Ssm Winter 2007 Engtech Lightbulb

Switching on the Light Bulb

by Caitlyn McCullough

Lighting the Way in Neural Circuit Dynamics and Depression

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e are all familiar with the proverbial light bulb inside our head that flickers with the ebb and flow of ideas. Dr. Karl Deisseroth, Assistant Professor in Stanford’s Department of Bioengineering and the Department of Psychiatry and Behavioral Sciences, has stepped beyond the figurative, developing a tool that may one day lead to the use of an LED implanted in the brain to stimulate neurons in the treatment of neurological diseases and psychiatric disorders. The spotlight of his work is the light-gated cation-selective membrane channel recently discovered in green algae called channelrhodopsin-2 (ChR2). Although photostimulation of neurons in direct clinical intervention is a distant goal with many challenges, the Deisseroth lab has demonstrated millisecond-timescale optical control of neurons in a genetically targetable and temporally precise manner using ChR2. Such unprecedented selectivity and precision may revolutionize how neuroscientists and bioengineers view neural circuit dynamics and neurological diseases.

In the Dark The brain consists of a dense architecture of heterogeneous neural cell subtypes, each presumed to play a specific role in gating of information and electrical rhythms. It is estimated that hundreds of neural subtypes exist, and understanding their respective contributions to neural processing is important in elucidating fundamental concepts of basic neuroscience, identifying aberrations in neurological diseases and psychiatric disorders, and developing interventions for treatment of disease. To probe neural circuits for subtype involvement, a means of selective excitation or inhibition of neural subtypes in a temporally precise, accurate, and physiologically relevant manner within an intact circuit is required. Current methods of probing neural circuits, through the use of electrodes or optical methods, have not achieved this combination of spatial selectivity and temporally precision. Optical methods such as light-mediated uncaging of signaling molecules, chemically modified ion channels and receptors, and the use of naturally occurring photosensitive proteins, have thus far been complicated by the difficult administration of exogenous cofactors or by requiring downstream machinery. Despite these challenges with optical control of neurons, the advantages of photostimulation have kept many, including Deisseroth and his colleagues, “carefully watching this literature for years.”

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Photo Credit: Karl Deisseroth

In the Light The gene was not initially known to neuroscience, having been identified in Chlamydomonas reinhardtii in 2001 by algae biologists in Germany. ChR2 is a seven-transmembrane protein with a light-sensitive cofactor called “all-transretinal” (ATR) bound at the core. Blue light isomerizes the ATR core, resulting in an open pore that allows selective cations to diffuse into the cell, causing depolarization. If the threshold potential is reached, voltage-gated ion channels open, initiating an action potential and the passage of an electrical signal across the synapse to another neuron. Could this directly light-gated ion channel be the means of optical control that neuroscientists have been in search of for decades? Deisseroth reached out to the algae experts (Nagel et al. in Germany), hypothesizing that this gene could be brought to neuroscience and bring great advances to the study of neural circuits. With such great gains, equal risk was involved. Deisseroth and his colleagues undertook this high risk project, investing time and effort, unsure if the Karl Deisseroth is an assistant channel would work without professor in both the Department of Bioengineering and the the addition of exogenous Department of Psychiatry at ATR. “If you pooled all the Stanford University. scientists involved, 99 out of 100 would have said, ‘No way, it’s not going to work,’” without addition of the cofactor; this had been the stumbling block for previous photostimulation attempts. Fortunately, the efforts have paid off. Retinoids present in mammalian systems allow for ChR2 to function properly. “We had a prepared mind and were able to bring [ChR2] to neurons first - (published in Nature Neuroscience in 2005),” Deisseroth comments. Photo Credit: Karl Deisseroth

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In the Lab The Deisseroth lab has demonstrated noninvasive, genetically targeted, millisecond-timescale activation in both in vitro cultured rat neurons and in vivo hippocampal regions of mice. Genetic modification of neurons is achieved using lentiviral vectors engineered to encode for the ChR2-

Deisseroth has stepped beyond the figurative, developing a tool that may one day lead to the use of an LED implanted in the brain to stimulate neurons in the treatment of neurological diseases and psychiatric disorders. yellow fluorescent protein (YFP) fusion protein, as well as a cell-specific promoter and components to improve longevity of expression (Fig 1a). Using a commonly found promoter, high levels of ChR2-YFP are expressed in the mouse hippocampus (Fig 1b). Excitation of in vitro neuron cultures with blue light induces rapid inward current (Fig 1c) and depolarization (Fig 1d). Five superimposed traces of a spike train indicate the temporal precision and accuracy of stimulation (Fig 1e). Depending upon the length of the light impulse, subthreshold potentials and action potentials can both be generated. Stimulation of in vivo neurons with ChR2 can also be achieved using an implanted fiberoptic cable. The success of ChR2 in mammalian systems poses great promise for basic neuroscience and bioengineering.

In the Future

Photo Credit: Karl Deisseroth

Selective excitation using ChR2 has widespread applications, including basic neuroscience concepts,

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guiding stem cell differentiation, ion channel drug discovery, and treatment of depression. “ChR2 allows us to do all optical interrogation of neural cells. There are existing [optical] voltage and calcium sensors that allow us to observe the activity of the cell; now we can also optically control them. This opens the door to high throughput all optical screening,” comments Deisseroth. It is estimated that ion channel drug discovery (presently done using the patch-clamp method, a physical interaction of a tiny pipette with an ion channel) could be accelerated by a factor of 1000 or more with this method. Neurological diseases and psychiatric disorders could be better understood, as well as improvement of treatments for such aberrations. Current treatment of Parkinson’s disease, epilepsy, and increasingly, depression, involve deep brain stimulation with an electrode. Excitatory cells, inhibitory cells, and other cells in the region are activated by the patchclamp method, creating a source of associated side effects and decreased efficacy. By using ChR2, the specific roles of neural subtypes in normal and abnormal functions can be determined. Excitation of targeted neural populations involved in the treatment of disease or psychiatric disorders can then be achieved using ChR2. Deisseroth imagines the use of an inductively controlled implanted wireless LED to activate ChR2 in genetically targeted neural cells to interfere with electrical rhythms involved in depression. By no means is this a trivial task; light and gene delivery are two inherent challenges that need to be further investigated. Deisseroth continues to make advances in the use of ChR2 with other photostimulation techniques in order to modulate and observe neural activity in an all optical system. He is opening channels, literally, for neuroscientists and bioengineers to understand neural circuit dynamics, neurological diseases and psychiatric disorders, and ways in which intervention may one day bring light to the brain. “[ChR2] is a very promising addition to our tool belt,” says Deisseroth. “It opens the door to a whole new way of doing things and we’re excited to be working on it here at Stanford.” S CAITLYN MCCULLOUGH is a first-year graduate student in Bioengineering. She enjoys racing bikes, and is a member of the Stanford Cycling Team and Team Tibco. To Learn More Visit the Department website of Dr. Karl Deisseroth: http://www.stanford.edu/groups/dlab

Channelrhospsin-2 (ChR2) is shown to be a valuable tool. A lentiviral vector is used for delivery of ChR2 (a), ChR2-YFP is expressed in mouse hippocampus dentate granule cells (b). Blue light induces inward current (c), depolarization (d), and temporally precise, accurate, and sustainable spike trains (e) in mouse neuron without exogenous cofactor.

layout design: Natasha Prats

Read Zhang F,Wang LP, Boyden ES, Deisseroth K. Channelrhodopsin2 and optical control of excitable cells. Nat Methods. 2006 Oct; 3(10):785-92. Read Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci. 2005 Sep;8(9):1263-8.

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