Rationale:
The nucleus reticularis pontis oralis (NRPO) is part of the brainstem reticular formation and has classically been associated with sleep-waking cycle. Although a few studies during the 80`s and 90`s pointed the NRPO as a site that modulates tonic components of motor seizures, little is known about its role in seizure susceptibility and control. Here, we used optogenetic neuromodulation, fiber photometry, EEG, and behavioral approaches to investigate the role of the NRPO in the control of absence seizures using the WAG/Rij model of genetic absence epilepsy, in which animals have hundreds of spontaneous seizures in a day.
Methods:
53 WAG/Rijs were submitted to stereotaxic surgery to implant bilateral cortical electrodes over the frontal, parietal, and cerebellum (reference and ground). Independent groups of male and female WAG/Rijs (n=8/group) received intra NRPO injection of AAV5-hSyn-ChR2-mcherry (activation opsin), AAV8-CAG-ArchT-GFP (inactivation opsin), or AAV8-CAG-GFP (no opsin-control). All ChR2 animals were tested under 5, 20, and 100 Hz (blue light 450nm); ArchT animals were tested under 100 Hz (green light 520nm). We performed additional test sessions to compare open loop (continuous) and closed loop (on-demand) light delivery. By the end of the protocol, animals were submitted to the open field test to assess the effects of NRPO manipulation on locomotion. 5 WAG/Rijs received intra NRPO pGP-AAV1-Syn-jGCaMP8m-WPRE injection to assess Ca2+ transients during seizure and inter-seizure intervals. Opsins were allowed to express for 3 weeks.
Results:
Closed-loop optogenetic activation (5, 20, and 100 Hz) of the NRPO in response to spike-and-wave discharge (SWDs) detection rapidly suppressed seizures in male and female WAG/Rijs (p< 0.05). NRPO open loop activation (continuous light delivery with 5, 20, and 100 Hz) suppressed SWDs manifestation in male and female WAG/Rijs (p< 0.05). Open and closed-loop inactivation of NRPO neurons did not modify SWDs manifestation in WAG/Rijs (p >