Journal: PLoS ONE

Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

doi: 10.1371/journal.pone.0087816

Figure Lengend Snippet: Western blotting was performed in order to detect MC1R protein expression in mouse and human tissue. Left panel: incubation with a rabbit polyclonal MC1R antibody (Alomone Labs). Right panel: incubation with MC1R antibody and control blocking peptide antigen (BP) in order to show antibody specificity. A) mouse whole glomerular lysate, B) cultured wild type mouse podocytes and C) a human malignant melanoma cell line A375. MC1R protein expression was detected at the expected size of 37 kDa (left panel).

Article Snippet: The following antibodies and dilutions were used for detection of specific proteins: anti-MC1R antibody 1∶500 (AMR-020, Alomone Labs, Ltd., Israel) and anti-actin goat polyclonal antibody 1∶1000 (sc-1615, Santa Cruz Biotechnology, Inc., Dallas, TX).

Techniques: Western Blot, Expressing, Incubation, Blocking Assay, Cell Culture

Journal: Molecular Pain

Article Title: Endothelin receptor type A is involved in the development of oxaliplatin-induced mechanical allodynia and cold allodynia acting through spinal and peripheral mechanisms in rats

doi: 10.1177/17448069211058004

Figure Lengend Snippet: Systemic administration of a dual ET A /ET B receptor antagonist prevented oxaliplatin-induced mechanical allodynia and cold allodynia: Mechanical allodynia (a, b) and cold allodynia (c) were examined using the von Frey test and the acetone test, respectively. Bosentan (50 mg/kg) was intraperitoneally administered for 8 consecutive days (A; from days −1 to 6) and for 2 consecutive days (b and c; days −1 and 0) before intraperitoneal oxaliplatin administration ( n = 6). (a) Mechanical allodynia was examined before each drug administration and 2 h, 8 h, 24 h, 4 days, 7 days, 11 days, and 14 days after oxaliplatin administration. (b) The dose–response relationship of bosentan was examined at day 1 after oxaliplatin administration. (c) Cold allodynia was performed before and 2 h, 8 h and 24 h after oxaliplatin administration. * p < 0.05 and ** p < 0.01, compared with the vehicle group using the Mann–Whitney U -test (a, c). ** p < 0.01, compared with the vehicle group using the Steel test (b). Pre: before administration of bosentan.

Article Snippet: Oxaliplatin (Yakult Corporation, Tokyo, Japan) was diluted in 5% glucose solution (1 mg/mL) and intraperitoneally administered at a dose of 5 mg/kg at day 0. , Bosentan, a dual ET A /ET B receptor antagonist (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), atrasentan, a selective ET A receptor antagonist (Sigma-Aldrich, St Louis, MO, USA) and BQ-788, a selective ET B receptor antagonist (Alomone Labs, Jerusalem, Israel) were dissolved in 60% dimethylsulfoxide and 40% propylene glycol.

Techniques: MANN-WHITNEY

Journal: Molecular Pain

Article Title: Endothelin receptor type A is involved in the development of oxaliplatin-induced mechanical allodynia and cold allodynia acting through spinal and peripheral mechanisms in rats

doi: 10.1177/17448069211058004

Figure Lengend Snippet: Systemic administration of an ET A receptor antagonist, but not an ET B receptor antagonist, prevented oxaliplatin-induced mechanical allodynia and cold allodynia: Mechanical allodynia (a, b, d) and cold allodynia (c, e) were examined using the von Frey test and the acetone test, respectively. An ET A receptor antagonist, atrasentan (a–c), or an ET B receptor antagonist, BQ-788 (d, e), was intraperitoneally administered for 2 consecutive days (days −1 and 0) before intraperitoneal oxaliplatin administration (5 mg/kg) on day 0 ( n = 5–8). (a, d) Mechanical allodynia was examined before each drug administration and 2 h, 8 h, 24 h, 4 days, 7 days, 11 days, 14 days, 21 days, and 28 days after oxaliplatin administration. (c, e) Cold allodynia was examined before atrasentan, BQ-788 and oxaliplatin administration and 2 h, 8 h, 24 h and 4 days after oxaliplatin administration. * p < 0.05 and ** p < 0.01, compared with the vehicle group using the Mann–Whitney U -test. (b) The dose–response relationship of atrasentan was examined at day 1 after oxaliplatin administration. * p < 0.05, compared with the vehicle group using the Steel test. Pre: before administration of atrasentan or BQ-788.

Article Snippet: Oxaliplatin (Yakult Corporation, Tokyo, Japan) was diluted in 5% glucose solution (1 mg/mL) and intraperitoneally administered at a dose of 5 mg/kg at day 0. , Bosentan, a dual ET A /ET B receptor antagonist (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), atrasentan, a selective ET A receptor antagonist (Sigma-Aldrich, St Louis, MO, USA) and BQ-788, a selective ET B receptor antagonist (Alomone Labs, Jerusalem, Israel) were dissolved in 60% dimethylsulfoxide and 40% propylene glycol.

Techniques: MANN-WHITNEY

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: Rise times of mEPSCs and SICs are differentially affected by mannitol. (A) Overall, event rise times were slightly but not significantly faster in the presence of mannitol compared to ∧ [K + ] o alone ( n = 9). (B) For individual 10-min applications, events occurring during the mannitol wash period were much slower than events during the preceding mannitol application period or the initial period in ∧ [K + ] o ( n = 8). (C) Mixed AMPA/NMDA receptor mEPSCs were significantly slower in ∧ [K + ] o + mannitol compared to those occurring in ∧ [K + ] o alone ( n = 9). (D) mEPSCs occurring during both mannitol applications were slower than events occurring during the initial ∧ [K + ] o application ( n = 8). (E) Unlike mEPSCs, SICs had faster rise times during periods of mannitol exposure compared to periods in ∧ [K + ] o ( n = 8). (F) The slowest SICs occurred during the mannitol wash period when cells were swelling the most, with faster events occurring during both mannitol applications ( n = 7). (G) Cumulative probability showed a leftward shift for event rise times in mannitol, suggesting that increasing the osmolarity of ∧ [K + ] o ACSF results in faster rise times overall ( n = 1962 events). (H) Cumulative probability analysis revealed no effect on SIC rise times ( n = 60 events). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: Increased frequency of all events and NMDA receptor mEPSCs during mannitol application, while SIC frequency increases upon mannitol removal. Average frequency of events was calculated for the combined 20-min applications of each solution, as well as for individual 10-min bins. (A) Average frequency of all event types was higher in the presence of ∧ [K + ] o + mannitol than in ∧ [K + ] o alone ( n = 13). (B) Average event frequency increased during both 10 min applications of mannitol as well as the mannitol wash period ( n = 13). (C) Average frequency of mEPSCs was higher during co-application of ∧ [K + ] o + mannitol than during application of ∧ [K + ] o alone ( n = 13). (D) Average frequency of mEPSCs increased during the second co-application of ∧ [K + ] o + mannitol relative to the initial application of ∧ [K + ] o alone, with non-significant fluctuations in frequency during the first ∧ [K + ] o + mannitol co-application and mannitol wash periods ( n = 13). (E) There was no significant difference between the frequency of SICs during ∧ [K + ] o + mannitol co-application vs. ∧ [K + ] o alone ( n = 13). (F) Frequency of SICs significantly increased during the mannitol wash period relative to both the initial ∧ [K + ] o application and the first ∧ [K + ] o + mannitol co-application ( n = 13). (G) Cumulative probability distribution of instantaneous frequency (calculated from inter-event intervals) showed a greater frequency of events in ∧ [K + ] o + mannitol compared to ∧ [K + ] o alone ( n = 208 events). (H) Cumulative probability distribution of instantaneous frequency showed no difference in SIC frequency across experimental conditions ( n = 48 events). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: Amplitude of all NMDA receptor events and SIC decreases with the addition of mannitol. (A) Average amplitude of all NMDAR-mediated events occurring during co-application of ∧ [K + ] o + mannitol was lower compared to ∧ [K + ] o alone ( n = 13). (B) Average amplitude of all events was significantly lower in the presence of mannitol than during the mannitol wash period ( n = 13). (C) There was no significant change in amplitude of mEPSCs with and without mannitol across all time periods ( n = 13) or (D) between separate 10-min time bins ( n = 11). (E) Average amplitude of SICs occurring during co-application of ∧ [K + ] o + mannitol was lower than in ∧ [K + ] o alone ( n = 11). (F) Amplitude of SICs was not significantly different in any specific 10 min recording stretch ( n = 4). The number of statistically usable cells was reduced due to the paucity of SICs in the + mannitol condition, when the extracellular volume expands. (G) Cumulative probability analysis reflected the reduced amplitude of all events during co-application of ∧ [K + ] o + mannitol when compared to application of ∧ [K + ] o alone ( n = 234 events). (H) Cumulative probability analysis failed to return any significant differences between SICs in the presence of ∧ [K + ] o + mannitol vs. ∧ [K + ] o alone ( n = 60 events). * p < 0.05 and ** p < 0.01.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: Rise times of all NMDA receptor currents and SICs, but not mEPSCs, become faster in mannitol. (A) Rise times for all NMDAR events were significantly faster during co-application of ∧ [K + ] o + mannitol compared to ∧ [K + ] o alone ( n = 13). (B) Rise times for all NMDAR events were considerably slower during the mannitol wash period compared to either 10 min period in + mannitol ( n = 13). (C) There were no significant differences in the rise times for NMDAR mEPSCs during ∧ [K + ] o application with and without mannitol ( n = 13). (D) There was no significant difference in rise times of NMDAR mEPSCs when analyzed in separate 10-min time bins ( n = 11). (E) Rise times for SICs were significantly faster during co-application of ∧ [K + ] o + mannitol than during ∧ [K + ] o application alone ( n = 11). (F) Rise times for SICs were not significantly different when compared across individual 10 min recording periods ( n = 4). The number of statistically usable cells was reduced due to the scarcity of SICs occurring in the + mannitol condition. (G) Cumulative probability analysis failed to return any significant difference in rise times of all NMDAR events in ∧ [K + ] o vs. ∧ [K + ] o + mannitol ( n = 234 events). (H) Cumulative probability revealed a significant leftward shift toward faster rise times for SICs in mannitol, suggesting that dilating the extracellular space sped up the rate of glutamate diffusion in the ECS ( n = 60 events). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques: Diffusion-based Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: Addition of DL-AP5 attenuates volume-related effects on neuronal excitability. (A) Generally, the amount of holding current to maintain voltage-clamp at –70 mV was reduced in ∧ [K + ] o + mannitol compared to ∧ [K + ] o alone in all experiments. Holding currents for mixed AMPA + NMDA receptor experiments (blue, n = 9) and those isolating NMDA receptor currents (+NBQX) (green, n = 13) appeared remarkably similar and had no points during which they were significantly different. Holding currents recorded during the NMDA receptor inhibition experiments (+NBQX/+DL-AP5) were less negative overall and were significantly less negative following the second ∧ [K + ] o + mannitol co-application period (yellow, n = 8). (B) Comparison of resting membrane potentials for the NMDA receptor isolation and NMDA receptor inhibition experiments. Shifts in resting membrane potential indicated that cells became depolarized relative to baseline during application of ∧ [K + ] o , while co-application of ∧ [K + ] o + mannitol triggered slight hyperpolarizing shifts. Resting membrane potentials recorded during the NMDAR inhibition experiments (yellow, n = 8) indicated significantly less depolarization compared to the NMDAR isolation experiments following the initial application of ∧ [K + ] o alone, the mannitol wash period, and the second co-application of ∧ [K + ] o + mannitol (green, n = 13). * p < 0.05 and *** p < 0.001.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques: Inhibition, Isolation

Journal: Frontiers in Cellular Neuroscience

Article Title: Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium

doi: 10.3389/fncel.2022.930384

Figure Lengend Snippet: DL-AP5 substantially attenuates NMDA receptor currents during application of ∧ [K + ] o . Approximately 1-min section of recording taken from the mannitol wash period for an experiment conducted in NBQX without DL-AP5 (A) compared to the mannitol wash period with 50 μM DL-AP5 (B) . Note the number of large SIC-like events in the absence, but not the presence, of DL-AP5. (C) Frequency of all NMDA receptor events was significantly lower in DL-AP5 (yellow, n = 8) compared to + NBQX alone (green, n = 13) during ∧ [K + ] o application both with and without mannitol. (D) When separating events into individual 10-min recording periods, NMDA receptor events were significantly reduced during the mannitol wash period and second ∧ [K + ] o + mannitol application period. (E) DL-AP5 also significantly inhibited the occurrence of SICs either with or without mannitol. (F) When grouping frequencies of SICs into 10-min time bins, DL-AP5 significantly inhibited SICs during the mannitol wash period and second ∧ [K + ] o + mannitol co-application period. SICs were blocked completely during the second ∧ [K + ] o + mannitol co-application period. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Additional experiments in ∧ [K + ] o ACSF + mannitol as described above were performed in the presence of 10 μM NBQX (Alomone Labs) to block AMPA receptor currents (NMDA receptor isolation), or with the addition of 50 μL DL-AP5 (Abcam) to block remaining NMDA receptor currents (NMDA receptor inhibition).

Techniques: