Perampanel reduces paroxysmal depolarizing shift and inhibitory synaptic input in excitatory neurons to inhibit epileptic network oscillations

Abstract

Background and purpose: Perampanel is a newly approved anticonvulsant uniquely targeting AMPA receptors, which mediate the most abundant form of excitatory synaptic transmission in the brain. However, the network mechanism underlying the anti-epileptic effect of the AMPAergic inhibition remains to be explored.

Experimental approach: The mechanism of perampanel action was studied with the basolateral amygdala network containing pyramidal-inhibitory neuronal resonators in seizure models of 4-aminopyridine (4-AP) and electrical kindling.

Key results: Application of either 4-AP or electrical kindling to the basolateral amygdala readily induces AMPAergic transmission-dependent reverberating activities between pyramidal-inhibitory neuronal resonators, which are chiefly characterized by burst discharges in inhibitory neurons and corresponding recurrent inhibitory postsynaptic potentials in pyramidal neurons. Perampanel reduces post-kindling "paroxysmal depolarizing shift" especially in pyramidal neurons and, counterintuitively, eliminates burst activities in inhibitory neurons and inhibitory synaptic inputs onto excitatory pyramidal neurons to result in prevention of epileptiform discharges and seizure behaviours. Intriguingly, similar effects can be obtained with not only the AMPA receptor antagonist CNQX but also the GABA_A receptor antagonist bicuculline, which is usually considered as a proconvulsant.

Conclusion and implications: Ictogenesis depends on the AMPA receptor-dependent recruitment of pyramidal-inhibitory neuronal network oscillations tuned by dynamic glutamatergic and GABAergic transmission. The anticonvulsant effect of perampanel then stems from disruption of the coordinated network activities rather than simply decreased neuronal excitability or excitatory transmission. Positive or negative modulation of epileptic network reverberations may be pro-ictogenic or anti-ictogenic, respectively, constituting a more applicable rationale for the therapy against seizures.

Keywords: AMPA receptors; GABAergic transmission; epileptic seizures; glutamatergic transmission; perampanel.

FIGURE 1

AMPA receptor antagonists ameliorate 4‐aminopyridine (4‐AP)‐induced seizure behaviours in free‐moving rats. Unilateral microinjection of 4‐AP (2 mM) directly into the basolateral amygdala (BLA) in free‐moving rats results in seizure behaviours, which are defined by the modified Racine's stages (see Section 2). The number of behavioural events corresponding to specific Racine's stages was counted within 60 min immediately after microinjection. Co‐injection of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (200 μM, n = 5 rats) or perampanel (80 μM, n = 5 rats) readily reduces both the occurrence and the severity of seizure behaviours. Rat Numbers 1–3 in the analyses of CNQX and perampanel are of the same three rats

FIGURE 2

Perampanel completely inhibits 4‐aminopyridine (4‐AP)‐induced recurrent inhibitory postsynaptic potentials (IPSPs) in pyramidal neurons (PNs) of basolateral amygdala (BLA). (a) Morphological and electrophysiological identification of PNs and interneurons (INs) in the BLA (see Section 2 for details). The photos show intracellular labelling of a BLA PN (left) and an IN (right) by sulforhodamine 101 in the recording pipette. The longest diameter of a PN (usually >20 μm) would in general exceed that of an IN (~10 μm, Asprodini, Moises, 1992, Rainnie, Shinnick‐Gallagher & Washburn, 1993). In response to suprathreshold depolarizing current injection, PNs usually show low‐frequency spikes with adaptation, while INs exhibit high‐frequency spikes with little adaptation. (b) Pair recording of a PN and an IN shows reverberating activities between the two types of neurons induced by 4‐AP (10 μM, top rows). In the presence of 4‐AP, the PN fires clusters of spikes that are followed by IN burst discharges (grey arrows). Recurrent IN burst discharges are synchronized with IPSPs in the PN (red arrows) that apparently interrupt PN spikes (traces in the red box are magnified to show the reverberant and synchronous activities). n = 26 and 28 PNs for analyses of spike and IPSP frequency, respectively (only those having baseline membrane potential more depolarized than −60 mV used in the analyses of mean spike frequency). INs: n = 18. Co‐application of perampanel (5 μM, n = 6 and 5 for PNs and INs, respectively) completely inhibits all IPSPs in PNs and burst discharges in INs but preserves PN spikes. In the study of perampanel effect, the analysis of the interspike interval (ISI) is also performed for the recordings with baseline membrane potential more positive than −55 mV. The plot of ISIs in 10‐s recording session of PNs (n = 5) demonstrates two clusters of ISIs, namely, a short ISI cluster (green) and a long ISI cluster (yellow), in the presence of 4‐AP. The two larger symbols represent the mean of long ISIs and that of short ISIs. Co‐application of perampanel abolishes the clustered ISIs so that ISIs are evenly spread (white). Events of burst discharges and excitatory postsynaptic potentials (EPSPs) are also summed as events of “excitatory waves”. ^N.S. P > 0.05; ^* P < 0.05, Kruskal–Wallis test followed by pairwise Mann–Whitney U test. (c) A PN–PN pair recording shows that application of 4‐AP (10 μM, n = 28) produces recurrent and coupled IPSPs in the two PNs in the current‐clamp recording at first and then synchronized IPSPs in a PN (No. 2, staying in current clamp) and inhibitory postsynaptic currents (IPSCs) in the other PN (No. 1, switched to the voltage‐clamp mode and held at −55 mV). Both IPSPs and IPSCs are abolished by GABA_A receptor antagonist bicuculline (10 μM, n = 5). The IPSP frequency in 4‐AP is from the same group of PNs in (b). ^* P < 0.05, Mann–Whitney U test

FIGURE 3

4‐Aminopyridine (4‐AP)‐induced network oscillation requires AMPAergic and GABAergic synaptic transmission. (a) AMPA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not NMDA receptor antagonist AP‐5 blocks 4‐AP‐induced oscillatory activities in pair recording of a pyramidal neuron (PN) and an interneuron (IN) (see red and grey arrows for reverberant and synchronous activities). 4‐AP‐triggered spikes, burst discharges, and excitatory postsynaptic potentials (EPSPs) in INs are abolished by CNQX (10 μM, n = 6) but not AP‐5 (10 μM, n = 7). In PNs, CNQX (10 μM) completely inhibits the recurrent inhibitory postsynaptic potentials (IPSPs) (n = 9) but preserves spikes induced by 4‐AP (n = 7, only those having baseline membrane potential more depolarized than −60 mV used in the analyses of mean spike frequency), while AP‐5 (10 μM, n = 6) has little effect on the recurrent IPSPs and mean spike frequency. Plots of interspike interval (ISI) in 10‐s recording session of PNs (n = 5) show 4‐AP‐induced two clusters of ISIs (yellow and green for long and short ISIs, respectively) eliminated by co‐application of CNQX (10 μM, n = 5, white; only those having baseline membrane potential more depolarized than −55 mV were included in ISI analyses). The two larger symbols represent the mean of long ISIs and that of short ISIs. ^N.S. P > 0.05; ^* P < 0.05, Kruskal–Wallis test followed by pairwise Mann–Whitney U test. Except for the analysis of ISI, the analyses of 4‐AP groups are of the same PNs (n = 26–28) and INs (n = 18) in Figure 2. (b) GABA_A receptor antagonist bicuculline reduces 4‐AP‐induced reverberating activities. A representative PN–IN pair recording shows recurrent and synchronous burst discharges in INs and IPSPs and clustered spikes in PNs induced by 4‐AP (grey and red arrows). We then define the “oscillatory activity” of INs by having excitatory waves (burst discharges or EPSPs) synchronized with IPSPs in PNs. With co‐application of bicuculline (10 μM), the IN would either show no spikes and no bursts (with the paired PN also firing no spikes, i.e. the “silence”) or exhibit discharges (with the paired PN also firing spikes that are not regulated by the IPSP, i.e. the “non‐oscillatory activity”). The proportion of time staying in different discharge modes is indicated at the top of the representative recordings. Analyses of continuous recording (200 s) of IN activities from the same PN–IN pairs (n = 5 pairs) reveal that co‐application of bicuculline completely eliminates the initial 4‐AP‐induced persistent oscillatory activity (~97.5 ± 2% of the time) and results in either silence (~49.7 ± 8% of the time) or non‐oscillatory activity (~50.3% of the time) of INs (the pie charts). Co‐application of bicuculline eliminates all synchronous events in PN–IN network (n = 5 PN–IN pairs) but preserves the mean spike frequency of PN. The analyses of the PN mean spike frequency in the 4‐AP group are of the same PNs (n = 26) in Figure 2, and in the 4‐AP plus bicuculline (n = 5) are from traces of PNs when the paired INs show non‐oscillatory activity. Plots of interspike interval (ISI) in 10‐s recording session of PNs (n = 5) show 4‐AP‐induced two clusters of ISIs (yellow and green for long and short ISIs, respectively) eliminated by co‐application of bicuculline (10 μM, n = 5, white). ^N.S. P > 0.05; ^* P < 0.05, Wilcoxon signed‐rank test for synchronized events, and Mann–Whitney U test for the mean spike frequency

FIGURE 4

Perampanel reduces electrical stimulation‐induced paroxysmal depolarizing shift and activities in pyramidal neurons (PNs) and interneurons (INs) of basolateral amygdala (BLA). In the representative recording (a) of an IN (top) and a PN (bottom), delivery of kindling‐mimicking stimulation causes large depolarization plateaus (orange and red boxes for IN and PN, respectively). The plateau is followed by an increase in spikes, burst discharges, and/or excitatory waves in all INs (n = 18 for analyses of burst discharges and n = 21 for analyses of spikes and excitatory waves) and also an increase in spikes and/or composite postynaptic potentials or “cPSP” (which include burst discharges, excitatory postsynaptic potentials [EPSPs], and inhibitory postsynaptic potentials [IPSPs]) in some PNs (n = 13; see analyses in b). See Section 2 for details. Application of perampanel (5 μM) reduces both the height and duration of the post‐stimulation depolarization plateau in PNs (see magnified overlaying traces in the red box, n = 8) and the duration of the plateau in INs (see magnified overlaying traces in the orange box, n = 6). Note that all post‐stimulation activities in both INs (n = 6) and PNs (n = 5 and 8 for the mean spike frequency and cPSP frequency, respectively, only those having pre‐stimulation baseline membrane potential more depolarized than −60 mV used in the analyses of mean spike frequency) are abolished by perampanel. ^N.S. P > 0.05; ^* P < 0.05, Kruskal–Wallis test followed by pairwise Mann–Whitney U test for post‐stimulation activities and Mann–Whitney U test for post‐stimulation plateau

FIGURE 5

Electrical stimulation‐induced pyramidal neuron (PN)–interneuron (IN) reverberating activities require AMPAergic transmission. (a) Representative recordings show that application of AMPA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (10 μM, bottom traces) rather than NMDA antagonist AP‐5 (10 μM, top traces) effectively abolishes the kindling‐mimicking stimulation‐induced recurrent burst discharges in INs. Orange box: magnified overlaying traces showing that AP‐5 does not significantly reduce the post‐stimulation plateau height. (b) A pair recording of an IN and a PN shows that delivery of kindling‐mimicking stimulation induces large depolarizing plateaus in both neurons, followed by repetitive IN burst discharges, which are synchronized with the repetitive excitatory postsynaptic potentials (EPSPs) and then inhibitory postsynaptic potentials (IPSPs) in the PN (indicated by red dashed arrows in partially magnified traces, blue and teal boxes). Application of CNQX (10 μM) reduces the post‐stimulation plateau and completely inhibits all other post‐stimulation activities. Orange and red boxes: magnified overlaying traces showing the effect of CNQX on the post‐stimulation plateau in IN and PN, respectively. In PNs, CNQX dramatically reduces both the height and duration of post‐stimulation plateau (the red box). In INs, CNQX shortens the post‐stimulation plateau duration (the orange box). (c) Quantitative analyses for the effect of AP‐5 (n = 10) and CNQX (n = 5 for INs and n = 8 for PNs) on the post‐stimulation events in INs (top row) and PNs (bottom row). ^N.S. P > 0.05; ^* P < 0.05, Kruskal–Wallis test followed by pairwise Mann–Whitney U test for INs and Mann–Whitney U test for PNs. The analyses in control (saline) were from the same group of PNs (n = 13) and INs (n = 18–21) in Figure 4