OPTOGENETICS

BY:

Akshay Goyal

Kunal Parmani

Akshat Bordia

INTRODUCTION

• Optogenetics is the integration of optics and genetics to achieve gain of previous loss of function of events within cells of living tissue

• Optogenetics (from Greek optos, meaning "visible") uses light to control neurons which have been genetically sensitised to light.

• It is a neuromodulation technique 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 the effects of those manipulations in real-time.

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 articulated by Francis Crick in his Kuffler Lectures at the University of California in San Diego in 1999.

• An early use of light to activate neurons was carried out by Richard Fork and later Rafael Yuste, 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 who employed Drosophila rhodopsin photoreceptors for controlling neural activity in cultured mammalian neurons.

• Then a major breakthrough occured in August 2005, when Karl Deisseroth's laboratory in the Bioengineering Department at Stanford 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).

Why study Optogenetics??

• 1-2 billion people worldwide suffer from:

stroke

addiction

chronic pain

anxiety disorders

epilepsy

Parkinson’s

Alzheimer’s ...

And these disorders of brain can be treated via targeted neuromodulation.

HOW IT WORKS??

• The technique makes use of a certain kind of reagents which are light-sensitive proteins.

• Like: for Spatially-precise neuronal control optogeneticactuators like channelrhodopsin, halorhodopsin, and archaerhodopsin are used.

• For temporally-precise recordings optogeneticsensors are used.

MECHANISM• The genes for light-activated ion channels are

introduced to a population of cells by a human engineered virus

• Which cells express these light-sensitive channels depends on the promoter region of the inserted DNA sequence

• Cells which contain a promoter that can recognize the promoter sequence will express these channels while cells that lack a promoter specific for the sequence will not

• Once the genes have been inserted, it can take 1-2 weeks for them to be fully expressed

• When studies are ready to be run, a fiber optic cable is surgically attached to the top of the skull or inserted near the brain area of interest depending on how close it is to the surface of the brain

• The channels, and in turn the neurons in which they are embedded, can now be controlled by light from the optic cable

SO THE SIX STEPS OF OPTOGENETICS ARE..

• Create a genetic construct

• Insert construct into virus

• Inject virus into mammal

• Insert optrode: fibreoptic cable+electrode

• Laser light opens ion channels in neurons

• Record behavioual results

CHALLENGES

• Transfection methods

The word transfection is a blend of trans- and infection.

Transfection is the process of deliberately introducing nucleic acids into cells. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of material.

• Transfection methods are focused on the construction of vectors and promoters. The molecular biology aspect is a challenge, because of the enormous variety of constructs, which have to be tested by time consuming screening. The virus approach is quick and efficient and has a biomedical implication, whereas the construction of transgenic animals are time consuming but have been proven to be ideal for a variety of different experiments in basic research.

• Improvement of the optogenetic tools

Although the wild type rhodopsin and halorhodopsin work quite well, an improved light sensitivity is important for experiments in the mammalian brain because of its low transmittance and because their activity is influenced by their surroundings.

• Improvement of appropriate light sources

Device like micro pipe lights are sufficient for light stimulation on the surface of the brain but for applications on the dense tissue in the brain, we need better excitation sources to increase the transmittance.

POTENTIAL BENIFITS

• Research is focused on neurological diseases, because all the advantages of the light stimulation contain a promising potential for gene therapy and other benefits.

• The optogenic approach offers for the future an enormous potential for basic research, because nerve excitation and silencing can be performed simply by light with high precision in a reversible manner

1. Cell culture, Network analysisachieved by growing cultured nerve cells on micro or nano patterned substrates. Cells can be stimulated or silenced simply by a light-beam with up to now unknown spatial precision.

2. Mapping of the brain and behaviorChR2 (Channelrhodopsin- retinylidene proteins, light-gated ion channels or sensory photoreceptors) can be used for remote control of neurons. Studies are possible on which certain areas of the brain are stimulated via light pipes. Examples : (i) movement of whisker of rodents; on the olfactory system where light replaces the ligands, and (ii) on the movement of animals after stimulation of the motor cortex.

3. Gene therapy

In the future gene therapy with the optogenetictools appears possible. Transduction via AdenoAssociated Viruses (AAV) (does not cause disease) has beenperformed successfully on the human eye tocure Lebers Congenital Amaurosis (dystrophy of retinadue to malnutrition or disease) , by transduction ofcells in the human retina to replace the missing retinalisomerase. In analogy to this, AAV´s could be loadedwith the microbial rhodopsins and could be used forgene therapy on the diseases like recovery of vision,parkinson disease.

4. Recovery of visionExperiments on photoreceptor deficient mice have

shown that light evokes potentials in the visual cortex after the transduction of the ON bipolar cells with ChR2 in the retina. This indicates that the retina of the animals regained photosensitivity, which is transmitted via the optic nerve to the brain. Trajectories of the movement of the animals in the dark and in the light show clearly an increased activity in the light as it is obtained for wild type animals. It is conceivable that such an approach might be possible for blind humans, suffering e.g. the dry or the wet macular (yellowish central portion of the retina) degeneration.

5. Parkinson disease, Epilepsy

Deep brain stimulation

Deep brain stimulation (or DBS) is a way to inactivate parts of the brain that cause Parkinson's disease and its associated symptoms without purposefully

destroying the brain.

In deep brain stimulation, electrodes are placed in the thalamus (to treat essential tremor and multiple sclerosis) or in the globus pallidus (for Parkinson's disease).

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