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Opto- and Pharmacogenetics: Young Field Gives Researchers New Control

Have neuroscientists entered the era of the programmable brain? Thanks to optogenetics and pharmacogenetics, which let scientists switch neuronal activity on or off with light or designer drugs, scientists can now control specific subtypes of neurons in research mice with millisecond precision. 

Opto- and Pharmacogenetics: New Methods Shed Light on Brain Processes

Have neuroscientists entered the era of the programmable brain? Thanks to the related techniques of optogenetics and pharmacogenetics, which allow scientists to turn the activity of neurons on or off using light or designer drugs, scientists can now control specific subtypes of neurons in research animals with millisecond precision. This allows them to functionally dissect complex brain circuitry and determine which cells cause specific behaviors. In a satellite conference held 11-12 October 2012 in New Orleans, Louisiana, just before the Society for Neuroscience 2012 annual meeting, over 300 neuroscientists from around the world gathered to share data obtained with these methods and showcased new technical advances in this young field. Talks were greeted with enthusiasm by a highly engaged audience, and more than one presenter commented on the community spirit and buzz they felt. “These talks have been truly inspirational,” said Brad Lowell at Beth Israel Deaconess Medical Center, Boston, Massachusetts, summing up the group mood. Presentations ranged from basic science and discussions of new methodology to disease research. Only a few talks touched on neurodegenerative diseases such as Alzheimer’s and Parkinson’s, but speakers noted the potential the techniques hold for elucidating pathological mechanisms.

“We’re experiencing a revolution in neuroscience. New methods are allowing us to do things we couldn’t have dreamed of five to 10 years ago,” said Gary Aston-Jones at the Medical University of South Carolina, Charleston, expressing a common theme. Striking another popular note, Karl Deisseroth at Stanford University praised the willingness of the community to share unpublished data, reagents, and new protocols. Presenters noted that the field has taken off in the last five years, with hundreds of published papers and thousands of labs worldwide now using these methods.

News Flash: How It Works...
Deisseroth first demonstrated optical control of neurons in 2005 (see Boyden et al., 2005). The technique involves expressing light-sensitive transmembrane proteins called opsins in specific neurons in rodent brains (see ARF related news story and ARF news story). These opsins come from simple organisms such as microbes, fungi, or algae, and form ion channels that open when activated by light. The two most commonly used are halorhodopsin, which responds to yellow light, and channelrhodopsin, which opens under blue light. Halorhodopsin acts as an off switch for neurons, as it pumps in chloride ions and hyperpolarizes cells, while channelrhodopsin boosts neural activity by letting in cations and depolarizing the cells. Because light penetrates only about one millimeter through brain tissue, researchers implant optic fibers into rodent brains to reach the target regions. Deisseroth noted that the limited penetrance of light is a positive, giving the technique fine spatial control on top of millisecond temporal control. In his keynote address, he pointed out that a huge variety of opsins exist in the microbial world, potentially providing researchers with a “vast number of tools.” Molecular engineering improves the properties of these proteins and makes them more useful for research, he added.

...Even in the Dark
The related field of pharmacogenetics complements these techniques. This approach, pioneered by researchers such as Bryan Roth at the University of North Carolina, Chapel Hill, involves engineering receptors to respond to specific drugs that have no other biological activity (see Armbruster et al., 2007, and Pei et al., 2008). These proteins, known as “Designer Receptors Exclusively Activated by Designer Drugs” (DREADDs) and “Receptors Activated Solely by Synthetic Ligands” (RASSLs), couple to specific G proteins that trigger downstream second messenger pathways that can have a variety of effects on cells.

In contrast to optogenetics, pharmacogenetics gives a longer-lasting response and can target a much broader brain area (since drugs penetrate better than light). John Neumaier at the University of Washington, Seattle, noted that designer receptors seem to modulate plasticity, rather than immediate behavior as optogenetics do. Optogenetic and pharmacogenetic approaches can be combined in the same animal to provide multiple levels of control, presenters noted. In the future, “Multiplexing will be de rigueur,” Roth suggested. He also pointed out that pharmacogenetic techniques have the potential to translate to people, since the drugs have no innate biological activity and therefore should have few, if any, side effects. However, just as in animals, therapeutic DREADDs would have to be introduced through a viral vector and be targeted to a particular neuron class.

Clarifying Circuitry: Behavioral and Disease Applications
The techniques could greatly advance understanding of brain circuitry. Scientists have struggled to map and decode circuits amongst the billions of neurons in the brain, partly because of the difficulty of functionally isolating specific types of neurons intermixed among many others. By injecting virally encoded opsins or designer receptors under the control of cell type-specific promoters, researchers can now single out neuronal classes. Moreover, they can manipulate the neurons to determine their direct effect on connecting cells. As one example, the hypothalamus, with its numerous anatomic nuclei and functions, has been a particular challenge to study, Lowell noted. With these new techniques, “the hypothalamus is now the land of opportunity,” he said. Although the hypothalamus is known to regulate appetite and feeding behavior, the wiring diagram remains a puzzle. Lowell used pharmacogenetic techniques to demonstrate an essential role for Agouti-related protein neurons found in the hypothalamus’ arcuate nucleus. Stimulation of these neurons triggered voracious appetite in rodents, while silencing them dropped feeding below normal, he reported.

Presenters described the use of optogenetics and pharmacogenetics to study a variety of complex processes such as depression, drug addiction, schizophrenia, satiety, arousal, and behavioral conditioning. “The level of sophistication in the coding of behaviors is stunning,” said Ann Graybiel of MIT. In many cases, researchers revisited classic studies that showed a role for a neuron type in a particular process but could not demonstrate causality. Using optogenetics, they determined which neurons directly act on others and gained new insight into complex circuitry.

For example, previous studies generated conflicting data about the role of cholinergic neurons in Parkinson’s disease. Some research showed that these neurons become hyperactive due to a loss of dopaminergic control (see, e.g., Pisani et al., 2003). In support of this, anticholinergic drugs were one of the first treatments for PD (see Brocks, 1999). However, other studies noted a loss of cholinergic neurons as the disease advances (see Bohnen and Albin, 2011) and reported that cholinesterase inhibitors, which increase cholinergic activity, improve PD symptoms (see, e.g., Aarsland et al., 2004).

To clarify the issue, Corinne Beurrier at Aix-Marseille University, France, selectively expressed channelrhodopsin and halorhodopsin in the striatal cholinergic neurons of mice, allowing her to activate or silence the cells with different-colored lights. Her preliminary data, presented in a poster session, suggests that turning on cholinergic neurons inhibits the majority of GABAergic medium spiny neurons (MSNs) in the striatum, while quieting cholinergic cells sparks MSNs, although she noted that more experiments are needed to confirm these results. She induced PD-like symptoms in the mice by either administering haloperidol, which blocks dopamine receptors, or lesioning the substantia nigra with the chemical 6-hydroxydopamine. In both models, silencing cholinergic neurons improved motor function. By contrast, activating these cells had no effect on motor deficits. Hyperactive cholinergic neurons do play a role in worsening motor symptoms in these models, Beurrier concluded. For data on how optogenetics and pharmacogenetics are being applied in AD and memory research, see Part 2.—Madolyn Bowman Rogers.

This is Part 1 of a two-part series. See also Part 2.


News Citations

  1. Flashy Technique Uses Light to Command Neural Firing
  2. PD Studies Suggest Motor Cortex as Treatment Target
  3. Opto- and Pharmacogenetics: New Ways to Study Memory and AD

Paper Citations

  1. . Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci. 2005 Sep;8(9):1263-8. PubMed.
  2. . Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5163-8. PubMed.
  3. . Engineered GPCRs as tools to modulate signal transduction. Physiology (Bethesda). 2008 Dec;23:313-21. PubMed.
  4. . Targeting striatal cholinergic interneurons in Parkinson's disease: focus on metabotropic glutamate receptors. Neuropharmacology. 2003 Jul;45(1):45-56. PubMed.
  5. . Anticholinergic drugs used in Parkinson's disease: An overlooked class of drugs from a pharmacokinetic perspective. J Pharm Pharm Sci. 1999 May-Aug;2(2):39-46. PubMed.
  6. . The cholinergic system and Parkinson disease. Behav Brain Res. 2011 Aug 10;221(2):564-73. PubMed.
  7. . Role of cholinesterase inhibitors in Parkinson's disease and dementia with Lewy bodies. J Geriatr Psychiatry Neurol. 2004 Sep;17(3):164-71. PubMed.

External Citations

  1. DREADDs

Further Reading

Opto- and Pharmacogenetics: New Ways to Study Memory and AD

By allowing researchers to turn on or off specific classes of neurons in mice at will, optogenetics and pharmacogenetics promise to revolutionize the field of neuroscience (see Part 1 of this series). In a satellite conference held 11-12 October 2012 before the Society for Neuroscience annual meeting in New Orleans, Louisiana, several researchers discussed how they have used the techniques to analyze basic memory mechanisms and to study Alzheimer’s disease.

Triggering and Synthesizing Memories: Real-World Inception?
In the Christopher Nolan movie Inception, Leonardo DiCaprio’s character inserted false memories into a man’s brain. Sound like science fiction? Perhaps not. Optogenetics might allow scientists to create fake memories, said Susumu Tonegawa from MIT in his talk. If that could be done, it would demonstrate that scientists understand how memories are made, Tonegawa noted. As a first step to figuring out how the brain stores experiences, Tonegawa wondered if he could artificially trigger a memory by activating the proper engram, which is a network of synaptically connected neurons believed to record experiences. To demonstrate this, he needed a way to label only the neurons involved in the memory, and only at the time of memory formation, as well as a means to selectively stimulate those neurons later. He turned to optogenetics.

Tonegawa planned to specifically label the memory engram neurons with channelrhodopsin-2, a light-activated cation channel (see Part 1 of this series). That would allow him to reactivate those neurons later. The challenge was to induce expression of the opsin only in the memory engram cells. As a first step, the researchers focused on hippocampal memories formed in a contextual fear conditioning paradigm. In mice, they expressed channelrhodopsin-2 in the dentate gyrus under control of an inducible TetO promoter. That promoter kept the gene silent as long as the mice received doxycycline in their diet. With the gene off, the researchers habituated the mice to a safe environment. Then they stopped doxycycline treatment, de-repressing channelrhodopsin synthesis in cells that also expressed the TetO activator tTA. Meanwhile, the tTA gene under the control of the c-fos promoter only turned on in response to activity.

The researchers waited two days to allow the doxycycline to clear from the mice, then placed the mice in a second context, where they received a shock. In theory, the cells that fired during this experience would turn on c-fos, thus activating tTA, which would cause channelrhodopsin to be made only in those cells. To test this, the researchers resumed doxycycline treatment, put the mice back in the safe context, and then activated channelrhodopsin-labeled cells with a pulse of blue light. The mice froze, showing that they were re-experiencing a fear memory without any external trigger (see Liu et al., 2012).

Numerous control experiments demonstrated that this response occurred only when channelrhodopsin labeled a fear memory. For example, if the mice did not get a shock in the fear context, they did not freeze later in response to light, even though their memories of the new environment got labeled with the opsin. The findings validate the theory of memory engrams, showing that a network of cells does store particular experiences, Tonegawa said.

In the future, Tonegawa will investigate whether there are multiple engrams in different brain regions for the same memory, and if each is sufficient to recall the memory. Examination of labeled cells will also help map engrams and their connections. Tonegawa wondered if scientists could go further and implant a false memory by using optogenetics. Though this sounds more like Inception sci-fi, Tonegawa believes it could be done by light-activating a previously formed memory engram in conjunction with exposure to a new, unconditioned stimulus, e.g., an odor. The animal would then have a false memory of receiving a foot shock along with the smell. Although one previous study found this method does not work (see Garner et al., 2012), Tonegawa suggested that study activated too broad a swath of brain to really test the theory.

New Approaches in Alzheimer’s Disease
Optogenetic and pharmacogenetic techniques are also being used to examine hypotheses and develop treatments for Alzheimer’s disease. Sylvie Claeysen at INSERM, France, discussed a pharmacogenetic strategy that shows therapeutic potential in AD mice. She introduced a point mutation into the serotonin receptor 5-HT4 to make it a Receptor Activated Solely by Synthetic Ligands, or RASSL (see also Part 1). The receptor also shows some basal activity in the absence of any ligand, Claeysen noted (see Claeysen et al., 2003). Claeysen first used this receptor to study Parkinson’s disease, but in recent studies found it beneficial in AD models as well.

When the researchers expressed this RASSL in mouse cortical neurons, the cells released more sAPPα, a product of the non-amyloidogenic α cleavage of amyloid precursor protein (APP). Levels of sAPPα climbed further in the presence of the synthetic ligand. Claeysen reported that 5-HT4 RASSL physically interacts with mature α-secretase ADAM10. The interaction probably occurs in the plasma membrane and serves to stimulate the secretase, Claeysen said. Knocking down ADAM10 levels sharply curtailed sAPPα release in this system. She is currently looking for more interacting proteins that might form a complex with ADAM10, APP, and the 5-HT4 RASSL.

In theory, stimulation of ADAM10 should reduce amyloidogenic processing of APP and could ameliorate pathology. To test this idea, the researchers expressed the RASSL in 5xFAD mice, which have particularly aggressive AD-like pathology, and treated them with the synthetic agonist at one or two months of age for several weeks. This stage corresponds to the prodromal phase of the disease, as the animals have amyloid plaques but do not yet show behavioral deficits, Claeysen said. Treated mice had fewer plaques and less inflammation compared to controls, with better effects the longer the treatment continued. Claeysen has not yet looked at behavior but plans to do that next.

In contrast to this pharmacogenetic approach, Li-Huei Tsai at MIT used optogenetics to look at the role of the cholinergic system in a different mouse AD model. Cholinergic neurons in the medial septum project to the hippocampus, and cholinesterase inhibitors improve cognitive function in mild AD. As cholinergic neurons are thought to mediate hippocampal θ rhythms, which have been linked to cognition (see ARF related news story), Tsai wondered if improved rhythms might explain some of the cognitive benefit of these drugs (see Yener et al., 2007).

To explore this, Tsai used CK-p25 mice, which accumulate amyloid plaques and neurofibrillary tangles, and exhibit profound neurodegeneration, and, in common with the 5xFAD mouse model, show dampened hippocampal θ and γ rhythms. She expressed channelrhodopsin-2 in cholinergic neurons of the medial septum, activated the cells with light, and measured oscillations in the hippocampus. A single pulse of light ramped up oscillations for the next 10 minutes. The effects on cognitive performance were dramatic. These animals perform poorly in associative learning tasks, but one minute of light stimulation improved their learning skills to wild-type levels. The boost lasted up to one week, Tsai reported. She also saw improvements in spatial memory and long-term potentiation.

What Lies Ahead
Presenters emphasized that current tools and methodologies for optogenetics and pharmacogenetics have only scratched the surface. Many talks highlighted technical advances. For example, Feng Zhang, previously in Karl Deisseroth’s lab at Stanford University, Palo Alto, California, and now at MIT, talked about finding novel opsins that respond to different wavelengths of light. He noted that channelrhodopsin-1 from the green algae Volvox activates with green or yellow light, while rhodopsin-3 from the alga Guillardia theta inhibits neurons in response to blue light (see Mei and Zhang, 2012). As green fluorescent protein and related proteins from jellyfish and other marine organisms have revolutionized the study of cell and molecular biology, these and other opsins will broaden the tools available to researchers and permit finer control of neuronal kinetics, Zhang said. Likewise, Ed Boyden at MIT noted that red light penetrates deeper into brain than other wavelengths, making opsins that respond to this wavelength particularly useful. He is developing several red light receptors, as well as other optogenetic tools such as a fiber array that can bend light 90 degrees for better targeting of specific brain regions (see website).

Other researchers focused on hardware. One limitation in recording electrical activity from mouse brain is that these small animals cannot support heavy equipment on their heads. Chris Moore at Brown University, Providence, Rhode Island, described the creation of an UltraLight FlexDrive, weighing less than two grams. The device allows recording from 32 or 64 channels and can also independently control several optical fibers, Moore said. In collaboration with Josh Siegle and Jakob Voigts at MIT, he is also building a system for real-time detection of electrical activity, which he said would be cheaper than current slower systems (see website for details of other hardware).

Optical fibers that protrude from the head and tether mice, interfering with free movement and behavior, present another problem for optogenetic studies. Jordan McCall, a doctoral student in the lab of Michael Bruchas at Washington University, St. Louis, Missouri, said providing light through an implantable micro-ILED (inorganic light-emitting diode) could circumvent that issue. The 20-micron-thick device (smaller than many neurons) can be inserted into the brain with a thin needle. The device causes less gliosis than a standard metal cannula does, McCall reported. The micro-ILED can emit multiple, independently controlled wavelengths of light in numerous directions. Researchers control the device by radio signals, which the animal receives through a tiny, removable antenna placed on the head. The system allows mice to scurry around freely while their neurons are stimulated.

Numerous resources exist to help researchers carry out optogenetic studies. These include The Jackson Lab websites for optogenetics and useful transgenic mouse strains, and a “cookbook” that describes how to combine optogenetics with functional MRI to look at the network impact of activating specific cell types (see Desai et al., 2011). In concluding remarks, Gary Aston-Jones at the Medical University of South Carolina, Charleston, praised the “amazing new technologies and devices presented” at the satellite meeting. He also invited attendees to submit papers to a special issue of Brain Research dedicated to optogenetics and pharmacogenetics that will appear next year.—Madolyn Bowman Rogers.

This is Part 2 of a two-part series. See also Part 1.


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News Citations

  1. Opto- and Pharmacogenetics: New Methods Shed Light on Brain Processes
  2. Off Key—Aβ Detunes the Theta Rhythms of the Hippocampus

Paper Citations

  1. . Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature. 2012 Apr 19;484(7394):381-5. PubMed.
  2. . Generation of a synthetic memory trace. Science. 2012 Mar 23;335(6075):1513-6. PubMed.
  3. . A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL). J Biol Chem. 2003 Jan 10;278(2):699-702. PubMed.
  4. . Increased frontal phase-locking of event-related theta oscillations in Alzheimer patients treated with cholinesterase inhibitors. Int J Psychophysiol. 2007 Apr;64(1):46-52. PubMed.
  5. . Molecular tools and approaches for optogenetics. Biol Psychiatry. 2012 Jun 15;71(12):1033-8. PubMed.
  6. . Mapping brain networks in awake mice using combined optical neural control and fMRI. J Neurophysiol. 2011 Mar;105(3):1393-405. PubMed.

External Citations

  1. 5xFAD mice
  2. CK-p25 mice
  3. website
  4. website
  5. optogenetics
  6. transgenic mouse

Further Reading