Sequencing the transcriptomes of more than 100 species of alga yields new channelrhodopsins with promising properties for optogenetics. A far red–shifted channelrhodopsin, Chrimson, opens up new behavioral capabilities in Drosophila, and alongside a fast yet light-sensitive blue channelrhodopsin, Chronos, enables independent excitation of two neuronal populations in brain slices. Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system–mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

Overcoming the limits of the microscope: The resolution of a light microscope is limited. Physicists have long since worked out what these limits are and which parameters determine the spatial resolution. Many groups have nevertheless made numerous attempts to overcome these resolution limits. Rather than improving the power and quality of the microscope, Chen et al. instead expanded the biological specimens under study (see the Perspective by Dodt). They introduced a polymer gel into fixed cells and tissues and chemically induced swelling of the polymer by almost two orders of magnitude. They could then produce much higher-resolution images of their samples, which included the mouse hippocampus. Science , this issue p. 543 ; see also p. 474

The experimental use of microbial opsins — light-sensitive ion channels — has ushered in a revolution in neuroscience, as they make it possible to modulate the activity of genetically targeted neurons in response to exogenous light. Now, Ed Boyden and colleagues have screened archaebacteria, bacteria, plants and fungi for opsins with novel properties and have found a fundamentally new mechanism for neural control: light-driven proton pumping. Although protons are not used natively as charge carriers by neural systems, light-driven proton pumping by archaerhodopsin-3 from Halorubrum sodomense mediates powerful neural silencing in response to light. And a proton pump from the fungus Leptosphaeria maculans enables neural silencing by blue light. The use of these reagents will facilitate the shutdown of neural circuits with light as a tool for studying the role of neural circuits in behaviour and pathology. If the activity of genetically specified neurons is silenced in a temporally precise fashion, the roles of different cell classes in neural processes can be studied. Members of the class of light-driven outward proton pumps are now shown to mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) enables near 100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch)1 from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins2,3 or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue–green light-drivable proton pump from the fungus Leptosphaeria maculans4 (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of ‘optogenetic’ voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.

We report a noninvasive strategy for electrically stimulating neurons at depth. By delivering to the brain multiple electric fields at frequencies too high to recruit neural firing, but which differ by a frequency within the dynamic range of neural firing, we can electrically stimulate neurons throughout a region where interference between the multiple fields results in a prominent electric field envelope modulated at the difference frequency. We validated this temporal interference (TI) concept via modeling and physics experiments, and verified that neurons in the living mouse brain could follow the electric field envelope. We demonstrate the utility of TI stimulation by stimulating neurons in the hippocampus of living mice without recruiting neurons of the overlying cortex. Finally, we show that by altering the currents delivered to a set of immobile electrodes, we can steerably evoke different motor patterns in living mice.

This paper reports the use of light-field microscopy for fast, large-scale imaging of neuronal activity in vivo. It is applied to image the entire animal in the worm and the whole brain in zebrafish. High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of ∼700 μm × 700 μm × 200 μm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.

Improved expansion microscopy method preserves signal from fluorescent proteins and antibodies using off-the-shelf reagents. Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.