Remote controlling the brain and functional optical imaging


So far, stimulation of cells, especially, of neurons, has only been possible by applying chemical agents (ions, ligands, metabolites) or by electricity. Electrical stimulation is either limited to one or a few cells by single cell clamp-type methods, or by generalized stimulation of larger areas in nervous tissue. Chemical stimulation (which includes inhibition), usually acts on many cells with limited specificity. All cells that have the appropriate reception mechanism will respond. Chemical stimulation is notoriously slow in response, due to diffusion barriers. The method of optogenetics [1] allows more specific targeting of the intended stimulation and is speedy enough to control action potentials in neurons.

Opto: Light-activated proteins affecting the membrane potential (opto-proteins)

The first protein to be described that connects light and membrane potential in a direct, i.e. single-component way is bacteriorhodopsin, found in Halobacterium halobium [2]. Bacteriorhodopsin is a proton pump that moves protons against the electrochemical gradient out of the bacterial plasma upon illumination by blue light. It has been possible to reconstitute this protein into artificial lipid vesicles together with mitochondrial ATP-ase (ATP-synthase). The system performed phosphorylation upon illumination, indicating that bacteriorhodopsin provides energy for the ATPsynthase, which is driven by a proton gradient. As the proton is charged, the protein is a device that has a direct influence on the membrane potential upon illumination with visible light.

A similar protein, that does not pump protons, but chloride ions, was found in other Archae, e.g. Halobacterium salinarum and Natronomonas pharaonis. Chloride is pumped into the bacterium upon illumination of this protein halorhodopsin by green-yellow light. The physiological effect of both types of "opto-pumps" is hyperpolarization, i.e. an increase of the negative potential inside the cell.

A different way to influence the membrane potential by illumination is used by a series of green algae. The light- sensitive proteins found in Chlamydomonas rheinhardii show an increased conductance upon illumination by blue light. These algae use channelrhodopsin as a light sensor for phototaxis– they avoid intense illumination. It turned out that different proteins are involved. Channelrhodopsin1 was identified as a light-activated proton channel, whereas Channelrhodopsin2 is a light-activated cation channel that will allow mono- and bivalent cations to pass the membrane following the gradient upon illumination. These "opto-channels" consequently decrease the transmembrane potential, i.e. depolarize the cell [3].  Meanwhile, other opto-channels have been identified, e.g. from the Volvox algae. Furthermore, so called "designer channelrhodopsins" are engineered that can be switched on and off by different colors of light, or in general have different spectral properties.

A consequent continuation of the theme "opto-proteins" was the design and development of light-sensitive receptors, which do not have any influence on the membrane potential, but mimic the presence of a ligand when illuminated. Such chimeric G-protein coupled receptors, fused with rhodopsin fragments, can trigger cell signaling pathways upon illumination [4]. The light-activated G-protein receptors are referred to as "optoXRs".

Fig. 1: Variations of light-activated proteins that are used to control cellular behavior. Affecting the classical membrane potential, channelrhodopsins increase membrane conductance for cations. Bacteriorhodopsins and halorhodopsins are ion pumps for protons or chloride ions against the electrical membrane potential. These proteins open or pump upon illumination with the appropriate color of light.

Genetics: Introduction of Opto-Proteins into Target Cells

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 engineerin g [5]: 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.

Activation and Response of Optogenetically Modified Cells

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 model [6]. 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 cells [7]. From here to control of a living mouse’s behavior by an implanted light fiber was a short step [8].

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.

Applications of Optogenetics in Combination with Microscopy

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 peace [10], as we can repair the mental damage technically?

Personal Growth Series – Karl Deisseroth on Cracking the Neural Code

YouTube video by GoogleTechTalks, November 25, 2008
Speaker: Professor Karl Deisseroth

Youtube Video: Karl Deisseroth on Cracking the Neural Code

The technological seeds of a Manhattan project-style scientific enterprise, the optical reverse-engineering of brain circuits to crack the neural code, have recently been planted at Stanford.

The brain is a high-speed dynamical system consisting of different players that are intertwined and that cannot be separately controlled using conventional methods. For this reason, until recently we have not been able to speak the language of the brain (with millisecond timescale and cell-specific resolution), and in 1979 Francis Crick called for a technology by which all neurons of just one type could be controlled, "leaving the others more or less unaltered".

Tools from the Deisseroth laboratory at Stanford over the past four years have responded to this challenge. These include optical technologies for controlling neural circuits, using precisely-targeted delivery of light energy of different colors that is captured by neurons using nanoscale protein-based antennae, resulting in controlled activity of just the targeted cell types with millisecond precision. Light is delivered by fiberoptics; while light encounters all cell types, only the desired cell type is light-sensitive and responds. Using different optogenetic probes, cells can be turned on or off with millisecond precision and in different combinations.

These tools have now been used to optically deconstruct Parkinsonian neural circuitry, setting the stage both for cracking the neural codes of normal brain function, and for re-engineering neural circuits in disease.

BioTechniques Podcast: Optogenetics - Shining a Light on Neuroscience

Listen to the podcast

Researchers Ed Boyden from MIT and Jean Bennett from the University of Pennsylvania discuss the development of optogenetics and recent clinical applications, especially for the treatment of retinal disease.

Podcast Date: October 26, 2012

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