Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

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

Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.Despite the central importance for both basic and translational research, most current technologies available for cell-type-targeting rely on transgenic animals, which limits their applicability. Either the genetic tool that senses or modulates brain function, or the enzyme, such as Cre recombinase, that allows the genetic tool to be conditionally expressed, is expressed from the animal's genome. The inclusion of a transgenic component in the cell-type-targeting strategy excludes its use in therapy for humans, limits its range of application in pre-clinical, non-human primate research, and complicates its use in model organisms such as mice. The development of transgenic non-human primates and mice is costly and slow, especially since cell-type targeting is often applied in the context of other genetic manipulations, such as double or triple gene knockouts, or when targeting different cell types with different tools.Viral vectors for cell-type-targeting may overcome such limitations. AAVs are the most frequently used vectors in both basic research and gene therapy, as they are safe for use in all tested species, including humans and non-human primates, and their production is simple, cheap, and fast (Planul and Dalkara, 2017). They have three important components: the capsid for cell entry, the promoter that drives transgene expression, and the gene of interest to be expressed in the transduced cells, and they drive expression episomally (Duan et al., 1998; Penaud-Budloo et al., 2008). Futhermore, many genetic tools are small enough to fit into AAVs, different AAVs can be injected together, and synthetic AAV capsids allow brain-wide delivery (Deverman et al., 2016).Cell-type-targeting by AAVs could be achieved by engineering the capsid and/or by using specific promoters. Capsid protein mutations can be used to tune the efficacy of