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

2019 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: seizure / cannabinoids / in-situ-hybridization / paraventricular nucleus / supraoptic nucleus / photostimulation / CB1RKO / JWH-018

Outline of Annual Research Achievements

In this research project we aim to identify brain regions and signaling pathways underlying cannabinoid-induced seizures. As we showed in our previous research natural and synthetic cannabinoids induce epileptic seizures in mice (Malyshevskaya et al., 2017), however their appearance is different form classic epileptic seizure. The difference of severity, seizure duration time, and the fact that these seizures are prevented by CB1R-blocker also suggest a completely distinct mechanisms from classical epileptic seizure models. Therefore using CB1RKO mice we confirmed their resistance to the cannabinoid convulsive effects. We also discovered certain brain regions are activated upon synthetic cannabinoid JWH-018 administration. With in-situ hybridization (ISH) method we found that c-fos (immediate early gene) mRNA expression is increased in the hypothalamic paraventricular (PVN) and supraoptic (SO) nuclei, 1 hour after cannabinoid administration, which were not activated in control mice. To verify brain regions involved in cannabinoid convulsive effects we used optogenetic activation of SO nuclei. Bilateral photostimulation of SO nuclei alone did not induce electrographic seizures. Therefore we are testing the possibility of importance of both PVN and SO nuclei in seizure induction process which requires mutual photostimulation. The experiment with complex AAV injection and photostimulation is ongoing.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

Our initial AAV injections of ChR2 with the activation of the supraoptic nucleus did not result in electrographic seizure. One possibility is that sole activation of SO nuclei is not efficient enough to generate seizures. Another possibility is the level of transfection or possible lacking of precision with the stereotactic injection.

Strategy for Future Research Activity

We are planing to perform set of surgeries with a higher titer of the AAV and increase the precision of the AAV injections. Additionally we will perform a transfection and activation of both SO and PVN nuclei bilaterally.