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EXP NEURO > Connectomes I > Flashcards

Connectomes I Flashcards

1
Q

current model of the brain:

A
  • brain like microprocessor
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2
Q

circuit function =

A

architecture of circuit + component properties

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3
Q

Santiago Ramon y Cajal: sig

A
  • defined neuron theory
  • using Golgi stain technique could discover neuron structure
  • also found many diff cell types
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4
Q

list (2) components which make up neuronal activity:

A
  • membrane kinetics

- neuropharmacology

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5
Q

membrane kinetics: features

A
  • AP

- ion channels

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6
Q

neuropharm: general features

A
  • synaptic transmission

- plasticity

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7
Q

hardware and software: what do we understand so far

A
  • hardware

- good models predicting biophysical and chemical processes governing neural activity

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8
Q

hardware and software: what don’t we understand yet

A
  • how simple circuits are organised

- therefore can’t predict how groups of connected neurons will behave

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9
Q

neurons: issues

A
  • very small synapses (1-10µm)
  • each communicate w 1-10 000 others
  • highly variable cell types/ brain regions: no generally applicable organisational scheme
  • huge divergence and convergence
  • # possible permutations > # atoms in the universe
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10
Q

define connectome:

A

structural architecture of NS connectivity in all animals at all resolutions

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11
Q

technology: list 3 diff resolutions

A
  • nano-scale
  • meso-scale
  • macro-scale
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12
Q

technology: give technique and eg. - nanoscale

A
  • brute force

- animal and human

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13
Q

technology: give technique and eg. - mesoscale

A
  • circuit tracing

- animal

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14
Q

technology: give technique and eg. - macroscale

A
  • low resolution non-invasive

- human

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15
Q

ultimate technology: 3 features

A
  • large scale
  • specific
  • functional
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16
Q

list the ideal technology: (5) points

A
  • allow selection of subpop of neurons for investigation
  • identify local/ distant connections
  • identify connected cells in the live animal
  • integrated w tools that allow investigation of behaviour
  • identify monosynaptically linked neurons
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17
Q

nanoscale: name technique

A
  • serial block face electron microscopy
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18
Q

serial block face electron microscopy: procedure

A
  • cut sample of tissue into v thin sections (nm), image on electron microscope, reconstruct in 3D vol
  1. prepare and image tissue
  2. trace individual neurons
  3. render, detect synapses
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19
Q

serial block face electron microscopy: pros

A
  • ultimate resolution

- images EVERYTHING

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20
Q

serial block face electron microscopy: cons

A
  • small field of view (<0.1 x 0.1mm)
  • very slow (2014: 1.5yrs/ mm3)
  • expensive (300 Tb/mm3(
  • purely anatomical: hard to relate structure to function
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21
Q

serial block face electron microscopy: satisfies which ideals (1)

A
  • identify monosynaptically linked neurons
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22
Q

mesoscale: name technique

A
  • conventional tract tracing
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23
Q

conventional track tracing: procedure

A
  • monitor axonal movement of dyes (tracers) through to identify long range connectivity
24
Q

conventional track tracing: tracer

A
  • substance that is endocytosed (taken up) and transported within neuron to allow identification of its body/axon/terminals
25
Q

conventional track tracing: list tracer types (6)

A
  • static
  • transynaptic
  • chemical
  • viral
  • anterograde
  • retrograde
26
Q

conventional track tracing: features- retrograde

A
  • enter synaptic terminals

- move backwards to cell body

27
Q

conventional track tracing: features- retrograde eg. (4)

A
  • cholera toxin B (CTb)
  • latex beads
  • rabies virus (SADB19)
  • pseudorabies virus (PRV)
28
Q

conventional track tracing: features- cholera toxin B (CTb)

A
  • static retrograde
29
Q

conventional track tracing: features- rabies virus (SADB19)

A
  • monosynaptically restricted retrograde
30
Q

conventional track tracing: features- pseudorabies virus (PRV)

A
  • polysynaptic retrograde
31
Q

conventional track tracing: features- anterograde

A
  • enter cell body

- move forwards towards synaptic terminals

32
Q

conventional track tracing: anterograde tracers- eg. (3)

A
  • phaseolus vulgaris leucoagglutinin (PHA-L)
  • most adeno-assoc viral vectors (AAV)
  • herpes strain 129 (H129)
33
Q

conventional track tracing: features- PHA-L

A
  • static anterograde
34
Q

conventional track tracing: features- H129

A
  • polysynaptic anterograde
35
Q

conventional track tracing: features- AAV (adeno-assoc viral vectors)

A
  • monosynaptically restricted anterograde
36
Q

conventional track tracing: theoretical steps and what retro/anterograde tracers label in cells

A
  • inject tracer in region of interest
  • look for labelled cells: retrograde tracers label cell bodies
  • anterograde tracers label axons and synaptic terminals
37
Q

conventional track tracing: list (5) caveats (warnings/precaution)

A
  • most tracers bidirectional (antero + retro)
  • tracers can be taken up by fibres of passage
  • labelled terminals not necessarily indicate synaptic connection (<50% under EM)
  • can’t be selectively targeted to particular cell types within injection site
  • both conventional/viral tracers good for identifying distant inputs, but can’t identify LOCAL connectivity adequately (due to indiscriminate tracer deposition at injections site)
38
Q

viral tracers: features

A
  • replication deficient viruses that deliver exogenous genes (transgenes) to target cells (neurons) driving transcription and expression of protein
  • by driving expression of fluoro ‘reporter’ proteins can now visualise ‘transduced neurons’
  • trajectories of transduced neurons can then be mapped
39
Q

viral tracers: allen brain connectivity atlas features

A
  • mesoscale connectome of mouse brain
  • online repository of whole brain images from 2911 experiments, AAV-reporter vectors injected at various sites in mouse brain
40
Q

targeted circuit discovery- transsynaptic viral tracing: features and eg

A
  • spread through CNS by jumping across synapses

- derived from pathogens (rabies, tetanus, herpes, pseudorabies, vesicular stomatitis virus)

41
Q

transsynaptic viral tracing: initial concept was to exploit

A
  • infection mechanism used by neuron specific (neurotrophic) viruses
  • identify cells that control Ad secretion using pseudorabies (Strack) 1989
42
Q

transsynaptic viral tracing: Strack’s experiment procedure

A
  • inject virus into adrenal gland
  • kill rat, process tissue to detect infected cells
  • count cells, make maps
43
Q

transsynaptic viral tracing: satisfies which ideals (1)

A
  • identify local and distant connections
44
Q

monosynaptic tracing using recombinant rabies: main features (2)

A
  • fancy tracer, not transsynaptic

- no connectome tracing

45
Q

monosynaptic tracing using recombinant rabies: procedure- 1. genetic modification of rabies virus

A
  • G-deleted fluoro reporter (exised g-protein gene, swap w reporter etc.)
  • cells genetically modified to synthesis rabies glycoprotein

= pseudotyped virus

46
Q

monosynaptic tracing using recombinant rabies: G-deleted rabies key features (3) retrograde/anterograde

A
  • replication competent (self-amplifying)
  • drives transgene (eg. reporter) expression at high levels
  • retrograde only: can only infect terminals
47
Q

monosynaptic tracing using recombinant rabies: satisfies which ideals (3)

A
  • identify distant connections
  • identify connected cells in live animal
  • identify monosynaptically linked neurons
48
Q

monosynaptic tracing from targeted subpop: strategy

A
  • coat rabies in envelope protein from avian sarcoma + leukosis virus and drive expression of the ASLV receptor (TVA) on your target population
  • restricts entry to cells expressing avian receptor protein (TVA)
  • drive non-mammalian viral envelope receptor on target pop of neurons
49
Q

restore transsynaptic spread: strategy

A
  • drive TVA + rabies glycoprotein on target pop of neurons

- exogenously expressed rabies glycoprotein reintegrates w rabies virion

50
Q

validation: evidence of synaptic connection

A
  • record both neurons: drive AP in presynaptic neuron- time-locked changed in membrane current in postsynaptic neuron denote connectivity
51
Q

validation: evidence of synaptic connection- +ve deflections =

A

inhibitory inputs

52
Q

validation: evidence of synaptic connection- -ve deflections

A

excitatory inputs

53
Q

monosynaptic tracing (?) from targeted subpop: satisfies which ideals (4)

A
  • allows selection of subpop of neurons for investigations
  • identify local and distant connections
  • identify connected cells in live animal
  • identify monosynaptically linked neurons
54
Q

connectome tracing: features incl cons

A
  • presence of reporter= synaptic connection exists
  • don’t need to image in ultra-high res, just enough to resolve labelled neurons
  • imaging /animal <1 wk
  • cells start to die after 14 days, major shortcoming
55
Q

mapping connectome that controls BP: strategy

A
  • rabies + herpes
  • seed= spinal projection + monosynaptic input
  • ID input neurons
  • MRI-based brain model
  • import neuron positions
  • volumetric brain reconstruction and mapping
  • pooled data

EXP NEURO (10 decks)

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