the ‘life cycle’ of neuromuscular synapses · 2010. 10. 18. · ch.2 2.5 mv 1 mv 10.00 ms vm...
TRANSCRIPT
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The The ‘‘Life CycleLife Cycle’’ of Neuromuscular Synapses of Neuromuscular Synapses
Homeostatic regulation of synaptic strength andthe safety factor for neuromuscular transmission
1. Structure and quantal analysis of function
2. Size-strength regulation and plasticity
3. Myasthenias
4. Insights from Drosophila
Frog
Rat
Man
The size of NMJ and the extent of junctional folding vary between species
Frog
Rat
Man
NMJ size and muscle mibre diameter are correlated
0 250 500 750 1000 1250 15000
50
100
150
200
Synaptic area
Frog
Rat
Man
Evoked release and NMJ area are correlated
Desaki & Uehara, 1981
Wood & Slater (1997)
2
Action potential
…add µ-conotoxinX
…add d-tubocurarine
Measuring EPP’s….
Ch.2
10 mV
5.00 ms
Latency
(1-2 ms)
Amplitude
(1-40 mV)
Rise Time(1-2 ms) Half-decay Time
(2-3 ms)
Typically-measured characteristics of the EPP (or MEPP)
Desaki & Uehara, 1981
IV
~
Voltage clamp
End-Plate Current (EPC)
2 ms
200,000 channels
20 mV
End-Plate Potential (EPP)
τ = RINCmR CR
R
EACh
Vm
Ch.2
2.5 mV
1 mV
10.00 ms
Vm
Ch.2
2.5 mV
1 mV
10.00 ms
Vm
Ch.2
2.5 mV
1 mV
10.00 ms
10 mV
2 nA
mf
0
-2
-4
-6
-8
-10
mV
AC
1
190 200 210 220 230 240 250 260 270 280 290
s
Keyboard31
6
5
4
3
2
mV
AC
1
85 90 95 100 105 110 115 120 125 130 135 140 145
s
Ch.2
10 mV
5.00 ms
Ch.2
10 mV
5.00 ms
Rin
MEPPs
EPPs
ntSynaptic size-strength regulation compensates for diameter-input resistance
20 ms
Actual m
Threshold m
Threshold
NMJ operate with a ʻsafety factorʼ of 3-5
Wood SJ, Slater CR. The contribution ofpostsynaptic folds to the safety factor forneuromusculartransmission in rat fast-and slow-twitch muscles.J Physiol. 1997Apr 1;500 ( Pt 1):165-76.PMID: 9097941
3
50 µm
Rewind to 1952…
Quantal Analysis
“The neuromuscularjunction... [is] anexperimentally favourableobject whose study couldthrow considerable lighton synaptic mechanismselsewhere”
Sir Bernard Katz, FennLecture, IUPS Glasgow,1993
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Desaki & Uehara, 1981, J Neurocytol 10,101
MEPPs
Synaptic recordings from the frog NMJ: B. Katz et al.
EPPs
Mini analysis
Fatt & Katz, 1952, JPhysiol
Amplitude
Interval
Ch0
-5 mV 5.00 ms
1
2
3
4
0
Quantal size = Effect of one vesicle release (MEPC/MEPP)
Quantal content = Number of vesicles released (EPC/EPP)
5
Binomial model:
Let: n=3p= 0.17(q=1-p)
m=n.p
P(0) = ?P(1) = ?P(2) = ?P(3) = ?
Binomial model:
Let: n=3p= 0.17(q=1-p)
m=n.p
P(0) = q3
P(1) = 3pq2
P(2) = 3p2qP(3) = p3
P(x) =n!
x!(n ! x)!px.q(n! x)
Let :x<<np<<1
Thenq(n-x) ~ exp(-np)
andn!
(n ! x)!" n
x
P(x) = exp(!m).m
x
x!
P(0) = ?P(1) = ?P(2) = ?P(3) = ?
Poisson Distribution
P(x) = exp(!m).m
x
x!Poisson Distribution
P(0) = exp(-m)P(1) = m.exp(-m)P(2) = m2.exp(-m)/2P(3) = m3.exp(-m)/6
Freq
uenc
y
Poisson distribution of QuantalContents of EPPs (n=100 trials)
0 1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
m=1
Quantal content
6
Freq
uenc
y
Poisson distribution of QuantalContents of EPPs (n=100 trials)
0 1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
m=2
Quantal content
Freq
uenc
y
Poisson distribution of QuantalContents of EPPs (n=100 trials)
0 1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
m=3
Quantal content
Freq
uenc
y
Poisson distribution of QuantalContents of EPPs (n=100 trials)
0 1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
m=4
Quantal content
Freq
uenc
y
Poisson distribution of QuantalContents of EPPs (n=100 trials)
0 1 2 3 4 5 6 7 8 9 10 11 12
0
10
20
30
40
m=5
Quantal content
“God does not play dice ”
--> Simulation:Excel
7
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
y = exp(!(x ! µ)2 / 2" 2 ) /(" 2# )
The Normal (Gaussian) Distribution
x
yy 5
x2!( )
2 0.25"exp# $
% &
0.5 2'=
(µ = 0; σ =0.5)
P(x) = exp(!m)m
x
x!k =1
n
" .1
2#k$ 2
! x ! kx ( )2
2k$ 2
%
& ' '
(
) * *
+
,
- -
.
/
0 0
m=3 quantaσ= 0.2 mvx =1.1mv
y 153!( )exp 3
x"x!# $
% &' ( 1
0.2 2)k
x 1.1k!( )2!
2k0.22# $
% &' (
exp# $% &' (
# $% &' (
k 1=
10
*=
q = MEPP
m =EPP
q
Quantal Size:
Quantal Content:
MEPPEPP
Stim.
MEPPs
EPPs
Quantal analysis
Px
=e!mm
x
x!
Methods of quantal analysis:
1. Direct method : m=EPP/MEPP (better, EPC/MEPPC)
2. Failures method: P(0)=exp(-m); m=Ln(Tests/Failures) ( for binomial: P(0)=(1-p)n)
3. Variance method: m = 1/(C.V.)2 i.e. m=EPP2 /var(EPP) (for binomial: var(m)=npq)
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Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
Desaki & Uehara, 1981, J Neurocytol 10,101
I
V
The ACh null-potential (reversal potential) is about -10 mV
McLachlan EM, Martin AR. Non-linear summation of end-plate potentials in the frogand mouse. J Physiol. 1981 Feb;311:307-24.PMID: 6267255
EPC’s sum linearly : EPP’s sum non-linearly
v' = v /(1! v /(Em! E
r)
m =v!
q(1 ! v!
(Em ! Er )
v' = v /(1! fv(Em ! Er )
Correction Factors
Martin (1955):
v= EPP amplitudeq= MEPP amplitudem = quantal content
McLachlan & Martin (1981)
Where f = an empirically determined ('fudge’) factor
For mouse muscle, long fibres: f=0.8For frog muscle, long fibres: f=0.55
For short muscle fibres (e.g. FDB) the correction is unknown, butf=0.3 gives a good fit to our data.
Activity dependent regulation of quantal size/content
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Inactivity triggers sprouting and upregulation of transmitter release
Tsujimoto et al.(1990) J Neurosci.
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
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…
Myasthenia
MG: AChR antibodies
X X
Myasthenia gravis and LEMS are autoimmune diseases
LEMS: Ca channelantibodies
X X
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Summary of electrophysiological changes inMyasthenia Gravis and Myasthenic Syndrome
(NI=Normal Individual)
Synaptic basal lamina and acetylcholinesterase
C h .0
5 m V
5 .0 0 m s
C h.0
5 m V
5 .0 0 m s
Half Decay Time
T1 T2 T3 T40
5
10
15Hepes Buffered Ringer Solution
Neostigmine (5µM)
EPP Train Number
Tim
e (m
S)
C h.0
5 m V
5 .0 0 m s
C h.0
5 m V
5 .0 0 m s
Neostigmine (5 µM)
Control
Kosala Dissanayake
Anticholinesterases enhance EPP amplitude and prolong EPP decay time
dTC dTC neo sux sux sux neo direct
Myasthenic Syndrome(LEMS):
EMG
EPP’s have low quantal contentand show facilitation
EPP
Normal
LEMS
11
0 Ca +Ca +4AP TTXDirectDirect +Mg +4AP
Congenital Myasthenic Syndromes
Palace & Beeson (2008) J Neuroimmunol
Plasticity/Homeostasis of Drosophila NMJ
Drosophila 3rd Instar fillet
Drosophila NMJ - Scanning EM
Yoshihara et al (1997) J Neurosci 17, 8408- 8426
Drosophila NMJKarunanithi, S. et al.(2002) J Neurosci 22,10267-10276.
Renger et al (2000) J Neurosci 20,3980-3992
Sub-Synaptic Reticulum
T-bar
PSD
Jiao et al (2010) J Struct Biol In press
Central Core
12
IN 0
5 mV
5.00 ms
Drosophila Larval NMJ
EPPS in 5 mM Mg 2+NC82 immunostainfor bruchpilot
IN 0
5 mV
50.00 ms
50 µm
Davis & Goodman, 1998 Nature 392,82-86
Davis & Goodman, 1998 Nature 392,82-86
Davis & Goodman, 1998 Nature 392,82-86
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SUMMARY
Statistical analysis of synaptic potential amplitudes shows that transmitter release is “quantized”. Neuromuscular junctions operate with a high “safety- factor”, secured in part by the endplate-size to fibre diameter ratio.
Defects in transmitter release, sensitivity and size- strength relationships lead to various ʻmyasthenicʼ syndromes, characterised by significant muscle weakness.
Studies on Drosophila NMJ may provide insight into reciprocal, independent, ʻhomeostaticʼ regulation of transmitter release and endplate sensitivity.