>Regressive and Progressive Changes during CNS Development>Synapse Loss and Regeneration after Lesion

R. Grantyn

Monday, March 14, 2005 - Afternoon

Developmental PhysiologyJohannes 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) FundamentalNeuroscience, 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

Optimal connectivity requires optimal numbers of connected cell populations.

During development neurons are eliminated by programmed cell death (PCD)

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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

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. Apoptotic MN (*) among healthy neurons

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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.

Developmental stages when PCD occurs

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?

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The normal PCD of spinal motoneuronscan be reduced by treatments that block the activity in the target. For instance, the block of muscle contraction increases the survival of MNs.

What happens if, during PCD, the activity of the target is blocked?

Motoneurons

Activity-dependent release or activity-dependent accessto a neurotrophic substance?

Production/activity-dependent release in the target

Access to the target

Classical neurotrophinhypothesis

More recent experiments pointin this direction

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The survival of a neuron depends on its access to trophic substances*

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Sensory ganglion explant after treatment with nerve growth factor(NGF 100 ng/ml)

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Data from R. Levi-Montalcini

Intracellular signalling pathways of NGF

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Developmental changes in the patterns of afferent distribution in the peripheral

nervous system

Ganglion cells in the rat submandibular ganglion with afferent axons and contacting axon terminals. Camera lucida drawings from >Lichtman JW (1977)

Optimal connectivity requires an optimal distribution of axons.

During development the number of afferent axons per cell is reduced

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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 neuro-muscular junctions. Thus, the transition from multiple to single innervation implies a spatial component.

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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).

Elimination of multiple innervation of striate muscle fibersoccurs both in form of axon elimination (if parent cell dies)or axon branch retraction (if parent cell stays alive)

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Scheme to illustrate the punishment signal concept.A: All receptors are synchronously activatedB: Receptors are differentiatially activated. Theactive 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 punishmentsignal and no synapse is lost.

Synapse elimination reflects competition and is activity-dependent

x

x x

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Developmental changes in the patterns of afferent distribution in the central nervous

system

Ocular dominance columns/stripes can be revealed by anterogradetracing 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)

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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.

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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 subcorticaltarget 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 rostralor 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 retinotopicmap requires neurotransmission.

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In the optic tectum excessive side branches of retinal axons undergo ‚pruning‘.

Pruning is under the control of NMDAR activation

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A: In the mature retina, retinal ganglion cells show diverse anduncorrelated 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 thecolored 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).

Horizontal network activity is a prerequisite for the removal of inappropriate synaptic connections

Neurons that fire together - wire together.

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What happens to the synaptic connections after axotomy?Example: Neuromuscular synapse

What happens when a motor axon is cut?

A: Typical MN in an adult vertebrateB: 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 pre-synaptic terminals may retract, and degenerative changes may occur in pre- and postsynaptic cells.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.

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Schwann cell promote axon re-growth in the peripheral nervous systemAfter 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.

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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.

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Synthesis and distribution of ACh receptors after denervation

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 siteof innervation.

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.

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Control of ACh receptor synthesis by calcium and neural factors

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-3-kinase (PI3-kinase) and ras/mitogen-activated protein (ras/MAP) kinase pathways, leading to expression of AChR α,β,γ,δ and ε subunits.

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What happens if in a motor nerve only a fraction of motor axons is cut?

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.

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What is the synaptic response of central neurons to lesion?

What happens if the axons of sensory neurons are cut at different sites?

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).

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Bridges bypassing the CNS enable CNS neurons to grow forprolonged distances

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

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(B)

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.

What happens in the CNS target if the outgrowth of central axonsis supported by a graft?

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Evidence for axonal sprouting in the hippocampal formation The entorhinal cortex lesion model.

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).

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.

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Granule cell of the fascia dentata=target of the perforant path from theentorhinal cortex

Thus, neighbouring afferents tend to occupy vacant sites on partially de-afferented neurons.

How is the balance between excitatory and inhibitory inputs regulated?

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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

Excessive depolarization (slicing) results in rapid increaseof inhibitory synapse number

0 h 2.5 h after slicing

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Overexpression/silencing of single genes can alter the balance between E and I inputs and thereby cause a disorder of brain development

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Ngn3 =Neurogenin, a bHLH transcriptionFactor;Is normally suppressed by the neurotrophinNGF

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.

To determine the influence of BDNF on the E-I balance of hippocampal neurons

will be the task of today‘s practical course

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