The document discusses the history and anatomy of the neuromuscular junction, including how red indians first discovered curare's ability to block the NMJ without understanding it. It then covers the parts of the NMJ, how acetylcholine is synthesized and released, the types of nicotinic acetylcholine receptors present, and the clinical significance of these components in neuromuscular transmission and the effects of neuromuscular blocking drugs.

1. Presented by
Dr Prathibha K T
PG in Anaesthesiology
Chairperson :
Dr Suma K V MD
Assistant Professor
13/08/2013

2. HISTORY
 Red Indians used arrow poison to hunt their food, using
Curare as the poison. Unknown to them they laid the
foundation of blocking the NMJ.
 On 23rd January 1942 Harold Griffith, assisted by his
resident Enid Johnson injected intocostrin IV, a
preparation containing curare, to aid muscle relaxation
in a 150 lbs male patient undergoing interval
appendicectomy under cyclopropane anaesthesia at the
homeopathic hospital (later the Queen Elizabeth
Hospital) in Montreal. This was an outstanding event of
supreme importance in the development of modern
anaesthesia.

3. The introduction of Neuromuscular blocking
agents into clinical anaesthesia in 1942 was one of the
milestones in the history of speciality. It is very
important for an anaesthesiologist to know about the
anatomy and physiology of neuromuscular junction
before producing a neuromuscular block.

4. DEFINITION
 The NMJ is a synapse at which an electrical impulse
travelling down a motor nerve, releases chemical
transmitter which cause the muscle to contract.
 A single nerve fibre runs from the ventral horn of the
spinal cord as a myelinated axon upto neuromuscular
junction. As it approaches the muscle it divides and
joins the muscle branches. The nerve fiber and the
muscle fibres it innervates form a functional unit ‘the
motor unit’.

6. PARTS OF NMJ
 The anatomy of NMJ consist of following parts:
1. Pre-synaptic membrane
2. Synaptic cleft
3. Post-Synaptic membrane
• The nerve is separated from the surface of the
muscle by a gap of about 20nm called junctional
cleft.
• Presynaptic membrane contains prejunctional
acetylcholine receptors and active zone.

7.  Synaptic cleft: Lies Between the muscle endplate and
nerve terminal which are held in tight alignment by
basal lamina.
 Post synaptic membrane – acetylcholine
receptors: At the post synaptic membrane the area
overlying the nerve terminal is called muscle end
plate. The membrane here is thrown into primary and
secondary clefts.
 At the shoulder of these clefts numerous acetylcholine
receptors are present.

10. CLINICAL SIGNIFICANCE
• One NMJ per cell
• Except extra ocular muscles – Tonic
muscles
• EOMs are multiply innervated with
several NMJs
• Depolarising relaxants affect them
differently.

11. Ach (Synthesis, storage, release)
 Synthesized in the Presynaptic terminal from substrate
Choline and Acetyl CoA.
CAT
CHOLINE + ACETYL CoA ACETYL CHOLINE
COMT
50% Carrier Facilitated Transport Release
CHOLINE + ACETYL CoA ACETYL CHOLINE
Synaptic Cleft

12.  Different pools of acetylcholine in the nerve terminal
have variable availability for release
a) The immediately releasable stores, VP2: Responsible
for the maintenance of transmitter release under
conditions of low nerve activity. 1% of vesicles
b) The reserve pool, VP1: Released in response to nerve
impulses. 80% of vesicles
c) The stationary store: The remainder of the vesicles.

13.  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.
 Contents of a single vesicle constitute a quantum of
acetylcholine.
 Release of acetylcholine may be
a) Spontaneous or
b) In response to a nerve impulse.

14.  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 from
50-100 vesicles.
 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.

15.  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 receptors.
 These are vesicle from the immediately releasable
stores

16.  Once the contents have been discharged, they are
rapidly refilled from the reserve stores.
 The reserve vesicles are anchored to actin fibrils in the
cytoskeleton, by vesicular proteins called synapsins
 Some calcium that enters the axoplasm, on the arrival
of the nerve impulse binds to calmodulin, which
activates protein kinase-2 which phosphorylates
synapsins, which, in turn dissociates the vesicle from
the actin fibrils allowing it to move forward to the
release site.

17. • ‘P’ Channel : Only in Nerve terminal, Voltage Dependent
• ‘L’ Channel : Slower, Cardiac fibers
• Eaton Lambert Myasthenic Syndrome : Autoimmune Disease
• Antibodies against Voltage gated Ca++ Channels
• Decreased Ca++ Channel Function -> Decreased Ach release ->
Muscle Weakness

18.  Docking of the vesicle and subsequent discharge of
acetylcholine by exocytosis, involves several other
proteins.
 Membrane protein called SNAREs ( Soluble N-
ethylmaleimide sensitive attachment proteins) are
involved in fusion, docking, and release of
acetylcholine at the active zone.
 SNARE includes – synaptic vesicle protein
synaptobrevin, synataxin and SNAP-25.

20. CLINICAL SIGNIFICANCE
• Botulinium Toxin & tetanus Neurotoxins
• Selectively digest one or all of these SNARE proteins
• Block exocytosis
• Muscle weakness or paralysis
• Used therapeutically – to treat spasticity or spasm

21.  The released acetylcholine diffuses to the muscle type
nicotinic acetylcholine receptors which are
concentrated at the tops of junctional folds of
membrane of the motor end plate.
 Binding of acetylcholine to these receptors increases
Na and K conductance of membrane and resultant
influx of Na produces a depolarising potential, end
plate potential.
 The current created by the local potential depolarise
the adjacent muscle membrane to firing level.

22.  Acetylcholine is then removed by acetyl cholinesterase
from synaptic cleft, which is present in high
concentration at NMJ.
 Action potential generated on either side of end plate
and are conducted away from end plate in both
directions along muscle fibre.
 The muscle action potential in turn initiates muscle
contraction

23. NICOTINIC ACETYLCHOLINE RECEPTORS
 The most important receptors in the neuromuscular
junction are the nicotinic acetylcholine receptors
(nAChRs).
 Being the most studied receptor, the nAChR is the
prototype for the cys-loop superfamily of ligand-gated
ion channels which also includes GABA, glycine and 5-
HT3 receptors. All members of the cys-loop ligand-
gated ion channel superfamily share a common
structure and function. They have a common
architecture with five subunits surrounding a central
pore .
 Activation of the nAChR by ACh leads to an influx of
cations (i.e. Na+ and Ca2+) that lead to depolarization
of the cell membrane

24.  The acetylcholine receptors are nicotinic and are of
following types:
A. Pre synaptic
B. Post synaptic
Junctional or mature
Extra junctional or immature
Neuronal α7 receptor

25. Presynaptic nAChRs

26. Nicotinic autoreceptors are localized at the presynaptic nerve
terminal and are responsible for the increased release of ACh into the
synaptic cleft during high frequency stimulation.
This cholinergic receptor has now been identified as the
neuronal nAChR 32. Targeted pharmacological inhibition of this
receptor during high frequency repetitive (i.e. tetanic) stimulation
causes a typical tetanic fade phenomenon.
It was recently demonstrated that non-depolarizing NMBAs in
clinical use, inhibit this 32 nAChR and this explains why TOF fade is
absent during depolarizing block.

27. Acetyl choline receptors/Post junctional 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 5 protein subunits
designated as alpha, beta, delta and epsilon joined to
form a channel that penetrates through and projects
on each side of the membrane.

29.  In the fetus, gama replaces epsilon subunit.
 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
• Upper voltage- dependent
• Lower time-dependent

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

31. 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 subunit is replaced by the fetal gamma 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 18hrs of injury and an altered
response to neuromuscular blocking drugs can be detected
in 24hrs of the insult.

32.  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
response with an exaggerated efflux of
intracellular potassium.
 The longer opening time of the ion channel on the
extrajunctional receptor also results in larger efflux.

33. 7 AchR characteristics :
• Resistant to NDMR – requiring  dosage
• No desensitisation seen.
•  sensitivity to depolarizing agents like Sch
• Leading to prolonged K+ efflux  hyperkalemia
and ventricular fibrillation.

34. Video

35. 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.
 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

36.  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 antiport system
 The sodium is eventually removed from the cell by
ATPase

37. ACETYLCHOLINESTERASE
 This 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 don’t interact with
receptors are released from the binding site & are
destroyed almost immediately by acetylcholinesterase,
in <1 ms, after its release into the junctional cleft.

38. Effects of Electrolytes on Ach release
 Ca2+   ECF Ca2+   Ach release
 Excitability of nerve membrane
 Mg2+   ECF Mg2+   Ach release
Postsynaptic membrane stabilized
 K+   ECF K+  Hyperpolarizing membrane 
Resistant to action of Ach/Sch
But potentiate NDMR blockade
 dosage requirement in
hypokalemia (K+<2.5mm)

39. PHYSICAL CHANNEL BLOCKADE
 Various drugs can block the neuromuscular junction
and prevent depolarisation.
 Blockade can occur in two modes
a) Blocked when open
b) Blocked when closed

40. OPEN CHANNEL BLOCK
 In this, the drug molecule enters a channel which has
been opened by acetylcholine.
 This is use dependent block
 Physical blockade by a molecule of an open channel relies
on the open configuration of the channel and the
development of this is proportional to the frequency of
channel opening.

41.  This mechanism may explain the synergy that occurs
with certain drugs such as local anaesthetic,
antibiotics and muscle relaxants.
 In addition, the difficulty in antagonizing profound
neuromuscular blockade may be due to open channel
block by the muscle relaxants

42. CLOSED CHANNEL BLOCK
 The drugs occupy the mouth of the channel and
prevents ions from passing through the channel to
depolarise the end plate.
 Tricyclic drugs and naloxone may cause physical blockade
by impending interaction of acetylcholine with the
receptor.
 For drugs interfering with the function of the
acetylcholine receptor, without acting as an agonist or
antagonist, the receptor lipid membrane interface may
also be another site of action.
Eg: Volatile agents, Local anaesthetic and Ketamine

43. NEURONS
The functional and
structural unit of the nervous
system specialized to conduct
information from one part of
the body to another
There are many
different types of neurons but
most have certain structural
and functional characteristics
in common.

44. Membrane potential
Electrical potentials exist across the
membranes of essentially all cells of the body.
Nerves and muscle cells – capable of self-
generating electrochemical impulse at their
membranes.
Used to transmit signals along the
membranes. Na, K and Cl are involved in the
development of membrane potentials.
Concentration difference of ions across a
selectively permeable membrane can, under
appropriate condition cause the creation of a
membrane potential.

45. Basic Physics of Membrane
Potentials:
The potassium concentration is great inside a nerve
fiber membrane but very low outside the membrane.
Because of the large potassium concentration gradient, there
is a strong tendency for extra numbers of potassium ions to
diffuse outward through the membrane.
As they do so, they carry positive electrical charges to
the outside, thus creating electropositivity outside the
membrane and electronegativity inside because of
negative anions that remain behind and do not diffuse
outward with the potassium.

46. Within a millisecond or so, the potential
difference between the inside and outside, called the
diffusion potential, becomes great enough to block
further net potassium diffusion to the exterior, despite
the high potassium ion concentration gradient.
In the normal mammalian nerve fibre, the
potential difference required is about 94 millivolts,
with negativity inside the fibre membrane.

47. Resting Membrane Potential
of Nerves:
The resting membrane potential of large nerve
fibres when not transmitting nerve signals is about –90
millivolts.
That is, the potential inside the fibre is 90
millivolts more negative than the potential in the
extracellular fluid on the outside of the fiber.

48. The important factors in the establishment of
the normal resting membrane potential of -
90mV:
# Contribution of potassium diffusion
potential.
# Contribution of sodium diffusion
through nerve membrane.
# Contribution of sodium-potassium
pump.

49. Stages of action potential:
Resting stage: membrane polarized during this
stage.
Depolarisation stage: potential rises rapidly in the
positive direction.
Repolarisation stage: rapid diffusion of potassium
ions to exterior re- establishes the normal negative
resting membrane potentials.

50. Factors involved in nerve
action potential:
Voltage-Gated Sodium and Potassium Channels:
The necessary factor in causing both
depolarization and repolarization of the nerve
membrane during the action potential is the voltage-
gated sodium channel.
A voltage-gated potassium channel also plays an
important role in increasing the rapidity of
repolarization of the membrane.
These two voltage-gated channels are in addition
to the Na+-K+ pump and the K+-Na+ leak channels.

51. Voltage gated sodium channel

52. Voltage-Gated Potassium
Channel and Its Activation:

53. Electrogenic Nature of the Na+-K+ Pump
• The fact that the Na+-K+ pump moves three Na+
ions to the exterior for every two K+ ions to the interior
means that a net of one positive charge is moved from
the interior of the cell to the exterior for each cycle of
the pump. This creates positivity outside the cell but
leaves a deficit of positive ions inside the cell; that is, it
causes negativity on the inside.
•Therefore, the Na+-K+ pump is said to be
electrogenic because it creates an electrical potential
across the cell membrane.
•This electrical potential is a basic requirement in
nerve and muscle fibers for transmitting nerve and
muscle signals.

54. Depolarising
Stimulus
Opening of
voltage gated Na
channels
Inactivation of Na
Channels
Increased P Na
Depolarisation of
membrane potential
Increased flow of Na into
the cell
START
STOP
Positive
feedbac
k
+
Feedback Control in Voltage
Gated Ion Channels

55. Depolarisation
by Na influx
Opening of
voltage gated K+
channels
Increased P K
Repolarisation of
membrane potential
Increased flow of K+ out
of the cell
START
Negativ
e
feedbac
k
-

56. Re-establishing Sodium and Potassium Ionic Gradients
After Action Potentials Are Completed:
Sodium ions that have diffused to the interior of the cell
during the action potentials and potassium ions that have diffused
to the exterior must be returned to their original state by the Na+-
K+ pump.
Because this pump requires energy for operation, this
“recharging” of the nerve fiber is an active metabolic process,
using energy derived from the adenosine triphosphate (ATP)
energy system of the cell.

58. Myelinated and unmyelinated nerve fibres:
 Large fibers are myelinated and small ones are
unmyelinated
 Central core of the fiber is the axon - the membrane of
the axon is actual conductive membrane for conducting
action potential.
 The length of the axon is interrupted by the node of
Ranvier.
Signal transmission in nerve trunks

59. Special Characteristics of Signal Transmission in
Nerve Trunks
“Saltatory” Conduction in Myelinated Fibers from
Node to Node.
Even though almost no ions can flow through the thick
myelin sheaths of myelinated nerves, they can flow with ease
through the nodes of Ranvier. Therefore, action potentials occur
only at the nodes.
The action potentials are conducted from node to node;
this is called saltatory conduction.

60. Saltatory conduction is of value for two reasons:
First, this mechanism increases the velocity of nerve transmission
in myelinated fibers as much as 5- to 50-fold.
Second, saltatory conduction conserves energy for the axon
because only the nodes depolarize, allowing 100 times less loss of
ions than would otherwise be necessary.

61. “Refractory Period” After an Action Potential:
A new action potential cannot occur in an excitable fiber as
long as the membrane is still depolarized from the preceding action
potential.
The reason for this is the sodium channels become
inactivated, and no amount of excitatory signal applied to these
channels at this point will open the inactivation gates.
The only condition that will allow them to reopen is for the
membrane potential to return to or near the original resting
membrane potential level.
The period during which a second action potential cannot be
elicited, even with a strong stimulus, is called the absolute
refractory period.

64. Effect of Synaptic Excitation on the Postsynaptic Membrane—
Excitatory Postsynaptic Potential
Figure shows a presynaptic
terminal that has secreted an
excitatory transmitter into the
cleft between the terminal and
the neuronal somal
membrane.
This transmitter acts on the
membrane excitatory receptor
to increase the membrane’s
permeability to Na+.

65. Effect of Inhibitory Synapses on the Postsynaptic
Membrane— Inhibitory Postsynaptic Potential:
The inhibitory synapses open mainly chloride channels,
allowing easy passage of chloride ions. Opening potassium channels
will allow positively charged potassium ions to move to the exterior,
and this will also make the interior membrane potential more
negative than usual.
Thus, both chloride influx and potassium efflux increase the
degree of intracellular negativity, which is called
hyperpolarization.
Therefore, an increase in negativity beyond the normal
resting membrane potential level is called an inhibitory
postsynaptic potential (IPSP).

66. Effect of Acidosis or Alkalosis on Synaptic Transmission:
Most neurons are highly responsive to changes in pH of the
surrounding interstitial fluids. Normally, alkalosis greatly
increases neuronal excitability. Conversely, acidosis greatly
depresses neuronal activity.
Effect of Hypoxia on Synaptic Transmission:
Neuronal excitability is also highly dependent on an adequate
supply of oxygen. Cessation of oxygen for only a few seconds can
cause complete inexcitability of some neurons.

68. REFERENCES
1. Millers text book of anesthesia,7th ed.
2. Clinical anesthesia, Barash. 6th ed.
3. Morgan`s principles of anesthesia.
4. Textbook of physiology, Ganong. 24th ed.
5. Snell`s Textbook of anatomy.
6. Guyton & Hall Textbook of physiology. 12th ed.