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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)
12
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.”