Connectomes I Flashcards
current model of the brain:
- brain like microprocessor
circuit function =
architecture of circuit + component properties
Santiago Ramon y Cajal: sig
- defined neuron theory
- using Golgi stain technique could discover neuron structure
- also found many diff cell types
list (2) components which make up neuronal activity:
- membrane kinetics
- neuropharmacology
membrane kinetics: features
- AP
- ion channels
neuropharm: general features
- synaptic transmission
- plasticity
hardware and software: what do we understand so far
- hardware
- good models predicting biophysical and chemical processes governing neural activity
hardware and software: what don’t we understand yet
- how simple circuits are organised
- therefore can’t predict how groups of connected neurons will behave
neurons: issues
- 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
define connectome:
structural architecture of NS connectivity in all animals at all resolutions
technology: list 3 diff resolutions
- nano-scale
- meso-scale
- macro-scale
technology: give technique and eg. - nanoscale
- brute force
- animal and human
technology: give technique and eg. - mesoscale
- circuit tracing
- animal
technology: give technique and eg. - macroscale
- low resolution non-invasive
- human
ultimate technology: 3 features
- large scale
- specific
- functional
list the ideal technology: (5) points
- 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
nanoscale: name technique
- serial block face electron microscopy
serial block face electron microscopy: procedure
- cut sample of tissue into v thin sections (nm), image on electron microscope, reconstruct in 3D vol
- prepare and image tissue
- trace individual neurons
- render, detect synapses
serial block face electron microscopy: pros
- ultimate resolution
- images EVERYTHING
serial block face electron microscopy: cons
- 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
serial block face electron microscopy: satisfies which ideals (1)
- identify monosynaptically linked neurons
mesoscale: name technique
- conventional tract tracing
conventional track tracing: procedure
- monitor axonal movement of dyes (tracers) through to identify long range connectivity
conventional track tracing: tracer
- substance that is endocytosed (taken up) and transported within neuron to allow identification of its body/axon/terminals
conventional track tracing: list tracer types (6)
- static
- transynaptic
- chemical
- viral
- anterograde
- retrograde
conventional track tracing: features- retrograde
- enter synaptic terminals
- move backwards to cell body
conventional track tracing: features- retrograde eg. (4)
- cholera toxin B (CTb)
- latex beads
- rabies virus (SADB19)
- pseudorabies virus (PRV)
conventional track tracing: features- cholera toxin B (CTb)
- static retrograde
conventional track tracing: features- rabies virus (SADB19)
- monosynaptically restricted retrograde
conventional track tracing: features- pseudorabies virus (PRV)
- polysynaptic retrograde
conventional track tracing: features- anterograde
- enter cell body
- move forwards towards synaptic terminals
conventional track tracing: anterograde tracers- eg. (3)
- phaseolus vulgaris leucoagglutinin (PHA-L)
- most adeno-assoc viral vectors (AAV)
- herpes strain 129 (H129)
conventional track tracing: features- PHA-L
- static anterograde
conventional track tracing: features- H129
- polysynaptic anterograde
conventional track tracing: features- AAV (adeno-assoc viral vectors)
- monosynaptically restricted anterograde
conventional track tracing: theoretical steps and what retro/anterograde tracers label in cells
- inject tracer in region of interest
- look for labelled cells: retrograde tracers label cell bodies
- anterograde tracers label axons and synaptic terminals
conventional track tracing: list (5) caveats (warnings/precaution)
- 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)
viral tracers: features
- 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
viral tracers: allen brain connectivity atlas features
- mesoscale connectome of mouse brain
- online repository of whole brain images from 2911 experiments, AAV-reporter vectors injected at various sites in mouse brain
targeted circuit discovery- transsynaptic viral tracing: features and eg
- spread through CNS by jumping across synapses
- derived from pathogens (rabies, tetanus, herpes, pseudorabies, vesicular stomatitis virus)
transsynaptic viral tracing: initial concept was to exploit
- infection mechanism used by neuron specific (neurotrophic) viruses
- identify cells that control Ad secretion using pseudorabies (Strack) 1989
transsynaptic viral tracing: Strack’s experiment procedure
- inject virus into adrenal gland
- kill rat, process tissue to detect infected cells
- count cells, make maps
transsynaptic viral tracing: satisfies which ideals (1)
- identify local and distant connections
monosynaptic tracing using recombinant rabies: main features (2)
- fancy tracer, not transsynaptic
- no connectome tracing
monosynaptic tracing using recombinant rabies: procedure- 1. genetic modification of rabies virus
- G-deleted fluoro reporter (exised g-protein gene, swap w reporter etc.)
- cells genetically modified to synthesis rabies glycoprotein
= pseudotyped virus
monosynaptic tracing using recombinant rabies: G-deleted rabies key features (3) retrograde/anterograde
- replication competent (self-amplifying)
- drives transgene (eg. reporter) expression at high levels
- retrograde only: can only infect terminals
monosynaptic tracing using recombinant rabies: satisfies which ideals (3)
- identify distant connections
- identify connected cells in live animal
- identify monosynaptically linked neurons
monosynaptic tracing from targeted subpop: strategy
- 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
restore transsynaptic spread: strategy
- drive TVA + rabies glycoprotein on target pop of neurons
- exogenously expressed rabies glycoprotein reintegrates w rabies virion
validation: evidence of synaptic connection
- record both neurons: drive AP in presynaptic neuron- time-locked changed in membrane current in postsynaptic neuron denote connectivity
validation: evidence of synaptic connection- +ve deflections =
inhibitory inputs
validation: evidence of synaptic connection- -ve deflections
excitatory inputs
monosynaptic tracing (?) from targeted subpop: satisfies which ideals (4)
- allows selection of subpop of neurons for investigations
- identify local and distant connections
- identify connected cells in live animal
- identify monosynaptically linked neurons
connectome tracing: features incl cons
- 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
mapping connectome that controls BP: strategy
- rabies + herpes
- seed= spinal projection + monosynaptic input
- ID input neurons
- MRI-based brain model
- import neuron positions
- volumetric brain reconstruction and mapping
- pooled data
