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. 2015 Oct 14;35(41):13917-26.
doi: 10.1523/JNEUROSCI.2916-15.2015.

All-Optical Interrogation of Neural Circuits

Valentina Emiliani  1 , Adam E Cohen  2 , Karl Deisseroth  3 , Michael Häusser  4
Affiliations

All-Optical Interrogation of Neural Circuits

Valentina Emiliani et al. J Neurosci. .

Abstract

There have been two recent revolutionary advances in neuroscience: First, genetically encoded activity sensors have brought the goal of optical detection of single action potentials in vivo within reach. Second, optogenetic actuators now allow the activity of neurons to be controlled with millisecond precision. These revolutions have now been combined, together with advanced microscopies, to allow "all-optical" readout and manipulation of activity in neural circuits with single-spike and single-neuron precision. This is a transformational advance that will open new frontiers in neuroscience research. Harnessing the power of light in the all-optical approach requires coexpression of genetically encoded activity sensors and optogenetic probes in the same neurons, as well as the ability to simultaneously target and record the light from the selected neurons. It has recently become possible to combine sensors and optical strategies that are sufficiently sensitive and cross talk free to enable single-action-potential sensitivity and precision for both readout and manipulation in the intact brain. The combination of simultaneous readout and manipulation from the same genetically defined cells will enable a wide range of new experiments as well as inspire new technologies for interacting with the brain. The advances described in this review herald a future where the traditional tools used for generations by physiologists to study and interact with the brain-stimulation and recording electrodes-can largely be replaced by light. We outline potential future developments in this field and discuss how the all-optical strategy can be applied to solve fundamental problems in neuroscience.

Significance statement: This review describes the nexus of dramatic recent developments in optogenetic probes, genetically encoded activity sensors, and novel microscopies, which together allow the activity of neural circuits to be recorded and manipulated entirely using light. The optical and protein engineering strategies that form the basis of this "all-optical" approach are now sufficiently advanced to enable single-neuron and single-action potential precision for simultaneous readout and manipulation from the same functionally defined neurons in the intact brain. These advances promise to illuminate many fundamental challenges in neuroscience, including transforming our search for the neural code and the links between neural circuit activity and behavior.

Keywords: calcium imaging; genetically encoded calcium sensor; genetically encoded voltage sensor; optogenetics; two-photon microscopy; wavefront shaping.

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Figures

Figure 1.
Figure 1.
The toolkit for all-optical interrogation of neural circuits. A schematic outline of the different ingredients required for the all-optical approach is shown. galvo, galvanometer; ACRs, anion channel rhodopsins; Arch, archaerhodopsin; YC, yellow chameleon; AOD, acousto-optic deflector.
Figure 2.
Figure 2.
All-optical electrophysiology. A, Comparison of fluorescence signals recorded simultaneously from a GECI, GCaMP6f, and a GEVI, QuasAr2, expressed as a fusion construct in a rat hippocampal neuron. Subthreshold depolarizations, such as indicated by the arrow, do not have a correlate in the Ca2+ signal. B, Spatially resolved all-optical electrophysiology in a cultured rat hippocampal neuron. The blue region indicates the optically stimulated patch of dendrite. The action potential initiated in an unstimulated process and propagated back into the soma and into the dendritic arbor. Movie frames were calculated by sub-Nyquist interpolation of data acquired at a 1 s exposure time. Scale bar, 50 μm. Bottom right, Immunostaining of the same cell with anti-EGFP (EGFP; green) and anti-AnkyrinG (AnkG; magenta). Scale bar, 25 μm. Magenta arrows, Site of action potential initiation; distal end of the axon initial segment. C, Single-trial optical recordings of APs initiated by pulses of blue illumination (10 ms, 7.5 mW/cm2). Signal represents whole-soma fluorescence without photobleaching correction or background subtraction (modified from Hochbaum et al., 2014).
Figure 3.
Figure 3.
Patterned photostimulation and functional imaging in freely behaving mice. A, Schematic of the holographic fiberscope composed of two illumination paths: one for photoactivation with CGH including a liquid-crystal spatial light modulator, and a second for fluorescence imaging including a DMD. Backward fluorescence was detected on a scientific complementary metal oxide semiconductor (sCMOS) camera. Both paths were coupled to the sample using a fiber bundle attached to a micro-objective (MO). L, Lens; BS, beam splitter; O, microscope objective. B, Left, Calcium signal triggered by photoactivation (blue line; p = 50 mW/mm2) with a 5 μm holographic spot placed on the soma of a ChR2-expressing cell recorded in a freely behaving mouse coexpressing GCaMP5-G and ChR2 in cerebellar molecular layer interneurons (MLIs). Right, Structure illumination image recorded in a freely behaving mouse and showing MLI somata and a portion of a dendrite (inset). Scale bars: 10 μm. C, Top, The same photoactivation protocol as in A was repeated every 30 s for 15 min (photostimulation power, 50 mW/mm2; imaging power, 0.28 mW/mm2). Bottom, Expansion of the top trace showing that spontaneous activity frequently occurs between evoked transients (adapted from Szabo et al., 2014).
Figure 4.
Figure 4.
Targeting manipulation to functionally defined ensembles of neurons. A, A field of view showing neurons coexpressing GCaMP6s (green) and C1V1–2A-mCherry (pink) in the C2 barrel of mouse somatosensory cortex. Scale bar, 50 μm. Symbols are as shown in B. B, Groups of individually identified neurons were selected for photostimulation based on their response to dorsoventral and rostrocaudal whisker stimulation. Five neurons that responded differently or not at all to sensory stimulation (gray shading) were simultaneously photostimulated (pink line; adapted from Packer et al., 2015). C, Two-photon fluorescence image of CA1 hippocampal neurons expressing GCaMP3 (green) and C1V1(E122T/E162T)-2A-EYFP (red) in an awake mouse. Inset, Images of unmixed GCaMP3 and enhanced yellow fluorescent protein (EYFP; top panels) and a pseudocolor merge (bottom; image sizes, 25 × 65 μm). Somatic GCaMP3 appeared to be annular from nuclear exclusion, whereas EYFP was diffuse. D, Schematic and experimental examples of place cell perturbation. A trained mouse ran along a 400 cm virtual reality track (top). A neuron with a place field in this environment (gray shaded region) was stimulated while the mouse ran through a different part of the track (red shaded region). Single-trial examples of place-cell activity (ΔF/F traces) are shown below for imaging-only (black; Ctrl) and stimulation (red; Stim) traversals. Place-specific stimulation mimicked the activity observed in the place field (adapted from Rickgauer et al., 2014).
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