Preclinical Approaches Flashcards
What is a disruptive technology?
“Disruptive” technology: typically displaces an established technology, ground-breaking
How do we make genetic manipulations?
Knock out models or knock in models
What is a knock out model?
Inactivate a targeted gene
E.g., the serotonin transporter knockout mouse lacks the gene that makes the protein key for re-uptake (transport) of serotonin – so serotonin levels are elevated
What are global knock outs critical for?
Critical in initial evaluation of role of a gene in brain function
Is there convincing evidence for knock out models?
Conflicting results have been obtained when using KO mice in research
What are the conflicting effects likely caused by?
Likely due to compensation effects
What are knock in models?
Replace original DNA sequence with a modified version, to alter function of coding gene
What are humanised mutations?
Mouse has a human gene inserted into its genome. Common in research of neurodegenerative diseases
What are some examples of humanised mutations?
The APP knockin mouse has an extra gene that causes overproduction of the protein amyloid – this has a prominent role in Alzheimer’s disease (hAPP mice have the human gene inserted)
What is a limitation of global gene manipulations?
A lack of specificity in time and space - compensation/fatal
What is another way to make genetic manipulations aside from knock out models?
Targeting specific cell types
- Induce or suppress expression of a gene of interest using a cell type specific promoter to control mutation - only expression in selective cell types
What are some common promoter lines for neurons?
CaMKII (calcium/calmodulin-dependent kinase II alpha gene): excitatory neurons in neocortex and hippocampus
Human synapsin 1
Platelet-derived growth factor beta chain
Is selection of neuron sub-types possible?
Yes ==
VGAT for GABAergic neurons
Is selection of non-neuronal cells possible?
Yes ==
GFAP for astrocytes
What does GFAP stand for?
Glial fibrillary acidic protein
What is conditional expression?
Conditionally expressed genes have the property that every individual in a population carries and transmits the gene, but only a fraction, , expresses the gene and exposes it to natural selection
What is an example of conditional expression?
Cre-lox recombination system
How does the cre-lox recombination system work?
Cre recombinase expression is under celltype-specific promoter (e.g. VGAT)
Cre recombinase recognises loxP sequences – removing genetic material in between
In cells lacking Cre – original function of gene is unchanged
Can combine with expression of reporter gene, e.g. GFP
Development of CreERT2 – inducible
What else is conditional expression used to express?
GCaMP
ChR2
loxP
Flank ‘STOP’ sequence - exposure to Cre recombinase leads to expression of gene in selected cells
What is the reason for the development of CreERT2?
Used so you can temporally control the gene expression by treating the animal with Tamoxifen
Cre only becomes active when treated with Tamoxifen
What is viral delivery?
DNA packaged into virus for efficient delivery into brain cells
What is the common system of viral delivery?
Adeno-associated virus (AAV - non-human pathogen)
What is the effect of AAV?
Provides long term transgene expression - expression in chosen cell type due to promotor expression
Can AAVs cross the BBB?
The development of AAV serotypes allow them to cross the BBB
Can be injected systemically via the tail vein
What are some examples of AAV serotypes?
AAV9
AAV-PHP.B
Can viral delivery be combined with other methods?
Yes = can combine with Cre-lox system - inject AAV vectors containing recombinase-dependent virus into recombinase-expressing mice
What methods can be used to monitor neural activity?
Electrophysiology
Optical imaging
Genetic manipulations
What does electrophysiology measure?
Records membrane potential fluctuations - directly reports neural activity
What is an advantage of electrophysiology?
High spatial and temporal resolution
High signal-to-noise ratio
What is a limitation of electrophysiology?
Only measures in vicinity of the electrode and the cell identity is unknown
Measurements are typically from a subset of relatively active neurons - if a neuron only fires often or isn’t very active, these neurons may not be picked up on the recording
What is optical imaging?
Optical imaging uses light and special properties of photons to obtain detailed images of organs, tissues, cells and even molecules
Often uses fluorescent sensors (dyes)
Measures changes in e.g., voltage or calcium
How does calcium imaging work?
Action potentials cause calcium influx through VGCC – can use calcium imaging to detect spike (output) activity in neuronal networks
Uses fluorescent calcium indicators which bind to calcium and then can de detected
Allows access to many neurons simultaneously (neural populations).
Possibility to track same neurons over time
How is calcium imaging used to record neural activity in vivo?
Use of calcium dyes
Bulk load dyes to assess populations of neurons
Can measure with single cell resolution (with 2 photon microscopy) and with single action potential sensitivity
What are the advantages of calcium dyes?
Wide range available with different Ca2+ affinities
Good sensitivity, signal-to-noise ratio and dynamic range
What are the limitations of calcium dyes?
Can’t easily distinguish between cell types
Cannot reliably target specific cell type or compartment
Typically load cell body rather than fine processes
Often single imaging session of limited duration
Delivery via whole cell patch clamp or bulk loading can damage cell
How can genetic manipulations be used to monitor neural activity?
Genetically Encoded Calcium Indicators : GECIs
Reports changes in calcium allowing cell types to be distinguished
How is cell type sensitivity achieved in GECIs?
Via transgenic mice or viral delivery - target genetically defined cell type
What is the most common GECI?
GCaMP
What is the structure of GCaMP?
Fusion of fluorescent protein (GFP) and endogenous calcium binding protein/buffer (calmodulin)
How does GCaMP work?
Increase in neural activity causes a rise in Ca2+
Calmodulin binds to calcium causing a protein conformational change causing an increase in GFP fluorescence
Measure fluorescence changes with a camera of 2 photon microscope
What are the uses of GCaMP?
Can monitor calcium changes in axon/synaptic terminals and also the ability to correlate neural activity with specific behaviours and phenotypes
How is GCaMP delivered into animals?
Can deliver via AAV
Transgenic mouse models e.g., Thy1-GCaMP6
What are the advantages of GECIs?
Can be targeted to cell type through genetic promoter or brain area by local viral injection
Can be targeted to specific compartment e.g., mitochondria
Can image over long time repeatedly in vivo
Cellular and subcellular resolution possible
What are the limitations of GECIs?
Typically narrower dynamic range and slower response time
Interpretation of signals can be challenging
What is a limitation of calcium imaging?
It is an indirect measure of neural activity (measures intracellular calcium)
What techniques are used to image the changes in calcium?
Fluorescence imaging
Multi-photon imaging
2-photon imaging
What is fluorescence imaging?
Uses a high-intensity light source that excites a fluorescent molecule called a fluorophore in the sample observed
The samples are labeled with fluorophore where they absorb the high-intensity light from the source and emit a lower energy light of longer wavelength
What are the two types of fluorescence microscopy?
1-photon microscopy
2-photon microscopy
What is the main difference between 1-photon and 2-photon microscopy?
Unlike 1-photon microscopy where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image
What is multiphoton imaging?
Another name for 2-photon imaging
What method is used to analyse brain activity in animals during naturalistic behaviour?
Miniscopes - microscopes are large and heavy and we need to head fix the animal so miniaturised fluorescence microscope have been developed so we can fit to the head of the animal
Neural activity can be monitored in freely behaving animals
What did Dombeck et al. (2010) study using GECIs?
Identification of place cells during navigation
What methods did Dombeck et al. (2010) use?
Awake head fixed mice on a spherical treadmill with a VR linear track
Mice had an AAV injection of AAV-Synapsin1-GCaMP3
Used a 2 photon microscope to image the hippocampus
What did Dombeck et al. (2010) find?
Optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit
What did Wirtshafter et al. (2022) investigate?
Investigation of place cells in freely moving rats
What methods did Wirtshafter et al. (2022) use?
Freely moving rat on a linear track with a reward at either end
Rats had an AAV injection into hippocampus with AAV-syn-GCaMP7c
Used a miniscope with a hippocampal imaging window
What did Wirtshafter et al. (2022) find?
Demonstrated that, in rats, hundreds of cells can be visualized and held across weeks
Showed that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement
Additionally showed that an extremely large percent of cells recorded during a navigational task are place cells and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map
What developments have been made in GECIs?
Variants of GCaMP have different molecular properties, e.g. GCaMP8s has high SNR, slow decay, GCaMP8f faster decay time (report neural activity on ms timescale) but lower SNR
Improvements being made – GCaMP6 allows single spike detectability
Red-shifted: RCaMP (RFP-based) - potential to combine with e.g. optogenetics
Increasing use of miniscopes allows calcium imaging in freely moving mice
