Abstract
SIGNIFICANCE: Two-photon optogenetics and simultaneous calcium imaging can be used to visualize the response of surrounding neurons with respect to the activity of an optically stimulated target neuron, providing a direct method to assess neuronal connectivity. AIM: We aim to develop a two-photon optogenetics-based method for evaluating neuronal connectivity, compare it to the existing indirect resting-state synchrony method, and investigate the application of the method to brain pathophysiology. APPROACH: C1V1-mScarlet was introduced into GCaMP6s-expressing transgenic mice with an adeno-associated virus. Optical stimulation of a single target neuron and simultaneous calcium imaging of the target and surrounding cells were performed. Neuronal connectivity was evaluated from the correlation between the fluorescence intensity of the target and surrounding cells. RESULTS: The neuronal connectivity in the living brain was evaluated using two-photon optogenetics. However, resting-state synchrony was not always consistent with two-photon optogenetics-based connectivity. Comparison with neuronal synchrony measured during sensory stimulation suggested that the disagreement was due to external sensory input. Two-photon optogenetics-based connectivity significantly decreased in the common carotid artery occlusion model, whereas there was no significant change in the control group. CONCLUSIONS: We successfully developed a direct method to evaluate neuronal connectivity in the living brain using two-photon optogenetics. The technique was successful in detecting connectivity impairment in hypoperfusion model mice.