in Vivo and ex Vivo Imaging Techniques
Compare adeno-associated virus (AAV) and Simple Retrovirus (MMLV).
AAV: most commonly used virus
Single stranded DNA
Neurotropic (if you inject it into the brain it will affect neurons/ preferentially affects nervous system)
Episomal: not integrated into the host genome, maintained at the cell
Insert size <5kb: small amount of cargo you can but into the virus (one or two proteins or small proteins)
Slow getting the expression into animals
Long-term expression
MMLV Retrovirus: Used for developmental neuroscience because it affects dividing cells
Once it's incorporated into the cell it will express it for the rest of the cell's life because it is integrated into the host genome
Single stranded RNA
Larger insert size (8kb)
Long term expression
Explain "serotype."
Refers to the surface structure of the virus. Different serotypes will confer different tropisms, specifically for different types of tissue.
Define "tropism."
The ability of different viruses to infect different cellular types ultimately to produce a successful infection
Explain "promoter."
Confers targeting specificity
Helps viruses target neurons (i.e., can help target inhibitory, glial cells, etc)
Explain "open reading frame."
"Cargo" to be expressed
Where you put the sequence for whatever protein you want to express in the cell
Can have a second open reading frame if you want to express additional proteins
Explain "linker."
Allows for expression of multiple open reading frames.
Allows genes to be transcribed and will separate them into two separate proteins
Explain "regulatory element."
Enchances viral expression and makes it work better. It is not required
Define "structural connectivity."
Existence of a physical connection between brain regions. Ex. axon projections, white matter tracts, synaptic contacts
Define "functional Connectivity."
Statistical dependencies between brain regions. Ex. correlated activity
Define "effective connectivity."
Stimulation of one region induces activation or inhibition in another region.
What consitutes a network?
Structural, functional, and effective connectivity.
In a fluorescent protein, what is responsible for light emission?
Chromophore. You can change this to get different wave lengths.
What is responsible for diversity of emission colours in fluorescent proteins?
Chromophore structural differences are responsible for diversity of emission colours.
Explain "GCaMP."
GCaMP is an engineered genetically encoded calcium indicator that reports changes in calcium based on a fusion between GFP and the calcium binding protein, calmodulin. When calmodulin binds to Ca, a conformational change causes an increase in GFP fluorescence.
Explain "GRAB sensors."
GRAB (GPCR Activation Based) sensors are designed to measure presence of different neurochemicals.
When neurochemicals bind to the GPCR coupled to the cpGFP molecule or cpRFP, it causes a conformational change in the GFP which causes an increase in fluorescence.
Explain "SnFR sensors."
SnFR sensors (sensing fluorescent reporter) are designed to detect glutamate. GFP molecules have a slit in them so when you attach the bacterial glutamate binding protein, glutamate comes in a causes a conformational change that pulls the GFP molecule together and increases fluorescence.
You can replace the glutamate sensor for other sensors to detect other things (GABA, Ach, Glucose).
Explain "genetically encoded voltage indicators."
GEVI sensors sense voltage. The typically exhibit a very small signal to noise signal making them difficult to use for in vivo and population based work.
Explain fiber photometry
A way to record fluorescence. Similar to optogenetics, but rather than stimulate neurons with light, we record emitted light from neurons. Take a fibre optic probe and implant it into the region of the brain that is expressing the virus, connect that fibre through a cable to an optical sensor/detector (i.e., a camera).
Explain mutli-region fiber photometry.
Fibers are implanted into multiple regions and can be used to correlate simultaneously collected signals during behavioural tasks. This is a way to see how functionally connected regions are. It is a small scale version of what you can get with an fMRI. Limited to the number of regions that can be targeted simultaneously.
Explain array based fibre photometry.
High density fibre arrays allow for correlated activity recordings from many regions or subregions. This allows for increased number of regions but lower flexibility of region choices. Integrate this activity to look at functional connectivity.
Explain multi-channel fibre photometry
Used to look at different populations of neurons in one region and how they interact.
List pros and cons of fibre photometry.
Pros:
Minimally invasive compared to other in vivo imaging techniques
Cell type specific targeting
Population activity
Cons:
Targets a relatively small tissue area
Population activity: What does a change in activity indicate? More cells active vs. cells more active?
Explain multiphoton imaging
It allows for large scale imaging of active neurons or cortex. You inplant a head bar in the animal, fix the animal to the imaging set up, put microscope over the head ad record the activity of the cells.
Limited to imaging cells with the animal in one place and not able to image deeper structures of the brain
Explain in vivo imaging in freely moving mice
This records single cell activity compared to photometry that looks at populations. To do this you implant a lens into the brain which will relay where spatial information is, use it to monitor single cell activity. Attach a microscope to the head too. This allows you to see changes in fluorescence of individuals neurons rather than populations. Pro: the animal can move around freely so you can see how neurons react with certain behaviours.
Explain place cell activity.
Place cells are cells in the hippocampus which are active when animals are in a certain place in the environment. They are important for spacial navigation and informing spatial maps of the environment. Different neurons have different place fields. Can be imaged with calcium indicators and mini scopes instead of using electrophysiology. Cons: Limited to how much you can see
List the pros and cons of two-photon microscopy.
Pros:
Can look at deep structures
Can look at many neurons at the same time
Can look at 3D tissue
Cons:
Behaviour you get with these approaches are still contrained, not accurate to how they would respond in real life.
Explain channelrhodopsins
They are blue light sensitive non-selective cation/ light-gated ion channel. If you shine light on it, it causes an influx of cations.
You can use ChR2 in mammalian neurons to activate them in vivo.
Explain how optogenetics work
Take a light sensitive protein from algae (protein is an ion channel that opens in response to blue light). Take the gene for this protein and insert the DNA into specific neurons in the brain. Now you can cause neurons to fire or not to fire just by flashing blue light.
Pro:
Has nice temporal control
Con:
Limited to small area where light hits
Explain chemogenetics
Chemical or drug based stimulation with cell specific targeting Ex. DREADDs.
DREADDs:
Artificial receptors designed based on GPCRs, there are excitatory and inhibitory versions, and can be expressed in transgenic mice or delivered using viruses. They are activated by administration of specific artificial ligands.
Cons:
Slower in response and not as potent as optogenetics
Doesn't completely shut down response, may inhibit neurons but they can still be activated.
Compare optogenetics and chemogenetics
Opto:
Activation or inhibition of neuronal activity
Much higher temporal precision
Stimulation area is typically quite limited
Potential side effects of light stimulation (e.g., heat)
Chemo: Modulation of neuronal activity
Better for chronix or long temporal activation
Larger stimulation area is possible
Potential side effects of agonists
Explain the steps for tissue analysis
1. Get tissue sample
2. Processing and embedding
3. Sectioning
4. Data and statistical analysis
5. Visualization and image analysis
6. Antigen retrieval and antibody staining
If you have lots of tissue to analyze you can go from step 1 to 5.
What is CLARITY and what is it used for in examining brain tissue?
It is a tissue clearning technique used to make the brain tissue transparent. It is a hydrogel mix. Hydrogel binds proteins, but not lipids. Take brain and put it in strong detergent and electrophoretic gradient. The lipids are driven out by electric field and you're left with a transparent brain. It preserves anatomical structures, proteins and nucleic acids.
What are the two general concepts of CLAIRITY tissue clearning method?
1. Decrease opacity of the tissue
Do that by removing lipids.
2. Refractive index matching
When light travels through something it will bend. To accomplish refractive index matching, you but the brain into a solution that has a refractive index that matches the brain tissue.
What are the pros and cons of tissue clearing?
Pros:
Whole organ quantification
Fiber/projection tracing
Neuron morphology (able to look at whole neurons without cutting them)
Visualization (able to look at full organs and organisms)
-Can use this to look at relative changes in volume of stuctures (i.e., how much nutrient deprivation affects different organs in development)
Cons:
Generates massive amounts of data
Can be very time consuming
Learge volumes of antibody required (if immuno-labelling)
Some tissue deformation occurs with all clearing techniques
Specialized imaging equipment needed
How do you image cleared tissue? Name the microscopes.
Choices are limited by what you have available and how much tissue you can fit under your microscope.
Lightsheet: often the best choice for large clearned samples but they aren't very common
2-Photon: can be used for deep imaging, enhanced by tissue clearing - slower than lightsheet
Confocal: can be used to image smaller pieces of cleared tissue but you may need specialized objectives to increase working distance
Epifluorescence: should proably be considered your last resort but can be used especially if combined with deconvolution
Explain single plane illumination microscopy (SPIM)
Epifluorescent microscope
Lightsheet microscope: Laser comes in from each side of the sample, it is narrowed in the middle so a thin sheet of light covers the specimine in XY plain and gives thin Z plain. Specimine is moved through thr sheet of light and excitation from that comes up through the lens. You can get the whole XY portion of the image by moving the specimine.
What is the process for whole-brain imaging of active neurons?
1. Have animal perform behavioural task
2. Perfuse the brain and clear the tissue
3. Label for CFOS
4. Image the brain using lightsheet microscopy (for example)
5. Quantification: Now you can see all the neurons active in that tissue
6. Atlas registration: Register tissue to an atlas
7. Analysis: Count number of active neurons in each region, which can help create a correlation matrix.
How do you measure functional activity after whole-brain imaging process?
Look at correlation matrix region by region, if no correlation than no connection.
If c-fos expression is significantly correlated across mice = functional connectivity.
Look at all regions that have functional connectivity and make a network graph of activity.