Neuromuscular junction anatomy, physiology and basics

1. ANATOMY AND PHYSIOLOGY OF
NEUROMUSCULAR
JUNCTION
Speaker-Dr. Prabhat Bid(2nd year Pgt)
Moderator-Dr. Satyajit Bera(R.M.O CT)
-Department of Anaesthesiology
-Medical College Kolkata

2. What is the NMJ
 The NMJ is a synapses at which electrical impulses travelling
down the motor nerve, releases the chemical
messengers/transmitter which causes the muscle to contract.
 The NMJ is the chemical synapse between an alpha motor
neuron and a muscle cell. The transmission of motor action
potentials or indeed their prevention is an obvious importance
to anaesthesists.
The α-motor neuron originates in the ventral horn of the spinal
cord. Its axon is myelinated, as the conduction of motor action
potentials needs to be rapid. Before the axon reaches the NMJ,
it branches to innervate several muscle cells. A motor unit
consists of an α-motor neuron and the muscle cells that it

3. For neuromuscular transmission there
are 3 essential components:
 1.PRESYNAPTIC PART(MOTOR NERVE
TERMINAL)
 2.SYNAPTIC CLEFT
 3 POSTSYNAPTIC MEMBRANE (MUSCLE END
PLATE)

4. ANATOMY

5. Neuro Muscular Junction

6. MOTOR UNITS
 Motor unit consist of an alpha- motor neuron and the
muscle fibre/fibres it innervate.
Each motor neuron has its origin in the ventral horn of
the spinal cord or medulla and runs an uninterrupted
course as a large, myelinated axon to the
neuromuscular junction. Each neuron branches and
supplies several muscle cells, which together form the
motor unit.

7.  The NMJ junction or the endplate is a highly specialized
synapse at which presynaptic motor nerve endings
meet the post synaptic membranes of skeletal muscles.
 Each motor neuron approaches its target muscle fiber,
its loses its myelin sheath and makes a contact with a
single muscle fiber to form a NMJ.
The more delicate the movements, the fewer muscle
fibres per motor neuron.
 More intense contraction = more motor unit & long
muscle fibres.

8. PRESYNAPTIC NERVE ENDING
 Presynaptic part consists of distal demyelinated part of the
motor nerve axon and is separated from the extra cellular fluid
by extension of the terminal Schwann cells and insulates the
entire structure.
 Contents of the presynatic end:
- Calcium channel (P type- fast)
- Acetylcholine vesicles(3 types)
- Mitochondria
- Active zones
- Proteins: Synaptotagmin,Synaptobrevin, Syntaxin,
Synaptosome associated protein 25(SNAP 25)
- Presynaptic nicotinic AchRs.

9. ACETYLCHOLINE VESICLES
 3 lacs vesicles in an end plate
 45 nm : bound by lipid bilayer membrane.
 Active zones
 5000-10,000 molecules of Ach in 1 vesicle, loaded
by
Mg++ dependent proton pumping ATPase
 1% - Releasable store(VP2)
80% - Reserve pool(VP1)
Rest - Stationary pool

11.  The SNARE (soluble N-ethylmaleimide-sensitive
attachment protein receptors) proteins are involved in
fusion, docking, and release of acetylcholine at the
active zone
 Synaptophysin is a glycoprotein component of the
vesicle membrane.
 Phosphorylation of another membrane protein,
synapsin, facilitates vesicular trafficking to the release
site.
 Synaptotagmin is the protein on the vesicular
membrane acts as a calcium sensor and localize the
synaptic vesicles to synaptic zones rich in calcium

12.  Synaptobrevin is a vesicle associated membrane
protein (VAMP). During depolarisation & entry of Ca
it unfolds & forms a ternary complex with
syntaxin/SNAP-25.
Assembly of this complex guides the vesicle to the
active zone.(Docking and Fusion)
Then ca influx sensed by Synaptotagmine leads to
burst release of Ach.

13. MODEL OF PROTEIN MEDIATED MEMBRANE
FUSION AND EXOCYTOSIS

14. Acetylcholine synthesis

15. Acetylcholine Storage
 Once synthesised Ach is packaged into vesicles. Each vesicle
contains around 5000 Ach molecules known as QUANTUM.
 THERE ARE FUNCTIONALLY 3 TYPES OF VESICLES :
 1.Vesicles in the active zones VP2(1%)-this vesicle are docked at
presynaptic membrane ready for immediate realease.
 2.Vesicles in the reserve pools VP1(80%)-this vesicles move
forwards to replace the vessicles in the active zone as they are
used.
 3.Vesicles in the stationary store(19%)-this vesicles cannot
released their Ach

16. Vesicle Types at presynaptic nerve
terminal

17. SYNAPTIC CLEFT
 The nerve is
separated from the
muscle by a gap of
20 – 50 nm, called
the synaptic cleft.
 Contents:
1. Extracellular Fluid
2.Acetylcholinestera
se

18. MUSCLE END PLATE
 The motor end plate is a specialized region of the
sarcolemma (muscle membrane). It is oval,
spanning an area of 3000 micrometre square. It is
heavily corrugated, with deep invaginations, called
the primary and the secondary clefts, to increase the
surface area. The nicotinic Ach receptors are
densely populated in the shoulders of the clefts (5
million AChR per junction), while the sodium
channels are located in the depths of the folds.

19. MUSCLE END PLATE
 1.nAchRs
 2.Na Channels
-Voltage dependent gate (VDG)
-Timed dependent gate (TDG)
VDG opens till depolarisation persists but TDG closes
and cuts off the flow of sodium. TDG does not
open again until VDG closes and reopens with a
fresh depolarisation.

20.  Synthesised in muscle cells
 Cylindrical receptor – central pore as ion channel
 MW-250000 Dalton.
 Each subunit consist 400-500 amino acids.
 One molecule of Ach binds with each α subunits at
amino acid sequence 192-193
ACh RECEPTORS

21. Acetylcholine Receptor
 2 TYPES :–
 MUSCARINIC ( M1 –M5)
 NICOTINIC-
Nm – skeletal muscle end plate.
Nn – adrenal medulla, ganglionic cells etc.

22. nAchRs
1.Adults nAchRs is a
pentameric complex consist of
2α, 1β, 1, 1ϵ.
2.Fetal/ Extrajunctional- 2α,
1β, 1, 1.
These subunits form a
transmembrane pore and a
pocket for acetylcholine
binding.

23. Adult versus Fetal nAchR

24. Depolarisation of Nr
terminal
↓
Opening of voltage gated
Ca channel
↓
Entry of Ca in nerve
terminal
↓
Mobiliation of synaptic
vesicles
↓
Binding to docking protein
↓
Fusion of vesicles
↓
Release of ACh into

25. Action Potential

26.  At the presynaptic zone….
 Nerve action potential causes membrane depolarization.
 Activation of voltage dependent p-type calcium channel occurs.
 Entry of calcium into the axoplasm.
 Release of vesicles (VP2) from active zone .
 Docking and pore formation occurs via SNARE protein.
 Release of Ach in the synaptic cleft.
Neuro muscular transmission

27.  Post-junctional zone……..
 Two molecules of Ach binds with the 2 alpha subunits of Ach
receptors .
 Conformational change of the receptors leads to opening of the
channel.
 Inward flow of Na+ and Ca2+ and outward flow of K+ starts.
 Change of membrane potential occurs.
 Activation of voltage sensitive Na+ channels.
Neuro-Muscular transmission
cont’d…

28.  Post-junctional zone……..
 Na entry across the muscle membrane via Na channel causes
initiation and propagation of action potential to the T-
tubules(upstokes of AP)
 DHPR(L-VGCC) types of ca channels get activated in the T-
tubules.(voltage sensor)
 Activates RyR1 receptor in sarcoplasmic reticulum.
 Muscle contraction occurs.
Neuro-Muscular transmission
cont’d…

29. Termination of Neurotransmission
 Ach rapidly removed from the synaptic cleft mainly
by degradation.
 1.Ach is rapidly hydrolysed by the enzyme AchE to
choline and acetic acid, this product are actively
transported into presynaptic membrane for
resynthesis of Ach.
 2.AchE is mainly found in the junctional fold of the
synaptic cleft.

31. Structures of skeletal muscle
 Two type of contractile filaments- a)thick – myosin
b)thin - 3 types
actin.
tropomyosin.
troponin.

32. Thick filaments
 Myosin – 2 heavy chains + 4 light chains.
 Heavy chain twist over each other and form a
double helix .
 Each heavy chain has 2 light chain heads.
 Light chain – ATP ase action.

33. Thin filaments
 Actin – two types – G actin and F actin.
 Tropomysoin – covers active site of actin molecules
to which myosin heads can attach.
 Troponin – 3 types :- I, T, C.

34. Clinical relevance
 Neurotransmission at the NMJ can be blocked by
following mechanism
 1. inhibision of Ach synthesis- Hemicholinium blocks
the uptake of choline in the nerve action.
 2.inhibition of vesical exocytosis –
a-Mg and amynoglycosides-block the presynapic
voltage gated ca channel.
b- Botulinum toxin-It degrades a protein known as
SNAP-25 required for vesicle docking.

35. Clinical relevance continue
 3.blockage of Ach receptor-
a.Depolarising MR
b.Non depolarising MR

36.  Depolarization of membrane opens the voltage dependent
gate of peri junctional Na+ channels.
 Influx of Na+ occurs.
 After some time, Time dependent gate of Na+ channel closes.
 But the voltage dependent gate remains open as there is
presence of Sch in the environment and
 Na+ channels remains in that state and propagation of
depolarization stops.
 This is also called Phase I block.
 Characterized by a) decrease in twitch tension, (b) no fade
during repetitive stimulation (tetanic or TOF), and (c) no post
tetanic potentiation.
Action of depolarizing MR

37. ILLUSTRATION OF SODIUM CHANNEL
1.At rest lower
gate is opened
and upper gate
is
closed.(resting
state)
2.When muscle
get depolarised the
upper gate opens
and sodiun
enter.(active state)
3.shortly after that
the lower gate
closed
(inactive state)

38.  It is a complex phenomenon associated with typical fade in muscle
during continuous exposure to depolarizing drug.
 Causes due to depolarizing action of Succinyl choline on pre
junctional neuronal Ach receptors.
 Higher concentration of succinyl choline is needed.
 During repetitive nerve stimulation post junctional receptors also
get involved.
 Repeated opening of channels causes Na+ inflow and K+ outflow
results in abnormal membrane potential.
 Inflow of Ca2+ causes disruption of receptors and sub end plate
structures.
 The activity of Na+-K+-ATPase is increased to restore membrane
potentials.
Phase II Block

39.  Normally, the two ACh molecules have to combine
with the two alpha 1 subunits of the nAChR to cause
conformational change resulting in opening of the
channel and flow of Na and Ca into the muscle and
potassium ions flow outside. The channel excludes
anions like chloride. The influx of ions depolarizes
the adjacent membrane and causes the muscle to
contract. When one or both ACh molecules detach
from the receptor, the channel closes by a reversed
mechanical conformation. This stops the
depolarization.
Actions of acetylcholine on end plate
receptor

40. Classic Action of NDMR
 Both α subunits of nAchR, must be occupied by
agonist for cationic flow across the membrane
 NDMRs  inhibits depolarization by competitive
inhibition of Ach from its binding site.
 Relative concentration and affinity determines the
effectiveness of a particular NDMR.
 When it binds to one or both of the binding site 
conformational change of Ach receptor  no
cationic flow across membrane occurs.

41. Continued....
 If choline esterase inhibitor is added - neostigmine 
Ach concentration remains high
 increase the chance of binding of 2 Ach even though
NDMR is present.
After large doses of NDMR / absence of clinical signs of
reversal (high concentration of NDMR)
 neostigmine ineffective  until the concentration of
the relaxant lowers in perijunctional area by elimination /
redistribution.
 Anticholinesterase is not indicated at deep block.

42. Actions of acetylcholine or Non depolarising
muscle relaxant(NDMR) on end plate receptor

44. Receptor desensitization
 AchRs Bind with agonist (also with antagonist) but
remains inactivated.
 Some Lipid soluble drugs also causes
desensitization of receptors.
 Desensitized receptors potentiate action of muscle
relaxants.
 The aberrant attachment of drugs to the recognition
site probably responsible for this type of block.

45. Few drugs causes receptor desensitization

46. NEURO MUSCULAR DISORDER
 1- MYASTHENIA GRAVIS
 2-LAMBERT EATON MYASTHENIC SYNDROME
 3-PERIODIC PARALYSIS

47.  Is a chronic autoimmune disorder .
 Autoantibody forms against the α subunit of muscle type of
nicotinic Ach receptors causes its destruction or inactivation.
 As many as 80% of functional receptors may be lost.
 Incidence varies between geographical regions
◦ Young age  female > male
◦ >60 yrs  male > female
 70%  thymic hyperplasia; 10%  thymomas
 May occur as a part of paraneoplastic syndromes
Myasthenia gravis

48.  Ocular, pharyngeal and laryngeal muscles are involved causing ptosis, diplopia and
dysphagia.
 Patchy distribution of muscle weakness
◦ Symptoms vary from day to day
◦ Remission of varying duration
 These patients are at the risk of pulmonary aspiration.
 Myocarditis may cause heart block, cardiomyopathy.
 Diagnosis :-
◦ Neurological examination
◦ Weakness, easy exhaustion of skeletal muscle and partial recovery with rest is
the hallmark.
 Confirmation :- Tensilon test
◦ Administration of an anti-cholinesterase e.g. edrophonium
◦ Improvement in 5 minutes and lasts for 10 minutes
 Electrophysiologic evaluation  a classic decrement in the compound muscle
action potential after repetitive nerve stimulation
Myasthenia gravis

49.  Treatment :-
◦ Oral pyridostigmine is the drug of choice, maximum dose is 120mg every 3 hourly.
◦ Immunosuppressive therapy with corticosteroid, azathioprine, cyclosporin, mycophenolate.
◦ Plasmapheresis is done for Myasthenic crisis.
◦ Thymectomy is one of the treatment option.
 Anaesthetic considerations :-
◦ Pulmonary function tests
◦ Risk for post-operative mechanical ventilation
◦ Succinylcholine for tracheal intubation
 Increased dose requirement
 Prolonged neuromuscular block
◦ Potent volatile anaesthetics :- decreased margin of safety  decreased requirements of
muscle relaxants
◦ Epidural/ Spinal anaesthesia :- need for post-operative monitoring of muscle function and
ventilation
Myasthenia gravis

50.  Frequently a part of Auto-antibody against P/Q type voltage sensitive
calcium channel at prejunctional membrane and ANS.
 paraneoplastic syndromes – usually Small cell lung cancer.
 Decrease in number of channels.
 Restricted calcium entry during AP.
 Muscle weakness and fatigability
◦ Proximal > Distal
◦ Lower extremities > Extraocular/Bulbar muscle groups
 Unlike Myasthenia gravis
◦ Worse in morning  Gradual improvement throughout the day
◦ Improvement of muscle function with exercise
Eaton-Lambert Myasthenic syndrome

51. Treatment  Plasmapheresis, Immunoglobulins, 3,4-
diaminopyridine  Temporary relief
 Anaesthetic implications :-
◦ Anticholinesterase drugs are ineffective as reversal of
neuromuscular blockade
◦ Risk of postoperative respiratory failure  need for prolonged
respiratory monitoring in the postoperative period
Eaton-Lambert Myasthenic syndrome

52. QUANTAL THEORY
 Spontaneous depolarizing potentials at neuromuscular junctions can
be seen. This potentials have only one hundredth the amplitude of the
evoked end-plate potentia produced when the motor nerve is
stimulated. These small-amplitude potentials are called miniature
end-plate potentials (MEPPs)

53.  Because MEPPs are too big to be produced by a single molecule of
acetylcholine, it was deduced that they are produced by uniformly sized
packages, or quanta, of transmitter released from the nerve (in the
absence of stimulation). The stimulus-evoked end-plate potential is the
additive depolarization produced by the synchronous discharge of quanta
from several hundred vesicles.
 The amount of Ach released by each nerve impulse is large, atleast 200
quanta of about 50,000 molecules each & the number of AchRs activated
by transmitter released is about 500,000

54. THANK YOU