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

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).

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).

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.

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.

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.

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.

Blockade of GABA_A did not block the propagations. In an alternative experimental protocol, GABA_A 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.

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

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.