Connectomes II Flashcards
in mesoscale circuit tracing tech: list (2) types of Qs asked
- where does input to this cell group come from?
- does cell group A receive input from cell group B?
in mesoscale circuit tracing tech: where does input to this cell group come from?
hypothesis-free discovery project: unlikely to definitively answer Qs regarding functional organisation of a circuit
- serves as platform for generation of more specific hypotheses (that need to be tested in later experiments)
in mesoscale circuit tracing tech: does cell group A receive input from cell group B?
- specific research Q (RQ)
- unambiguously addressed by presence of labelled neurons
mapping connectome controlling BP: quick procedure
- mapped inputs to group of spinally projecting brainstem neurons in RVLM (rostral ventrolateral medulla): known to control BP via excitatory effects on sym nn
mapping connectome controlling BP: findings
- found monosyn input neurons in many diff brain regions, many were already assoc w effects on BP
mapping connectome controlling BP: unexpected finding
- RVLM neurons receive input from cholinergic interneurons of intermediate reticular nucleus
mapping connectome controlling BP: hypothesis
- monosynaptic input from cholinergic IRt neurons responsible for post-inspiratory activation of sym nn
- functional experiments upholds the hypothesis
eg. accessory resp mm (2) exhibit third phase in respiration?
- post-inspiratory phase (post-i)
- eg. crural diaphragm, laryngeal adductors
respiratory pump mm and innervation eg. (2) exhibit what 2 phases?
- inspiration
- expiration
- eg. phrenic nn, diaphragm
what inn post-i activity
- sym nn which also controls BP
hypothesis: if IRt is silenced, what happens to post-i activity?
- testing if necessary for generation of post-i activity
= should disappear if so
limitations of G-deleted rabies: list (2)
- does not label all presynaptic neurons
- rabies virus is toxic to humans
limitations of G-deleted rabies: labelling of presyn neurons issues (3)
- each neuron 1000s synaptic inputs, many rabies virions required inside starter cell to infect sig portion of synapses
- trans-syn efficiency of each virion is stochastic (random prob dist can’t be predicted precisely) due to multiple factors (incl. interaction btw rabies glycoprotein and cellular components)
- overall <10% input neurons are labelled (possibly less)
limitations of G-deleted rabies: solutions: labelling efficiency issues (2)
- try g-proteins from diff strains of rabies to find highest efficiency
- PBG variant is highest no. of presyn n/ starter cell - optimise PBG gene sequence (oG) so more protein expressed per neuron
- manipulation glycoprotein lead to improved efficiency
limitations of G-deleted rabies: toxic rabies features
- standard (B19 strain) g deleted rabies starts killing neurons by 14 days
limitations of G-deleted rabies: solutions to toxic rabies
- try other strains of rabies:
- others engineered to drive same targeted retrograde labelling (eg. N2c) = higher infection efficiency, lower toxicity
or 2. make B19 strain less toxic
- manipulate rabies to prod protein turning replication off
Qs to ask: brain is more than its connectome! (2)
- what are response properties of neurons that provide input to cell group u target?
- what NT do they release?
incorporate functional toolboxes into transsynaptic tracers: modifed rabies variants used tools like? and effect
- channelrhodopsin2
- allowing identified input neurons to be switched on/off
- allows u to figure out what effect identified network components have overall network behaviour
incorporate functional toolboxes into transsynaptic tracers: variants that express genetically encoded Ca sensors features-
- Ca sensors enable u to visualise activity in pressyn neurons
- mod GFP genes change brightness in relation to intracellular Ca conc, optical recording of neuronal activity
- allows u to determine behavioural profile of input neurons
simultaneous id/recording of microcircuits in vivo: Q- how r microcircuits that discriminate visual stimuli organised? list approach (3)
- target single visual cortex output (pyramidal) neurons w genetics tools required for rabies entry and spread (and red fluorescent protein)
- infect single neuron w rabies encoding GCamp6 Ca sensor, install glass window over brain surface
- after infection of neuron, connectome record the responsiveness of all neurons in microcircuit to standardised visual stimuli
simultaneous id/recording of microcircuits in vivo: which ideals are satisfied (5)
- allow selection of subpop of neurons for investigation
- identify local and distant connections
- identify connected cells in the live animal
- integrated w tools that allow investigation of behaviour
- identify monosynaptically linked neurons
simultaneous id/recording of microcircuits in vivo: conclusion
- although powerful, approach not suitable for neural networks that can’t be imaged in vivo (eg. circuits that are v spread out/ reside deep in the brain)
simultaneous id/recording of microcircuits in vivo: final thought on monosynaptic viral tracers (4)
- transformative technology because coz targeted to selected cell groups and provide unambiguous connectivity data
- already work quite well (not for humans) and rapidly improving
- tools for analysis hav yet to catch up
- no equivalent anterograde monosynaptic tracers
whole brain human connectivity: MRI based imaging techniques for inferring connectivity in humans- list (2)
- diffusion weighted tractography
- dynamic functional connectivity
MRI imaging: pros (3)
- image lrg vol quickly
- human subjects
- subjects alive, conscious
MRI imaging: cons (5)
- poor spatial resolution (2-3mm)
- each voxel 8-27 mm3
- poor temporal res (10-30ms) will miss short latency responses
- doesn’t actually measure neuronal activity, rather fluctuations in brain blood flow
- size, cost
diffusion weighted tractography: concept
- MRI measures magnetic resonance of water molecules aligned within a magnetic fields
- water not evenly dist in brain - lots inside cells, not much in axons/fibre tracts (wrapped in fat)
= map axon tracts
diffusion weighted tractography: id of lrg fibre tracts BUT (cons)
- doesn’t tell u which direction the tracts r travelling and only id’s v lrg bundles of axons
diffusion weighted tractography: useful for?
- gross anatomy (eg. changes in brain connectivity after head injury) but maybe hard to directly relate structure to function
dynamic functional connectivity: concept
- parts of brain connected will have lvls of activity fluctuate together
- image whole brain activity in steady state conditions using fMRI, then analyse entire dataset to id voxels that have linked activity
dynamic functional connectivity: procedure
- image brain for prolonged period
- divided brain into regions of interest
- each region: determine how activity in region fluctuates w time compared to other regions of interest- connected regions should wax/wane together= connectivity matrix
connectomic insight: list specific brain circuits (4)
- breathing
- vision
- sex diff in human brain structure
- decision making in worms
connectomic insight: list some general rules that dictate circuit architecture etc. (2) and extent where
across diff species, diff res (micro/meso/macro) and diff brain regions, same rules:
- spatial embedding (neurons receive connections from nearby cells vs more distant cells)
- reciprocity (neurons provide feedback. to upstream circuit components)
connectomic insight: what does having these general rules mean?
- prob relatively simple rules govern brain self-assembly
- diff circuits prob hav more in common w each other than prev thought?
list some parallel dev which help increase accessibility, reduce cost of powerful connectomic tech (4)
- imaging
- vector biology
- analytics
- machine learning
how will we use all this connectomic knowledge?
- better understand the architecture of specific circuits
- dev better understanding of general rules that dictate brain organisation
define: PBG and oG
PBG: variant of G-deleted rabies strain w highest efficiency (highest no. pressyn neurons/starter cell)
oG: optimised gene, modified PBG which increased glycoproteins expressed/neuron
