Conclusions:
Prevention of off-target effects during optogenetic stimulation is fundamental for reducing misinterpretation of experimental results. The use of discontinuous light at low power levels and/or duty cycle can achieve this goal. However, such conditions could reduce the efficiency of optogenetic protocols in neurons expressing first-generation opsins, a limitation that can be overcome by using new-generation opsins such as ChRmine or ChroME (Marshel et al. , 2019; Sridharan et al. , 2022).