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Axon Outgrowth and Pathfinding
Additional notes
Growth Cones
• Nerve cell processes (both axons and
dendrites) grow specialized terminations,
called growth cones.
• This is the site from which neurite extensions
arise.
• Note the appearance in cortex and extensions
(lamellopodia, filipodia (finer).
• These extensions are filled with organelles
(e.g., mito, SER, vesicles), because synthesis of
new membrane is occurring rapidly.
Growth Cones (cont’d)
• Growth occurs distally (at the end):
cytoskeleton, microtubules, filamentous actin
and neurofilaments are important
components with the “+” ends (i.e., tubulin
polarizing) oriented distally.
• Tubules: provide highway for fast transport of
materials where needed (end).
• Fibriolar actin network: changes shape, which
drives the outward movement. (and
polymerization).
Mechanism of Advance
• Constant actin polymerization at the edge 
push forward with rearward flow of
polymerized actin and depolymerization
(“caterpillar tread”).
• This process can occur only on a permissive
substratum. These are…
- ECM molecules (e.g., laminin-1, fibronectin).
- Cell surface molecules (e.g., cadherins).
These substratum bind to cell surface receptors
(integrins), which link to cellular cytoskeleton.
Guidance Cues for Growth Cone Advance
and Axon Outgrowth
• The movement of growth cones is guided by both
attractive and repulsive cues, which either activate or
inhibit actin polymerization or the attachment of cell
surface receptors to this substrata.
• Two early theories:
i. Axons advance more or less randomly and were
“fine-tuned” as they reached their proper target
(either repelled or attachment strengthened).
ii. Axons grow along very pre-set (stereotyped)
trajectories and growth is highly directed and precise
throughout (not just at the target).
The latter has been most supported by research over the
last 50+ years.
Stepwise Migration
• How do axons make such a long journey (in
some cases, several cm)?
• Much like the way we tackle the material in
this class: breaking the process up into smaller
steps.
• So, the pathway they follow is divided into
discrete segments, bounded by guidepost
cells, which provide important guidance
information.
• What kind of study may have informed us
about the function of these cells?
• Answer: Ablation studies.
• In the development of nerve tracts, earlier
extending axons (“pioneers”) can serve as a
simple scaffold for the rest, simplifying and
accelerating the process.
Attractive and Repulsive Cues
• How do axons navigate each segment?
• Both local and long-range cues exist (analogous to
endocrine and paracrine signals), which act in concert
e.g., what keeps neural crest cells migratory in the
anterior compartment?
answer: repulsive cues are sent out from posterior
compartment cells “hem in”.
What are these repulsive cues?
Still an active area of research…examples can be
inhibition of actin polymerization or cell surface
receptor binding.
Note: repulsive cues can be strong enough to Δ the
morphology of growth cones ”growth cone collapse”.
• Long-range cues: Chemotrophic cues.
e.g., chemoattraction: diffusible factors secreted by
targets of the depolarizing axon, as in “come
hither”.
Cortex cell studies have provided much information
about this phenomenon;
e.g., work with NGF.
e.g. of chemorepulsion: a “push-from-behind” or
“get away” secreted by a group of cells near the
cell body propels the axon away.
Chemorepulsion can also deflect axons away from
cell groups they should avoid.
Molecules Mediating this Guidance
1. Local
a. ECM molecules; e.g., laminin-1 – a potent
stimulator of axon extension - a “positive
corridor” for several axons, as well as for
neural crest cells.
Others: fibronectin, tenascin.
Integrins are the major receptor type, present
on axons, which modulate inhibitory effects;
e.g., proteoglycans, tenascins (specific signal
transducing apparatus of the cell).
b. Cell surface molecules from local cells.
e.g., those from IgG genes, such as NCAM, bind to specific
homologous molecules on the surface of the axon and
allow it to adhere and extend.
e.g., cadherins
2. Long-range
Semaphorins: secreted proteins + transmembrane
domains; a repellant that leads to growth cone collapse .
Netrins: contributes a chemotropic (attractant) activity, as
well as repellant (push from behind) in specific situations.
Structure is highly analogous to ECM molecules;
so, distinction between long-and short-range cues is not
sharp.
Honing in on the Final Target:
Positional Information
• Recent active research has revealed that the
early theories of gradients of positional
information (which we know to be important
in establishing A/P polarity in neuronal
migration) appear to apply to the formation of
the precise topographical patterns of axonal
connections with their targets.
• One candidate: Ephrins (still an active area of
research)
Growth cones guide axons in the developing
nervous system
The structure and action of growth cones
The structure and action of growth
cones
[Ca2+]intracellular
The major classes of axon guidance molecules
(secreted and
transmembrane)
Signals in embryo affect growth cones and growing axons
Netrins chemotropically regulate pathway formation in a
developing spinal cord
wild type
netrin knockout
Mechanisms of topographic mapping in the vertebrate visual system
or
Nasal
or
Temporal
Mechanisms of topographic mapping in the vertebrate visual
system
(Anterior)
(Posterior)
Target-derived trophic support regulates survival of related neurons
Changes to synapses during early postnatal life in the
mammalian PNS
Effect of the neurotrophin NGF on the outgrowth of neurites and survival of
neurons
24 hr after
addition of
NGF
8 day chick sensory ganglion
without NGF
9 day mouse
embryo
sympathetic
ganglion
treated with
antibodies
to NGF
Neurotrophin receptors and their specificity for the neurotrophins
Signaling through the neurotrophins and their receptors
Hardwiring the Brain:
Endocannabinoids Shape Neuronal Connectivity
Paul Berghuis, Ann M. Rajnicek, Yury M. Morozov, Ruth A. Ross, Jan Mulder, Gabriella M.
Urbán, Krisztina Monory, Giovanni Marsicano, Michela Matteoli, Alison Canty, Andrew J.
Irving, István Katona, Yuchio Yanagawa, Pasko Rakic, Beat Lutz, Ken Mackie, Tibor
Harkany
The roles of endocannabinoid signaling during central nervous system development are unknown. We
report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of GABAergic
interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization
and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these
GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of
Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical
GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and
demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.
Science 25 May 2007
Vol. 316, pp. 1212 - 1216
Background
• Axon guidance is crucial to the embryonic wiring of the cerebral cortex
• G protein-coupled CB1 cannabinoid receptors respond to marijuana and
to endocannabinoids in the adult
• Endocannabinoids released from postsynaptic cells retrogradely suppress
transmitter release from presynaptic cells in adults
• CB1R are expressed on developing axonal projections, but whether
endocannabinoids are axon guidance molecules was unknown
The Approach
• Determine the spatial and temporal expression of CB1R and
endocannabinoids in vivo
• Examine the effects of CB1R agonists and antagonists on neurons in culture
• Analyze innervation of neurons in vivo in CB1R knockout mice
Figure 1
Temporal and spatial coincidence of CB1R localization
with endocannabinoid availability during corticogenesis
hippocampus
R
E
C
E
cerebral cortex
pyramidal neurons
hippocampus
P
T
O
R
S
GABAergic interneurons
DAGL and NAPE-PLD
are synthetic enzymes
for endocannabinoids
--------------- cerebral cortex ---------------
L
I
G
A
N
D
S
7 µm
pyramidal neurons
GABAergic interneurons
Figures 2&3
Agonists induce CB1R removal from filopodia of
GABAergic neurons in culture and cause repulsive turning
control
WIN 55,212-2 is a CB1R agonist
AM251 is a CB1R antagonist
AEA is a CB1R agonist
Physiological importance of CB1R-mediated growth cone repulsion:
gene knockout alters distribution and density of inhibitory innervation
wild type
knockout
Hardwiring the Brain:
Endocannabinoids Shape Neuronal Connectivity
Paul Berghuis, Ann M. Rajnicek, Yury M. Morozov, Ruth A. Ross, Jan Mulder, Gabriella M.
Urbán, Krisztina Monory, Giovanni Marsicano, Michela Matteoli, Alison Canty, Andrew J.
Irving, István Katona, Yuchio Yanagawa, Pasko Rakic, Beat Lutz, Ken Mackie, Tibor
Harkany
Science 25 May 2007
Vol. 316, pp. 1212 - 1216
Conclusions:
1) Endocannabinoids and their receptors are in the right place in vivo, they affect
growth cone turning in vitro, and they affect subsequent innervation in vivo.
2) Endocannabinoid signaling regulates growth cone steering and thus axon guidance.
3) Prenatal exposure to cannabis could affect neuronal development and account for
cognitive and behavioral deficits.