Physiology and Pathophysiology of Neuromuscular Transmission€¦ · 1 Physiology and Pathophysiology of Neuromuscular Transmission “The neuromuscular junction... [is] an experimentally

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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

2

http://en.wikipedia.org/wiki/Active_zone

Proteins of the Active Zone

Tools for studying synaptic form and function

3

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”

4

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

6

Gillingwater D. Thomson

5 ms

Facilitation

300 ms

10 mV

Short-term Depression

EPP’s show short-term plasticity


1. Botulism and Myasthenias

2. Characteristics of MEPPS and EPPS

3. Quantal analysis

4. Safety factor and size-strength relationships




3. Quantal analysis

4. Safety factor and size-strength relationships

7

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

9

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




3. Quantal analysis

4. Synaptic strength and safety factor

Desaki & Uehara, 1981, J Neurocytol 10,101

12

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

13




3. Quantal analysis


Action potential

…add µ-conotoxinX

…add d-tubocurarine

Measuring EPP’s….

14

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

17

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


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


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


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


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


- MEPP variance


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p=0.044 p=0.49

Binomial statistics are a better predictor orresponse variability when p>0.1

Problems


- MEPP variance


21

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


- MEPP variance


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.




3. Quantal analysis


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NMJ have a high ‘safety factor’




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

26

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),

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Physiology and Pathophysiology of Neuromuscular Transmission€¦ · 1 Physiology and Pathophysiology of Neuromuscular Transmission “The neuromuscular junction... [is] an experimentally