Traditional and contemporary lab techniques for mapping brain circuitry Flashcards
What is connectomics? Goal?
Branch of neuroscience research. The ultimate goal of connectomics is to generate a wiring diagram, similar to an electrical circuitry, and that this similarity is not a coincidence.
Which method gives similar results to the one with the Golgi method, but with the added benefit that neurons can be targeted with high specificity?
Biocytin staining using the patch-clamp technique.
Describe 4 steps of the Biocytin staining method.
Describe 1 limitation of the Biocytin staining method.
The process requires that cells are still alive at the time of injection, and so this is also a limitation of this technique because the brain has to be first sectioned, sections have to be kept alive in a culture medium, and subsequently patched and injected with biocytin.
What are lipophilic dyes?
Which variance is the most used?
These are fluorescent molecules, highly soluble in the fatty lipid bilayer of the cell membrane.
There are several variances of lipophilic dyes, with DiI the most widely used. But others with different emission wavelengths have also been developed so that different axonal tracks can be labelled within the same brain. DiI and its variance diffuse passively along the cell membrane, eventually lighting up even very long axonal projections, such as the one in the figure that depicts axons originating in the visual tunnels and extending all the way to the visual cortex.
How components A and B are formed after vibrio cholera ingestion?
A compound that is commonly used to trace axonal projections is derived from the pathogenic (causing disease) toxin of the bacterium vibrio cholera. The toxin normally affects cells in the intestine and leads to severe dehydration, which can be lethal. After ingestion, vibrio cholera colonises the small intestine and secretes cholera toxin, which is formed of two components known as A and B.
What is the difference between components A and B?
What is Cholera toxin subunit B (CTB)?
Now, in fact, toxicity requires only component A, because this activates iron channels on the cell membrane, leading up to water being extruded from the cell and resulting in dehydration.
The B component instead is required for the internalisation into a cell. So, it was rather ingenious to use just a cholera toxin B subunit to label axonal projections without having the pathological implications of the entire toxin.
After binding to receptors on the cell surface, the toxin is endocytosed and travels to the endoplasmic reticulum via a retrograde pathway.
How we can see the below axons?
So, in the figure, we can see cholera toxin B. CTB chemically conjugated with a green fluorescent molecule that has been injected into the hippocampus. n the hippocampus, it’s being picked up by axons from the entorhinal cortex and transported back to the respective neurons somas.
What is an anterograde tracer?
An anterograde tracer could be defined as one that spreads in the same direction as the flow information in a neuron within a network.
When an anterograde tracer is injected into area where neural cell bodies are present, it will spread towards the tip of the axon.
What is the retrograde tracer?
A retrograde tracer works moving against the direction of the information flow. So, when injected into a particular area of the brain, it would be picked up by axon terminals in that region, and over time, spread to neuronal cell bodies that send projections to that region.
Which compounds are strictly retrogradely transported (2)?
Retrobeads are latex microspheres;
FluoroGold is also known as hydroxystilbamidine
What do we see in the picture?
Retrobeads are injected into the hippocampus. The beads are picked up by axon terminals of neurons that innervate the hippocampus.
Their precise neuronal identity can be determined by immunoreaction for a noradrenergic marker (TH).
Some of these TH-positive neurons contain red labelled retrobeads in their soma, confirming that their axonal target is the hippocampus
What molecules are transported anterogradely?
Plant lectins. In this case, the compound belongs to a family of proteins normally found in plants and are known as lectins. Lectins bind ubiquitous receptors on the cell membrane and are then transported anterogradely to the tip of the axon.
How lectins best delivered?
As lectins are proteins, they’re best delivered by a genetic vector, such as a modified virus or a plasmid, that can express the mRNA for a particular lectin, in order for this to be translated into the anterograde transsynaptic protein within the neuron.
Which is the best-known vector to deliver exogenous* DNA sequences into neurons?
How does it work?
*(growing or originating from outside an organism)
The recombinant adeno-associated virus (rAAV).
The genome of the virus has been modified so that most of its original content is removed and replaced by our gene of interest. For instance, this could be a fluorescent protein, like the green fluorescent protein, GFP
Why AAVs are not thought to be pathogenic and are therefore a valid tool for gene therapy in humans?
Because recombinant AAVs are not transsynaptic and cannot infect other neurons other than those they entered at the first infection event.
What is CLARITY?
The innovative tissue-clearing technique is known as clarity, and works by removing lipids from tissue, therefore facilitating penetration of the scanning laser light deep into the tissue.
What is Cre? How does it work?
What is loxP?
Cre is a recombinase* which is normally found in simple viruses that infect bacteria. Cre works by recognising a specific DNA sequence known as the loxP site and cuts the double-strand DNA molecule as indicated in the figure by the two black arrows.
As you can see, the loxP sequence contains two left and right palindromic regions surrounding a unique region in between.
*Recombinases are enzymes that catalyse site-specific recombination events within DNA; for example, genetic recombination during meiosis in which recombination serves to generate new combinations of alleles on chromosomes.
What is Inversion?
What is deletion/excision?
Whats has been shown here?
The organisation of a transgene that contains cDNAs encoding green, yellow, red and blue fluorescent proteins.
The YFP and the CFP are in opposite orientation relative to the promoter (CMV/Thy1) and therefore cannot be translated into the respective proteins.
What has been depicted here?
So, without Cre activity, the transgene can only express GFP. Neurons that express this particular construct, but have no Cre, would therefore appear green under fluorescent light. If Cre activity is present, then several alternative outcomes can take place, depending on which loxP sites are selected for recombination and on their relative orientation.
We can see how two incompatible lox sites can be used in combination to induce transgenic expression of a fluorescent protein only when the Cre recombinase is also present in the same cell.
So here, the black and white triangles indicate the two types of incompatible lox sites. In absence of Cre, no fluorescent protein can be expressed. But if Cre is present, then the resolution of all possible recombination events leads to the inversion of the cDNA expressing the fluorescent protein, which can now light up those neurons that contain Cre, but actually remains silent in neurons that do not contain Cre.
What is non-transsynaptic tracing system?
Two sequential steps of tracer injection and retrograde tracing are required and false positives can be detected when unrelated neurons project to the site of the second injection so that one is led to believe that, say neuron D projects to region A, whilst in reality, only neuron C does.
What is transsynaptic tracing system?
With a transsynaptic viral tracer, only one injection is needed, as the virus will then cross from post to pre synapse infecting the afferents neurons only.
What modification steps are taken to be able to use the rabies virus?
- Removing one gene from the genome of the virus. The gene encodes for a capsid protein and is known as the G protein or glycoprotein. The gene is essential for viral infectivity and is also essential to spread across the synapse
- Removing the G gene, and replacing these with one encoding a fluorescent protein, say GFP, so that the infected neurons can easily be detected
What is viral pseudotyping?
Pseudotyping refers to the use of envelopes from different viruses. Viral pseudotyping changes the tropism of the virus. In the case illustrated, the rabies virus, which is a mammalian virus, changes tropism to avian cells because the envelope used is from an avian virus.
Monosynaptic retrograde tracing
Monosynaptic retrograde tracing 2
Whats is Channelrhodopsin (ChR2)?
So, the first molecule that we see on the left side of the slide is called channelrhodopsin, and channelrhodopsin is an ion channel that is opened or activated when it is illuminated by blue light, so in the blue light part of the spectrum. Upon opening, channelrhodopsin allows the entry of positive ions in the cell. So essentially, the end result of that is that cells become depolarised and can fire action potentials. So, this is an activator of neurons.
halorhodopsin
halorhodopsin has the opposite effect and in fact upon activation from light (and in this case, the channel is sensitive to yellow light), it will facilitate entry of negative ions in the cells. So, the end result will be hydropolarisation of the cell, and so suppression of firing and suppression of generation of action potentials.
What is optogenetics?
Is the process of controlling neurons using light.
the two channels respond to different wavelengths - channelrhodopsin to blue light, halorhodopsin to yellow light - and this means that they can be used in combination in the same neuron. This is nicely illustrated in the last panel, bottom right, where we see how individual pulses of blue light generate action potentials. But, if these are then overlapped with exposure to yellow light, then these action potentials are suppressed. So, as I mentioned previously, optogenetics has really revolutionised the way we study brain function because now we have a possibility to control activation or inhibition of neurons
