We are pleased to present in this special issue of Experimental Physiology a set of papers which summarize experiences of a number of internationally recognized laboratories working with the technology currently known as ‘optogenetics’. Contrary to popular belief, optogenetics is not all about using light-sensitive ion channels, such as channelrhodopsin, although this particular method has led to a flood of high-profile publications. Strictly speaking, optogenetics means experimentation which involves a combination of genetic manipulation and optics. Therefore, perhaps, its history should date back to the first days when scientists started experimenting with green fluorescent protein or even earlier. Genetic engineering enables targeted expression, in mammalian cells, of various constructs, which can be subdivided into reporters and effectors (or sensors and actuators, following Miesenbock & Kevrekidis, 2005). Unsurprisingly, this approach has been swiftly adopted particularly by neuroscientists. Brain represents a particularly difficult experimental target; there, the numerous cell types are intermingled and interconnected, they are hard to visualize and almost impossible to control selectively other than by using patch clamp on a cell-by-cell basis. Therefore, understanding the complex behaviours of neuronal networks is a formidable task. The recent appreciation of the active role of astroglia has added yet another dimension to it. At the same time, optogenetics deserves a wider application than only neuroscience, and we hope to excite our diverse readership with the possibilities it offers.
Papers within this volume explore applications of both families of optogenetic constructs.
Dreosti & Lagnado (2011) provide a general overview of various approaches for imaging synaptic activity such as probes for detecting vesicular exocytosis, free glutamate and membrane voltage. They make a special emphasis on the fast Ca2+ sensors targeted to presynaptic terminals, an approach recently used by these authors with a great success. Genetic probes for membrane potential monitoring are a specific focus of a paper from T. Knöpfel's group (Mutoh et al. 2011). Of all areas of optogenetic reporters, generation of membrane potential probes is perhaps the most challenging direction, because the only meaningful signal arises from a very fine, small subplasmalemma compartment (thinner than the theoretical resolution of the light microscopy), while any other fluorescence appears as background and decreases the dynamic range. Importantly, the authors also pay special attention to the optics required for successful implementation of this technology. Compared with the indirect indices of neuronal activity, such as Ca2+ probes, voltage sensors are the only currently explored approach that can possibly provide an accurate read-out of neuronal excitation within its full dynamic range. Note that optogenetic probes for many other important cellular signalling molecules have been developed (for example, cAMP), which are not covered in this themed issue.
The next set of papers (Lin, 2011; Mancuso et al. 2011; Schoenenberger et al. 2011) addresses various aspects of application of optogenetic effectors (many of which are based on channelrhodopsin) for studies of neuronal activity in isolation and within networks. Lin (2011) reviews the most popular variants of these probes and provides insider information on their performance. He also brings to the reader's attention some variants which are currently less frequently used but could, in fact, out-perform their more famous rivals in a head-to-head test. Readers might be interested in looking into these alternatives, because the optimal performance of optogenetic effectors is the key to success, especially in more challenging in vivo experiments.
G. J. Augustines's group (Mancuso et al. 2011) provides an overview of available channelrhodopsins and halorhodopsins and points the reader towards the most recent members of the latter family, which are not only more potent than the former family, but are also excitable by far-red light (>650 nm). This issue is very important for in vivo use of optogenetic effectors (actuators), since red light penetrates brain tissue much better than shorter wavelengths (blue and green). Without doubt, using less light is safer in the experiments where repetitive stimulation is required. The authors also highlight the use of the Cl−-sensing probe Clomeleon and its new upgrade, SuperClomeleon, which they expect to publicize shortly.
T. G. Oertner's group (Schoenenberger et al. 2011) focuses on application of channelrhodopsin-like constructs in a very hot topic area of neuroscience – studies of synaptic plasticity – which are often performed in brain slices. One interesting aspect of this review is the discussion of the differences between the ‘natural’ (spontaneous), electrically induced and light-induced action potentials. It should be remembered that Channelrhodopsin-2-induced currents have different kinetics from the native membrane Na+ channels and that Channelrhodopsin-2 is fairly Ca2+ permeable (Nagel et al. 2003). Therefore, the results need to be interpreted with caution, since light-induced action potentials are associated with increased calcium influx and a very high probability of neurotransmitter release.
Optogenetic experimentation on astrocytes is discussed in the paper from our group (Figueiredo et al. 2011). We have successfully used both optogenetic reporters and effectors for studies into the roles of these usually electrically non-excitable cells. While the Ca2+ permeability of channelrhodopsins may be a confounding factor for some studies, such as those mentioned above, it has turned out to be advantageous for astrocyte research, because it enabled us to activate these cells selectively in vitro and in vivo.
Finally, Masseck et al. (2011) review a very interesting family of optogenetic tools which enables control of intracellular signalling by mimicking G-protein-mediated signalling with light-sensitive chimeric G-protein-coupled receptors (GPCRs). Light-sensitive GPCRs allow something that has previously been even more difficult to achieve than electrical control of cellular populations in the brain. It is now possible to ‘switch on’ with light all three main pathways employed by these ubiquitous regulators of cellular metabolism, including the signalling cascades affected by Gαs, Gαq and Gαi subunits. It is difficult to overestimate the potential use of this technology for studies into the role of not only neuronal, but also astrocytic function. That review also highlights another technology, which in some cases may be used as an excellent alternative to optogenetics or in combination with it. Over the recent years, mutant GPCRs termed DREADDS (designer receptors exclusively activated by designer drugs) have been designed (Armbruster et al. 2007; Alexander et al. 2009). The DREADDS are solely activated by a synthetic biologically inert drug and, like the light-controlled GPCRs, exist in three varieties with different intracellular coupling. This allows researchers to activate the desired intracellular signalling pathways in a defined group of cells by systemic application of this drug without the need for implanting optrodes. One can envisage a situation where interplay between different cell types could be studied by controlling one of them by light and the other via DREADDS.
We hope that the readers will find the tips and ideas in the reviews collected in this volume interesting, useful and stimulating. Applicability of optogenetics is by no means limited to studies of the CNS, and we hope to draw the attention of the diverse readership of Experimental Physiology to this exciting new technology. As the field of optogenetics matures, the arsenal of available tools expands and so does the resolution and throughput of our experiments.