM arachidonic acid). † p

    Techniques Used:

    Effect of bupivacaine and/or LE on TREK-1 manipulated H9c2 cells. ( a ) Efficiency of TREK-1 or TREK-1 small interfering RNA (siRNA) transfection on H9c2 cells. pcDNA3.1 (vector), TREK-1 in pcDNA3.1 (TREK-1), scrambled siRNA (Sc-siRNA), or TREK-1 siRNA was transfected into H9c2 cells. PCR (upper panel) and immunoblotting (lower panel) assays were performed on day 2 after transfection. The TREK-1 siRNA transfection was performed twice (36 h apart). We analyzed 1 μg of cDNA and 30 μg of protein per lane by semi-quantitative PCR and immunoblotting, respectively. GAPDH and β-actin were used as loading controls for PCR and immunoblotting, respectively. ( b ) Altered cell viability in TREK-1 overexpressed and knocked-down cells by treatment with bupivacaine and/or LE. The relative cell viability was calculated by dividing each control, which is a group only transfected with vector, TREK-1, scrambled siRNA, or TREK-1 siRNA without bupivacaine and/or LE treatment. The signs of + and – represent treatment condition with and without chemicals, respectively. Each bar represents the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Effect of bupivacaine and/or LE on TREK-1 manipulated H9c2 cells. ( a ) Efficiency of TREK-1 or TREK-1 small interfering RNA (siRNA) transfection on H9c2 cells. pcDNA3.1 (vector), TREK-1 in pcDNA3.1 (TREK-1), scrambled siRNA (Sc-siRNA), or TREK-1 siRNA was transfected into H9c2 cells. PCR (upper panel) and immunoblotting (lower panel) assays were performed on day 2 after transfection. The TREK-1 siRNA transfection was performed twice (36 h apart). We analyzed 1 μg of cDNA and 30 μg of protein per lane by semi-quantitative PCR and immunoblotting, respectively. GAPDH and β-actin were used as loading controls for PCR and immunoblotting, respectively. ( b ) Altered cell viability in TREK-1 overexpressed and knocked-down cells by treatment with bupivacaine and/or LE. The relative cell viability was calculated by dividing each control, which is a group only transfected with vector, TREK-1, scrambled siRNA, or TREK-1 siRNA without bupivacaine and/or LE treatment. The signs of + and – represent treatment condition with and without chemicals, respectively. Each bar represents the mean ± SD of three independent experiments. * p

    Techniques Used: Small Interfering RNA, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Reduction in bupivacaine-induced changes in membrane potential in TREK-1 manipulated H9c2 cells. ( a ) Changes in plasma membrane potential (PMP) by bupivacaine and LE with time. Green FluoVolt ® membrane labeling dye was stained and quantified the intensity using the Fluoview software program. The scale bar represents 50 μm. Typical PMP changes in response to bupivacaine and LE treatment. The bar graphs show the net changes in PMP displayed as fluorescence intensity. Arrowheads represent addition of chemicals. * p
    Figure Legend Snippet: Reduction in bupivacaine-induced changes in membrane potential in TREK-1 manipulated H9c2 cells. ( a ) Changes in plasma membrane potential (PMP) by bupivacaine and LE with time. Green FluoVolt ® membrane labeling dye was stained and quantified the intensity using the Fluoview software program. The scale bar represents 50 μm. Typical PMP changes in response to bupivacaine and LE treatment. The bar graphs show the net changes in PMP displayed as fluorescence intensity. Arrowheads represent addition of chemicals. * p

    Techniques Used: Labeling, Staining, Software, Fluorescence

    Changes in TWIK-related potassium channel (TREK)-1 expression level in H9c2 cells by bupivacaine and LE. ( a ) No detection of TREK-2 and TWIK-related arachidonic acid-activated potassium channel (TRAAK) expression. Trigeminal ganglion (TG) was used as a positive control for expression of TREK-2 and TRAAK. ( b ) TREK-1 mRNA expression. Rat TREK-1 transfected HEK-293 cells and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were used as a positive control and a loading control, respectively. RT represents reverse transcriptase. The signs of + and – represent treatment condition with and without bupivacaine and/or LE, respectively. ( c ) TREK-1 protein expression. Protein obtained from rat TREK-1 overexpressed cells was used as a positive control, and α-tubulin was used as a loading control. ( d ) Localization of TREK-1 protein. Green and blue indicate TREK-1 expression and nucleus stained with DAPI, respectively. The scale bar represents 30 µm. Changes in TREK-1 mRNA expression level detected by ( e ) semi-quantitative PCR and ( f ) real-time PCR. ( g ) Changes in TREK-1 protein level by bupivacaine and LE. Cells were treated with bupivacaine and/or LE for 24 h. We analyzed 1 μg of cDNA and 30 μg of protein per lane by semi- and real-time quantitative PCR and immunoblotting, respectively. GAPDH and RPS12 were used as reference genes for real-time PCR. β-actin was used as a loading control for quantification of protein level. Each bar represents the mean ± SD of four independent experiments. * p
    Figure Legend Snippet: Changes in TWIK-related potassium channel (TREK)-1 expression level in H9c2 cells by bupivacaine and LE. ( a ) No detection of TREK-2 and TWIK-related arachidonic acid-activated potassium channel (TRAAK) expression. Trigeminal ganglion (TG) was used as a positive control for expression of TREK-2 and TRAAK. ( b ) TREK-1 mRNA expression. Rat TREK-1 transfected HEK-293 cells and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were used as a positive control and a loading control, respectively. RT represents reverse transcriptase. The signs of + and – represent treatment condition with and without bupivacaine and/or LE, respectively. ( c ) TREK-1 protein expression. Protein obtained from rat TREK-1 overexpressed cells was used as a positive control, and α-tubulin was used as a loading control. ( d ) Localization of TREK-1 protein. Green and blue indicate TREK-1 expression and nucleus stained with DAPI, respectively. The scale bar represents 30 µm. Changes in TREK-1 mRNA expression level detected by ( e ) semi-quantitative PCR and ( f ) real-time PCR. ( g ) Changes in TREK-1 protein level by bupivacaine and LE. Cells were treated with bupivacaine and/or LE for 24 h. We analyzed 1 μg of cDNA and 30 μg of protein per lane by semi- and real-time quantitative PCR and immunoblotting, respectively. GAPDH and RPS12 were used as reference genes for real-time PCR. β-actin was used as a loading control for quantification of protein level. Each bar represents the mean ± SD of four independent experiments. * p

    Techniques Used: Expressing, Positive Control, Transfection, Staining, Real-time Polymerase Chain Reaction

    Functional expression of TREK-1 in H9c2 cells. ( a ) Effects of bupivacaine and/or LE on TREK-1 overexpressed HEK-293 cells. Whole-cell currents were recorded in 5mM KCl, and the current levels at +60 mV were determined and analyzed. ( b ) Single-channel activity of TREK-1 in response to bupivacaine and/or LE under inside-out patch mode at +60 mV of pipette potential. ( c ) Inhibition and activation curves of TREK-1 channel by bupivacaine and LE, respectively. The dotted lines represent the IC 50 and EC 50 of bupivacaine and LE. ( d ) The single-channel recording of TREK-1-like channel in H9c2 cells. The TREK-1-like channels were activated by intracellular low pH and negative pressure. ( e , f ) H9c2 background whole-cell currents. Whole-cell currents were recorded from H9c2 cells in the presence of 1 mM 4-AP, 1 mM Ba 2+ , and 1 mM TEA to rule out contamination of other K + channels. Cell membrane potential was held at −80 mV, and ( e ) voltage ramps or ( f ) voltage steps from –120 to +60 mV in 20 mV intervals were applied for 1 s or 2 s durations, respectively. Pipette and bath solutions contained 150 and 5 mM K + , respectively. ( g ) Effect of different concentrations of LE on H9c2 background K + currents. ( h ) Combined effects of TREK-1 inhibitors and LE on H9c2 background K + channels. Each bar (data) represents the mean ± SD of three independent experiments ( n = 6). * p
    Figure Legend Snippet: Functional expression of TREK-1 in H9c2 cells. ( a ) Effects of bupivacaine and/or LE on TREK-1 overexpressed HEK-293 cells. Whole-cell currents were recorded in 5mM KCl, and the current levels at +60 mV were determined and analyzed. ( b ) Single-channel activity of TREK-1 in response to bupivacaine and/or LE under inside-out patch mode at +60 mV of pipette potential. ( c ) Inhibition and activation curves of TREK-1 channel by bupivacaine and LE, respectively. The dotted lines represent the IC 50 and EC 50 of bupivacaine and LE. ( d ) The single-channel recording of TREK-1-like channel in H9c2 cells. The TREK-1-like channels were activated by intracellular low pH and negative pressure. ( e , f ) H9c2 background whole-cell currents. Whole-cell currents were recorded from H9c2 cells in the presence of 1 mM 4-AP, 1 mM Ba 2+ , and 1 mM TEA to rule out contamination of other K + channels. Cell membrane potential was held at −80 mV, and ( e ) voltage ramps or ( f ) voltage steps from –120 to +60 mV in 20 mV intervals were applied for 1 s or 2 s durations, respectively. Pipette and bath solutions contained 150 and 5 mM K + , respectively. ( g ) Effect of different concentrations of LE on H9c2 background K + currents. ( h ) Combined effects of TREK-1 inhibitors and LE on H9c2 background K + channels. Each bar (data) represents the mean ± SD of three independent experiments ( n = 6). * p

    Techniques Used: Functional Assay, Expressing, Activity Assay, Transferring, Inhibition, Activation Assay

    14) Product Images from "Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction"

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.908792

    Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.
    Figure Legend Snippet: Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Techniques Used: Immunohistochemistry

    TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P
    Figure Legend Snippet: TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Techniques Used: Expressing

    The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).
    Figure Legend Snippet: The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Techniques Used:

    15) Product Images from "Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes"

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00319

    MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
    Figure Legend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Techniques Used: Staining, Labeling

    Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P
    Figure Legend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Techniques Used: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

    MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.
    Figure Legend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Techniques Used: Mouse Assay, Staining

    16) Product Images from "TREK-1 null impairs neuronal excitability, synaptic plasticity, and cognitive function"

    Article Title: TREK-1 null impairs neuronal excitability, synaptic plasticity, and cognitive function

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-019-01828-x

    TREK-1 KO depresses the ratio of paired-pulse facilitation, occludes long-term potentiation and impairs hippocampal-dependent space memory. a TREK-1 KO decreases the ratio of paired-pulse facilitation (PPR) of CA3–CA1 synapses. Left , in a WT and TREK-1 KO neuron as indicated, two consecutive EPSCs were induced by SC stimulation pulses at 40 ms interval. b LTP in the CA1 region was induced by high-frequency stimulation of SC (HFS, arrow). The LTP in TREK-1 KO mice was markedly occluded (n = 10) when compared to WT (n = 12). Left , Representative fEPSP traces CA1 region before (solid traces) and 40 min after HFS of SC afferents (dashed traces) in WT and TREK-1 KO mice. c Quantification of fEPSP slope change reveals a TREK-1 KO induced occlusion of LTP. The quantification was made by averaging normalized fEPSP slope from 36 to 40 min after HFS or low-frequency stimulation, as a short line indicated in the time course of B. d New object recognition. Left , schematic for test protocol. Right , the bar graph shows TREK-1 KO mice (n=11) spent less time on new objects than WT mice (n=10), as the discrimination index decreasing. Student’s t -test was used. * P
    Figure Legend Snippet: TREK-1 KO depresses the ratio of paired-pulse facilitation, occludes long-term potentiation and impairs hippocampal-dependent space memory. a TREK-1 KO decreases the ratio of paired-pulse facilitation (PPR) of CA3–CA1 synapses. Left , in a WT and TREK-1 KO neuron as indicated, two consecutive EPSCs were induced by SC stimulation pulses at 40 ms interval. b LTP in the CA1 region was induced by high-frequency stimulation of SC (HFS, arrow). The LTP in TREK-1 KO mice was markedly occluded (n = 10) when compared to WT (n = 12). Left , Representative fEPSP traces CA1 region before (solid traces) and 40 min after HFS of SC afferents (dashed traces) in WT and TREK-1 KO mice. c Quantification of fEPSP slope change reveals a TREK-1 KO induced occlusion of LTP. The quantification was made by averaging normalized fEPSP slope from 36 to 40 min after HFS or low-frequency stimulation, as a short line indicated in the time course of B. d New object recognition. Left , schematic for test protocol. Right , the bar graph shows TREK-1 KO mice (n=11) spent less time on new objects than WT mice (n=10), as the discrimination index decreasing. Student’s t -test was used. * P

    Techniques Used: Mouse Assay

    TREK-1 KO depolarizes membrane potential and increases input resistance of hippocampal pyramidal neurons. a TREK-1 (left) and neuronal marker NeuN (middle) immunostaining in the hippocampal CA1 region. Areas outlined in the square boxes are shown in higher magnification in the lower panels where TREK-1 and NeuN staining are nicely co-localized in neuronal soma and primary dendritic processes. Scale bar represents 25 μm. b TREK-1 KO depolarizes the membrane potential of CA1 pyramidal neurons. c TREK-1 KO increases the input resistance (R in ) of CA1 pyramidal neurons in K + -based physiological pipette solution ([K + ] p ) but not in Cs + -based pipette solution ([Cs + ] p ). d The membrane capacitance (C M ) is not altered in CA1 pyramidal neurons in TREK-1 KO mice. Numbers of neurons analyzed are shown in bars. Statistical significance was evaluated by Student’s t-test. (* P
    Figure Legend Snippet: TREK-1 KO depolarizes membrane potential and increases input resistance of hippocampal pyramidal neurons. a TREK-1 (left) and neuronal marker NeuN (middle) immunostaining in the hippocampal CA1 region. Areas outlined in the square boxes are shown in higher magnification in the lower panels where TREK-1 and NeuN staining are nicely co-localized in neuronal soma and primary dendritic processes. Scale bar represents 25 μm. b TREK-1 KO depolarizes the membrane potential of CA1 pyramidal neurons. c TREK-1 KO increases the input resistance (R in ) of CA1 pyramidal neurons in K + -based physiological pipette solution ([K + ] p ) but not in Cs + -based pipette solution ([Cs + ] p ). d The membrane capacitance (C M ) is not altered in CA1 pyramidal neurons in TREK-1 KO mice. Numbers of neurons analyzed are shown in bars. Statistical significance was evaluated by Student’s t-test. (* P

    Techniques Used: Marker, Immunostaining, Staining, Transferring, Mouse Assay

    TREK-1 KO increases the excitability of CA1 pyramidal neurons. a Left, a 100 pA/300 ms current step induced 4 and 7 spikes in a WT and TREK-1 KO CA1 pyramidal neuron, respectively. Cells were held at −70 mV. Right, a series of current steps, ranging from 10 pA to 230 pA at 300 ms duration and 20 pA increment, induced a steeper current input-spike output relationship in TREK-1KO (n = 14) than WT neurons (n = 9). b Rheobase current for neuronal firing threshold. The inset shows the current steps from 10 pA −100 pA steps at 10 ms duration and 10 pA increments for detecting of the rheobase current and threshold (dash line) for neuronal firing in a WT and TREK-1 KO neuron, respectively. c TREK-1 KO significantly reduces the rheobase currents. d TREK-1 KO decreases the threshold for neuronal firing. e TREK-1 KO does not affect the amplitude of neuronal firing spikes. Statistical significance was evaluated by Student’s t -test (bar graphs) or two-way ANOVA followed by a post-hoc Tukey HSD test (input-output plots). * P
    Figure Legend Snippet: TREK-1 KO increases the excitability of CA1 pyramidal neurons. a Left, a 100 pA/300 ms current step induced 4 and 7 spikes in a WT and TREK-1 KO CA1 pyramidal neuron, respectively. Cells were held at −70 mV. Right, a series of current steps, ranging from 10 pA to 230 pA at 300 ms duration and 20 pA increment, induced a steeper current input-spike output relationship in TREK-1KO (n = 14) than WT neurons (n = 9). b Rheobase current for neuronal firing threshold. The inset shows the current steps from 10 pA −100 pA steps at 10 ms duration and 10 pA increments for detecting of the rheobase current and threshold (dash line) for neuronal firing in a WT and TREK-1 KO neuron, respectively. c TREK-1 KO significantly reduces the rheobase currents. d TREK-1 KO decreases the threshold for neuronal firing. e TREK-1 KO does not affect the amplitude of neuronal firing spikes. Statistical significance was evaluated by Student’s t -test (bar graphs) or two-way ANOVA followed by a post-hoc Tukey HSD test (input-output plots). * P

    Techniques Used:

    TREK-1 KO does not alter astrocyte K + conductance or astrocyte syncytial isopotentiality. a TREK-1 immunostaining signal is negligible in astrocytes. TREK-1 (red), NeuN (blue) double staining in Aldh1l1-eGFP mouse in CA1 region. Scale bar: 25 μm. b TREK-1 OK does not alter the resting V M of hippocampal astrocytes at CA1 region (WT, n=33; TREK-1 KO, n=31). c TREK-1 KO does not alter the passive K + conductance of astrocytes. Astrocyte was held at −80 mV and the membrane currents were induced by command voltages from −180 to +20 mV with 10 mV increment (inset, command voltages). Astrocytes in both genotypes showed a similar linear I-V relationship membrane conductance (passive) and comparable amplitude of passive conductance. d The I-V curves that were constructed from the current amplitudes. The current amplitude at y1 (V +20 mV ) and y2 (V -180 mV ). e Astrocyte syncytial isopotentiality remains intact in TREK-1 KO mice. Left , a schematic illustration of the use of non-physiological [Na + ] p or [Cs + ] p to examine the existence of syncytial isopotentiality in an astrocyte network. Right , representative V M,ss recordings with [K + ] p , [Na + ] p and [Cs + ] p , respectively. Insets show an unchanged V M,ss in TREK-1 KO astrocytes. Student’s t -test was used.
    Figure Legend Snippet: TREK-1 KO does not alter astrocyte K + conductance or astrocyte syncytial isopotentiality. a TREK-1 immunostaining signal is negligible in astrocytes. TREK-1 (red), NeuN (blue) double staining in Aldh1l1-eGFP mouse in CA1 region. Scale bar: 25 μm. b TREK-1 OK does not alter the resting V M of hippocampal astrocytes at CA1 region (WT, n=33; TREK-1 KO, n=31). c TREK-1 KO does not alter the passive K + conductance of astrocytes. Astrocyte was held at −80 mV and the membrane currents were induced by command voltages from −180 to +20 mV with 10 mV increment (inset, command voltages). Astrocytes in both genotypes showed a similar linear I-V relationship membrane conductance (passive) and comparable amplitude of passive conductance. d The I-V curves that were constructed from the current amplitudes. The current amplitude at y1 (V +20 mV ) and y2 (V -180 mV ). e Astrocyte syncytial isopotentiality remains intact in TREK-1 KO mice. Left , a schematic illustration of the use of non-physiological [Na + ] p or [Cs + ] p to examine the existence of syncytial isopotentiality in an astrocyte network. Right , representative V M,ss recordings with [K + ] p , [Na + ] p and [Cs + ] p , respectively. Insets show an unchanged V M,ss in TREK-1 KO astrocytes. Student’s t -test was used.

    Techniques Used: Immunostaining, Double Staining, Construct, Mouse Assay

    TREK-1 KO increases excitatory and inhibitory synaptic transmission in CA1 pyramidal neurons. a The frequency but not the amplitude of mEPSCs increases in TREK-1 KO neurons. mEPSCs were recorded in the presence of 0.5 μM TTX and 100 μM PTX. Top , representative mEPSCs traces; Bottom , the cumulative probability of the mEPSC frequency (left) and amplitude (right). b Input-output relationship of EPSCs in CA1 neurons. Input refers to graded electrical stimulation of Schaffer collateral (SC), whereas output refers to SC stimulation-induced whole-cell EPSCs. The neuronal EPSCs outputs show an increase in TREK-1 KO neurons (n=19) compared to wild-type neurons (n=17). c Left , the SC stimulation-induced EPSCs and IPSCs were recorded from the same neuron by varying holding V M from −70 mV to 0 mV, respectively. TREK-1 KO resulted in an enhanced EPSCs and IPSCs compared to wildtype ( P
    Figure Legend Snippet: TREK-1 KO increases excitatory and inhibitory synaptic transmission in CA1 pyramidal neurons. a The frequency but not the amplitude of mEPSCs increases in TREK-1 KO neurons. mEPSCs were recorded in the presence of 0.5 μM TTX and 100 μM PTX. Top , representative mEPSCs traces; Bottom , the cumulative probability of the mEPSC frequency (left) and amplitude (right). b Input-output relationship of EPSCs in CA1 neurons. Input refers to graded electrical stimulation of Schaffer collateral (SC), whereas output refers to SC stimulation-induced whole-cell EPSCs. The neuronal EPSCs outputs show an increase in TREK-1 KO neurons (n=19) compared to wild-type neurons (n=17). c Left , the SC stimulation-induced EPSCs and IPSCs were recorded from the same neuron by varying holding V M from −70 mV to 0 mV, respectively. TREK-1 KO resulted in an enhanced EPSCs and IPSCs compared to wildtype ( P

    Techniques Used: Transmission Assay

    TREK-1 null alters dendritic sprouting and the number of spines. a Representative images of CA1 pyramidal neurons revealed by electrode biocytin-loading followed by Alexa 488-conjugated streptavidin staining. Scale bar: 100 μm. b-d Summary of the total dendritic length (B), branch numbers (C) in both apical and basal dendritic trees, and apical dendritic arborization in terms of the number of intersections (D). e Representative higher resolution 3D confocal dendritic segment images for spine detection, classification, and measurements. Scale bar: 3 μm. f Summary of the dendritic spine densities in WT and TREK-1 KO groups. g Quantification of dendritic spine subtypes. Left , images of the four different spine subtypes. Right , quantification of mature and immature dendritic spine subtypes of CA1 neurons. The numbers of the dendritic segments used for quantification are shown in the bars, and these segments were obtained from 4–7 mice in each group. h Western immunoblots show comparable levels of expression of presynaptic (synaptophysin), and postsynaptic (PSD-95) proteins in WT and TREK-1 KO mice. GAPDH was used as a protein quantity loading control. Data for all bar graphs were analyzed using Student’s t -test. In C , the comparison of the number of intersections between the two groups was analyzed using repeated-measures two-way ANOVA. (* P
    Figure Legend Snippet: TREK-1 null alters dendritic sprouting and the number of spines. a Representative images of CA1 pyramidal neurons revealed by electrode biocytin-loading followed by Alexa 488-conjugated streptavidin staining. Scale bar: 100 μm. b-d Summary of the total dendritic length (B), branch numbers (C) in both apical and basal dendritic trees, and apical dendritic arborization in terms of the number of intersections (D). e Representative higher resolution 3D confocal dendritic segment images for spine detection, classification, and measurements. Scale bar: 3 μm. f Summary of the dendritic spine densities in WT and TREK-1 KO groups. g Quantification of dendritic spine subtypes. Left , images of the four different spine subtypes. Right , quantification of mature and immature dendritic spine subtypes of CA1 neurons. The numbers of the dendritic segments used for quantification are shown in the bars, and these segments were obtained from 4–7 mice in each group. h Western immunoblots show comparable levels of expression of presynaptic (synaptophysin), and postsynaptic (PSD-95) proteins in WT and TREK-1 KO mice. GAPDH was used as a protein quantity loading control. Data for all bar graphs were analyzed using Student’s t -test. In C , the comparison of the number of intersections between the two groups was analyzed using repeated-measures two-way ANOVA. (* P

    Techniques Used: Staining, Mouse Assay, Western Blot, Expressing

    TREK-1 KO increases the active/silent ratio of SC-CA1 glutamatergic synapses. a In the presence of PTX, the AMPA-EPSC and NMDA-EPSC in CA1 pyramidal neurons were induced by SC stimulation at the holding V M of −70 and +40 mV, respectively. TREK-1 KO increases the AMPA-EPSC/ NMDA-EPSC ratio. b The amplitudes of SC stimulation-induced NMDA receptor currents are comparable between WT and TREK-1 KO neurons. The NMDA receptor currents were isolated by adding PTX and NBQX for inhibition of GABAa and AMPA receptors, respectively. c Minimal SC stimulation-induced AMPA-EPSC (holding at −70 mV) and NMDA-EPSC (holding at +40 mV) in WT and TREK-1 KO neurons. In each case, 6 consecutive traces are superimposed to show the variation from absence to the presence of SC stimulation-induced EPSCs. The NMDA-EPSCs were recorded in the presence of 10 μM NBQX. d Use of minimal SC stimulation, the induced and failed EPSC events are plotted over a 40 min duration in a WT and TREK-1 KO neuron as indicated. In each case, the AMPA-EPSC events were recorded first by holding the cell at −70 mV, and the NMDA-EPSC events were recorded afterward by switching the holding to +40 mV in the presence of 10 μM NBQX. e Summary of the data from D, a less failure rate without affecting the amplitude of minimal SC stimulation-induced AMPA-EPSCs occurred in TREK-1 KO neurons. f Summary of the data from D, no change in either the failure rate or the amplitude of minimal SC stimulation-induced NMDA-EPSCs in TREK-1 KO neurons. Student’s t -test was used. * P
    Figure Legend Snippet: TREK-1 KO increases the active/silent ratio of SC-CA1 glutamatergic synapses. a In the presence of PTX, the AMPA-EPSC and NMDA-EPSC in CA1 pyramidal neurons were induced by SC stimulation at the holding V M of −70 and +40 mV, respectively. TREK-1 KO increases the AMPA-EPSC/ NMDA-EPSC ratio. b The amplitudes of SC stimulation-induced NMDA receptor currents are comparable between WT and TREK-1 KO neurons. The NMDA receptor currents were isolated by adding PTX and NBQX for inhibition of GABAa and AMPA receptors, respectively. c Minimal SC stimulation-induced AMPA-EPSC (holding at −70 mV) and NMDA-EPSC (holding at +40 mV) in WT and TREK-1 KO neurons. In each case, 6 consecutive traces are superimposed to show the variation from absence to the presence of SC stimulation-induced EPSCs. The NMDA-EPSCs were recorded in the presence of 10 μM NBQX. d Use of minimal SC stimulation, the induced and failed EPSC events are plotted over a 40 min duration in a WT and TREK-1 KO neuron as indicated. In each case, the AMPA-EPSC events were recorded first by holding the cell at −70 mV, and the NMDA-EPSC events were recorded afterward by switching the holding to +40 mV in the presence of 10 μM NBQX. e Summary of the data from D, a less failure rate without affecting the amplitude of minimal SC stimulation-induced AMPA-EPSCs occurred in TREK-1 KO neurons. f Summary of the data from D, no change in either the failure rate or the amplitude of minimal SC stimulation-induced NMDA-EPSCs in TREK-1 KO neurons. Student’s t -test was used. * P

    Techniques Used: Isolation, Inhibition

    17) Product Images from "Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes"

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00319

    MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
    Figure Legend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Techniques Used: Staining, Labeling

    Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P
    Figure Legend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Techniques Used: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

    MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.
    Figure Legend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Techniques Used: Mouse Assay, Staining

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    Alomone Labs rabbit anti trek 1
    MOR and <t>TREK-1</t> co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P
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    MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    doi: 10.3389/fncel.2018.00319

    Figure Lengend Snippet: MOR and TREK-1 co-localizes in astrocytic soma and process. (A) Subcellular distribution (soma, process and microdomain) of MOR in astrocyte (left). MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and GFP, representing astrocyte, is stained with immunoperoxidase (dark amorphous deposits, arrows). Cellular distribution of double labeling of TREK-1/TWIK-1 (middle) and MOR/TWIK-1 (right) in astrocyte. TREK-1 or MOR is stained with immunogold with silver enhancement (dark specks, arrowheads), and TWIK-1 is stained with immunoperoxidase (dark amorphous deposits, arrows). The soma, process, and microdomain of the astrocyte was colored blue. Presynaptic axon terminal (pre) and postsynaptic dendrite (post) were colored red and green, respectively. N is nucleus. Scale bar indicates 500 nm. (B) Summary bar graph for the frequency of MOR detection in each subcellular structure of astrocytes. Numbers in the bar graph are the number of MOR-positive soma, processes or microdomains out of total number of profiles observed. The data was collected from three animals. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (*** P

    Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

    Techniques: Staining, Labeling

    Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    doi: 10.3389/fncel.2018.00319

    Figure Lengend Snippet: Astrocytic glutamate release upon MOR activation is mediated by TREK-1. (A) Left, schematic diagram of sniffer-patch with acutely dissociated astrocytes from pSicoR-TREK-1-shRNA-mCherry-injected hippocampus and GluR1LY-GFP-expressing HEK cells. Right, differential interference contrast image and fluorescent image of GluR1LY-GFP-expressing HEK cell (green) and TREK-1-shRNA-mCherry-infected hippocampal astrocyte (red). (B) Representative traces of Ca 2+ response and inward current in acutely dissociated hippocampal astrocyte of scrambled-shRNA and TREK-1-shRNA-injected mouse. (C) Summary bar graph for DAMGO-induced glutamate current normalized by the full activation current in scrambled shRNA and TREK-1-shRNA-injected mice. Data from at least three independent mice for each group. Unpaired two-tailed t -test with Welch’s correction (* P

    Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

    Techniques: Activation Assay, shRNA, Injection, Expressing, Infection, Mouse Assay, Two Tailed Test

    MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Activation of Astrocytic μ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes

    doi: 10.3389/fncel.2018.00319

    Figure Lengend Snippet: MOR and TREK-1 are co-expressed in hippocampal astrocytes of MOR-mCherry mouse. (A) A structured illumination microscopic (SIM) image of a hippocampal astrocyte of MOR-mCherry mouse, immunostained with antibodies against GFAP and TREK-1. (B) 2D histogram of mCherry intensity and TREK-1 intensity in the SIM image (A) . Pearson’s coefficient (r) is calculated within the colocalized pixels which were automatically thresholded by Imaris 9.2 program. (C) Representative confocal images of co-localization of MOR-mCherry and TREK-1 in astrocytes in hippocampal cornu ammonis 1 (CA1) of MOR-mCherry mice. (D) An example of ROI generation within a single astrocyte. (E) Representative 2D histogram of mCherry intensity and TREK-1 intensity in the confocal image. (F) Pearson’s coefficients (r) between mCherry and TREK-1 from confocal images of single astrocytes. (G) A confocal image of hippocampal tissues from WT and TREK-1 KO mice, stained with an antibody against TREK-1.

    Article Snippet: Primary antibody used are as follow: chicken anti-GFAP (1:500, ab5541, Millipore), rabbit anti-TREK-1 (1:100, APC-047, Alomone Labs), and rabbit anti-MOR (1:200, sc-15310, Santa Cruz Biotechnology).

    Techniques: Mouse Assay, Staining

    Genetic deletion of TWIK-1 and TREK-1 genes together does not alter the electrophysiological properties of astrocytes. (A,B) Bar graph summary of the V M and R in from WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current profiles from WT and TWIK-1 −/− /TREK-1 −/− astrocytes, respectively. (D) Averaged I-V plots from these two genotypes, where the whole-cell current amplitudes in both inward and outward directions were comparable. (E) The RI values were also comparable between the two genotypes.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: Genetic deletion of TWIK-1 and TREK-1 genes together does not alter the electrophysiological properties of astrocytes. (A,B) Bar graph summary of the V M and R in from WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current profiles from WT and TWIK-1 −/− /TREK-1 −/− astrocytes, respectively. (D) Averaged I-V plots from these two genotypes, where the whole-cell current amplitudes in both inward and outward directions were comparable. (E) The RI values were also comparable between the two genotypes.

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques:

    Quinine does not reveal the functional contribution of TWIK-1 and TREK-1 in single or double gene knockout mice. (A) Representative astrocyte V M recordings first in 100 μM BaCl 2 bath application for 5 min, followed by addition of 400 μM quinine for 20 min, from a WT, TREK-1 −/− , TWIK-1 −/− and TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 400 μM quinine-induced V M depolarization (Δ V M 1) and the total V M depolarization induced by BaCl 2 plus quinine from all four genotypes. (C) Representative whole-cell current recordings first in aCSF as control, then in 100 μM BaCl 2 plus 400 μM quinine, and washout. Representative I–V relationships were shown in the right panel. (D) Summary of RI values from four genotypes obtained from astrocyte recordings in the presence of 400 μM quinine and 100 μM BaCl 2 together in bath. The RI values were comparable among the four genotypes.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: Quinine does not reveal the functional contribution of TWIK-1 and TREK-1 in single or double gene knockout mice. (A) Representative astrocyte V M recordings first in 100 μM BaCl 2 bath application for 5 min, followed by addition of 400 μM quinine for 20 min, from a WT, TREK-1 −/− , TWIK-1 −/− and TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 400 μM quinine-induced V M depolarization (Δ V M 1) and the total V M depolarization induced by BaCl 2 plus quinine from all four genotypes. (C) Representative whole-cell current recordings first in aCSF as control, then in 100 μM BaCl 2 plus 400 μM quinine, and washout. Representative I–V relationships were shown in the right panel. (D) Summary of RI values from four genotypes obtained from astrocyte recordings in the presence of 400 μM quinine and 100 μM BaCl 2 together in bath. The RI values were comparable among the four genotypes.

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Functional Assay, Double Knockout, Mouse Assay, In Situ

    TREK-1 gene deletion does not alter the electrophysiological properties of astrocytes. (A) Identification of astrocyte in hippocampal slices based on cellular morphology and positive SR101 staining in CA1 region. (B,C) Membrane potential ( V M ), and input resistance ( R in ) in WT and TREK-1 −/− astrocytes. (D) Representative astrocyte whole-cell passive conductance from a WT and a TREK-1 −/− astrocyte are shown separately as indicated. The voltage commands used for whole-cell current induction are displayed on the left panel. (E) I-V plots show the averaged current amplitudes from WT and TREK-1 −/− astrocytes. The equation for rectification index (RI) is also illustrated and the resulted RI values are summarized in (F) .

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: TREK-1 gene deletion does not alter the electrophysiological properties of astrocytes. (A) Identification of astrocyte in hippocampal slices based on cellular morphology and positive SR101 staining in CA1 region. (B,C) Membrane potential ( V M ), and input resistance ( R in ) in WT and TREK-1 −/− astrocytes. (D) Representative astrocyte whole-cell passive conductance from a WT and a TREK-1 −/− astrocyte are shown separately as indicated. The voltage commands used for whole-cell current induction are displayed on the left panel. (E) I-V plots show the averaged current amplitudes from WT and TREK-1 −/− astrocytes. The equation for rectification index (RI) is also illustrated and the resulted RI values are summarized in (F) .

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Staining

    K ir4.1 inhibition does not reveal the functional contribution of TREK-1 in TREK-1 −/− astrocytes. (A) Representative V M response to K ir 4.1 inhibitor 100 μM BaCl 2 from a WT and a TREK-1 −/− astrocyte, as indicated in situ . Δ V M indicates the peak V M depolarization during a 5 min BaCl 2 bath application. (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. (D) I-V plots derived from recordings in (C) . The Ba 2+ -sensitive currents in I-V plots were obtained from sweep subtraction. The Ba 2+ -sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and TREK-1 KO, RI = 0.90, respectively. (E) Summary of RI values from WT and TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: K ir4.1 inhibition does not reveal the functional contribution of TREK-1 in TREK-1 −/− astrocytes. (A) Representative V M response to K ir 4.1 inhibitor 100 μM BaCl 2 from a WT and a TREK-1 −/− astrocyte, as indicated in situ . Δ V M indicates the peak V M depolarization during a 5 min BaCl 2 bath application. (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. (D) I-V plots derived from recordings in (C) . The Ba 2+ -sensitive currents in I-V plots were obtained from sweep subtraction. The Ba 2+ -sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and TREK-1 KO, RI = 0.90, respectively. (E) Summary of RI values from WT and TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Inhibition, Functional Assay, In Situ, Derivative Assay

    TREK-1 channels are predominantly located in cytoplasm. (A) Fractionation western blot results revealed the subcellular distribution of TREK-1 channels in cytoplasmic fraction vs . membrane fraction in two independent tests of mice hippocampal samples. Anti-glial fibrillary acidic protein (GFAP) (50 kDa) and ATP1α2 (112 kDa) were markers for cytoplasmic and membrane fractions, respectively. The blots shown in (A) were first incubated with anti-TREK-1 antibody and then re-probed with the rest of other primary antibodies sequentially after the original membranes were stripped with stripping buffer (see “Materials and Methods” Section). (B) Bar graph summary showing the relative ratio of TREK-1 proteins located in cytoplasmic vs . membrane fractions. Data are shown as mean ± SEM. Numbers indicate the times of observations. ** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: TREK-1 channels are predominantly located in cytoplasm. (A) Fractionation western blot results revealed the subcellular distribution of TREK-1 channels in cytoplasmic fraction vs . membrane fraction in two independent tests of mice hippocampal samples. Anti-glial fibrillary acidic protein (GFAP) (50 kDa) and ATP1α2 (112 kDa) were markers for cytoplasmic and membrane fractions, respectively. The blots shown in (A) were first incubated with anti-TREK-1 antibody and then re-probed with the rest of other primary antibodies sequentially after the original membranes were stripped with stripping buffer (see “Materials and Methods” Section). (B) Bar graph summary showing the relative ratio of TREK-1 proteins located in cytoplasmic vs . membrane fractions. Data are shown as mean ± SEM. Numbers indicate the times of observations. ** p

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Fractionation, Western Blot, Mouse Assay, Incubation, Stripping Membranes

    K ir 4.1 inhibition does not reveal the functional contribution of TWIK-1/TREK-1 in double gene knockout mice. (A) Representative V M response to K ir 4.1 inhibitor, 100 μM BaCl 2 , from a WT and a TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. I–V relationships were shown in (D) . (D) I–V plots derived from recordings in (C) . The Ba 2+ -sensitive currents, in I-V plots were obtained from sweep subtraction. The Ba 2+ - sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and double gene knockout mice, RI = 0.90, respectively. (E) Summary of RI values from WT and TWIK-1 −/− /TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: K ir 4.1 inhibition does not reveal the functional contribution of TWIK-1/TREK-1 in double gene knockout mice. (A) Representative V M response to K ir 4.1 inhibitor, 100 μM BaCl 2 , from a WT and a TWIK-1 −/− /TREK-1 −/− astrocyte as indicated in situ . (B) Summary of 100 μM BaCl 2 -induced V M depolarization, where the V M depolarization was comparable between WT and TWIK-1 −/− /TREK-1 −/− astrocytes. (C) Representative whole-cell current recorded first in control, then 5 min in 100 μM BaCl 2 , and washout. I–V relationships were shown in (D) . (D) I–V plots derived from recordings in (C) . The Ba 2+ -sensitive currents, in I-V plots were obtained from sweep subtraction. The Ba 2+ - sensitive currents were shown in expanded y -axis in the inset that showed a moderate inward rectification in both WT, RI = 0.91, and double gene knockout mice, RI = 0.90, respectively. (E) Summary of RI values from WT and TWIK-1 −/− /TREK-1 −/− astrocytes obtained from recordings in the presence of 100 μM BaCl 2 for K ir 4.1 inhibition; the RI values were comparable between the two groups.

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Inhibition, Functional Assay, Double Knockout, Mouse Assay, In Situ, Derivative Assay

    Expression of astrocyte K + channels in TREK-1, TWIK-1 single, and TWIK-1/TREK-1 double gene knockout mice. (A) Schematic illustrations of genetic deletion of four transmembrane domains, including two pore-forming regions, in TREK-1 knockout (TREK-1 −/− , upper), and TWIK-1 knockout (TWIK-1 −/− , lower) mice. (B) PCR genotyping confirmation of successful genetic knockout of the targeted genes in TWIK-1 −/− , TREK-1 −/− , and TWIK-1/TREK-1 double knockout (TWIK-1 −/− /TREK-1 −/− ) mice. (C) The relative quantity of mRNA of K + channels in freshly dissociated mature astrocytes. The results were examined from wild type (WT), TWIK-1 −/− , TREK- 1−/− , and TWIK-1 −/− /TREK-1 −/− astrocytes using qRT-PCR.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ

    doi: 10.3389/fncel.2016.00013

    Figure Lengend Snippet: Expression of astrocyte K + channels in TREK-1, TWIK-1 single, and TWIK-1/TREK-1 double gene knockout mice. (A) Schematic illustrations of genetic deletion of four transmembrane domains, including two pore-forming regions, in TREK-1 knockout (TREK-1 −/− , upper), and TWIK-1 knockout (TWIK-1 −/− , lower) mice. (B) PCR genotyping confirmation of successful genetic knockout of the targeted genes in TWIK-1 −/− , TREK-1 −/− , and TWIK-1/TREK-1 double knockout (TWIK-1 −/− /TREK-1 −/− ) mice. (C) The relative quantity of mRNA of K + channels in freshly dissociated mature astrocytes. The results were examined from wild type (WT), TWIK-1 −/− , TREK- 1−/− , and TWIK-1 −/− /TREK-1 −/− astrocytes using qRT-PCR.

    Article Snippet: The membranes were incubated with anti-TREK-1 antibodies (1:2000, Alomone Labs, Jerusalem, Israel) at 4°C overnight.

    Techniques: Expressing, Double Knockout, Mouse Assay, Knock-Out, Polymerase Chain Reaction, Genotyping Assay, Quantitative RT-PCR

    Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    doi: 10.12659/MSM.908792

    Figure Lengend Snippet: Immunohistochemistry showing the TREK-1 channel distribution in dorsal root ganglion. ( A, C ) Sections from control rats (magnification: [ A ] ×50; [ C ] ×200); ( B, D ) Sections from detrusor overactivity rats (magnification: [ B ] ×50; [ D ] ×200). Black arrows indicate immunopositive neurons.

    Article Snippet: The membranes were incubated with TREK-1 primary antibody (Alomone Labs, Israel), diluted at 1: 1000.

    Techniques: Immunohistochemistry

    TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    doi: 10.12659/MSM.908792

    Figure Lengend Snippet: TREK-1 protein expression in the bladder and dorsal root ganglion of control and detrusor overactivity rats. Data are presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Article Snippet: The membranes were incubated with TREK-1 primary antibody (Alomone Labs, Israel), diluted at 1: 1000.

    Techniques: Expressing

    TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    doi: 10.12659/MSM.908792

    Figure Lengend Snippet: TREK-1 mRNA expression in bladder, spinal cord and dorsal root ganglion of control and DO rats. The mRNA expression is normalized to β-action and presented as means (SD) of TREK-1/β-action. Asterisk indicates P

    Article Snippet: The membranes were incubated with TREK-1 primary antibody (Alomone Labs, Israel), diluted at 1: 1000.

    Techniques: Expressing

    The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Increased Expression of TREK-1 K+ Channel in the Dorsal Root Ganglion of Rats with Detrusor Overactivity After Partial Bladder Outlet Obstruction

    doi: 10.12659/MSM.908792

    Figure Lengend Snippet: The distribution of TREK-1 channel in the bladder and spinal cord of control and detrusor overactivity rats. ( A–D ) Sections of bladder showing TREK-1 channel located in the bladder mucosa ( A, B ) and smooth muscle ( C, D ). ( E–H ) Sections of the spinal cord showing TREK-1 located in the ventral horn ( E, F ) and dorsal horn ( G, H ).

    Article Snippet: The membranes were incubated with TREK-1 primary antibody (Alomone Labs, Israel), diluted at 1: 1000.

    Techniques:

    Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Deletion of TREK-1 aggravates microglia cell activation, neutrophil infiltration, and promotes the secretion of pro-inflammatory cytokine IL-1β and TNF-α on 1 day and 3 days after ICH. Immunofluorescent staining of MPO ( a ) and Iba-1 ( b ) with DAPI in the perihematoma region to detect activated microglia and infiltrated neutrophils on days 1 and 3 after ICH. Scale bar = 20 μm. c – d Statistical analysis of the percentage of activated microglia and infiltrated neutrophils. e – f ELISA result of TNF-α and IL-1β in the perihematoma zone on days 1 and 3 after ICH. Values are expressed as mean ± SEM ( n = 5–7), and data were evaluated by Student’s independent sample t test and one-way ANOVA with Tukey post hoc test. * p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay

    Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Deletion of TREK-1 promotes the ICAM-1, VCAM-1, and PECAM-1 protein expression after ICH. a Immunofluorescent staining of ICAM-1, VCAM-1, and PECAM-1 with DAPI in the perihematoma region on day 3 after ICH. Scale bar = 20 μm. b – d Semi-quantitative analysis of the mean immunofluorescent intensity of ICAM-1, VCAM-1, and PECAM-1. The data were represented as the mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). * p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Expressing, Staining

    Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Deletion of TREK-1 increases the necrosis and apoptotic of neurons accompanied by retarded functional recovery after ICH. a A representative Nissl staining picture shows that TREK-1 deficiency exacerbated the necrosis of neurons. Scale bar = 50 μm. b Counting of Nissl-stained neurons in the perihematoma region on days 1 and 3 after ICH. c Apoptotic neurons in the perihematoma region on days 1 and 3 were detected by double staining of TUNEL (green), NeuN (red), and DAPI (blue). Scale bar = 50 μm. d Statistical analysis of percentage of apoptotic neurons on days 1 and 3 post-ICH in WT ICH and TREK-1 KO ICH groups. e–g Statistical analysis of Longa scores ( e ), corner turn scores ( f ), and forelimb placing scores ( g ) in WT and TREK-1 KO groups. Values are expressed as mean ± SEM ( n = 10), and data were evaluated by Student’s independent sample t test. * p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Functional Assay, Staining, Double Staining, TUNEL Assay

    Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Expression of TREK-1 in the BBB of normal mice brain and perihematoma tissue after ICH. a The diagram of the experimental design in this study. b The localization of TREK-1 immunoreactivity was observed by immunofluorescent staining of TREK-1 (red) with GFAP (blue), and CD31 (green), respectively. Scale bar = 50 μm. c Representative western blot image of TREK-1 and GAPDH expression in perihematoma tissue. d Statistical analysis of western blots signals of TREK-1 in the perihematoma tissue of WT mice after ICH. The data were expressed as mean ± SEM and evaluated by one-way ANOVA with Tukey post hoc test ( n = 5). * p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Expressing, Mouse Assay, Staining, Western Blot

    TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: TREK-1-deficient mice possess a larger hematoma volume on day 3 after ICH and more brain water content on day 7 post-ICH. a A representative MRI image on days 1 and 3 after ICH. b Representative image of HE staining on days 1 and 3 after ICH. c Statistic analysis of hematoma volume according to the HE staining. d Statistical analysis of brain water content using the wet/dry weigh method on days 1, 3, and 7 after ICH. e Representative western blot image of AQP4 expression in the perihematoma tissue. f Quantification of AQP4 expression according to the β-actin expression on days 1 and 3 after ICH. Values were expressed as mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 5–7). # p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Mouse Assay, Magnetic Resonance Imaging, Staining, Western Blot, Expressing

    Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Deficiency of TREK-1 increases the MMP-9 protein expression but have no change on TJPs expression on days 1 and 3 after ICH compared with WT ICH group. a The immunofluorescent staining of the TJPs (ZO-1, occludin, claudin-5) on day 3 after ICH. Scale bar = 20 μm. b Representative western blots image of ZO-1, occludin, claudin-5, and MMP-9. c – f Quantifications of ZO-1, occludin, claudin-5, and MMP-9 protein levels in sham, ICH 1-day, and 3-day groups after ICH. All interest protein expression were normalized to β-actin. Values are expressed as mean ± SEM ( n = 5), and data were evaluated by one-way ANOVA with Tukey post hoc test. # p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Expressing, Staining, Western Blot

    Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p

    Journal: Journal of Neuroinflammation

    Article Title: Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

    doi: 10.1186/s12974-019-1485-5

    Figure Lengend Snippet: Deficiency of TREK-1 exacerbates the BBB impairment after ICH. a The representative brain slices show the extravasation of Evans Blue dye on day 1 after ICH. b The ultrastructure of the lumen (L), tight junctions (arrow) between endothelial cells (E) were observed under a TEM. c Statistic analysis of the extravasation of Evans Blue dye on days 1 and 3 after ICH. The values were represented as the mean ± SEM and data were evaluated by one-way ANOVA with Tukey post hoc test ( n = 4–5). * p

    Article Snippet: Then, these slices were incubated at 4 °C for 12–16 h with primary antibodies (Additional file : Table S2) including rabbit anti-TREK-1 (1:200; Alomone Lab, Jerusalem, Israel), goat anti-mouse glial fibrillary acidic protein (GFAP) (1:200; Abcam Shanghai, China), rabbit anti-Iba1 (1:200; Wako, Osaka, Japan), mouse anti-MPO (1:100; Santa Cruz, Biotechnology, TX, USA), rat-anti mouse CD31 (1:100, BD Bioscience , New Jersey, USA), rabbit-anti mouse ICAM1 (1:100, Proteintech, Wuhan, China), rabbit-anti mouse VCAM1 (1:100, Cell Signaling Technology, Beverly, MA, USA).

    Techniques: Transmission Electron Microscopy