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    Alomone Labs 1 ebio
    1 Ebio, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1 article reviews
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    1 ebio - by Bioz Stars, 2022-06
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    Alomone Labs nimodipine
    <t>Nimodipine</t> alters GnRH induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8 - figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male (A) wild-type and (D) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs-Ringer. Comparisons of AUC from a (B) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p
    Nimodipine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nimodipine alters GnRH induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8 - figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male (A) wild-type and (D) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs-Ringer. Comparisons of AUC from a (B) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: Nimodipine alters GnRH induced calcium responses in gonadotropes of wild-type and Ctail mice. The analysis in Figure 8 - figure supplement 1 was repeated but with the L-type calcium channel blocker nimodipine (Nim) applied prior to and during the second GnRH pulse. Raster plots of GnRH induced calcium responses in the absence (left) and presence of nimodipine (right) in gonadotropes from a representative adult male (A) wild-type and (D) Ctail mouse. The two stimuli were separated by 60 min wash with Krebs-Ringer. Comparisons of AUC from a (B) wild-type (275 cells; 11.5 ± 4.7 vs. 7.1 ± 2.8 a.u. for GnRH and GnRH/Nim, respectively; p

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Mouse Assay

    GnRH induced ERK1/2 phosphorylation is Gα q/11 - and PKC-dependent, and calcium-independent in homologous LβT2 cells. (A) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (DMSO) for 1 h, and then treated with vehicle (water) or 10 nM GnRH for 5- or 15-min. Whole cell protein lysates were collected and subjected to SDS-PAGE and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. (B) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085]. (C) LβT2 cells were pretreated for 20 min with vehicle (DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (D) Data from 3 independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. (E) LβT2 cells were pretreated for 20 min with vehicle (DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (F) Data from 3 independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999.

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: GnRH induced ERK1/2 phosphorylation is Gα q/11 - and PKC-dependent, and calcium-independent in homologous LβT2 cells. (A) LβT2 cells were pretreated with 10 µM FR900359 (Gα q/11 inhibitor) or vehicle (DMSO) for 1 h, and then treated with vehicle (water) or 10 nM GnRH for 5- or 15-min. Whole cell protein lysates were collected and subjected to SDS-PAGE and western blotting with phospho- (top) or total (bottom) ERK1/2 antibodies. Blots from one of two replicate experiments are shown. (B) Data from independent duplicate experiments exemplified in panel A were quantified by normalizing the densitometry for the pERK1/2 bands to the total ERK1/2 bands. Data are presented as fold phospho-ERK1/2 relative to the control condition. Bar height reflects the group mean. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Vehicle vs. FR900359: 0 min GnRH p > 0.9999; 5 min GnRH p = 0.0106; 15 min GnRH p = 0.0085]. (C) LβT2 cells were pretreated for 20 min with vehicle (DMSO) or 5 µM Gö6983 for 20 min followed by treatment with vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (D) Data from 3 independent experiments in exemplified panel C were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control (vehicle) vs. GnRH (vehicle) p = 0.0048; Control (Gö6983) vs. GnRH (Gö6983) p = 0.3470. Control (vehicle) vs. Control (Gö6983) p = 0.9472; GnRH (vehicle) vs. GnRH (Gö6983) p = 0.0389. (E) LβT2 cells were pretreated for 20 min with vehicle (DMSO), 20 µM BAPTA-AM, 5 µM or 10 µM nimodipine, or 10 µM U0126 for 20 min followed by vehicle (water) or 10 nM GnRH for 5 min. Western blots were performed as in panel A. (F) Data from 3 independent experiments in panel E were presented and quantified as in panel B. Data were analyzed by two-way ANOVA, followed by Sidak’s post-hoc comparison tests. Bars with different letters differed significantly. Control vs. GnRH: Vehicle p = 0.0103; BAPTA-AM p = 0.0229; Nimodipine (5 µM) p = 0.0377; Nimodipine (10 µM) p = 0.0350; U1026 p > 0.9999.

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: SDS Page, Western Blot

    GnRH-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. (A) Relative Fshb and (B) Lhb expression in LβT2 cells treated with vehicle (DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of 2 independent experiments. Data were analyzed with two-way ANOVAs, followed by post-hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Journal: bioRxiv

    Article Title: Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

    doi: 10.1101/2021.09.13.460073

    Figure Lengend Snippet: GnRH-induced Fshb and Lhb expression does not depend on calcium entry via L-type channels in homologous LβT2 cells. (A) Relative Fshb and (B) Lhb expression in LβT2 cells treated with vehicle (DMSO) or 10 µM nimodipine for 20 min followed by treatment with water (vehicle) or high frequency GnRH (10 nM) pulses. Gene expression was assessed by RT-qPCR and normalized to Rpl19 . Data reflect the means of 2 independent experiments. Data were analyzed with two-way ANOVAs, followed by post-hoc Tukey’s test for multiple comparisons. Bars with different letters differed significantly. Panel A: Control (vehicle) vs. GnRH (vehicle) p = 0.0178; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0232; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8545; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0235. Panel B: Control (vehicle) vs. GnRH (vehicle) p = 0.0061; Control (vehicle) vs. GnRH (Nimodipine) p = 0.0043; GnRH (vehicle) vs. GnRH (Nimodipine) p = 0.8770; Control (Nimodipine) vs. GnRH (Nimodipine) p = 0.0178.

    Article Snippet: To evaluate the contribution of voltage-gated calcium channels, after 1 h of recovery, tissue was incubated for 30 s with 20 μM nimodipine (ALOMONE LABS N-150 N150SM0250; Jerusalem, Israel) followed by a second application of 10 nM GnRH alone ( ) or in combination with 20 μM nimodipine ( ) for 30 s. Finally, to determine cell viability, high potassium solution (50 KCl mM, 120 NaCl mM, 10 HEPES mM, 2 CaCl2 mM, pH 7.4) was applied for 30 s. For each condition, the numbers of animals and cells analyzed are indicated in the figure legends.

    Techniques: Expressing, Quantitative RT-PCR

    CNQX and nimodipine significantly attenuate CX614-induced increases in BDNF mRNA content. Scatter graphs show BDNF cRNA-labeling densities (μCi/gm) in the stratum granulosum ( A, C ), the CA3 stratum pyramidale ( B ), and the entorhinal cortex layers

    Journal: The Journal of Neuroscience

    Article Title: Positive Modulation of AMPA Receptors Increases Neurotrophin Expression by Hippocampal and Cortical Neurons

    doi: 10.1523/JNEUROSCI.20-01-00008.2000

    Figure Lengend Snippet: CNQX and nimodipine significantly attenuate CX614-induced increases in BDNF mRNA content. Scatter graphs show BDNF cRNA-labeling densities (μCi/gm) in the stratum granulosum ( A, C ), the CA3 stratum pyramidale ( B ), and the entorhinal cortex layers

    Article Snippet: For experiments using CNQX (20 μ m; Tocris Cookson, Ballwin, MO), APV (100 μ m; Tocris Cookson), nimodipine (20 μ m; Alomone Labs, Jerusalem, Israel), or CoCl2 (5 m m; Sigma), cultured slices were first pretreated (1 hr for CNQX and APV, 30 min for nimodipine, and 10 min for CoCl2 ) with either the blocker or vehicle in media and then treated for 3 hr with either blocker alone, blocker + 50 μ m CX614, 50 μ m CX614 alone, or vehicle.

    Techniques: Labeling

    Ca 2+ channel inhibitors differentially affect firing frequency of SCN neurons between day and night. A , Representative extracellular recordings of spontaneous action potential activity recorded from SCN neurons in day and night. B , Daytime firing decreased in Cd 2+ (P=0.01), Nimodipine (P=0.03), ConoGVIA (P=0.01), AgaIVA (P=0.01), Ni 2+ (P=0.01), Dan (P=0.01) and CPA (P=0.04) compared to control day slices (Ctrl). C , Nighttime firing decreased in Cd 2+ (P=0.01), but increased in Dan (P=0.01) and CPA (P=0.01) compared to control night slices (Ctrl). TTAP2 had no effect on firing in day (P=1) or night (P=1). * P

    Journal: The Journal of physiology

    Article Title: Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing

    doi: 10.1113/JP278327

    Figure Lengend Snippet: Ca 2+ channel inhibitors differentially affect firing frequency of SCN neurons between day and night. A , Representative extracellular recordings of spontaneous action potential activity recorded from SCN neurons in day and night. B , Daytime firing decreased in Cd 2+ (P=0.01), Nimodipine (P=0.03), ConoGVIA (P=0.01), AgaIVA (P=0.01), Ni 2+ (P=0.01), Dan (P=0.01) and CPA (P=0.04) compared to control day slices (Ctrl). C , Nighttime firing decreased in Cd 2+ (P=0.01), but increased in Dan (P=0.01) and CPA (P=0.01) compared to control night slices (Ctrl). TTAP2 had no effect on firing in day (P=1) or night (P=1). * P

    Article Snippet: Drugs were used at final concentrations of 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel; T-550), 10 μM Nimodipine (Alomone Labs, Jerusalem, Israel; N150), 10 μM dantrolene (Dan; Sigma, Darmstadt, Germany; D9175), 1 μM cyclopiazonic acid (CPA; Alomone Labs, Jerusalem, Israel; C-750), 3 μM ω-conotoxin GVIA (ConoGVIA; Alomone Labs, Jerusalem, Israel; C-300), 3 μM ω-conotoxin MVIIC (ConoMVIIC; Alomone Labs, Jerusalem, Israel; C-150), 200 nM ω-agatoxin IVA (AgaIVA;Alomone Labs, Jerusalem, Israel; STA-500), 1 μM TTA-P2, (TTAP2;Alomone Labs, Jerusalem, Israel; T-155), 200 μM CdCl2 (Cd2+ ; Sigma; 529575), 30 μM NiCl2 (Ni2+ ; Sigma; 22387), and 5 μM Bay K8644 (Bay K; Sigma, Darmstadt, Germany; B133).

    Techniques: Activity Assay

    Action potential evoked Ca 2+ currents from day and night SCN cells. A , Action potential voltage commands recorded from SCN neurons during the day and night were used to evoke Ca 2+ currents. Action potential commands were delivered from a holding potential of −150 mV. Dotted lines represent 0 mV. B - C , Representative day and night Cd 2+ -sensitive ( B ) and Nimodipine-sensitive ( C ) current responses. D , Normalized peak current density for each Ca 2+ current subtype. *The day versus night Cd 2+ -, ConoGVIA, and Ni 2+ -sensitive currents are significantly different (P=0.001, P=0.04, and P=0.001, respectively; unpaired t -test). N’s are the number of neurons recorded: Cd 2+ -sensitive current (n=11 day, 10 night), Nimodipine-sensitive (11, 10), ConoGVIA-sensitive (9, 8), AgaIVA-sensitive (9, 9), Ni + -sensitive (9, 7), and TTAP2-sensitive (5, 3). Data were obtained from 3–4 slices per condition as follows (# slices day, # slices night): Cd 2+ (3, 3), Nimodipine, (4, 4), ConoGVIA (3, 4), AgaIVA (3, 3), Ni 2+ (3, 4), TTAP2 (3, 3).

    Journal: The Journal of physiology

    Article Title: Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing

    doi: 10.1113/JP278327

    Figure Lengend Snippet: Action potential evoked Ca 2+ currents from day and night SCN cells. A , Action potential voltage commands recorded from SCN neurons during the day and night were used to evoke Ca 2+ currents. Action potential commands were delivered from a holding potential of −150 mV. Dotted lines represent 0 mV. B - C , Representative day and night Cd 2+ -sensitive ( B ) and Nimodipine-sensitive ( C ) current responses. D , Normalized peak current density for each Ca 2+ current subtype. *The day versus night Cd 2+ -, ConoGVIA, and Ni 2+ -sensitive currents are significantly different (P=0.001, P=0.04, and P=0.001, respectively; unpaired t -test). N’s are the number of neurons recorded: Cd 2+ -sensitive current (n=11 day, 10 night), Nimodipine-sensitive (11, 10), ConoGVIA-sensitive (9, 8), AgaIVA-sensitive (9, 9), Ni + -sensitive (9, 7), and TTAP2-sensitive (5, 3). Data were obtained from 3–4 slices per condition as follows (# slices day, # slices night): Cd 2+ (3, 3), Nimodipine, (4, 4), ConoGVIA (3, 4), AgaIVA (3, 3), Ni 2+ (3, 4), TTAP2 (3, 3).

    Article Snippet: Drugs were used at final concentrations of 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel; T-550), 10 μM Nimodipine (Alomone Labs, Jerusalem, Israel; N150), 10 μM dantrolene (Dan; Sigma, Darmstadt, Germany; D9175), 1 μM cyclopiazonic acid (CPA; Alomone Labs, Jerusalem, Israel; C-750), 3 μM ω-conotoxin GVIA (ConoGVIA; Alomone Labs, Jerusalem, Israel; C-300), 3 μM ω-conotoxin MVIIC (ConoMVIIC; Alomone Labs, Jerusalem, Israel; C-150), 200 nM ω-agatoxin IVA (AgaIVA;Alomone Labs, Jerusalem, Israel; STA-500), 1 μM TTA-P2, (TTAP2;Alomone Labs, Jerusalem, Israel; T-155), 200 μM CdCl2 (Cd2+ ; Sigma; 529575), 30 μM NiCl2 (Ni2+ ; Sigma; 22387), and 5 μM Bay K8644 (Bay K; Sigma, Darmstadt, Germany; B133).

    Techniques:

    Relative contribution of voltage-gated calcium currents in the SCN. Macroscopic voltage-activated calcium currents recorded in whole-cell voltage-clamp mode during the day or night. From a holding potential of −90 mV, currents were elicited from 150-ms voltage steps in 10-mV increments. A , Representative total Ca 2+ currents before (control) and after addition of Cd 2+ . B , Current-voltage relationships for the total Ca 2+ current and Cd 2+ -sensitive components, versus L-, N-, P/Q-, R-, and T-type subtypes of the Ca 2+ current (Cd 2+ -sensitive, 200 μM; Nimodipine-sensitive, 10 μM; ConoMVIIC-sensitive, 3 μM; ConoGVIA-sensitive, 3 μM; AgaIVA-sensitive, 200 nM; Ni 2+ -sensitive, 30 μM; and TTAP2-sensitive, 1 μM). One Ca 2+ current subtype was isolated per cell by application of an inhibitor and subtraction from the baseline current (total Ca 2+ ). C , The relative contribution of each inhibitor-sensitive current to the total Ca 2+ current in day and night, calculated from the peak current. Due to the variation in current magnitudes from cell to cell (and overlapping specificities of the drugs), the sum of the individual inhibitors exceeds 100%. N’s are the number of neurons recorded (day, night): Cd 2+ (12, 10), Nimodipine, (21, 20), ConoMVIIC (7, 7), ConoGVIA (11, 8), AgaIVA (17, 18), Ni 2+ (9, 8), TTAP2 (4, 4). Data were obtained from 3–8 slices per condition as follows (# slices day, # slices night): Cd 2+ (3, 3), Nimodipine, (8, 8), ConoMVIIC (2, 2), ConoGVIA (4, 4), AgaIVA (7, 6), Ni 2+ (3, 3), TTAP2 (2, 4). Total Ca 2+ currents were obtained at baseline from all recordings (n=96 neurons in 35 slices for day and 82 neurons in 33 slices for night).

    Journal: The Journal of physiology

    Article Title: Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing

    doi: 10.1113/JP278327

    Figure Lengend Snippet: Relative contribution of voltage-gated calcium currents in the SCN. Macroscopic voltage-activated calcium currents recorded in whole-cell voltage-clamp mode during the day or night. From a holding potential of −90 mV, currents were elicited from 150-ms voltage steps in 10-mV increments. A , Representative total Ca 2+ currents before (control) and after addition of Cd 2+ . B , Current-voltage relationships for the total Ca 2+ current and Cd 2+ -sensitive components, versus L-, N-, P/Q-, R-, and T-type subtypes of the Ca 2+ current (Cd 2+ -sensitive, 200 μM; Nimodipine-sensitive, 10 μM; ConoMVIIC-sensitive, 3 μM; ConoGVIA-sensitive, 3 μM; AgaIVA-sensitive, 200 nM; Ni 2+ -sensitive, 30 μM; and TTAP2-sensitive, 1 μM). One Ca 2+ current subtype was isolated per cell by application of an inhibitor and subtraction from the baseline current (total Ca 2+ ). C , The relative contribution of each inhibitor-sensitive current to the total Ca 2+ current in day and night, calculated from the peak current. Due to the variation in current magnitudes from cell to cell (and overlapping specificities of the drugs), the sum of the individual inhibitors exceeds 100%. N’s are the number of neurons recorded (day, night): Cd 2+ (12, 10), Nimodipine, (21, 20), ConoMVIIC (7, 7), ConoGVIA (11, 8), AgaIVA (17, 18), Ni 2+ (9, 8), TTAP2 (4, 4). Data were obtained from 3–8 slices per condition as follows (# slices day, # slices night): Cd 2+ (3, 3), Nimodipine, (8, 8), ConoMVIIC (2, 2), ConoGVIA (4, 4), AgaIVA (7, 6), Ni 2+ (3, 3), TTAP2 (2, 4). Total Ca 2+ currents were obtained at baseline from all recordings (n=96 neurons in 35 slices for day and 82 neurons in 33 slices for night).

    Article Snippet: Drugs were used at final concentrations of 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel; T-550), 10 μM Nimodipine (Alomone Labs, Jerusalem, Israel; N150), 10 μM dantrolene (Dan; Sigma, Darmstadt, Germany; D9175), 1 μM cyclopiazonic acid (CPA; Alomone Labs, Jerusalem, Israel; C-750), 3 μM ω-conotoxin GVIA (ConoGVIA; Alomone Labs, Jerusalem, Israel; C-300), 3 μM ω-conotoxin MVIIC (ConoMVIIC; Alomone Labs, Jerusalem, Israel; C-150), 200 nM ω-agatoxin IVA (AgaIVA;Alomone Labs, Jerusalem, Israel; STA-500), 1 μM TTA-P2, (TTAP2;Alomone Labs, Jerusalem, Israel; T-155), 200 μM CdCl2 (Cd2+ ; Sigma; 529575), 30 μM NiCl2 (Ni2+ ; Sigma; 22387), and 5 μM Bay K8644 (Bay K; Sigma, Darmstadt, Germany; B133).

    Techniques: Isolation

    Effect of Ca 2+ channel inhibitors on SCN action potential rhythmicity. A-F , Representative spontaneous action potential activity recorded from organotypic SCN slices over 3 days of baseline control (Ctrl) and 3 days following the application of drugs: vehicle control (Veh) ( A ), 10 μM Nimodipine ( B ), 3 μM ConoGVIA ( C ), 200 nM AgaIVA ( D ), 30 μM Ni 2+ ( E ), 5 μM Bay K ( F ). Each line is firing rate recorded at a single electrode within the SCN. G , Percentage of recordings from electrodes within the SCN that exhibited rhythmic firing. Paired t -tests were used to compare baseline control to after drug values. The percentage of rhythmic recordings decreased in Nimodipine (P=0.02), ConoGIVA (P=0.04), AgaIVA (P=0.005), Ni 2+ (P=0.008) and BayK (P=0.02). H , χ 2 amplitude quantified from the rhythmic recordings. χ 2 amplitude of rhythmic recordings decreased in Nimodipine, (P=0.04) ConoGIVA (P=0.04), AgaIVA (P=0.03), Ni 2+ (P=0.04) and BayK (P=0.04). I , Period length from the rhythmic recordings. Period length was increased in Dan (P=0.04). Individual data points in panels G - I are the slice mean ± SEM values (from the recordings within one slice): Veh (n=198 recordings, 11 slices), Nimodipine (n=123, 8), ConoGVIA (n=91, 5), AgaIVA (n=63, 5), Ni 2+ (n=84, 5), TTAP2 (n=57, 3), Dan (n=101, 5), CPA (n=93, 5) and BayK (n=75, 4).

    Journal: The Journal of physiology

    Article Title: Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing

    doi: 10.1113/JP278327

    Figure Lengend Snippet: Effect of Ca 2+ channel inhibitors on SCN action potential rhythmicity. A-F , Representative spontaneous action potential activity recorded from organotypic SCN slices over 3 days of baseline control (Ctrl) and 3 days following the application of drugs: vehicle control (Veh) ( A ), 10 μM Nimodipine ( B ), 3 μM ConoGVIA ( C ), 200 nM AgaIVA ( D ), 30 μM Ni 2+ ( E ), 5 μM Bay K ( F ). Each line is firing rate recorded at a single electrode within the SCN. G , Percentage of recordings from electrodes within the SCN that exhibited rhythmic firing. Paired t -tests were used to compare baseline control to after drug values. The percentage of rhythmic recordings decreased in Nimodipine (P=0.02), ConoGIVA (P=0.04), AgaIVA (P=0.005), Ni 2+ (P=0.008) and BayK (P=0.02). H , χ 2 amplitude quantified from the rhythmic recordings. χ 2 amplitude of rhythmic recordings decreased in Nimodipine, (P=0.04) ConoGIVA (P=0.04), AgaIVA (P=0.03), Ni 2+ (P=0.04) and BayK (P=0.04). I , Period length from the rhythmic recordings. Period length was increased in Dan (P=0.04). Individual data points in panels G - I are the slice mean ± SEM values (from the recordings within one slice): Veh (n=198 recordings, 11 slices), Nimodipine (n=123, 8), ConoGVIA (n=91, 5), AgaIVA (n=63, 5), Ni 2+ (n=84, 5), TTAP2 (n=57, 3), Dan (n=101, 5), CPA (n=93, 5) and BayK (n=75, 4).

    Article Snippet: Drugs were used at final concentrations of 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel; T-550), 10 μM Nimodipine (Alomone Labs, Jerusalem, Israel; N150), 10 μM dantrolene (Dan; Sigma, Darmstadt, Germany; D9175), 1 μM cyclopiazonic acid (CPA; Alomone Labs, Jerusalem, Israel; C-750), 3 μM ω-conotoxin GVIA (ConoGVIA; Alomone Labs, Jerusalem, Israel; C-300), 3 μM ω-conotoxin MVIIC (ConoMVIIC; Alomone Labs, Jerusalem, Israel; C-150), 200 nM ω-agatoxin IVA (AgaIVA;Alomone Labs, Jerusalem, Israel; STA-500), 1 μM TTA-P2, (TTAP2;Alomone Labs, Jerusalem, Israel; T-155), 200 μM CdCl2 (Cd2+ ; Sigma; 529575), 30 μM NiCl2 (Ni2+ ; Sigma; 22387), and 5 μM Bay K8644 (Bay K; Sigma, Darmstadt, Germany; B133).

    Techniques: Activity Assay

    Effects of Bay K8644 on Ca 2+ current during the day and night. A - B , Representative day and night Bay K-sensitive Ca 2+ currents. C - D , Current-voltage relationships for day and night total Ca 2+ current components. E , Comparison of day and night Bay K-sensitive currents showing that increasing L-type channel activity activated currents of similar magnitude during both the day and night, thereby eliminating the day versus night difference in current magnitude. F , Bay K-sensitive currents normalized to total current, to account for variability in Ca 2+ current levels between cells. Both day and night currents were increased with Bay K application. However, no significant difference between day and night current was observed (unpaired t -test). N’s are the number of neurons recorded (day, night): total Ca 2+ (96, 82), Nimodipine-sensitive (21, 20), and Bay K-sensitive (9, 9). Data were obtained from 3–8 slices per condition as follows (# slices day, # slices night): Nimodipine-sensitive (8, 8), and Bay K-sensitive (5, 3). G , Bay K-sensitive current normalized to the Nimodipine-sensitive current, showing the relative increase for nighttime L-type current is greater than daytime.

    Journal: The Journal of physiology

    Article Title: Diurnal properties of voltage-gated Ca2+ currents in suprachiasmatic nucleus and roles in action potential firing

    doi: 10.1113/JP278327

    Figure Lengend Snippet: Effects of Bay K8644 on Ca 2+ current during the day and night. A - B , Representative day and night Bay K-sensitive Ca 2+ currents. C - D , Current-voltage relationships for day and night total Ca 2+ current components. E , Comparison of day and night Bay K-sensitive currents showing that increasing L-type channel activity activated currents of similar magnitude during both the day and night, thereby eliminating the day versus night difference in current magnitude. F , Bay K-sensitive currents normalized to total current, to account for variability in Ca 2+ current levels between cells. Both day and night currents were increased with Bay K application. However, no significant difference between day and night current was observed (unpaired t -test). N’s are the number of neurons recorded (day, night): total Ca 2+ (96, 82), Nimodipine-sensitive (21, 20), and Bay K-sensitive (9, 9). Data were obtained from 3–8 slices per condition as follows (# slices day, # slices night): Nimodipine-sensitive (8, 8), and Bay K-sensitive (5, 3). G , Bay K-sensitive current normalized to the Nimodipine-sensitive current, showing the relative increase for nighttime L-type current is greater than daytime.

    Article Snippet: Drugs were used at final concentrations of 1 μM tetrodotoxin (TTX; Alomone Labs, Jerusalem, Israel; T-550), 10 μM Nimodipine (Alomone Labs, Jerusalem, Israel; N150), 10 μM dantrolene (Dan; Sigma, Darmstadt, Germany; D9175), 1 μM cyclopiazonic acid (CPA; Alomone Labs, Jerusalem, Israel; C-750), 3 μM ω-conotoxin GVIA (ConoGVIA; Alomone Labs, Jerusalem, Israel; C-300), 3 μM ω-conotoxin MVIIC (ConoMVIIC; Alomone Labs, Jerusalem, Israel; C-150), 200 nM ω-agatoxin IVA (AgaIVA;Alomone Labs, Jerusalem, Israel; STA-500), 1 μM TTA-P2, (TTAP2;Alomone Labs, Jerusalem, Israel; T-155), 200 μM CdCl2 (Cd2+ ; Sigma; 529575), 30 μM NiCl2 (Ni2+ ; Sigma; 22387), and 5 μM Bay K8644 (Bay K; Sigma, Darmstadt, Germany; B133).

    Techniques: Activity Assay

    LTCC activity is reduced in Kv2.1 KO hippocampal neurons. (A) Representative Ba 2+ current traces recorded from WT (left) and Kv2.1 KO CHNs (right) recorded at +10 mV in vehicle or in the presence of the LTCC inhibitor nimodipine (10 µM). (B) Representative raw tail current records from a WT (left) and Kv2.1 KO (right) CHN induced by a step to -70 mV from a 10 mV prepulse, recorded in vehicle or in the presence of 10 µM nimodipine. C-F. Comparison of WT (red) and Kv2.1 KO (black) CHNs. (C) Maximum tail current amplitudes measured at -70 mV from a 10 mV prepulse. Each point represents one cell. (D) As in C but recorded in the presence of 10 µM nimodipine. (E) Maximum nimodipine-sensitive tail current amplitudes obtained from each cell by subtracting maximum tail current amplitudes measured in vehicle from those measured in the presence of nimodipine. (F) Representative nimodipine-sensitive LTCC gating and tail currents recorded from WT and Kv2.1 KO CHNs. (G) Quantification of nimodipine-sensitive LTCC Q on (left), I tail (center), and Q on vs. I tail (right) recorded from WT and Kv2.1 KO CHNs. Each point corresponds to a single cell (*p=0.019, Student’s t -test).

    Journal: bioRxiv

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in neurons

    doi: 10.1101/702514

    Figure Lengend Snippet: LTCC activity is reduced in Kv2.1 KO hippocampal neurons. (A) Representative Ba 2+ current traces recorded from WT (left) and Kv2.1 KO CHNs (right) recorded at +10 mV in vehicle or in the presence of the LTCC inhibitor nimodipine (10 µM). (B) Representative raw tail current records from a WT (left) and Kv2.1 KO (right) CHN induced by a step to -70 mV from a 10 mV prepulse, recorded in vehicle or in the presence of 10 µM nimodipine. C-F. Comparison of WT (red) and Kv2.1 KO (black) CHNs. (C) Maximum tail current amplitudes measured at -70 mV from a 10 mV prepulse. Each point represents one cell. (D) As in C but recorded in the presence of 10 µM nimodipine. (E) Maximum nimodipine-sensitive tail current amplitudes obtained from each cell by subtracting maximum tail current amplitudes measured in vehicle from those measured in the presence of nimodipine. (F) Representative nimodipine-sensitive LTCC gating and tail currents recorded from WT and Kv2.1 KO CHNs. (G) Quantification of nimodipine-sensitive LTCC Q on (left), I tail (center), and Q on vs. I tail (right) recorded from WT and Kv2.1 KO CHNs. Each point corresponds to a single cell (*p=0.019, Student’s t -test).

    Article Snippet: Caffeine (Sigma Cat# C0750), thapsigargin (Millipore Cat# 586005), nimodipine (Alomone Cat# N-150), Bay K8644 (Alomone Cat# B-350), and tetracaine (Sigma Cat# T7508) were dissolved in warm KRB at 2x the final concentration and added to cells during imaging by a pipette.

    Techniques: Activity Assay