AI News, One-Step Optogenetics for Hacking the Nervous System

One-Step Optogenetics for Hacking the Nervous System

The 12-year-old technique, which enables scientists to control brain cells with light, typically requires a multi-step process and several surgeries on animal models.

Once the genetic modifications have taken hold, researchers implant a device that delivers light—usually with silica optical fibers or light-emitting diodes—to the modified cells.

The smallness of the electrodes left room for the other two elements: a polymer waveguide to deliver light and two microfluidics channels to deliver the gene-carrying virus.

In one study, they delivered a light-sensitive gene construct into an area of the mouse brain called the medial prefrontal cortex, where activating neurons is known to make mice run faster.

German and Swiss researchers four years ago developed an all-in-one optogenetics probe, and published a report onit in the journalLab on a Chip.But that design hasn’t been adopted by many optogenetics research labs.

“It was a really great pioneering demonstration,” but the design process isn’t conducive to production in large quantities, and the materials aren’t well suited for optogenetics, she says.

By contrast, Anikeeva’s probe is made by a thermal drawing process, in which they fabricate a large scale version of the device, and the heat and stretch the structure hundreds of meters long.

Anikeeva says that since she first presented at a conference in July 2016 an early version of the probe, she has received several requests from researchers wanting to use the device for a variety of applications: studying nerve circuitry linked to anxiety and addiction, peripheral nerves, and even motor nerves used to control prosthetics.

A Better Way to Probe the Brain

The brain is often described as the most complex structure known: a multitude of cells, joined into networks and abuzz with electrical and chemical activity.

Though her lab is less than four years old, prominent journals have published a string of her group’s papers demonstrating new technologies, including thin, flexible polymer-fiber probes for stimulating and recording activity from neurons, as well as magnetic nanoparticles that could be used to stimulate them with no wires at all.

She began her doctoral research in 2004 in the lab of Vladimir Bulović, then an associate professor of electrical engineering and computer science, who was developing new electronic and optical devices using nanotechnology.

But “before I would arrogantly go and try to solve problems that I didn’t know existed,” she says, “I decided that I needed to actually spend some time in a biological environment.” That decision led her to Karl ­Deisseroth’s neuroscience lab at Stanford University.

“It’s something that I have some chance of understanding.” Deisseroth’s group needed hardware to send light into specific areas of mouse brains while also taking electrical recordings from the illuminated cells.

They were too large and too bulky and didn’t have enough capability.” The biologists Anikeeva worked with were manipulating individual wires under microscopes, a far cry from sophisticated fabrication techniques used in the electronics industry.

better hammerIn a basement lab at MIT, Andres Canales, SM ’13, a PhD student in Anikeeva’s group, is watching a physical transformation take place: a cylinder of polymers and metal is being slowly melted and pulled into a long, vermicelli-like wire from a tall tower in one corner of the room.

One reason ­Anikeeva was keen to return to MIT was to work with Yoel Fink, the director of the Research Laboratory of Electronics and a leading innovator in this technique of fiber drawing, in which materials are assembled together, heated, and pulled like taffy into ultrathin fibers that preserve the original structure and functionality.

Thanks to this collaboration, her team has incorporated optical waveguides, electrodes, and drug delivery channels into a single fiber that can be as thin as a human hair and flexible enough to wrap around a finger.

With a thin, multifunctional device, he says, “you can have all the capability with minimal perturbation or damage to the brain tissues.” The devices can also be used in the spinal cord, which is challenging to access and requires a flexible device because it is often moving and stretching.

Although electrical stimulation of the spinal cord can evoke movement in paralyzed animals and has been used clinically in humans with modest results, Chet Moritz, a professor of rehabilitation medicine at the University of Washington in Seattle, says that optical stimulation could allow more precise control of specific cells.

“With optogenetics, you can have fairly high confidence that you are activating a specific circuit.” Moritz works on stimulating the upper spinal cord—ultimately in order to restore movements like reaching and grasping, which require more finesse than walking.

In a recent paper in , her group demonstrated a technique that uses magnetic fields and injected nanoparticles to activate cells deep within the brains of mice.

The particles are made from iron oxide (commonly used as a contrast agent in MRIs) and coated in polymers to keep the body’s immune system from whisking them away.

And while this study used genetic engineering to get a heat-sensitive protein into mouse cells, she says that TRPV1 is prevalent in the human brain, so such tinkering may not be necessary to use the technique in humans.

She envisions using soft polymer probes to precisely map the brain, or to deliver a drug or optical stimulation and then monitor its effect on cell activity.

To better understand neuromuscular diseases and how the body recovers from spinal cord injuries, scientists require tools that can record and modulate neural activity in the spine during normal movement.

Described recently in Science Advances, the probes, made of thermally drawn elastomer fibers coated with a mesh of silver nanowires, could accurately record the neural activity in the spines of freely moving mice and elicit leg movements in anesthetized mice through optical impulses.

“We now have a stretchable optoelectronic fiber, which is able to perform chronic probing and interrogation in spinal cord circuits in a freely moving subject,” says study lead author Polina Anikeeva, an MIT materials scientist.

Being able to record neural activity in the spine during optical or electrical stimulation could allow scientists to discover neural pathways important for recovery from spinal cord injuries, which frequently cause loss of organ function or voluntary limb control.

Naturally, the rubber-like properties of elastomers make them the perfect material to turn into fibers for the spinal cord, but elastomers are not suitable for the thermal fiber drawing technique, which involves heating and pulling the material through a furnace.

To provide the fibers with electrical connections for electrical stimulation and monitoring, the team deposited uniform 1-mm-thick conductive mesh layers of silver nanowires using dip coating.

Seung Hwan Ko, a materials scientist at Seoul National University, said the work was “very interesting and somewhat exciting,” adding that the difficulty in making stretchable optoelectronic stimulators lies mainly in finding highly stretchable materials that are good electrical conductors.

“The authors showed a very smart way to combine a highly stretchable optical stimulator and a highly stretchable metal electrode in a highly stretchable fiber form,” says Ko, who was not involved in the study.

Optogenetics

The earliest genetically targeted method that used light to control rhodopsin-sensitized neurons was reported in January 2002, by Boris Zemelman (now at UT Austin) and Gero Miesenböck, who employed Drosophila rhodopsin cultured mammalian neurons.[8] In 2003, Zemelman and Miesenböck developed a second method for light-dependent activation of neurons in which single inotropic channels TRPV1, TRPM8 and P2X2 were gated by photocaged ligands in response to light.[9] Beginning in 2004, the Kramer and Isacoff groups developed organic photoswitches or 'reversibly caged' compounds in collaboration with the Trauner group that could interact with genetically introduced ion channels.[10][11] TRPV1 methodology, albeit without the illumination trigger, was subsequently used by several laboratories to alter feeding, locomotion and behavioral resilience in laboratory animals.[12][13] [14] However, light-based approaches for altering neuronal activity were not applied outside the original laboratories, likely because the easier to employ channelrhodopsin was cloned soon thereafter.[15] Peter Hegemann, studying the light response of green algae at the University of Regensbug, had discovered photocurrents that were too fast to be explained by the classic g-protein-coupled animal rhodopsins.[16] Teaming up with the electrophysiologist Georg Nagel at the Max Planck Institute in Frankfurt, they could demonstrate that a single gene from the alga Chlamydomonas produced large photocurents when expressed in the oocyte of a frog.[17] To identify expressing cells, they replaced the cytoplasmic tail of the algal protein with the fluorescent protein YFP, generating the first generally applicable optogenetic tool.[15] Zhuo-Hua Pan of Wayne State University, researching on restore sight to blindness, thought about using channelrhodopsin when it came out in late 2003.

In August 2005, Karl Deisseroth's laboratory in the Bioengineering Department at Stanford including graduate students Ed Boyden and Feng Zhang (both now at MIT) published the first demonstration of a single-component optogenetic system in cultured mammalian neurons,[20][21] using the channelrhodopsin-2(H134R)-eYFP construct from Nagel and Hegemann.[15] The groups of Gottschalk and Nagel were first to use Channelrhodopsin-2 for controlling neuronal activity in an intact animal, showing that motor patterns in the roundworm Caenorhabditis elegans could be evoked by light stimulation of genetically selected neural circuits (published in December 2005).[22] In mice, controlled expression of optogenetic tools is often achieved with cell-type-specific Cre/loxP methods developed for neuroscience by Joe Z.

An example of this is voltage-sensitive fluorescent protein (VSFP2).[37] Additionally, beyond its scientific impact optogenetics represents an important case study in the value of both ecological conservation (as many of the key tools of optogenetics arise from microbial organisms occupying specialized environmental niches), and in the importance of pure basic science as these opsins were studied over decades for their own sake by biophysicists and microbiologists, without involving consideration of their potential value in delivering insights into neuroscience and neuropsychiatric disease.[38] Light-Activated Proteins: Channels, pumps and enzymes The hallmark of optogenetics therefore is introduction of fast light-activated channels, pumps, and enzymes that allow temporally precise manipulation of electrical and biochemical events while maintaining cell-type resolution through the use of specific targeting mechanisms.

Building on prior work fusing vertebrate opsins to specific G-protein coupled receptors[42] a family of chimeric single-component optogenetic tools was created that allowed researchers to manipulate within behaving mammals the concentration of defined intracellular messengers such as cAMP and IP3 in targeted cells.[43] Other biochemical approaches to optogenetics (crucially, with tools that displayed low activity in the dark) followed soon thereafter, when optical control over small GTPases and adenylyl cyclases was achieved in cultured cells using novel strategies from several different laboratories.[44][45][46][47][48] This emerging repertoire of optogenetic probes now allows cell-type-specific and temporally precise control of multiple axes of cellular function within intact animals.[49] Hardware for Light Application Another necessary factor is hardware (e.g.

Most commonly, the latter is now achieved using the fiberoptic-coupled diode technology introduced in 2007,[50][51][52] though to avoid use of implanted electrodes, researchers have engineered ways to inscribe a 'window' made of zirconia that has been modified to be transparent and implanted in mice skulls, to allow optical waves to penetrate more deeply to stimulate or inhibit individual neurons.[53] To stimulate superficial brain areas such as the cerebral cortex, optical fibers or LEDs can be directly mounted to the skull of the animal.

Longer photostimulation of mitral cells in the olfactory bulb led to observations of longer lasting neuronal activity in the region after the photostimulation had ceased, meaning the olfactory sensory system is able to undergo long term changes and recognize differences between old and new odors.[76] Optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology have been integrated to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition.

The few cholinergic neurons present in the nucleus accumbens may prove viable targets for pharmacotherapy in the treatment of cocaine dependence[41] In vivo and in vitro recordings (by the Cooper laboratory) of individual CAMKII AAV-ChR2 expressing pyramidal neurons within the prefrontal cortex demonstrated high fidelity action potential output with short pulses of blue light at 20 Hz (Figure 1).[34] The same group recorded complete green light-induced silencing of spontaneous activity in the same prefrontal cortical neuronal population expressing an AAV-NpHR vector (Figure 2).[34] Optogenetics was applied on atrial cardiomyocytes to end spiral wave arrhythmias, found to occur in atrial fibrillation, with light.[78] This method is still in the development stage.

In addition, this approach has been applied in cardiac resynchronization therapy (CRT) as a new biological pacemaker as a substitute for electrode based-CRT.[79] Lately, optogenetics has been used in the heart to defibrillate ventricular arrhythmias with local epicardial illumination,[80] a generalized whole heart illumination[81] or with customized stimulation patterns based on arrhythmogenic mechanisms in order to lower defibrillation energy.[82] Optogenetic stimulation of the spiral ganglion in deaf mice restored auditory activity.[83] Optogenetic application onto the cochlear region allows for the stimulation or inhibition of the spiral ganglion cells (SGN).

Moreover, this technique has been shown to extend outside neurons to an increasing number of proteins and cellular functions.[58] Cellular scale modifications including manipulation of contractile forces relevant to cell migration, cell division and wound healing have been optogenetically manipulated.[88] The field has not developed to the point where processes crucial to cellular and developmental biology and cell signaling including protein localization, post-translational modification and GTP loading can be consistently controlled via optogenetics.[58] While this extension of optogenetics remains to be further investigated, there are various conceptual methodologies that may prove to immediately robust.

There is a considerable body of literature outlining photosensitive proteins that have been utilized in cell signaling pathways.[58] CRY2, LOV, DRONPA and PHYB are photosynthetic proteins involved in inducible protein association whereby activation via light can induce/turn off a signaling cascade via recruitment of a signaling domain to its respective substrate.[89][90][91][92] LOV and PHYB are photosensitive proteins that engage in homodimerization and/or heterodimerization to recruit some DNA-modifying protein, translocate to the site of DNA and alter gene expression levels.[93][94][95] CRY2, a protein that inherently clusters when active, has been fused with signaling domains and subsequently photoactivated allowing for clustering-based activation.[96] Proteins LOV and Dronpa have also been adapted to cell signaling manipulation;

treating PC12 cells with epidermal growth factor (inducing a transient profile of ERK activity) leads to cellular proliferation whereas introduction of nerve growth factor (inducing a sustained profile of ERK activity) is associated with a different cellular decision whereby the PC12 cells differentiate into neuron-like cells.[102] This discovery was guided pharmacologically but the finding was replicated utilizing optogenetic inputs instead.[103] This ability to optogenetically control signals for various time durations is being explored to elucidate various cell signaling pathways where there is not a strong enough understanding to utilize either drug/genetic manipulation.[58]

Implantable devices for optogenetic studies and stimulation of excitable tissue

The paper deals with the currently available implants used in optogenetic experiments on laboratory animals in vivo.

The device can be used in medical diagnostics, prosthetics, myostimulation, neurostimulation and cardioacceleration, for instance, at neurological and rehabilitation medical institutions.

It is necessary to provide a point generation and layer-by-layer scanning of excitation pulse through integration of individual microelectrode arrays into a single test-system.