Optogenetic Induction of Neuronal Seizures

Adult CD1 mice were used for these experiments after approval was obtained by the IACUC of Weill Cornell Medical College. Mice were induced with isoflurane (2–4%) in 70% N₂:30% O₂ by facemask. After induction, the animal was maintained under isoflurane anesthesia using a small mask at ~1.5%. Animals were mounted in a stereotaxic frame. The heart rate, arterial blood oxygen saturation, and end-tidal CO₂ were monitored and maintained at normal values throughout the experiment. Temperature was monitored rectally and maintained at 37°C with a homeothermic blanket system (Harvard Apparatus, Holliston MA). A ~ 4 × 5 mm cranial window was opened over one hemisphere between Lambda and Bregma to expose the neocortex. The dura was carefully removed. Ruthenium-bipyridine-triphenylphosphine caged 4-aminopyridine (RuBi-4-AP) (200 μl, 10 mM) was topically applied or microinjected into neocortex at least 30 min before uncaging. For the focal illumination, 2 μl RuBi-4-AP solution was injected in neocortex at the depth of 300 μm using an UltraMicroPump (UMP-3, WPI, Sarasota, FL) via a glass electrode controlled at a speed of 100 nl/min. For control experiments, 4-AP ictal discharges were induced by injecting 4-AP (Sigma, 15 mM, 0.5 μl) at the same location using a Nanoject II injector as previously described (Schwartz and Bonhoeffer, 2001 (link); Zhao et al., 2009 (link)).