Interneurons spark seizure-like activity in the entorhinal cortex

Abstract

Excessive neuronal synchronization is presumably involved in epileptiform synchronization. However, the respective roles played by interneurons (GABAergic) and principal (glutamatergic) cells during interictal and ictal discharges remain unclear. Here, we employed tetrode wire recordings to establish the involvement of these two cell types in 4-aminopyridine-induced interictal- and low-voltage fast (LVF) onset ictal-like discharges in the rat entorhinal cortex in an in vitro slice preparation. We recorded a total of 90 single units (69 putative interneurons, 17 putative principal and 4 unclassified cells) from 36 slices, and found that: (i) interneurons (66.7%) were more likely to fire during interictal discharges than principal cells (35.3%); (ii) interneuron activity increased shortly before LVF ictal onset, whereas principal cell activity did not change; (iii) interneurons and principal cells fired at high rates throughout the tonic phase of the ictal discharge; however, (iv) only interneurons showed phase-locked relationship with LVF activity at 5-15Hz during the tonic phase. Finally, the association of interneuron firing with interictal discharges was maintained during blockade of ionotropic glutamatergic transmission. Our findings demonstrate the prominent involvement of interneurons in interictal discharge generation and in the transition to LVF ictal activity in this in vitro model of epileptiform synchronization.

Keywords: 4-Aminopyridine; Interictal discharges; Interneurons; Low-voltage fast onset ictal discharges; Principal cells.

Fig. 1

Interictal and ictal activity patterns. A: Schematic diagram of a horizontal slice showing the location of tetrodes (squares) in the EC. Each square represents the 4 channels of a tetrode. B: Representative example of an isolated interictal discharge (*, a), the last interictal spike preceding an ictal discharge (**) and the sentinel spike (b). An ictal discharge (dashed line) is also shown with the low-voltage fast (5–15 Hz) at onset, which is identified by a rectangle; arrows represent the time points used as onset to calculate single-unit ictal and interictal relationships. C: Field potentials (filtered between 300 and 3000 Hz) are shown to illustrate multi-unit activity that was used to identify single units. D: Parameters used to cluster single units as putative interneurons or principal cells. The width of the action potential at 50% amplitude and the trough to peak were calculated separately and combined to calculate the peak amplitude asymmetry. E: K-mean clustering analysis showing the two clusters of cells (i.e., putative interneurons and putative principal cells). Red circles indicate the centroid of each cluster.

Fig. 2

Single unit sorting. Representative features of an interneuron (A) and of a principal cell (B). Action potential waveforms recorded on the four channels of a tetrode are shown for an interneuron (Aa) and a principal cell (Ba). Note that the amplitude of action potentials differs between channels and that interneurons show a more symmetric action potential whereas in principal cells, a peak during action potential repolarization was not observed. Three dimensional representations of the clusters obtained with the cluster cutting algorithm are shown in Ab and Bb. Multiunit activity (gray circles) are separated from single units (black circles). Note in Bb that another single unit (principal cell) is visible (gray circles). Auto-correlograms for each unit are shown in Ac and Bc. Note that interneurons tended to show a slow decay between 0 and 100 ms lags whereas this decay was faster in principal cells.

Fig. 3

Single unit activity during interictal discharges. A: Spike train of an interneuron (A) and of a principal cell (B) and their corresponding field potential. The firing of interneurons was tightly coupled to the occurrence of interictal discharges, although they could also fire outside of them. Principal cell firing was also tightly coupled to the occurrence of interictal discharges, but compared to interneurons, they were less likely to fire outside of interictal discharges. C: Raster plot of an interneuron showing activity 2 s before and after slow interictal discharges (n = 20 interictal discharges). The perievent time histogram of interneurons (n = 46) is also shown. Note the increase in firing rates at the peak of the spike component of interictal discharges. D: Raster plot of a principal cell at the onset of interictal discharges (n = 20 interictal discharges). Note that action potentials from principal cells mainly occurred during interictal discharges. The perievent time histogram of principal cells (n = 17) is also shown.

Fig. 4

Single unit activity during isolated interictal and pre-ictal spikes. A: Firing rates of interneurons during isolated interictal discharges, during the last interictal discharge that precedes an ictal discharge and during the “sentinel” spike that leads to LVF activity. Note that the “sentinel” spike is associated to significantly higher firing rates before its onset compared to the last interictal spike preceding ictal discharges and isolated interictal discharges (* p < 0.05) (a). The last interictal discharge that preceded ictal discharges was also associated to significantly higher firing rates from interneurons compared to isolated interictal discharges (* p < 0.05) (a).

Fig. 5

Single unit activity during ictal discharges. A: Field potential recording obtained from EC and filtered between 300 and 3000 Hz shows single unit activity. Note that the amplitude and waveforms of action potentials did not change significantly throughout the ictal discharge. B: Single unit activity from interneurons (n = 35) and principal cells (n = 12) during the pre-ictal, ictal and post-ictal periods. Note the increase in firing rate of interneurons before ictal onset (*p < 0.01). After ictal onset, both interneurons and principal cells showed increased firing rates (*p < 0.01). This period was associated to high synchrony between pairs of interneurons (a). Such effect was not observed at the end of ictal discharges (b) (n = 16 pairs). Note the high synchrony near zero time lag (*). The solid line represents the average of the cross-correlogram and the dashed line the threshold for significance (2 SD). C: Spike trains of two interneurons (INT 1 and INT 2) recorded on the same tetrode and the corresponding field potential showing an ictal discharge. Note that interneurons (INT) fired at high rates before and at the onset of the ictal discharge, during the tonic phase. The bottom trace shows a principal cell (PC) and two interneurons recorded during an ictal discharge. Principal cells were less likely to fire during ictal discharges.

Fig. 6

Single unit relationship with low-voltage fast onset activity A: Spike train of an interneuron and its corresponding field potential with power spectral analysis. Low-voltage fast oscillations during the tonic phase (5–15 Hz) are highlighted (gray square). Note the progressive decrease in frequencies over time. B: Interneurons show a phase-locking relationship with the 5–15 Hz oscillations recorded during the tonic phase, and tend to fire closed to the trough of field oscillations (peak at 0°, inset), between 90° and 150° (polar plot). C: Spike train of a principal cell and its corresponding field potential with power spectral analysis. D: Principal cells do not show any preferred phase during low voltage fast oscillations (5–15 Hz) occurring during the tonic phase.

Fig. 7

Single unit activity during blockade of ionotropic glutamatergic transmission. A: Action potential waveform of an interneuron recorded under 4AP and CPP/NBQX. No difference was observed between the two conditions. B: K-mean clustering analysis performed with values obtained for the trough to peak, the halfwidth duration and the peak amplitude asymmetry, for single units recorded after the addition of CPP/NBQX. Single unit features were similar as the ones obtained under 4AP (see Fig 1E). C: Raster plot of an interneuron recorded under CPP/NBQX. Note the increase in firing rates after the first deflection (onset) of interictal discharges. The perievent histogram for interneurons (n = 27) recorded under CPP/NBQX is also shown. D: Spike train of an interneuron and its corresponding field potential under 4AP and under CPP/NBQX application. Note that CPP/NBQX abolished ictal discharges, leaving only interictal discharges to occur. Interneurons under CPP/NBQX also tended to show prolonged discharges of action potentials. The inset (a) shows the initiation of a long-lasting discharge in coincidence with the occurrence of an interictal spike. E Average field potential centered on the first action potential of discharges recorded under CPP/NBQX (n = 7 interneurons, 93 discharges). Note that the first action potential of a discharge was associated to the occurrence of an interictal discharge. F: Average field potential centered on the last action potential of long-lasting discharges. Note that the last action potential of a discharge was not related with the occurrence of an interictal discharge.