Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo

Abstract

We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. Concurrent two-photon optogenetic activation and calcium imaging is enabled by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical crosstalk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states, and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mammalian brain in vivo.

Principles of designing interpretable optogenetic behavior experiments

“…In addition, optogenetic manipulations that are even more precise, from opsins that target specific cell compartments to light delivery systems that drive more naturalistic patterns of activity, may both reduce unintended network side effects and reveal how more specific or complex patterns of activity alter the network. For instance, two-photon holographic excitation of opsin-expressing neurons may support manipulation of activity with near-single cell resolution (Andrasfalvy et al 2010;Papagiakoumou et al 2010;Packer et al 2012Packer et al , 2015. In sum, while optogenetic tools allow for the temporally and spatially precise recruitment of well-defined sets of cells, these tools may lead to potential confounds due to protein expression, ions conducted, heat and light, and network effects.…”

Regulation of Cellular Molecular Signaling by Light (Invited Paper)

“…This idea was thus named as optogenetics, and now it has been developed as the most powerful tool to stimulate cells, especially in neuroscience to control neural excitability. dyes such as Fluo-5F [57], or genetically encoded calcium sensors like G-CaMP [58][59][60] for real-time Ca 2+ dynamics detection with a temporal resolution of around 10 ms. Generally, this technique has been developing very fast and contributing greatly to neuroscience [61][62][63], especially to in vivo research [64][65][66]. Recently, it was found that proteins from optogenetics could be activated by two-photon excitation and thus combined with two-photon microscopy system [67,68].…”

“…Recently, it was found that proteins from optogenetics could be activated by two-photon excitation and thus combined with two-photon microscopy system [67,68]. Further with fluorescent readouts methods by genetically encoded calcium sensors, simultaneous cellular-resolution optical stimulation and imaging can achieved [59,60]. However, it should be noted that cations such as Na + , K + and H + were also activated due to the non-specificity of those optogenetic proteins [69].…”

Optogenetic Approaches to Target Specific Neural Circuits in Post-stroke Recovery

“…Such in vivo deep brain recording of neural activity could be important to study the role of thalamocortical and cerebellothalamic projections during stroke recovery. Notably, both optogenetic toolsets-stimulation and recording of neural circuit activity-have been combined in an all-optical setting for photostimulation and fast calcium imaging without spectral overlap in vivo [110,111].…”