Overcoming off-target optical stimulation-evoked cortical activity in the mouse brain in vivo

Genetic engineering of exogenous opsins sensitive to a wide range of light wavelengths allows the interrogation of brain circuits to an unprecedented temporal and spatial precision. In particular, red-shifted opsins offer access deeper within the brain tissue. It is however crucial to consider the potential unintended back-activation of endogenous opsins due to laser light striking the back of the retina. Here, we found that in complete darkness and with no expression of exogenous opsins, optical fiber laser stimulation at wavelengths of 637 nm (red), 594 nm (orange), or 473 nm (blue) from within the ipsilateral mouse visual cortex resulted in a strong neuronal response in its contralateral counterpart. This neuronal activation occurred even at low laser intensities (1 mW at the fiber tip, ~31.8 mW/mm ² ) and was most pronounced using red wavelengths. We therefore took advantage of retinal light adaptation using external illumination with a relatively dim ambient light source (20 lux) which was found to completely abolish orange and blue laser-evoked neuronal activation from within the brain, even at high laser intensities (15 mW, ~477.3 mW/mm ² ). To prevent red laser-evoked retinal activation, however, only much lower intensities (2.5 mW, ~79.6 mW/mm ² ) combined with external illumination (20 lux) could be used. These findings demonstrate the critical need for careful selection of light wavelengths and intensities for laser stimulation during optogenetic experiments in the mouse brain in vivo . Additionally, light adaptation of the retina through ambient light exposure offers an effective solution to minimize unintended retinal activation.