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Systems Neuroscience > Optogenetics 2 > Flashcards

Optogenetics 2 Flashcards

1
Q

What is disruptive technology?

A

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

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2
Q

What do we use to monitor neural activity?

A

Previously electrophysiology – now genetically encoded fluorescent sensors/indicators

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3
Q

How do we manipulate neural activity?

A

Previously direct electrical stimulation/sensory stimulation – now optogenetics and chemogenetics

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4
Q

What is a knock-out model?

A

If DNA is the instruction manual or ‘blueprint’ for an animal then a gene is a specific instruction
The gene is an instruction to manufacture a specific protein
Proteins are the building blocks of cellular function

It has become possible to ‘knock-out’, or ‘knock-in’ specific genes into the DNA of either the whole animal, or specific cells.

‘Knock-out’: inactivate a targeted gene.

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5
Q

What is ciritical in initial evaluation of role of a gene in brain function?

A

Global knock outs

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6
Q

What are compensation effects?

A

Give conflictiing results

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7
Q

What is a knock-in model?

A

Knock-in’: replace original DNA sequence with a modified version, to alter function of coding gene.

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8
Q

What are humanized mutations?

A

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

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9
Q

What is a limitation of global gene manipulations?

A

Lack of specificity in time and space

This can be fatal

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10
Q

What can we do to overcome lack of specificity in time and space?

A

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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11
Q

What are 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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12
Q

Can we use promoters to examine sub-types of neurons?

A

Yes

e.g. VGAT for GABAergic neurons

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13
Q

Can we use promoters to examine non-neuronal cells?

A

Yes

e.g. GFAP (glial fibrillary acidic protein) for astrocytes.

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14
Q

What is Cre-lox recombination?

A

Cre recombinase expression is under a celltype-specific promoter (e.g. VGAT)

Cre recombinase recognises loxP sequences and removes genetic material in between two loxP sites

In cells lacking Cre – original function of gene is unchanged

Its used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells

It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus

Can combine with expression of reporter gene, e.g. GFP

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15
Q

What has been an important develoment for cre-lox recombinase?

A

Development of CreER^T2
Can induce expression of cre and induce the expression of the gene your interested in by treating the animal with Tamoxifen

This allows us to have temporal control

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16
Q

What can cre-lox recombinase also be used to express?

A

GCaMP
ChR2

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17
Q

How can cre-lox recombinase express e.g. GCaMP?

A

loxP sites flank ‘stop’ sequence- exposure to Cre recombinase leads to expression of gene in selected cells (e.g Ai38 mouse – GCaMP3 is floxed).

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18
Q

What is viral delivery?

A

DNA packaged into virus for efficient delivery into brain cells

Adeno-associated virus (AAV, non-human pathogen) is commonly used as delivery system in neuroscience

Provide long term transgene expression – expression in chosen cell type due to promoter expression

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19
Q

Why is viral delivery good?

A

Allows long term expression of the gene of interest

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20
Q

What has been an important development for viral delivery?

A

Development of AAV serotypes (AAV9, AAV-PHP.B)

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21
Q

Why is the development of AAV serotypes incredibly important?

A

Important because it can be injected systemically (tail vein) (e.g. Deverman et al., 2016) and it can cross the BBB so we can get expression of the gene of inteest in the brain itself without having to directly inject into the brain

22
Q

Can we combine viral delivery with cre-lox?

A

Yes

Inject AAV vectors containing recombinase-dependent virus into recombinase-expressing mice.

23
Q

How can we use genetics in neuroscience?

A

Combine it with electrophysiology

24
Q

What is electrophysiology used for?

A

Record membrane potential fluctuations – directly reports neural activity

High spatial and temporal resolution, high SNR

25
Q

What is a disadvantage of electrophysiology?

A

Can only measure in the vicinity of the electrode

Its really difficult to see what the cell identity is

Measurements typically from subset of relatively active neurons so neurons that dont fire frequently may be missed

26
Q

What is optical imaging?

A

Fluorescent sensors (dyes) – activity in many cells, high spatial resolution
Changes in e.g. voltage, calcium possible
Focus on calcium imaging

27
Q

How can we use ca2+ imaging

A

When we have action potentials we get influxes of Ca2+ so we can use calcium imaging to detect spike (output) activity in neuronal networks

Allows access to many neurons simultaneously (neural populations).
- Possibility to track same neurons over time.

28
Q

Hoe can we do in vivo imaging of neural activity?

A

Using calcium dyes

29
Q

How do we use calcium dyes?

A

Bulk load dyes to assess populations of neurons

Can measure with single cell resolution (with 2 photon microscopy) and single action potential sensitivity

But can’t easily distinguish between cell types

30
Q

What is a limitation of using calcium dyes?

A

Injecting dyes into the brain but we have no control what cells take up the dyes e.g. astrocytes take up lots of dyes

31
Q

How else can we monitor neural activity?

A

genetic manipulations e.g. GECIs

32
Q

What does GECI stand for?

A

Genetically Encoded Calcium Indicators

33
Q

How do GECIs report Ca2+?

A

Some GECIs report calcium by direct emission of photons (luminescence), but most rely on fluorescent proteins as reporters, including the green fluorescent protein GFP and its variants (eGFP, YFP, CFP).

34
Q

What do GECIs allow for?

A

Cell type specificity: via transgenic mice or viral delivery – target genetically defined cell type

Reports change in calcium, allows cell types to be distinguished

GCaMP: Fusion of fluorescent protein (GFP) and endogenous calcium binding protein/buffer (calmodulin)
Neural activity –> rise in [Ca2+]i –> calmodulin binds calcium –> protein conformational change causes increase in GFP fluorescence

Monitor fluorescence changes with e.g. camera or 2P microscope, routinely applied in vivo

35
Q

What is the purpose of GECIS?

A

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

36
Q

How can GECIs be delivered?

A

Via AAV

37
Q

What is a limitation of GECIS?

A

Its an indirect measure of neural activity, we are measuring intracellular Ca2+

and calcium can be filtered or delayed compared to neural responses

38
Q

What are the advantages of synthetic indicator dyes?

A

Wide range available with different Ca2+ affinities

Good sensitivity, signal to noise ratio, dynamic range

39
Q

Whate are some limitations of synthetic indicator dyes?

A

Cannot reliably target specific cell type or compartment

Typicallu load cell body rather than fine proceses

Often single imaging session of limited duration

Delivery via whole cell patch clamp or bulk loading (can damage cell?

40
Q

What is a strength of GECIS?

A

Can be targetting to cell type (though genetic promoter) or brain area (by local viral injection)

Can be targeted to specific compartment e.g. mitochondria

Can image over a long time, repeatedly in vivo

Cellular and subcellule resolution possible

41
Q

What is a limitation of GECIs?

A

Typically narrower dynamic range and slower response time(improving)

Interpretation of signals can be challening

42
Q

What is fluorescence microscopy?

A

Fluorescence microscopy has become an essential tool in cell biology.

Allows researchers to visualize the dynamics of tissue, cells, individual organelles, and macromolecular assemblies inside the cell.

43
Q

What is 2photon microscopy?

A

also called multiphoton microscopy

can be used for live cell imaging of thick biological specimens, as it has several advantages over confocal microscopy. Molecules can be visualized deeply within the specimen with a maximal penetration depth of about 1 mm.

44
Q

What are miniscopes?

A

Development of miniaturized fluorescence microscope – can fit to head of animal - neural activity can be monitored in freely behaving animals

Single-photon fluorescence or two-photon fluorescence imaging

45
Q

What did Dombeck et al. (2010) do?

A

Used awake head-fixed mice on a spherical treadmill

On a VE linear track

Used 2PHOTON microscopy

Hippocampal imagin window

AAV injectioon- Synapsin-1-GCAMP3

46
Q

What did Dombeck et al. (2010) find?

A

Identification of place cells during navigation

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.

also found activation of the dendrites

47
Q

What did Wirtshafter et al. (2022) do?

A

Freely moving rat
Linear track, reward at either end
Miniscope
Hippocampal imaging window
AAV injection into hippocampus
AAV-syn-GCaMP7c

48
Q

WHat did Wirtshafter et al. (2022) find?

A

When the rats had periods of locomotion there was increases in Ca2+

So calcium reflecting neural activity was reflecting period of locomotion along the track

Different cells have different preferential firing based on location but also direction

49
Q

What did Wirtshafter et al. (2022) conclude?

A

Place cell firing is sufficient to decode animal position

50
Q

How have GECIs been developed?

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 other approaches e.g. optogenetics

Increasing use of miniscopes allows calcium imaging in freely moving mice

Systems Neuroscience (16 decks)

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