Widefield imaging of rapid pan-cortical voltage dynamics with an indicator evolved for one-photon microscopy.

Widefield imaging with genetically encoded voltage indicators (GEVIs) is a promising approach for understanding the role of large cortical networks in the neural coding of behavior. However, the limited performance of current GEVIs restricts their deployment for single-trial imaging of rapid neuronal voltage dynamics. Here, we developed a high-throughput platform to screen for GEVIs that combine fast kinetics with high brightness, sensitivity, and photostability under widefield one-photon illumination. Rounds of directed evolution produced JEDI-1P, a green-emitting fluorescent indicator with enhanced performance across all metrics. Next, we optimized a neonatal intracerebroventricular delivery method to achieve cost-effective and wide-spread JEDI-1P expression in mice. We also developed an approach to correct optical measurements from hemodynamic and motion artifacts effectively. Finally, we achieved stable brain-wide voltage imaging and successfully tracked gamma-frequency whisker and visual stimulations in awake mice in single trials, opening the door to investigating the role of high-frequency signals in brain computations.

Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition.

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of 23 makes H. catenoides KCR1 (HcKCR1) a powerful hyperpolarizing tool to suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. HcKCR1 enables optogenetic control of K+ gradients, which is promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinson's disease and long-QT syndrome and other cardiac arrhythmias.

Synaptojanin cooperates in vivo with endophilin through an unexpected mechanism.

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin-Amphiphysin-Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.