Download Synaptogenesis

>Regressive and Progressive Changes during CNS Development
>Synapse Loss and Regeneration after Lesion
R. Grantyn
Monday, March 14, 2005 - Afternoon
Developmental Physiology
Johannes Müller Center for Physiology, Charité, CCM
http://www.charite.de/ch/physio/sep/access.htm
Recommended reading:
1) Kandel ER, Schwartz JH, Jessel TM (2000) Principles of Neural Science, McGraw-Hill, ch. 55,
pp. 1087-1114.
2) Squire LR, Bloom FE, McConnell SK, Roberts JL, Spitzer NC, Zigmond MJ (2003) Fundamental
Neuroscience, Academic Press, ch. 19, pp. 525-532, ch. 20, pp. 533-554.
3) Nicholls JG, Martin AR, Wallace BG, Fuchs PA (2001) From Neuron to Brain, Sinauer, ch. 23, pp.
516-524, ch. 24, pp. 525-548.
Synaptic adjustment to changing neuron numbers
Classical experiments of Victor Hamburger 1934:
Removal of a wing bud leads to motoneuron death.
Conversely, transplantation of a supernumerary limb
rescues a substantial fraction of the motoneurons that
would have otherwise died.
Squire et al 2003-19.1
Optimal connectivity requires
optimal numbers of connected cell populations.
During development neurons are eliminated
by programmed cell death (PCD)
Apoptotic MN (*) among healthy neurons
For review:
>Hamburger V (1992) History of the dicovery uronal death in embryos, J Neurobiol 23:1116-1123
>Oppenheim RW (1991) Annu Rev Neurosci 14:453-501
Squire et al 2003-19.2
Developmental stages when PCD occurs
Once synaptic connections are
stabilized, normal PCD ceases.
Following the period of PCD, axonal
pathways and synaptic connections are
refined by collateral and synapse
elimination.
Red lines: elements to be eliminated.
The functions of programmed cell death during
development
- Removal of cells that have no function
- Removal of cells of an inappropriate phenotype
- Removal of harmful cells (DNA defects)
- Removal of transient guidance aids: Transient targets
- Connectivity matching: Creation of optimal levels of innervation
- Connectivity errors: Removal of neurons with inappropriate connections
What is the mechanism of connectivity-related cell death?
Motoneurons
The normal PCD of spinal motoneurons
can be reduced by treatments that block
the activity in the target.
For instance, the block of muscle contraction
increases the survival of MNs.
Squire et al 2003-19.16
What happens if, during PCD, the activity of the target is blocked?
Production/activity-dependent
release in the target
Classical neurotrophin
hypothesis
Access to the target
More recent experiments point
in this direction
Squire et al 2003-19.16
Activity-dependent release or activity-dependent access
to a neurotrophic substance?
Squire et al 2003-19.15
The survival of a neuron depends on its access
to trophic substances*
Squire et al 2003-19.03
Sensory ganglion explant after treatment with nerve growth factor
(NGF 100 ng/ml)
Data from R. Levi-Montalcini
Squire et al 2003-19.08
Intracellular signalling pathways of NGF
Developmental changes in the patterns of
afferent distribution in the peripheral
nervous system
Squire et al 2003-20.01
Optimal connectivity requires an optimal
distribution of axons.
During development the number of afferent axons per cell is reduced
Ganglion cells in the rat submandibular ganglion with afferent axons and contacting axon terminals.
Camera lucida drawings from >Lichtman JW (1977)
Squire et al 2003-20.05
During development initially overlapping axonal projections segregate
Combining cells from mice in which axons express different fluorescent proteins
(CFP and YFP), the innervation pattern is visualized. During development the
synapses of competing axons segregate from each other at individual neuromuscular junctions.
Thus, the transition from multiple to single innervation implies a spatial component.
Squire et al 2003-20.03
Elimination of multiple innervation of striate muscle fibers
occurs both in form of axon elimination (if parent cell dies)
or axon branch retraction (if parent cell stays alive)
Neuromuscular junction at different stages of development. Red: AChRs labelled with α-bungarotoxin.
Green: motor axons expressing GFP. NMJ1 undergoes transition from multiple to single innervation, as
an axon branch retracts. The eliminated branch terminates in a "retraction bulb".
For details >Keller-Peck... Lichtman JW (2001).
Scheme to illustrate the punishment signal concept.
A: All receptors are synchronously activated
B: Receptors are differentiatially activated. The
active receptors are protected, the inactive ones (x)
not.
C:The inactive receptors are then withdrawn from
the membrane surface, and the corresponding terminal
is also withdrawn.
D: When all receptors are silent, there is no punishment
signal and no synapse is lost.
x
x
x
Squire et al 2003-20.04
Synapse elimination reflects competition and is activity-dependent
Developmental changes in the patterns of
afferent distribution in the central nervous
system
Squire et al 2003-20.07
Ocular dominance columns/stripes can be revealed by anterograde
tracing methods
Ocular dominance columns of the neonatal monkey primary visual cortex,
at the level of layer 4c,
revealed by injecting [3H]proline into the vitreous body of one eye.
Light stripes represent the anterogradely transported label
from the injected eye. Dark regions are occupied by axons driven by the other eye.
From >Hubel T et al (1971)
Squire et al 2003-20.06
The segregation of axons of the visual pathway is the basis for
the formation of ocular dominance columns
In the immature CNS the visual projections from the left and right eye overlap.
During development, the afferents segregate in the dorsal lateral geniculate
nucleus (dLGN) and in layer 4c of the visual cortex.
In the mature visual system RGCs from each eye connect different cells in the dLGN and the visual cortex.
Thus, pathways representing the left and the right eye separate spatially and functionally.
A
B
C
B: In the mature rat visual system,
the axon terminals of temporal (T)
retinal ganglion cells are highly
restricted to the rostral (R) part of
the superior colliculus, a subcortical
target of the ganglion cells.
Conversely, the terminal arbors of
nasal (N) axons occupy predominantly
the caudal (C) part of the colliculus.
A: At earlier stages of development,
however, individual temporal and nasal
axons are not confined to the rostral
or caudal parts of the colliculus, but
instead elaborate many side branches,
which are eliminated with maturation.
C: Blockade of postsynaptic activity
by an in vivo infusion of D-APV, an
NMDA receptor antagonist, prevents
the elimination of side branches at
inappropriate sites.
Thus, sharpening of the retinotopic
map requires neurotransmission.
Squire et al 2003-20.09
In the optic tectum excessive side branches of retinal axons
undergo ‚pruning‘.
Pruning is under the control of NMDAR activation
A: In the mature retina, retinal ganglion cells show diverse and
uncorrelated patterns of action potential activity. Action
potentials (vertical lines) of three cells are schematized here.
B: Before eye opening, retinal ganglion cells generate rhythmic
bursts of action potentials that are synchronized between
neighboring cells. The synchrony is not perfect, as shown by the
colored spikes at an expanded time scale. This is because the
activity propagates across the retina, during which some ganglion
cells are activated before others. A wave of activity (W1) is
evident when calcium indicator dyes are used to monitor spike
activity (colored image). In this example, the wave propagated
from green to yellow to red cells (images obtained once every
second).
Neurons that fire together - wire together.
Squire et al 2003-20.11
Horizontal network activity is a prerequisite for the
removal of inappropriate synaptic connections
What happens to the synaptic connections after axotomy?
Example: Neuromuscular synapse
A: Typical MN in an adult
vertebrate
B: After axotomy, the distal
segment of the axon degenerates.
Schwann cells dedifferentiate,
proliferate, and,
together with invading microglia
cells and macrophages, phagozyte
the axonal and myelin remnants.
The axotomized neuron may
undergoe chromatolysis, the presynaptic terminals may retract,
and degenerative changes may
occur in pre- and postsynaptic
cells.
Nicholls et al 2001-24.3
What happens when a motor axon is cut?
C: The axon regenerates along the
column of Schwann cells within
the endoneural tube and sheath of
basal lamina that had surrounded
the original axon.
After axotomy, the distal
portion of the axon and
the myelin degenerate and
are phagocytized. Schwann
cell proliferation is
stimulated by two
cytokines: leukemia
inhibitory factor (LIF)
from macrophages and
Reg-2 from axon terminals.
Expression of Reg-2 is
enhanced by LIF.
Proliferating Schwann cells
synthesize two
neurotrophic factors,
BDNF and NGF, which bind
to the low-affinity
neurpotrophin receptor
p75 and help sustain
regenerating axons and
guide them to their
targets. Schwann cells and
macrophages also
synthesize apolipoprotein
E (ApoE), which may help
promote neuron survival
and axon regrowth.
Nicholls et al 2001-24.11
Schwann cell promote axon re-growth in the peripheral nervous system
Nicholls et al 2001-24.4
New ACh receptors appear after muscle denervation
Application of ACh ito a cat striate muscle after
transsection of its motor axon reveals changes in
the ACh-induced potentials.
A: Experimental paradigm
B: In a muscle fiber with intact innervation, a
response is seen only in the vicinity of an endplate.
C: After 14 days of denervation, a deafferented
muscle fiber responds to ACh along its entire
length.
A: Fetal muscle fiber. mRNAs for
the α2,β,γ,δ subunits of the AChR are
expressed in nuclei all along the
length of the myofiber. The
embryonic α2βγδ of the receptor is
found over the entire surface of the
myofiber and accumulates at the site
of innervation.
Nicholls et al 2001-24.4
Synthesis and distribution of ACh receptors after denervation
B: In adult muscles, mRNAs for the
α,β,δ and ε subunits are expressed
only in nuclei directly beneath the
end plate. The adult α2βδε subunits
of the ACh receptor are highly
concentrated at the junctional folds.
C: In denervated adult muscles,
nuclei directly beneath the endplate
express α,β,γ,δ and ε subunits; all
other nuclei re-express the fetal
subunit pattern of α2βγδ. Embryonic
AChRs are found all over the surface
of the myofiber, producing
denervation supersensitivity. The
adult form of the receptor is
restricted to the end plate region.
D: If denervated muscles are stimulated, the AChR pattern resembles that of innervated muscle fibers.
In extrasynaptic regions of a vertebrate
skeletal muscle fiber, influx of calcium through
voltage-activated Ca channels (VACCs) activates
protein kinase C (PKC), which phosphorylates and
inactivates myogenin. This keeps the production
of extrasynaptic AChRs low.
At the synapse, the neuregulin ARIA is released
from nerve terminals and interacts with erbB2/3
receptors. This activates phosphatidylinositol-3kinase (PI3-kinase) and ras/mitogen-activated
protein (ras/MAP) kinase pathways, leading to
expression of AChR α,β,γ,δ and ε subunits.
Nicholls et al 2001-24.7
Control of ACh receptor synthesis by calcium and neural factors
A: Normal pattern of innervation of a mammalian
skeletal muscle
B: Some fibers are denervated by cuting a few of the
motor axons
C: Axons sprout from the terminals and from nodes
along the preterminal axons of undamaged motoneurons
to innervate the denervated fibers.
D: After 1-2 month, sprouts that have contacted
vacant end plates are retained, while other sprouts
disappear.
Nicholls et al 2001-24.9
What happens if in a motor nerve only a fraction of motor axons is cut?
What is the synaptic response of central neurons to lesion?
A: Normal state.
B: Different sites of axonal lesions.
C: Extent of regeneration. Axons of dorsal root
ganglion neurons and motoneurons regenerate through
lesion sites in the peripheral nerves (blue). However,
regeneration of dorsal root axons stops when they
reach the astrocytic processes that delimit the
surface of the spinal cord. Axons of dorsal root
ganglion neurons also do not regenerate through the
glia scars that form at lesion sites in the CNS white
matter (red).
Nicholls et al 2001-24.16
What happens if the axons of sensory neurons are cut at different sites?
The grafted bridge consists of
adult rat sciatic nerve in which
axons have degenerated, leaving
Schwann cells. These act as a
lane along which central axons
can grow.
A: Sites of insertion of the
graft.
B: Regenerating neurons are
labelled by injecting a tracer
substance into the graft.
for more information see
>Lund R
>Aguayo AJ
>Schwab ME
>Björklund A
Nicholls et al 2001-24.17
Bridges bypassing the CNS enable CNS neurons to grow for
prolonged distances
A: The optic nerves were cut, and one was replaced by
an ischiadicus bridge (yellow). Regeneration was
tested by injecting the tracer [3H]HRP into the graft,
or recording field potentials in the superior colliculus
in response to light.
B: EM image showing a [3H]HRP-labelled synaptic
terminal with round vesicles forming an asymmetric
contact.
(B)
Nicholls et al 2001-24.18
What happens in the CNS target if the outgrowth of central axons
is supported by a graft?
Evidence for axonal sprouting in the hippocampal formation
A: A typical granule cell in the
dentate gyrus receives dense
synaptic input from the ipsilateral
entorhinal cortex, and sparse input
from the contralateral cortex and
medial septum on its dendrites in the
outer molecular layer (OML).
Granule cell of the fascia dentata
=target of the perforant path from the
entorhinal cortex
B: Following ablation of the
ipsilateral enthorhinal cortex, axons
from the contralateral entorhional
cortex sprout extensively and
replace the ipsilateral entorhinal
input in the OML.
C, Example of a terminal arbor of an
axon from the contralateral
entorhinal cortex
D: Same after lesion of the
ipsilateral entorhinal cortex.
Thus, neighbouring afferents tend to occupy vacant sites on partially de-afferented neurons.
Nicholls et al 2001-24.10
The entorhinal cortex lesion model.
How is the balance between
excitatory and inhibitory inputs regulated?
Turrigiano & Nelson (2004) Nature Neurosci Rev 5:97
The activity of CNS neurons displays homeostatic plasticity
- Chronic (2 d) deactivation leads to up-regulation of discharge
- Chronic activation (block of inhibition) leads to down-regulation of discharge
0h
2.5 h after slicing
Meier, Akyeli, Kirischuk, Grantyn (2003) Mol Cell Neurosci 23:600
Excessive depolarization (slicing) results in rapid increase
of inhibitory synapse number
Ngn3 =
Neurogenin, a
bHLH transcription
Factor;
Is normally
suppressed by
the neurotrophin
NGF
Salama-Cohen, Arevalo, Grantyn, Rodríguez-Tébar (submitted)
Overexpression/silencing of single genes can alter the balance between
E and I inputs and thereby cause a disorder of brain development
To determine the influence of BDNF on the E-I balance
of hippocampal neurons
will be the task of today‘s practical course
Singh, Henneberger, Meier,Arevalo, Rodríguez-Tébar, Grantyn (submitted)
Brain-derived neurotrophic factor (BDNF) is known to be epileptogenic.
We have recently found out that it also changes the balance between
E and I synapses.