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Target Selection
Chapter Six
Axon Growth And Guidance
Axonal growth dependents on the
a ability of the growth cone to
navigate in a very complex
environment
Axonal growth occurs when the
axon encounter the appropriate
environment generated by
adhesive and extracellular matrix
molecules, as well as diffusible
signals that may promote axonal
attraction or repulsion
From Neuron to Brain, IV ed.
Target Selection
How does the growth cone choose one or
two target cells out of thousands of
similar cells in a region?
1. Axons Defasiculate
2. Axons enter target location
•
Based on growth signals and repulsion
3. Axon terminals slow down their growth
and branch in different directions
Target Selection
4. Different strategies get axon to target:
a) Molecular barriers block alternative routes
b) Topographical maps have predetermined
physical locations
c) Axons find appropriate 3-D layer
5. Axon chooses specific dendrites
6. Nervous system tests connections
1. Keeps connections that work
2. Remove connections that don’t
Target Selection
1. Defasiculation
In Drosophila, each segmental
ganglion contains ~40 neurons
that innervate 30 different types
of muscles. How does each
growth cone know what muscle
to innervate?
It appears that each muscle fiber
has a variety of adhesion,
growth-promoting and repulsive
signals that allow proper axonal
projection
1. Defasiculation
In Drosophila, a variety of different
target-derived molecules regulate
fasciculation including:
1) Netrin
2) Connectin (mediates homophilic
cell adhesion)
3) Fasciclin II and III (mediates
homophilic cell adhesion)
Disruption of normal pattern of
netrin, connectin and fasciclin
expression disrupts normal target
projection in the fly
From Neuron to Brain, IV ed.
1. Defasiculation
• Beat 1a = anti-adhesion factor
• Without it axons fail to defasiculate
• Mutants can be corrected by removing adhesive
factor (Beat 1c) or by decreasing adhesion in general
2. Target Recognition
• Target cells can be:
– Organs, sensory cells, muscles, other
neurons, etc
• Signal is usually derived from target tissue
itself
• Example – Neurotrophins
– Derived from target growth factors
– Attract neurons into target tissue
2. Target Recognition
• Blood vessels release NGF
– Neurotrophin
• Axons grow along vasculature
• In the absence of NGF
– Axons fail to enter (innervate) target tissues
• If NGFs are overexpressed
– Get increase in innervation
• GDNF (another neurotrophin) attracts cRet expressing neurons
Role of Growth and Neurotrophic Factors in
Target Selection
In the mouse, lack of expression of
the neurotrophin NT3 in the ears
prevent sympathetic neurons from
innervating the pineal gland and the
external ear
Application of exogenous NT3 can
restore normal sympathetic
innervation in NT3 knockout mice
2. Target Recognition
In Drosophila, the TN nerve normally
does not innervate muscle fibers 6 or
7, whereas fiber RP3 innervates both
If FasII expression is increased in
muscle fiber 6, both TN and RP3
nerves project to that fiber and that
fiber only
2. Target Recognition
Decreased expression of Netrin or increased
expression of semaphorin II (a repulsive molecule)
prevents normal projection of RP3 nerve to fibers 6 and
7
2. Target Recognition
Increased expression of Netrin or FascII in both
muscles result in TN nerve innervating both muscle
fibers
The Net/FascII-induced projections of TN nerve to
fibers 6 and 7 can be prevented by semaphorin II (a
repulsive molecule) expression in both muscle fibers
3. Slowing down and branching
Growth cones change when
they enter the target. Once
they enter the target, growth
cones become more
complex structurally and
slow down in order to
sample the environment
For example, retinal
ganglion cells grow at a rate
of 60 um/h in the optic tract,
but slow to around 15 um/h
as they enter the tectum
3. Branching
• Axons slow and begin to branch when
they enter target area
3. Branching
• Branching of axons begins behind the
axon’s tip
• Seems to be a connection between the
axon stopping and branching occurring
– Branches occur at pause points
• In vitro observations have seen that
branching occurs whenever axon is
repulsed and collapses
– Cytoskeleton must be fragmented at this
position
3. Branching
• Link between repulsion and branching
4a. Molecular Barriers
• Repulsion set up at the boundaries of a
target tissue
• This can do two things:
– Block exit of target area
– Corral axon to correct target synapse
• Examples of repulsive signals:
– Sema3A
– EphrinA5
4a. Molecular Boundaries
• Experimentally remove the repulsive
signal
• Axons will incorrectly innervate different
target
4a. Molecular Boundaries
• Repulsive signal coming from target tissue
• Mouse will “hear” the visual world now
4b. Topographic Maps
• The spatial positioning of neurons are
mapped in one very specific set of
locations in the brain
• The starting position of the innervating
neuron will determine the position of it’s
terminal within the target
• Topographic maps exist to set up an
continuity of visual (or auditory) space in
three dimensions
Topographic Maps
Topographic maps are
present in the cerebral
cortex, spinal cord, cochlea,
visual system and others
Layer structure of the lateral geniculate nucleus
From Neuron to Brain, IV ed.
Topographic Maps
Topographic maps are
orderly representations of
some physical properties
(like sensory, motor, visual
information) of the world
Layer structure of the lateral geniculate nucleus
In the visual system, retinal
ganglion cells send axons to
particular layers of the
thalamus and the primary
visual cortex
From Neuron to Brain, IV ed.
How Are Topographic Maps Generated?
Stimulate rostral
preganglionic roots of the
superior cervical ganglia
(SCG) stimulate pupil
dilation
Stimulate more caudal
preganglionic roots of SCG
stimulate vasoconstriction of
blood vessel in the ears
Neurons must encode
rostral-caudal origins and
innervate accordingly
Topographic
organization of the SCG
How Are Topographic Maps Generated?
Cutting the sympathetic
axons above T1 eliminate
all reflexes. However, with
time these reflexes recover
and involve the same spinal
segments
This suggests that
preganglionic fibers
innervate appropriate SCG
neurons in such a way that
the topographic map is
conserved
How Are Topographic Maps Generated?
Transplantation experiments
also indicate that
transplanted ganglia receive
the right projection from the
corresponding spinal cord
segment
Transplantation of a T5
ganglion to more rostral
locations does not prevent
T5 from receiving axonal
projections from more distal
spinal cord segments
Visual Maps and Chemospecificity
Rotation of the eye 180 degrees
results in the formation of an
inverted image in the frog’s brain
because dorsal (or ventral) axons
always project to one specific area
of the tectum
It appears that retinal fibers are
guided to the tectum by
biochemical tags present across
the retina and the tectum. These
are based on a chemical gradient
of factors across the tectum
Visual Maps and Chemospecificity
• Therefore the spectral coordinates of the
visual field are mapped onto specific brain
regions
• Occurred based on how retinal axons
innervated the tectum
• Innvervation was controlled by a chemical
gradient of some sort
– Stamps each target cell with exact latitude
and longitude
• Stripped carpet
experiment
• Retinal axons
grow on
Anterior cell
types
• Nasal axons
are insensitive
• Cells are denatured or treated with PLC to
remove membrane bound proteins
• Now retinal axons will grow anywhere
– Therefore signal must have been repulsive
4b. Topographical Mapping
• Two repulsive signals were isolated from
stripped carpet experiment
– Ephrin A5 and Ephrin A2
• Both are expressed posterior (high) to
anterior (low)
• Repulse the retinal axons Æ pushing them
towards the anterior cell types
• Thereby producing the start of a
topographical map of the visual system
Ephrins
Ephrins are divided into two
family Ephrin A and B that
interact with Eph receptor
tyrosine kinases
Ephrin A family ligands are
guanosyl-phosphatidylinosito linked receptors
Ephrin B family ligands are
membrane bound (involved
in the formation of blood
vessels)
Holder & Klein, 1999
Ephrin A5, a ligand
for the Eph receptor
tyrosine kinases has
a graded expression
in the superior
colliculus and is
involved in the
formation of
topographic maps
In mice lacking Ephrin
A5, axons project to
the posterior
colliculus
Frisén et al., 1998
4b. Topographical Mapping
• Visual map based on Ephrin expression
• Wild type:
– There is one center were all RGCs go within
the tectum
• In double knockout of Ephrin A2 and A5:
– No center and no vision
• If Eph receptor is overexpressed:
– Two centers and double vision
4b. Topographical
Mapping
Reciprocal Expression
• Growth cones can regulate the amount of
receptor that they express
• Thereby controlling their response to the
amount of ligand in the environment
• EphB receptors expressed in gradient:
– Ventral (high) to dorsal (low)
• EphrinB ligand expressed in gradient:
– Medial (high) to lateral (low)
Reciprocal Expression
RGC axons travel from dorsal retina to lateral tectum
And from ventral retina to medial tectum
Forward vs. Reverse signaling
• Reverse signaling
– When the ligand expressed from the axons
– Attracted to the receptor expressed in the
target tissue
– Xenopus example
• Forward signaling
– When receptor expressed on axons
– Attracted to the ligand expressed in the target
– Mouse example
Reciprocal Expression
Reverse signaling – ligand in axons go to receptor
Forward signaling – receptor in axons goes to ligand
Reciprocal Expression
• This reciprocal expression of Ephrin
receptors and ligands seems to be a
consistent model of setting up topographic
maps
• Used in:
– Retina cells – forming visual map
– Body surface – forming somatosensory map
– Cochlea cells – forming auditory map
Visual Maps
There is a somatotopic
representation of retinal
axons in the tectum:
z Dorsal axons project to
more ventral areas of the
tectum
z Ventral axons project to
more dorsal area of the
tectum
z Anterior axons project to
posterior areas
z Posterior axons project to
anterior areas
Somatotopic Maps in the Cerebral Cortex
Mapping experiments
indicate that each part of
the body is represented in
the primary sensory and
motor cortex in a very
exact location
Topographic map
Each body part (such as
lips, back, eyes) is
represented to different
degrees.
Somatosensory Map
• Some locations on the somatosensory
map are enlarged
• Because these areas of the body have
more innervation than others
• Example in humans:
– Lips, fingers, tongue have many sensory
neurons
• Locations are flexible
– Due to injury or activity
Somatotopic Maps in the Cerebral Cortex
Somatotopic representation in
the cortex is very plastic and
depends on the level of use of
a particular body part. This
plasticity is particularly evident
during early periods of
development but can occur in
the adult brain as well
Disuse or surgical removal of
a particular area of the body
can result in rearrangement of
the somatotopic maps
Specificity of Somatopic Maps Is Also
Evident in the Cortex
The cerebral motor cortex is
topographically connected to
motor neurons targets in the
spinal cord.Particular areas of
the motor cortex project to
cervical and hindlimb spinal
cord segments
This specificity in the projections
is maintained in vitro. Cultured
explants from forelimb cortex
prefer to send axons into
explants of the cervical cord
Shifting Connections
The brain is plastic and can shift or fine tune
these connections
1. Testing branches:
•
Maintain correct branches and remove
ectopic branches
2. Adjust with levels of activity
•
As seen in somatosensory map and
amputation
3. As brain grows – axons can expand
Activity
Mouse barrels in the
cortex of the mouse
brain
Changing due to
activity of the whiskers
Growth
A = Normal growth
B = Compression with lost target
C = Expansion with lost axon
Any Questions?
Read Chapter Six