Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Neuronal Dynamics Regulating Brain and Behavioral State Transitions.

Prolonged behavioral challenges can cause animals to switch from active to passive coping strategies to manage effort-expenditure during stress; such normally adaptive behavioral state transitions can become maladaptive in psychiatric disorders such as depression. The underlying neuronal dynamics and brainwide interactions important for passive coping have remained unclear. Here, we develop a paradigm to study these behavioral state transitions at cellular-resolution across the entire vertebrate brain. Using brainwide imaging in zebrafish, we observed that the transition to passive coping is manifested by progressive activation of neurons in the ventral (lateral) habenula. Activation of these ventral-habenula neurons suppressed downstream neurons in the serotonergic raphe nucleus and caused behavioral passivity, whereas inhibition of these neurons prevented passivity. Data-driven recurrent neural network modeling pointed to altered intra-habenula interactions as a contributory mechanism. These results demonstrate ongoing encoding of experience features in the habenula, which guides recruitment of downstream networks and imposes a passive coping behavioral strategy.

Assessment of Murine Retinal Acuity Ex Vivo Using Multielectrode Array Recordings.

Purpose: Visual acuity, measured by resolution of optotypes on a standard eye chart, is a critical clinical test for function of the visual system in humans. Behavioral tests in animals can be used to estimate visual acuity. However, such tests may be limited in the study of mutants or after synthetic vision restoration techniques. Because the total response of the retina to a visual scene is encoded in spiking patterns of retinal ganglion cells, it should be possible to estimate visual acuity in vitro from the retina by analyzing retinal ganglion cell output in response to test stimuli. Methods: We created a method, EyeCandy, that combines a visual stimulus-generating engine with analysis of multielectrode array retinal recordings via a machine learning approach to measure murine retinal acuity in vitro. Visual stimuli included static checkerboards, drifting gratings, and letter optotypes. Results: In retinas from wild-type C57Bl/6 mice, retinal acuity measurement for a drifting grating was 0.4 cycles per degree. In contrast, retinas from adult rd1 mice with outer retinal degeneration showed no detectable acuity. A comparison of acuities among different regions of the retina revealed substantial variation, with the inferior-nasal quadrant having highest RA. Letter classification accuracy of a projected Early Treatment Diabetic Retinopathy eye chart reached 99% accuracy for logMAR 3.0 letters. EyeCandy measured a restored RA of 0.05 and 0.08 cycles per degree for static and dynamic stimuli respectively from the retina of the rd1 mouse treated with the azobenzene photoswitch BENAQ. Conclusions: Machine learning may be used to estimate retinal acuity. Translational Relevance: The use of ex vivo retinal acuity measurement may allow determination of effects of mutations, drugs, injury, or other manipulations on retinal visual function.

Photopharmacologic Vision Restoration Reduces Pathological Rhythmic Field Potentials in Blind Mouse Retina.

Photopharmacology has yielded compounds that have potential to restore impaired visual responses resulting from outer retinal degeneration diseases such as retinitis pigmentosa. Here we evaluate two photoswitchable azobenzene ion channel blockers, DAQ and DAA for vision restoration. DAQ exerts its effect primarily on RGCs, whereas DAA induces light-dependent spiking primarily through amacrine cell activation. Degeneration-induced local field potentials remain a major challenge common to all vision restoration approaches. These 5-10 Hz rhythmic potentials increase the background firing rate of retinal ganglion cells (RGCs) and overlay the stimulated response, thereby reducing signal-to-noise ratio. Along with the bipolar cell-selective photoswitch DAD and second-generation RGC-targeting photoswitch PhENAQ, we investigated the effects of DAA and DAQ on rhythmic local field potentials (LFPs) occurring in the degenerating retina. We found that photoswitches targeting neurons upstream of RGCs, DAA (amacrine cells) and DAD (bipolar cells) suppress the frequency of LFPs, while DAQ and PhENAQ (RGCs) had negligible effects on frequency or spectral power of LFPs. Taken together, these results demonstrate remarkable diversity of cell-type specificity of photoswitchable channel blockers in the retina and suggest that specific compounds may counter rhythmic LFPs to produce superior signal-to-noise characteristics in vision restoration.