The authors describe an "all-optical" approach for imaging and optical stimulation of neural circuits in vivo to image neural activity and to simultaneously manipulate this activity during behavior of an awake animal. Optogenetic interventions were targeted to individual neurons in the mammalian brain on the basis of functional signatures recorded with the same microscope. The approach depends upon the resolution of two-photon imaging and two-photon optical stimulation with a spatial light modulator (SLM) together with software that enables flexible targeting of readout and manipulation of visualized neurons.
A Bruker Ultima in vivo dual-beam path multiphoton microscope was adapted for closed loop simultaneous all-optical imaging (using a genetically encoded calcium indicator, GCaMP6) and optical stimulation (using an optogenetic actuator, C1V1). Two-photon imaging of layer 2/3 mouse cortex (about 100–300 μ m deep) was performed by resonant-galvanometer raster scanning. A reflective multilevel SLM was coupled to the microscope for the optical stimulation of groups of around ten neurons in the current study (although stimulating more neurons is possible using the SLM). Raw data from the image acquisition card of the microscope was made available to a custom closed-loop interface using the PrairieLink feature of the PrairieView software.
Several different activity-guided neural circuit manipulations in the brains of living mice were performed using this “all optical” multiphoton method. The ability to directly manipulate activity patterns using the closed-loop approach will enable improved tests of models of circuit connectivity, dynamics, and plasticity. Goals for future applications of the technique include the correction of aberrant activity patterns in conditions such as epilepsy and Alzheimer’s disease, and the implementation of a new generation of optical brain-machine interfaces.