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EXP NEURO > Calcium imaging, fibre photometry > Flashcards

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
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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

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3
Q

brain relo referred to as:

A
  • algorithm/ computation
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4
Q

features: algorithm/computation

A
  • constant summation of diff neurochemical signals, ultimately cause behaviour expressed/emotion experienced
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5
Q

eg. if NT x too high, Y too low

A

= anxiety/addiction/depression

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6
Q

why do we watch neurons?

A
  • help us understand their function during pathological behaviours
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7
Q

If we understand dysfunction, its possible..

A
  • possible to dev better treatments, even prevent disease
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8
Q

name (2) ways historically to watch neurons during behaviour:

A
  • histology for immediate early genes

- electrophysiology

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9
Q

histology for IEGs: aka and why use?

A
  • immediate early genes are activated rapidly, temporarily in response to neuron becoming active (APs)
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10
Q

histology for IEGs: eg. and used for

A
  • cFos

- used as proxy for neuronal activation

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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
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12
Q

histology for IEGs: cFos expression where after cocaine injection?

A
  • D1 + D2 receptor-expressing neurons in NAc
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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
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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)

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15
Q

electrophysiology: features

A
  • implant electrodes in brain to record electrical activity of neurons during behavioural events
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16
Q

criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is met in electrophysiology (2)

A
  • temporal sensitivity: during stimulus

- longitudinal sampling: weeks

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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
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18
Q

calcium imaging: why use?

A
  • interrogate brain function during translational models of psychological dysfunction
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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
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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)
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21
Q

calcium imaging: fluorescent molecules- define fluorescence

A
  • molecules absorb photons of one wavelength, and emit photons of another wavelength
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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)
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23
Q

calcium imaging: fluorescent molecules- GFP features (3)

A
  • green fluorescent protein
  • isolated from jelly fish
  • excited by 475nm emits 510nm
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24
Q

calcium imaging: fluorescent molecules- GFP excitation colour

A

blue colour

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25
Q

calcium imaging: fluorescent molecules- GFP emits colour

A

green colour

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26
Q

calcium imaging: fluorescent molecules- RFP features

A
  • red fluorescent protein
  • isolated from discosoma
  • when excited by 560nm, emits ~600nm
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27
Q

calcium imaging: fluorescent molecules- RFP excitation colour

A

green colour

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28
Q

calcium imaging: fluorescent molecules- RFP emits colour

A

orangey yellow colour

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29
Q

calcium imaging: fluorescent molecules- crucial point?

A
  • diff in excitation and emission
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30
Q

calcium imaging: Ca transients- v close correlate to?

A

AP

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31
Q

calcium imaging: Ca transients- features

A
  • temporary (transient) passage of Ca through neuron

- vital in Ca imaging and fibre photometry

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32
Q

calcium imaging: Ca transients- gradients outside vs inside

A

outside ~1500 000M

inside ~100nM

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33
Q

calcium imaging: GCaMP- fusion of (3)

A
  • GFP (G)
  • calmodulin (CaM)
  • myosin light chain kinase M13 peptide (P)
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34
Q

calcium imaging: GCaMP- calmodulin function

A
  • symmetrical hinged protein that binds to Ca
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35
Q

calcium imaging: GCaMP- Ca present causes calmodulin to

A
  • calmodulin fold at hinge
  • GFP is whole (functional)
  • green photons emitted and now quantifies measure of Ca binding
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36
Q

calcium imaging: viral mediated gene transfer- how to put GCaMP inside neuron

A
  • protein can be prod by neurons if given the gene

- viruses used and can be targeted towards specific cell types, projection sites

37
Q

calcium imaging: viral mediated gene transfer- virus procedure

A
  • 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
Q

calcium imaging: imaging Ca w camera- location

A
  • small cameras implanted on brain surface

- or shallow brain regions (eg. cortex)

39
Q

calcium imaging: imaging Ca w camera- features (3)

A
  • 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
Q

calcium imaging: imaging Ca w camera- difficult for? (2)

A
  • conducting in behavioural experiments: animal rarely freely moving
  • can’t reach deep brain regions
41
Q

calcium imaging: fibre optics- how to send and receive light to and from the brain?

A
  • 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
Q

calcium imaging: fibre optics- ‘scramble’?

A
  • fibres scramble image of cells, but enable free movement of animal
43
Q

calcium imaging: fibre optics- define fibre photometry

A
  • use fibre optics to measure light
44
Q

criteria: cellular specificity, temporal sensitivity + longitudinal sampling- which is met in Ca imaging/fibre photometry?

A
  • all 3
45
Q

calcium imaging: summary of process

A

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
Q

revision reward circuitry: dopamine prod? and released?

A
  • prod by VTA

- released into NAc

47
Q

revision reward circuitry: dopamine circuit features

A
  • 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
Q

revision reward circuitry: NAc med spiny neurons express on what receptors?

A
  • either D1 or D2 receptors
49
Q

revision reward circuitry: patterns of D1/2 during acute drug exposure?

A
  • activates D1

- suppresses D2

50
Q

revision reward circuitry: patterns of D1/2 during withdrawal from chronic cocaine

A
  • enduring increase in D1

- decrease in D2 activity

51
Q

revision reward circuitry: eg. drugs acting on dopamine pathway (4)

A
  • stimulants (meth, MDMA, cocaine, nicotine)
  • cannabinoids (THC)
  • alcohol
  • opiates (heroin, morphine, oxycodone)
52
Q

unique applications of fibre photometry in behavioural neurosci: list (4)

A
  • 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
Q

why is timing important?

A
  • similar neural events may have diff behavioural consequences
54
Q

social interaction is simple/complex behaviour? and often impaired?

A
  • complex behaviour

- impaired in various psychiatric disorders

55
Q

describing patterns of brain activation distinguish? behaviour and may lead to?

A
  • distinguish social from non-social behaviour

- lead to targeted therapies

56
Q

social approach paradigm: features

A
  • detailed vid recording of approach, interaction and withdrawal
  • 3 chambers: middle, neutral and social chamber
57
Q

timing: social situation dopamine cells?

A
  • VTA dopamine cells most active during approach + interaction w novel mouse
    (minimal activity at withdrawal)
58
Q

timing: novel object situation dopamine cells?

A
  • VTA dopamine cells
  • most active during withdrawal from novel object
  • (minimal activity at interaction)
59
Q

summary: during social interaction, peak activity in cells encodes?

A
  • appetitive approach behaviours
60
Q

summary: during novel object investigation, peak activity encodes?

A
  • withdrawal behaviours
61
Q

summary: timing important as withdrawal and interaction?

A
  • differ by half a sec

- traditional brain analysis methods (cFos) cannot detect this

62
Q

summary: cell type/pathway specificity important as?

A
  • VTA activity not always coincide w VTA-NAc activity

- electrophysiology can’t distinguish btw these 2 pop

63
Q

cell type important: D1 + D2 in NAc respond similarly/differently to rewards?

A
  • may respond differently
64
Q

cell type important: how do D1/2 cells encode for expression of drug induced conditioned behaviours?

A
  • 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
Q

cell type important: process of injecting cocaine and activity of D1/2 cells

A
  • 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
Q

summary: fibre photometry helped identify D1/2 activity by? (2)

A
  • 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
Q

predicting behaviour from pre-existing neural signatures: why only sml proportion of stressed people -> depression?

A
  • 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
Q

predicting behaviour from pre-existing neural signatures: mice that dev depressive-like symptoms (avoid social contact) after chronic stress have changes in ? activity

A
  • changes to D1/2 receptor activity = ‘vulnerable’ or ‘susceptible’ phenotype
69
Q

predicting behaviour from pre-existing neural signatures: using fibre photometry we can?

A
  • record D1 and D2 activity BEFORE behaviours (unlike before)
70
Q

predicting behaviour from pre-existing neural signatures: ‘bullying’ experiment

A
  • 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
Q

predicting behaviour from pre-existing neural signatures: result of ‘bully’ experiment

A
  • 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
Q

predicting behaviour from pre-existing neural signatures: lower D1 activity =

A

susceptible to stress

73
Q

predicting behaviour from pre-existing neural signatures: higher D1 activity =

A

resilient to stress

74
Q

predicting behaviour from pre-existing neural signatures: baseline D2 acitivity

A
  • not related to later stress susceptibiility
75
Q

predicting behaviour from pre-existing neural signatures: resocialising- resilient mice D1 activity

A
  • greater D1 activity during new social interaction in prev fearful IZ vs. susceptible mice
76
Q

predicting behaviour from pre-existing neural signatures: resocialising- D2 activity

A
  • during new interactions doesn’t differ btw two phenotypes
77
Q

summary: unsure whether it preceded chronic stress or consequence of chronic stress so using fibre photometry (2)

A
  • 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
Q

should we give cocaine to vulnerable people to boost D1 activity?

A
  • nope
  • reward, stress, addiction, depression involve vast sys beyond VTA -> dopamine to D1/2
  • need to look at broader, interconnected circuits
79
Q

circuit-wide/ sys-wide measurement of reward induced activity: multifibre photometry enables

A
  • recording from many distant and deep brain regions at the same time in same animal
80
Q

circuit-wide/ sys-wide measurement of reward induced activity: GCaMP injected and fibres implants in? (7)

A
  • prefrontal cortex PFC
  • hippocampus (CA1)
  • basolateral amygdala (BLA)
  • lateral hypothalamus (LH)
  • bed nucleus of stria terminalis (BNST)
  • NAc
  • VTA
81
Q

circuit-wide/ sys-wide measurement of reward induced activity: record?

A
  • record and cross-correlate sys-wise activity when alone and when interacting w novel mouse
82
Q

circuit-wide/ sys-wide measurement of reward induced activity: define functional connectivity:

A
  • correlated activity btw brain regions during a stimulus
83
Q

circuit-wide/ sys-wide measurement of reward induced activity: functional connectivity when alone vs. w novel mouse?

A
  • minimal when alone

- considerable functional connectivity when interacting w novel mouse

84
Q

circuit-wide/ sys-wide measurement of reward induced activity: VTA sends dopamine where else? (3)

A
  • PFC
  • NAc
  • BLA
85
Q

circuit-wide/ sys-wide measurement of reward induced activity: how do we know these are dopamine projections from VTA?

A
  • GCaMP exclusively expressed in cells w dopamine transporter (DAT)
  • VTA also has glutamate and GABA
86
Q

circuit-wide/ sys-wide measurement of reward induced activity: VTA dopamine cells fire softly/intensely to sugar rewards?

A

intensely

87
Q

circuit-wide/ sys-wide measurement of reward induced activity: which VTA dopamine axons not active during sugar reward?

A

in PFC

88
Q

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?

A
  • intensely fire

- BLA only slightly activated

EXP NEURO (10 decks)

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