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Systems Neuroscience > Preclinical Approaches > Flashcards

Preclinical Approaches Flashcards

1
Q

What is a disruptive technology?

A

“Disruptive” technology: typically displaces an established technology, ground-breaking

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2
Q

How do we make genetic manipulations?

A

Knock out models or knock in models

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3
Q

What is a knock out model?

A

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

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4
Q

What are global knock outs critical for?

A

Critical in initial evaluation of role of a gene in brain function

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5
Q

Is there convincing evidence for knock out models?

A

Conflicting results have been obtained when using KO mice in research

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6
Q

What are the conflicting effects likely caused by?

A

Likely due to compensation effects

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7
Q

What are knock in models?

A

Replace original DNA sequence with a modified version, to alter function of coding gene

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8
Q

What are humanised mutations?

A

Mouse has a human gene inserted into its genome. Common in research of neurodegenerative diseases

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9
Q

What are some examples of humanised mutations?

A

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)

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10
Q

What is a limitation of global gene manipulations?

A

A lack of specificity in time and space - compensation/fatal

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11
Q

What is another way to make genetic manipulations aside from knock out models?

A

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

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12
Q

What are some common promoter lines for neurons?

A

CaMKII (calcium/calmodulin-dependent kinase II alpha gene): excitatory neurons in neocortex and hippocampus

Human synapsin 1

Platelet-derived growth factor beta chain

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13
Q

Is selection of neuron sub-types possible?

A

Yes ==

VGAT for GABAergic neurons

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14
Q

Is selection of non-neuronal cells possible?

A

Yes ==
GFAP for astrocytes

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15
Q

What does GFAP stand for?

A

Glial fibrillary acidic protein

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16
Q

What is conditional expression?

A

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

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17
Q

What is an example of conditional expression?

A

Cre-lox recombination system

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18
Q

How does the cre-lox recombination system work?

A

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

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19
Q

What else is conditional expression used to express?

A

GCaMP
ChR2
loxP

Flank ‘STOP’ sequence - exposure to Cre recombinase leads to expression of gene in selected cells

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20
Q

What is the reason for the development of CreERT2?

A

Used so you can temporally control the gene expression by treating the animal with Tamoxifen

Cre only becomes active when treated with Tamoxifen

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21
Q

What is viral delivery?

A

DNA packaged into virus for efficient delivery into brain cells

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22
Q

What is the common system of viral delivery?

A

Adeno-associated virus (AAV - non-human pathogen)

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23
Q

What is the effect of AAV?

A

Provides long term transgene expression - expression in chosen cell type due to promotor expression

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24
Q

Can AAVs cross the BBB?

A

The development of AAV serotypes allow them to cross the BBB
Can be injected systemically via the tail vein

25
Q

What are some examples of AAV serotypes?

A

AAV9
AAV-PHP.B

26
Q

Can viral delivery be combined with other methods?

A

Yes = can combine with Cre-lox system - inject AAV vectors containing recombinase-dependent virus into recombinase-expressing mice

27
Q

What methods can be used to monitor neural activity?

A

Electrophysiology
Optical imaging
Genetic manipulations

28
Q

What does electrophysiology measure?

A

Records membrane potential fluctuations - directly reports neural activity

29
Q

What is an advantage of electrophysiology?

A

High spatial and temporal resolution
High signal-to-noise ratio

30
Q

What is a limitation of electrophysiology?

A

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

31
Q

What is optical imaging?

A

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

32
Q

How does calcium imaging work?

A

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

33
Q

How is calcium imaging used to record neural activity in vivo?

A

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

34
Q

What are the advantages of calcium dyes?

A

Wide range available with different Ca2+ affinities

Good sensitivity, signal-to-noise ratio and dynamic range

35
Q

What are the limitations of calcium dyes?

A

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

36
Q

How can genetic manipulations be used to monitor neural activity?

A

Genetically Encoded Calcium Indicators : GECIs

Reports changes in calcium allowing cell types to be distinguished

37
Q

How is cell type sensitivity achieved in GECIs?

A

Via transgenic mice or viral delivery - target genetically defined cell type

38
Q

What is the most common GECI?

A

GCaMP

39
Q

What is the structure of GCaMP?

A

Fusion of fluorescent protein (GFP) and endogenous calcium binding protein/buffer (calmodulin)

40
Q

How does GCaMP work?

A

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

41
Q

What are the uses of GCaMP?

A

Can monitor calcium changes in axon/synaptic terminals and also the ability to correlate neural activity with specific behaviours and phenotypes

42
Q

How is GCaMP delivered into animals?

A

Can deliver via AAV
Transgenic mouse models e.g., Thy1-GCaMP6

43
Q

What are the advantages of GECIs?

A

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

44
Q

What are the limitations of GECIs?

A

Typically narrower dynamic range and slower response time

Interpretation of signals can be challenging

45
Q

What is a limitation of calcium imaging?

A

It is an indirect measure of neural activity (measures intracellular calcium)

46
Q

What techniques are used to image the changes in calcium?

A

Fluorescence imaging
Multi-photon imaging
2-photon imaging

47
Q

What is fluorescence imaging?

A

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

48
Q

What are the two types of fluorescence microscopy?

A

1-photon microscopy
2-photon microscopy

49
Q

What is the main difference between 1-photon and 2-photon microscopy?

A

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

50
Q

What is multiphoton imaging?

A

Another name for 2-photon imaging

51
Q

What method is used to analyse brain activity in animals during naturalistic behaviour?

A

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

52
Q

What did Dombeck et al. (2010) study using GECIs?

A

Identification of place cells during navigation

53
Q

What methods did Dombeck et al. (2010) use?

A

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

54
Q

What did Dombeck et al. (2010) find?

A

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

55
Q

What did Wirtshafter et al. (2022) investigate?

A

Investigation of place cells in freely moving rats

56
Q

What methods did Wirtshafter et al. (2022) use?

A

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

57
Q

What did Wirtshafter et al. (2022) find?

A

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

58
Q

What developments have been made in GECIs?

A

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

Systems Neuroscience (16 decks)

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