Written by Hilary Bayer & Ali Cetin
Cell type classification is a fundamental challenge in biological sciences. To determine the identity and understand the biological significance of a cell we necessarily consider morphology, physiology, and genetic and proteomic profiles.
The mammalian brain comprises vast numbers of intricately interconnected neurons with diverse molecular, anatomical, and physiological identities. We wanted to understand how the fine-scale connectional architecture of the neocortex was related to brain function and resulting observable behavior. We needed tools to analyze the roles played by individual cells of diverse types.
Site-specific DNA recombinases (SSRs) have the ability to precisely modify DNA within cells by binding to specific short segments of DNA and either inverting or excising the intervening sequences. Because of this, they have proven extremely useful in exploring functional and genetic components of the nervous system in numerous model organisms. Researchers have previously reported several light-inducible SSRs, paving the way for precision single-cell targeting. We carried this work forward significantly by regulating SSRs with spatiotemporal accuracy sufficient to target individual neurons within intact neural circuitry.
To enable accurate single-cell targeting, we modified three SSRs, Cre, Dre and Flp, by combining them with a fungal-based light-inducible protein, Vivid, so that light triggered activity of the enzymes (Fig. 1a, b). In our initial experiments we demonstrated light regulation of recombination induced by these enzymes, RecVs. We named this method optogenomics: genome modification using light.
Encouraged by our successes, we used RecVs to generate rAAV viral vectors with which we infected genetically modified mouse lines. With this technique we further restricted the light-induced recombination to achieve neuronal subtype specificity (Fig. 1c).
Figure 1. Design and application of the RecVs. a, Schematic of the RecV system. b, Alignment of amino-acid sequences of Cre, Dre and Flp recombinases with split sites noted by arrows. c, Schematic of the RecV rAAV construct. Somatostatin FlpO mouse line, Sst-IRES-FlpO, crossed with a Cre/Flp double-dependent tdTomato reporter mouse line, Ai65, was retro-orbitally injected with AAV-PHP.eB EF1a-iCreV and light was delivered to the left hemisphere. We observed recombination in somatostatin-positive inhibitory interneurons at the hemisphere that received light induction as revealed by immunohistochemistry (100% of reporter positive cells (119) were Sst positive (313); 38.1% of Sst cells were reporter positive).
While experimenting with enhanced blood-brain-barrier-permeable PHP.eB capsids1, we serendipitously discovered that intracerebroventricular delivery of these viruses generates brain-wide genetic alterations like those we obtained consistently with retro-orbital injection.
We wondered whether we might employ these enzymes to achieve targeted optogenomic manipulations of single cells. Using 2-photon excitation, we selected individual cells and induced expression of fluorescent reporters. We showed that functional imaging can be performed without interfering with optogenomics. Sparse yet strong labeling of neurons allowed us to reconstruct their individual axonal and dendritic details throughout the entire mouse brain.
Our method might be used in combination with other technologies to generate an organized classification of neurons. Researchers may also use it to categorize neurons that are active during different phases of behavior. Monosynaptic rabies tracing is an effective method for dissecting circuits one synapse at a time2. We foresee using 2-photon excitation combined with optogenomics in single-cell rabies tracing experiments3 to functionally profile behaviorally relevant neurons—as well as neurons immediately presynaptic to them—in any part of the brain. We anticipate that such research may yield important insights into brain structure and function.
Optogenomics can be used in diverse model organisms during developmental or adult stages to study systems at a single-cell level. Our demonstration of the intracerebroventricular route for whole-brain infections may be applicable to modification of embryos, to promoting infection of the nervous system, and to overcoming obstacles related to intravenous delivery. It may also help avoid immune-response interference with therapeutic or research-related viral delivery, including gene therapy.
We anticipate that optogenomics may provide a foundation for future detailed analysis of neuron type-specific circuits to establish links among genetic identity, morphology, connectivity, and function. Readers will find a more thorough exposition of this work at: https://www.nature.com/article....
1 Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol 34, 204-209, doi:10.1038/nbt.3440 (2016).
2 Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M. & Callaway, E. M. Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus. Proc Natl Acad Sci U S A 107, 21848-21853, doi:10.1073/pnas.1011756107 (2010).
3 Marshel, J. H., Mori, T., Nielsen, K. J. & Callaway, E. M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562-574, doi:10.1016/j.neuron.2010.08.001 (2010)
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