Authors :
Presenting Author: Su-youne Chang, PhD – Mayo Clinic
Karni Bar-Or, MS – Modulight Bio
Filip Mivalt, MS – Mayo Clinic
Daniela Maltais, BS – Mayo Clinic
Yoav Kfir, PhD – Modulight Bio
Jiwon Kim, BEng – Mayo Clinic
Inyong Kim, BEng – Mayo Clinic
Matan Hershko, PhD – Modulight Bio
Ofir Levi, PhD – Modulight Bio
Ofer Yizhar, PhD – Weizmann Institute of Science
Yotam Eldar, MD – Modulight Bio
Gregory Worrell, MD, PhD – Mayo Clinic
Rationale:
Optogenetics offers unmatched cellular specificity and control over cellular activity. Various opsins have been tested in animal models of epilepsy, each contributing to our understanding of seizure circuit dynamics. We evaluated eOPN3, a red-shifted, inhibitory type-2 opsin (GPCR opsin), to explore its potential for epileptogenic circuit modulation in a swine seizure model, advancing integrated electrical and optogenetic therapies, and paving the way for drug-resistant epilepsy treatments.
Methods:
MRI-guided stereotactic surgery was used to deliver 15–60 µL of AAV-eOPN3 (AAV5/9-CaMKII-eOPN3-mScarlet) into the anterior thalamic nucleus (ANT) and hippocampus (HPC) of three Göttingen micropigs. Each hemisphere received either an active or a control viral vector (AAV5/9-CaMKII-mScarlet) with gadolinium to enable visualization of injection sites and diffusion volume via postoperative MRI. Two to three months post-injection, bilateral DBS electrodes integrated with optic fibers were implanted into the ANT and HPC to assess: 1) opsin expression using fiber photometry, 2) optogenetic modulation of electrical stimulation response potentials (ERPs), 3) induction and propagation of seizure-like activity via HPC kainic acid (KA) injection, and 4) optogenetic modulation of KA-induced seizure activity. After the electrode implantation following fiber photometry, local field potentials (LFPs) were continuously monitored during electrical and optical stimulations and pre- and post-KA injection. Brains were harvested for histological analysis to evaluate opsin expression, quantify viral expression volume, and estimate eOPN3 axonal transport distances.
Results:
AAV5 and AAV9 achieved efficient transduction in the pig brain, with eOPN3 expression confirmed during surgery via fiber photometry. ANT stimulation elicited intensity-dependent ERPs in the HPCs, which were attenuated by HPC illumination. HPC stimulation induced ERPs in the contralateral HPC. The HPC-induced ERPs were significantly reduced by illuminating the recording side HPC contralateral to the stimulation side, demonstrating the optogenetic modulation of the axonal projection between HPCs. KA injection into the HPC induced 20-30 Hz seizure-like activity. Seizure induction latency and efficacy improved under ketamine/xylazine anesthesia compared to isoflurane. Early stage, localized KA-induced seizure activity was effectively suppressed by ANT-HPC illumination. However, at later stages, ANT-HPC illumination lost efficacy in controlling the wider spread KA-induced seizures. Histology confirmed eOPN3 expression in the HPC, ANT, and other Papez circuit nodes.
Conclusions:
Our study highlights eOPN3-mediated inhibition alters the latency and spread of seizure-like activity. We developed a platform, incorporating pre- and postoperative MRI for precise viral vector delivery, real-time fiber photometry for quantifying opsin expression, and integrated electro-optical stimulation and signal recording to assess optogenetic efficacy. This large animal model provides a solid foundation for future translational research to develop optogenetic therapies for human epilepsy.
Funding:
NIH R01 NS092882, Birdel Fund Mayo Clinic Philanthropy, and Modulight Bio