an insight to the NMJ

1. THE NEUROMUSCULAR PHYSIOLOGY
PRESENTER - DR. SOURAV MONDAL
MODERATOR – DR. HANSRAJ BAGHEL, MD
DEPT OF ANAESTHESIOLOGY , SSMC, REWA, MP, INDIA

2. PARTS OF NMJ
The anatomy of NMJ consist of following parts:
 Pre-synaptic membrane
 Synaptic cleft
 Post-Synaptic membrane

4. STEPS IN NORMAL NM TRANSMISSION
Sodium and calcium flow through the open receptor channel generating an end-plate potential.
Opening of receptor channels. Receptors do not open unless both α receptors are occupied by ach
Ach molecules bind to the α subunits of the ach receptor on the post junctional membrane,
generating a conformational change and
Ach is released from storage vesicles at the nerve terminal. Enough Ach is released to bind
500,000 receptors.
Nerve action potential is transmitted, and the nerve terminal is depolarized.

5. After unbinding ACh, the receptors’ ion channels close, permitting the end-plate to
repolarize. Calcium is resequestered in the sarcoplasmic reticulum, and the muscle cell
relaxes.
ACh is rapidly hydrolyzed into acetate and choline by the substrate-specific enzyme
acetylcholinesterase .
when a threshold voltage is developed across them, a muscle action potential
(MAP) is generated
When between 5% and 20% of the receptor channels are open and a threshold
potential is reached, ....Voltage-gated sodium channels within perijunctional portion
of the muscle membrane open

6. ROLE OF CALCIUM
• The concentration of calcium and the length of time during which
it flows into the nerve ending, determines the number of quanta
release.
• If Ca+2 is not present , then depolarisation of the nerve , even by
electrical stimulation will not produce the release of transmitter
whereas doubling the extracellular Ca+2 results in 16-fold increase
in the quantal content of an end-plate potential
• Calcium current is normally stopped by the out flow of potassium.
• Calcium channels are specialized proteins, which are opened by
voltage change accompanying action potentials

7. • Part of calcium is captured by proteins in the
endoplasmic reticulum & are sequestrated.
• Remaining part is removed out of the nerve by the
Na+/Ca+2 antiport system
• The sodium is eventually removed from the cell by
ATPase

8. THE ACETYLCHOLINE
• Synthesized in the Presynaptic terminal from substrate
Choline and Acetyl CoA.
Choline Acetyltransferase
CHOLINE + ACETYL CoA ACETYL CHOLINE
Carrier
Facilitated Transport Release
CHOLINE + ACETYL CoA ACETYL CHOLINE
Acetylcholinesterase
Synaptic Cleft

9. SYNAPTIC VESICLE AND RECYCLING
• Different pools of acetylcholine in the nerve terminal have
variable availability for release :
 The immediately releasable stores, VP2: Responsible for
the maintainance of transmitter release under conditions
of low nerve activity. 1% of vesicles.
 The reserve pool, VP1: Released in response to nerve
impulses. 80% of vesicles.
 The stationary store: The remainder of the vesicles.
• The vesicles in the VP2 pool are bit smaller and limited to an
area very close to the nerve membrane , where they are
bound to the active zones.

10. • Majority of the synaptic vesicles (VP1) are
sequestered in the reserve pool and tethered to
cytoplasmic skeleton in a filamentous network made
up of primarily actin , synapsin , synaptotagmin and
spectrin.
• Each vesicle contains approx 12,000 molecules of
acetylcholine, which are loaded into the vesicles by
an active transport process in the vesicle membrane
involving a magnesium dependent H+ pump ATPase.

11. • Contents of a single vesicle constitute a quantum of
acetylcholine.
• Release of acetylcholine may either be
 Spontaneous , or
 In response to a nerve impulse.
• When a nerve impulse invades the nerve terminal, calcium
channels in the nerve terminal membrane are opened up.
• Calcium enters the nerve terminal and there is calcium
dependant synchronous release of the contents .

12. • The number of quanta released by each nerve impulse is very
sensitive to extracellular ionized calcium concentrations.
Increased calcium concentration results in increased quanta
released
• To enable this, vesicle must be docked at special release sites
(active zones) in that part of the terminal where the axonal
membrane faces the postjunctional acetylcholine receptor.
• The soluble N-ethylmaleimide-sensitive attachment protein
receptor (SNARE) protein are involved in fusion , docking and
release of Acetylcholine at the active sites. The SNARE
proteins involve the synaptic –vesicle protein, synaptobrevin;
the plasmalemma-associated protein, syntaxin; and the
synaptosome-associated protein of 25-kd (SNAP-25).

13. • These are vesicle from the immediately releasable
stores
• Once the contents have been discharged, they are
rapidly refilled from the reserve stores. Repeated
stimulation requires the nerve ending to replenish its
store of vesicles filled with transmitter , a process
known as mobilization.
• Uptake of Choline and the activity of Choline
acetyltransferase are probably the rate limiting
steps.

14. PROCESS OF EXOCYTOSIS
• When there is an action potential and calcium ions
enter , synapsin becomes phosphorylated , which
frees the vesicle from its attachment to the
cytoskeleton.
• Syntaxin and SNAP-25 are complexes attached to the
plasma membrane . After an initial contact ,the
synaptobrevin on the vesicle forms a ternary
complex with syntaxin and SNAP-25.

15. • Synaptotagmin is the protein on vesicular membrane
that acts as a calcium sensor , localizes the synaptic
vesicles to synaptic zones rich in calcium channels ,
and stabilizes the vesicles in the docked state.
• Assembly of the ternary complex forces the vesicles
to move close to the underlying nerve terminal
membrane and the vesicle is then ready for release.

16. ACETYLCHOLINESTERASE
• Acetylcholinesterase is a type B carboxylesterase enzyme.
• The protein enzyme is secreted from the muscle, but remain
attached to it by thin stalks of collagen, attached to the
basement membrane.
• Acetylcholine molecules that do not immediately react with a
receptor or those released after binding to the receptor are
almost instantly destroyed by acetylcholinesterase, in <1 ms,
after its release into the junctional cleft.

17. CHOLINERGIC RECEPTORS (CHOLINOCEPTORS)
Two families of cholinoceptors,
designated:
• MUSCARINIC receptors
• NICOTINIC receptors,
Types of cholinergic receptors

18. MUSCARINIC RECEPTORS
• There are five subclasses of
muscarinic receptors: M1,
M2, M3, M4, and M5.
• Only M1, M2 and M3,
receptors have been
functionally characterized.
• These receptors, in addition
to binding acetylcholine,
also recognize muscarine.
• Muscarine is an alkaloid that
is present in certain
poisonous mushrooms.

19. LOCATIONS OF MUSCARINIC RECEPTORS
Although all five subtypes have been found on
neurons, M1 receptors are also found on
gastric parietal cells, M2 receptors on cardiac
cells and smooth muscle, and M3 receptors on
the bladder, exocrine glands, and smooth
muscle.

20. When M1 and M3 receptors are
activated, the receptor undergoes a
conformational change and interacts
with a G protein, designated Gq , which
in turn activates phospholipase C (PC).
This leads to the hydrolysis of
phosphatidylinositol-(4,5)-bisphosphate-
P2 to yield diacylglycerol and inositol
(1,4,5)-trisphosphate , which cause an
increase in intracellular Ca2+ .
This action can then interact to
stimulate or inhibit enzymes, or cause
hyperpolarization, secretion, or
contraction.
MECHANISMS OF ACETYLCHOLINE SIGNAL
TRANSDUCTION ON M1 & M3 RECEPTORS

21. MECHANISMS OF ACETYLCHOLINE SIGNAL
TRANSDUCTION ON M2 RECEPTOR
M2 subtype on the cardiac muscle stimulates a G
protein, designated Gi , that inhibits adenylyl cyclase
and increases K+ conductance, to which the heart
responds with a decrease in rate and force of
contraction.

23. NICOTINIC RECEPTORS
The nicotinic receptor is composed of
five subunits, and it functions as a ligand-
gated ion channel.
Binding of two acetylcholine molecules
on α subunits elicits a conformational
change that allows the entry of sodium
ions, resulting in the depolarization of
the effector cell.
 Another isoform of Ach contains a γ
subunit instead of the ε subunit known as
fetal or immature receptor, because this
form initially expressed in fetal muscle,
often referred to as extrajunctional
receptors.

24. LOCATION OF NICOTINIC RECEPTORS
Nicotinic receptors are located in the CNS,
adrenal medulla, autonomic ganglia, and
the neuromuscular junction. Those at the
neuromuscular junction are sometimes
designated NM and the others NN.

26. THE ACETYLCHOLINE RECEPTORS

27. POST JUNCTIONAL RECEPTORS
Three isoforms of
postjunctional
niconitinic AChRs
exist :
 A junctional or
mature receptors
 an extrajunctional
or immature (fetal)
receptor
 the recently
described neuronal
α7 receptor

28. JUNCTIONAL OR MATURE RECEPTORS
• Present in the post junctional membrane of the
motor end plate & are of nicotinic type. These
receptors exist in pairs.
• It consists of protein made up of 1000 amino acids,
made up of 5 protein subunits designated as alpha
(α), beta (β) , delta (δ) and epsilon (ε) joined to form
a channel that penetrates through and projects on
each side of the membrane.

29. • Each receptor has central funnel shaped core which
is an ion channel, 4 nm in diameter at entrance
narrowing to less than 0.7nm within the membrane.
• The receptor is 11 nm in length and extends 2nm
into the cytoplasm of the muscle cell.
• The receptor has 2 gates, an Upper voltage-
dependent and a Lower time-dependent.

30.  When acetylcholine receptors bind to the pentameric
complex, they induce a conformational change in the
proteins of the alpha (α) subunits which opens the
channel and it occurs only if it binds to both the alpha
(α) binding sites.
 For ions to pass through the channel both the gates
should be open.
 Cations flow through the open channel, Na+ and Ca+2 in
and K+ out, thus generating end plate potential.
 Na+ ions are attracted to the inside of the cell which
induces depolarisation.

31. THE SODIUM CHANNELS
• Sodium channels are present in muscle membrane.
• Perijunctional areas of muscle membrane have a higher
density of these sodium channels than other parts of the
membrane.
• These sodium channels have two types of gate
- voltage dependent
- time dependent
• Sodium ions pass only when both gates are open.

32. The channel therefore
possesses three
functional states.
 A...At rest, the lower
gate is open but the
upper gate is closed
 B...reaches threshold
voltage depolarization,
the upper gate opens
and sodium can pass
 C...Shortly after the
upper gate opens the
time dependent lower
gate closes

33. POSSIBLE CONFIGURATION OF Na CHANNELS
• Resting state: Voltage gate closed
Time gate open
Channel closed
• Depolarization: Voltage gate open
Time gate open
Channel open
• With in a few milliseconds: Voltage gate open
Time gate closed
Channel closed
• End of depolarization: Voltage gate closed
Time gate open
Channel closed

34. EXTRAJUNCTIONAL RECEPTOR
• These tend to be concentrated around the end plate,
where they mix with post junctional receptors but may be
found anywhere on the muscle membrane. In them, the
adult epsilon (ε) subunit is replaced by the fetal gamma
(γ) subunit.
• They are not found in normal active muscle, but appear
very rapidly after injury or whenever muscle activity has
ended.
• They can appear within 18 hrs of injury and an altered
response to neuromuscular blocking drugs can be
detected in 24 hrs of the insult.

35. • When a large number of extrajunctional receptors are
present, resistance to non-depolarising muscle
relaxants develops, yet there is an increased sensitivity
to depolarising muscle relaxants.
• In most extreme cases, increased sensitivity to
succinylcholine results in lethal hyperkalemic receptors
with an exaggerated efflux of intracellular potassium.
• The longer opening time of the ion channel on the
extrajunctional receptor also results in larger efflux.

36. THE NEURONAL α7 RECEPTORS
• The neuronal α7 AchRs consists of five α7 subunits. Each of all
receptor subunits consists of approximately 400 to 500 amino acids.
• The receptor protein complex pass entirely through the membrane
and protudes beyond the extracellular surface of the membrane
and into the cytoplasm.
• In α7 AChRs , however , even when three subunits are bound by an
antagonist , the two other subunits are still available for binding by
agonist and cause depolarisation . This feature may account for
some of the resistance to muscle relaxants when α7 AChRs are
expressed In muscle and in other tissues during pathologic states
like sepsis, denervation , immobilisation , burns etc.

37. MAINTENANCE OF MATURE
NEUROMUSULAR JUNCTIONS
• Multiple factors including electrical activity, growth
factor signaling and the presence or absence of
innervation , control the expression of the three type
of receptor isoforms.
• The nerve releases several growth factors that
influence the synaptic apparatus of nearby nuclei.

38. FINALLY
Nerve-derived growth factors ,including AGRIN and ARIA/NEUREGULIN , cause the
receptors to cluster in the subsynaptic area and prompt the expression of mature isoform.
SECONDLY
The nerve-induced electrical activity results in the repression of receptors in the
extrajunctional area.
FIRSTLY
Nerve-supplied factors induce the synaptic nuclei to increase synthesis of AChRs.

39. • Before innervation , as in the fetus , AChRs are
present throughout the muscle membrane. After
innervation , AChRs become more and more
concentrated at the postsynaptic membrane and are
virtually absent in the extrasynaptic area after birth.
• In the active adult, and normal innervated muscle ,
just the nuclei under and very near the end plate
direct the synthesis of the receptor . Nuclei beyond
the junctional area are not active , and therefore no
receptors are expressed anywhere in the muscle cells
beyond the perijunctional area.

40. PREJUNCTIONAL RECEPTORS
• These are nicotinic receptors that control ion channel
specific for Ca+2 which is essential for synthesis and
mobilization of acetylcholine and are composed of
alpha (α) and beta (β) subunits only.
• They contain protein subunits that are blocked by
non depolarising muscle relaxants resulting in tetanic
fade and train-of-four fade.

41. NEUROMUSCULAR BLOCKING AGENTS –
CLASSIFICATION ,MECHANISM & DURATION OF
ACTION
DEPOLARIZING
Short-acting
• Succinylcholine
NONDEPOLARIZING
Short-acting
• Gantacurium
• Mivacurium
Intermediate-acting
• Atracurium
• Cisatracurium
• Vecuronium
• Rocuronium
Long-acting
• Pancuronium
• Pipecuronium
• Doxacurium

42. MECHANISM OF ACTION of
DEPOLARIZING NMBA
1
• Depolarizing muscle relaxants closely resemble ACh and readily bind to ACh
receptors, generating a muscle action potential.
2
• Unlike ACh, however, these drugs are not metabolized by acetylcholinesterase,
and their concentration in the synaptic cleft does not fall as rapidly, resulting in a
prolonged depolarization of the muscle end-plate.
3
• Continuous end-plate depolarization causes muscle relaxation
4
• opening of perijunctional sodium channels is time limited (sodium channels rapidly
“inactivate” with continuing depolarization)

43. PHASES OF BLOCK IN
DEPOLARIZING NMBA
Phase I block
• Perijunctional sodium channel cannot reopen until the
end-plate repolarizes.
• The end-plate cannot repolarize as long as the
depolarizing muscle relaxant continues to bind to ACh
receptors; this is called a phase I block.
Phase II Block
• After a period of time, prolonged end-plate
depolarization can cause changes in the ACh receptor
that result in a phase II block.

44. MECHANISM OF ACTION OF NON-
DEPOLARIZING NMBA
Nondepolarizing muscle
relaxants function as
competitive antagonists.
Nondepolarizing muscle
relaxants bind ACh receptors
but are incapable of inducing
the conformational change
necessary for ion channel
opening.
Because ACh is prevented
from binding to its
receptors, no end-plate
potential develops.
Neuromuscular blockade
occurs even if only one α
subunit is blocked.

45. NEUROMUSCULAR JUNCTION AT
EXTREMES OF AGE
• NEWBORN
 Just before birth , the AChRs are all clustered around the junctional
area, and minimal extrajunctional AChRs are present.
 The newborn postsynaptic membrane ,itself , is not specialised ,
having almost no synaptic folds , a widened synaptic space , and a
reduced number of AChRs.
 The early postnatal AChR clusters appears as an oval plaque.
 Within a few days simplified folds appear.

46.  With continued maturation , the plaque is transformed to a
multiperforated pretzel-like structure.
 The polyinnervated end plate is converted to a singly innervated
juntion because of a retraction of all but one terminal.
• OLD AGE
 Anatomic changes involve increased preterminal and axonal
branching within the individual neuromuscular junction, either with
or without an increase in the junctional size.
 The points of contact between the junctional and post-junctional
membrane decrease , resulting in a decline in trophic interactions
between nerve and muscle and stimulus transmission which in turn
results in age-associated functional denervation, muscle wasting
and weakness.