Modulating Neural Activity Using Light
Modulation of neural activity has been used for hundreds of years to combat neurological impairments. Traditional neuromodulation techniques used have been either electrical stimulation or genetic or pharmacologic intervention. However the heterogeneous nature of brain tissue presents major challenges for selectively controlling subsets of well-defined neuron circuits. Most of these traditional neuromodulation techniques simultaneously affect surrounding cells and processes in addition to the target neurons. This lack of specificity in modulation often results in a limited strength of conclusions drawn from conventional neuromodulation experiments. To overcome the spatial and temporal limitations of electrical, pharmacologic, and genetic neuromodulation approaches, various microbial and engineered opsins have been developed to control electrical and biochemical activities of neurons with cell-type selectivity, high temporal precision, and rapid reversibility.
Optogenetics, a term coined by Karl Deisseroth in 2005, is a neuromodulation technique that combines optics and genetics to control and monitor the activities of individual cells in living tissue, typically neurons. Because most neurons in the brain are not naturally light sensitive, these cells are genetically modified to express light-sensitive ion channels (cell membrane proteins) that regulate electrical activity in the cell by gating the flow of ions. These proteins, known as Opsins, allow control of neural population activity when illuminated by specific wavelengths of light.
The neural control is not only limited to in-vitro (static) tissue observation but also includes control freely moving animals. Along with neuromodulation, the aim of optogenetics is also to precisely measure the effects of those manipulations in real-time. The fast on– off kinetics of microbial opsins makes it possible to evoke or inhibit neural activities within milliseconds, on a time scale relevant to physiologic brain functions. (Zhang et al 2012)
The key reagents used in this method are Opsins, which function as optogenetics actuators like channelrhodopsin, halorhodopsin and archaerhodopsin that enable neural control, while optical recording of neuronal activities can be made with the help of optogenetic sensors for calcium (GCaMP), vesicular release (synaptopHluorin), neurotransmitter (GluSnFRs), or membrane voltage (Arclightning, ASAP1). Control or recording is confined to genetically defined neurons and performed in a spatiotemporally precise manner by light.
The recent emergence of optogenetic tools — genetically encoded opsins that allow neurons to be turned on or off with bursts of light — promises to revolutionize the study of how neurons operate singly and as members of larger networks, and could ultimately offer new hope for patients suffering from vision impairment or neurological disorders such as epilepsy or Parkinson’s disease.
In 2010, optogenetics was chosen as the "Method of the Year" across all fields of science and engineering by the interdisciplinary research journal Nature Methods. At the same time, optogenetics was highlighted in the article on "Breakthroughs of the Decade" in the academic research journal Science.
Turn-ons and turn-offs
At a basic level, the nervous system can be thought of as a highly complex electrical circuit. Every neuron contains a variety of pump and channel proteins that control the flow of ions across its membrane, maintaining a negative membrane potential in the resting neuron. Activation signals, for example from neurotransmitters, cause positively-charged ions to flow into the cell from the external environment via these channel proteins, resulting in membrane depolarization. At a certain threshold, this triggers an action potential — a rapid influx of sodium ions that effectively reverses the voltage inside the cell, initiating a chain reaction of sodium-ion influx that propagates down the length of the axon, eventually causing the release of neurotransmitters that stimulate or inhibit the production of electrical impulses in neighboring neurons.
The development of ‘caged’ neurotransmitters — chemically modified to remain inactive unless triggered (‘uncaged’) by laser illumination — and chemically modified photo-switchable ion channels have allowed notable improvements in precision for functional studies, although with limited possibilities for application.
However, the real revolution came with the discovery of the algae protein channelrhodopsin, which allows influx of positive ions in response to illumination with blue light to act as an ‘on’ switch. A few years later, scientists recognized the potential of the archaeal protein halo- rhodopsin, which triggers influx of negatively-charged chlorine ions in response to yellow light and thereby hyperpolarizes the cell, to act as an ‘off’ switch. Both of these proteins can readily be introduced into target cells by various techniques, allowing scientists to rapidly and accurately turn individual neurons on and off without the need for additional drugs or chemicals.
Breakthroughs in materials science now allow the cultivation of neurons in complex predetermined patterns. The capacity to stimulate or silence individual cells selectively within these engineered cultures, in conjunction with reagents that allow the direct visualization of neuronal activity, promises to yield insights about brain structure and function.
Even the complex environment of the living brain is within reach of Optogenetics. For example, it is now possible to use light channels to deliver illumination to certain areas of the mouse brain model, activating olfactory circuits, inducing muscle movement or switching on motor centers to trigger physical activity. These successes in animal models are merely a prelude to a longer-term goal: optogenetic gene therapy in humans. Studies have already shown that light sensitivity can be restored to photoreceptor-deficient animal models by delivering the channelrhodopsin gene into retinal cells. Eventually, viral-delivery systems could allow similar therapeutic strategies in humans to treat blindness resulting from macular degeneration and other disorders — a promising alternative to more invasive implant-based therapeutic approaches.
Early work is also underway using optogenetics to improve the treatment of Parkinson’s disease. A similar approach could be applied to treat epilepsy, using halorhodopsin to enable selective inhibition of brain regions involved in the onset of seizures. Treatment of these and other neurological disorders could, for example, theoretically entail the pairing of a carefully crafted gene-therapy strategy and an implantable device for optogenetic stimulation.
Optogenetics already offers great opportunities for basic neuroscience research, as has already been demonstrated by many laboratories worldwide; although biomedical applications still face unpredictable challenges and risks, these areas of research offer great promise for redefining neurological therapeutic strategies in the future.
Selecting a Light Source for Optogenetics
There are a number of factors that impact the efficiency of Optogenetic control of neural networks. These include:
- Absolute amount of light reaching a neuron
- The number of opsins present in the plasma membrane
- The light sensitivity of the opsins
- The intrinsic neuron membrane physiological properties and that of its surrounding neural network environment
While the intrinsic membrane physiological properties of a neuron cannot be controlled by an experimenter, the other three factors can. The absolute amount of light reaching a neuron depends upon the light power at the tip of light source and the pattern of light propagation in the tissue. Light propagation is determined by the optical properties of the brain tissue, with major considerations being tissue absorption and tissue scattering that reduce the intensity of light as it propagates away from the light source.
At locations within the close proximity of the light source, where light intensity is at the saturation level for opsin action, the efficiency of optically controlling neurons will not vary by location. But at locations further away from the light source, where light intensity falls below the saturation level, the efficiency of controlling neurons will decrease with distance, and eventually at locations where light power falls below the threshold for opsin activation, light will be unable to modulate neural activities. Light propagation in brain tissue general falls off nonlinearly, to ∼1% at locations ∼1 mm away from the tip of the light source.
Current opsins operate at the visible light wavelength, ∼450–640 nm, where hemoglobin’s oxygenated (HbO2) and deoxygenated (Hb), are the major light absorbers.
Lasers or LEDs as Light Sources?
The efficiency of neural modulation is greatly impacted by the type of light source. There are three critical parameters in selecting a light source:
- Source type
The two options for source type include diode or diode-pumped solid state (DPSS) lasers and light-emitting diodes (LEDs).
Lasers as Light Source
Lasers are widely used in Optogenetics both because they permit the application of narrow bandwidth light (facilitating multimodal optical control with more than one opsin) and because they can be efficiently coupled to optical fibers. This last characteristic is a particular advantage in deeper brain structure manipulation with an implanted fiber optic.
Diode-pumped solid state (DPSS) lasers with a maximum power of 100 mW are an appropriate choice in Optogenetics (Adamantidis et al., 2007; Aravanis et al., 2007). The low divergence and high power of the laser beam enables it to be steered by multiple optical components, and it can be combined with other techniques to manipulate the activity of single neurons (Prakash et al., 2012) or achieve patterned stimulation (Packer et al., 2013).
Optical fibers can be used to deliver light to specific intracranial locations and permit optical control of deep brain structures. Small diameter optical fibers (~200 μm) minimize tissue damage and can be coupled efficiently to laser light sources (Warden et al., 2014). The fiber can be cut to the appropriate length to target a specific brain region and can either be fixed directly to the skull or inserted through a cannula to facilitate simultaneous pharmacological manipulations (Warden et al., 2014).
DPSS lasers are easy to use and powerful enough to ensure maximal activation of opsin proteins. They are characterized by the wavelength and intensity of light they emit as well as whether they can driven by both digital and analog signals. Since a laser emits light at a specific wavelength, it is important to use the appropriate laser to activate a specific opsin protein.
Channelrhodopsin (ChR2) is maximally activated by 473 nm blue light. The inhibitory protein halorhodopsin is maximally activated by 593 nm yellow light; however it can be activated by a 532 nm green laser, which is significantly less expensive than a yellow laser.
Lasers can be driven by digital computer signals (TTL pulses) or analog signals. TTL driven lasers are easy to use and they often come with power knobs that allow the laser intensity to be adjusted. Analog controlled lasers require more complex electronics to activate them, but they allow for the delivery of more complex waveforms, such as sinusoids.
Light-Emitting Diode Light Sources
Light-emitting diodes (LEDs) represent an alternative to lasers for optical stimulation primarily because of their smaller foot print, lower cost, and diverse color options (Warden et al., 2014). However, the low efficiency of LED-optical fiber coupling limits the utility of LED light sources for some wavelengths of light due to low resultant power from the fiber tip, and heat generation. There is promising work dedicated to overcoming this technical hurdle, but it is presently not practical to use LEDs for most in vivo applications.
However, their small size and low power requirements make LED light sources very useful for in-vitro multisite illumination and portable wireless Optogenetic devices (Wentz et al., 2011; Stark et al., 2012). LEDs are typically attached through a dual lamphouse adapter on the back of the electrophysiology microscope. The LED light shines through the optics of the microscope and illuminates the tissue immediately underneath the objective. The diameter of the light beam can be adjusted using the field stop diaphragm on the microscope.
Lasers for OptogeneticsLaser vs. LEDs - based on the inherent properties of lasers, they are a normally the preferred choice of light source for many researchers. It is important that the light source be appropriately powered, giving enough light to provide activation of opsin proteins while minimizing tissue heat. Typical opsin proteins like ChR2 are maximally activated by less than 1 mW of wavelength-specific light. To achieve this level of light at the opsin protein it is safe to use a laser capable of producing much more intense light. Light losses occur at any junction (collimators, connectors, splitters) as well as when the light passes through tissue. 5–30 mW lasers are sufficient for in vitro preparations. 50–200 mW lasers are typically used for in vivo work to enable maximal opsin protein activation. The light that comes out of DPSS lasers has to be collimated into optical fibers. The collimating devices are called fiber couplers and they can be purchased as a package with the lasers. Furthermore, laser systems penetrate through tissue with greater efficiency due to their low angle of incidence and high numerical aperture.