Optogenetic activation of the neural circuit between dorsal raphe nucleus and the pre-Bötzinger complex contributes to inhibition of the seizure-induced respiratory arrest in the DBA/1 mouse SUDEP model

Sudden unexpected death in epilepsy (SUDEP) is the fatal cause leading to the death of epilepsy patients, especially those with anti-epileptic drug resistance. However, the underlying mechanism of SUDEP remains elusive. Our previous study demonstrated that enhancement of serotonin (5-HT) synthesis by intraperitoneal (IP) injection of 5-hydroxytryptophan (5-HTP) significantly reduced the incidence of seizure-induced respiratory arrest (S-IRA) in DBA/1 mice SUDEP models. Given that 5-HT2A receptor (5-HT2AR) plays an important role in mediating the respiration system in brain, we hypothesized that 5-HT2AR is of great significance to modulate S-IRA and SUDEP. To test this hypothesis, we examined whether the decreased incidence of S-IRA evoked by either acoustic stimulation or PTZ injection by administration with 5-HTP will be blocked by administration with ketanserin (KET), a selective antagonist of 5HT2AR, in DBA/1 mice SUDEP models to examine the role of 5-HT2AR in modulating S-IRA. Our results suggested that the decreased incidence of S-IRA by 5-Hydroxytryptophan (5-HTP), a precursor for central nervous system (CNS) serotonin (5-HT) synthesis, was significantly reversed by IP or intracerebroventricularly (ICV) injection of ketanserin in our models. Furthermore, given that 5-HT2AR locating in the pre-Botzinger complex (PBC) which plays a key role in regulating the respiratory rhythm, we wonder whether the lower incidence of S-IRA by IP with 5-HTP was significantly reversed by KET which inhibits 5-HT2AR in the above models and acts on the 5-HT2AR in pre-Botzinger complex .To test our hypothesis, we activated the neural circuit between dorsal raphe nucleus and the pre-Botzinger complex by optogenetics technology. Our results indicated that the lower incidence of S-IRA evoked by PTZ by stimulating the TPH2-ChETA, a variant of channelrhodopsin-2 (ChR2) neurons in the dorsal raphe nucleus (DR) in DBA/1 mice and the suppressant effects was significantly reversed by administration of KET in bilateral pre-Botzinger complex without changing electroencephalogram (EEG) activity. The neural circuit between DR and the pre-Botzinger complex was confirmed by injection of CTB555, a nerve tracer, in DR or the pre-Botzinger complex, respectively. Taken together, our findings suggested that targeting the neural circuit between the DR and the pre-Botzinger complex in which 5-HT2AR plays a critical role in regulating this process would be promising to prevent SUDEP. Keywords: sudden unexpected death in epilepsy; 5-Hydroxytryptophan (5-HTP); ketanserin (KET); pre-Botzinger complex; dorsal raphe nucleus; 5HT2A receptor; respiration rhythm.