The elucidation of brain regions and signaling pathways underlying cannabinoid-induced seizures

2020 Fiscal Year Research-status Report

• Project/Area Number: 19K19479
• Research Institution: University of Tsukuba
• Principal Investigator: マリシェフサカヤ オリガ 筑波大学, 国際統合睡眠医科学研究機構, 研究員 (20739429)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: photostimulation / supraoptic nucleus / paraventricula nucleus / channelrhodopsin-2 / NREM sleep / Delta SWS / wakefullness / arousal

Outline of Annual Research Achievements

We previously published, that agonists of cannabinoid-receptor-1 (CB1R) induce seizures in mice and this convulsive effect is mediated by CB1 receptors. In addition, our data suggest that CB1R agonists activate specific brain area, which could be involved in the development of cannabinoid seizure. Therefore, we aim to identify brain region and signaling pathway involved in the pro-convulsive effect of cannabinoids. As we found out last year bilateral photostimulation of SO nuclei alone did not induce electrographic seizures, we did some additional experiments involving bigger area of virus transfection (expanding to both PVN and SO nuclei). The area of injection was confirmed by immunohistochemical staining and despite the sufficient level of transfection we couldn’t observe electrographic or behavioral seizures. On the other hand, we discovered that prolonged photostimulation (4 hours) results in increased wakefulness (time in wake) with complete absence of sleep (NREM and REM) during stimulation. This effect was highly significant and resulted sleep rebound accompanied by increased NREM sleep amount and Delta NREM amount immediately after end of stimulation. This suggests that SO nucleus is strongly involved in inducing and possibly maintaining arousal state. To our knowledge this data has not been reported by anyone yet. We are planning to investigate the effect of longer photostimulation of SO nuclei and to anatomically dissect inputs and outputs of such arousal effect.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

The research work is progressing as planned with a modification regarding unexpected experimental result. This surprising finding modified research hypothesis and currently expanded for the sleep and wake inducing center in the CNS.

Strategy for Future Research Activity

According to our initial proposal we were planning to investigate signalling pathway involved in the seizure-inducing effect of cannabinoids. Because virus injection and activation of candidate nuclei (SON, PVN) resulted in quiet unexpected findings we modified and expanded our hypothesis. We are planning to deeply investigate how activation of SO and PVN nuclei produces arousal and what mechanism underlie this phenomenon.

Causes of Carryover

We are planing to continue animal surgeries for the transfect ion with AAV, which requires transgenic colony maintenance, virus production, surgeries consumables (wires, sockets etc.) and antibody source for the IHC. In addition, there are expenses associated with data dissemination to public (publication fee, conference participation fee etc.)