The basics for symbiosis of Optics and Genetics have been explained in this presentation. " How light can change the very way of life?" .This question has been addressed using relevant web content, consultations from book and through nature videos. This presentation was awarded the highest score in PHM805 at Dayalbagh Educational Institute, Agra.

2. OPTOGENTETICS
Presented By:
Zeeshan
M.Sc. Physics
Understanding the bond of Light with Life

3. Presentation Overview…
•Introduction
•History
•Implementation
•Applications
•Summary

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.

9. Presentation Overview…
Introduction
•History
•Implementation
•Applications
•Summary

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.

13. Presentation Overview…
Introduction
History
•Implementation
•Applications
•Summary

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.

22. Presentation Overview…
Introduction
History
Implementation
•Applications
•Summary

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.

29. Presentation Overview…
Introduction
History
Implementation
Applications
•Summary

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…..