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:

Journal: Scientific Reports

Article Title: Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABA A receptor clustering induced by inhibitory synaptic plasticity

doi: 10.1038/s41598-017-14210-5

Figure Lengend Snippet: Correlative light-high resolution scanning electron microscopy (CL-HRSEM) localization of GABA A Rα1 in control (CTRL, a–e) and iLTP (NMDA, f–i) primary hippocampal neurons growing on photo-etched coverslips. The BSE signal (pseudo-coloured in yellow) is superimposed on the grey-scale SE images. ( a ): low magnification HRSEM images of a CTRL neuron immunolabelled for GABA A Rα1. Inset: same neurons imaged by CFM. ( b ): CFM image of part of the neurite bundle boxed in a. Note the presence of GABA A Rα1 clusters (bright spots) along the neurite. The boxed regions are magnified in c (insets) and in d–e. ( c ): HRSEM image showing the same region boxed in a. The yellow spots on the neurite bundle are GABA A Rα1 receptor clusters. The double inset shows higher magnifications of the single neurite boxed in (c) (bottom left) imaged respectively at the CFM (above) and at the HRSEM (below). The arrowheads point to neurites without gold nanoparticles; ( d ): HRSEM image of the bundle of neurites boxed in c (upper right); ( e ): CFM image of the same bundle of neurites imaged in d. The arrow points to a fluorescent spot not observed in d; ( f ): low magnification HRSEM images of a NMDA stimulated neurons immunolabelled for GABA A Rα1. Inset: the same neurons imaged by CFM; ( g ): HRSEM image showing a portion of the cell body of the neuron imaged in f (upper Inset). Left inset: the same region imaged at the CFM. Right Inset: HRSEM higher magnification of the region boxed in g. ( h ): HRSEM image of the region boxed in f (bottom Inset). The arrowheads point to neurites without gold nanoparticles; ( i ), CFM image of the same region imaged in h. The arrow points to a fluorescent spot not observed in h. Circles and brackets point to the same sub-regions. Scale bars are 10 µm in a, a inset, f, f inset; 5 µm in b and g right inset; 1 µm in c–e and g–i; 0.2 µm in g right inset.

Article Snippet: During the recovery period, 12 minutes after the end of the NMDA treatment, live immunolabelling of GABA A Rα1 subunits was performed, namely cells were incubated 13 minutes in a solution containing primary antibody against GABA A Rα1 (Alomone Labs, Israel) diluted 1:30 in 0.5% bovine serum albumin (BSA), 350 mM sucrose in PBS, followed by 13 minutes incubation with gold-conjugated secondary antibody.

Techniques: Electron Microscopy

Journal: Scientific Reports

Article Title: Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABA A receptor clustering induced by inhibitory synaptic plasticity

doi: 10.1038/s41598-017-14210-5

Figure Lengend Snippet: Subcellular GABA A Rα1 receptor distribution on soma and neurites in CTRL and NMDA stimulated hippocampal neurons. ( a ): bar plot showing gold clusters density in CTRL and NMDA stimulated neurons; ( b ): bar plot showing clusters size (number of gold particles/cluster) in CTRL and NMDA stimulated neurons; ( c ): bar plot showing the number of clusters formed respectively by n ≤ 5 and n > 5 gold particles on soma and neurites in CTRL and NMDA stimulated hippocampal neurons. * Indicates significant differences (*p < 0.05, Student’s t-test). Values are mean ± s.e.m.

Article Snippet: During the recovery period, 12 minutes after the end of the NMDA treatment, live immunolabelling of GABA A Rα1 subunits was performed, namely cells were incubated 13 minutes in a solution containing primary antibody against GABA A Rα1 (Alomone Labs, Israel) diluted 1:30 in 0.5% bovine serum albumin (BSA), 350 mM sucrose in PBS, followed by 13 minutes incubation with gold-conjugated secondary antibody.

Techniques:

Journal: Scientific Reports

Article Title: Correlating Fluorescence and High-Resolution Scanning Electron Microscopy (HRSEM) for the study of GABA A receptor clustering induced by inhibitory synaptic plasticity

doi: 10.1038/s41598-017-14210-5

Figure Lengend Snippet: Post-synaptic surface and post-synaptic GABA A Rα1 distribution at inhibitory synapses of CTRL and NMDA stimulated hippocampal neurons. ( a ): 3D model of an inhibitory synapse immuno-labelled for the GABA A Rα1 in a hippocampal neuron in control conditions (CTRL). Slice1 and slice2 are two tomographic slices through the dotted lines in a. Arrowheads point to gold clusters; ( b ): 3D model of an inhibitory synapse immunolabelled for the GABA A Rα1 in a hippocampal neuron after the induction of plasticity by NMDA stimulation. Slice1 and slice2 are two tomographic slices through the dotted lines in b. Arrowheads point to gold clusters; note that in NMDA treated samples, more gold clusters are visible. ( c ): bar plot showing the area of the post-synaptic membrane on GABA A Rα1 immunolabelled synapses in both CTRL and NMDA stimulated neurons; ( d ): bar plot showing the number of gold clusters/synapse in CTRL and NMDA stimulated neurons; ( e ): bar plot showing the gold clusters volume normalized over the post-synaptic area in CTRL and NMDA stimulated neurons. Values are mean ± s.e.m. * Indicates significant differences (p < 0.01, Student’s t-test); ns = statistically not significant. Colour codes for the 3D models: post-synaptic membrane (blue), pre-synaptic membrane (green), gold clusters (yellow), and neurotransmitter vesicles (cyan). Scale bars: 200 nm.

Article Snippet: During the recovery period, 12 minutes after the end of the NMDA treatment, live immunolabelling of GABA A Rα1 subunits was performed, namely cells were incubated 13 minutes in a solution containing primary antibody against GABA A Rα1 (Alomone Labs, Israel) diluted 1:30 in 0.5% bovine serum albumin (BSA), 350 mM sucrose in PBS, followed by 13 minutes incubation with gold-conjugated secondary antibody.

Techniques: