1

The importance of being earnest….

Behaviour

Experience

….or at least active...

The Life of Neuromuscular SynapsesThe Life of Neuromuscular Synapses

1. Growth/aging of muscle, NMJ andsynaptic strength

2. Synapse elimination: the NMJ as a‘Hebbian’ synapse

3. Synaptic degeneration

Activity influences:

2

http://myathleticlife.com/2012/01/sarcopenia-doomed-age-related-muscle-loss/

40 year oldtriathlete

74 year oldsedentarymale

3

Buford, TW et al.(2010) Aging Res Rev 368-283

Exercise confers little benefit on sarcopenia

Monoinnervated NMJ’s are stable and grow 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)

4

Li, TI & Thompson, WJ (2011) J Neurosci 31:14910 –14919

Davis, Ann Rev Neuro 2006. 29:307-23

Impair neurotransmitter release

Increased sensitivity retargets setpoint muscle excitation

Set point muscle excitation

Upregulation of quantal content in α-BTX treated rats

Plomp et al (1992) J Physiol 458,487-499;Plomp et al (1992) J Physiol 478, 125-136

BTX

CON

5

Duchen, 1970

Paralysis (neuromuscular block) promotes sprouting

C E

Exercise modestly increases endplate size and quantal content

%

Fahim, M.A.(1997) J App Physiol 83,59-66Dorlochter et al.(1991) J Physiol 486, 283-292

Normal▼ ▲

(Sprouting) (Hypertrophy)

So, activity may have a biphasic effect…

6

Denervation changes muscle phenotype

Most of these properties are reversed, restoring theinnervated phenotype, by direct electrical stimulation

Conclusion:

Activity (exercise), nutrition and aging have cleareffects on metabolism and muscle growth; but theeffects are complex.

Based on D. Purves & J.W. Lichtman, Principles of Neural Development.

Cyto-plasm

Genes

Molecular

CellularInteractions

Cellular

Behaviour

Experience

Systems

7

1. Growth/aging of muscle, NMJ andsynaptic strength

2. Synapse elimination: the NMJ as a‘Hebbian’ synapse

3. Synaptic degeneration

Activity influences:

Levay, Stryker & Shatz. J. comp. Neurol. 179,223-244.(1978)

8

“When an axon of cell A isnear enough to excite a cell Band repeatedly andpersistently takes part infiring it, some growth processor metabolic change takesplace in one or both cellssuch that Aʼs efficiency asone of the cells firing B isincreased.”

D.O.Hebb (1949) TheOrganization of Behaviour.

B

A

B

A

9

Paraphrasing…

“Cells that fire together, wire together”

“Use it or lose it”

“When an axon of cell A is near enough to excite a cell B and repeatedly and persistentlytakes part in firing it, some growth process or metabolic change takes place in one or bothcells such that Aʼs efficiency as one of the cells firing B is increased.”D.O.Hebb (1949) The Organization of Behaviour.

"When the presynaptic axon of cell A repeatedly and persistently fails to excite thepostsynaptic cell B while cell B is firing under the influence of other presynaptic axons,metabolic change takes place in one or both cells such that A's efficiency, as one of thecells firing B, is decreased." G.S. Stent (1972) PNAS 70, 997-1001

FromPurves,D.(1988) Body &

Brain: a trophic theory ofneural connections. Harvard

University Press.

Motor terminals compete for synaptic occupancy duringsynapse elimination

Walsh & Lichtman (2003) Neuron 37,67-73

10

Synapse elimination is influenced by activity

0 10 20 300

20

40

60

80

100StimulationParalysisNormal

Age (days)

MPN LPN SN

4DL

10 µm

Rodent 4th Deep Lumbrical Muscles (4DL) provide opportunities toexplore activity-dependent plasticity of NMJ

Betz & Ridge, 1984

Stim

Stimulated

Unstimulated

Control

Selective stimulation biases synapse elimination

11

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)

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Is activity necessary for synapse elimination?

α-BTXCostanzo, E.M., Barry, J.A. & Ribchester, R.R. (2000)

Competition at silent synapses in reinnervated muscle.Nature Neuroscience. 3,694-700.

TTX-only TTX/α-BTX

Buffelli M et al. Physiology 2004;19:85-91©2004 by American Physiological Society

Replacing asynchronous activity with synchronous activity….

13

Buffelli M et al. Physiology 2004;19:85-91

…leads to a greater level of polyneuronal innervation

Conclusion:

Activity is influential, but not decisive indetermining the fate of neuromuscularsynapses

1. Growth/aging of muscle, NMJ andsynaptic strength

2. Synapse elimination: the NMJ as a‘Hebbian’ synapse

3. Synaptic degeneration

Activity influences:

14

WldSasamodelofslowsynap.cdegenera.on

Wong et al., 2009,Mol Cell Neurosci

X

YFP16:WldS - 5d axotomy

Gillingwater/ D.Thomson

Ch.2

5 mV

10.00 ms

Ch.2

5 mV

10.00 ms

Ch.2

5 mV

10.00 ms

Synaptic degeneration precedes axonal degeneration in WldS mice

Tatyana Dias

5d Axotomy5ms

10 mv

Synapses degenerate in WldS homozygotes over 3-10 days(YFP does not interfere with the WldS phenotype)

0 1 2 3 4 5 6 7 8 9 100

25

50

75

100

Days post axotomyYFP Non-YFP

0

25

50

75

100 Mann-Whitney: P=0.49; NS

15

•WldS,30hinculture

•S/mula/onat1Hzcon/nuous,10Hzfor1severy10s,100Hzfor1severy100s

Effects of stimulation on synaptic degeneration in vitro

Control 1Hz 10Hz 100Hz0

20

40

60

80

100

resp

onsi

ve fi

bres

(%)

*

Control 100 Hz

0

20

40

60

80

100

Occu

pie

d E

nd

pla

tes (

%)

*

Does activity influence (prime or protect)synaptic degeneration in WldS mice?

2-3 weeks 5d

Condition

Cutsciatic nerve

Challenge

ICR/CFM

Assay

2- 4 weeks of voluntary wheel running does not delay synapticdegeneration in heterozygous WldS mice

contra

latera

l (n=1

9)

2 wee

ks (n

=6)

4 wee

ks (n

=5)

contro

l (n=8

)

0

20

40

60

80

100

% in

nerv

ated

fib

res

Rosalind Brown

16

ElectrophysiologicalanalysisofthenumberofresponsivefibresintheFlexorDigitorumBrevismuscle

Inactivity inhibits the WldS phenotype: Physiology

7 days TTX 7 days Saline Unoperated Control0

20

40

60

80

100

resp

onsi

ve fi

bres

(%)

•YFP16Wldsmice;Tritc‐α‐BTXstaining•Numberofoccupiedendplatescounted

TTX

Con

YFP16: WldS /Tritc-BTX

Inac.vityinhibitstheWldSphenotype:Morphology

TTX block+cut No block +cut Unoperated Control

0

20

40

60

80

100

occu

pie

d e

nd

pla

tes (

%)

***

*****

Conclusion

Inactivity sensitises neuromuscular junctions totriggers of synaptic degeneration in WldS mice.

17

Motor neurone disease (eg ALS)

http://emedicine.medscape.com/article/2111360-overview#aw2aab6b7

Hypertrophy and Atrophy in ALS

http://emedicine.medscape.com/article/2111360-overview#aw2aab6b7

Grouping of Slow (Type I) and Fast MHC (Type II) muscle fibres in ALS

18

Activity mitigates onset but not progression of ALS in female SOD1 mice

Veldink et al (2003)Neuromuscular Disorders 13737–743

19

Kirkinezos et al. Ann Neurol 53:804–807 2003

Or is it males…

Deforges S et al. J Physiol 2009;587:3561-3572©2009 by The Physiological Society

Swimming is better than running..?

thy1.2:YFP16/SOD1G93A

13 week old - asymptomatic

50 µm

Confocal Z-seriesprojection

Preterminal axons atrophy, then fragment, before motor nerveterminals

(Wheel running does not influence this process)

20

Summary

Activity (exercise), nutrition and aging have clear effects on metabolism and muscle growth; but the effects are complex.

Activity is influential, but not decisive in determining the fate of neuromuscular synapses

Inactivity sensitises neuromuscular junctions to triggers of synaptic degeneration in WldS mice.

“Exercise regimes [may] merit more detailed clinical evaluation in ALS.”