One can imagine the difficulty of trying to examine a single neuron among the tens of billions in the tangled web of the brain, with a topography as dynamic as Hogwarts castle (but with electricity, of course).
Shrewd researchers have recently circumvented roadblocks caused by the extreme complexity of the mammalian brain using a technique called optogenetics, which is an innovative experimental method to hone in on specific neurons and control their processes down to the millisecond. In this article we will review the field of optogenetics, its origins and how the technique is revolutionising neuroscience as well has having a much broader impact.
The Dark Ages of Neuromodulation
Neuromodulation methods preceding optogenetics relied on the use of microelectrodes inserted into the brain at an approximate location of interest. This imprecise setup electrically stimulates all of the cells at the site of insertion, causing a crossing of lines that makes it impossible to identify the specific neurons involved in a particular process. Because of this, the electrode method only enables the general study of specific brain regions. Other issues with microelectrodes include that they cannot be controlled on the same timescale of neuronal events, and they restrict the mobility of the animals being studied. The lack of spatial and temporal resolution combined with the restriction of normal animal behavior severely limits the utility and applications of electrode-based neuroscience studies.
The Dawn of Optogenetics
Neuroscientists forged ahead to address these limitations and discovered that, in an entirely separate branch of biology, researchers were controlling cellular responses on much faster timescales, and with significantly more spatial acuity, using light (Oesterhelt and Stoeckenius, 1971).
Ecologists have known for more than 40 years that microbes and algae have evolved mechanisms to capture photons of light in order to glean information about their environment and generate energy (Oesterhelt and Stoeckenius, 1971). Opsin proteins, including channel rhodopsins, halorhodopsins, and bacteriorhodopsins, confer specific cellular responses by controlling the flow of ions across the cell membrane when exposed to a narrow range of wavelengths (Zhang and Feng, 2011).
In a high-risk, high-reward endeavor to improve our understanding of clinical psychiatry, Karl Deisseroth and his team found that they could force mammalian cells to manufacture light-sensitive opsin proteins and, with the application of light, were able to turn neurons on and off (Boyden et al., 2005). They could also direct only selected types of neurons to produce the opsin gene by linking it to the promoter of a cell-type specific gene, thus enabling high spatial resolution with control of only one type of neuron (Zhang et al., 2010, Diester et al., 2011).
Other groups had also previously attempted to control neurons using light, with a two-component system using a light-sensitive chemical in conjunction with a neuron-controlling protein, which is responsive to the light-sensitive chemical. (Banghart et al., 2004, Zemelman et al., 2002). Deisseroth’s methods streamline this complicated process into a single-component system that can be broadly applied across a variety of scientific fields.
Refining Optogenetic Techniques
Researchers continue to improve upon these optogenetic methods, for example by introducing multiple opsin genes with sensitivity to different wavelengths of light to simultaneously control multiple selected cell populations (Han and Boyden, 2007). Additionally, they have fine-tuned the temporal response so that particular opsins confer faster, slower, or even reversible action potentials (Gunaydin et al., 2010). There is still plenty of scope for improvement, including discovering additional opsin genes, improving methods of delivery and gene expression, and enhancing fiber-optic light delivery and simultaneous electrical signal recording tools (optrodes).
Using optogenetics, researchers are only just beginning to uncover the complex neural circuitry that controls everything from involuntary breathing, sleeping, learning and emotion to neurological diseases like Parkinson’s and psychiatric disorders such as substance abuse (Abbott et al., 2009, Alilain et al., 2008, Bellmann et al., 2010, Kanbar et al. 2010).
Tools of the Optogeneticist
As with any trade, optogenetics apprentices have at their disposal a variety of tools for the job, but the overall strategy using a single-component approach is the same: delivery of genetic material encoding a light-sensitive protein (an opsin), expression in a particular subset of cells, exposure of these neurons to light in order to modulate their activity, and accurately recording responses.
Optogenetics is considered to be a single-component system because the opsin is both the ion channel responsible for activating or inactivating the neuron, and the light-responsive element. Thus, the opsin is the sole party responsible for converting photons of light into a biologically recognizable signal by altering the flow of ions, a response known as the effector function.
Delivering the Opsin
The most common delivery method of the opsin is via a viral vector that is itself not directed toward any particular cell type, but is rather delivered regionally. Precise control over specific cell populations comes in the form of a Trojan horse promoter camping out in front of the opsin gene. This triggers opsin expression only in cells where that promoter is active, enabling precise spatial control of cells.
To overcome the fact that some promoters for specific cell types are weaklings, researchers often turn their attention to pre-existing strong promoters that recognize Cre (Zeng et al., 2012). There is an abundance of readily available transgenic Cre/lox-recombinase mice that express Cre only in specific cell types of interest. Subsequent infection of the opsin virus with the Cre-responsive promoter amplifies response of the initial cell-type specific promoter.
A key to the opsins’ ability to exhibit light-responsive activity expressed in mammalian cells, is the natural presence of the opsin co-factor ‘all trans-retinal’ in vertebrate tissues (Surya and Knox, 1998). This serendipitous finding greatly improved the feasibility of the single-component approach.
Fine-tuning Optogenetics Using Alternative Opsins
Channelrhodopsin-2 (ChR2), the first opsin to be discovered, allows positive sodium (Na+) ions to flow into the cell when stimulated by blue light (at a wavelength of 470 nm) (Nagel et al., 2002, Nagel et al, 2003). This ion influx causes neurons to fire an action potential. On the other hand, halorhodopsin (NpHR) has the opposite effect, causing a negative chloride (Cl-) ion flux into the cell when stimulated with yellow light (580 nm), blocking any neuronal firing from taking place (Zhang et al., 2007, Gradinaru et al., 2008). Two other opsins, Archaerhodopsin-3 (Arch) and Mac, pump protons into cells when stimulated by light and so also inhibit neuron activity (Boyden, 2011). In this way, ChR2 and NpHR/Arch/Mac can be used to stimulate or block a neuronal response, respectively, enabling gain- or loss-of-function studies of a precise neuronal population. The choice of which opsin to use depends on the wavelength of light and the direction, amplitude, and timescale of the desired ion channel response.
Light Delivery and Response Recording
Optimized technologies for light delivery and response recording are vital for optogenetics, given the quick timeframe in which responses in neurons occur. For optogenetic experiments in vivo, light is delivered by either a fiber optic cable or a solid-state light source through an implanted device or an embedded window (Zorzos et al., 2010). Wavelengths that can penetrate deeper into tissues are ideal so that researchers can study even those cell types that are deep within tissues. For example, recent modifications to the NpHR opsin have enabled its ability to respond to red/far-red light (Zhang et al., 2008).
The responses of the cells simulated with light are recorded with optrodes that enable fast readouts of electrical signals (Nakamura et al., 2013). It is of great importance that these optrodes can record electrical signals as quickly as the light is delivered, down to the millisecond, to ensure that the timescales of the response are captured accurately.
As more researchers begin tinkering with optogenetics, the toolbox grows and evolves. We look forward to exciting new findings in neuroscience and beyond that will continue to emerge from the darkness.
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