Mini Symposium Summary week 8.
1) The Contribution of Postsynaptic Folds To The Safety Factor for Neuromuscular Transmission
in Rat Fast- and Slow-Twitch Muscles (Wood S.J. & Slater C.R., 1997).
Aim:
The aim of the paper was to assess the contribution of junctional folds to the safety factor.
Method:
- Analysed the MEPPs, EPPs, MEPCs and EPCs
- Used the data obtained to calculate the quantal content released in normal neuron-evoked
responses, at the junctional folds, in extra-junctional regions and at threshold levels.
- Used fast twitch (EDL) and slow twitch (soleus) muscles from rat’s legs for these experiments.
Results:
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Soleus
EDL
Fast-twitch muscles have a greater quantal content to slow-twitch muscles by 40%.
The number of quanta needed to produce an action potential is 13 in both muscle types.
Difference between junctional and extra-junctional regions: in the soleus muscle, less charge and
voltage is needed to evoke an action potential in the junctional area compared to the extrajunctional area. This was not the case for EDL.
However, the peak amplitude needed to evoke an action potential is significantly lower in
junctional regions of both types of muscle fibre, and EDL muscles required 15% more charge in
both these areas to reach threshold.
These differences can be explained by the differences in inactivation of VGSCs between muscle
types.
Calculated the number of quanta needed to reach threshold in junctional and extra-junctional
regions for both types of muscle: the junctional regions require fewer quanta and the soleus
muscle requires fewer than the EDL muscle.
The table below shows the number of quanta released for the different regions of the two types of
muscle.
Junctional
20.8
22.1
Extra-Junctional
26.6
26.5
So, in summary, far more quanta are released than necessary to produce an action potential.
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Attempted to quantify the differences in folding between the fast and slow twitch muscles: in
both types of muscle the surface area is increased 5 times, suggesting there is no difference in the
amount of membrane between the two muscles.
The synaptic contact of the EDL muscle is 64% of that of the soleus muscle but we already know
that it has a 40% greater quantal content. This suggests that the EDL muscle releases twice as
much transmitter per unit area that the soleus muscle.
Conclusion:
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The rat motor nerve terminal releases 3.5-5 times as much neurotransmitter as necessary to
produce an action potential.
Calculated the safety factor of these muscles as a ratio of full quantal content to threshold quantal
content.
Both the results for voltage and charge show that the safety factor of the fast twitch muscle is
greater, which is consistent with earlier findings.
Role of the junctional folds in the safety factor: as the amount of membrane at the NMJ does not
vary between the two muscle types, they concluded that the junctional folds increase the safety
factor by a factor of two, but the differences in the safety factor between fast- and slow-twitch
muscles is due to the increased neurotransmitter release in fast-twitch muscles.
BBQ: How do junctional folds amplify the EPP?
The influx of current at the top of the folds and the high resistance of the cleft enhances the depolarisation
of the membrane in the depths of the folds, where the VGSCs are. This should theoretically depolarise the
folds by as much as 10mV more than the EPP, causing more VGSCs to open.
2) Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis.
(Frank C.A. et al., 2006).
Background: Homeostasis is an essential type of feedback regulation that enables a biological system to
maintain a constant functionality within a changing environment. It was generally believed that
homeostatic signalling is slow, and takes several hours – days to occur. Retrograde signalling occurs
when a signal travels from the postsynaptic membrane to the presynaptic compartment; the components
of retrograde signalling involved are not really known.
Aim: To demonstrate that homeostatic signalling at the Drosophila NMJ can potentiate presynaptic
transmitter release within 5-10min following pharmacological blockade of postsynaptic neurotransmitter
receptors.
Method: Injection of glutamate receptor antagonist PhTox.
Results and Conclusions:
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Homeostatic signalling can occur within 5-10mins after postsynaptic receptor blockade.
Homeostasis can occur in the absence of motor neuron activity and thus does not require evoked
neurotransmission.
- mEPSPs can induce homeostatic signalling at the NMJ.
- Homeostatic signalling system controls a homeostatic change of presynaptic transmitter release:
investigated the involvement of presynaptic Ca2+ channels:
 Cac = α-subunit of Cav2.1 channel
• Only in presynaptic terminal of nervous system at the active site
• Required for stimulus-evoked NT release at the NMJ
 Mutations of Cav 2.1 block rapid induction and sustained expression of homeostasis - perhaps crucial
for retrograde signalling.
 Calcium channels are required for synaptic homeostasis and also presynaptic Cacophony (α-subunit of
Cav2.1 channel) is required for rapid homeostatic modulation of transmitter release.
– Homeostatic signalling can be independent of synaptic growth and development at NMJ.
– It is a form of plasticity, capable of tuning pre-synaptic transmitter release to offset even small
changes in postsynaptic excitability.
– Homeostatic signalling could be retained throughout life to counteract stress, disease or injury
related perturbations that would alter neural function – protective mechanism.
– Altered channel activity and mutations of pore forming Cav 2.1 have been linked to migraine
and ataxia in humans - perhaps there is a link between impaired homeostatic signalling and neurological
diseases?
3) Pre- and post- synaptic abnormalities associated with impaired neuromuscular transmission in a
group of patients with “limb-girdle myasthenia.” (Slater C.R. et al., 2006).
Aim: To identify any structural and functional abnormalities of the NMJ, and if possible to determine
whether the defects are predominantly pre- or post synaptic.
Methods:
- Initial Methods (on the LGM patients) included repetitive nerve stimulation, single fibre-EMG.
- LGM patients were then compared to 8 control subjects. Muscle biopsy sample were taken from
the VL muscle and then intracellular recordings were made (to investigate amplitude and decay
time of EPPs/mEPPs and EPCs/mEPCs, and quantal content).
- Electron and Light microscopy was also used to investigate the muscle samples.
Conclusions:
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LGM patients had impaired neuromuscular transmission - mean EPP amplitude was only 46%
that of the controls in the LGM patients. mEPP amplitude and quantal content was also reduced
in LGM subjects. mEPC amplitude and decay time were not reduced in LGM subjects. This
suggests the local abundance of AChR is normal, as is AChE activity.
General appearance of NMJs under electron and light microscopy was generally normal. Muscle
fibre diameter was larger in LGM subjects compared to control subjects.
Light Microscopy- motor nerve terminals were generally smaller and less compact, BgTx binding
site number was reduced in LGM subjects, and AChE high activity areas were smaller in the
LGM subjects.
Electron Microscopy - post synaptic folding was sig. reduced in LGM subjects.
Structure-Function Relationships
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Sig. positive correlation between quantal content and synaptic area, but no sig. difference
between quantal content: synaptic area ratio in LGM and control groups. This implies quantal
release per unit area is not decreased (even though quantal content is lower than usual in
LGM subjects).
The reduction in mEPP amplitude is largely due to increased muscle diameter, with a
reduction in local AChR density also playing a role.
Overall Conclusion:
Two main abnormalities observed:
1) reduced NMJ size
2) reduced post-synaptic folding.
 The neuromuscular transmission impairment seen in LGM subjects involves both pre- and postsynaptic factors.
- Overall conclusion: “The clinical weakness in these LGM patients is primarily a result of structural
abnormalities of the NMJ, rather than defects in the process of neuromuscular transmission itself”
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Strengths:
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A very comprehensive, novel study in to the neuromuscular transmission defects in LGM subjects
Clear aim, with an ultimate conclusion to the aim
Indicate clear further work areas
Weaknesses:
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A lot of raw data presented in the paper- may have been a better way of presenting it?
The authors do acknowledge that this experimental data was obtained from a small sample size,
from one area of Britain. A larger, more varied sample size could have added more weight to
their conclusions.
BBQ: What is the genetic component to myasthenia gravis and other myasthenic syndromes and how will
this determine effective treatments?
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DNA analysis was made on genes implicated in myasthenic disorders - AChR subunits, rapsyn (a
protein associated with intracellular AChR domains), ColQ (AChE collagenic tail peptide) and
MusK (involved in NMJ organisation).
No abnormalities/mutations were observed in any of these genes.
Further studies have indicated that a mutation in DOK7 ( a muscle protein essential for
neuromuscular synaptogenesis, that interacts with MusK) can lead to LGM
Currently, there is no uniform response to treatments for this syndrome- further work into
treatments targeting the DOK7 protein would evidently be beneficial.
Quantal analysis: Key Points
Methods of quantal analysis:
I) The Direct method: m=EPP/mEPP
II) Failures method: P(0)= exp(-m) or m=Ln(Tests/Failures)
III) Variance method: m=1/(C.V.)² or m=EPP²/var(EPP)
Where m=mean quantal content
1) The quantal Hypothesis
The quantal hypothesis of synaptic transmission delineates a relationship between the amplitudes of
mEPPs and EPPs. Importantly:
• The quantum underlying the smallest EPP and the spontaneous mEPP are one and the same;
• The release of each quantum is probabilistic and occurs independently of the next;
• An evoked EPP is caused by the synchronous release of several quanta, due to the large increase
in the probability of release of individual quanta.
According to the binomial distribution, for any number of quanta/vesicles n with a release probability of
p, a certain stimulus will release x quanta:
Since p and n are not estimated easily, this equation can be derived to obtain an equation conforming to
the Poisson distribution:
And m=Ln(Tests/Failures)
2) The safety factor at the NMJ
Wood & Slater (1997)
According to this graph, we can assume that:
- The minimal quantal content (QC) required to reach threshold ~ 15
- The quanta content released under normal physiological conditions ~ 45
 Having calculated the mean QC m, we can therefore calculate how far synaptic transmission at this
NMJ falls short of both i) the minimum QC required to reach threshold and ii) the amounts of
neurotransmitter released under normal physiological conditions by subtracting m from the values above.
3) Non-linear summation
Each quantal component of the EPP depolarises the membrane potential towards the reversal potential by
a small amount, but as the amount of overall depolarisation becomes greater each successive quantal
component has a weaker effect on further depolarisation.
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Direct method tends to underestimate mean QC
Variance method tends to overestimate quantal content – why?
Using the variance method, consider the formula: m=EPP²/var(EPP)
- When the QC is low, summation tends to be linear and each quantal component depolarises the
membrane by a certain amount, leading to a high variance (thus ↓m according to the equation).
- When the QC is high, summation tends to be non-linear and the effectiveness of each quantal packet
declines, leading to a smaller variance (thus ↑m and overestimation).
 Relationship between observed amplitude of EPP and the amplitude which would be obtained if
transmitter quanta produced linear summation (McLachlan, 1981):
V’=V/(1-f(V/E))
V’ = predicted amplitude
V = observed amplitude
f = ‘fudge factor’ – long muscle fibres have an f-factor of 0.8
E = (resting membrane potential – reversal potential)
As QC increases, there is a bigger discrepancy between the predicted and observed amplitudes, V’ and V
respectively (as shown in the Excel table during the seminar), indicating non-linear summation.
4) Possible explanations for a low quantal content:
- Botulinum Toxin
- Lambert-Eaton disease
- Congenital myasthenic syndrome
- Abnormal size due to degeneration or in response to injury
- Neonatal neurons
- Role of the postsynaptic folds in forming a high-resistance pathway and increasing the density
of VGSCs – amplification of released neurotransmitter (Martin, 1994).
-…
References
Martin A.R. (1994). Amplification of neuromuscular transmission by postjunctional folds. Proc. R. Soc.
Lond., 258, 321-326.
McLachlan E.M. & Martin A.R. (1981). Non-linear summation of end-plate potentials in the frog and
mouse. J Physiol., 311, 307-324.
Wood S.J. & Slater C.R. (1997). The contribution of postsynaptic folds to the safety factor for
neuromuscular transmission in rat fast- and slow-twitch muscles. Journal of Physiology, 500.1, 165-176.