Experimental methods (I.e. N2) Flashcards
(24 cards)
What is the dead salmon study? What does it show?
Danger of drawing conclusions from high dimensionality data analysed with low power (few repeats):
* High risk of false positives
* Statistic tests struggle to differentiate signal from noise
A dead salmon was exposed to an emotional valence task (i.e. presented with images of different emotional value).
* Image processing through a particular method identified an area with signficant BOLD signal change…
* This is not possible
What is the GRASP technique?
GFP reconstitution across synaptic partners.
* One GFP fragment injected with anterograde tracer (AAV1)
* Complementary fragment injected with retrograde tracer (e.g. AAV9) in other area
* Only becomes fluorescence when they meet, revealing a direct connection
How can functional connectivity maps be used to infer potential causes of disease (e.g. Alzheimer’s)?
Can compare functional connectivity map to gene expression map.
For example: link between Alzheimer’s disease development and immune activation in brain
* Is it cause or correlation?
What are the main ways that connectivity maps could be used in diagnosis and treatment of brain disorders?
- Diagnosis: e.g. particular patterns of activity/structural differences. Good for autism
- To predict who might respond to a partiuclar treatment based on part of the network which is dysfunctional or matching activity to gene expression maps.
- Tracing disease progression e.g. Alzheimer’s: predict which areas will be damaged next or do retrospecitive analysis to examine root causes
What fundementally limits the use of connectomics in understanding of psychiatric disease? (3 overarching points)
Variability between individuals:
* Comparison between control and case groups requires ignoring heterogeneity
Experimental design
* Disconnect between those used in research and frontline reality (e.g. many university students used in research but minority of cases)
* Mostly male studies
* Some groups cannot be measured e.g. severely autistic people cannot remain still in a scanner…
Tools available to get data:
* Resolution
* Capacity
* Noise and artifact removal
How could you selectively manipulate the connection between two brain regions using optogenetics?
- Inject Cre-recombinase carrying virus into one region (e.g. anterograde tracer)
- Inject cre-dependent optogene into other region (e.g. with retrograde tracer)
- Only neurons receiving projections from one to other will express optogene but others in the exposed area will not.
What are the main limitations to Ca2+ imaging? (Provide study name)
- Reporting molecule can change spiking pattern by opening K+ channels or chelating Ca2+ (Mckiernan et al 2013)
- Ca2+ is a proxy to voltage (i.e. highly correlated) not a direct measure
What does the AX-CPT test for? How can it be translated for mice?
Test for attention:
* Series of letters shown, only signal if X preceeded by A.
* False alarm rates (BX or AY) measured showing a lack of attention. Misses also suggest lack of attention.
* Measure how it changes over repeats
Translated to 5C-CPT task in rodents
* 5 target apertures (lights) presented
* Rodents are trained to nose-poke an illuminated aperture
* But must not poe when the other apertures are also illuminated
* A miss is no nose-poke despite one illuminated aperture (or incorrect selection) a false alarm is a nose-poke when all 5 lights are illuminated
What rewards can be used (i..e how can a response be induced)? DOes this change across species?
Primary reward:
* Associating a stimulus with a reward (i.e. Pavlovian conditioning) such as food or sex
* In animals
Secondary reward:
* Money, praise
* Humans
Problem: primary and secondary rewards shown to activate different brain areas (so are animal experiments really valid?)
What functions do all behavioural tests require to some degree? Why is this a problem?
Perception, action and attention.
Problem is testing another function when these are deficient - can skew results.
How can response to reward be measured?
Measuring unconditional responses:
* Salivating before food
* Rearing behaviour in rodents
* Measuring dopamine release
Measuring expectation of reward:
* Orienting towards associated stimulus (CS)
* Moving towards US (e.g. sight of food)
What role does the amygdala have in response to reward? How was this discovered?
Experiments in rodents to food reward.
CeN: controls attention to CS of reward:
* Rodents with CeN lesion no longer orient towards light cue
BLA: necessary for stimulus-reward association learning.
* BLA lesions prevent adjusting response after devaluation (food replaced by LiCl).
Which brain regions are involved in flexibility? Which tasks target them?
Rule shifting: vlPFC
* WCSA
* Attentional set-shifting tasks (e.g. shapes with lines overlaid, first trained to select particular line than ignore lines and choose particular shape)
Reversal learning: (l)OFC
* Reverse the association between a stimulus and the reward/punishment
What are the different ways to measure anxiety (categories - give example of each)?
Physiological measures:
* BP, HR, GSR
* Brain activity
Behavioural response:
* Innate e.g. freezing in rodents, blinking in humans
* Adaptive: moving to edges in OFT, egg-calling in marmosets
* Need to be made quantifiable (e.g. reaction time, time spent, startle magnitude).
Lay out the key points for an essay on whether new technology has fundementally changed neural circuit analysis.
- What is neural circuit analysis? - Understanding of structure, function and effects of neural circuits and their components through experimentation
- Relies on correlational and causal investigation requiring ability to observe and manipulate neural circuits
- New technology has increased the precision of investigation - allowing increased resolution of measurement at higher capacity.
HOWEVER: All these tools are still subject to same caveats:
* That the act of observing does not change the function of the system
* That the variable used t oobserve represents what is being measured (accuracy of proxy)
* That the act of manipulation only changes the variable targeted.
Therefore there has been a shift in the practicalities of analysis but not the fundemental principles.
Will look at this through analysis of observation tools, manipulation tools and the use of computation.
Lay out the key points for an essay on the merit of using different animal species in the study of neural circuits.
- Why do we study neural circuits? - Better understanding to model for use in prediction (of human function)
- Understanding requires correlation and causation experimentation
- Causation study requires manipulation which is very limited in humans
- Animals provide good model - similarities in neurobiology with more ability to manipulate and observe (higher resolution). True in different animals to different extent (often a trade off)
- Problem: animals are not hte same as humans. Therefore experimental methodoogy is different
- When results are different from humans is this due to a fundemental difference in neurobiology (reducing translatability to humans) or from the difference in experimental set up?
- Will explore these problems looking at commonly used animal models - drosophila, rodents and non-human primates.
How can you measure the amount of glutamate release
SuperGluSnFR – fluoresces when glutamate bound.
Observed using microscopy.
What is the problem with CNO for chemogenetics? How has this been tackled?
CNO can be reverse metabolised into clozapine (‘reliable phenomenon - Manvich et al)
* Clozapine interacts with DA/5-HT
Improvement:
* Desochloroclozapine (DCZ) has much lower reverse metabolism.
* 1000x lower dose required for same manipulation capacity
What are 4 main uses of organoids for understanding neurobiology (general)?
- Brain formation/development investigation
- Evolutionary differences
- Development of diseaes
- Investigating importance of variability
What are chimeroids? Why are they useful?
Brain organoids made up of multiple different cell types (one system or different individuals).
Useful for:
* Testing different cell types in the same condition
* Testing effect of variability (e.g. cells from different patient of the same tumours)
What are ALI-COs? Why are they useful?
Air-liquid interface-culture organoid
Benefits:
* Extended life of culture (as exposed to nutrients and oxygen (reduces necrosis)
* Extends longevity of studies (important for developmental or progressive disease modelling)
* More robust and sustained neural activity seen (e.g. more mature dendritic morphology)
What are some general pros and cons of organoid models?
Pros:
* Use of human cells (relevant)
* 3D rather than 2D cultures allows more representative structure
* High reproducibility/throughput
* Easy to GE/record from/profile
Cons:
* Artificial environment - high cellular stress, hypoxia
* Severely limited interaction with surrounding tissue (e.g. no ephaptic communication, immune interaction)
* Limited longevity (e.g. for study of mature structures)
What is the TRAP system? How does it work?
Targeted recombination in active populations (TRAP):
* Cells express a cre-recombinase promotor with opsin/fluorescence reporter
* But recombination of cre is dependent on tamoxifen (an oestrogen receptor drug)
* Administration of tamoxifen can be tightly temporally controlled
* Only cells active in that window are labelled.
What is the Tet-OFF system? Why is it useful?
Tetracycline indicible conditional expression system (Tet-OFF):
* Transgene (carrying ChR2-EYFP) is only expressed when there is no administered doxycycline
* Removing doxycycline for the training window means only those active during training (involved in learning) express opsin.
* These cells can then be selectively manipulated (optogeneic activation)
E.g. Kim et al 2018 looking at memory engram (memory storing) cells
