Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to human blindness conditions. We show that the cone-specific disruption of DGCR8 in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. However, the number of cones remained unchanged. The loss of the outer segments occurred gradually over 1 month, and during this time the genetic signature of cones decreased. Reexpression of the sensory-cell-specific miR-182 and miR-183 prevented outer segment loss. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro.

SummaryNeuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.Video Abstract

Inferring the direction of image motion is a fundamental component of visual computation and essential for visually guided behavior. In the retina, the direction of image motion is computed in four cardinal directions, but it is not known at which circuit location along the flow of visual information the cardinal direction selectivity first appears. We recorded the concerted activity of the neuronal circuit elements of single direction-selective (DS) retinal ganglion cells at subcellular resolution by combining GCaMP3-functionalized transsynaptic viral tracing and two-photon imaging. While the visually evoked activity of the dendritic segments of the DS cells were direction selective, direction-selective activity was absent in the axon terminals of bipolar cells. Furthermore, the glutamate input to DS cells, recorded using a genetically encoded glutamate sensor, also lacked direction selectivity. Therefore, the first stage in which extraction of a cardinal motion direction occurs is the dendrites of DS cells.