Optogenetics and GECIs Flashcards
What is disruptive technology?
A disruptive technology is one that displaces an established technology and shakes up the industry or a ground-breaking product that creates a completely new industry
What is optogenetics?
It is the insertion of microbial proteins (ion channels) into the target cells of choice - these proteins are called opsins and they respond to light of a specific wavelength to open the channel
These can be used to cause a cell to depolarise and become active OR they can be used to prevent a cell from firing - inhibition
What causes excitation and inhibition in optogenetics?
Excitation = channelrhodopsin (responses to blue light)
Inhibition = Halorhodopsin (responses to yellow light)
What allows cell identification in optogenetics?
As well as expressing the opsin you express a fluorescent protein to allow cell identification
How do you get the opsins you want into the cell of choice?
Best way to do it is use a virus called Adeno-associated virus (AAV)
Very small virus known to infect humans and animals not known to cause any disease
It is possible to ‘load’ up this virus with the genetic information to allow the expression of the opsin once inside the cell of choice
How do you get the virus into the correct system using injections?
Method 1 = direct injection into a wildtype animal - the virus targets only one type of cell by expressing promoters that are only expressed in the cell population of choice
Method 2 = direct injection into a cre-recombinase transgenic animal - you have a mouse that only expresses an enzyme called cre-recombinase in a specific set of neurons e.g., somatostatin interneurons
The AAV will go into all neurons in the injected area but the expression of the opsin will only happen in cells where cre-recombinase is present
What promoter is used for excitatory cells in the cortical region?
CaMKIIa
What promoter is used for inhibitory cells in the cortex?
VGAT - targets all GABA inhibitory neurons
What method of getting the virus into the correct system involves no injection?
Method 3 = no injection - breeding of transgenic mouse strains
Cross a cre-recombinase somatostatin-expressing mouse with a mouse with that expresses channelrhodopsin/EYFP protein following exposure to cre-recombinase
By crossing these two strains of mice, 50% of all offspring will have channelrhodopsin and EYFP in SOM interneurons
What is another method of getting the virus into the correct system?
Method 4 = in-utero electroporation
Injection of your genetic material directly into the embryo’s ventrical system
The electric current forces the DNA to move through the embryo and into the cells that are differentiating
Doesn’t use AAVs - so the promotor or targeting ability can be increased in size
Timing is really important - the cells transfected are the ones differentiating at the time of infection
What has research by Lee found about optogenetic stimulation of excitatory cells in BOLD responses?
Injected AAV - CaMKIIa - expressing excitatory neurons in the rat motor cortex
Waited 10 days for transfection to take place then put the animal in the magnet to do BOLD fMRI imaging with a laser to stimulate the cells via fibre optic cable
Did electrophysiology first to show that when the light was on it caused spiking of cells at the same frequency
The BOLD signal was very similar to sensory stimulation - excitatory cells produces positive BOLD response
Even showed a post-stimulus undershoot
What else did Lee show in excitatory cells?
Caused BOLD changes along top down projections
Showed that projections of the motor cortex were also activated by the motor cortical stimulation
Shows the potential power to map circuits in the brain by combining optogenetics and fMRI - but it didn’t show much on mechanism of NVC - NVC comes from sensory stimulation
What did Lee find about inhibitory cells?
Parvalbumin (PV) interneurons (GABAergic) produced a central positive BOLD response with a surround negative region - similar to our rat sensory stimulation experiments
What did Urban et al find about the role of interneurons in NVC?
First experiments were in slice experiments - Used Parvalbumin - cre mouse and injected AAV vector to place channelrhodopsin and EYFP (fluorescent) into PV interneurons at the site of injection - then took this area of the brain out and performed slice experiments
Used light stimulation to activate the PV neurons
Again like all slice research the responses were very slow but it does seem to fit with Lee’s surround negative finding
How have other groups (Anenberg et al) tested the role of interneurons in NVC?
Focussed on activating all GABAergic interneurons in the brain in vivo - using the VGAT labelling approach
Used VGAT-ChR2-YFP mice bought from Jax labs
Measured blood flow responses with Laser Speckle imaging and used a single channel electrode to measure neuronal activity
Showed with electrophysiology that stimulation of the GABA cells stopped spontaneous cortical firing
Blood flow showed robust increases in response to a brief stimulation
They then used pharmacology to see if they could block the optogenetic response
Tested the glutamate antagonists on the forepaw response first - worked well - good proof of the principal - strongly reduced the neuronal responses and the subsequent haemodynamics
However the optogenetic response was largely preserved - they suggested it might be nitric oxide
