Rationale: Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with uncontrolled epilepsy. One potential SUDEP risk factor is duration of post-ictal generalized EEG suppression (PGES), a period of low amplitude EEG activity after some seizures. Mechanisms underlying PGES are unknown. Our previous work demonstrated that increased serotonergic (5-HT) tone or stimulation of dorsal raphe nucleus (DRN) 5-HT neurons before and during a seizure reduces PGES duration. A potential intermediary downstream of the DRN is the pedunculopontine tegmental (PPT) nucleus, a largely cholinergic component of the ascending arousal system critical for sleep-wake regulation and EEG modulation. We hypothesized that pre- and post-ictal excitation of DRN 5-HT terminals in PPT will reduce PGES duration.
Methods: Adult (8-16 wks) male and female
Pet1-Cre (express Cre recombinase in 5-HT neurons) and WT (n = 8 per sex/genotype/condition) mice were implanted with EEG/EMG electrodes and a bipolar stimulating/recording electrode in the right basolateral amygdala (in mm from bregma: AP: -1.3 ; ML: -2.8; DV: -4.7), and received an injection of AAV allowing Cre-mediated expression of Channelrhodopsin2-mCherry, Archaerhodopsin-YFP, mCherry, or YFP into the DRN (AP: -4.6; ML: 0; DV: -3.0), and an optical fiber to the PPT (AP: -4.46; ML: ± 1.12; DV: -3.5). After recovery and viral transfection, mice underwent afterdischarge threshold determination, and were kindled with the threshold stimulation (2x/day, 80–540 µA, 1 s train of 1 ms biphasic square wave pulses at 60 Hz) until the mouse exhibited consistent generalized tonic-clonic seizures. During trials, mice underwent laser excitation (473 nm, 4 Hz, 10 mW) or inhibition (593 nm, constant, 10 mW) of DRN 5-HT terminals in the PPT prior to or after seizure induction during wake or NREM. Seizure and PGES duration were determined and EEG frequency characteristics (
delta, 1-4 Hz; theta, 4-8 Hz; alpha, 8-12 Hz; beta, 12-30 Hz) were analyzed during recovery from PGES. Trials in female mice were conducted during diestrus or metestrus. AAV transfection and electrode placement were verified post-hoc.
Results: Pre-ictal excitation of DRN 5-HT terminals in the PPT decreased PGES duration without affecting seizure duration. Resumption of baseline EEG activity was delayed by inhibition of DRN 5-HT terminals in the PPT. There were no significant sex differences. There was no effect of laser treatment in groups that received AAV mCherry or YFP. Afterdischarge threshold and kindling rate did not differ between genotypes, sexes, or AAV groups.
Conclusions: These data suggest that the PPT may be a node in a DRN network influencing PGES duration. The identity of the specific neurons and 5-HT receptors in the PPT mediating our findings is unknown. Future work will combine pharmacology and immunohistochemical assays to address this. Elucidating these mechanisms could have important implications for development of prophylactic strategies for SUDEP.
Funding: Please list any funding that was received in support of this abstract.: AES and NIH/NINDS F31NS113479 to A.N.P. NIH/NINDS R01NS095842 to G.F.B.