Targeting subsets of neurons within a genetically defined population. Credit: Kim Caesar/Nature Publishing Group

The year 2015 marked the ten-year anniversary of the introduction of channelrhodopsin 2 (ChR2) into neuroscience. ChR2 has proven to be a powerful tool not only for stimulating neurons, but also for inspiring the development of other optogenetic tools. Typically, these tools are excited with wide-field illumination, and specificity is imparted by their expression in genetically defined neurons. But for a more detailed dissection of neural circuitry, patterned illumination schemes come in handy. These allow one to manipulate the activity of particular subsets of neurons within a genetically defined population.

The desired illumination patterns can be generated in different ways. Illumination with fast scanning mirrors is an option, or computer-generated holographic patterns may be produced with spatial light modulators. In addition, illumination schemes need to be integrated with the optical readout of neural activity that is often combined with optogenetic activation. Linking these two goals is not necessarily easy and requires optimization because of optical cross-talk between the optogenetic activators and the activity sensors.

Several recently introduced methods exemplify the various strategies available to researchers. Near-simultaneous illumination of neurons can be accomplished by focusing a two-photon laser to the size of a soma and switching between different neurons with the help of a fast scanning mirror (Nat. Neurosci. 17, 1816–1824, 2014). One can also achieve simultaneous illumination by splitting a two-photon beam into multiple beamlets and targeting them to the neurons of interest with a spatial light modulator (Nat. Methods 12, 140–146, 2015). In both studies, the use of a red-shifted optogenetic actuator (C1V1) reduced optical cross-talk with the green calcium indicators that reported neural activity. In freely behaving animals, fiberscopes can deliver one-photon illumination patterns generated in a similar manner (Neuron 84, 1157–1169, 2014).

Cellular-resolution optogenetics will continue to further our understanding of neural-subtype diversity and function. However, these approaches allow the stimulation of neurons in only a single 2D plane within the field of view so far, whereas neurons communicate and function in the 3D environment of the living brain. Expanding these optical stimulation approaches into three dimensions is likely to open up exciting possibilities for probing neuronal function.