Optogenetics
- 2. OPTOGENTETICS Presented By: Zeeshan M.Sc. Physics Understanding the bond of Light with Life
- 4. Introduction… What is Optogenetics? Optogenetics is a biological technique which involves the use of light to control cells in living tissue, typically neurons, that have been genetically modified to express light-sensitive ion channels.
- 5. • It is a neuromodulation method employed in neuroscience that uses a combination of techniques from optics and genetics to control and monitor the activities of individual neurons in living tissue—even within freely-moving animals—and to precisely measure these manipulation effects.
- 6. • The key reagents used in Optogenetics are light-sensitive proteins. • Neuronal control is achieved using optogenetic actuators like channel rhodopsin, halorhodopsin, and archaerhodopsin. • Optical recording of neuronal activities can be made with the help of optogenetic sensors for calcium, vesicular release (synapto-pHluorin), Neurotransmitter (GluSnFRs), or membrane voltage . • Control or recording is confined to genetically defined neurons and performed in a spatiotemporal-specific manner by light. How it is done??
- 7. Understanding Opsin And Rhodopsin….. Opsins: Opsins are a group of light-sensitive proteins found in photoreceptor cells of the retina. Opsins are involved in vision, mediating the conversion of a photon of light into an electrochemical signal.
- 8. Rhodopsins: Rhodopsin (also known as visual purple) is a light- sensitive receptor protein involved in visual phototransduction. It is named after ancient Greek ῥόδον (rhódon) for “rose”, due to its pinkish color, and ὄψις (ópsis) for “sight”. Rhodopsin is a biological pigment found in the rods of the retina. Rhodopsin is extremely sensitive to light, and thus enables vision in low- light conditions. When rhodopsin is exposed to light, it immediately photobleaches.
- 10. History… • The "far-fetched" possibility of using light for selectively controlling precise neural activity (action potential) patterns within subtypes of cells in the brain was thought of by Francis Crick in his Kuffler Lectures at the University of California in San Diego in 1999. • An earlier use of light to activate neurons was carried out by Richard Fork, who demonstrated laser activation of neurons within intact tissue, although not in a genetically-targeted manner. • The earliest genetically targeted method, which used light to control genetically-sensitised neurons, was reported in January 2002 by Boris Zemelman and Gero Miesenböck, who employed rhodopsin photoreceptors for controlling neural activity in cultured mammalian neurons. • In 2003 Zemelman and Miesenböck developed a second method for light- dependent activation of neurons in which single ionotropic channels were gated by caged ligands in response to light.
- 11. History… • 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. However, these earlier approaches were not applied outside the original laboratories, likely because of technical challenges in delivering the multiple component parts required. • Dr. Zhuo-Hua Pan of Wayne State University, researching on restore sight to blindness, thought about using channelrhodopsin when it came out in late 2003. • By February 2004, he was trying channelrhodopsin out in ganglion cells — the neurons in our eyes that connect directly to the brain — that he had cultured in a dish. • They became electrically active in response to light. Over the moon with excitement, Pan applied for a grant from the National Institutes of Health. The NIH awarded him $300,000, with the comment that his research was "quite an unprecedented, highly innovative proposal, bordering on the unknown." Continued…
- 12. History…. • 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, beginning in cultured mammalian neurons using channelrhodopsin, a single-component light-activated cation channel from unicellular algae.
- 14. Implementation….
- 15. Channelrhodopsin-2 (ChR2) induces temporally precise blue light-driven activity in rat prelimbic prefrontal cortical neurons. a) In vitro schematic (left) showing blue light delivery and whole-cell patch-clamp recording of light-evoked activity from a fluorescent CaMKllα::ChR2-EYFP expressing pyramidal neuron (right) in an acute brain slice. b) In vivo schematic (left) showing blue light (473 nm) delivery and single-unit recording. (bottom left) Coronal brain slice showing expression of CaMKllα::ChR2-EYFP in the prelimbic region. Light blue arrow shows tip of the optical fiber; black arrow shows tip of the recording electrode (left). White bar, 100 µm. (bottom right) In vivo light recording of prefrontal cortical neuron in a transduced CaMKllα::ChR2-EYFP rat showing light-evoked spiking to 20 Hz delivery of blue light pulses (right). Inset, representative light-evoked single-unit response. Implementation….
- 17. Well one more clip to get the feel of…. A nematode expressing ChR2 in its gubernacular-oblique muscle group responding to stimulation by blue light. Blue light stimulation causes the gubernacular-oblique muscles to repeatedly contract, causing repetitive thrusts of the spicule, as would be seen naturally during copulation
- 18. Technique…
- 19. • The technique of using optogenetics is flexible and adaptable to the experimenter's needs. For starters, experimenters genetically engineer a microbial opsin based on the gating properties (rate of excitability, refractory period, etc..) required for the experiment. • There is a challenge in introducing the microbial opsin, an optogenetic actuator, into a specific region of the organism in question. A rudimentary approach is to introduce an engineered viral vector that contains the optogenetic actuator gene attached to a recognizable promoter such as CAMKIIα. This allows for some level of specificity as cells that already contain and can translate the given promoter will be infected with the viral vector and hopefully express the optogenetic actuator gene. • Another approach is the creation of transgenic mice where the optogenetic actuator gene is introduced into mice zygotes with a given promoter, most commonly Thy1. Introduction of the optogenetic actuator at an early stage allows for a larger genetic code to be incorporated and as a result, increases the specificity of cells to be infected.
- 20. • A third and rather novel approach that has been developed is creating transgenic mice with Cre-Recombinase, an enzyme that catalyzes recombination between two lox-P sites. Then by introducing an engineered viral vector containing the optogenetic actuator gene in between two lox-P sites, only the cells containing the Cre-Recombinase will express the microbial opsin. This last technique has allowed for multiple modified optogenetic actuators to be used without the need to create a whole line of transgenic animals every time a new microbial opsin is needed. • After the introduction and expression of the microbial opsin, depending on the type of analysis being performed, application of light can be placed at the terminal ends or the main region where the infected cells are situated. Light stimulation can be performed with a vast array of instruments from light emitting diodes (LEDs) or diode-pumped solid state (DPSS). These light sources are most commonly connected to a computer through a fiber optic cable. Recent advances include the advent of wireless head-mounted devices that also apply LED to targeted areas and as a result give the animal more freedom of mobility to reproduce in vivo results
- 21. Story so far Nut-Shell…. Three primary components in the application of Optogenetics are as follows (A) Identification or synthesis of a light- sensitive protein (Opsin) such as Channelrhodopsin-2 (ChR2),Halorhodopsin (NpHR), etc... (B) The design of a system to introduce the genetic material containing the opsin into cells for protein expression such as application of Cre- Recombinase or an Adeno-Associated-Virus (C) Application of light emitting instruments.
- 23. Applications… • The field of optogenetics has furthered the fundamental scientific understanding of how specific cell types contribute to the function of biological tissues such as neural circuits in vivo. • Moreover, on the clinical side, optogenetics-driven research has led to insights into Parkinson's disease and other neurological and psychiatric disorders. • Indeed, optogenetics papers in 2009 have also provided insight into neural codes relevant to autism, Schizophrenia, drug abuse, anxiety, and depression.
- 25. Examples : Amygdala • Optogenetic approaches have been used to map neural circuits in the amygdala that contribute to fear conditioning. • One such example of a neural circuit is the connection made from the basolateral amygdala to the dorsal- medial prefrontal cortex where neuronal oscillations of 4 Hz have been observed in correlation to fear induced freezing behaviours in mice.
- 26. Examples: Olfactory Bulb • Optogenetic activation of olfactory sensory neurons was critical for demonstrating timing in odour processing and for mechanism of neuromodulatory mediated olfactory guided behaviours (e.g. aggression, mating). In addition, with the aid of optogenetics, evidence has been reproduced to show that the "afterimage" of odours is concentrated more centrally around the olfactory bulb rather than on the periphery where the olfactory receptor neurons would be located
- 27. Example: Heart • Optogenetics was applied on atrial cardiomyocytes to end spiral wave irregular heartbeats, found to occur in irregular heart beat, with light. • This method is still in the development stage. A recent study explored the possibilities of optogenetics as a method to correct for arrhythmias and resynchronize cardiac pacing.
- 28. Example: Spiral ganglion • Optogenetic stimulation of the spiral ganglion in deaf mice restored auditory activity.Optogenetic application onto the cochlear region allows for the stimulation or inhibition of the spiral ganglion cells (SGN). • In addition, due to the characteristics of the resting potentials of SGN's, different variants of the protein Channelrhodopsin-2 have been employed such as Chronos and CatCh. • Chronos and CatCh variants are particularly useful in that they have less time spent in their deactivated states, which allow for more activity with less bursts of blue light emitted. • The result being that the LED producing the light would require less energy and the idea of cochlear prosthetics in association with photo-stimulation, would be more feasible.
- 30. Summary In this presentation We learnt- • Basic idea of Optogenetics. • Historical development. • How is it implemented. • Techniques involved. • Real Life Examples.
- 31. Reviewing What we learnt……..
- 32. Thank You Special Thanks to : Prof. Sukhdev Roy Dept. of Physics and Comp Sci. DEI Dr. K.S. Daya Dept. of Physics and Comp Sci. DEI Motivated by talks of Prof. Vibha R. Satsangi
- 33. Questions and Doubts are Welcome…..