381 Furley's Lecture

  • Created by: Sarah
  • Created on: 07-01-19 09:12
what is the function of axons?
to carry information away from the cell body in action potentials
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what do dendrites do?
integrate incoming information, can have multiple inputs into the dendrites
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what is the difference between axons and dendrites MTs?
axons have highly polarised MTs whereas dendrites have less ordered mixed orientation MTS
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how are MT stabilised?
crosslinking by MT-associated proteins
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what are MAPs in dendrites?
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What is the MAPs for axons?
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what is the difference between Tau and MAP2?
Tau = axons + MAP2 = dendrites (somatodendritic), used as molecular markers, the way they cross link MTs is different
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how are axons highly polarised MT organised?
with the + end towards the synapse and the - end towards the cell body
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where is GluR1 restricted to?
the cell body and dendrites
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where is L1 restricted to?
the axons at the growth cone
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what is L1?
a cell surface adhesion molecule restricted to axons
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what experiment did they do to show that the plasma membrane is compartmentalised?
coated beads with antiboides against L1 cannot be dragged with optical tweezers across the boundary into the cell body
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what experiment did they do to show GluR1 is compartmentalised to the cell body and dendrites?
antibodies against GluR1 on beads can be dragged into dendrites from the cell body
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what are the different domains for the membrane compartments called
axons= axonal domain, cell body + dendrites = somatodendritic domain
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how are cell surface molecules held in place?
by the underlying actin cytoskeleton
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how do optical tweezers work?
highly organised polarised streamed laser light, laser light can trap beads and move them around- see how much force is required to move the bead = how bound the bead is to the thing on the CS
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what is the relevance of cell division to neuronal polarity?
asymmetric cell division comonents used to partition things asymetrically in cell division are also used to parition things in neurons
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why is the shape of sensory neurons in the DRG weird?
not obvious which ones the sensory terminal and which is the axon
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what do we mean by neuronal polarity?
the fact that the cell is a different shape at one end to the other, have polarity dendrites and axons
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how are dendrites MTs organised?
go in both directions- mixed orientation and less ordered
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what did antibody staining for Tau-1 and MAP2 in hipoocampal neurons in a dish show?
Tau localised to axons and MAP2 in dendrites
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from the axon intitial segment onwards what is restricted to the axon?
L1 adhesion molecule
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what dies it mean that the membrane is compartmentalised?
there are components that are differentially localised within neurons
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why is L1 localised to the axon?
because it's synthesised and shipped down the axon and inserted into the membrane at the growing axon tip (this is the GC)
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what kind of barrier maintains the membrane compartments?
some kind of actin-dependent diffusion barrier (from the cytoskelton) means things can be put on specific places on the membrane
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how do we show that barrier/that the membranes compartmentalised?
axon initial segment0 coat beads with antibodies against L1 and find that these can't be dragged across the boundary
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what are optical tweezers used for?
see how much somethings bound to the CA, track things on the surface and move things around
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what was used to move the L1 antibody coated beads?
optical tweexer
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Work by Gary Banker on mouse/rat hippocampal neurons in culture showed what?
there's six stages of neuron development- from a spherical cell to several sprouts to immature neurites and then one neurite is chosen to become an axon
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what is a neurite?
any projection from the cell body. An immature neurite is neither an axon or dendrite- have no identity yet
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how do you see a neurite becoming an axon?
one neurite becomes progressively longer until one of them is noticeably longer than the other and it forms a prominent GC and becomes the axon, in culture you see axons froming on the dendrites + other synapses formed with axons and other axons
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how can we observe neurite selection into an axon?
Movie of GFP labelled + end directed kinesin Kif-1. Watch over time the GFP is flipping around different neurites testing them out and then eventually becomes localised to the neurite that becomes the axon (becomes concentrated)
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what is the principle behind neurite formation?
there is a choice going on in the cell, the cell explores different neurites then chooses one of them to become the axon
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the choice of neurite to become the axon appears to be stochastic, what does stochastic mean?
random but acknowledges there's order underneath we don't understand, neurites are tried out
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how can we identify neurites?
they contain dynamic MTs and are tyrosinated so highly unstable and change
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what is critical for MT stabilisation?
axon initiation
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what do you start to see when you get an axon proper forming?
tyrosinated unstable MT (neurites) -> acetylated MTs, stabilised + crosslinked (axon)
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when are MTs stabilised and crosslinked?
when an axon proper is formed- axon initiation
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how does the state change in axon initiation?
go from a dynamic tyrosinated MT dynamic state to choosing one of these neurites to stabilise its MTs
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how did they show that MT stabilisation is required for axon initiation?
artificially stabilise the MTs by taxol treatment. Needle pipette in taxol into one of the neurites and we can predict that neurite will become the axon
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how do you force the choice of neurite to become an axon?
taxol treatment- artificially stabilise MTs
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what does the taxol treatment experiment suggest?
there must be competition between the neurites to become axons by stabilising the MTs and maybe there's some feedback loop to suppress MT stabilisation in the neurites that aren't chosen so only one becomes the axon
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how can you show that there's competition and suppression of MTs in other neurites?
taking hippocampal cells that have begun to entend an aoxn then chop it the cell will choose a different neurite to become an axon- suggests something in the axon chosen suppressing other neurites becoming axons
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could the feedback loop to just get one axon initiated and stop other neurites becoming axons be +ve or -ve?
could be either- +ve start to become an axon so diminishing components idea you get all the components or -ve something in the axon that suppress other neurons shown by cutting it
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what are the 3 theoretical models of how you could have +ve and -ve feedback loops in axon initiation?
1) Diffusible inhibitor 2) Limiting component 2) mechanical tension
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what is the diffusible inhibitor idea?
there's a positive feedback loop in the GC reinforcing the signal at the leading edge of the cell that's groeing but a negative inhibitor diffusing through the rest of the cell to stop them becoming the leading edge
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what is the limiting component model?
you have limiting components- only sufficient amounts of molecules involved in creating the leading edge so when you get a leading edge you run out of components and can't make it elsewhere
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what is the mechanical tension model?
mechanical tension to prevent the leading edge from forming elsewhere
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what is the common point with all 3 models?
there's a requirement for a +ve feedback loop operating in the GC some kind of activator and some kind of inhibitor
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what proteins are thought to be involved in a +ve feedback loop?
Harvey Ras proteins and PI3 kinase
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what happens of you overexpress Hras or PI3 kinase in hippocampal neurons in culture?
multiple axons form
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what experiment did they do to show Hras is involved in axon formation?
if you transfect Hras along with a fluorescent protein you can see by the fluorescence they have great long axons
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how can you block Hras overexpression giving multiple axons?
with LY294002- a PI3 kinase inhibitor
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what does LY294002 (PI3K inhibitor) stopping multiple axons by Hras suggest?
Hras is working by activating PI3 kinase in some way.
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where does Hras accumulate?
in the growth cone as the symmetries broken
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what was the inhibitor experiemnt?
Hras overexpression gives multiple axons and Hras accumulation in the GC -> PI3K inhibitor prevents this and Hras disappears
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what happens if you activate PI3K in cells?
it drives expression of Hras
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what does this tell us about PI3K and Hras?
they work in a +ve fedback loop- they activate each other
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how is Hras involved in a negative feedback loop?
Hras is depleted from non-axonal regions, early hippocampal cell no particular area with high Hras protein but after the cell becomes polarised Hras becomes concentrated in the GC
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if you graft the high concentrated Hras to around the cell body and in the neurites what happens?
there's a decrease in Hras around the cell body- you **** Hras out of the cell as it's a limiting component
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how is Hras transferred from the cell body?
its actively transferred from the cell body down to the GC- you can see it being shipped down to the GC- consistent with there being a limiting compontnet (Hras)
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how is Hras shown to be a limiting component?
it goes from being broadly distributed in the cell body to concentrated in the growth cone
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What does PI3 kinase lead to the accumulation of?
PIP3 at the membrane
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what happens after PI3 kinase leads to an accumulation of PIP3 at the membrane?
PIP3 phosphorylated AKt and you get downstream effects- affects MT and actin dynamics
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what does acitvation of Akt inhibit?
activated AKt inhibits GSK3 beta activity which normally destabilised MTs so PI3K/PIP/Akt activation leads to MTs becoming stabilised
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where does phosphorylated Akt accumulate?
in the tip of the growing GC
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What are actin dynamics affected by?
Rac later on which is affected by PIP3
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what does inhibiting GSK3 beta in hippocampal neuron cultures do?
forms multiple axons as you are promoting MT stabilisation by inhibiting GSKB from destabilising MTs = consitent with this pathway being important in axon choice
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what proteins are involved in asymmetric division?
Parr proteins (partioning proteins)
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what does the Par-3 complex establish?
establishes cell polarity in general and is important in establishing neuronal polarity
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how do hras levels change before and after polarisation (neurite selection)?
distributed in all the neurites to lots more at the axon tip when polarised
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where is evidence of elevated PIP3 (phosphorylated AKt) found?
in nascent chosen axon but not in naive neurites
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how are MTS affected by molecules?
MTs are affected by Akt and Gsk3 beta, which inhibits Akt but is inhibited by PIP3
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describe the ras pathway?
Ras -> PI3 kinase -> coverts PIP2 to PIP3 -> PIP3 phopshorylates/activates Akt - and inhibits Ras and Gsk3beta -> Akt then inhibits Gsk3 beta
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how does Gsk3 beta regulate MT stability?
by differentially modulating MAP activities
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how do they show that GSK 3 beta is involved in MT stability?
results in multiple axons when you inhibit GSK beta as it usually destabilises MTs
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what else foes PI3K also affect?
the Par-2 complex and SAD kinases
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what happens in par-titioning defective genes?
mutants fail to develop asymmetric divisions, genetic screens identified mutations that affected the way cell division occur
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how does the 2 cell stage c elegan normally divide?
in different planes
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paritinoning defective genes affect what?
affect the planes of cell division and asymmetric localisation of proteins within those cells
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what are parititioning genes required for?
to establish polarity of the egg after sperm entry including cortical contraction and that initiates polarisation through the mutually antagonistic interactions of the Par3 complex with the Par1/2 complex
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what is part of the Par-3 complex?
CDC-43, PAR-6, PAR-3, PKC-3
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What is pat of the Par1/2 complex?
PAR-4 = LKB1, PAR-2-> PAR1 = SAD Kinase
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what are these complexes important for?
Polarisation, important for paritition and downstream of these you affect MT organisation and this in turn differentially localises components of the cell
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what is an example of paritioning in C elegans?
P granules are paritioned in the WT animal but not positioned int he par mutant- this is how we found out about the Par-3 complex
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where is an example of the Par-3 complex being differentially localised?
polarised in mammalian epithelial cells (apical-basal), par-3 is at the apical surface
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what cells is Par-3 usually seen in?
crawling cells like leukocytes at the leading edge. You see it in enurons localised to the GCs
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what Par-related molecules promote axonogenesis?
LKB1 and SAD kinases
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define axonogensis?
: De novo generation of a long process of a neuron, including the terminal branched region.
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what are related to paritioning defective (PAR) kinases?
SAD kinases
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what does a loss of SAD kinase result in?
a failure of acetylated (stable) tubulin to predominate over tyrosinated tubulin (Dynamic). In hippocampal neurons if you don't have SAD kinases = loss of axons, can't make them
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what are SAD kinases important in?
making axons
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what is SAD kinase a part of?
the Par1/2 complex
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what is the other component of the Par1/2 complex?
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what does a loss of LKB1 result in?
a failure to make axons
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what experiment did they do to show a loss of LKB1 results in a failure to make axons?
antibody agaisnt tublin shows neurons and they transfected in a fluroscently tagged siRNA against LKB1 now see with the axon marker that neurons that got the siRNA didn't form an axon, no LKB1 = no axon formation
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what is required to make axons?
SAD kinases and LKB1
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what is wrong with most of the experiments we've discussed?
neurons don't differentiate and become polarised in a dish. Hippocampal neurons are born into complex environments in the cortex with radial glia cells with interkinetic nuclei movement. Come to the apical surface (ventricular zone) and divide.
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where is the apical surface in the brain?
where the ventricles are
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where does cell division of neural stem cells/radial glia occur?
at the apical surface
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what becomes more and more as development progresses?
more and more asymmetric division
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what is there lots of in the environment for radial glia cells?
information- that means that polarisation might actually be biased. they have multiple neurites then start to extend a leading process and a trailing process and the trailing process becomes the axon
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What's the evidence that EC cues affect the initiation of polarity in vivo?
components eg LKB1 are diferentially localised in there diferentially modified forms- LKB1 or phops lkb1. Phosphorylated LKB1 is first in the trailing process then in the axon= no phos then becomes evident as the trailing process forms
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what is LKB1 downstream of?
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what is LKB1 antagonist with?
Par-3 complex
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what is LKB1 phosphorylation doing?
LKB1 phosphorylation if stopping LKB1 from inhibiting the Par-3 complex and at the same time leading to the activation of SAD kinase
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what is LKB1 phosphorylated by?
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what does phosphorylated LKB1 (by PKA) do?
activates the SAD kinases
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what does the Par-3 complex and PI3 kinase normally do?
acts to inhibit the inhibition of the axon by GSK3B (inhibition of an inhibition)
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Pathway from EC -> PKA?
EC cues -> PKA -> Phosphorylates LKB1 -> phosphorylated LKB1 sctiavtes SAD kinases -> axon specification
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what do SAD kinases affect?
MT associated protein stability and axon initiation
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what happens in parallel to the PKA pathway?
Par-3 complex and PI3 kinase inhibit the inhibition of axon formation by GSK3B also affecting MAP so results in axon formation
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How did they show that loss of LKB1 in the cortex leads to a loss of axon initiation?
in utero electroporation of LKB1 siRNA in mice
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what are the polarised EC cues that activate SAD kinases and inhibit GSKB inhibition of axon specification?
clues from the clinic several genes underlying mental retardation affect cortical development disrupting neuronal progenitor migration
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what are the diseases and some mutations associated with mental retardataion because of disruption to neuronal progenitor migration?
Lissencephaly (smooth brain), spectrum of mental retardations, tubulinopathies (TUBA1A (MTs involves))
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what are milder mental retardation associated with?
TGFB receptor mutation
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What do TGF betas do?
expressed in the VZ of the developing cortex (right place, right time), TGFB can initiate axons in vitro, TGFBR KD neurons do not develop axons in vitro or in vivo
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what is lissencephaly?
no gyri or sulci form have a smooth brain
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several factors promote axonal polarity in vitro what are these?
BDNF, IGF1, Wnts
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what do BDNF, IGF1, Wnts do in vitro?
promote axonal polarity
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what did the genetic KOs of BDNF, IGF1 and Wnts show?
they had no effect so maybe they're not the EC initiators of polarity
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what are semaphorins?
a family of inhibitory guidance cues
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what does biochemical purifaction of semaphorins forom the retina do to sensory axons in a dish?
causes the collapse of sensory axons
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what are the 2 forms of semaphorins?
membrane-bound (retinal axons) or secreted (Sema3A)
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what does Sema3a (secreted) do to dendrites?
1) attracts dendrites basally as it's expressed in a gradient from basal to apical (high apically repel dendrites bsasally) + 2) promotes dendrite formation at the expense of axons in vitro
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how does Sema3A feed into the pathways?
it increases cGMP and suppresses cAMP so it inhibits PKA phosphorylation of LKB1 + GSK3B
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what is GSK3B usually inhibited by?
unphosphorylated LKB1 -> inhibits Par 3 complex -> inhibits GSK3B so now GSK3B not inhibited?
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what has opposite effects on axon and dendrite formation?
manipulation of cAMP and cGMP
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what happens apically?
TGFB phosphorylates Par6 (part of par-3 complex) to promote axon specification
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what happens basally?
Sema3A increases cGMP and supresses cAMP thus inhibiting LKB1 phosphorylation and axon formation
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what do many signals effect?
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why did they do in utero electroporation of LKB1 siRNA in mice and not just KO the whole thing?
particular parts of the cortex targeted, don;t want to KO these components in the whole animal because they have functions in other places. Inject siRNA into ventricle and then electroporate drives into cells of the ventricles KO in a selective way
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what gene is involved in lissencephaly?
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how do they show lissencephaly?
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how was it shown that the TGFB superfamily are expressed in the ventricular zone?
in situ hybridisation of the developing cortex
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how did they show that TGF betas could initiate axons in vitro?
put down stripes of components eg TGF and no TGF and you put on hippocampal neurons you see axons from over the TGF beta stripe showing it can initiate it+ KD of the TGFb rec in neurons means they dont dev axons in vitro or in vivo
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what uses the same pathway as TGF betas? (are TGF beta family)
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what do TGF betas phosphorylate?
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what kind of receptors do TGF betas use?
single pass serine/threonine kinase receptors
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axon formation is promoted by what?
TGF betas
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what did a Sema3A stripe assay show?
axons form where Sema3A is not and dendrites formation where the Sema3A is
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what dendrite and axon marker did they use?
Map2- dendrites Smi-312- axons
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on the stripe assay what do you get if you put on a cAMP promoting drug?
force axons
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on the stripe assay what do you get if you put on a cGMP promoting drug?
force dendrites
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how is what cGMP doing equivalent to what Sema3A is doing?
sema increases cGMP levels intracellularly and here we're putting on a cAMP analogue on the stripe which promotes dendrites in a similar way
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many of the pathways for neuronal polarity are used and relevant for what?
axon guidance
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what is the axon promoting factor?
TGF beta
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what is the dendrite promoting factor?
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How does TGFB and Sema3A work against each other? how do semas make dendrites?
In opposing gradients in the developing cortex, they feed into pathways, TGF beta affects directly the Par-3 complex, Sema inhibits part of the pathway by cGMP so inhinits axon specification so get dendrites
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what did the movie of a rat sensory neuron growing with the GC tip show?
GC moves in a crawling sense. The filopodia are not really attached and they are exploring the environment so it's like the palm of my hand is attached and the filopida explores, lamella move around env- motile + dynamic structure exploring the env
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why do we use aplysia to study growth cones?
they are easy to culture and unlike rat sensory GCs they lay flat out on the dish so easier to study
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how is actin arranged in the GC?
as filamentous polymerised actin- it's in bundles
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what domains are the MTs in?
the central and transitional domains
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how are filopodia and lamellae different?
filopodia- polarised actin to form larger bundles lamella- actin bundles are crosslinked into a net
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are filopodia or lamella stuck donw?
no netheir are stuck down instead they are both highly motile
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where is filamentous actin?
in the peripheral domain
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what happens to the filamentous actin as it goes from the peripheral domain to the transitional domain?
it flows in from the outside and actin subunits are being added at the tips. Actin filaments are being chopped up in the transitional domain
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what us tubulin doing in the GC? what happens when it comes into contact with an attractive cue?
It shoots up the back of the F actin filaments and explores bits of the filopodia. In contact with an attractive cue , tubulin is dragged sporadically into the filopodia, this happens much more dramatially
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what is it called when tubulin are being dragged sporadically into the filopodia in a resting GC?
microtubule capture
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how did they show that tubulin is dragged into the filopodia much ore dramatically when it comes into contact with an attractive cue?
bead coated with an aplysian cell adhesion molecule (attractive cue) and then add with a pipette you get GC in the direction, filopodia coming out and tubulin redirected into that bit
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what is happening when the GC comes into contact with the cues?
tubulin is rapidly dragged into the filopodia and from the fixed tissue you can see filamentous actin building up at the point of contact with the bead and tubulin invading, fingers explore touch and rearranges cytoskeleton when something intersting
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do GC turn?
they don't turn , they reorganizse- cytoskeleton reorganises itself to the point of the cue
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when is a molecular clutch engaged?
When the GC comes into contact with a positive cue, clutch is engaged arrests backwards flow of F-actin as rec binds cue in env stops backwards flow but continues polymerising so move forward
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how are MT linked to actin?
by an actin-tubilin link, an actomyosin-based actin tubulin link captures MT in the extending filopodia
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where does N cadherin predominate?
in the nervous system
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how do cadherins bind?
homophically so like-like interactions between cell membranes
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how did they show evidence for a molecular clutch
micropatterned a glass with islands of N-cadherin protein then put neurons down onto that substrate and have N-cadherin-GFP fusion in the neurosn so you can see where N-cadherin is inside and outside the cell
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what result did they get?
GC over these patterns and the GFP-Ncadherin accumulates over where N-cadherin is on the substrate
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what did this show us?
N-cadherins bind homophillically
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how did they follow this up?
they followed up what happens to actin inside the cell. Actin moves towards the middle of the GC and the treadmilling of actin
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how did they monitor actin?
PALM technique- single particle tracking, plots the motion of these particles (actin molecules)
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what was the result of following up actin?
some actin move more slowly or at all they are where the N cadherin substrates are
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what did this suggest?
actin moves more slowly over the N-cadherin, something is arresting the actin flow at points of contact of N-cadherin with the substrate- evidence there's a clutch operating
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what was the idea that came from this experiement?
that a cell surface receptor (N-cadherin) can bind to a ligand (homophillic binding with N-cadherin on the substrate) and that engages a clutch which arrests the actin flow
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how does the clutch bind?
the likelihood is it's a direct interaction of things on the IC domain of the axon with the F-actin cytoskeleton- so there's a literal clutch binding
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how else could the clutch be controlled?
by X-linked actin (don't need a direct junction between the substrate and F-actin so no recs)
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what does an SEM od the lamellae show?
chaortic and interweaved F-actin in the lamellae, weaving F actin gives strength
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what is the possibility of what's happening with the clutch here?
binding of the receptor on the surface to the substrate generates a signal and that activates intracellular crosslinking via 2nd messengers so crosslinks the F actin cytoskeleton
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what does crosslinking F-actin do?
stop backward flow and force a forward flow, actin polymerisation at the tips continues so you push forward and arrest backwards flow with the clutch acrosslinking F-actin
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what is the crosslinking protein likely to be?
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what happebs with actin when you have positive cues?
F-actin builds up
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what happens to the amount of filamentous actin in the cell when you pippette in semas?
it decreases
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what happens in GC collapse? what causes GC collapse?
GC collapse caused by semas- F-actin is desatbilised
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how is the actin cytoskeleton being controlled?
may be the clutch + other pathways (Rho GTPases etc)
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what are Rho GTPases?
regulators of the actin cytoskeleton, many different catergories. General characterstic is they bind to GTP in on state and GDP in the off state.
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what are Rho GTPases controlled by?
GEFs (Guanine exchange factor) and GAPS (guanine activating factor)
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what do GEFs do?
turn on GTPases they swap GDP for GTP, adds phosphate
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what do GAPS do?
deactivate GTPases ***** off thw phosphate GTP -> GDP
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whichm particular RhoGTPase is involved affects whta?
what effect they have on the cytoskeleton
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how do we know different Rho GTPases give different effects?
transfect fibroblasts with different members of the RhoGTPase damily you get different effects. Make CA dorms of GTPASEs, CA RHoA, Rac+Cdc43
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what is a constitutively active mutation?
mutation that puts it in the active on state all the time
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what did CA RhoA induce?
induces stress fibres- associated with cell attachment
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what does CA Rac give?
induces lamellipodia
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what does CA Cdc43 induce?
cdc43 induces filopodia
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what happens if you put in dominant negative versions of these?
you suppress the formation of these structures
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is whats going on in fibroblasts the same for neuron GCs?
no its slightly different
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what does modulation of Rho GTPases control?
axon growth
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what are positive regulators of axon growth in their activated state?
Rac + Cdc43- stimulate axon growth
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what is a negative regulator of axon growth when activated?
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what is important to whether an axon is going to grow?
the balance between RhoA inhiniting axon growth and Rac+Cdc43 stimulating it
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what experiments with RhoA showed this balance was important?
1) expression of CA RhoA causes neurite retraction which might be the same as sema3A on (collapse) whereas dominant negative RhoA blocks colpase response so you need RhoA+Semas to collapse the GC
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what experiments did they do with Rac + Cdc43 to show this was important?
DN Cdc42 blocks formation of axons and dendrites, DN Rac affects only axon growth
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what does CA Rac or Cdc43 do to axon growth?
blocks axon growth (doesn't make sense as they normally stimulate axon growth but if always on they don't)
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what did this tell us about the way Rac + Cdc43 function?
when you're building the cytoskeleton you need to build it and disassemble it- polymerising and depolymerising it and treasdmilling must happen for the axon growth
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what does growth require?
assembly and disassembly of actin structures
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what do you see in aplysia growth cone guidance towards an attractive cue 2 GCs colliding on a dish?
collision is an attractive collision- the actin cytoskletion wqas grwoing in one dirrection but then F-actin is disassembled and re-assembled towards the attractive cue
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what does it mean that CA Rac and Cdc42 prevents axon growth?
the structure they nromally promote must be disassembled as well as assembled for growth (Actin treadmilling)
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if you don't disassemble F-actin what can't you do?
change the direction of growth
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why does CA Rac+Cdc42 block axon growth?
bocks disassembly so all filopodia is stabilised and doesn't grow out
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what are the 2 different ways Rac + Rho could be working?
permissive: beeded fir disassm=embly+assemby to grow and turning happening downstream. Instructive: they are downstream of attractive or repulsive cues
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if they are instructive what are they important for?
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what is the evidence that Rho GTPases are instructive?
1)Factors that collapse GC activate Rho and downreg Rac 2) several guidance factor recs either bind and modulate Rho GTPases directly or they bind to GEFs+GAPs to regulate them
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what is an example of a guidance factor receptor binding and modulating Rho GTpases?
Plexin B semapgorin recptor binds directly to Eac
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What does semaphorin signalling alter?
the RhoA/Rac balance
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where did the first example that semaphorin signalling alters the RhoA/Rac balance come from?
from drosophila muscles- in flies all semaphorins have plexins for receptors
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where do the motor neurons usually innervate in the muslces?
innervate the muscles that do not express semaphorins
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what do you need to alter the levels of to affect the muscle innevration in drosophila?
the levels of Rac, PlexinB and RhoA- different levels change what's happening
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how did they show that altering the levels of a receptor will alter muscle innervation?
used genetics of the fly to elevate plexinB receptor levels in the motor neurons- greater sensitivity to semas so don't extend as far into the environment as they usually do, double rec- hypersensitive don't go near the muscles
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what happens if you make less Rac in the motor neurons of drosophila but elevate PlexinB levels?
Rac normally promotes axon growth so when it's reduced in the MNs + elevated PlexinB they become more sensitive to semas
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what does more rac and elevated PlexinB (so less innervation) do to the sema sensitivity?
so less innevration bcos plexinB but more Rac means you can restore WT situation can get more innervation, makes them less sensitive to semas promotes axon growth
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what does decreasing the levels of RhoA in the MNS do to sema sensitivity when plexinB makes less innervation because of increased sema sensitivity?
less sensitive to semas
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what directly binds to plexinB?
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what is the 2 pieces of evidence that suggest Rho GTPases are instructive?
1) several guidance factor receptors either: a) bind and modulate RhoGTPases directly (Rac+PlexinB) or b) bind to GEFs and Gaps to regulate Rho GTPases 2) factors that cause GC collapse upreg Rho and downreg Rac
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how can guidance signals regulate Rho GTPases?
1) direct receptor interaction with GTPases 2)indirect regulated through GEFs and GAPS
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what model was suggested by these studies altering Rho GTPases in MNs as well as direct binding of rac-GTP to PlexinB suggest?
no semas- plexinB not activated and the levels of free Rac outweigh the free activated RhoA and you get axon growth
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what happens when the sema binds the receptor?
PlexinB receptor becomes activated -> sequestration of Rac-GTP takes out of env = more free/activated RhoA outweighs Rac so get GC collapse
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what happens to the PlexinB when sema binds?
crosslinks the receptor
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what is the balance that determines the outcome to the semaphorins?
the balance between free-RhoA and Rac
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what happens to Rac-GTP when semas bind to PlexinB?
it gets sequestered at the membrane, so less is free in the neuron
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what is an example of an axon guidance receptor?
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what was the semas, PlexinB and Rac/Rho concentrations an example of?
where GTPases bind directly to the axon guidance receptor (PlexinB)
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how else may guidance signals regulate Rho GTPases?
indirectly- via GEFs or GAPS
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why is it important to think of guidance signals working through GEFs+GAPS?
Rho GTPases family is very small and they are broadly expressed so not specific expression but GEFs+GAPs much larger families + more restricted expression so greater specificity
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what does it suggest that there the GEFs+GAPs have a much bigger family than the Rho GTPases and much more restricted expression?
Regulation by GEFs+GAPs give much greater specificity
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what is an example of where GEFs+ GAPs bind directly to guidance receptor?
EphA4 receptor binding ephexin which is a GEF
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what is ephexins role?
a GEF that can turn onRhoA and turn off Rac+Cdc42
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how many families of there of GEFs and GAPs in flies?
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how many RhoA GTPase family is there in the fly?
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how might other cues act?
via 2nd messengers such as calcium
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example of where guidance cues regulate Rho GTPases indirectly?
through ephexin GEF- binds to EphA4 and turns on RhoA and turns off Rac+Cdc42 which gives GC collapse
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what can turn GCs?
a localised ca flux
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what experiments did they do to show ca fluxes can turn g?
Gomez (zebrafish?) where they put a calcium indicator into GCs in vivo. GCs of these MNs go out to the periphery light up as having high ca lebvels
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what happened with the netrin pippette experiement by Hong to GC turning?
GC in vitro squirting netrin out of a pipette- netrin stimulates the level of IC calcium shown by ca indicator dye
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what do you see if you monitro ca levels in real time in filopodia?
you see fluxes in the filopodia themselves suggesting that ca responses can be seen in filopodia
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what trick do we use to induce calcium fluxes in a GC to induce turning?
calcium cages
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how do calcium cages work?
they are light activated cages with molecules like o-nitrophenyl EGTA, when you flash light on them they release the ca that's bound- load GC with caged Ca focus laser and release ca in that spot
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what does the ca release from the cage do to the GC?
calcium flux induces GC to turn that way
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what can calcium transients regulate?
Rho GTPases
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how did they reveal the ca flux in the caging experiment?
Fluo-3 dye
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what can ryanodine released from a pipette onto the GC do?
release calcium from IC stores and turn the GC towards it, you see ca fluxes within the turning GC
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what did the WB show ryanodine was activating and suppressing?
activates Rac/cdc42 (axon growth) and supresses RhoA (axon growth inhibitors)
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what was this ryanodine a good example of?
that there's a direct connection between calcium (released by ryanodine) to the Rho GTPases- RhoA/Rac balance that's important
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what can be regulated at several places?
the treadmilling process of F-actin, growing at the tip and broken down at the far end
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key points of regulation of how Rho GTPases modulate the actin cytoskeleton?
1) filament disassembly control 2) branching regulation 3) termination of branch extension 3) regulate filament assembly 5) regulate the amount of crosslinking between branches
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how could you regulate filament disassembly?
by cofilin
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how could branching be regulated?
initiated by the clutch idea and the branching involves the Arp2/3 complex that is activated in response to WASP proteins
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what do capping proteins do?
regulate the termination of branch extension
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how could you regulate that filament assembly (polymerisation)
prolifin and thymosin
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what is actomyosin contractility interaction involved in?
regulating the amount of crosslinking between branches
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key point about regulation of the actin cytoskeleton?
many points of modulation, Rho GEFs_ Gaps via Rho, Rac and Cdc42 feed into different points and different guidances cues affect different parts of the pathway
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what else other than actin is really important?
the regulation of microtubules- MTs are really important for making the growth happen
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what is thought to act first to a cue in the env MTs or actin?
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what is the evidence that MT react faster?
a bead put onto an aplysian GC and track the actin + MT treadmilling. When the beads put on there's a flash of MT concentration long before there is the same thing with actin.
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Where do MTs gather?
around the site of contact straight away but happens later with actin
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what was the message from this experiment?
when cues are encountered the MTs react first- this is robably something to do with bringing in goodys to the site of contact
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How does MT invasion recruit goodies?
via CRMP - collapsin response mediator protein
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what might invading MTs bring?
important signal transduction elements/and or factor that are required for membrane protrusion into the point of contact with the cue
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what are the goodies?
monomeric actin (to build the filamentous actin), collapsin response mediator protein
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what does collapsin response mediator protein do?
promotes MT polymerisation and drives WAVE complex proteins into filopodia in a kinesin dependent manner
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what pathways are Rac activated in?
the PI3K pathway (Actin dynamics) and the Rac Rho pathway (Elongation, lamellipodia)
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what is there lots of between pathways? evidence?
cross talk, Rac is activated in both pathways, Cdc42 affects initial outgrwoth, guidacnce factors can act at any point in the pathway
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what changes the response to guidance cues by affecting polarity?
PKA/PKG via cAMP and CGMP levels
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how is polarity formation thought to be involved in axon guidance?
thought to continue from polarity process to guidance, attraction enhances axonal identity and repulsion inhibits it
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what brings in Rho GEFs?
After MTs begin to concentrate they bring in things to the point of contact with the cue- bring in Rho GEFs by vesicles
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why would you get MTs before actin monomers?
you need to bring in monomeric actin subunits to build filamentous actin
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what is the first response to a cue?
MTs engaged come into the site of contact and deliver things ot the area- they may deliver things that affect the way the GC responds to cues
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what axons are attracted to the floorplate by netrin?
commisural axons from the dorsal spinal cord
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what did Mark demonstrate?
they could take cells expressing netrin and these cells would attract C axons towards them
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what distance is netrin porbably acting over in the rat/mouse?
0.5mm- the embryonic spinal cord is about 1mm over the DV axis. The BMPs from the roof plate repel he C axons so netrin is probably working over about 500um (0.5mm)
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in vertebrate nervous systems where is netrin expressed?
along the ventral midline of the NS
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how did they find netrin?
biochemical purification led to cloning og the gene encoding the FP chemoattractant
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in mammals C axons turn axons turn anteriorly (rostrally) after crossing the FP in rodents at least what could be doing this?
1) A-P gradient of an attractant making them turn anteriorly 2) there's a repellant sitting posteriorly which drives them anterior = long range diffisuble cues or 3) short range diffusible cues
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what's an example of a short range non-diffusible cue?
ephrin cell surface molecules that could be set up in gradients as seen in the tectum
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how do you get gradients of short-range non diffusbile molecules?
by a gene expression gradient
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how did they test if it was a long range diffusible cue or short range non-diffusible cue?
with different sizes of explant, if diffusible molecule a big explant means the gradient will be maintained in the middle as the diffusible mol won't diffuse out but a smaller explant means diffusible mol will diffuse into the collagen+gradient lost
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what will happen if its a long range diffusible cue thats causing the C axons to turn anteriorly?
the gradients and turning will stay the same in both explants becuase the cue is not lost to the env
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how did they do this experiment?
took small explant pieces and put them in a culture assay and then put a large explant in another culture assay+see difference, Open book preparation of the FP- dissected out SC and lay flat on collagen gel culture grow fix+lipophillc dye(di-i)
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when did they do this?
before axons have begun to extend
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why did they use Di-i a lipophillic dye?
Di-i marks axons membranes so can track them
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what was the result of this experiemnt?
in a big explant axons turn normally but if its a small axons axon turning is confused
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what was suggested from this result?
this was consistent with a long range diffusible cue- means there is an attractant at the anterior end
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how would you know if it's a repellant or attractant in this experiment?
you'd have the most effect at the edges where there's most diffusion outwards - diffusion out didn't affect growth of the posterior but affected anterior growth
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how did theyknow it was an attractant?
if an attractant had diffused out of here the levels of the attractractant will be lower than it is in the middle (diffusion less) reversal of grad, high point will always remain in middle
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if it was a repellant where would the gradient be highest?
at the posterior
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if it was a repellant what would you see with a large explant?
axons pushing axons to turn anterior but by time 2 diffusion away at the end but was low already at ant but has become low at post now too so would expect at post end axons will turn down grad but at ant end not affected so marge
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what would you see if it was an attractant?
high point at ant, over time diffusion out of ends get reversed at ant but unaffected at post so axons growth abberant, turns axons back the other way high point in middle
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what is expressed in a gradient to turn C axons?
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how was it discovered that Wnt4 was expressed in a gradient to turn C axons?
noticed an AP gradient of Wnt4 at the time the C axons were being guided (right place, right time)
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what do Wnts bind to?
frizzled 7 TM pass receptors
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what experiment was done to show wnt4 was in a gradient to turn C axons?
transfected cos cells with a control or wnt4 vector (forcing expression of wnt4) and placed them at the anterior or posterior end of the small explant
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what result did they get?
depending on where Wnt4 was placed it could dictate where the C axons would turn- Wnt4 causes turning
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what distance was this over?
embryonic day 11.5 in mice, spinal cord is 4mm long at that time so wnts acting over 4mm = long distance
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what other experiment showed wnts were importnat?
the frizzled KO mouse have confused turns once they get to the floorplate (FP attraction by netrin not affected)
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what is wnt an example of?
early patterning molecules (used to pattern neuronal progenitors) are also usedin axon guidance
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what other patterning molecules used again?
BMPs + Shh- early on they're used for patterning and with shh
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what works redundantly with netrin to guide C axons?
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what are the 2 different ways to detect gradients?
1) temporal detection- change in concentration over time 2) spatial detection- change concentration across cell/GC
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how do you look at temporal detection?
compare the amount of ligand concentration at 2 different time points
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how do you look at spatial detection?
compare the ligand concentration at 2 different points of the cell
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what is likely to be the most important gradient detection for eukaryotic cells? why?
spatial detection. Eukaryotic cells don't move around
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what are studies to do with detecting gradients usually done in?
studies of dictyostellium or neurophils and leukocytes in culture- eg neutrophils chase bacteria in culture clearly polarised leading edge
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what is dicytostelium?
slime mould- normally grows as individual amobae but when resources are scarce the cells make a chemotactic factor which draws them together
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how do amboeae disperse to other environemnts?
make a fruiting body which makes spores to project to other places
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dicytosetelium experiment?
cAMP chemotactic factor in a pippette draws individual amboae in to make spores then disperse
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accuracy of oritentation towards a source depends on the absolute concentration of the chemoattarctant which can be determined when you are closest to the dissociation constant (Kd) of the rec, why?
at Kd- receptors are half occupied, you have a better chance at detecting change because it could go in either direction- up or down, if all occupied can't detect a increase if you're completely unoccupied can't detect a decrease
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what else determines the accuracy of orientation towards a source?
steepness of gradient (what concentration change detecting),
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how do we express gradients?
as a % change per micron
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when can leuokcytes orient with accuracy?
when there's more than a 2% change across a 10um diameter (when gradient is greater than 0.2% per micron)
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what does this mean for the size of the cell?
large diameter cells can orientate more accurately than small cells using spatial detection (why bac use temporal as they're very small)
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across the width of a GC what difference in NGF molecules can be sensed?
a difference in a gradient of 1 in a thousand NGF molecules
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what is required in even relatively steep gradients (2% over 10um) to detect a change in the gradient?
steep gradients require small differences in receptor occupancy to be detected across the GC
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why is it a problem that they detect such small differences across the cell?
it suggests you need to amplify it in some way
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if you had 1000 receptors across the GC, 500 on each side and wanted to detect 2% change how many receptors of the 250 will need to be changed?
5 receptors
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what are the 2 methods GCs use to enhance their sensitivity to gradients?
amplification and adaptation
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how do you amplify a gradient? (general idea)
enhance the signal locally at the point of contact and inhibit signal reception in other parts of the cell
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what is a mechanism of amplifying the gradient?
he clustering of the receptors/signalling components near to the signal. Transporting stuff to where the signal is and removing it from the rest of the cell- so inhibiting responses elsewhere
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what is the receptor/signalling -components clustering idea similar to?
the limiting component idea of neuronal polarity
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what does clustering of receptors or downstream happen in?
chemotactic dictyostelium- involves the PI3 kinase pathway
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what experimental procedure did they use to show recpetor/component clustering?
cAMP (chemoattractant) activates GPCR receptor in dicytostellium and activates the PI3 kinase pathway- detected as PIP2 is bound by proteins& has pleckstrin homology domains
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how can you visualise pleckstrin homology domains (PHD)?
hook up the PHD to GFP - this will tell you if its being activated
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what molecule is a PHD protein?
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what is PI3 kinase activity antaginized by?
PTEN phosphatase
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what determines how much PIP3 kinase depends on?
the balance of PI3K and PTEN phosphatase activity, PIP3 kinase accumulates+disappears
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what converts PIP2 -> PIP3?
PI3 kinase
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what converts PIP3 -> PIP2?
PTEN phosphatase
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what does the PHD do?
bind lipids to the membrane
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where are the PH domain proteins recruited to?
the leading edge of chemotactic cells
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how did they show that PH domain proteinsa re recruited to the leading edge of chemotactic cells?
PH domain hooked up to GFP and added a chemoattractant source. Dictyo cells have been transfected with a reporter to see PH domains it accumulates along the leading edge of the cell disasppears and comes back, PIP3 orientated to where attractant is
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what are these complexes?
highly labile- they disappear and reappear
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how do we know similar things happen in neurons (PI3K way)?
PI3 kinase inhibitors (so no PIP3 formation) disrupts GC turning induced by netrin
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what other experiment showed neurons to be similar?
have a PH domain fused to GFP, this construct is in the neuron and add BDNF and see a flash of PHD domain in the filopodia (and cell body)
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what did the Tojima paper look at?
vesicles being orientated towards where cues are accumulating- things being directed towards the chemoattractant
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how did they do this experiment? what did they find?
Used a membrane dye (FM1) to show vesicles distributed in GC, if you put on a positive cue (Adhesion molecule substrate) the vesicles go in the direction of the cue
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where are vesicles being transported in this experiment?
towards the point of contact
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what marks exocytitic vesicles?
marker of vesicles VAMP3 venus
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what did the VAMP3 venus marker show?
we're putting out compoents that's coing to be put into cell membranes
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what is this all showing?
there is amplification happening by bringing in resources such as vescsicles or membrane components
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vesicles associated with membrane dye are tightly associated with what in the presence of a cue?
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if there is less ligand concentration (Shallow gradient) around then how much receptor occupancy is needed if you're 250um away from the optimal ligand concentration?
90% receptor occupancy
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when do GCs act most distriminatively?
when the chemoattractant is closest to Kd (half receptor occupancy so it can go up or down)
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how do the responses come less good at the extremes- eg stauration or no occupancy?
all filled- won't respond any more, and if you go all the way to the bottom of the gradient you won't respond as no occupancy
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what does signal saturation usually lead to in synaptic transmission?
desensitisation- the thresholds are reset so you need more to get the same response
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what is the frog turning assay that was performed by Mu-Ming Poo's lab
in the xenopus SC- take out spinal cord, put neurons in culture and look at how they respond to diff things, can inject things into frog at 1 cell stage- can manipulate what's going on
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what experiment did they do with this assay to show the response of the GC to netrin?
took frog spinal cord neurons growing in a dish and pipette in netrin/inactivated netrin (denatured protein) and show the movement of the GC response over time. Pulse these netrin
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what was the result?
the GC doesn't turn towards inactivated netrin but turns towards normal netrin
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how did they show GC response adaptation?
kept pulsing out netrin and GC turns towards it but then 20 mins later turns away 20 mins later turns towards, does a wiggling pathway
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what could the wiggling pathway mean?
the GC is desensitizing and resensitizing to netrin
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how did they set up a netrin gradient?
pulse netrin into a dish
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how did they observe the netrin gradient?
with a fluorescent dye tagged to netrin
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what was the wiggling pathway described as?
Zig-zag trajectory
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how did they show this wasn't due to twhay way netrin was being pulsed?
repeated the experiment and then repeated the experiment where they've added netrin to the bath before they pipette in netrin
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what happens if the GC had already been exposed to netrin in the bath?
the low level of netrin present in the bath doesn't turn immediately but give it time and it starts to turn
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what is the fact that GCs don't turn immediately if they've been pre-exposed to netrin mean?
they desensitise and resensitise as they going up increasing attractant gradients
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where are there similar responses to GC adapting to chemoattractatnts like netrin?
similar responses when GCs respond to repellants like Sema3A
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what happens over time to make it not turn imemdiately to turn to netrin and then to make it slowly turn?
over time it is regaining sensitivity to netrin
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what is this evidence for?
the GC can reset the thresholds of chemoattractants/chemorepellants which it's sensitive to
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what is the distance GC can be guided by gradients determined by?
the shape of the gradient, which depends how it is set up
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where are netrins and wnts set up?
they're diffusible molecules set up as gradients. Netrins = FP Wnts = along AP axis
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non-diffusible molecule gradients like ephrin tend to be more what?
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what can act over a longer distance diffusible or non-diffusible cues?
non-diffusible cues- they are more linear
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what is the theoretical maximum range of a diffusible molecule gradient? why?
1mm from where the point source is acting- situation in the SC, its exponential
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what is the theoretical maximum of non diffusible cues? example? why?
1cm- they're more linear (no sink/source) example is ephrin expression in the tectinal superior colliculus
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where was a gradient acting over a long distance observed in vivo?
the tectinal to superior colluculus- correspond with the distances we see these gradients acting
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what suggests a point source of netrin from the FP is not the case?
RNA for chick netrin localised to FP = point source, but there's 2 netrins!!
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what is the differences of where the 2 netrins are expressed?
netrin 1- expressed at the FP but netrin 2 is expressed at low levels throughout 2/3rds of the SC
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why does this not suggest a point source?
netrin 2 expressions same distribution throughout does not suggest a point source
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in mice how many netrins are there?
one netrin
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where is netrin in the mouse? what does this tell us?
not at the FP, around the VZ and elsewhere, not consistent with a point source
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what is it about the netrin structure that suggests its not consistent as a poitn source from the FP?
it's similar to laminin as it can associate with the ECM, it's found associated with the basement of the SC so unexpected distribution of netrin protein
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what have they shown about needing netrin in the floorplate?
2 recent papers shown you odn't need netrin specifically in the floorplate to get guidance of C axons- they still get to the FP
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what does this suggest?
netrin might be more of a linear gradient and it might be bound to the ECM to have its action
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how is the WNt4 gradient thought to be set up?
has graded RNA expression from anterior to posterior of wnt4 mRNA. This is at E11.5 but expression changes over time.
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what do we expect to happen to the wnt4 gradient as time proceeds?
it changes as there is a temproal component as well as a spatial component. There's a AP progression as development proceeds
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what is the main idea behind Wnt4 and netrins explantation?
the idea of a simple diffusible molecule is probably not correct as there's actually quite a lot of components
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how would you expect a point source gradient to be regulated?
would expect there to be a sink to sustain it. Needs to take cues out of the system.
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how does adaptation and amplification work in vivo?
not sure we only understand it from in vitro experiments where pumping out stuff from pipettes
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what is reprogramming?
the idea that when you're sending an axon to a distant target in the embryo it will go via intermediate targets or choice points. As you hit these choice points the set of cues you use will change or old cues will be used in a different way
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what happens when intermediate targets (choice points) are encountered?
axons reprogramme
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How is the pathway taken by C axons in the hindbrain different?
C axons cross the FP the axons don't turn immediately they go round and up the other side- allowed to do exper
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what is an example of reprogramming as the FP is crossed?
took a big open book preparation with the FP in the middle and they put an ectopic FP on one side and not the other to see if axons responsiveness to the FP changes after it's crossed the midline
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what were they testing?
whether the response of the C axons to the FP change after GCs had crossed the midline
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what happened to axons exposed to the ectopic FP before reaching the midline?
they turn towards the ectopic FP
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what happened tro the axons exposed to the midline after crossing the midline?
they no longer respond to the ectopic FP
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what did this mean?
the sensitivity of C axons to the FP (netrins) change after the midline is crossed
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what does this explain in the hindbrain?
explains in the hindbrain why C axons continue past the FP without turning
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what is the difference between C axons in the hindbrain and SC?
hindbrain- C axons don't turn towards FP, spinal cord- C axons turn after crossing the FP
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in the rodent spinal cord C axons can travel through stripes of semas and slits (repellants) but how?
there is a change in sensitivity to repellants as you cross the floorplate.
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how does the sensitivity to repellants change in the SC when C axons cross the FP?
after crossing the midline C axons become sensitive to semaphorins and slits in the FP
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where else are semas and slits expressed?
in the FP and in the ventral spinal cord
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why does it matter that slits and semas are expressed in the ventral SC?
it creates a channel through which C axons can grow
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how do sensitivities change in the rodent SC?
initially C axons go towards the FP attracted by netrins then they become sebsitive to semas+slits as they cross the FP moving them away
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what experiment did they do to show they lose response to netrins after crossing hte FP?
had an ectopic FP, pre-crossing axons go towards the ectopic FP out of the explant but post-crossing neurons do not
402 of 976
what is expressed in the midline glial cells in flies?
diffusible attractractants = netrins and CS repellants = slits
403 of 976
what determines whether axons cross the midline?
the level of Robo (receptor for slit) at the cell surface
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what is slit?
a repellant
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what is robo?
the receptor for slit
406 of 976
where is slit found?
in midline glial cells in the fly
407 of 976
where is robo found to be high?
on axons that don't cross the midline- they get repelled away by slits in the midline
408 of 976
what level of robo do C axons express?
initially low levels of robo so they can cross & not be repelled and then high robo levels so they don't re-cross
409 of 976
what happens in the roundabout/robo mutant (no robo)?
doesn't detect slit so can cross the midline and go back and forth roundabout of axons
410 of 976
where is commisureless expressed?
in the midline cells and in neurons that normally cross the midline
411 of 976
what happens when in commisureless protien is missing?
Robo protein is expressed at high levels in cells that would normally cross the mid-line (shouldn't be there but is) and now extend their axons longlitudinally and don't cross
412 of 976
if commisureless is forced in all neurons what happens?
Robo is lost everywhere resulting int he robo mutant phenotype
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what do these experiments tell us?
commisureless must be controlling/regulating robo expression.
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what is comissureless?
it's a membrane-associated protein found at the cell surface of neurons but also in IC compartments
415 of 976
what were the mutants found in a genetic screen that looked at how guidance across the midline was messed up?
roundabout mutant- kept crossing the midline, commisureless muant- high robo no axons cross midline extend longlitudinally
416 of 976
what does antibody staining show about robo levels?
axons that don't cross- high robo, axons that cross: initially low robo then high after crossing
417 of 976
what are the 2 possibilities of how comm is regulating robo?
could be involved in trafficking robo the membrane or could regulate it by being a TF
418 of 976
what is possibility was right?
comm involved in transporting robo to the membrane- controls robo getting to the cell surface
419 of 976
what are the 2 models of how comissureless is trafficking robo?
1) sorting model 2) clearance mechanism
420 of 976
what is the sorting model?
comm causes newly synthesized robo to be sent into endosomes for the degradation pathway so you fail to get the protein to the CS
421 of 976
What is the clearance model?
robo is sent down the axon but it doesn't find it's way to the surface. Suggested this might be happening as comm is as CS ssociated protein and it binds to comm in the env homophillic
422 of 976
why did they suggest comm in neurons binding to comm in the env might be stopping pre-made robo getting to the CS?
comm is expressed in neruons are that crossing but also in cells of the midline so though homophillic interaction
423 of 976
what is there a homophillic interaction of comm between?
between comm on the GC + comm on the cells of the midline membrane
424 of 976
what would comm be doing in the clearance model?
making sure robo protein was unable to be sorted to the surface
425 of 976
what happens once axons have crossed?
comm no longer functions and robo can get sorted to the cell surface to give a repulsive response to stop axons crossing back over
426 of 976
what did they set up these 2 models based on?
cell biology in vitro experiments
427 of 976
why was the clearance model more attractive?
it gave a clear mechanism for how you suppress robo until you've crossed and then upregulate robo after crossing
428 of 976
what would allow robo to the surface?
homophillic binding of comm ceases as the GC emerges to the other side and this allows robo to the surface
429 of 976
how did they try and test the models?
tried to see- is comm required on midline cells for crossing because it would be needed in the clearance model for homophillic interaction but not for the sorting model
430 of 976
how did they try and test the models?
tried to see- is comm required on midline cells for crossing because it would be needed in the clearance model for homophillic interaction but not for the sorting model
431 of 976
How did they see if comm was required on midline cells?
used a neuron specific promoter to drive a marker then same promoter to drive comm just in comm mutant neurons and you see a WT rescueq
432 of 976
what was this rescue as good as?
as good as when comm is turned on in midline cells-so was the same regardless of whether comm in midline
433 of 976
how did they turn on more comm in midline cells?
using a midline specific promoter- slit
434 of 976
maybe having it in the midline is important to get full recovery, how did they eliminate this criticism?
they drove comm in the midline as well as in the neurons but this was as good of as a rescue as it gets
435 of 976
what did this tell us?
you don't need comm in the midline to rescue midline crossing
436 of 976
what model for how comm is regulating robo was right?
sorting model
437 of 976
what is the difference between the clearance and sorting model?
in the sorting model robo should not be shipped down the neuron in the presence of como but in the clearance model it should be cleared from the CS
438 of 976
how did they test the sorting model (ie if robo was being shipped down the axon when comm was around)?
using a transgenic animal- fused GFP to robo so Robo-GFP fusion
439 of 976
what did they find in an axon not expressing comm?
robo transports down the axon
440 of 976
what did they find in an axon expressing comm? what was this consistent with?
robo doesn't transport its all in the cell body and not sent down the axon- consistent with the sorting model
441 of 976
what did this tell us?
comm is involved in trafficking robo down the axon
442 of 976
what model appears to be correct?
the sorting model
443 of 976
what still don't we understand about the sorting model?
why comm is in the midline (coincidental or involved in early patterning/other purpose?), why robo levels get upreg after crossing
444 of 976
what is upregulation of robo likely to involve?
contact with the midline
445 of 976
why would you not make robo when you contact the midline why would you have robo in vesicles?
robo is made before axons cross and stored in vesicles, it can appear at the CS rapidly on contact with midline
446 of 976
why would you not have a TF to upreg robo, why would it evolutionalry be a memrbae associated protein at the CS?
TF to upreg robo would take too long. GCs are a distance from the cell body so would have to wait to know to become sensitive to repellants, GC communicate to CB, CB makes robo transcription+translation long then back to GC = long.
447 of 976
what is the faster way to do it?
have robo already pre-made and have it sensitive to changes in the env so you can divert robo down the axon
448 of 976
what are examples of proteins that expression change on C axons as they hit the FP?
cell adhesion molecules- tag1 + L1
449 of 976
how does Tag1s expression change?
expressed as approach the FP but rapidly turned off after they corss it
450 of 976
how does L1 expression change?
is upregulated after crossing the FP
451 of 976
in vertebrates what robo molecules are there?
robo1+2 (same molecule in this)
452 of 976
what is rig1?
it's a robo-like receptor
453 of 976
what is rig1 also called?
454 of 976
what is the comm homologue in vertebrates?
we don't have one
455 of 976
when is Rig1 expressed?
pre-crossing axons, its at high levels as axons approach the midline
456 of 976
when are robo1+2 at low levels?
until the axons cross the midline they're at low levels
457 of 976
if you KO rig1/robo3 what happens?
prevents midline crossing by C axons
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why was this suprising?
KO a robo like molecule and the result is that we don't cross the midline- normally no robo means you can cross as much as you like (fly)
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what wasn't it because of?
slits are still on the midline here (the ligand for robo) and they do become sensitive to slits still as they cross the midline so strange
460 of 976
what are the 2 possibilities of why the Rig1/robo3 KO would mean axons don't cross the midline?
1) Rig1 rec for an attractant at the midline- so wouldn't cross as you don't have the receptor 2) Rig1 might be preventing premature sensitivity to a FP repellant so without it you sense repellant and don't cross
461 of 976
how did they test what Rig1 KO was doing?
used the classic FP assay in mouse tissues where axons grow towards the dorsal SC explant. Control + took dorsal SC from a Rig1 KO mouse
462 of 976
what was the difference between the control + the Rig1 KO?
axons extend towards the FP in the control but the axons are not attracted towards the FP in the Rig1 KO so consistent with Rig1 being a rec for an attractant
463 of 976
what happened when they took this assay and added a soluble EC domain of robo (including the slit binding domain) where slit function is blocked as there's no IC domain mops up slit, at the same time as they put in the ronbo explant in?
they found axons could grow now despite Rig1 KO if slit was blocked
464 of 976
how did they investigate this further?
instead of putting FP in the assays they put cells expressing different proteins (mainly Slit2) and they put netrin in the bath
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what did they find?
if you took WT pre-crossing C axons they will ignore slit if netrin is present in the bath so they grow in both sides and take no notice of cells expr slits
466 of 976
if they took the dorsal SC from the Rig1 KO mouse how do Rig1 respond to slits?
they no longer ignore slits in the presence of netrin they find axons are repelled by slit2
467 of 976
what does this suggest?
responsiveness to slit is dependent on rig-1 being expressed
468 of 976
so what is the difference in sensitivities to slit if rig1 is present or abnsent?
rig1 present = C axons can ignore slit2 Rig1 absent = sensitive to slit (both with netrin)
469 of 976
when they did a control with semas was the same true?
no it was only specific for slits (slits+ semas are both expressed in the midline)
470 of 976
when can netrin override the effect of slit?
only when Rig1 is present
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what is Rig1 doing?
blocking responsiveness to slits
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why is rig-1 analagous to comm even tho they work in different ways?
they are preventing premature sensitivity to repellants (comm- robo+slits and rig1- slits)
473 of 976
what is doing rig1 doing that is the same as comm?
it prevents robo signalling before the axons cross the midline
474 of 976
where are slits?
in the floorplate yet rig1 allows C axons to approach the FP even when slits are there
475 of 976
how is it thought that rig-1 is preventing to sensitivity to slits in the FP?
thought that rig-1 is preventing the function of robo1/2 receptors from responding and generating a signal
476 of 976
how is rig1 diferent from comm?
comm prevents robo from getting to the cell surface but rig1 is not doing that to robo1/2, it's shutting down its function
477 of 976
what assay did they look at how the netrin response is lost?
the xenopus spinal cord assay- looked at turning in response to netrin
478 of 976
what did they find?
the effect of netrin can be negated when slit is present- (equivalent to post crossing axons), see a netrin response when we don't have slits in the env. slit and netrin together = lose the positive response to netrin
479 of 976
why was it suprising that netrin positive response could be abolished by slit presence?
thought of as post-crossing axons so normally have crossed the midline so have already lost responsiveness to netrin but here they do not
480 of 976
What control did they do to check this was specific for netrin?
used BDNF- BDNF was not affected by slit
481 of 976
what was the 2 models to explain why C axons in the SC respond to netrin but that response is negated by slit?
1) ligand-ligand interaction 2) receptor mediated silencing
482 of 976
what is the ligand-ligand interaction model?
slit in the env in some way stops netrin binding to its receptor (DCC) eg slit and netrin might be binding together
483 of 976
What is the receptor mediated silencing model?
slit is binding to its own rec (robo) might be silencing the netrin rec
484 of 976
what model was right?
Receptor mediated silencing
485 of 976
what is the only thing required to block the netrin response?
only the Robo-IC domain is required to block the netrin response
486 of 976
how did they use the frog embryo to show this?
inject RNA into the 1 cell frog embryo and let it develop, culture the neurons and then test different RNAs for their effect on guidance
487 of 976
how did they show only the Robo-IC domain was required to block the netrin response/the receptor-mediated silencing model was right?
made hybrid versions of robo- eg take robo chop off EC domain and subsitute with TrkA rec (NGF rec) EC domain
488 of 976
what were they asking when they made these hybrid versions?
is it important that the ligand can bind to its own receptor (slit binds to robo) or is the ligand itself inteferring with netrin
489 of 976
what did they find when they did this?
IC robo domain with EC TrkA (NGF) rec = NGF inteferes with the netrin turning response, NGF normally attractant but add it to neurons with IC robo with TrkA it blocks the netirn turning response
490 of 976
which model was this in favour of?
the receptor-mediated silencing model
491 of 976
what is the idea behind this?
binding of a ligand to a receptor which has the IC robo domain is enough to give you silencing of the netrin response
492 of 976
what was the silencing of the netrin response due to?
an interaction of the IC robo domain with the IC DCC rec domain (the netrin receptor)
493 of 976
where are examples of crosstalk between receptors being important to prevent things operating in the wrong part of the pathway?
prevents robo (Slits), then DCC (netrin) from operating in the wrong part of the pathway
494 of 976
what happens on pre-crossing axons?
Rig-1 is high at the CS so prevents response to slits in the environment, robo1+2 inhibited by rig1. Respond to netrin to attract the axons towards the midline by the DCC receptor
495 of 976
what happens when you hit the FP?
now switch become sensitive to slits as rig-1 disappears and robo1/2 can bind slits and generate a signal
496 of 976
what is the switch in sensitivities before and after the FP is crossed?
first sensitive to netrin then switch to being sensitive to slits
497 of 976
how does robo1/2 prevent sensitivity to netrin after crossing?
robo1/2 IC domain blocks the IC domain of DCC so prevents a response to netrin
498 of 976
what could be causing the switch from attraction to repulsion of the FP?
1) something happens when you touch the FO 2) time mechanism- something happens as neurons mature
499 of 976
where is tag1 expressed?
on the C axons
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where is Nrcam expressed?
ont he floorplate
501 of 976
what experiment was done to show contact with the FP affects responsiveness?
messed around some adhesion molecules- tag1 which binds Nrcam. Open book exper out Di-i see axons crossing the FP then add antibodies against tag1 or Nrcam when axons coming to the FP
502 of 976
what did they find if you blocked Tag1 (on C axons) or Nrcam (FP) with antibodies?
lots of axons turn on the wrong side
503 of 976
what might this suggest?
contact with the FP might be important
504 of 976
what happens if they KO'd Nrcam?
abberant/messing up of the normal crossing pattern
505 of 976
what did they find whent they bound Nrcam to the surface f C axons by dropping soluble Nrcam into cultures?
it changes the expression of a midline repellant- looked at expression of semaphorins as they're expressed at the midline so looked at the PlexinA1 rec found adding Nrcam to c axons before they hit midline stabilised PlexinA1 on the CS
506 of 976
what is stopping responsiveness to the semas on the midline before axons reach the FP?
there's a degradation mechanism that is chopping up Plexin A1 on C axons before they get to the midline
507 of 976
what does contact with Nrcam on the midline do?
stops that degradation and allows pleckstrin A1 to get to the surface so become sensitive to semas
508 of 976
why do the mechanisms work that you;ve got something up and running but it's inhibited until you encouter choice points to release inhibition and switch sensitivities?
because the CB is a long way away from the GC so takes a long time to communicate
509 of 976
evidence that contact with the FP affects responsiveness?
2 adhesion moelcules- Tag1 on C axons + Nrcam on the FP bind to each other if you stop them functioning by adding antibodies you can interfere with when the GCs turn
510 of 976
what do antibodies to Tag1 give?
have premature turning on the wrong side of the FP and similarly with anitbodies to Nrcam
511 of 976
how much pleckstrinA1 is usually at the CS on axons that haven't crossed the FP?
very little sema rec so they are not repelled by the FP so can reach it
512 of 976
how is plexinA1 degraded?
by a protease called caplain
513 of 976
what happens when the GC comes into contact with Nrcam or you artifically dump Nrcam ontot he neurons on the dish?
you stabilise plexin A1 on the CS
514 of 976
what is this evidence for?
contact with the FP can enact the switch in responsiveness that we've been talking about.
515 of 976
what else can switch responsiveness and is a component of the FP?
516 of 976
responses to semas are also switched on by what?
517 of 976
what experiment showed that shh can also switch on responses to semas?
took semaphorin expressing cells (sema3F or sema3B) in collagen gel + put them in with a dorsal spinal cord explant and you put netrin in
518 of 976
what did they find?
no semas present- get netrin induced growth c axons out of the explant. If you have either Sema3F or Sema3B with netrin in the dish C axons are not repelled by the sema
519 of 976
what happened when they then stuck shh in the dish?
in spite of netrin being present the axons are now repelled away from the semas
520 of 976
where is shh normally expressed?
the FP
521 of 976
what does this tell you that shh is doing to the c axons?
makes the C axons responsive to semaphorins
522 of 976
what 2 things is shh doing?
attracting C axons towards the FP (works with netrin) then makes C axons sensitive to semas
523 of 976
how do we know shh can attract C axons towards the FP?
C axons are attracted to cells expressing shh, if you block shh with cyclopamine C axons are not attracted
524 of 976
what is this a direct effect of?
a direct effect of shh on the C axons rather than an effect on the patterning that happened earlier
525 of 976
what happens if you KO smo in the C axons?
then you see abberations in the pathway- so same effect of netrin KO
526 of 976
how do we do a conditional specific KO of smo?
use a wnt1 promoter (as expressed int he dorsal SC) which drives cre and then cross with a smo floxed allele mouse so cre turns on by wnt1 promoter and will cut out smo
527 of 976
where is an example where there might be an intrinsic switch (changing responsiveness with time)?
expression of adhesion molecules L1 + Tag1. Tag1 expressed as you go towards FP then turned off if you take DSC neurons before they reach FP and dissociate them and follow expression of there expression changes with time
528 of 976
how does Tag1 expression change with time?
tag1 disappears over time- present as approach FP then turned off
529 of 976
how does L1 expression change with time?
L1 low as you go towards FP and then expressed once you've crossed it
530 of 976
what is L1 and Tag1 expression independent of?
floorplate independent change in expression as there's no FP in these cultures
531 of 976
what does this tell us?
neurons mature so there are cell intrinsic mechanisms that switch the responsiveness
532 of 976
what experiment was done to show that there are cell intrinsic changes to shh itself?
spinal cord C axons in a dish and they tested their response to a gradient of shh
533 of 976
what did they find?
on avergae C axons 2 days in vitro are attracted to shh but at 2-4 days in culture you get a reversal of the response so get repulsion from shh = over time just C axons on their own in a dish reverse their response to shh
534 of 976
what is the reversal to shh mediated by?
a par-5 like protein (paritioning gene) (part of Par-2 complex?)
535 of 976
what is a par5 like protein?
536 of 976
what happened when they did the same experiment with a shh gradient but blocked the epression of 14-3-3 with siRNA?
you maintain the attraction to shh- so 14-3-3 makes shh repulsive
537 of 976
when is 14-3-3 increased?
in post-crossing axons so that it becomes less attractive to shh in the FP. Low approaching FP then elevated levels once crossed
538 of 976
what does 14-3-3 appear to be doing?
makes axons repulsive towards shh
539 of 976
if you overespress 14-3-3 what happens?
you get premature turning
540 of 976
what does 14-3-3 appear to be doing?
appears to be modulating when C axons become sensitive to FP (shh) and what that response is
541 of 976
what are the 2 switches?
contact with the FP and intrinsic cell changes over time like in response to shh- neurons maturing over time
542 of 976
which of these mechanisms is more predominant?
unclear which is more predominant but theres a lot of redundancy- several things going on to ensure C axons change the sensitivity at the right time
543 of 976
how is shh used in different ways?
1) attracts C axons to the FP 2) changes response to semas from attraction -> repulsion and that involves 14-3-3
544 of 976
how are shh levels graded in the spinal cord?
anterior (low) to posterior (high). posterior = in the FP
545 of 976
what does a loss of shh response in C axons lead to?
1) a failure to be attracted in vitro (redudant with netrin in vivo) 2) failure of C axons to become repelled by the FP (semas) axons wander so shh used to attract to FP then turn on sensitivity to semas
546 of 976
shh 4 roles?
1) attract to FP 2) switch to sema repulsion 3) AP guidance with shh gradient 4) involved in patterning early on-hypo and mesoderm
547 of 976
how are we getting a push and pull in both axes?
BMPs dorsally attracting and shh/netrin ventrally thats involved in getting C axons to the FP in the 1st place, once crossed midline wnt4 gradient anteriorly and then the push from shh in the other direction
548 of 976
what are shh and wnts both affected by?
signalling components which are associated with the par polarity complex
549 of 976
what does 14-3-3 affect?
shh is affected by 14-3-3 which affects PKA.
550 of 976
how is shh response reversed?
by intrinsic increase in 14-3-3 leads to shh being a repellant.
551 of 976
what is 14-3-3 part of? what does it inhibit?
sh14-3-3 is a par-5 homologue which inhibits PKA
552 of 976
how are wnts related to the Par-3 complex?
wnts signal through frizzled, GSK3B and dishevelled and that affects the par-3 complex
553 of 976
what are these interactions showing?
mutual inhibition acting to drive the GCs in their pathways
554 of 976
what is very central to the way the growth cone responds to the environment?
the cGMP and cAMP levels
555 of 976
why do responses to cues need to be integrated?
different responses depending on the combination of cues that is received so the cell has multiple cues in the env has to integrate info coming and decide what the result should be in that context
556 of 976
what are examples of combos of cues working together?
netrin + slits- effect of slits is negated by netrin (add more)
557 of 976
what is critically important in determining the polarity of a GC response to chemotropic molecules?
intracellular cyclic nucleotide levels
558 of 976
what is polarity in the guidance context?
how do you decide whether a response to a cue is going to be attraction or repulsion- they're opposites = the direction of the GCs response
559 of 976
why is knowledge of signals in axon growth helpful?
to get therapies for nerve regeneration and spinal cord injury
560 of 976
what model did they find a central role for cyclic nucleotides in?
561 of 976
what did they find dicytostelium GCs respond directly to?
cAMP, cyclic nucleotides in a pipette
562 of 976
how did they find dicytostelium are responsive to cyclic nucleotides?
they were looking at the effect of NGF- was testing which pathway NGF was using (including cAMP)
563 of 976
why was it not exactly like dicytostelium when they looked at the NGF pathway?
they used analogues of cyclic nucleotides that are able to go through membranes
564 of 976
what is the receptor for cAMP in dicytostelium?
GPCR 7 TM receptor
565 of 976
in these experiments what is happening with cAMP?
cAMP is going into the cell and hence affecting the change
566 of 976
whose lab really put the cyclic nucleotides at the centre of our understanding of axon guidance signalling?
Mu-Ming Poos lab
567 of 976
what did Mu Mings Poo lab do here?
using this membrane permeable analogue of db-cAMP and showing the GCs turning towards it using the classic xenopus GC assay
568 of 976
what experiments did Mu-Ming Poos lab do to show how important cAMP was?
exper with netrin in a pipette. if you block PKA (cAMP pathway) with this inhibitor see GC is turning away from the netrin source. Reversal of polarity- the way the GCs turning has been reversed.
569 of 976
overall finding?
turns out a response of a number of guidance cues is reversed by manipulating the concentrations of IC cyclic nucleotides
570 of 976
what is this response?
all or nothing
571 of 976
what do you see when you manipulate using an agonist or inhibitor of cAMP?
changing the levels of cAMP- the curve is very steep the change is flipping over a very narrow concentration so cAMP levels are critically important in affecting the polarity of the GC response
572 of 976
what is this turning dependent on?
the turning was ca dependent irrespective of the way it was turning- is dependent on there being a ca flux within the GC
573 of 976
what are polarity of GC responses affected by?
cAMP and cGMP ratio/levels
574 of 976
what response do you get when cAMP is high + cGMP low?
cues that cause this give attraction
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what response do you get if cAMP is low + cGMP is high?
repulsion- as caused by semas and ephrins
576 of 976
how can you reverse the response to a cue?
by the combination of receptors present
577 of 976
what is an example of reversing the response to a cue by the combination of receptors present?
netrin is the classic example- if just DCC present on the cell cAMP levels go up + attraction but if DCC is with Unc5 then you get cGMP levels going up so repulsion
578 of 976
what affects the polarity of the response caused by that cue?
the combination of recs for that cue
579 of 976
what repulsive cues inhibit cAMP and regeneration?
580 of 976
how is there different ways to end up with the same effect?
if you change the ratio of cAMP:cGMP
581 of 976
what is confusing about calcium fluxes in the GC?
higher ca concentrations are observed on the side of the GC facing the source regardless of whether its attractive or repulsive
582 of 976
localised ca fluxes do what to GCs?
ca fluxes turn GCs
583 of 976
when you released ca into the cell by caging what did they get?
attraction or you could get growth cones to turn
584 of 976
where are there fluxes?
in the filopodia of the GCs so calcium is important as GCs naviagte in vitro and in vivo
585 of 976
what is suggested to play a role in response outcome?
differential activation of ca channels
586 of 976
describe what happens when an attractive cue is encountered?
1) an attractive cue comes along, elevates cAMP and they activate TRP on the surface let EC Ca in and/or they elevate IP3 2) Both ca and IP3 trigger further release of ca from IC stores 3) feedback&elevates cAMP which in turn casuses further ca
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what is this more and more elevated ca an example of?
a positive feedback loop
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what does that positive feedback loop result in?
a high amplitude Ca flux which activates extentension of the GC
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what are attractive cues?
BDNF, NAG and netrin
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what are repulsive cues?
semas, slits or netrins (when DCC rec with Unc5)
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what happens in response to a negative cue?
1) elevate cGMP and activate TRP channels at the surface to let in EC ca 2) you get a ca flux but at the same time cGMP inhibits ca IC and inhibits a rise in cAMP 3) no release from IC stores no +ve feedback loop, low amplitude flux
592 of 976
what does the amplitude ca flux activate?
repulsion and GC collapse
593 of 976
how is it similar and different with an attractive and repulsive cue?
both activate TRP C channels to let in EC ca, in both cases you get calcium fluxes in one case its a high flux and you get a +ve feedback loop the other things dampened down by cGMP
594 of 976
what flux do you get for an attracitive and repulsive cue?
low flux ca- repulsive cue and high flux ca- attractive cue
595 of 976
what are the different amplitude of ca fluxes initiating?
endocytosis vs exocytosis
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what does a repulsive low amplitude ca flux initiate?
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what does an attractive low amplitude ca flux initiate?
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why would an attractive cue initiate exocytosis?
add components to the membrane so it can grow
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what do you see when you encounter a positive cue?
you get the transport of vesicles towards where the +cue is, get membrane insertion to get growth on one side so insert new membrane this happens in response to an attractive cue (high ca flux)
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how did they see clathrin vesicles forming for endocytosis with a repulsive cue?
used time laser turf microscopy
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what is there an increase in, in response to a repulsive cue?
increase in clathrin mediated endocytosis
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describe what happens when you encounter a repulsive cue after cGMP has dampened everything down?
clathrin vesicles were form, you get endocytosis on the side closest to the repulsive cue and the removal of the membrane to take the GC away from that point
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what kind of exocytosis is initiated by an attractive cue (high ca flux)?
VAMP2 mediated exocytosis (new membrane) growth + attraction
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what kind of endocytosis do you get with a repulsive cue (low ca flux)?
clathrin mediated endocytosis (take membrane away) -> GC moves away (repulsion)
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what may modulators that ffect cNMPs be doing?
altering the polarity of response (whether it's attractive or repulsive)
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is it just the direct receptors of cues that affect IC levels of cNMPs?
no there's lot of other modulators, cyclic nucleotides are affecte ddownstream so lots of things can affect there levels
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what does it mean for the GC that there are many modulators of cNMP?
the GC has a means by which it can monitor multiple different cues and make a decision about which way to respond on the basis of the relative concentrations of cyclic nucleotides
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what can inhibit cAMP levels increasing?
laminin and glutamate
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what can inhibit cGMP increasing?
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Why is it not just the cues that do the guidance?
you can have other inputs into the pathway
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what binds o laminin to lower cAMP levels?
integrin binds to laminin to lower cAMP levels
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how may specific environments algter guidance cues responses?
by modulating cNMPs concentrations
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where was an example that the environment might change the response by cNMP levels?
RGCs in the retina navigating to the tectum. Early pathway use netrin up to the optic nerve head but then contact with laminin in the optic nerve reverses the response to netrin which guides axons away from optic nerve head
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what does 14-3-3 in the environment do?
14-3-3 can alter PKA activity and that changes responses because it affects the cAMP pathways
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specifically in nerve regenration/injury how is it useful to know you can alter the response to cues by adding endogenous agents?
altering the response to cues by adding endogenous agents suggests that we might be able to promote axon growth in situations where growths inhibited
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what can affect the responses to molecules know to inhibit axon regeneration?
manipulation of IC cyclic nucleotide concentrations
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what agents prevent mammalian CNS axons regenrating?
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how do MAG + NOGO affect cAMP levels?
they affect cAMP levels by activating RhoA
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what has there been sucess in when manipulating cAMP + RhoA?
Success in repairing spinal cord injury
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desrcibe PNS nerve injury?
macrophages are rapidly recruited and they mop up the myelin debris. You get expression of growth related genes. Gene expression pattern of the damaged neuron changes dramatically, proliferation of schwann + glial cells to promote regeneration
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what gene become expressed in PNS nerve injury?
growth related genes
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what cells proliferate to promote regeneration in PNS nerve injury?
schwann cells and glial cells
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what happens in CNS nerve injury?
you don't see as rapid clearing of the myelin debris, you have inhibitory factors remaining from the myelin that inhibit and disrupt axon extension, no promotion of axon regeneration but get a glial scar forming which acts to inhibit the regeneration
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what factors mean the CNS nerve injury fails to regenerate?
1) failure to activate the growth promoting programme in the injured neuron 2) presence of inhibitory factors like MAG + NOGO 3) formation of a glial scar which presents a physical barrier
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do CNS axons just not have the ability to regenerate?
they can regenerate but it is the inhibitory environment in the damaged CNS that stops them from regenerating
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what did Aguayo et al hypothesize about CNS azon rgeneration?
hypothesized that CNS axons might not regenerate because they don't have the right environment
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what did they want to find out?
if CNS axons had the capacity to regenerate
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what experiment was done to test this?
thought CNS axons could regenerate if given the right substrate so if you graft in sciatic nerve sheath from the periphery that might allow growth to occur. Cut the optic nerve& grafted sciatic nerve to see if RGCs project axons with sciatic nerve
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what did they find?
RGCs could send their axons to the colliculus via the peripheral nerve graft
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what did this suggest?
given the right environment CNS axons can regenerate
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what does the DRG have?
an axon that goes out to the periphery for propiception etc and a central nerve branch going into the spinal cord (CNS)
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what test was done to show given the right substrate and/or giving the right genes being activated allowed regeneration?
conditioning lesion exper in the SC. Know peripheral nerve branch can generate but the central axon going into the SC can't regenerate.
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how did they do the conditioning lesion?
they cut the peripheral branch first, waited and then cut the central branch inside the spinal cord.
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what did they find when they did the CL?
you get both the peripheral and central branches regenerating
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what was the thought behind this experiment?
thought that the peripheral branch could regenerate as when its cut you get growth because it activates a programme of gene expression so if you turn that programme on maybe you could get regrowth anywhere
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what did this tell us about activating the cell bodies of the DRG neuron?
something about activating the cell bodies of the DRG neurons makes them capable of being able to regenerate even in a non-permissive CNS environment
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what is there thought to be in the PNS to allow regeneration?
there are substrates/factors in the periphery that can activate a regrowth programme & that programme can ignore inhibitory CNS factors
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what is the common feature of many different factors that inhibit growth in the CNS?
they are acting through RhoA
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where are most inhibitory CNS factors?
in myelin (or oligodendrocytes and astrocytes)
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what molecules activate RhoA to inhibit regeneration?
Mag, OMgp and NOGO
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do MAG, OMgp and NOGO all activate RhoA through the same receptor complex? how?
all activate RhoA through the same receptor complex. Complicated receptor has multiple different components- central one is NOGO receptor itself
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what do Mag, OMgp and NOGO all bind to? what do they act through?
the NOGO receptor itself and then act through the Rho GEF/GDI pathway -> activates RhoA -> GC collapse
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what do RHo GDIs do?
sequester inactive Rho (Rho-GDO) in the cytoplasm dampening the cells ability to activate (increases RhoA)
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how does cAMP antagonize GC collapse?
elevating cAMP activates PKA -> phosphorylates RhoA to inactivate it
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where does regeneration after aoxn injury occur?
lower vertebrates (ZF), the embryonic CNS and in the adult mammalian CNS
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where does axon regeneration not occur?
in the adult mammalian CNS
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what does activating the NOGO receptor change the levels of?
activates RhoA so changes the RhoA/Rac balance which leads to GC collapse
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why is it a good thing there's so many different ways that prevent regeneration?
several oppurtunities for therapeutic intervention to suppress the inhinitory effects of myelin when regenerating
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where does C3 transferase, ibuprofen and cethrin inhibit in the pathway?
Incrrease in RhoA leves -> Rho activation
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where does the ROCK inhibiotr Y27632 inhubit?
rho activation (ROCK) -> GC collapse
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what do db-cAMP and C3 transferase do to rho activation?
blocks rho activation
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what does Y27632 do?
blocks Rho kinase from causing GC collapse
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what does db-cAMP promote?
regrowth of central sensory afferents
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how did they test whether db-cAMP can enhace he growth of sensory afferents in the spinal cord?
using the pre-conditioning paradigm (the CL experiment)
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why did they want to see if db-cAMP promotes regrowth of sensory neurons?
they noticed cAMP levels go up after a peripheral lesion in the DRG so wanted to see if cAMP elevation activates regeneration programme in sensory neuron that happens downstream of conditioning
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what did they do in this experiment to test whether db-cAMP is involved in growth of sensory afferents?
put sensory fibres in a dish (in vitro) and grew them and put on db-cAMP showed that they could grow even in the presence of inhibitory CNS myelin
657 of 976
how did they show db-cAMP if injected at the right time can get axon regrowth in the central branch (CNS)
they inject db-cAMP into the DRG prior to the lesion in the CNS and this enhances regrowth in the drosal columns. elevate cAMP levels by injecting db-cAMP and do the central lesion and see axons regrow
658 of 976
why is it not very usefully therapeutically hthat injecting db-cAMP prior to lesioning was able to help regrowth in the CNS?
you've got to know when you'll get CNS damage to treat the DRG prior to the lesion- in accidents don't know when they'll happen
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how is the functional recovery you get with db-cAMP?
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how do we know increased cAMP is not enough?
if you compare that to the CL the CL is more effective in promoting growth than the db-cAMP treatment alone
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when they did transcriptomics after the CL what did they find?
large array of gene expression changes after the CL- found a whole bunch of regeneration-associated genes
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what is very important in determing the way that GCs respond?
the cyclic nucleotide levels
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what can manipulatioon of pathways that guide GCs be used for?
regenerating axons so we can repair injury
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what where they trying to mimic with the db-cAMP (soluble cAMP form)?
the pre-CL, as sensory fibres in vitro can grow when you put db-cAMP onto them even in the presence of CNS inhibitory myelin
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what did they find in the pre-CL experiment?
if you cut the peripheral branch of the DRG then you cut the CNS branch you find that the pre-conditioning allows the CNS branch to regrow0 activating a growth programme in the CNS despite MAG +NOGO
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in regeration associated genes (RAGs) that are turned on in the DRG after a CL what did they see a global increase in?
global increase in trafficking of numerous IC components into the injured central branch
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when is the only time you see this trafficking happening?
when you have a CL occuring- just doing cAMP alone doesn't get this trafficking
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things being trafficked?
mitochondria, MAPS, 14-3-3 proteins, RhoGDI, CRMP eg things that control Rho GTPaes
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what is a chimera graph and how is it made?
allows you track the movement of particles in the axon either in anterograde or retrograde direction, you track the passage of particles along an axon and you put that together over time in strips
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what do you see happen to particles in the CL?
there is lots of particles trafficking back and forth. It's mostly anterogrde transport
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what is anterograde transport?
towards the CNS and towards the tip of the axon
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what does this trafficking tell us about the CL?
there is a lot more going on in the CL than just elevating cAMP levels
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what do some of these components look like they might be involved in? what does this suggest the CL might be doing?
components involved in resetting the polarity of the cell (where axon+dendrite is), CL might be resetting the overall polarity of the cell
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what is right in the heart of all the inhibitory signalling responses?
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where did they look at Rho-kinase (ROCK) inhibition?
in the descending corticospinal tract, in the cervical spinal cord, they lesioned in the dorsal columns just below cervical levels
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what is C3 transferase?
an enzyme = a RhoA inhibitor
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what did they do to show that RhoA inhibition can affect the growth of cultured neurons growing in the presence of myelin?
they put an enzyme called C3 transferase (a RhoA inhibitor)
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what did this suggest?
inhibiting RhoA meant the CST could regrow
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what happened when they did this in vivo?
did not effect the regrowth of neurons in vivo
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why did they think it didn't work in vivo?
might be a delivery problem with C3 transferase- its an enzyme (protein) so delivering proteins into the body of a living animal is quite difficult
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how did they overcome the delivery problem with C3 transferase?
made a derivative that is able to get across membranes = cethrin/VX-210
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what is Cethrin/VX-210?
a derivative of C3 transferase that can get across cell membranes
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what has been shown with cethrin/VX-210?
this has been shown to work in animals and is now entering phase 2/3 clinical trials in humans
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what do we know now its passed phase 1 clinical trials? what do we not know?
phase 1 climical trials- testing whether a drug is safe to use but don't know about the efficacy
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why might manipulating the Rho-A pathway be useful?
its already been shown to improve CST regeneration in animals
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What did they dind if they used a ROCK inhibitor (Y27632?
if the ROCK inhibitor was administered at the same time as the lesion it does promote regrowth through the DC lesion
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how was the ROCK inhibitor used?
in vitro ROCK inhibitor could promote neurite growth on myelin-associated inhibitors in vitro. It also worked in vivo with enhanced sprouting of CST fibers in vivo and enhanced locomotor recovery after CST lesions in adult rats
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what is another manipulation of the RhoA pathway but with a different reagent?
can manipulate the same pathway with a non-steroidal anti inflammatory (NSAIDs) = ibuprofen
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what does ibuprofen do to DRG growth?
1) Ibuprofen enhances DRG growth on myelin&CSPGs 2) it inhibits RhoA activation at the injury site 3) enhances recovery dital to spinal cord hemi section (CST) lesion
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what are myelin and CSPGS?
both inhibitory components present in glial scars and myelin
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how did they show ibuprofen inhibits RhoA activation at the injury site in the animla dorsal columns?
injected with a control: highly elevated activated RhoA after SC injury. Animal treated with ibuprofen: RhoA activity doesn't appear after spinal cord injury
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what is shown to be enhanced with ibuprofen?
enhanced recovery to the distal SC in a similar dorsal column lesion- looking at the CST
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when they used ibuprofen how was mild enhanced recovery o motor function shown?
by longer stride length and shorter stride width in control (assay for CST function- effect on walking) stand mice in ink have them walk across papoer
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how was the control mice walking different to the ibuprofen treated mice?
widely spaced after SC injury in WT but after treatment with ibuprofen more narrow well-coordinated walking
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how do we know this is not a function of the cox inhibition by NSAIDs?
naproxen doesn't work in the same way in the SC injury experiment so its not the nromal NSAID pathway involved
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how is ibuprofen thought to be enhancing recovery and DRG growth + inhibiting RhoA?
ibuprofen appears to activate a TF, PPARY, that activates a phosphatatse (SHP-2) which inhibits RhoA GEF to suppress RhoA activation- but unclear
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what does all this evidence suggest?
another way of manipulating RhoA good evidence that manipulating RhoA function may work for SC function- diff drugs but good possiblities there
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why is there a lot of competition in this field?
the prize of being able to repair CNS spinal cord injury is high
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what did they do to see if the NOGO pathway is important in SC repair?
KO components of the pathway including the ligands (NOGO, OMgp and MAG) and different components of the receptor
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what did they find?
each paper finds different things eg KO mice still don't see any ability to regrow following injury in a triple mutant mouse no regen of axonal tract
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what was good pathways to look at and why?
cell growth pathways- if they regulate cell growth they may also regulate the axon growth ability
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how did they test cell growth pathways?
they did conditional KOs in a pool of mice for major growth genes and looked at their ability to regrow after optic nerve injury
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what major growth control genes did they do conditional KOs in?
Retinoblastoma, P53, Smad4, LKB1 and PTEN
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what are all the major growth genes they tested involved in?
all in pathways that control cell growth- maybe these genes will be able to manipulate whether axons can grow
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how did they damage the CNS in this experiment?
they damaged the optic nerve by an optic nerve crush
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how did the conditionally KO the genes?
they floxed the alleles of each of these different genes in a mouse and then they injected into the retina (VH) an adenovirus carrying a gene in which cre was expressed under a strong ubiquitous promoter
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what does the adenovirus do once its injected into the vitreous humerus?
infects the RGCs and turns on the cre which will KO the particular gene they're looking at
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what did they put in with the cre virus?
GFP to allow them to see axons that had been hit with the cre virus
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what was the control?
adenovirus carrying GFP alone
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what did they find?
control: optic nerve crush with just GFP- you get growth stopping at the site of injury, only positive result of all the genes KO'd was PTEN
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what gene made a difference to the optic nerve regrowth?
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What is PTEN? what does it antagonise?
a phosphate that antagonises PI3 kinase (PIP3 -> PIP2)
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in normal development what does PTEN regulate?
the mTOR pathway (mammalian target of rapamycin)
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how does PTEN feed into the mTOR pathway?
conversion of PIP3 -> PIP2 by mTOR stops the activation of Akt (by PIP3) activation of Akt means you relieve the inhibitions so get activation (inhibition of an inhibition)
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go through the pathway with Akt and mTOR?
inhibits PIP3 which activates Akt which inhibits TSC1+2 -> activates the mTOR pathway -> activates S6K. so inhibit Akt inhibit activation of mTor
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what is Akt inhibiting? how does PTEN stop this inhibition?
PTEN stops PIP3 activating Akt so then Akt stops inhibiting TSC1+2 which normally inhibits Rheb from activating mTOR pathway so no PIP3 Akt doesn't inhibit Tsc1/2 so Rheb can activate mTOR
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what did PTEN KO do?
significantlty enhanced neuronal survival and allowed RGC axon regrowth in adult mice
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in normal development what happens to the mTOR pathway?
it is progressively shut down/inhibited as development ceases and axons are goewn out so pathways turned off
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how do you see the decrease in mTOR pathway as dev ceases?
dowregulation of phospho-S6 kinase (marker of the mTOR pathway as mTOR activates S6 kinase)
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what do you see if you compare S6 kinase at E14.5 to postnal day 60?
the amount of S6 kinase present in axons is much lower.
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what happens if you take adult axons and injure them?
you get a downregulation of PS6 K so even small amounts there is in the adult gets switched off (in the retina)
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when is there downregulation of the mTOR pathway?
in development and ther is a superdownregulation of it if you have a nerve injury
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what does it mean that the mTOR pathway is shut down in nerve injury?
its as if the nerves are shutting themselves down and stopping themselves from regenerating
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where is PTEN KO working?
its stopping downregulation of PS6 K occuring and allowing the MTOR pathway to be activated and that seems to be enough to get axon regrowth in the optic nerve crush
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how did they show that it was this branch of the pathway (bit of Akt)?
did a similar thing to the PTEN KO by KO a downstream component = TSC1
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what did they find in the TSC1 KO?
you get axon regrowth (as Akt usually inhibits TSC1 from inhibiting Rheb) and Rheb activates mTOR so no TSC1 to inhibit Rheb = can activate mTOR
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if PS6K levels therefore the mTOR pathway is high what does this mean?
increased axon regeneration
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what does PTEN appear to be key in regulating?
retinal axon regeneration, PTEN regulating mTOR is important for suppressing axon regeneration
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what does PTEN deletion also enhance?
enhances spinal axon regrowth
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who looked at PTEN deletion in the spinal cord CST?
the same group who did the conditional KOs in cell growth genes
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what did they find if they did conditional PTEN KOs but looked at the CST?
they could get some regrowth across the dorsal column injuries in vivo and they could see that some of the axons form synpases
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what weren't they able to get?
any functional recovery
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what are they now looking for as a result of PTEN deletion enhancing spinal axon regowth across the DCs in vivo?
a drug that inhibits PTEN
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why is there a lag in finding small molecules to inhibit PTEN?
cancer drugs activate PTEN (to shut down cell growth) so all the drugs on the market are for activating it, not inhibiting it
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what are we getting to the point of because we've looked at pathways in terms of GC and turning?
we're getting to the point where we're able to design drugs that look hopeful for repairing SC injury
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what pathways are being looked at for repairing SC injury?
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what are the maps of things in the environment?
spatial maps
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what are non-spatial maps?
things you don't have a sense of but can detect- taste and smell
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in what animals is there inverse mapping of the retina onto the superior colliculus and tectum?
in frogs and maps
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what kind of map is this in the retinotectal system? why?
a topographic (spatial) map- what you see in the outside world is represented in the brain in a way that related to the outside world
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what was the stripe assay?
had a ***** of retina (nasal to temporal) and then had alternating *****es of anterior and posterior tectum and see where the axons grew
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in mammals what is the tectum called?
the superior colliculus
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where do temporal retinal axons go to?
the anterior (AT)
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where do nasal retinal axons go to?
the posterior (NP)
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what did the stripe assay show?
cells from the posterior tectum make a non-permissive tectum that repels temporal retinalwhe axons
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how did they know that the temporal axons were avoiding a repellant in the posterior?
the activity is abolished by heat treatment of the posterior membranes but not the anterior membranes and posterior membranes cause temporal GCs to collapse in vitro
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what is the inhibitory factor in the posterior tectum that the temporal axons avoid?
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what is the ephrin expression in the tectum?
high in the posterior, low in the anterior
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what are the receptors in retinal axons and which axons are they high in and which low?
Ephrin receptor for ephrins A2 + A5 is expressed in the retina in a gradient from high (temporal axons) to low (nasal axons)
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if you KO both ephrin A2+A5 what happens?
the topographic map is disorderd as temporal axons project to the posterior tectum
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what did theis tell us about how non-permissive repellant factors can be used?
they can be used instructively, they direct GCs to specific places to form topographic maps
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so what did the stripe assay show?
cells in the tectum make a non-permissive factor that repels temporal retinal axons
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how did they show that temporal -> anterior and nasal -> posterior?
a mouse expeiriment where we've injected into an adult mouse either into temporal or nasal retina lipophillic dyes that go down membranes to show where axons are mapping
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if they use lipophillic dyes to track axons but now KO ephrin A2/A5 (high in post + low in ant)?
temporal + nasal axons go all over the tectum = ephrins are important for topographic mapping
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is this what you would've expected?
got a grad of ephrins + ephrin receptors, you KO the ephrins you would NOT expect the nasal ones to be scattered throughout. Temporal axons kept in ant by repulsive gradient you'd expect them to go the posterior
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what happens if you KO ephrin A2 + A5 in the tectum?
topographic maps disordered, temporal axons can go to the posterior
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what did they find out to explain why they were going to particular spots and why nasal axons are scattered?
in rodents (mammals) the fibres (Temp+nasal) both grow all the way to the posterior tectum (not like in fish +chick), then you get branching+synaptogenesis- it's this process that is sensitive to the ephrin gradient
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what is different about mammlas (rodents) to chick and fish?
in rodents nasal and temporal axons grow all the way to the posterior
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what process is sensitive to the ephrin gradient?
the branching and synaptogenesis of axons
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how do axons get eliminated?
there is competition between axons for synaptic partners involving electrical activity so that cells that have weaker synapses get eliminated and axons get pruned back so temporal axons are pruned back
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what does the competition for synaptic partners require?
electrical activity
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what happens if temporal axons have no competitive advantage because the ephrins are missing?
they can compete all throughout the tectum (in the posterior too) for synapses so compete with nasal axons therefore affecting them
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what is electrical activity important for?
electical activity is important in mapping of the visual system in later stages for refining connections
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so what happens overall with the mapping?
the axons get guided to the tectum via labels but the actual refinement of the map is label and activity dependent
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what can electrical acticity (EA) do to guidance cues?
EA can modulate responses to guidance cues
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what happens in the Mu Ming Poo GC turning assay when you use different concentrations of netrin?
high concentrations- turning towards it, 5 fold lower netrin concentration- don't get turning
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how did they show that electical stimulation can enhance or reverse the response ot guidance cues?
Mu-Ming Poo GC turning assay and we have netrin in the pipette with different concentrations of netrin. Then add electrical stimulation using electrodes of the GC or CB then you can enhance the response to netrin
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what did the adding EA with electrodes do?
if you keep low concentration of netrin (that normally doens't give turning) but add electrical stimulation then you enhance the response to netrin and get turning
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what happened when they did the same experiment with MAG?
no electical dtimulation- MAG repels the GV away but add electrical stimulation of the neuron and you get attraction instead so changes the way GCs are responding
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how is EA changing the reponse of GCs to cues?
changes/modulates the cyclic nucleotide and ca levels
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how do we know EA modulates ca?
neuron in a dish is loaded with ca indicator dye, if you turn on the electircal stimulus you get elevation of ca levels and thats dependent on the level of activity (more EA pulses per min more ca)
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how do we know cyclic nucleotides are important in EA modulating responses to guidance cues?
cNMPs activity can be messed with by stimulating it or KO. Turn towards MAG with EA (attraction) but if you block components of the cAMP pathway it blocks the response
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what were similar things shown with?
netrin responses nad cAMP pathway components and ca
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how did they show EA affects ca with MAG?
MAG attracts with EA (when it normally repels) but if you drop the ca levels we just get repulsion with EA
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so what is electrical stimulation doing to ca and cAMP?
turning up the levels of cAMP in a ca dependent way and when you increase the EA by increasing pulses/min the effect (response) carries on for longer
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why does it hang around for longer if you do more electrical pulses?
spike in ca levels- if low levels (small num of pulses) it goes to baseline whereas high pulses keeps ca high and it doesn't go to baseline so continued effect
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what are chelating agents for?
uncaging to release ca to get GCs to turn and now we're using it to detect
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what is EDTA doing?
its chelated- hidden the metal inside its structure
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what happens in uncaging?
EDTA is flashed with high intensity laser light which breaks the bonds and releases the calcium
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what did they use instead of EDTA?
an EDTA like molecule called BAPTA and derivatives of it
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what did they do with BAPTA (chelating agent)?
BAPTA+derivatives hooked up to flurophores the way the flurophores lie over each other when ca is bound is different to when ca is not bound and the result of that is the response to illuminating light is much stronger when ca isn't bound
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what happens when ca binds to BAPTA and induces structural changes?
changes fluroscence intensities of flurophores
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what is refinement of connections dependent on?
activity dependent
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even in lower vertebrates (chick/frog) where the initial map in the tectum is tophographic (goes to right poles) even there what is it
still quite coarse-grained and axons are synapsing over a large area and over time maps become more precise and it becomes refined with EA
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what is activity dependent refinement involving?
competition between axons- contacts are lost over time and maps become more precise
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where is an example of where a map starts out coarse then becomes refined?
in the occular dominance columns- mapping from the LGN to the cortex
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if you block activity in one eye what happens?
these maps become disrupted
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what was Hebbians rule?
cells that fire together wire together (form synapses)
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what will happen if things are mapping onto the same place and firing at the same time?
synapses will be reinforced
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what happens if they're mapping onto different places and synpase firing is asynchronous?
synpase won't be reinforced
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where is the EA coming from in the mammals as the embryo doesn't see any light?
the embryonic retina is spontaneously active so when imaged for ca you see get waves of ca spontaneously emerging
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how do you block ca activity dependent waves?
with activity blockers
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when is there some kind of activity occuring?
before the retina sees light as axons map onto the tectum
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what could it be the activity in the retina? what is this consistent with?
could be that hebbian events are already operating- treatment with a sodium channel blocker (tetrodotoxin) blocks the map refinement
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how else did they show that hebbian events may be operating?
they used a genetic block of nicotinic acetlycholine receptors which blocks AP firing in neurons
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what is required for topographic map refinement?
EA and neural activity
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what do you see with mapping initially in the retinotectal system?
in the mosue you get early exubverant outgrowth of nasal and temporal axons throuhgout the tectum. they filled the temporal RGCs so over time you see branches being pruned back so you end up with localisation to the anterior
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what happens in the mouse that lacks the b2 subunit of the nAch recepotr?
you get initial exuberant growth trhoughout the tectum and then over time it doensn't refine properly
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what is this showing?
activity in this case firing (as blocked when lack nAch rec function) is involved in refining the connections in the tectum
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if activity is important for early wiring how is it that in the embryo when they do not see light so how do they get activity?
loaded with ca indicator saw spontaneous waves of ca activity across
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what did the spontaneous ca waves suggest?
hebbian principles may already be working there despite no light on the retina
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what is the 2 pieces of evidence that are consistent with hebbian principles already operating?
1) tetrodoxin (blocks Na channels- APs) block the refinement of the map anf 2) B2 nAch rec subunit KO doesn't refine the map in the tectum
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why are acetylcholine receptors important on RGCs?
they innervate the tectum
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what is the normal pattern of innervation in the mouse?
early exuberance anterior and temporal axons get pruned back but then there is competition which nasal axons win in post as temporal axons have ephrin disadvantage then axons being pruned back- temporal axons to ant colliculus/tectum
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what animal is there early exubarance- RGCs axons go eveytwhere and then get pruned back?
mouse (mammals)
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what happens if you block this activity in the mouse by KO beta 2 subinit of the ach rec?
uncorrelated RGC activity so the responses to retinal waves are disrupted and maps do not refine
807 of 976
Where is Eph A3 strongly and less strongly expressed?
Eph A3 is expressed strongly in temporal RGCs and low in nasal RGCs
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why is Eph A3 expressed strongly in temporal axons?
so they will be repelled from the posterior tectum
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how was Sperry right about these ephrins?
Sperry predicted that gradients would have positional info in the target and you would have a gradient of recs for the target gradient
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they thought this gradient to be a guiding gradient but what else does it provide? how did Sperry describe this?
guides but also gives positional info- Sperry described as having latitude and longlitude coordinates. The axons are aware of there positional value know where they are within the colliculus
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when would it make sense for GCs to extend branches?
at a specific position according to the amount of singal they receive
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what could be the signal to make them branch and form synapses at a particular position be?
could be a specific level of Ephrin receptor activation
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what is an example of how this might be working with temporal RGCs?
high Eph A3 recptors on the membrane- the signal that says to the axon you can branch & make synapses requires 2 recs to be occupied by ligand, can fill recs in low density of ligand because there's lots of recs so branching signal
814 of 976
what happens as temporal RGCs move up the ephrin gradient?
as the recs are high density getting them filled is easier -> generate a signal --> allows branching to occur
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how is this differeent with nasal RGCs?
they have a low density of EphA4 rec so will ignore low ephrin densitities in the anterior tectum and its only when you grow further in you generate a signal that starts branching
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what are the axons being able to sense?
where it is in the colliculus- so there's an element of positional info
817 of 976
is it true that you get a signal then branch?
in vitro assays suggest ephrins act within a specific range of concentrations to control branching
818 of 976
what experiment did they do to show positional information from a 'branch now' signal?
took explants from the retina (nasal to temp) and put strips of retina down onto cells expressing diff amounts of ephrins so some not expressing any ephrins and then cells all expr ephrins = ephrin concentration gradient from 0 to 100%
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what did they find when they put retina onto cells not expressing ephrins (the control)?
see some outgrowth
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at a low ephrin A2 (15%) concentration how much outhgrowth does that give compared to the control?
gives more outgrowth compared to the control with no ephrins suprising as repellants but low concs ephrins promoting outgrowth
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what did they find at high ephrin concentrations?
switches no longer promotes outgrowth but now inhibits it- at the highest concentration you get complete inhibition of the outgrowth
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how did they determine outrgowth?
probably in terms of the lengths and branches branches being generated here relative to control growth
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whats the difference between outgrowth at high and low ephrin concs?
low ephrin conc- promote axon outgrowth high ephrin conc- inhinit axon outgrowth
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why do temporal axons stop before nasal axons?
high concentrations of the receptor means you get inhibited before other people do (nasal axons) - ghet responses to the ephrin gradienr according to where you are in the nasal-temporal sequences
825 of 976
what will your final position depend on?
when promotion = inhibition- no net effect so the axon stops, axons from diff parts of the retina will branch at diff points in the gradient bcos of gradient of recs+ cues
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what is an example of inhibition = promotion to give branching?
BDNF signal = ephrin signal -> get branching in between the thresholds for branch promotion and branch inhibition
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what molecules does EA work through?
cNMPs, Ca AND neurotrophins
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what does EA do to cues?
enhances the response to cues
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which neurotrophin is particularly activated in the embryonic retina by EA?
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when there's enhanced retinal EA what is upregulated
BDNF in the environment
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what does BDNF being upregulated in the env do?
promotes RGC axon branching but only in tregions whhere eph-ephrin interactions are low
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how do they know that EA in the embryinic retina leads to upreg of BDNF in the env and increased branching when no ephrins?
stripe assay stripes of eprhin concentrations low and high- then put on ganglion cell
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what do you see?
branching happens when the ephrin concentrations are ow
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so how is BDNF being controlled?
BDNF is controlled by EA and that changes the response to ephrin signals
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what happens when you downregulate ephrin A5?
increased branching
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how do ephrins and the BDNF receptor (Trks) interact?
they interact physically in a neutrophin dependent (BDNF) manner
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so how does BDNF and ephrin change branching?
BDNF turned on, causes branching to start, at intermediate concentrations of ephrins we get high levels of branching induced (promotion=inhibition) but as the eph-EphA interactions increase get inhibition overpowering promotion branching so inhibit
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how could you get eph-EphA interactions to increase?
by high concentrations of ephrin or because axons have high concentration of ephA
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what is there an interaction between that allows you to make these fine maps?
EA and cues in the environment (like BDNF that allows you to make fine maps)
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where else do we see these maps?
in lots of other places in the brain like other places in the visual system- unlike chick+frog in mammals we have mapping of RGCs from the LGN + SC
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what is mapping of the different layers of the LGN also controlled by?
ephrin gradient
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where is this idea of ephrin gradients working?
several parts of the visual system and other areas where there's spatial maps = topographic maps of the body (somatopic maps- sensory maps and barrel cortices)
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how is somatosensory info maintained in the body?
in a topological order- is mapped all the way up to the sensory cortex where medial is the feet and lateral is the head the body is represented along to map onto the sensory cortex
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where is an example of different species having different kinds of maps?
the barrel cortex in the mouse for whiskers
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how are ephrins involved in the somatosensory cortex?
at the same time axons come from the thalamus to the sensory cortex ephrins are graded in their expression in a countergradient
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why is the ephrin gradients so important in neruoscience?
so much info about the outside world is put into maps within the cortex and other structures so ephrins expression give us a big clue to how thats set up
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how do we set up these gradients in the first place? what TF is important?
in the cortex a TF EMX2 is expressed in a gradient from high (caudal) to low (rostral) across the developing cortex and you get maps
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what happens when we overexpress EMX2 in mice?
it distorts the gradient and distorts the map and gradients from the visual + auditory cortex normally occupy a space but they expand out and crush the somatosensory cortex
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what controls EMX2 expression?
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what does changing the levels of FGF8 expression that is normally in the rostral pole of the cortex do?
changes the areas devoted to each region eg the auditory and visual region or somatosensory cortex, decrease- expansion or increase- push away or duplicate it by another FGF8 source
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what does EMX2 eregulate?
areal mapping in the neocortex
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when is EMX2 gene expressed high-caudal to low-rostral?
during cortical development
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what is thought to underlie the ephrin gradient?
the relationship between FGF8 and TMX2 TF
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what kind of maps do ephrin gradients usually underlie?
spatial gradients eg visual or somatosensory things
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what is a non-spatial map?
olfaction (smell)
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why is olfaction not spatial?
can work out where in the env its coming from just about but it's not where the smell is that's important it's what the association with that smell, association becomes important for mapping olfaction
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where are odours detected?
by the olfactory epithelium in the nasal cavity, there are olfactor receptor cells (OR) that are scattered throughout to detect odours
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what are the receptors for odourants?
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how many OR are there?
over 1000+ olfactory receptors- 1000+ genes encoding GPCRs each that detect a different odour
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why do dogs have even more olfactory genes than humans?
smell is even more important for them
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how many olfactory receptors does each olfactory neuron in the olfactory epithelium express?
1 olfactory receptor per neuron
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are receptors for a particular odour grouped?
they are not grouped within the nasal epithlium ORs are scattered throughout but then they become organised by axons from neurons expressing the same receptor all converging onto the same glomerulus in the olfactory bulb
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axons from neurons expressing the same OR go to the same place in where?
the olfactory bulb (they all converge onto one glomerulus)
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how is the olfaction mapping different to reinaltectal mapping?
retinaltectal mapping have a tophographic map that reflects the topological organisation of axons. Here info is scattered in the nasal epithelium and then gathered together onto glomeruli in the OB.
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what are P2 + M12?
different olfactory receptors- is just the naming system
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how did they do experiments that allowed them to visualise that neurons expressing the same receptor converge on the same glomerulus?
they took one of the OR genes and they've knocked into it a bacterial LacZ gene which allows you to visualise it with a blue dye
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what is driving the LacZ gene in the P2 OR gene?
the P2 gene promoter
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normally P2 exon woukd encode the OR coding region and the stop codon at the end means RNA pol falls off why can it re-enter and translate LacZ?
they put an IRE so the ribosome can re-enter and translate the rest of the structure
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does adding LacZ KO p2 gene?
no doesn't KO P2 gene by knocking in LaxZ gene just adding a bit which allows us to see where P2 is expressed
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what did this experiment find?
scattered P2 OR expression in the nasal epithelium but then they all converge onto the a single point on one or 2 glomerulus in the OB
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what gene showed the same distribution?
the M12 gene- scattered throughout olfactory epithelium converge onto glomerulus in OB
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what is this principle?
the one glomerulus-one receptor principle
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what do IREs allow us to do?
express 2 coding regions from one RNA transcript
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what is interesting about the organisation of related odourants in the OB?
glomeruli responding to related to related odourants are clustered in the OB eg cherry and lemon
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does any single odour have one receptor responding to it?
no, an odor can bind to a number of different receptors but them receptors tend to cluster together int he OB
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how is odor wuality encoded by spatial arrangement of xons?
nu where they end up on glomeruli on the OB
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the axons with the same OR go to the same place in the OB but when are the recs turned on?
at an early time when axons are navigating from the epithelium to the OB
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what does this timing suggest?
receptors may play a part in the navigation to the right glomeruli in the OB
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how did they test whether olfactory recs were important in navigation?
messed with the expression of the recs. Replaced the receptor with a diff rec- eg took the P2 promoter and coding regions and using KO techniques replace the P2 coding region with P3 or M71 coding region
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what happens when you do this?
there's a shift in where axons navigate to in a predictable manner- so now go to where the gene you're coding for would go = rec swap experiments demonstrate where sxons go is determined by which receptor is expressed
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what kind of olfactory recs are OR?
GPCRs 7 TM spanning domains
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in the absence of the ligand (odour) each GPCR has what?
a characteristic basal level of activity- they fire at a particular rate without a signal
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what does OR acticity determine?
guidance response state
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what does the basal characterstic level of firing affect?
the amounts of cAMP in the cell. GPCRs -> adenylate cyclase -> increases cAMP
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why is there basal activity?
normally binding ofa molecule to GPCR changes the receptor conformation but in the membrane there's continual molecular flux so GPCRs are moving even when there's no ligand bound
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so each rec generates a characterstic amount of flux that generates what?
a characterstic amount of cAMP that gives a characterstic state of cell in terms of the amount of cAMP
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Neurons expressing the same OR have similar levels of what?
have similar basal cAMP levels (dependent on adenyltate cyclase)
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what does the similar basal level of cAMP signalling determine?
the level of TF in the cell mediated by cAMP response element binding protein CREB -> trasncriptional activation -> sets levels of expression of cues + recs depending on which OR is opresent
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so in different OR neurons you have different what?
variation in the amount of cues and recs being expressed
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what are cues + recs?
type 1 molecules
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what is the result of this?
the type 1 protein levels (recs + cues) are characteristically associated with expression of a particular olfactory rec which in turn determines the mapping to the OB
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what molecule did they show there was a gradient with in the olfactory bulb?
neuropilin 2- gradient across the OB
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what has graded expression in the olfactory neurons and the OB?
neuropilin receptors in the axons and semas3s (ligands) in the OB
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what is the result of a graded expression of recs and ligand?
axons go to the point in the gradient where you have no net attraction or repulsion and will stop there
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what does level of cAMP signalling determine?
the level of trasncription (via CREB) of guidance cues + rec (robos/slits + semas/neuropilin)
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what is the difference between axons with high levels of neuropilin (rec) to low levels?
axon with high neuropilin levels will stop at a lower level in the gradient but an axon with low levels of neruopilin will go higher up the gradient
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what is the result of the recs and cues being expr in a gradient?
you get sorting of the axons according to the combination of receptors and ligands that they're expressing into these different levels and it means you gather together with axons that express the same OR according to the levels you're expr
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what can you do to disrupt where diff OR axons go in the OB?
mess with the level of cues
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what is an example of messing with the cues?
2 olfactory genes different colours put into locci to see where they go- mor128 + Mor10s go to 2 different glomeruli then change neuropilin 2 expr in the mor10 class then they congregate closer to the mor28 area
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what does this tell us about manipulating hthe levels of neuropilin 2?
allows you to push where axons navigate to in the OB
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why is this in some way like the visual system?
have a gradient of cues and recs
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why is the early guidance activity dependent in the olfactory system?
because there is a basal activity of the ORs
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what do we call these cues that are activity independent?
type 1 molecules
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how do we go from continous to discrete maps?
the set of cues used by olfactory neurons change so the ones coming in early are using eg robo + slit to guide but later on they switch to semas and neuropilins to guide which gives broad sorting of axons into diff parts of the OB
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how is this like retinotectal system?
you use gradients to sort them into different areas- diff glomeruli
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what is the effect of the characterstic basal acticity + same level of rec/cue expression?
effect is to put neurons expressing the same OR into the same region of the olfactory bulb before synapsing onto the glomerulus
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what is the final sorting into glomerulu dependent on?
second part becomes activity dependent
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how do we know glomeruli sorting is activity dependent?
if you block it then you affect the final sorting
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what is the effect of activity?
to elevate cAMP levels --? drive higher cAMP levels -> turn on expression of type 2 cues
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what are type 2 cues?
contactin and kirrels or ephrins (mutual repellants)
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what are contactins and kirrels
homophillic adhesion molecules
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what could be used to make axons with the same OR come together?
homophillic adhesion molecules so they bind together to sort them into the same glomeruli
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if you look at expression of diff members of the kirrel family what do you find?
kirrel2 and kirrel 3- see axons expressing the same kirrels end up in the same glomeruli and they don't mix with axons expressing other kirrels
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what else has an expression like this?
ephrins and ephs- this interactions lets us sort out axons and synapse on the same glomerulus
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what is the principle?
we get OR neurons with the same OR synapsing on the same glomerulus
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what are the 2 phases in mapping to the OB?
an early activity independent phase (rec basal activity expr type 1 rec + cues) 2) activity dependent phase
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where does the activity for the later stages come from?
is not clear yet
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so how do axons start off roughly in the same place in the OB but then become congregated specifically in 1 target area?
type 1 molecules sort them roughly into the right place but then type 2 adhesion moelcules bind axons with the same OR together and ephrins and ephs keep diff OR neurons apart
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whgat is the one neuron one olfactory receptor principle?
one neuron is expressing one type of odorant receptor
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what are type 1 molecules?
semas/neuropilins + slits/robos
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what causes the change to type 2 molecules?
cAMP levels change and you turn on diff sets of genes of type 2 molecules (contactins +kirrels)
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where do the olfactory bulb mitral cells go to?
various diff centrs- antenrior olfactor nucleus (AON), the piriform cortex, the entrohinal cortex and the amgydala
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what is the difference between visual info going into the visual cortex and olfactory ingfo going into the piriform cortex?
the projection of mitral cells (OB) axons into the piriform cortex doesn't exhibit any spatial organisation
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how do we know that the projection of mitral cell axons into the cortex doesn't exhibit any spatial organisation?
used transsynpatic tracing- put viruses into the olfactory epithelium that can jump synapses. Viruses carry markers in them to see them which allows you to trace the circuitry to the cortex
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what did they conclude from the transynpatci tracing?
particularly in the piriform cortex the axons from the OB go all over the place with no apparent organisation
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where is this not as true?
amygdala and AON
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what else did they show from this paper?
showed that individual odourants are activating subpopulations of neurons within the piriform cortex- mapping of axons here is reflected in where activation occurs according to the odourant in the olfactory epithelium
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what is the difference between the glomeruli in the OB and the piriform cortex?
different odorants don't activate one kind of rec eg lemon and cherry. In the PC they react to structurally dissimilar odours but in the OB lemon activates a set of glomeruli that are spatially similar to cherry
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how are the odorants grouped at each level?
odorants activate a distributed set of sensory neurons in the nasal epithelium but OR converge to the same glomeruli but these activate a dispersed set of neurons in the PC
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in mammals when do odors drive behaviours?
after learning- the signficance of an odor is learnt by association but at the time this work was done we doidn;t know what brain region was involved in association so what they were trying to find out
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what technique did they use to test if the PC is the site of odorant learning?
optogenetic activation of subsets of PC neurons
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what is channelrhodopsin? why can we use it very specifically?
channelrhodopsin comes from light sensitive bacterium. It's specific as the frequency of light that activates channelrhodopsin does not activate mammalian neurons so can activate just channelrhodopsin
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why did they want to get channelrhodopsin into piriform cortex neurons?
can drive APs in those neurons using light to fire channelrhodopsin and APs
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what did they pair APs (activation of channelrhodopsin) with?
pair the activity with an appetitive or aversive stimulus in naive (unconditioned) animals so we get classic associative learning
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what is classical associative conditioning?
a learning process that occurs when two stimuli are repeatedly paired: a response which is at first elicited by the second stimulus is eventually elicited by the first stimulus alone.
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what is classical associative conditioning in this context?
the light stimulus alone can elicit the appropriate behavioural response
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how did they get channelrhodopsin into the PC? Injected a virus into the PC in 3 diff ways, describe the simplest way?
1) simplest: use synapsin promoter (on in neruons) to drive expression of channelrhodopsin. Channelrhodospin on th same gene as XFP (fluroscent protein)- use IRE to get both proteins expressed from the same RNA. hit 50% of cells at the injection site
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what did they do which involved floxing?
infect a floxed channelrhodopsin into the mouse in which cre is driven by Emx1 promoter which is restricted to excitatory neurons- so now testing whether specifically activity in excitatory neurons is importnat. Emx1 driving cre
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why did they put in an IRE in Emx1?
so that we don't disrupt expression of emx1 but now it can drive cre
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what gene is expressed specfically in exciattory neurons?
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the mouse will make cre in any cell expressing what?
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what happens when they superinfect the virus that contains the floxed version of channelrhodopsin?
we get it turned on
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how come instead of cutting out channelrhodopsin cre turns channelrhodopsin on?
in this case have 2 different lox sites which are in reverse orientations with channelrhodopsin in the middle so because of loxp site orientation flip and turn around
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why does it matter that the loxp sites were facing in?
the net result is when the virus with floxed channelrhodopsin comes in under the control of the Emx1 promoter you drive expression of cre because you flip it around so turing on channelrhodopsin
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what was the last way they got channelrhodopsin in?
infect in a floxed channerhodopsin at the same time as a virus containing synapsin driving cre and a fluroscent protein. (3 flrusocent proteins). cre with EFP and channelrhodopsin-YFP and a cherry marker
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what is the result of this last method?
you turn on expression of channelrhodopsin in synapsin expressing cells but in a much lower % of neurons and they did that deliberately- wanted a lower num of neurons expressing channelrhodopsin
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how did they set the experiment up in the mouse?
mouse has a fibre optic cable on the top of its head- drive a hole through its skull to five fibre optic access to the PC so you can flash light onto the PC after channelrhosopsin infection with a virus
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what was photostimulation of channelrhodopsin expressing neurons in the PC cortex paired with?
paired stimulation of channelrhodopsin with foot shock
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how did they set up the foot shock?
2 sided chamber- only 1 area gives the mouse a foot shock (unconditioned stimulus)
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when did animals exhibit flight response to the foot shock? how many neurons had to be infected with channelrhodopsin 2?
the animals leave the area where they get a foot shock but only when channelrhodopsin was present in the PC neurons and you need at least 200 PC neurons infected with channelrhodopsin to get this response
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how they did they condition the mice?
firstly you do the light flash to stimulate the PC neurons and foot shock together then you show that the light flash alone is enough to induce flight
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how did they apply to this to olfaction?
they did lash flight and odorants together and showed that either photostimulation or odorants could elicit a flight response
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what was the unconditioned stimulus?
foot shock
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what is the conditioned stimilus?
the ligth flash- PC neurons stimulated
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how did they associate channelrhodopsin 2 photostimulation with appetive behaviours?
train the mice to take water in response to an odorant so the mice smell the odorant and take water But if you do that associately with photostimulation then you can re-train them to respond to the light flash
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how did they incorporate female mice into this?
mice trained to associate the presence of a female with an odor and that can be the conditioned stimulus (odor) or photostimulation
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what does this tell us?
you train mice with PC stimulation both aversive (foot shock) or appetitive behaviours (female presence or water) tells you neurons int he PC have enormous plasticity in terms of training, PC neurons are plastic in their associative capability
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how do we know that PC neurons are plastic in their associative capability?
the same set of Chr2 expressing PC neurons can be re-trained in either direction]
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how did they re-train them in multiple responses?
train them initially to the appetitive response to water by photostimulation then you take the same moure (therefore the same set of neurons) and then you can retrain them to associate the foot shock and flight with photostimulation and then back aga
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what does this suggest?
PC neurons are mediating these associative responses- there's a very strong behavioural driver in PC neurons
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how can this get very specific?
distinct set of chr2-expressing PC neurons can be trained and retrained to elicit different behaviours & you can retrain those different behaviours
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how did they show this?
they have 2 different fibre optic cables going into diff regions and you change each one independently& you can retrain and reverse the training independently showing you that diff parts of the PC are capable of diff sets of training
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what is this all telling you?
there's extraordinarly plasticity in the PC cortex = it's a very plastic substrate
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does this prove that the PC is the site of odorant learning?
it shows that PC is plastic and can be used for associative neurons but doesn't prove its necessary for associative odourant learning
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what would prove that PC neurons are necessary for associative learning?
inactive KO PC and show you can't get odourant learning- hasn't been doing
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what shows that the PC is very plastic?
any group of approx 200 neurons can be used to elicit diverse behavioural associations reversibly (can be retrained)
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summary of PC?
its plastic and can demonstrate associative learning but it doesn't prove that its the site of odourant learning
968 of 976
how do we know that the plasticity is a specific property of the PC?
similar experiments done in other regions of the brain (eg somatosensory cortex) elicit a specific behavioural output according to where you are in that (topologically constrained) eg stimulate medial foot sense.
969 of 976
in other regions what are specific behaviours associated with?
specific behaviours associated with specific locations- not PC its distinct in its capabilities
970 of 976
all responses to odourants learned?
no- a small subset of odours elicit innate responses but it is species specific
971 of 976
what is an innate response of an odourant that is specific specific?
trimethyl-thiazole is a compound from fox that elicits fear in naive mice
972 of 976
are projections into the PC random or specific?
973 of 976
what are connections into the amgydala like?
highly specific you see predominance of particular odorant projections from particular glomeruli- particular types, particular glomeruli predominate in the amygdala
974 of 976
what did they show about the connections from the OB into the PC?
conncections from the bulb were dispersed through the PC nad they showed it wasn't steriotypically- the distribution of connections vary from animals to animals (random connections)
975 of 976
what do they suggest about random connections?
these random connections are used to associate odours with particular experiences so can elicit associative behaviours
976 of 976

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Card 2


what do dendrites do?


integrate incoming information, can have multiple inputs into the dendrites

Card 3


what is the difference between axons and dendrites MTs?


Preview of the front of card 3

Card 4


how are MT stabilised?


Preview of the front of card 4

Card 5


what are MAPs in dendrites?


Preview of the front of card 5
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