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Systems Neuroscience > Optogenetics > Flashcards

Optogenetics Flashcards

1
Q

Name 5 common neuroscience research methods:

A

5 from:
-MRI
-fMRI
- Optogenetics
- Cognitive Experiments
- Genetic Manipulation
- EEG
- in vitro slice
- in vivo electrophys
- TMS
- animal studies
- Calcium imaging
- optical imaging (light microscopy/OIS)
-pharmacological experiments
- DBS (direct brain stimulation)
- DTI (diffusion tensor imaging, form of MRI)

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2
Q

Name the 4 main invasive methods of measurement in neuroscience.

A
  • electrophysiology (electrode recordings)
  • tracers
  • disrupt connectivity
  • lesion
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3
Q

what are the 2 types of electrophysiological recordings?

A

intracellular and extracellular

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4
Q

describe intracellular recordings

A

target single cells with the tip of the electrode inside the cell

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5
Q

describe extracellular recordings

A

record from larger populations with the tip of the electrode outside cells

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6
Q

what do intra and extracellular recordings show?

A

the connectivity between structures, where stimulating electrodes are inserted into one area and recording electrodes are in another part to determine connectivity

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7
Q

what are the 2 types of tracers?

A

Anterograde and Retrograde

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8
Q

what do anterograde tracers do?

A

map out connection by injecting the tracer into one region and measuring the direction travelled by the tracer via synapses (forward signal travel)

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9
Q

what do retrograde tracers do?

A

map out backwards along axon to determine where a region gets its input from

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10
Q

draw a diagram of an anterograde tracer

A

look on mindmap/notes

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11
Q

draw a diagram of a retrograde tracer

A

look on mindmap/notes

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12
Q

describe the disruption of connectivity research in neuroscience

A

where the connection between areas is cut to determine the effects on circuit function, both direct and downstream looking at structure/behaviour/activity

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13
Q

draw a diagram of the disruption of connectivity

A

look in mindmap/notes

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14
Q

describe lesion research

A

to lesion structures in order to inform of what the function of the structure is and see what the downstream effects are

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15
Q

what is psychopharmacology?

A

a non-invasive way to study neural systems by looking at the effect of drugs on cognition/behaviour through their action on specific receptors on specific cell types. They can act by blocking or activating receptors/ removing NT or affecting NT reuptake

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16
Q

weakness of disrupting connectivity?

A

results can be difficult to interpret

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17
Q

what are the 2 key genetic-based approaches used In systems neuroscience research?

A

Optogenetics and Chemogenetics

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18
Q

define optogenetics

A

the combination of genetic and optical methods to achieve gain/loss of function in well-defined events in specific cells of living tissue

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19
Q

what are the 4 key features of optogenetics?

A

1) light-sensitive proteins (microbial opsins)
2) ability to target strong and specific expression of opsin in cells
3) ability to guide light to specific areas/cells at precise times
4) compatible readout approach

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20
Q

what are the 3 key types of microbial opsin?

A

1) Archeo/Bacterio-rhodopsin
2) Halorhodopsin
3) Channelrhodopsin

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21
Q

describe Archeo/Bacterio-rhodopsin

A

H+ pump (out of cell)
hyperpolarises
inhibitory

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22
Q

describe Halorhodopsin

A

Cl- pump (into cell)
hyperpolarises
inhibitory

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23
Q

describe channelrhodopsin

A

Cation pump (into cell)
depolarises
excitatory

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24
Q

discuss further development into opsins

A

opsins have been further developed/engineered to change their kinetics/spectra sensitivity/conductance.

For example, once the crystal structure of the channelrhodopsin was understood, scientists could engineer it to make it a Cl- -pump, changing its conductance

Red-shifted opsins allow an all-optical approach where the opsin is activated by red light so GECI or another optical measurement can work using a different wavelength

OptoXRs are light-sensitive GPCRs, instead of ion channels, to drive intracellular signalling pathways

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25
Q

what are the 3 ways that genes for the opsins can be delivered to target cells?

A

viral delivery, cre-recombinase and transgenic animals

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26
Q

discuss viral delivery of opsins

A

AAV (adeno-associated viral vectors) containing the DNA of the opsin (with promoters to target expression in specific areas) is injected directly into the animal’s brain

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27
Q

discuss cre-recombinase delivery of opsins

A

transgenic mouse that expresses cre-recombinase in specific cells, then are injected with cre-recombinase -dependent opsin virus to only activate opsins in cre-recombinase cells

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28
Q

discuss transgenic mouse delivery of opsins

A

where we want to express channelrhodopsin in certain cell types everywhere in the brain can be done by breeding a cre-mouse with a recombinase-dependent opsin mouse.

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29
Q

how is light guided to specific areas/cells at specific times?

A

an LED/laser optic cable emitting the required wavelength for opsin activation is placed on brain region , allowing local stimulation to target the opsin and give downstream effects

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30
Q

what is meant by the compatible readout approach required for optogenetics?

A

another measure being used to observe the effect of optogenetics is having to manipulate neural activity.

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31
Q

define disruptive technology

A

a new technology which displaces established technology (groundbreaking)

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32
Q

what technique was traditionally used to monitor neural activity?

A

electrophysiology

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33
Q

what is being used more in modern times to monitor neuron activity?

A

genetically encoded fluorescent sensors/indicators

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34
Q

what is a knockout model?

A

genetic manipulation technique to inactivate a target gene

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35
Q

typically are knockout and knockin models global or local?

A

typically global- knocking out the gene throughout the animal

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36
Q

what are the limitations of global gene knockout?

A
  • you can obtain conflicting results
  • the gene knocked out may be critical in development and can lead to the animal being non-viable (fatal)
  • may get compensation effects, perhaps due to redundancy in proteins/pathways, giving unexpected results
  • lack spatial and temporal specificity, important when genes may have different roles in development compared to adulthood etc.
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36
Q

what is a knock-in model?

A

where you replace the original DNA sequence with a modified version to alter the function of the coding gene

37
Q

what kinds of mutations are often used in knock-in mouse models?

A

‘humanised genes’ where a human gene is inserted into the genome (common in neurodegenerative disease studies)

38
Q

give an example of a knock-in humanised genes used in mouse models.

A

APP KI mouse gives an AD model with the overproduction of beta-amyloid, which forms plaques- a hallmark of AD

39
Q

define promoters

A

DNA sequence that allow the induction or suppression of gene expression in specific cell types (gives control)

40
Q

give an example of a promoter and its role

A

1 of:
- CAMKII - promotes gene expression in excitatory neurons in the neocortex and hippocampus
- VGAT- promotes gene expression in GABAergic neurons
- GFAP (glial fibrillary acidic protein)- promotes gene expression in astrocytes

41
Q

outline the Cre-lox recombination system

A

it allows conditional expression or removal of genes, can be combined with a promoter

to create it, a cre mouse with cre gene section and stop codon is crossed with a lox-p (floxed) mouse with target gene flanked by loxp, maybe with reporter gene e.g. GFP.
This produces mixed offspring, some are cre-recombinase which recognises the loxp and deletes the target gene between the loxp (reporter gene e.g. GFP before stop codon expressed). Some are naive/WT and don’t express cre-recombinase, with the target gene function untouched. This variation is within the same litter

42
Q

draw a diagram of cre-lox recombination breeding

A

see notes

43
Q

what’s the benefit of adding a promoter to the cre-lox model?

A

it allows increased specificity of gene expression to cell type (some spatial resolution)

44
Q

what’s the con of adding a promoter to the cre-lox model?

A

it doesn’t get round the temporal resolution issue- e.g. if gene acts differently between development and adulthood

45
Q

what helps researchers get time specificity in the Cre-lox model?

A

they can use CreER^T2 which allows time specificity where cre is bound to an oestrogen receptor and when tamoxifen is given the recombinase activates to KO target gene at a given time. The cre-oestrogen-binding domain is originally in cytoplasm but with tamoxifen, it’s translocated to nucleus to KO target gene

46
Q

how would the recombination system be used to express an exogenous gene?

A

loxp sites flank a stop codon upstream of the gene you’re trying to express, then when cre-recombinase is present (in cells producing cre), it brings the loxp sites together and cuts out the stop codon, so the target gene can be expressed

47
Q

what are limitation of cre-recombinase/transgenic mice models?

A

mouse breeding is costly
unpredictable
takes time
often global in brain

48
Q

what method of delivery can help get around some of the weaknesses of cre-recombinase methods?

A

viral delivery

49
Q

outline viral gene delivery?

A

DNA is put into AAV viral vector then injected into a specific brain region

50
Q

how can viral gene delivery be brainwide?

A

some AAV virus strains can effect the whole brain, e.g. AAV9

51
Q

can viral vector and cre-recombinase be combined?

A

yes- so the DNA is only expressed in cells with cre-present

52
Q

weaknesses of viral delivery?

A
  • limits length of DNA (often promoters too as they are long chains)
  • damage/neuroinflammation
53
Q

strengths of gene KO:

A
  • you can easily identify that the effect is due to the gene loss
  • less invasive
  • global (is also a weakness sometimes)
54
Q

weaknesses of KI:

A
  • human genes may not be easily expressed into mouse or have a different effect
  • breeding takes time
  • global (strength and weakness)
55
Q

strengths of KI:

A
  • good for human disease research
  • easily identify the effects of gene addition
  • not o invasive
56
Q

strengths of viral delivery

A

locational and temporal specificity
can combine with cre-lox system

57
Q

outline fMRI BOLD

A

BOLD signal measures for neural activity due to increased BF to bring O2 to the area and afterwards increase in deoxy blood.`

58
Q

weaknesses of fMRI BOLD

A
  • a surrogate measure of neural activity
  • low temporal resolution (in secs where neural activity is over ms)
  • poor spatial resolution
59
Q

outline EEG

A

done on awake humans to measure the electrical activities in neurons, giving different frequency bands to relate to different neurophysiological processes

60
Q

strength of EEG

A
  • high temporal resolution
  • monitors ongoing/current activity
  • non-invasive
61
Q

strength of fMRI

A
  • whole brain
  • monitors ongoing activity
  • non-invasive
62
Q

weakness of EEG

A
  • low spatial resolution
63
Q

what is the gold standard of monitoring neural activity?

A

electrophsyiology

64
Q

strengths of electrophysiology?

A

-high spatial resolution (long electrode with channels down the length so can measure at different depths)
- high temporal resolution

65
Q
A
66
Q

outline the next generation of electrodes:

A

neuropixels
monitor the activity of multiple neurons across multiple brain regions with many more channels in a long shank
can be used during free-movement as its chronically implanted
gives loads of data so difficult to analyse

67
Q

strengths of optical imaging

A
  • less invasive
  • high spatial resolution
  • high temporal resolution
68
Q

briefly outline fluorescence microscopy

A

the fluorescent molecule is inside cells excited by a specific wavelength of light, and relaxes to ground state by releasing a photon which can be imaged

69
Q

outline/compare 1-photon and 2-photon microscopy

A

1 photon is traditional as it records a single photon fluorescence, however, it gives a lot of field excitation outside ROI as light travels through, it has a short wavelength and higher energy, hence limited penetration, whereas 2P is double wavelength, half the energy, 2 photons arrive at areas at the same time to excite molecule, it’s much more specific and deeper penetration

70
Q

outline research into imaging neocortical network activity with calcium imaging

A
  • an increase in calcium occurs when there’s an AP
  • calcium dye was bulk loaded into cells to look at neural activity alongside patch clamps (electrophysiology)
  • They found that when a spike was detected, Ca2+ fluoresced, hence Ca2+ transient increase can indicate spiking
71
Q

what’s the downside of bulk-loaded calcium indicator?

A

it can be difficult to identify between cell types/specify loading into certain cell types

72
Q

how are GECIs applied?

A

either via transgenic mice or viral delivery

73
Q

outline how GECIs work

A

when neural activity increases, as does Ca2+, hence there is fluorescence

74
Q

what is the most common GECI and how does it work?

A

GCaMP- a fusion of calmodulin and GFP. Calmodulin binds calcium, causing a conformational change and leading to GFP fluorescence

75
Q

are GECI’s generally used in or ex vivo?

A

in vivo

76
Q

strength of GECIs

A
  • allows one to see cell body as well as axonal/dendritic/synaptic activity
  • unlike synthetic dye, they can target specific cell types and allow imaging over a long time, you don’t have to keep reloading the dye
77
Q

weakness of GECIs

A

calcium imaging is an indirect measurement of neural activity

78
Q

outline study which used GECIs

A

using AAV GCaMP GECI in the hippocampus and 2P microscope.
study found that as mice ran along a VR track place cells in the hippocampus would fire when they were in a particular location. This was seen in Ca2+ fluorescence and confirmed with electrophysiology

79
Q

outline miniscopes

A

microscopes are traditionally large and heavy so when used on animals fixes their head, whereas miniscopes allow freely moving animals

80
Q

compare optogenetic techniques to electrical stimulation techniques, e.g. TMS

A

optogenetics allows control in defined cell populations with better spatiotemporal resolution and can occur with spontaneous electrophysiological recordings possible

81
Q

compare optogenetics to lesion studies

A

optogenetics is reversible/temporary, unlike lesion studies and can up or down-regulate activity. It is also better at detecting causal relationships, rather than just correlations

82
Q

compare optogenetics with pharmacology studies

A

optogenetics allows better temporal precision and the possibility to investigate causal relationships- not just correlational

83
Q

what is chemogenetics?

A

a technique using inert ligands to activate engineered receptors. It can be used to reversibly alter the function of neurons in a circuit and behaviour

84
Q

strengths of chemogenetics:

A
  • using pharmacology instead of light works on a longer time scale
  • less invasive than optogenetics
  • the engineered ligand only works on engineered receptors so well-controlled/precise
  • reversible
85
Q

what are DREADDs?

A

designer receptors exclusively activated by designer drugs
they are designed similar to GPCRs to interact ith the designer otherwise-inert ligand to allow non-invasive cell control

86
Q

what’s CNO

A

clozapine-N-oxide, is a designer ligand that activates DREADDs

87
Q

how are DREADDs expressed?

A

similar to opsins:
- can be combined with a viral vector and injected with or without a promoter (limits DNA length/ damage/neuroinflammation)
- can be bred into transgenic mice/cre-recombinase mice to have control over cell-type specificity

88
Q

how can designer ligands, such as CNO for chemogenetics be inserted?

A

can be applied with injection into the body (to get to the brain in the blood), or orally

89
Q

what’s the downside of CNO

A

it can metabolize to clozapine which can cross BBB

90
Q

strengths of optogenetics?

A
  • high spatial and temporal control of neuronal activity
  • can be used in many model systems
  • disruptive technology (groundbreaking)
91
Q

what are the general constraints to consider in research methods (give 5)

A

5 from:
- expertise e.g. having to write an MRI sequence
- facilities (access to a technique)
- time
- money
- ethics (cost vs benefit), 3Rs etc.
- the trade-off between spatial and temporal resolution
invasiveness

Systems Neuroscience (3 decks)

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