Nathan Cao Klapoetke

A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex.

Technologies for silencing the electrical activity of genetically targeted neurons in the brain are important for assessing the contribution of specific cell types and pathways toward behaviors and pathologies. Recently we found that archaerhodopsin-3 from Halorubrum sodomense (Arch), a light-driven outward proton pump, when genetically expressed in neurons, enables them to be powerfully, transiently, and repeatedly silenced in response to pulses of light. Because of the impressive characteristics of Arch, we explored the optogenetic utility of opsins with high sequence homology to Arch, from archaea of the Halorubrum genus. We found that the archaerhodopsin from Halorubrum strain TP009, which we named ArchT, could mediate photocurrents of similar maximum amplitude to those of Arch (∼900 pA in vitro), but with a >3-fold improvement in light sensitivity over Arch, most notably in the optogenetic range of 1–10 mW/mm2, equating to >2× increase in brain tissue volume addressed by a typical single optical fiber. Upon expression in mouse or rhesus macaque cortical neurons, ArchT expressed well on neuronal membranes, including excellent trafficking for long distances down neuronal axons. The high light sensitivity prompted us to explore ArchT use in the cortex of the rhesus macaque. Optical perturbation of ArchT-expressing neurons in the brain of an awake rhesus macaque resulted in a rapid and complete (∼100%) silencing of most recorded cells, with suppressed cells achieving a median firing rate of 0 spikes/s upon illumination. A small population of neurons showed increased firing rates at long latencies following the onset of light stimulation, suggesting the existence of a mechanism of network-level neural activity balancing. The powerful net suppression of activity suggests that ArchT silencing technology might be of great use not only in the causal analysis of neural circuits, but may have therapeutic applications.

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance

UNLABELLED An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity. VIDEO ABSTRACT

Sub-millisecond optogenetic control of neuronal firing with two-photon holographic photoactivation of Chronos

Optogenetic neuronal network manipulation promises to unravel a long-standing mystery in neuroscience: how does microcircuit activity relate causally to behavioral and pathological states? The challenge to evoke spikes with high spatial and temporal complexity necessitates further joint development of light-delivery approaches and custom opsins. Two-photon (2P) light-targeting strategies demonstrated in-depth generation of action potentials in photosensitive neurons both in vitro and in vivo, but thus far lack the temporal precision necessary to induce precisely timed spiking events. Here, we show that efficient current integration enabled by 2P holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice. These results pave the way for optogenetic manipulation with the spatial and temporal sophistication necessary to mimic natural microcircuit activity. SIGNIFICANCE STATEMENT To reveal causal links between neuronal activity and behavior, it is necessary to develop experimental strategies to induce spatially and temporally sophisticated perturbation of network microcircuits. Two-photon computer generated holography (2P-CGH) recently demonstrated 3D optogenetic control of selected pools of neurons with single-cell accuracy in depth in the brain. Here, we show that exciting the fast opsin Chronos with amplified laser 2P-CGH enables cellular-resolution targeting with unprecedented temporal control, driving spiking up to 100 Hz with submillisecond onset precision using low laser power densities. This system achieves a unique combination of spatial flexibility and temporal precision needed to pattern optogenetically inputs that mimic natural neuronal network activity patterns.

Hearing the light: neural and perceptual encoding of optogenetic stimulation in the central auditory pathway

Optogenetics provides a means to dissect the organization and function of neural circuits. Optogenetics also offers the translational promise of restoring sensation, enabling movement or supplanting abnormal activity patterns in pathological brain circuits. However, the inherent sluggishness of evoked photocurrents in conventional channelrhodopsins has hampered the development of optoprostheses that adequately mimic the rate and timing of natural spike patterning. Here, we explore the feasibility and limitations of a central auditory optoprosthesis by photoactivating mouse auditory midbrain neurons that either express channelrhodopsin-2 (ChR2) or Chronos, a channelrhodopsin with ultra-fast channel kinetics. Chronos-mediated spike fidelity surpassed ChR2 and natural acoustic stimulation to support a superior code for the detection and discrimination of rapid pulse trains. Interestingly, this midbrain coding advantage did not translate to a perceptual advantage, as behavioral detection of midbrain activation was equivalent with both opsins. Auditory cortex recordings revealed that the precisely synchronized midbrain responses had been converted to a simplified rate code that was indistinguishable between opsins and less robust overall than acoustic stimulation. These findings demonstrate the temporal coding benefits that can be realized with next-generation channelrhodopsins, but also highlight the challenge of inducing variegated patterns of forebrain spiking activity that support adaptive perception and behavior.