Seizure-Inducible Risks of In Vivo Optogenetic Manipulations

Optogenetic manipulation is pivotal in basic neuroscience research and in studying neuropsychiatric disorders, enabling the activation or inhibition of neuronal populations with millisecond precision. However, this technique can induce artificial neuronal hypersynchronization that is rarely observed under physiological conditions. Given that epileptic seizures arise from abnormally synchronized neuronal discharges, the potential for optogenetic stimulation to trigger seizures and confound experimental outcomes warrants close examination. Here, using electrophysiological and behavioral recordings, we demonstrate that even single-trial optogenetic stimulation of CaMKII-positive neurons in the hippocampal CA1 region, anterior piriform cortex (APC), or lateral/medial entorhinal cortex (LEnt or MEnt) can induce seizure-like discharges and behaviors in adult male C57BL/6 mice. Repeated stimulation in the APC, LEnt, or MEnt elicited more severe seizure-like activity. Furthermore, stimulation protocols characterized by high power, long duration, high frequency, and medium pulse width were more prone to inducing such events. Additionally, we found that CA1 stimulation could impair subsequent contextual fear memory. These findings provide critical cautions and practical guidelines for the design and implementation of in vivo optogenetic experiments in neuroscience research.