Calcium imaging, fibre photometry Flashcards
1
Q
what ‘tools’ do we need rn?
A
- those that can distinguish diff types of cells, compatible w translational animal models of disease
2
Q
contemporary neurosci assumes ‘normal, healthy’ behaviour emerges from?
A
- ‘normal, healthy’ brain function
- thus abnormal/unhealthy behaviours must be from abnormal brain activity
3
Q
brain relo referred to as:
A
- algorithm/ computation
4
Q
features: algorithm/computation
A
- constant summation of diff neurochemical signals, ultimately cause behaviour expressed/emotion experienced
5
Q
eg. if NT x too high, Y too low
A
= anxiety/addiction/depression
6
Q
why do we watch neurons?
A
- help us understand their function during pathological behaviours
7
Q
If we understand dysfunction, its possible..
A
- possible to dev better treatments, even prevent disease
8
Q
name (2) ways historically to watch neurons during behaviour:
A
- histology for immediate early genes
- electrophysiology
9
Q
histology for IEGs: aka and why use?
A
- immediate early genes are activated rapidly, temporarily in response to neuron becoming active (APs)
10
Q
histology for IEGs: eg. and used for
A
- cFos
- used as proxy for neuronal activation
11
Q
histology for IEGs: cFos technique
A
- apply stimulus to animal (drug injection, behavioural event)
- 90min later, euthanise, remove brain, dissect, apply antibody + visualise w microscope
12
Q
histology for IEGs: cFos expression where after cocaine injection?
A
- D1 + D2 receptor-expressing neurons in NAc
13
Q
criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is met in using histology for IEGs? (1)
A
- cellular specificity: can see D1 vs D2 receptors
14
Q
criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is NOT met in using histology for IEGs? (2)
A
- temporal sensitivity: 90min after stimulus
- longitudinal sampling: only 1 time point (euthanised)
15
Q
electrophysiology: features
A
- implant electrodes in brain to record electrical activity of neurons during behavioural events
16
Q
criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is met in electrophysiology (2)
A
- temporal sensitivity: during stimulus
- longitudinal sampling: weeks
17
Q
criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is NOT met in electrophysiology (1)
A
- cellular specificity: can’t dissociate btw D1/D2
18
Q
calcium imaging: why use?
A
- interrogate brain function during translational models of psychological dysfunction
19
Q
calcium imaging: what is it?
A
- combo of genetics, light physics, neurochem = enables real time measurement of targeted cells/projections during meaningful behaviours
20
Q
calcium imaging: list (6) things used
A
- fluorescent molecules
- Ca transients
- GCaMP (fluorescent Ca sensor)
- viral mediated gene transfer
- imaging Ca activity w sensor (camera)
- fibre optics (specifically for fibre photometry)
21
Q
calcium imaging: fluorescent molecules- define fluorescence
A
- molecules absorb photons of one wavelength, and emit photons of another wavelength
22
Q
calcium imaging: fluorescent molecules- black lights features and wavelengths
A
- shine almost invisible light (365nm) onto special phosphorescent paint will emit back visible wavelength (500-600nm)
23
Q
calcium imaging: fluorescent molecules- GFP features (3)
A
- green fluorescent protein
- isolated from jelly fish
- excited by 475nm emits 510nm
24
Q
calcium imaging: fluorescent molecules- GFP excitation colour
A
blue colour
25
calcium imaging: fluorescent molecules- GFP emits colour
green colour
26
calcium imaging: fluorescent molecules- RFP features
- red fluorescent protein
- isolated from discosoma
- when excited by 560nm, emits ~600nm
27
calcium imaging: fluorescent molecules- RFP excitation colour
green colour
28
calcium imaging: fluorescent molecules- RFP emits colour
orangey yellow colour
29
calcium imaging: fluorescent molecules- crucial point?
- diff in excitation and emission
30
calcium imaging: Ca transients- v close correlate to?
AP
31
calcium imaging: Ca transients- features
- temporary (transient) passage of Ca through neuron
| - vital in Ca imaging and fibre photometry
32
calcium imaging: Ca transients- gradients outside vs inside
outside ~1500 000M
| inside ~100nM
33
calcium imaging: GCaMP- fusion of (3)
- GFP (G)
- calmodulin (CaM)
- myosin light chain kinase M13 peptide (P)
34
calcium imaging: GCaMP- calmodulin function
- symmetrical hinged protein that binds to Ca
35
calcium imaging: GCaMP- Ca present causes calmodulin to
- calmodulin fold at hinge
- GFP is whole (functional)
- green photons emitted and now quantifies measure of Ca binding
36
calcium imaging: viral mediated gene transfer- how to put GCaMP inside neuron
- protein can be prod by neurons if given the gene
| - viruses used and can be targeted towards specific cell types, projection sites
37
calcium imaging: viral mediated gene transfer- virus procedure
- infect cells, deliver genetic payload
- enter cells by binding to viral receptors located on cell surface
- virus survives ~24hrs before getting killed by host immune sys
- virus delivers gene for GCaMP so native cells begin to
38
calcium imaging: imaging Ca w camera- location
- small cameras implanted on brain surface
| - or shallow brain regions (eg. cortex)
39
calcium imaging: imaging Ca w camera- features (3)
- enables visual recording of individual neurons
- ! for understanding micro-circuitry: how neurons A + B coordinate activity w each other
- also good for neuroplasticity: how does neuron A change its pattern of activity over time?
40
calcium imaging: imaging Ca w camera- difficult for? (2)
- conducting in behavioural experiments: animal rarely freely moving
- can't reach deep brain regions
41
calcium imaging: fibre optics- how to send and receive light to and from the brain?
- fibre optic cables made from hair-thin fine glass: carry LED or laser light
- cables mirrored tubes, enabling light to transfer efficient despite twisting and curving
42
calcium imaging: fibre optics- 'scramble'?
- fibres scramble image of cells, but enable free movement of animal
43
calcium imaging: fibre optics- define fibre photometry
- use fibre optics to measure light
44
criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is met in Ca imaging/fibre photometry?
- all 3
45
calcium imaging: summary of process
virus targets projection/cell types - prod GCaMP - AP cause influx of Ca - binds to GCaMP making GFP 'active' - implanted fibre optic/camera shines excitatory 470nm light onto active molecules which emit 510nm
- (if no Ca present, no light emitted)
- emitted light transferred from brain - fibre optic cable - camera = quantify using photoreceivers or cameras + software
46
revision reward circuitry: dopamine prod? and released?
- prod by VTA
| - released into NAc
47
revision reward circuitry: dopamine circuit features
- v old, conserved pathway
- mediate responses to natural rewards: food, sex, social interaction
- all drugs of abuse products effects this pathway, despite diff chemical structures
48
revision reward circuitry: NAc med spiny neurons express on what receptors?
- either D1 or D2 receptors
49
revision reward circuitry: patterns of D1/2 during acute drug exposure?
- activates D1
| - suppresses D2
50
revision reward circuitry: patterns of D1/2 during withdrawal from chronic cocaine
- enduring increase in D1
| - decrease in D2 activity
51
revision reward circuitry: eg. drugs acting on dopamine pathway (4)
- stimulants (meth, MDMA, cocaine, nicotine)
- cannabinoids (THC)
- alcohol
- opiates (heroin, morphine, oxycodone)
52
unique applications of fibre photometry in behavioural neurosci: list (4)
- enables measurement of 2 diff neural signals occurring closely in time (temporal sensitivity)
- enables measurement of 2 diff cell types located v closely together (spatial sensitivity)
- measurement of neural activity prior to behavioural event
- enables simultaneous measurement of multiple brain sys
all in freely moving behaving animal
53
why is timing important?
- similar neural events may have diff behavioural consequences
54
social interaction is simple/complex behaviour? and often impaired?
- complex behaviour
| - impaired in various psychiatric disorders
55
describing patterns of brain activation distinguish? behaviour and may lead to?
- distinguish social from non-social behaviour
| - lead to targeted therapies
56
social approach paradigm: features
- detailed vid recording of approach, interaction and withdrawal
- 3 chambers: middle, neutral and social chamber
57
timing: social situation dopamine cells?
- VTA dopamine cells most active during approach + interaction w novel mouse
(minimal activity at withdrawal)
58
timing: novel object situation dopamine cells?
- VTA dopamine cells
- most active during withdrawal from novel object
- (minimal activity at interaction)
59
summary: during social interaction, peak activity in cells encodes?
- appetitive approach behaviours
60
summary: during novel object investigation, peak activity encodes?
- withdrawal behaviours
61
summary: timing important as withdrawal and interaction?
- differ by half a sec
| - traditional brain analysis methods (cFos) cannot detect this
62
summary: cell type/pathway specificity important as?
- VTA activity not always coincide w VTA-NAc activity
| - electrophysiology can't distinguish btw these 2 pop
63
cell type important: D1 + D2 in NAc respond similarly/differently to rewards?
- may respond differently
64
cell type important: how do D1/2 cells encode for expression of drug induced conditioned behaviours?
- drug conditioned placed preference (classical/pavlovian conditioning) where mice choose which chamber it prefers to spend time in (saline vs. cocaine)
- uses transgenic mice, virus targets D1 or D2 neurons
65
cell type important: process of injecting cocaine and activity of D1/2 cells
- injection w cocaine in one context prod enduring preference for spending time in context
- D1 cells increase activity
- D2 cells reduce activity (minimal effect/ slight reduction)
66
summary: fibre photometry helped identify D1/2 activity by? (2)
- cell type specificity (D1 vs D2 due to transgenic mice + virus targeting)
- temporal precision (cocaine vs. saline chamber entries may occur only secs apart)
67
predicting behaviour from pre-existing neural signatures: why only sml proportion of stressed people -> depression?
- strong genetic component for both humans and rats
- certain strains of rodents more resilient to stress, others more vulnerable
- despite same life experience, 'vulnerable' mice more affected by stressors
68
predicting behaviour from pre-existing neural signatures: mice that dev depressive-like symptoms (avoid social contact) after chronic stress have changes in ? activity
- changes to D1/2 receptor activity = 'vulnerable' or 'susceptible' phenotype
69
predicting behaviour from pre-existing neural signatures: using fibre photometry we can?
- record D1 and D2 activity BEFORE behaviours (unlike before)
70
predicting behaviour from pre-existing neural signatures: 'bullying' experiment
- mice w fibres in NAc (D1 or D2) left to explore box and spend similar time in 'interaction zone' (IZ)
- mice repeatedly 'socially defeated' (bullied by larger mouse) in IZ
- then mice left explore box freely afterwards
71
predicting behaviour from pre-existing neural signatures: result of 'bully' experiment
- retrospectively mice grouped as 'resilient' or 'susceptible' depending on how much they avoided 'bully zone'
- resilient: spent more time in IZ than before,
- susceptible: avoid spot
72
predicting behaviour from pre-existing neural signatures: lower D1 activity =
susceptible to stress
73
predicting behaviour from pre-existing neural signatures: higher D1 activity =
resilient to stress
74
predicting behaviour from pre-existing neural signatures: baseline D2 acitivity
- not related to later stress susceptibiility
75
predicting behaviour from pre-existing neural signatures: resocialising- resilient mice D1 activity
- greater D1 activity during new social interaction in prev fearful IZ vs. susceptible mice
76
predicting behaviour from pre-existing neural signatures: resocialising- D2 activity
- during new interactions doesn't differ btw two phenotypes
77
summary: unsure whether it preceded chronic stress or consequence of chronic stress so using fibre photometry (2)
- baseline D1 activity predicts whether resilient/susceptible to chronic stress (higher activity = more resilient)
- resilient mice more willing to interact w new partner in same env of prev bully and have higher D1 activity (not D2) during interaction
- could help humans if record neural signature before chronic stress, targeted therapies to prevent depression?
78
should we give cocaine to vulnerable people to boost D1 activity?
- nope
- reward, stress, addiction, depression involve vast sys beyond VTA -> dopamine to D1/2
- need to look at broader, interconnected circuits
79
circuit-wide/ sys-wide measurement of reward induced activity: multifibre photometry enables
- recording from many distant and deep brain regions at the same time in same animal
80
circuit-wide/ sys-wide measurement of reward induced activity: GCaMP injected and fibres implants in? (7)
- prefrontal cortex PFC
- hippocampus (CA1)
- basolateral amygdala (BLA)
- lateral hypothalamus (LH)
- bed nucleus of stria terminalis (BNST)
- NAc
- VTA
81
circuit-wide/ sys-wide measurement of reward induced activity: record?
- record and cross-correlate sys-wise activity when alone and when interacting w novel mouse
82
circuit-wide/ sys-wide measurement of reward induced activity: define functional connectivity:
- correlated activity btw brain regions during a stimulus
83
circuit-wide/ sys-wide measurement of reward induced activity: functional connectivity when alone vs. w novel mouse?
- minimal when alone
| - considerable functional connectivity when interacting w novel mouse
84
circuit-wide/ sys-wide measurement of reward induced activity: VTA sends dopamine where else? (3)
- PFC
- NAc
- BLA
85
circuit-wide/ sys-wide measurement of reward induced activity: how do we know these are dopamine projections from VTA?
- GCaMP exclusively expressed in cells w dopamine transporter (DAT)
- VTA also has glutamate and GABA
86
circuit-wide/ sys-wide measurement of reward induced activity: VTA dopamine cells fire softly/intensely to sugar rewards?
intensely
87
circuit-wide/ sys-wide measurement of reward induced activity: which VTA dopamine axons not active during sugar reward?
in PFC
88
circuit-wide/ sys-wide measurement of reward induced activity: VTA dopamine axons fire softly/intensely to sugar reward and which only slightly activated by sugar?
- intensely fire
| - BLA only slightly activated
