Optogenetics is a newly developed technique based on a group of light-sensitive proteins called channelrhodopsins, which were isolated recently from various species of micro-organism. Although relatively new, this technique has already proven to be extremely powerful, because channelrhodopsins can be targeted to specific cells, so that their activity can be controlled by light, on a millisecond-by-millisecond timescale.
A group of researchers from Stanford University now report a new addition to the optogenetic toolkit, and demonstrate that it can be used to precisely control biochemical signalling pathways in the mouse brain and to manipulate complex reward-related behaviour. They have also used the existing channelrhodopsins to probe the neural circuitry implicated in Parkinson’s Disease and thus gain a better understanding of why deep brain stimulation is effective in treating the disease.
The new work comes from Karl Deisseroth‘s lab, which has been instrumental in developing channelrhodopsins as tools for studying the nervous system. In the first of two papers, which appeared online last week in the journal Nature, Deisseroth and his colleagues describe how they manipulated the learning behaviour of mice using chimeric α1- and β2-adrenoceptors. These receptors are coupled, on the inner surface of the cell membrane, to small enzyme molecules called G-proteins; when a catecholamine neurotransmitter (adrenaline, noradrenaline or dopamine) binds to the receptor, the G-protein is released, and sets off to initiate multiple complex biochemical signalling pathways inside the cell.
The researchers replaced the neurotransmitter binding site with the light-sensitive part of rhodopsin, the vertebrate homologue of the microbial proteins, then injected the chimeric receptors directly into the nucleus accumbens of mice, which lies underneath the cerebral cortex and is commonly referred to as the “reward centre” or “pleasure centre”. The animals were then made to perform a conditioned place preference test. This normally involves placing the animals into a chamber, and giving them a reward in a specific location. After repeated pairings of the location and the reward, the animals learn an association between the two, and subsequently spend more time in that location than in others.
In this case though, the researchers did not reward the mice with food pellets or a sugar solution. Instead, they used pulses of light, delivered to the nucleus accumbens via an optical fibre (below). Thus, whenever the mice moved into the designated location, the light activated the chimeric adrenergic receptors, and switched on the biochemical signalling pathways which would normally be initiated when the animals are given a real reward. Remarkably, the optical stimulation was sufficient to replace a real reward, such as pellets of food or a sugar solution, and as a result, the mice spent more time the following day in the location at which the stimulation had been delivered than in other parts of the chamber.
The second paper describes a set of experiments designed specifically to address the question of how deep brain stimulation (DBS) exerts its therapeutic effect on patients with Parkinson’s Disease, the movement disorder associated with degeneration of dopamine-producing neurons in the midbrain. DBS is an experimental technique involving the implantation of stimulating electrodes into the brain. In Parkinsonian patients, the subthalamic nucleus is targeted. However, the circuitry in this region of the brain is complex, and neurons within it can respond to stimulation by either increasing their activity, decreasing it, or, over prolonged periods of time, both. Exactly why DBS is so effective in alleviating the symptoms of some Parkinsons’ patients therefore remains unclear.
This set of experiments, described in the current issue of Science, was carried out in mice lacking dopaminergic neurons in the right half of the midbrain. The cells were destroyed with 6-hydroxydopamine, a neurotoxin which enters neurons via the dopamine and noradrenaline reuptake transporters, and therefore selectively destroys cells which synthesize these neurotransmitters. The mice treated in this way exhibited deficits in the limbs on the left side of their body, which are controlled by the right side of the brain, and as a result display rightward rotations when they move.
Deisseroth’s group expressed channelrhodopsins in various cell types in and around the subthalamic nucleus, to investigate the effect of stimulating each of them in turn. First, one type of channelrhodopsin, which inhibits nerve cell activity when activated, was targeted to excitatory neurons in the subthalamic nucleus. These cells are thought to become overactive in Parkinson’s, and it is widely believed that DBS may effectively ameliorate the symptoms of Parkinson’s by acting on them. However, supressing their activity with light of the appropriate wavelength had no effect on the behaviour of the animals.
A second possibility is that DBS works by stimulating bundles of nerve fibres which enter the ubthalamic nucleus. To test this hypothesis, the researchers introduced another channelrhodopsin, which increases neuronal activity when activated by a different wavelength of light, into neurons in layer 5 of the primary motor cortex. These cells project down into the spinal cord, but their axons also send branches into the subthalamic nucleus. High frequency pulses of light delivered to these cells rescued the movement deficits of the mice, so that their behaviour was indistinguishable from that of normal mice. By contrast, low frequency stimulation of the same cells was found to worsen the animals’ symptoms.
These studies contribute significantly to optogenetic technology and to our understanding of why DBS is an effective treatment for Parkinson’s. The first paper demonstrates that optogenetics can be used to activate specific signalling cascades in neurons, and therefore provides a powerful new method for further dissection of the numerous biochemical pathways which modulate neuronal activity. The findings reported in the second paper raise questions about the hypothesis that DBS is effective in alleviating Parkinsonian symptoms because it exerts an effect on excitatory neurons in the subthalamic nucleus, and instead implicate the primary motor cortex as a primary target. They also suggest that DBS is at least partly efficacious because it induces oscillatory activity in the subthalamic nucleus, and that targeting white matter tracts (the bundles of nerve fibres which connect distant regions of the brain) would be effective in producing widespread therapeutic effects.
As for the future of optogenetics technology, the next logical step would be to develop wireless implants. This is exactly what researchers at Brown University’s Nanophotonics and Neuroengineering Laboratory are aiming to do. They have already developed a dual-use “optrode” consisting of an array of 100 electrodes which can deliver light to specified neurons and simultaneously record the electrical activity of the cells, and a wireless brain implant which can record neuronal activity and convert the signals into a digital stream of light pulses. Combining the properties of these devices could therefore lead to a wireless optogenetic implant, which, if composed of light-emitting carbon nanotubes, might eventually result in smaller neural prostheses with increased biocompability and hence a longer lifespan than existing devices.
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Airan, R. et al (2009). Temporally precise in vivo control of intracellular signalling. Nature DOI: 10.1038/nature07926.
Gradinaru, V. et al (2009). Optical Deconstruction of Parkinsonian Neural Circuitry. Science DOI: 10.1126/science.1167093.