Brainstem spreading depolarization and cortical dynamics during fatal seizures in Cacna1a S218L mice

Abstract

Sudden unexpected death in epilepsy (SUDEP) is a fatal complication of epilepsy in which brainstem spreading depolarization may play a pivotal role, as suggested by animal studies. However, patiotemporal details of spreading depolarization occurring in relation to fatal seizures have not been investigated. In addition, little is known about behavioural and neurophysiological features that may discriminate spontaneous fatal from non-fatal seizures. Transgenic mice carrying the missense mutation S218L in the α1A subunit of Cav2.1 (P/Q-type) Ca2+ channels exhibit enhanced excitatory neurotransmission and increased susceptibility to spreading depolarization. Homozygous Cacna1aS218L mice show spontaneous non-fatal and fatal seizures, occurring throughout life, resulting in reduced life expectancy. To identify characteristics of fatal and non-fatal spontaneous seizures, we compared behavioural and electrophysiological seizure dynamics in freely-behaving homozygous Cacna1aS218L mice. To gain insight on the role of brainstem spreading depolarization in SUDEP, we studied the spatiotemporal distribution of spreading depolarization in the context of seizure-related death. Spontaneous and electrically-induced seizures were investigated by video monitoring and electrophysiological recordings in freely-behaving Cacna1aS218L and wild-type mice. Homozygous Cacna1aS218L mice showed multiple spontaneous tonic-clonic seizures and died from SUDEP in adulthood. Death was preceded by a tonic-clonic seizure terminating with hindlimb clonus, with suppression of cortical neuronal activity during and after the seizure. Induced seizures in freely-behaving homozygous Cacna1aS218L mice were followed by multiple spreading depolarizations and death. In wild-type or heterozygous Cacna1aS218L mice, induced seizures and spreading depolarization were never followed by death. To identify temporal and regional features of seizure-induced spreading depolarization related to fatal outcome, diffusion-weighted MRI was performed in anaesthetized homozygous Cacna1aS218L and wild-type mice. In homozygous Cacna1aS218L mice, appearance of seizure-related spreading depolarization in the brainstem correlated with respiratory arrest that was followed by cardiac arrest and death. Recordings in freely-behaving homozygous Cacna1aS218L mice confirmed brainstem spreading depolarization during spontaneous fatal seizures. These data underscore the value of the homozygous Cacna1aS218L mouse model for identifying discriminative features of fatal compared to non-fatal seizures, and support a key role for cortical neuronal suppression and brainstem spreading depolarization in SUDEP pathophysiology.

Figure 1

Survival of implanted homozygous Cacna1a^S218L mice and fatal seizure duration in implanted and naïve mice. (A) Survival plot of 26 implanted homozygous Cacna1a^S218L mice showing that the majority of mice died within 1 week after surgery. Note that during recovery from surgery (shaded grey), approximately half of the mice (n = 12) died. In the remaining mice (n = 14), which were monitored in the ECoG set-up for 2 weeks, fatal seizures occurred in all, except one that survived the recording period. Note, one additional mouse died following status epilepticus and was excluded from further analysis and not included in the plot. (B) Duration of spontaneous fatal seizure behaviour was not different for naïve and implanted mice (P = 0.50; data shown as mean ± SD).

Figure 2

Electrophysiological recordings during spontaneous fatal seizures in homozygous Cacna1a^S218L mice. (A) Example of cortical MUA and ECoG (DC and AC) recordings during a fatal seizure (behavioural onset indicated by the vertical dashed line). MUA (histogram) decreased during seizure behaviour (clonic behaviour in grey, tonic behaviour in black). MUA and AC ECoG were attenuated in the minute preceding the terminal depolarization (TD; indicated by an arrow). Note that the pre-ictal transition of AC ECoG amplitude parallels vigilance state (non-REM sleep followed by wakefulness). (B) ECG recordings during a fatal seizure in another mouse showing cardiac electrical activity following TD. Electrocardiographic arrest (not shown) occurred 7 min after the terminal depolarization. The onset of seizure-related behaviour preceded the plotted time series. AC = alternating current; bps = beats per second; DC = direct current; IHR = instantaneous heart rate.

Figure 3

Frequency of spontaneous non-fatal and fatal seizures (stage 5) and (pre-)ictal behaviour in implanted homozygous Cacna1a^S218L mice. (A) The distribution of non-fatal seizures (black dots) in implanted mice over the 24 h preceding the fatal seizure (grey dots; cross). (B) Fatal seizures were associated with a proportional increase of clonic behaviour during the second half of the seizure at the cost of tonic and other behaviours, when compared to non-fatal seizures (n = 11 per group; *P = 0.001; data are shown as median, interquartile intervals, minimum and maximum). (C) Vigilance state preceding non-fatal and fatal seizures (percentages indicate fraction of total time) was not significantly different (P > 0.99). FS = fatal seizures; NFS = non-fatal seizures.

Figure 4

Different ECoG and cortical multi-unit activity during spontaneous non-fatal and fatal seizures in homozygous Cacna1a^S218L mice. (A) Example cortical MUA and AC ECoG recordings of a fatal seizure [behavioural onset indicated by a red dashed line; clonic behaviour is shown in grey, tonic behaviour in black; terminal depolarization (TD) indicated by an arrow]. Details of ictal AC ECoG and MUA indicated by dashed boxes are shown in B and C. (B) Detailed inspection of the AC ECoG reveals oscillatory activity corresponding to theta band frequencies and (C) spike-wave complexes (indicated by arrowheads) not exceeding normal AC ECoG amplitude, that coincide with loss of cortical MUA. (D–F) Spectral analyses of primary sensorimotor (M1/S1) AC ECoG during non-fatal (red) and fatal (black) stage 5 seizures (n = 9) for total power (D), delta/total ratio (E) and theta/total ratio (F). Power during the first 60 s of all seizures (indicated by black bars) was compared with baseline power (1 h pre-ictal; baseline power levels for non-fatal/fatal seizures are indicated by red/black dashed lines). In addition, non-fatal and fatal seizures were compared over the same ictal period (insets; data are shown as median, interquartile intervals, minimum and maximum). Normalized total power and delta/total power ratio were attenuated (D and E; P = 0.001 and P = 0.010 for non-fatal and fatal seizures, respectively). Theta/total ratio was increased only for fatal seizures, compared to both baseline and non-fatal seizures (F; P < 0.001 and *P = 0.003, respectively). For all fatal seizures, cessation of seizure-related behaviour is indicated by vertical blue bars. (G and H) Normalized cortical MUA dynamics in relation to onset [G(i)] and end [G(ii)] of seizure-related behaviour. Black bars indicate 120-s time windows used for comparison of non-fatal and fatal seizures in (H). MUA decreased during fatal seizures (G) and was significantly reduced when compared to non-fatal seizures during the last 120 s of seizure-related behaviour (H; *P = 0.018; data are shown as median, interquartile intervals, minimum and maximum). AC = alternating current.

Figure 5

Multiple cortical spreading depolarizations associated with induced seizures followed by death in homozygous Cacna1a^S218L mice. Representative ECoG (AC and DC) recordings showing that stimulation (shaded grey) was followed by afterdischarges in all wild-type (WT) mice (7/7; example in A) and heterozygous Cacna1a^S218L (het S218L) mice (9/9; example in B), while only in a minority of homozygous Cacna1a^S218L (hom S218L) mice [2/11; example in C(i)], as indicated by insets (corresponding to the shaded red time windows). Cortical spreading depolarization (indicated by arrowheads in example traces) was observed directly following afterdischarges in all but one heterozygous Cacna1a^S218L mice. In homozygous Cacna1a^S218L mice, multiple cortical spreading depolarizations occurred, followed by a terminal depolarization [C(ii); TD; indicated by an arrow]. Fatal outcome was observed in 7 of 11 homozygous Cacna1a^S218L mice but none of the wild-type and heterozygous Cacna1a^S218L mice. AC = alternating current; DC = direct current.

Figure 6

DW-MRI visualization of spreading depolarization following induced seizures in anaesthetized homozygous Cacna1a^S218L mice. (A) Coronal brain maps corresponding to DW-MRI images in B–D. (B) Representative DW-MRI data following an induced fatal and (C) non-fatal seizure in a homozygous Cacna1a^S218L (hom S218L) mouse, and (D) a non-fatal seizure in a wild-type (WT) mouse. Data are shown for a single representative time point in each slice, capturing a snapshot of spreading depolarization (SD). Note that in B, all coronal maps are shown at three time points to demonstrate spatiotemporal propagation of spreading depolarization, whereas in C and D different sections at three time points are depicted to show the limited subcortical spread of spreading depolarization in non-fatal seizures. (E) Time course data of wild-type mice (n = 3; seven spreading depolarizations) and Cacna1a^S218L mice (n = 7; 13 non-fatal spreading depolarizations; four fatal spreading depolarizations) in relation to seizure onset (dashed line) are shown for distinct brain regions obtained from DW-MRI data. In Cacna1a^S218L mice, spreading depolarization appeared in the brainstem only during fatal seizures and was constrained to striatum, amygdala, hippocampus and colliculi during non-fatal seizures. Cortical spreading depolarization occurred in all wild-type and Cacna1a^S218L mice. A.u. = arbitrary units; Cb = cerebellum; Cx = cortex; DC = direct current; Hc = hippocampus; Md = medulla oblongata; Po = pons; SC = superior colliculus; St = striatum; Th = thalamus.

Figure 7

Physiological function following induced seizures during DW-MRI in anaesthetized homozygous Cacna1a^S218L mice. (A) Representative example from a single animal of respiratory rate, heart rate and spreading depolarization aligned temporally in relation to seizure onset (dashed line). (B) Physiological parameters (aligned with spreading depolarization dynamics shown in Fig. 6E) from homozygous Cacna1a^S218L (hom S218L) mice, following non-fatal (n = 10) and fatal seizures (n = 4), and wild-type (WT) mice, following non-fatal seizures (n = 15). Simultaneous respiratory (upper trace) and heart rate (lower trace) data acquired during seizures from the same animals showing that, for fatal seizures, respiratory rate decreased until complete arrest and preceded cardiac arrest. A.u. = arbitrary units. Data shown as mean ± SD.

Figure 8

Brainstem DC recordings during a spontaneous fatal seizure in a homozygous Cacna1a^S218L mouse. Example of a spreading depolarization (indicated by arrowheads) that occurred during seizure behaviour in the oral pontine reticular nucleus (PnO) and subsequently in the medullary reticular formation (MRF), followed by visual cortex (V1) AC ECoG suppression, bradycardia and cortical terminal depolarization (TD; indicated by an arrow). Note that ictal V1 AC ECoG amplitude was greatly reduced following cortical spreading depolarization. AC = alternating current; bps = beats per second; DC = direct current; IHR = instantaneous heart rate.