1

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

4

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)

8

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

9

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

10

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

13

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.