Opto‐ and Chemogenetic Dissection of Ictal Apnea Neural Circuitry

Sudden Unexpected Death in Epilepsy (SUDEP) is defined as the sudden, unexpected and unexplained death of a person with epilepsy and accounts for between 8 and 17% of epilepsy‐related deaths, rising to 50% for patients with refractory epilepsy. In a mouse model of SUDEP, we have recently shown that death is due to seizure‐induced respiratory arrest. In addition, apnea is initiated during the tonic phase and tonic respiratory muscle contraction is a possible mechanism of apnea. In the present study, we explore 1) whether tonic activity of the inspiratory rhythm generator in the brainstem and/or 2) upper motor neuron activity in the motor cortex drives ictal apnea. We recorded video, electrocorticogram (ECoG), electrocardiogram (ECG), and breathing via whole body plethysmography in adult mice carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; “D/+ mice”) during audiogenic seizures. This mutation was identified from a patient that died from SUDEP and D/+ mice have severe convulsive seizures with apnea and many suffer seizure‐induced death. To test the hypothesis that tonic activity from the inspiratory oscillator results in apnea, we implanted fiberoptic ferrules bilaterally into the Bötzinger Complex (BötC) of mice that express Channelrhodopsin2 (ChR2) under the vesicular GABA transporter (VGAT; “VGAT‐ChR2” mice) that were crossed with D/+ mice (Fig. 1A & B). The goal of the experiment is to photostimulate BötC during ictal apnea to inhibit tonic inspiratory activity and produce expiration (Fig. 1C). Seizures were evoked using a 15 kHz pure tone, as we have done before (Fig. 1D). Trains of light pulses (50 ms pulses, 5 mW of 473 nm light) were evoked repetitively during ictal apnea (Fig. 1E & F). However, this did not recover normal breathing rhythm and apnea duration was no different for any photostimulation paradigm versus control (p = 0.7892, F = 0.1747, One‐Way ANOVA; Fig. 1G). Although breathing was not affected during seizures, the effects on baseline breathing were substantial; for example, inspiration was inhibited for a full 10 second photostimulation train (Fig. 1H). To test the necessity of ictal activity from upper motor neurons in the motor cortex are required for generating ictal apnea, we expressed iDREADD receptors in cortical excitatory neurons of D/+ mice and injected CNO i.p. prior to inducing seizures with a 15 kHz pure tone (Fig. 2A). Under control conditions, seizures presented with the usual tonic phase apnea and spike wave discharges (SWDs) in the motor cortex (Fig. 2B). CNO was able to robustly inhibit the SWDs, but the tonic phase and apnea were not affected (Figs. 2C‐E). The effect of 3 mg/kg CNO on ECoG power was significantly greater than the effect on apnea (p = 0.0059, paired t‐test). In sum, we found that the core inspiratory oscillator circuitry in the brainstem is likely bypassed to create tonic inspiratory activity. Furthermore, inhibition of cortical upper motor neurons has no effect on apnea. Thus, our interpretation is that other pools of upper motor neurons must drive the tonic inspiratory activity and apnea.