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Research ArticleResearch Article: New Research, Neuronal Excitability

Propagating Activity in Neocortex, Mediated by Gap Junctions and Modulated by Extracellular Potassium

Christoforos A. Papasavvas, R. Ryley Parrish and Andrew J. Trevelyan
eNeuro 25 February 2020, 7 (2) ENEURO.0387-19.2020; DOI: https://doi.org/10.1523/ENEURO.0387-19.2020
Christoforos A. Papasavvas
Institute of Neuroscience, Newcastle University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom
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R. Ryley Parrish
Institute of Neuroscience, Newcastle University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom
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Andrew J. Trevelyan
Institute of Neuroscience, Newcastle University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom
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  • Figure 11.
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    Figure 11.

    Simulation of the high K+ propagation and comparison with the 4-AP propagation. A, Schematic of the model cell and of the model network of electrically connected cells. The cell has a simple morphology with a soma and two dendrites, one on the left and one on the right. The cells are randomly placed in a three-dimensional virtual slice, and they form a network through gap junctions. The left dendrite for each cell is used to connect it with other cells located on its left, whereas the right dendrite is used to connect it with cells on the right. The left side of the network is stimulated and the activity propagates to the right. B, The probability of connection between two cells is linearly decreased as the distance between them increases. C, Example of a connectivity matrix of a randomly generated network where the yellow color indicates a connection. The cell i.d. is derived from the ordering of the cells on the x-axis, from left to right. Notice that the leftmost cells do not have any direct connection with the rightmost cells due to the limited length of their dendrite (200 μm). D, Typical results of the simulation under three conditions, as follows: control, high K+, and 4-AP. In the control case, there is no propagation; only the cells in the stimulation area fire. In the high K+ case, there is a fast propagation to the right side of the network, but not all cells are participating. In the 4-AP case, there is qualitatively different propagation where the speed is lower but almost every cell in the network participates. E, Distribution of propagation speeds for the high K+ and 4-AP cases. The propagation under high K+ conditions is significantly faster (*p < 0.001, two-sided Wilcoxon rank sum test). F, Distribution of participation percentages for the high K+ and 4-AP cases. The participation under 4-AP conditions is significantly higher (*p < 0.001, two-sided Wilcoxon rank sum test). Box plots, indicating the median (red), 25–75th percentiles (blue), and the range extending to 1.5× the interquartile range (whiskers - extreme outliers beyond that range are shown as individual points). The code is available in Extended Data 1.

  • Figure 1.
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    Figure 1.

    Schematic of the experimental setup and an example of the control experiment. A, Extracellular recordings were taken using a 16-electrode linear MEA placed along layer V in a mouse cortical brain slice in which ChR2 was expressed in PV+ cells. The distance between adjacent electrodes was 100 μm. Photostimulation (blue LED) was delivered using a patterned illuminator through the microscope objective. B, The recordings were taken from the dorsal area of the slice, targeting the primary visual area. C, Example of an illumination pattern (four-electrode-wide area) that was drawn in the middle of the array using the patterned illuminator software. D, A typical control experiment during which repeated illumination was delivered for 31 min while the extracellular K+ remained at normal levels. The evoked activity was only seen at electrodes within the illumination area, and there was no propagating activity, a pattern that remained stable for the entire duration of the recording (31 min).

  • Figure 2.
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    Figure 2.

    Propagating activity arising with raised extracellular K+. The area around a subset of the electrodes was illuminated (marked with blue) with 3-s-long pulse trains (20 Hz, 50% duty cycle), repeated every 20 s. PV+ cells around these electrodes responded with four to five spikes per pulse (see zoomed-in traces). After raising the extracellular K+ concentration (from 3.5 to 7.5 mm), induced activity was recorded in a distant electrode as well (150 μm away). The activity recorded outside the illumination area was time locked to the activity inside that area, albeit with a short delay. Considering that the photostimulated cells are GABAergic in nature, the activity at the distant electrode is hypothesized to propagate through the electrical synapses between PV+ cells. In 6 of the first 12 slices, we saw evidence of rebound bursting at the end of the train of optogenetic stimuli—such an example is seen in the right traces (black arrowhead). Notably, this bursting characteristically involved regular spiking units, and so differed qualitatively from the unit activity seen in the time-locked bursts (Fig. 10).

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    Figure 3.

    Propagation is facilitated by raised extracellular K+ and the spatial spread of the propagations detected. The cumulative proportion of detected propagations at different levels of extracellular K+ concentration. The majority of propagations (16 of 24, 66.7%) were observed with an increase of the extracellular K+ up to 8.5 mm. The threshold (at the 50% point) was calculated at 8.0 mm after fitting a sigmoidal function (red dashed curve). Inset shows the distance at which time-locked firing was recorded, including instances at up to 550 μm away from the stimulation area.

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    Figure 4.

    Propagating activity is sensitive to drugs that block glutamatergic receptors and also gap junction blockers. The underlying mechanisms of the propagating activity were investigated by the application of pharmacological agents. This example is the continuation of the example in Fig. 2. First, the glutamate receptors were blocked by applying NBQX and d-APV (left). In this example, the spontaneous activity was decreased but the propagated activity at the distant electrode remained strong. This indicates that glutamate release (e.g., through disinhibition) was not involved in the propagation. Then, the gap junction blocker quinine was applied (right). The activity, recorded 250 μm away, was gradually suppressed, and eventually silenced altogether, as is evident from the decreasing amplitude and spike rate.

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    Figure 5.

    Propagating activity is facilitated by, but is not dependent on, glutamatergic transmission. Repeated propagation assays in the same slice were conducted under different pharmacological conditions. In this example, we first saw propagation at 4.5 mm [K+]o, but this was suppressed by the addition of glutamatergic blockers (filled arrowheads). Propagating events then resumed once [K+]o was increased further to 5.5 mm (open arrowheads). The propagating activity was subsequently blocked entirely by mefloquine.

  • Figure 6.
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    Figure 6.

    Propagating activity is prevented by multiple different gap junction blockers. The propagating activity silenced in Figure 4 was recovered by washing out the blockers (left). Stable propagating activity with increasing amplitude was recovered at the electrode 350 μm away. This activity remained strong for several minutes until another gap junction blocker was applied, namely mefloquine (right). The propagating activity was once again blocked, validating the involvement of gap junctions by using two different blockers.

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    Figure 7.

    Blockade of GABAA did not block the propagations. In an alternative experimental protocol, GABAA receptors were blocked using gabazine after the blockade of the glutamate receptors. The propagating activity outside the stimulation area remained strong, and the same qualitative result was found in all three slices of this protocol.

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    Figure 8.

    Summary plot of the effects of different pharmacological interventions on the spatial extent of propagating activity.

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    Figure 9.

    Summary plots of the effects of different pharmacological interventions on multiunit activity (MUA) outside the stimulation area. A, Modulation of the firing rate, by optogenetic stimulation, in different pharmacological conditions. In two recordings, the activity appears to drop significantly during the stimulation. This was actually because these slices had very high baseline activity, but notably, during the stimulation, the activity became tightly time locked in the distal electrodes, indicative of a propagating effect, that was not evident in low K+. B, Median z score of firing rate modulation, ±95% confidence interval, relative to the baseline condition (3.5 mm [K+]o). C, Median z score of firing rate modulation, ±95% confidence interval, relative to the previous condition. Note that although there were instances of glutamatergic blockade reducing the spread, across all recordings, there was no consistent effect, relative to the prior high [K+]o condition. D–F, Equivalent plots showing the modulation of the time-locked rhythmicity in the distant electrodes.

  • Figure 10.
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    Figure 10.

    Spike waveform analysis and calculation of propagation speed. A, The spike waveforms of all 20 pharmacologically manipulated propagations were analyzed in terms of their spike width and their amplitude ratio between valley and peak. Both regular- and fast-spiking waveforms were observed (see bottom panels for average spike examples). Notice that none of the hypothesized gap junction-mediated activity was found to exhibit a regular-spiking waveform (i.e., spike width >0.55 ms). B, Example analysis of the speed of propagation calculation, for the gap junction-mediated propagations. The spike time histograms from two different electrodes were plotted: one inside the illumination area (close to the border) and one outside the illumination area where propagating activity was detected. The period covered by the histogram matches the period of the illumination (50 ms). The first peak in each histogram is marked by an asterisk. The propagation speed was directly calculated from the time difference between these peaks and the distance between the respective electrodes. The inset shows the distribution of the calculated propagation speeds (only for propagations blocked by gap junction blockers). It reveals high variability (SD, 17.2 mm/s) and a median speed of 59.1 mm/s.

Tables

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    Table 1

    Biophysical mechanisms used in the model and their conductance values

    MechanismSomaDendrite
    Na+ conductance (S/cm2)0.0450.06
    Delayed rectifier K+ 0.0180.009
    N-type Ca++ 0.0003N/A
    D-type K+ 0.0000725N/A
    H-current0.00001N/A
    A-type K+ 0.0480.48
    fAHP0.0001N/A
    Ca++ diffusionYesNo
    Cm (μF/cm2)1.21.2
    Ra (Ω/cm)150150
    Rm (kΩ cm2)1010
    • Extended Data 1. Python code for the simulations shown in Figure 11. fAHP = fast after-hyperpolarization; Cm = membrane capacitance; Ra = axial resistance; Rm = membrane resistance.

Extended Data

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  • Extended Data 1

    Python code for the simulations shown in Figure 11. Download Extended Data 1, ZIP file.

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eneuro: 7 (2)
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March/April 2020
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Propagating Activity in Neocortex, Mediated by Gap Junctions and Modulated by Extracellular Potassium
Christoforos A. Papasavvas, R. Ryley Parrish, Andrew J. Trevelyan
eNeuro 25 February 2020, 7 (2) ENEURO.0387-19.2020; DOI: 10.1523/ENEURO.0387-19.2020

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Propagating Activity in Neocortex, Mediated by Gap Junctions and Modulated by Extracellular Potassium
Christoforos A. Papasavvas, R. Ryley Parrish, Andrew J. Trevelyan
eNeuro 25 February 2020, 7 (2) ENEURO.0387-19.2020; DOI: 10.1523/ENEURO.0387-19.2020
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Keywords

  • electrotonic
  • excitability
  • gap junction
  • parvalbumin
  • potassium
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