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Development and Remodelling of NMJ

Remodelling Neuromuscular SynapsesRemodelling Neuromuscular Synapses

Synapse formation

Presynaptic

Postsynaptic

2

Synapse elimination

J.F. Tello Polyneuronal innervation in fetal human muscle

(1917)

PI= [(A+B)-AB]/A

AB

AB

A B A BPI= AB/A

Physiological methods of measuring PI

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Loss of motor neurones?Or elimination of connections?

Loss of motor neurones..XElimination of connections ✓

Keller-Peck, C. et al.(2001) Neuron 31,381-394

Neurones retract some of their synapses while stabilising others

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Nguyen QT, Parsadanian AS, Snider WD, Lichtman JW (1998) Science 279:1725–1729.

Transgenic expression of a growth factor, GDNF, delays elimination

Monoinnervated NMJ’s are stable throughout life

Balice-Gordon & Lichtman (1990) J Neurosci 10, 894

Quantal Content (variancemethod) at NMJ of rat HD

0 100 200 300 4000

50

100First EPPPlateau EPP (10 Hz)

Age

(Based on Kelly & Roberts, 1977 and Kelly, 1978)

Synapse elimination is competitive: or is it?

MPN LPN SN

4DL

5

~ 1month after partial denervation

Compare motor unit sizes at the time of partialdenervation and a few weeks later

neonateBetz,W.J.,Caldwell,J.H. & Ribchester,R.R.(1979). The size of motor units during postnataldevelopment of rat lumbrical muscle. J.Physiol. 297,463-478.

Fladby & Jansen (1987) Acta Physiol Scand. 129,239-246.

Betz, Caldwell & Ribchester (1980) J.Physiol. 303,265-279

“Competition” or “Intrinsic Withdrawal”? We can determine motor unit size in thy1.2-YFP mice bycounting the number of muscle fibres that are innervated

Adrianna Teriakidis

Neonate~ 1 month after

partial denervation

Adrianna Teriakidis

Motor unit sizes 2 days after partial denervationare larger than motor unit sizes 4-6 weeks later.

Adrianna Teriakidis

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Synaptic regeneration: a recapitulation of development

Remodelling Neuromuscular SynapsesRemodelling Neuromuscular Synapses

J. Boeke, 1916

π

Sprouts

Axon

10 µm

µ

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J.A.Barry & R.R. Ribchester (1995) J.Neurosci. 15,6327-6339

FM1-43

RH414

Polyneuronal innervation in reinnervated muscle

MPN LPN SN

4DL

Ribchester (1988) J Physiol. 401, 53-75

WallerianDegeneration

X X

Collateralsprouting

Regeneration Synapseelimination

π µ

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Axonal sprouting is preceded by Schwann cell sprouting

Son et al (1996) TINS 19,280F.Court et al. J Cell Sci 121,3901-3911

Four cell-types at mammalian NMJ

NFSV2/AChR S100/AChR

Kranocytes sprout before terminal Schwann cells

Felipe Court

Paralysis and muscle atrophy provide strong stimulifor kranocyte proliferation and spread

Felipe Court

Synapse elimination is activity-dependent: or is it?

Synapse elimination is influenced by activity

0 10 20 300

20

40

60

80

100StimulationParalysisNormal

Age (days)

9

Inactivity upregulates transmitter release and promotes sprouting

OsmoticPump(i.p.)

Tubing

Lumbrical(4DL)Muscle

MPN LPN Sural Nerve

SN

LPN

L+SCuff

TTX

Ribchester,R.R. & Taxt,T.(1983). Motor unit size and synaptic competition in rat

lumbrical muscle reinnervated by active and inactive motor axons. J.Physiol. 344,89-111.

LPN Blocked Control

TTX

Active motor units have a competitive advantage over inactive units

Is activity sufficient for synapse elimination?

J.A.Barry & R.R. RibchesterJ.Neurosci. 15,6327-6339(1995)

E.M. Costanzo, J.A. Barry & R.R. RibchesterJ.Physiol. 521.2,365-374 (1999)

0 2 4 6 8

20

40

60

0

%Dual

Weeks Recovery

Polyinnervation persists at some NMJ after activity resumes

Brown et al., (1981) J Physiol

π persists in some adult muscles after neonatal paralysis

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Synaptic degeneration

Remodelling Neuromuscular SynapsesRemodelling Neuromuscular Synapses

Augustus Waller, ca. 1860

“Wallerian” Degeneration

24 hrs

Slater, 1966, Nature 209,305-307.

Miledi & Slater, 1970: “It is suggested that end-plate degeneration istriggered by a signal which passes from the site of sciatic nerve injuryto the nerve terminal. The duration of the period after transcectionwhen end-plates appear to be normal would then reflect the timerequired for this signal to travel the length of the isolated nervestump.” J Physiol 270, 508

X X

Gillingwater

Synapses degenerate first in Wallerian Degeneration

24-48h

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Am not I a flylike thee?Or art not thouA man like me?

- William Blake

Drosophila life cycle

Firstinstar

SecondinstarThird instar

Drosophila neuromuscular junction

There are three types of There are three types of boutons boutons or varicosities: or varicosities: IbIb, Is and II. Glutamate, Is and II. Glutamateis the excitatory neurotransmitter mainly released by the type Iis the excitatory neurotransmitter mainly released by the type Iboutonsboutons. Peptides such as . Peptides such as octopamine octopamine are released by the type IIare released by the type IIboutons boutons and they have a and they have a modulatory modulatory role.role.

Anatomy of the Drosophila NMJ

Type II boutons

Type I boutons

Synaptogenesis

During lateembryogenesis themotor neuron growthcone contacts itstarget muscle.Filopodial processesare initially formed butas the junctionmatures, boutons thatresemble those inlarvae becomeapparent

Embryo

3rd instar larva

Watching a Synapse Grow…

By using a non invasive, confocal imaging of synapses in live, Zito etal., 1999 showed that boutons BUD from branches or from pre-existingboutons at the Drosophila NMJs.

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The UAS/GAL4 Binary SystemThe expression of the gene downstream to theUAS is driven by the yeast transcriptionalactivator GAL4 in a developmental and/ortissue-specific manner

~1 mm

Drosophila 3rd Instar Larva : Motor Unit

Soma diameter ~ 10 µm : Volume ~ 500 µm3

Axon length ~0.3-3 mm : Volume ~ 750 - 3000 µm3

NMJ length ~ 100 µm x 3 = 100 boutons: Volume ~ 500µm3

Segment A5: fibre 6/7 NMJSegment A3: fibre 7/6/ 13/ 12 NMJs

VNC

OK6-Gal4:UAS-myr-RFP

20 µm

VNC

T Mosca, T Schwartz& RR Ribchester (onhis sabbatical…)

SUMMARY Synapses are induced after growth cones contact muscle fibres but the steps differ in “FaSyn” and “DeSyn” muscles

Synapse elimination accompanies a postnatal reduction in average motor unit size

Synapse elimination is partly competitive

Monoinnervated NMJ’s are normally stable

Synaptic regeneration partly recapitulates development

Synapse elimination in neonatal muscle and in reinnervated adult muscle is partly activity-dependent.

Activity is not sufficient for synapse elimination at all NMJ

Studies of the Drosophila NMJ may yield molecular insights into mechanisms of synaptic degeneration.