Optogenetics And GECIs Flashcards
Optogenetics
Activate a target of specific neuronal population and other cells of the neurovascular unit
It’s a disruptive technology
Now the go to technology for looking at NVC and neurophysiology across the whole brain in terms of looking at the system of neuroscience
What does optogenetics do?
Insert microbial proteins (ion channels aka opsins) into target cells which respond to light of a specific wavelength and open the channel
Can be used to cause a cell to depolarize and become active OR cause inhibition
Excitation = channelrhodopsin; responds to blue light; forces positive ions into cell and cause it to fire, activating cell of choice with an AP
Inhibition = halorodopsin; responds to yellow light; forces chloride ions into cells, hyper polarising it and stops it firing
As well as expressing the opsin you often express a fluorescent protein to allow cell identification
How do you get opsins into target cell?
Use a virus ie adeno-associated virus (AAV) - can inject it into the brain, not known to cause any disease
Possible to load the virus with genetic information to allow the expression of the opsin once inside the target cell
How do you get the virus into the correct system?
Direct injection of AAV into wild type animal brain
Direct injection of AAV into cre-recombinase transgenic animal
No injection - breeding of transgenic mouse strains
In-utero electroporation
1 - Direct injection of AAV into wild animal brain
Virus targets only 1 type of cell by expressing promotors that are only expressed in cell population of choice
CaMKIIa - virus biased to excitatory cortical cells
VGAT - target all GABA inhibitory neurons
2 - Direct injection into a cre-recombinase transgenic animal
Transgenic mouse only expresses cre-recombinase in specific set of neurons and expression of injected opsin will only happen in cells where this is present
Well targeted expression in cell of choice and way to guarantee only have 1 population of neurons labelled
But if used in AD mice the injection can cause artefacts (ie inflammation) and change the disease
3 - Breeding of transgenic mouse strains
No injection
Cross cre-recombinase in somatostatin-expressing neurons with mice with improved channelrhodopsin expression
Crossing these will mean most offspring are ready for blue light stimulation - same as 2 just breeding together rather than injecting
4 - In-utero electroporation
Directly inject genetic material into embryo’s ventricular system
Electrical current forces DNA to move through embryo into cells that are differentiating
Doesn’t use AAVs - targeting ability can be increased in size
Timing = very important - cells transferred are the ones differentiating at time of infection
Optogenetic stimulation can drive fMRI BOLD signals
Inject AAV expressing excitatory neurons in rat motor cortex
After 10 days, transfection has taken place so put animal in the magnet to do BOLD fMRI imaging with a laser to stimulate cells via fibre optic cable
Before the fMRI did electrophysiology to show that when the light was on it caused spiking of cells at the same frequency = proof of principle excitatory neurons are active in rats when do this experiment
Showed if excite excitatory neurons it causes the BOLD signal, had the typical triphasic signal - compared to saline controls
Nice positive linkage between excitatory neurons and positive BOLD - shows potential to map brain circuits by combining optogenetics and fMRI but didn’t show much on NVC mechanisms
Optogenetics of excitatory cells produces positive BOLD responses, what about inhibitory cells?
Identified PV interneurons and where they were activated there was a positive BOLD signal with a surround negative BOLD signal = inhibitory cell firing still drove dilation in blood flow and and negative surround BOLD signal suggested PV interneurons did something to the surrounding area to cause constrictions and and increase in deoxyH
Role of interneurons in NVC - 1
Combined mouse with cre-recombinase and the AAV injection with PV interneurons at site of injection before taking this area out and doing slice experiments
Used light stimulation to active PV neurons = prolonged activation = more proof inhibitory interneurons can cause negative BOLD signals and constrictions
Role of interneurons in NVC - 2
VAGT interneurons
Measured blood flow responses with laser speckle imaging and single channel electrodes to measure neuronal activity
Stimulation of GABA interneurons caused inhibition of excitatory cells - found this is true as lack of firing when stimulation = proof optogenetics is working
Then tested if glutamate antagonists could block optogenetics response - forepaw response; inhibited relapse of glutamate so neuronal activity changed dramatically which inhibited haemodynamic response
^ when did this on optogenetics the response was the same = optogenetic response is not linked to glutamatergic activity of cells - release of NOS from these cells may be causing dilation?
Combining optogenetics with 2-photon imaging
Titratef responses against the size of a whisker stimulation
VGAT interneurons stimulation and optogenetics, got post stimulus undershoot so what’s causing this?
Looked at this mechanism by blocking receptors for one type of cortical interneurons and suggested the post stimulus undershoot maybe be under the control of cortical interneurons
Astrocyte activation induces large CBF increases
Blood flow measured by laser speckle imagine
When astrocytes are activated there were large increases in CBF
Suggests astrocytes are important for NVC - but not many studies investigating this
Channelrhodopsin activation contracts pericytes and reduces blood flow
Prolonged stimulated of a pericytes produced slow decrease in CBF - Argues that pericytes aren’t involved in NVC but instead cause slow changes in blood flow which could be important for disease - cause changes in baseline blood flow which contribute to the disease
Stimulation of cortical pericytes leads to a slow constriction of capillaries
Strongly argues pericytes aren’t involved in NVC to a stimulus but instead control baseline blood flow which could be important for disease
Potential confounds of optogenetics?
Specificity of the targeting - highly unlikely you’d just be activating 1 cell population
Using a laser to activate optogenetics causes strong increases in heat which can cause artefacts in the brain in MRI and generate positive and negative BOLD signals just by changing the heat signals in the brain
Animals without injected opsins had a response when used a titrated laser (less power) - an inherent opsin in blood vessels (light sensitive protein) that is already activated when shine light on them without artificial injected ones
Summary on optogenetics to understand neuroimaging and NVC
Offers unprecedented opportunity to understand which cell type drives the BOLD response and how cortical circuits operate
All experiments so far have labelled larger populations of cells so labelling more specific populations will lead to greater understanding
Optogenetics have been done in parallel with sensory stimulation - more gain or loss of function experiments are needed to tease apart the roles different cells play in NVC
Genetically Encoded Calcium Indicators
Involves insertion of green fluorescent protein (GFP) which binds to calmodulin of target cell
When calcium increases in a cell (it’s active) it binds to calmodulin which causes conformational changes in protein, activating GFP and causes cells to fluoresce
This can be measured in a genra or 2-photon microscope - don’t need electrode to measure neural activity
Breakthrough compounds is known as GCAMP6
GCAMP6 imaging simultaneously with 2D-OIS
Allows you to be non-invasive and do it in awake animals
GCAMP signal has haemodynamic artefact so by measuring blood we can remove this effect
Connectivity based research
Allows more research into understand physiology basis of cortical connectivity as neuronal signal cannot be measured in same spatial way as haemodynamics
Astrocytes can be labelled with GCAMP
Only astrocytes will fluoresce - and do it before blood flow increase suggesting astrocytes are involved in NVC (at least in olfactory bulb where this was studied)
Nice use of these technologies to drive neurovascular response and measure it
The future of an all optical approach to experimentation
At present both optogenetics and GCAMP6 use the same wavelength of light as the illumination source so can’t do both at same time
New compounds that are activated or fluoresce in high wavelengths will allow experiments using all optical approaches - ie recording GCAMP in blue light and manipulation (optogenetics) in red light.
Allows remote stimulation and recording in the same experiment
Gives great control in terms of looking at neurophysiology
