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Physiology and Pathophysiology ofNeuromuscular Transmission
“The neuromuscularjunction... [is] anexperimentally favourableobject whose study couldthrow considerable lighton synaptic mechanismselsewhere”
Sir Bernard Katz, FennLecture, IUPS Glasgow,1993
http://www.ricercaitaliana.it/prin/dettaglio_completo_prin_en-2004053317.htm
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http://en.wikipedia.org/wiki/Active_zone
Proteins of the Active Zone
Tools for studying synaptic form and function
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Nishimune et al. (2004) Nature 432:580-587.
‘Bassoon’ immunostaining localises to active zones
Pseudocoloured
Surface Plot
Ca imaging during high frequency stimulation
Greg Lnenicka
OH
N N
O
OHN
OH
O-
N N
O
OHN
OH
- H+ + H+
λabs = 397 nm
λabs = 475 nm
chromophore
Measuring exocytosis with “synaptopHluorin”
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Recycling vesicles take up fluorescent styryl (“FM”) dyes
hνHydrophobic Hydrophilic
Bewick
Desaki & Uehara, 1981
5
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)
Ch0
-5 mV 5.00 ms
1
2
3
4
0
Action potentials are “all-or-nothing” signals...… but EPPs are variable responses
Quantal size = Effect of one vesicle released
Quantal content = Number of vesicles released
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Gillingwater D. Thomson
5 ms
Facilitation
300 ms
10 mV
Short-term Depression
EPP’s show short-term plasticity
Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Safety factor and size-strength relationships
Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Safety factor and size-strength relationships
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Botulinum toxins cleave SNARE proteins
MG: AChR antibodies
X X
Myasthenia gravis and LEMS are autoimmune diseases
LEMS: Ca channelantibodies
X X
EPP’s teeter on the brink of the muscle fibreaction potential firing threshold in myasthenias
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• Weight loss • Slurred speech• Weak shoulder abduction and hip flexion• Reflexes absent, but re-appear after exercise• Sensation normal
Lambert-Eaton Myasthenic Syndrome
Complete recording from one LEMS patient demonstrating an initial small restingcompound muscle action potential(CMAP), followed by a 10-second period of maximal
voluntary contraction and subsequently 30 CMAPs illustrating augmentation andexponential decay.
Maddison P et al. Neurology 1998;50:1083-1087
0 Ca +Ca +4AP TTXDirectDirect +Mg +4AP
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Myasthenia GravisBefore
After edrophonium(Tensilon Test)
• Bilateral ptosis• Double vision in all directions• Fatiguable weakness• Reflexes disappear after exercise• Sensation normal
dTC dTC neo sux sux sux neo direct
Pre- and post-synaptic abnormalities have distinctive effects on EPPs
- Normal presynaptic function Normal quantal content (impaired postsynaptic function)
- Impaired presynaptic function Low quantal content (normal postsynaptic function)
Synaptic Depression
Synaptic Facilitation
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Lambert-Eaton Myasthenic Syndrome
EMG
EPPs have low quantal contentand show facilitation
EPP
Normal
LEMS
Myasthenia gravisEMG
Intracellular recording - NMJ
Summary of electrophysiological changes inMyasthenia Gravis and Myasthenic Syndrome
(NI=Normal Individual)
50
11
C h.0
5 m V
5 .0 0 m s
C h .0
5 m V
5 .0 0 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 increase EPP amplitude and prolong EPP decay time
Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor
Desaki & Uehara, 1981, J Neurocytol 10,101
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MEPPs (aka ‘minis’) are independent eventsthat occur with low release probability. Thisgenerates and exponential, Poisson distributionof intervals between events.
If the mean frequency is m (s-1), then thefrequency of a given number of MEPPs, x, ineach one second raster sweep is given by:
P(x) = exp(!m).m
x
x!
Mini analysis
Fatt & Katz, 1952, JPhysiol
Amplitude
Interval
y = exp(!(x ! µ)2 / 2" 2) / (" 2# )
MEPP
EPP
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Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor
Action potential
…add µ-conotoxinX
…add d-tubocurarine
Measuring EPP’s….
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5 mV
10.00 ms
Ca2+
Mg2+
EPP’s (muscle action potential blocked)
EPP’s in low Ca/high Mg
5 mV
10.00 ms
Binomial model:
Let: n=3p= 0.17(q=1-p)
m=n.p
P(0) = ?P(1) = ?P(2) = ?P(3) = ?
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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
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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
“God does not play dice ”
Simulation:Excel
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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
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
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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
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
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
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p=0.044 p=0.49
Binomial statistics are a better predictor orresponse variability when p>0.1
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
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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
*=
22
q = MEPP
m =EPP
q
Quantal Size:
Quantal Content:
MEPPEPP
Stim.
MEPPs
EPPs
Quantal analysis
Px
=e!mm
x
x!
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
The ACh null-potential (reversal potential) is about -10 mV
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Desaki & Uehara, 1981, J Neurocytol 10,101
I
V
~
Rm CmR
Ri
EACh
End-Plate Current (EPC)
2 ms
200,000 channels
20 mV
End-Plate Potential (EPP)
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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.
Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor
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NMJ have a high ‘safety factor’
Physiology and Pathophysiology ofNeuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor- Nerve terminal size- Quantal content per unit area- MEPP amplitude- “Input resistance”
EPP amplitude is proportional to nerve terminal area
10 ms 20 µmCostanzo et al.(1999) J Physiol 521:365-74
RH414/FM1-43
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NMJ size and muscle mibre diameter are correlated
sub super
% O
ccu
pa
ncy
0
20
40
60
80
100
sub-sub sub-super super-super
m/a
(µ m
-2)
0.01
0.1
1
A B
m/a(µm-2)
%O
ccup
ancy
Specific quantal content (m/µm2) is constant
Costanzo et al.(1999) J Physiol 521:365-74
Frog 200
Rat, mouse 50-75
Man 20-30
Species Quantal content
Frog
Rat
Man
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0 250 500 750 1000 1250 15000
50
100
150
200
Synaptic area
Frog
Rat
Man
Evoked release and NMJ area are correlated
Frog
Rat
Man
The size of NMJ and the extent of junctional folding vary between species
Frog
Rat
Man
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
Rin=1
!
RmRi
d3
V
I
=
At! d
m
m ! At
q ! Rin
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RinHarris & Ribchester (1979) J Physiol 296, 245-265
Endplate area, fibre diameter and MEPP amplitude, frequency are correlated
Quantal size (q) = response to a single vesicular release (i.e. the amplitude of the spontaneous MEPP)
Abnormalities in quantal size indicate a postsynaptic problem
Quantal content (m) = amount of transmitter released (i.e. the number of synaptic vesicles producing an EPP)
Abnormalities in quantal content indicate a presynaptic problem
http://neuromuscular.wustl.edu/musdist/dag2.htm
Neuromuscular Junction: postsynaptic
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Congenital Myasthenic Syndromes
Palace & Beeson (2008) J Neuroimmunol
SUMMARY
1. Variation in MEPP interval and EPP amplitudeconforms to a Poisson Distribution
2. Quantal content of EPP’s can be estimated byDirect, ‘Failures’, and Variance Methods.Remember to make allowance, if necessary, fornon-linear summation of synaptic potentials
3. Quantal content at rodent NMJ’s is about 50 andthe ‘safety factor’ is about 3.
4. Determinants of synaptic strength and safety-factor at the NMJ include Ca sensitivity (LEMS),ACh receptor density (MG), endplate size(CMS), and muscle fibre size (input resistance),