Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.

The extraction of the direction of motion is an important computation performed by many sensory systems and in particular, the mechanism by which direction-selective retinal ganglion cells (DS-RGCs) in the retina acquire their selective properties, has been studied extensively. However, whether DS-RGCs simply relay this information to downstream areas or whether additional and potentially de novo processing occurs in these recipient structures is a matter of great interest. Neurons in the larval zebrafish tectum, the largest retino-recipent area in this animal, show direction-selective (DS) responses to moving visual stimuli but how these properties are acquired is still unknown. In order to study this, we first used two-photon calcium imaging to classify the population responses of tectal cells to bars moving at different speeds and in different directions. Subsequently, we performed in vivo whole cell electrophysiology on these DS tectal neurons and we found that their inhibitory inputs were strongly biased toward the null direction of motion, whereas the excitatory inputs showed little selectivity. In addition, we found that excitatory currents evoked by a stimulus moving in the preferred direction occurred before the inhibitory currents whereas a stimulus moving in the null direction evoked currents in the reverse temporal order. The membrane potential modulations resulting from these currents were enhanced by the spike generation mechanism to generate amplified direction selectivity in the spike output. Thus, our results implicate a local inhibitory circuit in generating direction selectivity in tectal neurons.

Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca 2ϩ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices.

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Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca 2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area maps, spanning cortex and striatum, of the effects of antiepileptic drugs on neural excitability and on the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices. ResultsA spectrally orthogonal Ca 2+ sensor and channelrhodopsin for 1-photon AON AON requires a spectrally orthogonal optogenetic actuator and activity reporter ( Fig. 1a). Examination of channelrhodopsin action spectra and Ca 2+ reporter excitation spectra suggested that the best approach for 1-photon AON was to use a blue-shifted channelrhodopsin and a red-shifted genetically encoded Ca 2+ indicator (RGECI). We thus set out to identify protein pairs suitable for this purpose.We began by comparing the single action potential responses of RGECIs in cultured neurons. jRGECO1a was the most sensitive (∆F/F = 54 ± 10%, n = ~120 neurons), followed by R-CaMP2 and jRCaMP1a, consistent with previous reports (Supplementary Fig. 1a-b, Supplementary Table 1) 15 . R-CaMP2 had the fastest kinetics (τon = 26 ± 10 ms, τoff = 270 ± 20 ms, n = ~120 neurons), followed by jRGECO1a (τon = 47 ± 1 ms, τoff = 440 ± 40 ms, n = ~120 neurons) and jRCAMP1a ( Supplementary Fig. 1ab, Supplementary Table 1). In HEK293T cells, under basal Ca 2+ conditions, jRGECO1a had the longest photobleaching time constant (τbleach = 81 ± 5 s, I561 = 44 W/cm 2 , n = 9 cells), followed by R-CaMP2 and jRCaMP1a ( Supplementary Table 1).Under typical imaging conditions (I561 = 0.1 W/cm 2 ), photobleaching of jRGECO1a was thus < 10% during 1 hr of continuous imaging. While photobleaching is often a concern for 1P imaging, these results established that this effect was minor for wide-area imaging of jRGECO1a. We selected jRGECO1a for its superior sensitivity and photostability.mApple-based fluorescent sensors, including jRGECO1a, are known to undergo photoswitching under blue light illumination 14,16 . We thus sought a blue-shifted channelrhodopsin that could drive spikes in jRGECO1a-expressing neurons at blue intensities low enough to avoid optical crosstalk. TsChR is the most blue-shifted published ChR (Fig. 1a), but was initially reported to produce only ~40% as much ph...

In the version of this Brief Communication originally published online, ref. 21 included details for a conference paper (Pegard, N. C. et al. Paper presented at Novel Techniques in Microscopy: Optics in the Life Sciences, Vancouver, BC, Canada, 12-15 April 2015). The correct reference is the following: Pégard, N. C. et al. Optica 3, 517-524 (2016). This error has been corrected in the print, HTML and PDF versions of the paper.

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