Parvalbumin-Positive Inhibitory Interneurons Oppose Propagation But Favor Generation of Focal Epileptiform Activity

Optogenetic activation of Pv interneurons does not prevent ictal event generation. A, Schematic of the experimental configuration also showing the typical firing of patched pyramidal neurons. Calibration: 0.2 s, 30 mV, 200 pA. B, Differential interference contrast image of the cortical region showing the NMDA-containing pipette that is used to both apply NMDA pulses and record extracellular local field potentials, the optical fiber used to activate ChR2 Pv interneurons, and the patch pipette onto a pyramidal neuron located at ∼400 μm from the ictal initiation site. A confocal z-stack maximal projection showing fluorescent ChR2-mCherry expressing Pv interneurons from the same region is also reported. C, Representative voltage-clamp recordings (V_h = −50 mV) from a pyramidal neuron and simultaneous local field potential recordings of ictal events in the absence (left) and in the presence (right) of pulsed light stimulation. A light-evoked IPSC is reported at enlarged scale. Calibration: 40 s, 500 pA, 0.01 mV; inset, 1 s, 100 pA. D, Percentage of ictal events evoked by double NMDA pulses in the presence and in the absence of pulsed stimulation applied at 0.5 Hz (n = 16, 10 mice), 2 Hz and 4 Hz (n = 8, 4 mice). E, Representative voltage-clamp recordings from a pyramidal neuron and simultaneous field potential recordings of ictal events in the absence (left) and in the presence (right) of a pulsed light stimulation at 0.5 Hz (n = 30 ictals, 13 mice) or 2 Hz (n = 5 ictals, 3 mice) that initiated after ictal onset. Calibration: 40 s, 0.01 mV, 500 pA. F, Ictal duration distribution in the absence (black) and in the presence (cyan) of pulsed light stimulation (n = 16, 10 mice). ***p < 0.001.

Optogenetic activation of Pv interneurons at the epileptogenic focus promotes ictal event generation. A, Schematic of the experiment and (B) representative voltage-clamp recording (V_h = −50 mV) from a pyramidal neuron 700 μm from the focus and simultaneous local field potential recording in response to a single NMDA pulse in the absence (left, right) and in the presence (middle) of light stimulation at 0.5 Hz, which was initiated 30 s before the NMDA challenge. Note the ictal response evoked by a single NMDA pulse in the presence of optogenetic stimulation. Calibration: 40 s, 0.01 mV, 500 pA. C, Distribution of normalized event duration in double NMDA pulses (black circles, n = 6, 4 mice) and single NMDA pulse experiments in the absence (green circles, n = 12, 4 mice) and in the presence (cyan, n = 6, 4 mice) of light stimulation. As in the representative experiment reported in B, in the six experiments the effect of a single NMDA pulse was checked twice, both before and after the ictal event evoked by the single NMDA pulse coupled with light stimulation. All data points are normalized to the mean duration of double NMDA-evoked events. D, Paired distribution of the delay in ictal onset measured from local field potentials recorded at the focus in a subset of the experiments (n = 6 ictals, 4 mice) that are reported in E. E, Distribution of the delay between the first NMDA pulse and the recruitment of the pyramidal neuron, as measured by t_IE (see Materials and Methods), in ictal events evoked by double NMDA pulses in the absence of light stimulation (black, n = 14, 7 mice), and single NMDA pulses in the presence of light stimulation (cyan, n = 20, 7 mice). F, Bar histogram of the percentage of ictal events evoked by a single NMDA pulse in slices from Pv-Cre mice injected with saline solution (n = 9 trials, 3 mice) and from ChR2 Pv mice in the absence (−, green bar, n = 33 trials, 9 mice) and in the presence (+, cyan, n = 23 trials, 9 mice) of pulsed light stimulation. G–I, Schematic of the experiment (top) and representative voltage-clamp (V_h = −50 mV) recordings (bottom) of the response to a single NMDA pulse in a pyramidal neuron located 250 μm from the focus in the absence of light stimulation (G), in the presence of light stimulation at the focus (H), and in the presence of light stimulation in distant regions 1 mm from the focus (I) (n = 3 ictal events, 2 mice). **p < 0.01. ***p < 0.001.

Individual Pv interneuron activation at the epileptogenic focus promotes ictal event generation. A, Schematic of the experiment also showing the typical firing discharge of an FS interneuron expressing GFP in a TCx slice from a G42 mouse. Calibration: 0.5 s, 20 mV, 500 pA. B, Representative current-clamp recordings at resting potential from a Pv interneuron located at the focus and simultaneous local field potential recordings in response to a single NMDA pulse in the absence (left, right) and in the presence (middle) of 1 s intracellular current pulses at 0.5 Hz (dashed line). The intense firing activity in the interneuron facilitates the generation of a full ictal event (middle). Calibration: 40 s, 30 mV, 0.01 mV. C, Percentage of ictal events successfully evoked by a single NMDA pulse in the absence (−, n = 11, 3 mice) and in the presence (+, n = 10, 3 mice) of current pulse stimulation. D, Distribution of event duration evoked by double NMDA pulse (black circles, n = 5, 3 mice) and by single NMDA pulse in the absence (green, n = 11, 3 mice) and in the presence (cyan, n = 5, 3 mice) of evoked Pv interneuron firing activity. In each experiment, single NMDA pulses without light stimulation were performed at least twice as in the representative experiment reported in B. All data points are normalized to the mean duration of double NMDA pulse-evoked ictal events. E, Paired distribution of ictal onset delay measured from local field potential recorded at the focus (n = 5, 3 mice). *p < 0.05. **p < 0.01.

Optogenetic activation of Pv interneurons induce postinhibitory rebound spiking in pyramidal neurons. A, B, Representative simultaneous current-clamp recordings from a pyramidal neuron, depolarized to action potential threshold by steady-state current injection, and a Pv interneuron in the absence (A, left panel) and presence of pulsed light stimulation at 0.5 Hz (B, left panels). Recordings were performed in the presence of 4-AP and 0.5 mm Mg²⁺. Histograms of the relative firing rate as a function of time, calculated over 50 ms time bins, obtained by the analysis of 300 consecutive sweeps (A, B, right panels). Open arrowheads indicate the main spike clustering. Red line indicates the multipeak fitting of the firing rate over time (reduced χ² = 0.99). Calibration: 200 ms, 20 mV. C, Distribution of normalized firing rate changes during rebound spiking both in basal conditions (empty circles, n = 10) and in the presence of 4-AP (full circles, n = 14). The mean value of both datasets is represented by the empty square (284 ± 28.1%, n = 24 cells, 14 slices, 6 mice). ***p < 0.001 (Wilcoxon signed-rank test).

Optogenetic activation of Pv interneurons promotes synchronous firing in pyramidal neurons. A₁, A₂, Spike trains recorded in 2-s-long sweeps from two neighboring pyramidal neurons in the absence (A₁) and in the presence (A₂) of light pulse stimulation at 0.5 Hz. Cyan bars represent 150 ms light pulses. The rhythmic activation of Pv interneurons causes a temporal modulation of spikes in both pyramidal neurons. Calibration: 200 ms, 20 mV. B₁, B₂, Raster plots showing the spike timing of the two pyramidal neurons (red and green bars) and a randomized trial (blue bars). C₁, C₂, Probability distribution in the 2 s period in the absence (C₁) and in the presence (C₂) of rhythmic optogenetic activation of Pv interneurons. D₁, D₂, For each spike produced by the pyramidal neuron 2 (PyN2), the distance from the closest spike produced by PyN1 was computed. The observed cumulative distribution of the nearest neighbor distances is plotted in red (n = 230 spikes). The distribution was computed again after randomization of the spike train of PyN1 (blue trace, n = 230 spikes). In the presence of optogenetic stimulation (D₂), the difference between the observed cumulative distribution and the cumulative distribution obtained after randomization is statistically significant (n = 447 spikes, *p < 0.05, Kolomogorov–Smirnov test). E, The integral of the difference between observed and simulated distributions has been computed in an interval centered ∼0, returning a measure of the synchronization between the two pyramidal neurons. F, Summary report for the increase in pyramidal neuron synchrony induced by Pv interneuron activation (n = 7 pairs, 5 mice) in the presence (black circles) and in the absence (cyan circles) of pulsed light stimulation both in basal ACSF and 4-AP. **p < 0.01. G, Representative local field potential recording of an NMDA-evoked ictal event in the presence of light pulse stimulation. The ictal event onset between two light pulses is shown at enlarged scales. Green bars represent the firing suppression period in pyramidal neurons induced by light stimulation of ChR2-expressing Pv interneurons (cyan bars, 150 ms at 0.5 Hz). Calibration: 40 s, 0.01 mV; inset, 500 ms, 0.01 mV. H, Distribution of both the ictal onset probability (orange line, n = 31 ictal events in 100 ms time bins, 17 mice) and the pyramidal neuron mean spiking probability in 4-AP (light gray represents SEM; n = 24 cells, 6 mice; 50 ms time bins) as a function of time during pulsed light stimulation of Pv interneurons. The 0 value is set as the end of the light stimulus.

Optogenetic activation of Pv interneurons distant from the focus blocks ictal event propagation and shortens ictal event duration at the focus. A, Schematic of the experiment. B, Representative voltage-clamp recordings (V_h = −50 mV) from a pyramidal neuron located >1 mm from the focus and simultaneous local field potential recordings at the focus in the absence (left and right) and in the presence (middle) of light stimulation starting 60 s before the double NMDA pulse. Calibration: 40 s, 0.01 mV, 500 pA. C, Percentage of propagating ictal events in the absence (n = 14, 3 mice) and in the presence of light stimulation (n = 8, 3 mice). D, Paired distribution of ictal event duration evoked by double NMDA pulses in the absence (empty and full black circles, n = 8, 3 mice) and in the presence (empty and full cyan circles, n = 8, 3 mice) of light stimulation, as measured from local field potentials recorded at the focus. The duration of the two ictal events that were not blocked by local optogenetic activation of Pv interneurons was not reduced. Mean values of black and cyan full circles are represented by full squares. Paired sample t test was performed on the values obtained from the six successful experiments (3 mice). **p < 0.01.

Individual Pv interneuron activation in penumbra regions blocks ictal event propagation. A, Schematic of the experiment and differential interference contrast image illustrating the neuronal clusters progressively recruited into the ictal event in Ca²⁺ imaging experiments. B, Representative single current-clamp recordings from a Pv interneuron and simultaneous mean Ca²⁺ signal from two neuronal clusters (red and blue traces; soma position of these neurons is reported in A according to their different time of recruitment into the propagating ictal event. Steps of current were repeatedly injected to evoke firing activity (inset) that prevented the occurrence of a full ictal event. Calibration: 40 s, ΔF/F₀ 40%, 40 mV; inset, 5 s, 20 mV. C, Bar histogram of the percentage of NMDA evoked ictal events without (−, n = 23, 8 mice) or during (+, n = 12, 8 mice) stimulation of individual Pv interneurons. D, Bar histograms reporting the number of recruited neurons (left) and ictal event duration (right) measured in red neuron Ca²⁺ signals, in the absence and in the presence of current pulse stimulation of Pv interneurons (n = 3 ictal events, 2 mice). *p < 0.05. ***p < 0.001.