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Optogenetics: Illuminating the Path toward Causal Neuroscience

Harvard Medical School・141 minutes read

The 2019 Warren Alpert Foundation Prize Symposium honored pioneers in genetics, neuroscience, and bioengineering, highlighting the transformative impact of optogenetics on neuroscience and brain research. Optogenetics revolutionized our understanding of neural circuits, paving the way for potential therapies for vision restoration, spinal cord injuries, and neuropsychiatric disorders.

Insights

  • The Warren Alpert Foundation Prize Symposium honors pioneers in genetics, neuroscience, physiology, and bioengineering, particularly focusing on optogenetics' development.
  • Optogenetics, a groundbreaking technology, enables the visualization and manipulation of neurons using light, revolutionizing neuroscience research.
  • The work of Edward Boyden, Karl Deisseroth, Peter Hegemann, and Gero Miesenbock has significantly advanced our understanding of the brain's inner workings and neural circuits through optogenetics.
  • Optogenetics has reshaped modern neuroscience, offering potential therapeutic applications for vision restoration, spinal cord injuries, and neuropsychiatric disorders.
  • The field of optogenetics combines diverse scientific disciplines, from biophysics to technology development, providing new insights into brain function and potential treatments for neurological diseases.
  • Optogenetics has led to significant advancements in neuroscience research, allowing for precise experiments on neural activity and the testing of theories on brain computation and behavior.
  • The Warren Alpert Foundation's support has been instrumental in recognizing transformative scientific achievements, including the development and application of optogenetics in neuroscience.

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Recent questions

  • What is optogenetics and its impact on neuroscience?

    Optogenetics is a revolutionary technology that allows scientists to visualize and modulate neurons using light, transforming our understanding of the brain's inner workings and neural circuits. Pioneered by researchers like Edward Boyden, Karl Deisseroth, Peter Hegemann, and Gero Miesenbock, optogenetics has reshaped modern neuroscience by enabling precise experiments, controlling neural activity, and testing theories on brain computation and behavior. This innovative approach has paved the way for potential therapies to restore vision, treat spinal cord injuries, and address neuropsychiatric disorders, offering new insights into brain function and potential treatments for various diseases.

  • How did Peter Hegemann contribute to optogenetics?

    Peter Hegemann played a crucial role in the development of optogenetics by conducting research on light-sensitive molecules in algae, laying the foundation for this groundbreaking technology. His work on channelrhodopsins, light-gated ion channels found in algae, led to the incorporation of these proteins into nerve cells, demonstrating the possibility of using light to modify neural activity. By identifying key elements like the retinal chromophore and amino acids crucial for light sensitivity and ion selectivity, Hegemann's research significantly contributed to the advancement of optogenetics, allowing for precise control of neural activity using light.

  • What is the significance of the Warren Alpert Foundation in scientific achievements?

    The Warren Alpert Foundation has been instrumental in recognizing transformative scientific achievements, including the development of optogenetics. Established by Warren Alpert, the Foundation has played a crucial role in acknowledging groundbreaking research in genetics, neuroscience, physiology, and bioengineering. Through its support, the Foundation has contributed to the celebration of scientific pioneers like Edward Boyden, Karl Deisseroth, Peter Hegemann, and Gero Miesenbock, whose work has reshaped modern neuroscience and opened new avenues for potential therapies and treatments for various diseases.

  • How does trial and error learning relate to basal ganglia function?

    Trial and error learning, a process of associating sensory inputs with motor outputs through repeated attempts, is influenced by the function of the basal ganglia in the brain. Damage to the basal ganglia, as seen in individuals with Parkinson's disease, can impair trial and error learning due to the crucial role this brain region plays in linking cues to motor outputs. Studies in model species have confirmed the importance of the basal ganglia in trial and error learning tasks, highlighting the significance of this brain area in adapting behavior based on past outcomes and feedback.

  • What role does the striatum play in decision-making and learning?

    The striatum, a component of the basal ganglia, plays a vital role in decision-making and learning processes in the brain. Through experiments using optogenetics to activate specific brain areas in mice, researchers have shown that the striatum is necessary during learning but not after, providing fast incremental updates to behavior. Inhibiting the striatum disrupts the mice's ability to learn through trial and error, indicating its involvement in linking cues to motor outputs and updating behavior based on past outcomes. The striatum's ability to store accumulated learning within a day and update behavior on different timescales suggests its crucial role in shaping decision-making processes and adaptive behavior.

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Summary

00:00

"Optogenetics: Revolutionizing Neuroscience Through Light Control"

  • The 2019 Warren Alpert Foundation Prize Symposium celebrates the achievements of four pioneers in genetics, neuroscience, physiology, and bioengineering, leading to the development of optogenetics.
  • Optogenetics allows visualization and modulation of neurons using light, revolutionizing neuroscience.
  • Edward Boyden, Karl Deisseroth, Peter Hegemann, and Gero Miesenbock's work transformed our understanding of the brain's inner workings and neural circuits.
  • Peter Hegemann's research on light-sensitive molecules in algae laid the foundation for optogenetics, followed by Karl Deisseroth's experiments activating neurons in the mammalian brain.
  • Gero Miesenbock demonstrated the possibility of using light to modify neural activity by incorporating light-sensing proteins into nerve cells.
  • Edward Boyden refined optogenetics, developing tools for controlling multiple cell types in the brain and achieving neuronal silencing.
  • Optogenetics has reshaped modern neuroscience, paving the way for potential therapies to restore vision, treat spinal cord injuries, and address neuropsychiatric disorders.
  • The Warren Alpert Foundation's support has been instrumental in recognizing transformative scientific achievements, including optogenetics.
  • Warren Alpert's impulsive decision to establish the Foundation led to significant contributions to science and the recognition of groundbreaking research.
  • Optogenetics has enabled precise experiments in neuroscience, allowing for the control of neural activity and testing of theories on brain computation and behavior.
  • The field of optogenetics combines diverse scientific approaches, from biophysics to technology development, offering new insights into brain function and potential treatments for neuropsychiatric diseases.

15:59

"Peter's groundbreaking research in optogenetics"

  • Peter has dedicated his career to studying how unicellular organisms sense, detect, and react to light.
  • Through curiosity-driven research, Peter identified central proteins that led to the development of optogenetic activators.
  • Peter expresses gratitude to the Warren Alpert Foundation for the honor of speaking.
  • He emphasizes the importance of starting research projects with wonder and curiosity.
  • Peter's lab focuses on Chlamydomonas reinhardtii, a green model organism.
  • Andrei Famintsyn's 1866 publication on Chlamydomonas behavior influenced Peter's work.
  • Peter's lab discovered light-activated ion channels in Chlamydomonas, mediated by rhodopsin.
  • Collaborating with Roger Chen, Peter identified channelrhodopsins as light-gated ion channels.
  • Channelrhodopsins were successfully expressed in animal cells, leading to groundbreaking research in neuroscience.
  • The technology involves using DNA from microorganisms like Chlamydomonas to manipulate neurons for various studies.

30:48

"Channelrhodopsin: Light Sensitivity and Optogenetics"

  • Karl and Ed will speak about autism, addiction, anxiety, Parkinson, and related topics.
  • The focus returns to understanding photoreceptors, particularly channelrhodopsin.
  • Current knowledge on channelrhodopsin is based on mutagenesis studies, biophysics, and the exostructure provided by Osamo Diwaki in Japan.
  • The retinal chromophore and amino acids are crucial for light sensitivity, color tuning, conductance, and ion selectivity.
  • The protein's key elements are the gates, closed in darkness, indicating a dark state.
  • Information on the open state of channelrhodopsin is still lacking.
  • Photoreceptors undergo numerous conformational changes post-light absorption, detectable through absorption wavelength changes.
  • The decision to return to the dark state or proceed in the photo cycle product is made on a picosecond timescale.
  • Mutations in channelrhodopsin residues can alter currents, open state lifetime, and photo cycle speed.
  • Two-component optogenetics combines a photo-activated enzyme with cyclic AMP or cyclic GMP-activated potassium channels for hyperpolarization, showing promise in inhibiting action potentials and heart beating.

45:24

"Trial and Error Learning in Squash"

  • The individual signed up for a squash class to try a new racquet sport but struggled initially.
  • Despite practicing and attending all classes, the individual still couldn't hit the ball with the racquet.
  • Squash exemplifies learning through trial and error, associating sensory inputs with motor outputs.
  • People with Parkinson's disease struggle with trial and error learning due to basal ganglia damage.
  • Damage to the temporal lobe causes amnesia but doesn't affect practice-based trial and error learning.
  • Basal ganglia are crucial for trial and error learning, confirmed in various model species.
  • A task was developed for mice to learn through trial and error, involving sensory and motor components.
  • Optogenetics was used to activate specific brain areas in mice to study trial and error learning.
  • The striatum in the basal ganglia was focused on to understand its role in linking cues to motor outputs.
  • Inactivating the striatum during learning disrupted the mice's ability to learn through trial and error.

01:00:04

Striatum's Role in Rapid Learning Mechanisms

  • Low learning index values indicate no cued reaching, middle values show the mouse starting to do cued reaches, and high values mean the animal is performing stereotyped cued reaches.
  • Animals with the intact striatum learn the task, but inhibiting the striatum prevents mice from learning.
  • A recovery experiment shows that stopping the manipulation allows animals to learn again, suggesting the striatum's role in learning cued reaching.
  • The hypothesis suggests the striatum may use feedback from past behavior outcomes to update future behavior on different timescales.
  • Learning can occur rapidly, as seen in a squash racket swing, or over minutes to hours, like a student cramming for an exam.
  • By measuring learning across trials within a day, faster cognitive mechanisms can be examined, such as reaction time improvement.
  • Mice learn within a day by increasing cued reaching and decreasing non-cued reaching, suggesting the striatum stores accumulated learning within a day.
  • Shutting off the striatum at the end of the day results in behavior reverting to the beginning of the day, indicating the striatum does not store within-day improvement.
  • Disengaging the striatum early in the day prevents animals from accumulating improved performance, supporting the hypothesis that the striatum acts on a fast timescale to update behavior.
  • Activating specific neurons in the brain can teach mice a cued response, with the striatum being necessary during learning but not after, providing fast incremental updates.

01:14:06

Optogenetic Tools Enhance Control Over Neural Activity

  • Light-driven proton pumps can be used to silence neural activity in neurons by genetically expressing them and shining green or yellow light.
  • Halorhodopsins can also silence neural activity by pumping chloride in response to green or yellow-orange light, hyper-polarizing the neuron.
  • Channelrhodopsin 2, when expressed in neurons and exposed to blue light, allows positive charge in, activating the neurons.
  • Arch, a light-driven proton pump, was found to silence neural activity effectively, leading to the discovery of more powerful molecules like ArchT.
  • Jaws, a red light-sensitive molecule derived from Crook's halorhodopsin class, can shut down neurons deep into the brain when exposed to red light.
  • Crimson, a channelrhodopsin found to respond well to red light, can drive neural activity when activated.
  • Cronos, another channelrhodopsin with fast kinetics, is useful in areas where speed is crucial, like in the auditory system.
  • Combining crimson and Cronos allows for differential control over neural spiking in response to blue and red light, enabling the study of multiple synaptic inputs.
  • CRCHR, a powerful molecule found in a screen, can be targeted to the cell body using a specific peptide, enhancing control over neural activity.
  • Efforts have been made to push the limits of optogenetic tools by maximizing amplitude, speed, color shifts, and spatial precision for better control over neural activity.

01:26:11

"Evolution of Genes for Neural Imaging"

  • Genes can be obtained from the wild or as mutants of an old gene for evolution.
  • Mutants are transfected into cultured mammalian cells using an automated microscope.
  • A robotic arm developed by a collaborator is used to extract interesting cells and genes.
  • Archaerhodopsin 3 was discovered to be a weakly fluorescent voltage indicator.
  • A large-scale direct evolution screen was conducted with almost 10 million mutants in two rounds.
  • Multiple parameters were screened for brightness, localization, safety, and photo-stability.
  • Directed evolution led to the discovery of a well-localized membrane indicator named archon.
  • Archon was used for imaging synaptic events in brain slices and awake behaving mammals.
  • A method was developed to expand brain tissue for detailed imaging of neural connections.
  • The expanded brain tissue imaging technique allows for high-resolution visualization of neural circuits.

01:37:31

"Neuroscience techniques reveal brain circuitry secrets"

  • Different cells received different viruses, with one cell receiving one copy of each.
  • Zooming in on axons in the mouse hippocampus reveals a resolution limit of the microscope.
  • Machine-learning techniques are being developed to trace neural circuits color-coded with Brainbow strategy.
  • Expansion allows for clear resolution of individual axons within a bundle.
  • Expansion mapping is used to create comprehensive brain circuit maps.
  • Optogenetics can be used to observe neural activity and test patterns in neural circuits.
  • Various species, not just brain cells, are being studied using expansion techniques.
  • Optogenetics has unlocked experimental doors in neuroscience, allowing for precise behavioral studies.
  • Sleep homeostasis in fruit flies is controlled by specific neurons in the brain.
  • The output arm of the sleep homeostat in fruit flies is represented by a small group of neurons in the central complex.

02:20:25

"Optical Control of Neurons in Sleep Regulation"

  • Neurons can be controlled optically to measure electrical activity, with one recording electrode used to monitor a population of neurons in an experiment lasting half an hour.
  • A fly initially awake and moving becomes still when the lights are switched on, activating the neuron being recorded, and resumes movement when the lights are turned off.
  • Sleep-inducing neurons can be in two states: one responsive to depolarizing currents, the other unresponsive even when depolarized, with distinct membrane properties.
  • The unresponsive state is characterized by a current leak or shunt, evident in smaller voltage deflections and quicker equilibrium membrane potential settling.
  • Dopamine is identified as a potential signal that silences sleep-inducing neurons, leading to awakening, with reversible effects on electrical activity.
  • The action of dopamine on sleep-control neurons is direct, mediated by a dopamine receptor, impacting passive membrane properties and electrical activity reversibly.
  • Two potassium channels, Kv1-channel shaker and Sandman, are modulated antagonistically between active and silent states, influencing the electrical activity of sleep-inducing neurons.
  • The regulation of the shaker potassium channel involves a beta subunit with an enzymatic nature, sensing changes in cellular-redox chemistry integral to sleep control.
  • Mutations in the hyperkinetic protein, a Kv beta subunit, lead to insomnia in flies, with its catalytic activity tied to its sleep-regulatory role.
  • Changes in redox chemistry in sleep-control neurons are linked to changes in sleep pressure, suggesting a role in monitoring energy metabolism through mitochondrial electron transport.

02:35:39

Mitochondrial Oxidative Stress Impacts Sleep Duration

  • ATP demand is high, and there is a balance between ADP levels and NADH fuel supply.
  • When there is an excess of NADH, full ATP reserves, and a large proton motive force, ATP synthase slows down.
  • Electrons accumulate in the ubiquinone pool and transfer to molecular oxygen, producing superoxide.
  • Neurons may experience mitochondrial oxidative stress due to excess electrons in the transport chain and low ATP demand.
  • Mitochondria of sleep-inducing neurons were filled with MitoTimer, indicating oxidative stress levels.
  • Sleep-deprived flies showed higher oxidative stress levels compared to well-rested flies.
  • Introducing a plant-derived molecule into neurons reduced sleep by capping mitochondrial reactive oxygen species production.
  • Manipulating superoxide dismutases in flies affected sleep duration, with specific mutations increasing or decreasing sleep.
  • Potassium channel beta subunit was found to couple mitochondrial electron transport to sleep in flies.
  • Lipid prooxidation products from mitochondrial membrane lipids may act as a signaling chain to induce sleep.

02:51:44

Neuron Control Through Light: Optogenetics Research

  • The kinetics of the A-type current in neurons is influenced by the oxidation process.
  • Sleep pressure needs to be dissipated when animals sleep, achieved through enzymatic activity of the beta subunit and voltage-driven rearrangements of the alpha subunit.
  • Voltage sensors in neurons move when Sandman moves out of the membrane, leading to conformational changes transmitted to the beta subunit, allowing for the replacement of NADP+ with NADPH.
  • Optogenetics, a technology allowing for the control of neuron firing with light, was envisioned by Francis Crick and later realized through experiments with hippocampal neurons transfected with opsins.
  • Crick provided constructive feedback on early optogenetic experiments, encouraging further work and improvements in the field.
  • Key individuals in the research include Boris Zemelman, Susana Lima, Jeff Donlea, Diogo Pimentel, Anissa Kempf, and Michael Song.
  • Dr. Charlotte Arlt's research focuses on using light to manipulate neuron activity in mice to understand decision-making processes in the brain.
  • Decision-making processes in the brain involve sensory input combined with internal knowledge or experience to guide actions, known as cognition.
  • Mice are trained to make decisions in a virtual reality setup by running to the left or right in mazes based on visual cues.
  • Brain circuits for decision-making are studied by inhibiting different brain areas in mice using channelrhodopsin-expressing neurons and optical methods.

03:05:14

Context Influences Brain Areas in Decision-Making

  • 0.5 or 50% signifies chance performance in an animal, either guessing randomly or consistently choosing one side.
  • In the control case, the animal's performance is significantly above chance level, indicating few mistakes and familiarity with the task.
  • Inhibiting the outer sensory cortex shows a similar performance pattern to the control case, suggesting the animal doesn't rely on spiking activity in that area for navigation decisions.
  • Inhibiting the retrosplenial cortex results in frequent mistakes, indicating the animal depends on this area for decision-making tasks.
  • Inhibiting the parietal cortex surprisingly doesn't affect the animal's task performance, suggesting this area is dispensable in this context.
  • Creating a flexible context where the animal must switch associations reveals that the animal adapts its strategy after making initial mistakes, performing well towards the end of each block.
  • Animals trained in the flexible context show similar decision-making patterns to those in the simple context, indicating adaptability.
  • Inhibiting the retrosplenial cortex in the flexible context leads to a significant drop in performance, highlighting the area's crucial role in decision-making.
  • Inhibiting the parietal cortex in the flexible context also results in a performance drop, suggesting its involvement in decision-making alongside the retrosplenial cortex.
  • Context plays a significant role in determining which brain areas are utilized for decision-making, with different contexts requiring different brain regions for optimal performance.

03:18:54

Advancements in Optogenetics and Channelrhodopsins

  • The progress in understanding the channelrhodopsin protein has been rapid, with significant advancements made in the past six years.
  • The retinol binding pocket and ion pore of the channelrhodopsin protein have been identified, including specific residues like glutamates E1, E2, E3, E4, E5.
  • Structural determinations and molecular modeling have allowed for fundamental changes in the properties of channelrhodopsins, such as increasing speed up to 200 Hertz and achieving bistable operation.
  • Collaborative efforts have led to the discovery of crystal structures of channelrhodopsins, enabling modifications to change ion selectivity and create chloride conducting channelrhodopsins for blue-light-based inhibition.
  • The discovery of naturally occurring chloride conducting channelrhodopsins furthered understanding of the structure and function of engineered channelrhodopsins.
  • The principle of surface electrostatic potential within the pore has been crucial in converting anion conducting channelrhodopsins to cation selective and vice versa.
  • The development of red-light-driven channelrhodopsins, particularly the Volvox carteri-derived variant, has enabled control of multiple individual neurons, advancing optogenetics.
  • The creation of C1V1, a chimera of channelrhodopsin-1, allowed for single-cell resolution control in vivo in mammals, leading to significant advancements in optogenetics.
  • The combination of Volvox-derived opsins and blue-light-actuated calcium sensors facilitated all-optical interrogation of neural circuits, eventually leading to control of mammalian behavior at the level of multiple single cells.
  • Targeting specific brain structures like the orbital frontal cortex with single-cell resolution control has provided insights into the interaction and competition of primary survival drives, such as feeding and social interaction.

03:32:13

"Optogenetic control enhances social and feeding responses"

  • Driving 20 to 25 feeding cells enhances and extends the feeding response to droplets, with each tick representing a lick by the animal.
  • Leaving out the Volvox-derived opsin eliminates the feeding response, indicating it's not a light artifact.
  • Targeting feeding cells specifically affects the appetitive drive, distinct from other cells.
  • Socially responsive cells were identified in head-fixed social behavior experiments.
  • Social cells do not respond to shocking stimuli, indicating their specificity to social interactions.
  • Driving social cells suppresses the feeding response initially in the orbital frontal cortex.
  • Volvox-derived opsins allow control at the individual cell level over mammalian behavior.
  • Care must be taken with light power to avoid damaging cells when controlling more cells.
  • Spatial light modulators enable holograms projected over large areas of the brain for experiments.
  • Optogenetic stimulation of specific cells in visual cortex elicits population dynamics resembling natural visual stimuli, impacting behavioral performance positively even in darkness.

03:45:56

"Neurons and Optogenetics in Brain Control"

  • Neurons can be stimulated to detect and classify stimuli, with layer 5 cells showing more potency than layer 2, 3 cells.
  • Optogenetics can elicit naturalistic brain-wide responses, as shown by experiments using neuropixels probes in different trajectories in animals.
  • A simple task of animals licking for water when thirsty was used to understand brain-wide representations of behavior.
  • More than half of all recorded neurons were modulated by the task, recruiting virtually the whole brain.
  • Properly targeted optogenetic stimulation of thirst neurons led to naturalistic behavioral responses across the brain.
  • Optogenetic targeting can elicit naturalistic dynamics locally and brain-wide, raising questions about brain controllability and structure.
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