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: Application of mannitol in the presence of ∧ [K + ] o decreases the volume of both astrocytes and neurons. (A) Representative confocal images of a stratum radiatum astrocyte (top) and CA1 pyramidal neuron (bottom) used in volume imaging experiments (far left). The second column shows the baseline volume of the astrocyte and neuron somata in binarized thresholded images (white). The next three columns show overlaid images of the astrocyte and neuron cell soma during each of the specified conditions. Magenta regions indicate increases in cell volume, while green regions indicate reductions in cell volume. Application of ∧ [K + ] ACSF triggered noticeable astrocyte volume increase compared to baseline, while neuronal volume remained relatively constant. In the continued presence of ∧ [K + ] o , introduction of mannitol triggered astrocyte and neuron volume decrease compared to ∧ [K + ] o alone. During the washout of mannitol, astrocytes dramatically increased in volume, while neuronal volume mostly recovered to baseline. (B) Baseline soma volume was determined as the average soma area calculated from three z-stacks taken at minutes 8, 9, and 10 in control ACSF. Graph depicts average percent volume change from baseline (“B”) of astrocytes (blue, n = 11) and neurons (red, n = 13) during continuous exposure to ∧ [K + ] o ACSF (orange bar) and two 10-min exposures to mannitol (green bar). One z-stack of the cell soma was collected every minute during exposure to ∧ [K + ] o. During combined exposure to ∧ [K + ] o and mannitol, a z-stack was collected at minutes (5) and (10). Note significant shrinkage of astrocyte somas by nearly 10% ( p < 0.001) compared to approximately 3% ( p < 0.01) by neurons during the 1st application of dual exposure to ∧ [K + ] o and mannitol. Washout of mannitol resulted in a rebound of astrocyte volume to approximately 3% above baseline ( p < 0.01), while neuron volume increased minimally. Asterisks denote significant differences between astrocyte and neuron volume at each timepoint. * 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: Imaging

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: Application of hyperosmolar ∧ [K + ] o ACSF triggers changes in both tonic and phasic excitatory currents. (A) Representative recording of a neuron voltage-clamped at –70 mV for the duration of the experiment in Mg 2+ -free ACSF + 1 μM TTX. Gaps in recordings coincide with periodic measurement membrane potential and holding current in order to reassess patch-clamp quality between alternating 10-min ACSF treatments: Control ACSF, ∧ [K + ] o , ∧ [K + ] o + mannitol, mannitol washout, and ∧ [K + ] o + mannitol (10 min ea.). (B) Partial section of ACSF baseline, 10-min application of ∧ [K + ] o , and 10 min application of ∧ [K + ] o with mannitol added to show greater detail of changes in activity. Shaded sections correspond to changes in holding current indicating depolarization or hyperpolarization during each application. The inset shows the value of holding current measured at the end of each application period. (C) An ∼1 min section of the recording in (B) showing individual events during exposure to ∧ [K + ] o . (D) An ∼1 min section of the recording in (B) showing individual events during exposure to ∧ [K + ] o with mannitol. Note the prominence of large SIC events in ∧ [K + ] o and smaller mEPSC events in hyperosmolar ∧ [K + ] o . * p < 0.05.

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: Patch Clamp, Activity 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: 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: Application of hyperosmolar ∧ [K + ] o triggers changes in neuron membrane excitability despite block of AMPARs. (A) Representative recording of a neuron voltage-clamped at –70 mV in Mg 2+ -free ACSF + 1 μM TTX and 10 μM NBQX. Gaps in recordings coincide with periodic measurement membrane potential and holding current in order to assess patch-clamp quality between alternating 10-min ACSF treatments: control ACSF, ∧ [K + ] o , ∧ [K + ] o + mannitol, mannitol wash, and ∧ [K + ] o + mannitol. (B) Section of recording showing 10-min mannitol wash and 10-min second application of ∧ [K + ] o + mannitol. Inset shows the value of the holding current measured at the end of each application period ( n = 13). (C) An ∼ 4-min section of the recording in (B) , showing events during the mannitol wash period in ∧ [K + ] o . (D) An ∼ 4-min section of the recording in (B) , showing events during the second ∧ [K + ] o + mannitol application. Note the large SICs during the mannitol wash period compared to the smaller mEPSCs present during the second ∧ [K + ] o + mannitol co-application. (E) A comparison of averaged SIC traces from the first application of ∧ [K + ] o with the first ∧ [K + ] o + mannitol co-application (left) and between the mannitol wash and the second ∧ [K + ] o + mannitol co-application (right). * p < 0.05.

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: Blocking Assay, Patch Clamp