Exam 2: Chapter 5 Flashcards
Axon growth occurs from the
growth cone
How do axons find targets?
Mechanical and chemical cues
Growth cones use ___ to changes shape
mobilization of cytoskeletal proteins
Short distance
interneurons
Long distance
projection neurons
Challenges faced by early axons
find their own path
Challenges faced by late axons
traverse complex environment
Zebrafish: look at axons, what happens
16-36 hr, nothing to a lot, real quick and efficiently
Grasshopper Ti Cells
axons use guidepost cells. If you ablate the guidepost cells, they lose their way
Hibbards Mauthner neurons
rotated salamander hindbrain 180 degrees
Normal: cross midline and go caudal
Rotate Soma 180: go rostral, but then hit the barrier and go back (little loop)
Significance: External cues too, not just intrinsic program
What happens if you cut through a frog tectum as it develops, cutting axon from soma?
still grows for a while, but won’t get to target
Significance: growth cone sufficient for environment response
What is faster: early or late axons?
Late, they have a path to follow
Growth cone shape depends on
filopodia
Growth cone speed (optic tract –> Tectum)
Slows down when it gets to the target (slows for tectum)
Growth cone at target
flattens and collapses
Pioneer (leader) axons
active filopodia
few lamellipodia
elaborate growth cones
Follower axons
simple, bullet shaped, few filopodia
Growth cones are complex at
midline
What happens at growth cone midline crossing?
Leaders become followers
Time lapse imaging
Method using GFP gene: make own floursecent protein, shows axons en route tectum. Watch growth in tissue
How do growth cones elongate?
Material added distally, Ca2+ dependent.
At actin/microtubules at axon tips
Experiment: FRAP fluorescence with bleach
bleach is still as growth cone advances, suggest distal assembly. If it had moved, then it would be soma.
Experiment: beads on axons, actin in axons
Beads: some interstitial growth, they move apart a little
Actin: at tips, goes back into the axon
P-zone
periphery, actin
C-zone
Central zone, microtubues
___ tethers actin/microtubules to use for transport
myosin
Growth Cone Guidence needs ___ and ___ interaction
filopodia and microtubules
Cytochalasin
inhibits filopodia formation
actin depolymerizing agent
Cytochalasin Experiment
Control: axon to tectum
Cytochalasin treated: can’t find tectum
Significance: actin filaments critical for growth cone navigation
How does 1 filopodia move growth cone?
Tension
Myosin pulls actin cables: clutch release mechanism
disconnected tubulin pulled forward
actin added at + end, disassembled at - end.
Axon turns in direction of polymerization and stabilization, as experimentally shown with
taxol
Axon turns away from depolymerization as experimentally shown with
cytochalasin and nocodazole
1) Mechanical Guidance
Physical Aspects of environment
Follows grooves/physical terrain
Mechanical Guidance experiment
cut corpus C.
Can guide around aspar region blocking
Artificial sling can get to other side
2) Adhesive Guidance (CAMS)
Filopodia adherence
Adhesive stuff
L1, polylysine
Non Adhesive stuff
cadherin, laminin, plain glass, plastic
Shape of growth cone on adhesive stuff
flat, slow growth cone
Shape/speed of growth cone on less adhesive stuff
fluffy and fast
Is adhesion enough? Sometimes. What is the systems
Less adhesion: fast
Adhesion: where you wanna go, it’s a balance
test for it by blasting air, see if it will detact
Homologous
Cadherin/Some CAM
Heterologous
Some CAM/Integrins
CAM heterophilic example
TAG-1 in interneurons binds to NrCAM in glial floorplate
CAMs
big protein, membrane bound, gives neurons preferences
Cadherins
Ca2+ dependent, need exact match
Integrins
Alpha/Beta subunits
connect to other stuff like ECM, Fibronectin, and Laminin
Vertebrate CNS adhesive path vs. PNS
CNS: laminin (retinal axons like it)
PNS: fibronectin
Chemotaxis
Molecules attract and repulse
Examples of chemoattraction
Nerve growth factor (NGF)
Example of Chemorepulsion
Semaphorin
Drosophilia Fas2
bundles axons together.
Fas2 in CAM, sticks together
KO Fas2- wandering axons
increase Fas2- stick too long
Significance: on and off selectively
LAMP- CAM in limbic system
needed for limbic connections, if LAMP antibodies in mouse brain, abnormal fiber projection.
Enhances neurite outgrowth
3x IgG domains
Neural Cell Adhesion Molecules (NCAM)
Some internal domains
Extracellular modified by carb residues, reduce adhesion
Nonsialyated with sialic acid
Very adhesive (less is more)
Sialyated
not adhesive
Sialic acids controls
defasiculation of certain nerve pathways
Polysailaic acid = less sticky CAM
Experiment: Endo-N, what does it do?
Digests sialic acid to make things more sticky
Labelled Pathway in the Grasshopper: What happens if P axons ablated?
G can’t follow P anteriorly.
Need P-axons before G crosses midline.
Axons have specific molecules on surface, lay down pathways which help other neurons find their way
Sometimes need to change CAM on surface
CNS: longitudinal tract (Fas2 homolog) Horizontal commissure (Fas1) Allows midline crossing
Looks like a ladder. CAM tells where to cross
Chemorepulsion: what does the semaphoring family do
Repulse DRG neurons to allow targeting
DRG neurons want dorsal
Collapsin is a semaphoring, shown to repulse growth cone
Semaphorin in ventral to keep DRG away
Experiment: DRG and dorsal and ventral tissue
DRG goes dorsal
Experiment: Olfactory and Septum
Olfactory grows away from septum
Experiment: DRG and notochord and dermomytune
DRG grows in between them
Vertebrates: KO Semaphorin
overgrowth in projections from both motor and sensory nerves –> peripheral targets
What determines if a molecule is attractive/repulsive?
2nd messengers
Local Cue test:
1) remove tectum
2) rotate epithelium square
1) still grow towards it
2) correct once they exit the rotated piece
Significance: local cues matter
Ligand/Receptor: Neterin (unc-6)
Receptors:
DCC (Unc-40 in C. Elegans)
Attractive
L/R: Slit
Receptors:
Robo = repulsive
L/R: Wnt
Receptor:
Frazzeled = attractive
Ryk = repulsive
Wnt is pro-
Anterior
Experiment: add cos cells with wnt in wrong place
grow posteriorly instead of anteriorly
Experiment: Ko Frazzeled receptors
not A or P after crossing
If it’s anterior commissural interneurons, what wnt receptors?
Frazzeled
If it’s posterior descending neuron, what wnt receptor?
Ryk
Netrin location
Netrin/Unc-6: midline
Netrin-1: floorplate
Experiment: Unc-6 mutant
growth defect
Experiment: Unc-40 mutant
Can’t orient
Experiment: Netrin-1 KO
Dorsal commissural interneurons can’t get to ventral midline
In vertebrates, DCC gets axons to ____-
Floorplate
Slit is a ligand found in
ventral midline
Olfactory bulb and motor neurons have ___ receptor
Robo, repulsive
Experiment: fuse robo and frazzeled
attracted to slit, repelled by netrin
intra Fraz + Extra Robo
Intracellular domain = attraction driver
Experiment:
1) Commissurless mutant
2) Roundabout mutant
1) no Comm to destroy Robo before crossing, so it never crosses
2) only crosses, nothing to stop robo from crossing
Slit at midline.
Drosophila Axon midline crossing
DCC-netrin gets axon to across
Once netrin leaves, slit sensitive, barrier up
Vertebrate midline crossing:
Types of Robos
Rig 1 = Robo 3 = Comm
Robo 3A = against Robo
Robo1/Robo 3B - repulsive, regular Robo
Vertebrate midline crossing: Before crossing
Robo 3A is high , so inhibits Robo1
Meanwhile, Netrin –> DCC, so attraction
Vertebrate midline crossing: After crossing
Robo 3B is high
Slit binds to Robo 1, which inhibits DCC and causes repulsion
If its in a vertebrate, and never meant to cross
no Robo3A
KO misexpress Robo in vertebrates
Shifts intermediate to medial and vis versa
Drosophila Experiment:
1) normal motor neuron (AVM) with Unc-40
2) Unc-40 and Unc-5
3) Unc-6 Mutant with any Unc-40/5 combo
1) Goes ventral
2) Goes dorsal
3) no preference
Why? unc-5 hates netrin even though unc-40 loves it.
Also: downstream signaling (cAMP levels)
Netrin + DCC = more cAMP, actin polymerizaition, attraction
Low cAMP = depolymerization, now repulsive
Retina –> Tectum Pathway
1) Laminin: force axon in certain direction, low cAMP so repulsive, causes turn for netrin
2) Slit/Shh repulsive, but leaves chaism as pathway for dorsal/nasal axon to cross. Slit is guiding RGC.
3) Ephrin-B attracts to midline
4) Sema3A- repulses to tectum
5) decrease in FGF to show destination reached
6) Gradient of eph/ephrin/wnt = where to plug in
Axons vs. Dendrites: Semaphorin
Axon- repelled (white matter)
Apical Dendrite: attracted
+cGMP = Sema attractive now
If you cut axon process on developing neuron
another minor process will replace if done early enough
Axon proteins
viral HA, HaemR1-distal, GAP43, Tau
Dendrite Proteins
GluR1, HA with GLuR1 C-terminal Tag, HaemR1 and HaemR1-proximal, MAP2
