Fast spiking interneuron control of seizure propagation in a cortical slice model of focal epilepsy

Abstract

In different animal models of focal epilepsy, seizure-like ictal discharge propagation is transiently opposed by feedforward inhibition. The specific cellular source of this signal and the mechanism by which inhibition ultimately becomes ineffective are, however, undefined. We used a brain slice model to study how focal ictal discharges that were repetitively evoked from the same site, and at precise times, propagate across the cortex. We used Ca(2+) imaging and simultaneous single/dual cell recordings from pyramidal neurons (PyNs) and different classes of interneurons in rodents, including G42 and GIN transgenic mice expressing the green fluorescence protein in parvalbumin (Pv)-fast spiking (FS) and somatostatin (Som) interneurons, respectively. We found that these two classes of interneurons fired intensively shortly after ictal discharge generation at the focus. The inhibitory barrages that were recorded in PyNs occurred in coincidence with Pv-FS, but not with Som interneuron burst discharges. Furthermore, the strength of inhibitory barrages increased or decreased in parallel with increased or decreased firing in Pv-FS interneurons but not in Som interneurons. A firing impairment of Pv-FS interneurons caused by a membrane depolarization was found to precede ictal discharge onset in neighbouring pyramidal neurons. This event may account for the collapse of local inhibition that allows spatially defined clusters of PyNs to be recruited into propagating ictal discharges. Our study demonstrates that Pv-FS interneurons are a major source of the inhibitory barrages that oppose ictal discharge propagation and raises the possibility that targeting Pv-FS interneurons represents a new therapeutic strategy to prevent the generalization of human focal seizures.

Figure 6. Hyperactivity and block in Pv-Fs interneurons precede the transition to ictal discharge

Pv-FS interneurons are a main source of the inhibitory barrages. A and B, dual current-clamp recordings from a Pv-FS (A) or a Som (B) interneuron and voltage-clamp recording from a neighbouring PyN in a TC slice from a G42 mouse in the 4-AP or low Mg²⁺ model (A, middle panel). Dual recording from a Pv-FS and a PyN (both in current-clamp mode) is also reported (A, lower panel). Response of the LTS-Som interneuron to current injections is also reported (B, inset, scale bars, 20 mV and 500 ms). C, linear regression showing the correlation between the f_w of Pv-FS interneuron firing and the normalized inhibitory charge transferred in the PyNs in the 4-AP model (circles; R= 0.79; P < 0.001; 39 inhibitory barrages from 9 ictal discharges, 5 pairs) and in the low Mg²⁺ model (triangles; R= 0.81; P < 0.001; 23 inhibitory barrages from 6 ictal discharges, 3 pairs). Asterisk marks the presence of 8 circles and 5 overlapping triangles. D, linear regression showing the absence of correlation between the f_w of Som interneuron firing and the normalized inhibition (R=−0.25; P= 0.08; n= 50 inhibitory barrages from 9 ictal discharges in 3 pairs). E, distribution of the latency of depolarization block in the Pv-FS interneurons (blue circles; 25 ictal discharges, 10 pairs) and in the Som interneurons (red diamonds; 15 ictal discharges, 3 pairs) with respect to the t_IE in the PyNs. Distribution of values in a time window of ±100 ms are also reported.

Figure 7. Hyperactivity and block in Pv-FS interneurons precede the transition to ictal discharge (cell-attached mode)

A, dual patch-clamp recordings from a GFP-expressing Pv-FS interneuron firing activity (in the cell-attached mode) and a neighbouring PyN (in whole-cell, voltage-clamp mode) in a TC slice from a G42 mouse in the 4-AP model. B, linear regression showing the correlation between the f_w of GFP-expressing Pv-FS interneuron firing and the normalized inhibitory charge transferred in the PyNs in the 4-AP model (circles; R= 0.83; P < 0.001; 44 inhibitory barrages from 7 ictal discharges, 3 pairs). C, distribution of the latency of depolarization block in the Pv-FS interneurons (9 ictal discharges, 3 pairs) with respect to the t_IE in the PyNs.

Figure 1. Modular propagation of focally evoked ictal discharges

A and B, current-clamp recordings from a PyN close (A) or distant (B) from the site where NMDA was applied to evoke an ictal discharge. In this and the other figures the black arrowheads mark the timing of the double NMDA pulse. The drawing reports the patch and the NMDA-containing pipettes, the epileptogenic focus (grey circle) and the region of neuronal Ca²⁺ imaging (blue box). Rf, rhinal fissure. The lower trace in B illustrates at expanded time scale the hyperpolarizing events that preceded ictal discharge propagation to this PyN. C, mean delays after the NMDA pulse of ictal discharge propagation to PyNs located close (<400 μm, 16 ictal discharges from 6 experiments) or distant (>700 μm, 19 ictal discharges from 10 experiments; *P < 0.001) from the focus. D and E, recruitment diagrams reporting in a pseudocolour scale the temporal derivative of the Ca²⁺ signal in each neuron (see Methods) from two different regions (<400 μm in D, >700 μm in E) outlined by the blue boxes in A and B. Lower traces in E show average Ca²⁺ changes from the three main clusters of neurons. F, OGB1 fluorescence image illustrating in different colours the three main clusters. Scale bar, 100 μm

Figure 2. GABAergic inhibitory barrages restrain propagating ictal discharges

A, voltage-clamp recording in a PyN (black trace; V_h=−50 mV) and averaged Ca²⁺ signal (blue trace) from the putative PyNs of the same region (also shown in B). B, schematic diagram of the experiment and OGB1 fluorescence image from the same experiment showing in red the cluster of neurons recruited at the time of t_IE in the patched neuron. Scale bar, 100 μm. C, voltage-clamp recordings from a PyN pair showing a full ictal discharge in the PyN close to the focus and only inhibitory barrages in the more distant neuron. D, voltage-clamp recordings from a distant PyN before and after bicuculline applied locally before the NMDA pulses. E, recruitment diagrams from a region distant from the focus before (upper panel) and after bicuculline applications (lower panels).

Figure 5. Early recruitment into propagating ictal discharges of GFP-expressing Pv-FS interneurons in G42 mice

A, fluorescence image of a layer V–VI region distant from the focus in a slice loaded with Rhod-2 showing GFP-expressing cells (white arrows; bar, 50 μm). A fifth GFP-expressing Pv-FS interneuron present in the field of view was not considered because it was not loaded with the Ca²⁺ dye. B, recruitment diagram from the region imaged in A. Lower traces: four GFP-expressing (green traces and green cells in panel 1) and two GFP-negative neurons (orange traces and orange cells in panel 1) exhibited a Ca²⁺ increase soon after NMDA stimulation and before two different clusters of putative PyNs were recruited (blue traces and cyan cells in panels 2 and 3). The lower right traces show the Ca²⁺ change in these cells during the whole ictal discharge. C, bar histogram of the mean interval (Δt) between the timing of PyN recruitment (n= 135) and the Ca²⁺ increase in GFP-expressing Pv-FS interneurons (n= 32, from 5 experiments) and GFP-negative neurons (n= 20).

Figure 8. The timing of the depolarization block in Pv-FS interneurons depends on the distance of these cells from the focus

A, dual current-clamp recording from two adjacent Pv-FS interneurons showing simultaneous depolarization block. B, two Pv-FS interneurons located at different distances from the focus entered into the depolarization block at different times. C, dual recording from a PyN and a Pv-FS interneuron located in TC layer V–VI at different distances from the focus. The depolarization block in the Pv-FS interneuron closer to the focus occurred several seconds before the t_IE in the PyN. The recording periods outlined by the dashed boxes are expanded on the right.

Figure 3. Activity of different classes of interneurons during propagating ictal discharges

A, representative response to hyperpolarizing and depolarizing current steps (scale bars, 20 mV and 200 ms) and current-clamp recordings from different classes of layer V–VI interneurons during propagating ictal discharges in the EC. The enlarged trace also reports the firing instant frequency (IF, red dots) of the FS interneuron. B, bar histogram reporting the mean delay of the inhibitory barrages in PyNs and of the increase in firing activity from the different interneuron types after the NMDA pulse (22 ictal discharges from 12 PyNs; 11 ictal discharges from 7 FS interneurons; 14 ictal discharges from 7 ANP interneurons; 4 ictal discharges from 3 IS interneurons and 6 ictal discharges from 3 LTS interneurons; **P < 0.001).

Figure 4. GFP-expressing Pv-FS interneurons during ictal discharge propagation in G42 mice

A and D, upper panels: schematic diagrams of the experiments in EC (A) and TC (D), current-clamp recording from Pv-FS interneurons (black traces) and simultaneous average Ca²⁺ signal (D, blue trace) from putative PyNs surrounding the patched Pv-FS interneuron. Superimposed is the instant frequency (red dots) of the Pv-FS interneuron firing activity. The response of the interneurons to current injections is also reported (insets; scale bars, 10 mV and 200 ms). Lower panels: bar histogram of the mean and maximal instant frequency in Pv-FS interneurons before (black bars) and after (grey bars) the NMDA pulse in EC (A; n= 4, *P= 0.012 and P= 0.017 for the mean and the maximum values, respectively) and in TC (D, n= 6, *P= 0.011 and P= 0.019 for the mean and the maximum values, respectively). B, left panel: confocal image of the GFP expression pattern in the TC of a G42 mouse in the different cortical layers. Scale bar, 100 μm. Right panels: double immunostaining showing that the GFP-expressing cells (green) are Pv immunopositive (red). Scale bar, 50 μm. C, bar histogram summarizing GFP-expressing cells in layer V–VI of the TC and the EC expressed as a percentage of the total number of Pv-immunopositive cells in G42 mice (10 and 11 slices from 3 mice, and 144 and 406 confocal sections, respectively). E, light microscope images of biocytin-filled PyN and GFP-expressing Pv-FS interneuron from layer V of a TC slice (left) and reconstruction of dendritic (black) and axonal (red) arborization of the latter cell (right). Scale bars, 100 μm.