Physiology and Pathophysiology of Neuromuscular Transmission

Physiology and Pathophysiology of
Neuromuscular Transmission
“The neuromuscular
junction... [is] an
experimentally favourable
object whose study could
throw considerable light
on synaptic mechanisms
elsewhere”
Sir Bernard Katz, Fenn
Lecture, IUPS Glasgow,
1993
http://www.ricercaitaliana.it/prin/dettaglio_completo_prin_en-2004053317.htm
1

Proteins of the Active Zone
http://en.wikipedia.org/wiki/Active_zone
Tools for studying synaptic form and function
2

‘Bassoon’ immunostaining localises to active zones
Nishimune et al. (2004) Nature 432:580-587.
Ca imaging during high frequency stimulation
Pseudocoloured
Surface Plot
Greg Lnenicka
Measuring exocytosis with “synaptopHluorin”
O
OH
N N λ abs = 397 nm
OH
O HN
- H + + H +
O
O -
N N λ abs = 475 nm
OH
O HN
chromophore
3

Recycling vesicles take up fluorescent styryl (“FM”) dyes
Hydrophobic
hν
Hydrophilic
Bewick
Desaki & Uehara, 1981
4

Typically-measured characteristics of the EPP (or MEPP)
Rise Time
(1-2 ms) Half-decay Time
(2-3 ms)
Amplitude
(1-40 mV)
Ch.2
Latency
(1-2 ms)
10 mV
5.00 ms
Action potentials are “all-or-nothing” signals...
… but EPPs are variable responses
4
3
2
Ch0
1
0
-5 mV
5.00 ms
Quantal size
= Effect of one vesicle released
Quantal content = Number of vesicles released
5

EPP’s show short-term plasticity
Facilitation
5 ms
Short-term Depression
10 mV
Gillingwater
300 ms
D. Thomson
Physiology and Pathophysiology of
Neuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Safety factor and size-strength relationships
Physiology and Pathophysiology of
Neuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Safety factor and size-strength relationships
6

Botulinum toxins cleave SNARE proteins
Myasthenia gravis and LEMS are autoimmune diseases
LEMS: Ca channel
antibodies
X
X
X
X
MG: AChR
antibodies
EPP’s teeter on the brink of the muscle fibre
action potential firing threshold in myasthenias
7

Lambert-Eaton Myasthenic Syndrome
• Weight loss
• Slurred speech
• Weak shoulder abduction and hip flexion
• Reflexes absent, but re-appear after exercise
• Sensation normal
Complete recording from one LEMS patient demonstrating an initial small resting
compound muscle action potential(CMAP), followed by a 10-second period of maximal
voluntary contraction and subsequently 30 CMAPs illustrating augmentation and
exponential decay.
Maddison P et al. Neurology 1998;50:1083-1087
0 Ca Direct +Ca +4AP +Mg +4AP Direct TTX
8

Myasthenia Gravis
Before
• Bilateral ptosis
• Double vision in all directions
• Fatiguable weakness
• Reflexes disappear after exercise
• Sensation normal
After edrophonium
(Tensilon Test)
dTC dTC neo sux sux sux neo direct
Pre- and post-synaptic abnormalities have distinctive effects on EPPs
Synaptic Facilitation
- Impaired presynaptic function
Low quantal content
(normal postsynaptic function)
Synaptic Depression
- Normal presynaptic function
Normal quantal content
(impaired postsynaptic function)
9

Lambert-Eaton Myasthenic Syndrome
EMG
EPP
Normal
LEMS
EPPs have low quantal content
and show facilitation
Myasthenia gravis
EMG
Intracellular recording - NMJ
Summary of electrophysiological changes in
Myasthenia Gravis and Myasthenic Syndrome
50
(NI=Normal Individual)
10

C h.0
5 m V
C h .0
5 m V
5 .0 0 m s
5 .0 0 m s
C h .0
C h .0
5 m V
5 m V
5 .0 0 m s
5 .0 0 m s
Anticholinesterases increase EPP amplitude and prolong EPP decay time
Control
Neostigmine (5 µM)
Kosala Dissanayake
Physiology and Pathophysiology of
Neuromuscular 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
11

MEPPs (aka ‘minis’) are independent events
that occur with low release probability. This
generates and exponential, Poisson distribution
of intervals between events.
If the mean frequency is m (s -1 ), then the
frequency of a given number of MEPPs, x, in
each one second raster sweep is given by:
P(x) = exp(!m). m x
x!
Mini analysis
Amplitude
y = exp(!(x ! µ) 2 / 2" 2 ) / (" 2# )
Interval
Fatt & Katz, 1952, JPhysiol
MEPP
EPP
12

Physiology and Pathophysiology of
Neuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor
Measuring EPP’s….
X
Action potential
…add µ-conotoxin
…add d-tubocurarine
13

EPP’s
(muscle action potential blocked)
EPP’s in low Ca/high Mg
Mg 2+
Ca 2+
5 mV
10.00 ms
5 mV
10.00 ms
Binomial model:
Let:
n=3
p= 0.17
(q=1-p)
m=n.p
P(0) = ?
P(1) = ?
P(2) = ?
P(3) = ?
14

Binomial model:
Let:
n=3
p= 0.17
(q=1-p)
m=n.p
P(0) = q 3
P(1) = 3pq 2
P(2) = 3p 2 q
P(3) = p 3
P(x) =
n!
x!(n ! x)! p x (n ! x)
.q
Let :
x

P(x) = exp(!m). m x
Poisson Distribution
x!
P(0) = exp(-m)
P(1) = m.exp(-m)
P(2) = m 2 .exp(-m)/2
P(3) = m 3 .exp(-m)/6
“God does not play dice ”
Simulation:Excel
16

Poisson distribution of Quantal
Contents of EPPs (n=100 trials)
40
Frequency
30
20
10
m=1
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Quantal content
Poisson distribution of Quantal
Contents of EPPs (n=100 trials)
40
m=2
Frequency
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Quantal content
Poisson distribution of Quantal
Contents of EPPs (n=100 trials)
40
m=3
Frequency
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Quantal content
17

Poisson distribution of Quantal
Contents of EPPs (n=100 trials)
40
m=4
Frequency
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Quantal content
Poisson distribution of Quantal
Contents of EPPs (n=100 trials)
40
m=5
Frequency
30
20
10
0
0 1 2 3 4 5 6 7 8 9 10 11 12
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=EPP 2 /var(EPP)
(for binomial: var(m)=npq)
18

Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
19

Binomial statistics are a better predictor or
response variability when p>0.1
p=0.044
p=0.49
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
20

The Normal (Gaussian) Distribution
y = exp(!(x ! µ) 2 / 2" 2 ) / (" 2# )
y
(!x 2 )
# % exp $
&
2"
0.25
y = 5
0.5 2'
(µ = 0; σ =0.5)
x
n
+
P(x) = " exp(!m) mx
.-
k =1 x! ,-
1 % !( x ! kx ) 2 (.
2#k$ 2 '
0
& 2k$ 2 *
)/
0
10 ' exp (! 3)
" 3 x
y 15 % ' &
( 1 '!
( x ! 1.1k) 2 (
% ' exp
# x! $
0.2 2)k # %
2k0.2 2 $ & &
( (
= * % &
# # $ $
k = 1
m=3 quanta
σ= 0.2 mv
x =1.1mv
21

MEPP
EPP
Quantal analysis
P x
= e!m m x
x!
MEPPs
EPPs
Stim.
Quantal Size:
Quantal Content:
q = MEPP
m = EPP
q
Problems
- Non-Poisson conditions
- MEPP variance
- Non-linear summation
The ACh null-potential (reversal potential) is about -10 mV
22

V
~
I
Desaki & Uehara, 1981, J Neurocytol 10,101
R
R m
C m
E ACh
R i
2 ms
200,000 channels
End-Plate Current (EPC)
End-Plate Potential (EPP)
20 mV
23

EPC’s sum linearly : EPP’s sum non-linearly
McLachlan EM, Martin AR. Non-linear summation of end-plate potentials in the frog
and mouse. J Physiol. 1981 Feb;311:307-24.PMID: 6267255
Correction Factors
Martin (1955):
v' = v /(1! v /(E m
! E r
)
m =
q(1 ! v ! (E m
! E r
)
v= EPP amplitude
q= MEPP amplitude
m = quantal content
v' = v /(1! fv(E m
! E r
)
v !
McLachlan & Martin (1981)
Where f = an empirically determined ('fudge’) factor
For mouse muscle, long fibres: f=0.8
For frog muscle, long fibres: f=0.55
For short muscle fibres (e.g. FDB) the correction is unknown, but
f=0.3 gives a good fit to our data.
Physiology and Pathophysiology of
Neuromuscular Transmission
1. Botulism and Myasthenias
2. Characteristics of MEPPS and EPPS
3. Quantal analysis
4. Synaptic strength and safety factor
24

NMJ have a high ‘safety factor’
Physiology and Pathophysiology of
Neuromuscular 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
RH414/FM1-43
20 µm
10 ms
Costanzo et al.(1999) J Physiol 521:365-74
25

NMJ size and muscle mibre diameter are correlated
Specific quantal content (m/µm 2 ) is constant
A
B
100
80
1
60
40
-2
) mµ
m/a
(µm -2 ) 0.1
%Occupancy
% Occupancy
20
0
sub super
m/a (
0.01
sub-sub sub-super super-super
Costanzo et al.(1999) J Physiol 521:365-74
Species
Quantal content
Frog
Rat
Frog 200
Rat, mouse 50-75
Man 20-30
Man
26

Vm
2.5 mV
Vm
Ch.2
2.5 mV
1 mV
Ch.2
31 Keyboard
AC
mV
1
6
1 mV
5
4
3
2
Ch.2
10 mV
10.00 ms
10.00 ms
85 90 95 100 105 110 115 120 125 130 135 140 145
5.00 ms
s
Vm
2.5 mV
Ch.2
1 mV
10.00 ms
AC
mV
1
0
-2
-4
-6
-10
-8
Ch.2
10 mV
5.00 ms
190 200 210 220 230 240 250 260 270 280 290
s
Evoked release and NMJ area are correlated
200
Frog
150
100
50
0
0 250 500 750 1000 1250 1500
Synaptic area
Rat
Man
The size of NMJ and the extent of junctional folding vary between species
Frog
Frog
Rat
Rat
Man
Man
Synaptic size-strength regulation compensates for diameter-input resistance
nt
mf
R in
= 1 R m
R i
A ! d 3
t
! d m
V
10 mV
2 nA
I
=
R in
20 ms
q ! R in
MEPPs
m ! A t
EPPs
27

R in
Harris & 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
Neuromuscular Junction: postsynaptic
http://neuromuscular.wustl.edu/musdist/dag2.htm
28

Congenital Myasthenic Syndromes
Palace & Beeson (2008) J Neuroimmunol
SUMMARY
1. Variation in MEPP interval and EPP amplitude
conforms to a Poisson Distribution
2. Quantal content of EPP’s can be estimated by
Direct, ‘Failures’, and Variance Methods.
Remember to make allowance, if necessary, for
non-linear summation of synaptic potentials
3. Quantal content at rodent NMJ’s is about 50 and
the ‘safety factor’ is about 3.
4. Determinants of synaptic strength and safetyfactor
at the NMJ include Ca sensitivity (LEMS),
ACh receptor density (MG), endplate size
(CMS), and muscle fibre size (input resistance),
29