The optogenetic (r)evolution

Abstract

Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By heterologous expression of light-sensitive microbial membrane proteins, opsins, cell type-specific depolarization or silencing can be optically induced on a millisecond time scale. What started in a petri dish is applicable today to more complex systems, ranging from the dissection of brain circuitries in vitro to behavioral analyses in freely moving animals. Persistent technical improvement has focused on the identification of new opsins, suitable for optogenetic purposes and genetic engineering of existing ones. Optical stimulation can be combined with various readouts defined by the desired resolution of the experimental setup. Although recent developments in optogenetics have largely focused on neuroscience it has lately been extended to other targets, including stem cell research and regenerative medicine. Further development of optogenetic approaches will not only highly increase our insight into health and disease states but might also pave the way for a future use in therapeutic applications.

Fig. 1

The optogenetic principle: changing the membrane voltage potential of excitable cells. a Activating tools—channelrhodopsins: channelrhodopsin-2 from Chlamydomoas reinhardtii (ChR2) and channelrhodopsin-1 Volvox carteri (VChR1) from nonselective cation channels leading to depolarization of target cells. Silencing tools—ion pumps: archaerhodopsin-3 (Arch) from Halorubrum sodomense works as a proton pump and leads to hyperpolarization of the target cell such as the chloride pump NpHR (NpHR) from Natronomonas pharaonis. b Spectral working properties of light-sensitive membrane proteins

Fig. 2

Targeted delivery of opsins to the mouse brain based on adeno-associated viruses (AAV) or transgenic approaches. a Opsins can be expressed in specific types of neurons or glia using gene-specific promoter elements (e.g., CamKIIα). AAV-based expression vectors integrating such promoter elements are delivered via stereotactic injection into desired brain areas. b Stereotactic injection of AAV vectors, which combine a strong ubiquitous promoter (e.g., Ef1α or CAG) either with a “STOP” cassette (top) or with an AAV-FlEx/AAV-DIO system (bottom), into specific cre mouse lines will result in a strong opsin expression selectively in neurons expressing the cre recombinase. c Classical transgenesis via pronucleus injection using a gene-specific promoter (top) or targeted knock-in strategies in embryonic stem cells (bottom) will result in a neuron- or glia-specific opsin expression in transgenic mice. d Alternatively, the combination of a ubiquitous promoter with a “STOP” cassette (top) or with an AAV-FlEx/AAV-DIO system (bottom) can be applied also in a transgenic approach either in a random or targeted fashion, e.g., to the ubiquitously expressed ROSA26 locus. Only after breeding to desired cre driver lines a brain region- or cell type-specific expression of opsins will be activated. Mice expressing opsins can be recognized by the highlighted brain and an inset in the background showing hippocampal neurons expressing opsin-GFP fusion protein. AAV adeno-associated virus, BGHpA bovine growth hormone poly A signal, GOI gene of interest, GSP gene-specific promoter, IRES internal ribosomal entry side, ITR inverted terminal repeat, STOP transcriptional terminator, UBP: ubiquitous promoter, WPRE woodchuck hepatitis virus posttranscriptional regulatory element