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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
what are the 3 ways that genes for the opsins can be delivered to target cells?
viral delivery, cre-recombinase and transgenic animals
26
discuss viral delivery of opsins
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
27
discuss cre-recombinase delivery of opsins
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
28
discuss transgenic mouse delivery of opsins
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.
29
how is light guided to specific areas/cells at specific times?
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
30
what is meant by the compatible readout approach required for optogenetics?
another measure being used to observe the effect of optogenetics is having to manipulate neural activity.
31
define disruptive technology
a new technology which displaces established technology (groundbreaking)
32
what technique was traditionally used to monitor neural activity?
electrophysiology
33
what is being used more in modern times to monitor neuron activity?
genetically encoded fluorescent sensors/indicators
34
what is a knockout model?
genetic manipulation technique to inactivate a target gene
35
typically are knockout and knockin models global or local?
typically global- knocking out the gene throughout the animal
36
what are the limitations of global gene knockout?
- 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.
36
what is a knock-in model?
where you replace the original DNA sequence with a modified version to alter the function of the coding gene
37
what kinds of mutations are often used in knock-in mouse models?
'humanised genes' where a human gene is inserted into the genome (common in neurodegenerative disease studies)
38
give an example of a knock-in humanised genes used in mouse models.
APP KI mouse gives an AD model with the overproduction of beta-amyloid, which forms plaques- a hallmark of AD
39
define promoters
DNA sequence that allow the induction or suppression of gene expression in specific cell types (gives control)
40
give an example of a promoter and its role
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
outline the Cre-lox recombination system
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
draw a diagram of cre-lox recombination breeding
see notes
43
what's the benefit of adding a promoter to the cre-lox model?
it allows increased specificity of gene expression to cell type (some spatial resolution)
44
what's the con of adding a promoter to the cre-lox model?
it doesn't get round the temporal resolution issue- e.g. if gene acts differently between development and adulthood
45
what helps researchers get time specificity in the Cre-lox model?
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
how would the recombination system be used to express an exogenous gene?
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
what are limitation of cre-recombinase/transgenic mice models?
mouse breeding is costly unpredictable takes time often global in brain
48
what method of delivery can help get around some of the weaknesses of cre-recombinase methods?
viral delivery
49
outline viral gene delivery?
DNA is put into AAV viral vector then injected into a specific brain region
50
how can viral gene delivery be brainwide?
some AAV virus strains can effect the whole brain, e.g. AAV9
51
can viral vector and cre-recombinase be combined?
yes- so the DNA is only expressed in cells with cre-present
52
weaknesses of viral delivery?
- limits length of DNA (often promoters too as they are long chains) - damage/neuroinflammation
53
strengths of gene KO:
- you can easily identify that the effect is due to the gene loss - less invasive - global (is also a weakness sometimes)
54
weaknesses of KI:
- human genes may not be easily expressed into mouse or have a different effect - breeding takes time - global (strength and weakness)
55
strengths of KI:
- good for human disease research - easily identify the effects of gene addition - not o invasive
56
strengths of viral delivery
locational and temporal specificity can combine with cre-lox system
57
outline fMRI BOLD
BOLD signal measures for neural activity due to increased BF to bring O2 to the area and afterwards increase in deoxy blood.`
58
weaknesses of fMRI BOLD
- a surrogate measure of neural activity - low temporal resolution (in secs where neural activity is over ms) - poor spatial resolution
59
outline EEG
done on awake humans to measure the electrical activities in neurons, giving different frequency bands to relate to different neurophysiological processes
60
strength of EEG
- high temporal resolution - monitors ongoing/current activity - non-invasive
61
strength of fMRI
- whole brain - monitors ongoing activity - non-invasive
62
weakness of EEG
- low spatial resolution
63
what is the gold standard of monitoring neural activity?
electrophsyiology
64
strengths of electrophysiology?
-high spatial resolution (long electrode with channels down the length so can measure at different depths) - high temporal resolution
65
66
outline the next generation of electrodes:
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
strengths of optical imaging
- less invasive - high spatial resolution - high temporal resolution
68
briefly outline fluorescence microscopy
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
outline/compare 1-photon and 2-photon microscopy
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
outline research into imaging neocortical network activity with calcium imaging
- 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
what's the downside of bulk-loaded calcium indicator?
it can be difficult to identify between cell types/specify loading into certain cell types
72
how are GECIs applied?
either via transgenic mice or viral delivery
73
outline how GECIs work
when neural activity increases, as does Ca2+, hence there is fluorescence
74
what is the most common GECI and how does it work?
GCaMP- a fusion of calmodulin and GFP. Calmodulin binds calcium, causing a conformational change and leading to GFP fluorescence
75
are GECI's generally used in or ex vivo?
in vivo
76
strength of GECIs
- 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
weakness of GECIs
calcium imaging is an indirect measurement of neural activity
78
outline study which used GECIs
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
outline miniscopes
microscopes are traditionally large and heavy so when used on animals fixes their head, whereas miniscopes allow freely moving animals
80
compare optogenetic techniques to electrical stimulation techniques, e.g. TMS
optogenetics allows control in defined cell populations with better spatiotemporal resolution and can occur with spontaneous electrophysiological recordings possible
81
compare optogenetics to lesion studies
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
compare optogenetics with pharmacology studies
optogenetics allows better temporal precision and the possibility to investigate causal relationships- not just correlational
83
what is chemogenetics?
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
strengths of chemogenetics:
- 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
what are DREADDs?
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
what's CNO
clozapine-N-oxide, is a designer ligand that activates DREADDs
87
how are DREADDs expressed?
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
how can designer ligands, such as CNO for chemogenetics be inserted?
can be applied with injection into the body (to get to the brain in the blood), or orally
89
what's the downside of CNO
it can metabolize to clozapine which can cross BBB
90
strengths of optogenetics?
- high spatial and temporal control of neuronal activity - can be used in many model systems - disruptive technology (groundbreaking)
91
what are the general constraints to consider in research methods (give 5)
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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