The activated synaptic terminals beyond the light illumination range affect the results of optogenetics

Optogenetics is widely applied to study complex brain networks. However, recent studies have found that light alone can produce effects that are unrelated to optogenetics, and it is still unclear whether this can affect the results of optogenetic experiments.We explored the characteristics of projection of interneurons to excitatory neurons in the auditory cortex with optogenetics, transgenic mice and patch-clamp recording.We discovered that postsynaptic responses can be induced when we stimulated a blank area adjacent to the edge of brain slice. Similar results can be observed after blocking the polysynaptic responses by drugs. Together with the results of control experiments, we found that the false response is caused by activating the synaptic terminals beyond the range of the blue light (470 nm). Also, there was a linear relationship between the response and the stimulus distance for all data, which suggested that these false responses may be related to other factors, such as light scattering.The LED-light-evoked response cannot reflect microcircuit of the recorded neuron and the activated neurons within the illumination range accurately. Together, these results confirm that light alone can affect neural activity, but this can be unrelated to the genuine 'optogenetic effect'.