Synthesis. Storage. Physiology and Pathophysiology of Neuromuscular Transmission. Release. Action. Inactivation. Myasthenia Gravis Before

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1 Synthesis Physiology and Pathophysiology of Neuromuscular Transmission Storage Release Action Inactivation Myasthenia gravis and LEMS are autoimmune diseases Myasthenia Gravis Before LEMS: Ca channel antibodies X X X X Case 1 Bilateral ptosis Double vision in all directions Fatiguable weakness Reflexes disappear after exercise Sensation normal MG: AChR antibodies After edrophonium (Tensilon Test) dtc dtc neo sux sux sux neo direct Lambert-Eaton Myasthenic Syndrome Case 2 Weight loss Slurred speech Weak shoulder abduction and hip flexion Reflexes absent, but re-appear after exercise Sensation normal 1

2 Congenital Myasthenic Syndromes Ca Direct +Ca +4AP +Mg +4AP Direct TTX Palace & Beeson (28) J Neuroimmunol Physiology and Pathophysiology of Neuromuscular Transmission Physiology and Pathophysiology of Neuromuscular Transmission 1. Characteristics of MEPPS and EPPS 2. Quantal analysis 3. Synaptic strength and safety factor 1. Characteristics of MEPPS and EPPS 2. Quantal analysis 3. Synaptic strength and safety factor Amplitude Mini analysis Interval Desaki & Uehara, 1981 Fatt & Katz, 1952, JPhysiol 2

3 C h. 5 m V C h. 5 m V 5. m s 5. m s C h. C h. 5 m V 5 m V 5. m s 5. m s Measuring EPP s. Typically-measured characteristics of the EPP (or MEPP) Rise Time (1-2 ms) Half-decay Time (2-3 ms) X Action potential add µ-conotoxin add d-tubocurarine Amplitude (1-4 mv) Latency (1-2 ms) Synaptic basal lamina and acetylcholinesterase Anticholinesterases increase EPP amplitude and prolong EPP decay time Control Myasthenia gravis EMG Neostigmine (5 µm) Kosala Dissanayake Intracellular recording - NMJ 3

4 Physiology and Pathophysiology of Neuromuscular Transmission 1. Characteristics of MEPPS and EPPS 2. Quantal analysis 3. Synaptic strength and safety factor 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 Synaptic recordings from the frog NMJ: B. Katz et al. MEPPs EPPs Desaki & Uehara, 1981, J Neurocytol 1,11 4

5 EPP s (muscle action potential blocked) EPP s in low Ca/high Mg Ch Mg 2+ Ca mv 1. ms Action potentials are all-or-nothing signals... but EPPs are variable responses 5 mv 1. ms -5 mv 5. ms Quantal size = Effect of one vesicle release (MEPC/MEPP) Quantal content = Number of vesicles released (EPC/EPP) Binomial model: Binomial model: Let: n=3 p=.17 (q=1-p) m=n.p Let: n=3 p=.17 (q=1-p) m=n.p P() =? P(1) =? P(2) =? P(3) =? P() = q 3 P(1) = 3pq 2 P(2) = 3p 2 q P(3) = p 3 5

6 P(x) = n! x!(n! x)! p x.q (n! x) Let : x<<n p<<1 Then q (n-x) ~ exp(-np) P(x) = exp(!m). m x Poisson Distribution P() =? P(1) =? P(2) =? P(3) =? x! and n! (n! x)! " n x P(x) = exp(!m). m x Poisson Distribution P() = exp(-m) P(1) = m.exp(-m) P(2) = m 2.exp(-m)/2 P(3) = m 3.exp(-m)/6 x! Frequency Poisson distribution of Quantal Contents of EPPs (n=1 trials) Quantal content m=1 Poisson distribution of Quantal Contents of EPPs (n=1 trials) Poisson distribution of Quantal Contents of EPPs (n=1 trials) 4 m=2 4 m=3 Frequency 3 2 Frequency Quantal content Quantal content 6

7 Poisson distribution of Quantal Contents of EPPs (n=1 trials) Poisson distribution of Quantal Contents of EPPs (n=1 trials) 4 m=4 4 m=5 Frequency 3 2 Frequency Quantal content Quantal content --> Simulation:Excel God does not play dice Problems - Non-Poisson conditions - MEPP variance - Non-linear summation Problems - Non-Poisson conditions - MEPP variance - Non-linear summation 7

8 The Normal (Gaussian) Distribution y = exp(!(x! µ) 2 / 2" 2 ) /(" 2# ) y (!x 2 ) # % exp $ & 2".25 y = 5.5 2' (µ = ; σ =.5) x Quantal analysis n + P(x) = " exp(!m) mx.- k =1 x!,- 1 %!( x! kx ) 2 (. 2#k$ 2 ' & 2k$ 2 * )/ MEPP EPP P x = e!m m x x! 1 ' exp (! 3) " 3 x y 15 % ' & ( 1 '! ( x! 1.1k) 2 ( % ' exp # x! $.2 2)k # % 2k.2 2 $ & & ( ( = * % & # # $ $ k = 1 MEPPs m=3 quanta σ=.2 mv x =1.1mv EPPs Stim. Quantal Size: Quantal Content: q = MEPP m = EPP q Methods of quantal analysis: 1. Direct method : m=epp/mepp (better, EPC/MEPPC) 2. Failures method: P()=exp(-m); m=ln(tests/failures) ( for binomial: P()=(1-p) n ) 3. Variance method: m = 1/(C.V.) 2 i.e. m=epp 2 /var(epp) (for binomial: var(m)=npq) Problems - Non-Poisson conditions - MEPP variance - Non-linear summation 8

9 The ACh null-potential (reversal potential) is about -1 mv 2, channels 2 ms Endplate Current 2 mv Endplate Potential Action Potential EPC s sum linearly : EPP s sum non-linearly V ~ I Desaki & Uehara, 1981, J Neurocytol 1,11 McLachlan EM, Martin AR. Non-linear summation of end-plate potentials in the frog and mouse. J Physiol Feb;311:37-24.PMID: 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 Physiology and Pathophysiology of Neuromuscular Transmission 1. Characteristics of MEPPS and EPPS 2. Quantal analysis 3. Synaptic strength and safety factor For mouse muscle, long fibres: f=.8 For frog muscle, long fibres: f=.55 For short muscle fibres (e.g. FDB) the correction is unknown, but f=.3 gives a good fit to our data. 9

10 Vm 2.5 mv Vm 2.5 mv 1 mv 31 Keyboard AC mv mv mv 1. ms 1. ms ms s Vm 2.5 mv 1 mv 1. ms AC mv mv 5. ms s NMJ size and muscle mibre diameter are correlated The size of NMJ and the extent of junctional folding vary between species Frog Frog Rat Rat Man Man Evoked release and NMJ area are correlated Synaptic area Frog Rat 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 q! R in m! A t 1 mv 2 na 2 ms I = R in MEPPs EPPs Pre- and post-synaptic abnormalities have distinctive effects on EPPs Synaptic Depression 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 - Normal presynaptic function Normal quantal content (impaired postsynaptic function) Quantal content (m) = amount of transmitter released (i.e. the number of synaptic vesicles producing an EPP) Synaptic Facilitation Abnormalities in quantal content indicate a presynaptic problem - Impaired presynaptic function Low quantal content (normal postsynaptic function) 1

11 Summary of electrophysiological changes in Myasthenia Gravis and Myasthenic Syndrome 5 (NI=Normal Individual) 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 5 and the safety factor is about Determinants of synaptic strength and safetyfactor at the NMJ include endplate size (CMS), Ca sensitivity (LEMS), ACh receptor density (MG), and muscle fibre size (input resistance.) 11