Optogenetics – Remote Controlling the Brain and Functional Optical Imaging

Optogenetics is obviously concerned with light, more precisely the activation of cellular functions upon illumination. Bacterial rhodopsins and channelrhodopsins are proteins that are naturally found in single cell organisms. In order to study the properties and function of these proteins, they were expressed in mammalian cells. To introduce the opto-proteins into the desired cells, genetic engineering is required.

This is possible by meanwhile standard techniques in genetic engineering⁵: the coding DNA for the protein is introduced in the desired cell type by extracting the DNA sequence with appropriate restriction endonucleases. This package of information is then introduced into the target cell by a variety of methods, e.g. retrovirus-mediated transformation, electroporation, precipitation methods, micro-injection or “biolistic” transformation by “gene guns” that use DNA-coated gold particles.

To achieve selectivity for specific cells, the gene for the opto-protein must be cotransfected with an appropriate promoter. Differentiated cells of the type under investigation usually express a specific pattern of proteins. The expression of proteins is controlled by promoters that are switched on (or off) during differentiation and control the cell type-specific spectrum of proteins. In order to activate the opto-protein only in the desired cell type, the promoter for the opto-protein has to be chosen from the specific promoter spectrum that controls the cell type-specific protein expression. The gene for the opto-protein is then only translated in cells which have the chosen promoter activated. These are specifically only those cells that show the desired differentiation (cell type). All other cells do not promote the translation of that gene and consequently will not respond to illumination.

The technique of optogenetics was named after the genetic engineering involved. It is not, as one might first suppose, a method of altering or manipulating genetic information by the application of light.

As mentioned above, the effect of illumination of a membrane containing opto-proteins is depolarization or hyperpolarization – depending on whether the protein increases the conductance (depolarization) or pumps ions against the already built-up electrical gradient (hyperpolarization). All known cells and cell compartments show an electrical potential under physiological conditions. There is, however, a very special type of cell which also uses the membrane potential in a dynamic way to transport information: the neuron. (Action potentials are also known from other excitable cells, e.g. muscle cells, endocrine cells, some plant cells and some single celled organisms).

An action potential in neurons is initiated by a depolarization of the membrane potential above a threshold. As soon as the threshold is passed, potassium channels will open and amplify the depolarization. The complex sequence of dynamic variations of membrane conductance as a function of the membrane potential is described by the Hodgkin-Huxley model⁶. Usually, the initial depolarization is a consequence of synaptic activity in the dendrites of the neuron, causing excitatory postsynaptic potentials. The opto-channel has exactly the same consequence. And indeed, it is possible to evoke action potentials in cells transfected with channelrhodopsin, just by flashing blue light onto the cells⁷. From here to control of a living mouse’s behavior by an implanted light fiber was a short step⁸.

As the activation of halorhodopsin hyperpolarizes the membrane potential, this protein will suppress the onset of an action potential under otherwise sufficient conditions, very much like inhibitory postsynaptic potentials evoked by inhibitory synapses. In a cell that contains both the channelrhodopsin and the halorhodopsin, the generation of action potentials by blue light pulses can be suppressed by simultaneous illumination  with yellow light.

These experimental concepts allow a huge variety of new experiments for studying and understanding the mechanisms of excitable cells and cell signaling pathways. They also open a window for more specific and exact diagnostics and are targeted to cure many diseases that are related to malfunction of neurons or other excitable cells. One striking example is the cure of blindness caused by deficiencies in the retina.

An immediate application for optogenetics in combination with microscopy is research on layers of cultivated excitable cells. Monitoring the membrane potential of the activated cells, or cells that have direct or indirect contact with the activated cells, reveals insights into the general biology of action potential generation, axonal signal transmission and synaptic activities. Connectivity of neurons and processing of signals in neuronal circuitries is another target for such experiments. Monitoring of the responses is possible with electrical electrodes, usually patch-clamp techniques, or fluorescent sensors – increasingly fluorescent biosensors based on FRET concepts.

To understand wiring and signal processing in real circuits, brain slices or tissue slices in general are frequently used. By introducing optogenetically excitable cells, it becomes possible to study these circuits in the natural context. Shining light onto a region of a brain slice will allow the response to be recorded in other regions, even if they are anatomically remote. Also, a mechanical response, e.g. muscle contraction, is a parameter that can be used to measure the effect of light- induced activation of various target cells. The possibility to transfect only very specifically dedicated cell types sets optogenetic techniques apart from traditional experiments with unspecific electrical or chemical stimulation.

The final step in applying optogenetic techniques to investigations by microscopy is of course the use of living whole animals and studying their response to light-induced alterations of membrane potential or activation of cell signaling pathways. Small animals, like fruit flies, nematodes or maggots, might be illuminated as a whole to trigger the optogenetic responses. The central nervous systems of mammals, very often rat or mouse brain, are studied through cranial windows in living animals. Here, two-photon microscopy is the method of choice, as one is interested in deep imaging – and chemical or physical clearing is not possible in vital specimens. In continuation of the intrinsic optical sectioning feature of two-photon imaging, two-photon illumination is the tool to very specifically activate only single cells in a complex 3D arrangement of neurons like the brain. Also, fast scanning concepts in combination with two-photon activation enable fast alterations in the stimulation of various cells to shed light on their wiring.

At the moment, ideas, concepts, suggestions and results that mention optogenetics are exploding and moving biological sciences forward at an unprecedentedly dizzy speed. Try to keep track!⁹

On the other hand, the method also has its critics. Will humans be remote-controlled in future? Is the experience of joy something that might be induced artificially in order to keep human beings efficient (figure in the introductory paragraph)? And does this mean that there’s no need to treat our fellow men with respect in order to live together in peace¹⁰, as we can repair the mental damage technically?