1. NEUROMUSCULAR JUNCTION
Dr. Mohammad Alqudah, Ph.D.
Department of Physiology and Biochemistry
School of Medicine , M2 5th floor

2. a functional connection between a neuron and another cell
Synapse:
a functional connection between a neuron and another cell
Axon of motor neuron
Two types of synapses
Electrical synapse
Chemical synapse, i.e. neuromuscular junction
evidences for the intermediate step (Chemical synapse):
Synaptic delay of .2 msec
Unidirectional stimulation
Curare blocks the transmission even though the muscle and the nerve still independently capable of firing.
Neuromuscular junction

3. NEUROMUSCULAR JUNCTION
Is the junction (synapse) between a nerve (motor neuron) and a muscle cell (muscle fiber)
Motor neuron is the neuron that innervates a muscle fiber
Motor unit : single motor neuron and the muscle fibers it innervate

4. Motor neurons and motor units
As axon approaches muscle , it divides into many terminal branches and loses its myelin sheath
Each of these axon terminal forms special junction ,a neuromuscular junction with one of the many cells that form the whole muscle
Motor unit 2

5. Neuromuscular Junction
Components of neuromuscular junction
Motor neuron
End plate region
Presynaptic terminal ( mitochondria and synaptic vesicles
10,000 Ach per vesicle)
Synaptic cleft (or gap) (Cholinesterase)
Postsynaptic membrane ( neurotransmitter’s receptors)
Is the location of neural stimulation
Action potential (electrical signal)
Travels along nerve axon
Ends at synaptic terminal
Synaptic terminal:
releases neurotransmitter (acetylcholine or ACh)
into the synaptic cleft (gap between synaptic terminal and motor end plate)

6. Presynaptic terminal contains: Mitochondria
Synaptic vesicles storing Ach about 10,000 Ach molecule per vesicle. Released into the cleft by exocytosis
Choline acetyltranferase

7. Physiological anatomy of the neuromuscular junction
Terminal button
Motor end plate

8. Sequence of events at the neuromuscular junction

9. 1 2 Sequence of events at the neuromuscular junction
Arrival of the action potential: causes depolarization of the terminal that opens voltage gated calcium channels allowing calcium influx.
Calcium influx 1 2

10. Sequence of events at the neuromuscular junction
4. Calcium influx into the terminal causes synaptic vesicles to fuse with the synaptic membrane and to release its contents (neurotransmitter acetylcholine ) into the synaptic cleft by exocytosis

11. Sequence of events at the neuromuscular junction
5. Ach diffuses in the synaptic cleft and binds its receptor on the motor end plate
Motor end plate contains nicotinic receptors for Ach , which are ligand gated ion channels
Ach binds to the nicotinic receptors and causes conformational change.
When conformational changes occurs ,the central core of channels opens & permeability of motor end plate to Na+ & K+ increases

12. Acetylcholine Opens Na+ Channel
Acetylcholine binds with receptors in the muscle membrane to allow sodium ions to enter the muscle.

13. Sequence of events at the neuromuscular junction
Motor end plate
causing local depolarization at the motor end plate called end plate potential EPP

14. End plate potential and excitation of the skeletal muscle fiber
Ach activation of its receptors leads to local membrane potential at the end plate called EPP.
The magnitude of this EPP usually about 50 – 75 millivolts. This is more than sufficient to depolarize the muscle cell and to initiate an action potential.
The action potential is all or none phenomenon, the depolarization must reach the threshold(20 – 30 millivolts) in odder for an action potential to take place.
While the EPP is graded potential depends on the strength of the stimulus and the amount of Ach released.

15. End plate potential EPP
Small quanta (packets) of Ach are released randomly from nerve cell at rest, each producing smallest possible change in membrane potential of motor end plate, the MINIATURE EPP (MEPP).
MEPP is about .5 millivolts
dependent upon the amount of transmitter contained in an individual vesicle. Opens about 1000 Ach sensitive channels.
When nerve impulse reaches the ending, the number of quanta release increases by several folds and result in large EPP.
EPP then spread by local current to adjacent muscle fibers which r depolarized to threshold & fire action potential

16. Acetyl cholinesterase AchE terminates the effect of Ach at neuromuscular junction
To ensure purposeful movement, muscle cell electrical response is turned off by acetylcholinestrase(AchE), which degrade Ach to choline & acetate
About 50% of choline is returned to the presynaptic terminal to be reused for Ach synthesis.
Now muscle fiber can relax, if sustained contraction is needed for the desired movement another motor neuron AP leads to release of more Ach

17. Drugs that affect the transmission at the neuromuscular junction
Ach release :
Ca+2
Mg+ and Mn+
Botulin toxin
Bind to the receptors
D – tubocurare ( curare) inhibits transmission
Carbachole
Methacholine Ach like effect
Nicotine
Cholinesterase inhibitors:
Irreversible – nerve gas ( diisopropyl flluorophosphate) and insecticides
Reversible - neostigmine and physiostigmine
Respiratory muscles

18. Ach release :
Ca+2 increases Ach release and thus prolonged its effect.
Mg+ and Mn+ decreases Calcium influx thus decreases Ach release
3. Botulin toxin:
Botulinum toxin: exerts its lethal effect by blocking the release of Ach .
Clostridium botulinum poisoning most frequently result from improperly canned food contaminated with clostridia bacteria
Death is due to respiratory failure caused by inability to contract diaphragm .

19. Bind to the receptors
D – tubocurare ( curare) inhibits transmission: competes with Ach by binding to Ach receptor sites; blocks neuromuscular transmission and thus paralyze the skeletal muscle
Carbachole
Methacholine Ach like effect but they are not deactivated by AChE
Nicotine

20. Cholinesterase inhibitors:
Irreversible – nerve gas ( diisopropyl flluorophosphate) and insecticides
Reversible - neostigmine and physiostigmine

21. Myasthenia gravis:
autoimmune disease where antibodies against the Ach receptors are produced. Which consequences do you expect? … weakened EPP and thus muscular weakness
How do you think you can ameliorate the situation?...

22. Fatigue of the neuromuscular junction
The impulse that reaches the neuromuscular junction causes three times as much end plate potential as that required to stimulate the muscle fiber, this is called the safety factor for transmission.
But stimulation with a frequency more that 100 times/ second for several min. often diminishes the number of Ach vesicles so much that impulses then fail to pass into muscle fiber, this is called fatigue of the neuromuscular junction

24. Spread of the action potential to the interior of the muscle fiber by way of a transverse tubule system
Muscle fiber is large … action potential can’t spread deep into the muscle fiber
T tubules penetrate deep in the muscle from one side to the other.
T tubule action potential causes release of calcium ions in the vicinity of all the myofibrils.
Calcium ions cause contraction
And this is what's called excitation-contraction coupling

25. Triad Structure

26. Excitation –Contraction Coupling
Links action potential to contraction
Motor neuron excitation
- action potential in the nerve cell
- action potential in muscle cell
- the T tubule conducts the action
potential deep into the muscle
2. Ca+2 release from the SR into the
myoplasm…
The process by which depolarization of the T-tubule
Is converted to an intracellular calcium signal and the
Subsequent activation of contraction is called
Excitation-Contraction Coupling

27. Release of calcium ions from the sarcoplasmic reticulum (SR)
T tubules depolarization is sensed by a voltage sensors called the dihydropyridine receptors DHPR.
DHPR is in direct contact with the calcium release channel, the ryanodine receptor located on the SR.
Calsequestrin is a protein in the SR that augments SR calcium storage ( low affinity high capacity.
Judith A. Heiny

28. SERCA Ca-ATPase pump ends the Ca2+ transient by pumping Ca2+ back into the SR
To relax a muscle, Ca2+ must return back to the SR
It is returned by the action of the smooth endoplasmic reticulum calcium ATPase (SERCA)
SERCA links the hydrolysis of ATP with the pumping of 2 Ca2+ ions back into the lumen of SR

29. NMJ of Smooth Muscle

30. NMJ of Smooth Muscle
No recognizable end plates or other postsynaptic specializations
Nerve fibers run along the membrane of muscle cells (no direct contact)
Nerve fibers have multiple varicosities containing synaptic vesicles
Form diffuse junctions that secrete NT (many NTs) into the matrix coating of the muscle

31. Structure of the Smooth muscle:
Contraction of smooth muscle
Structure of the Smooth muscle:
1. Size and Shape
2. Plasma Membrane and Caveolae
3. Dense Bands
4. Intermediate and Gap Junctions
5. Contractile Proteins

32. Intracellular Structures of Smooth Muscle
Contractile filaments are anchored diagonally, causing smooth muscle to contract in a corkscrew manner.

33. Smooth Muscle Contraction

34. Mechanisms of Smooth Muscle Contraction
MLC
Kinase
Phosphatase
MLC kinase (Ca2+/CaM)
MLC phosphatase (PP-1cd)
MLC-p
Contraction

35. MLC
MLC
MLCK
MLCPase
MLCK
MLCPase
MLC-p
MLC-p
Contraction
Relaxation

36. Contraction is biphasic and has different
mechansims mediating contraction
Agonist
Initial/Ca dependent
Sustained/Ca independent

37. G proteins, effector enzymes and second
messengers in smooth muscle contraction
Receptor
PIP2
DAG
PLC-b1
Gaq
PKC
IP3
IP3R-I
IP3 SR
Ca2+
Ca2+/CaM

38. Contraction Depolarization VO channel Ca2+ Influx [Ca2+]i Ca2+/CaM MLC
RyR SR
MLCK
MLCPase
MLC-p
[Ca2+]i +
Contraction
Ca2+/CaM

39. Smooth muscle contraction
m3 receptors g
a13.GTP b
p115RhoGEF
RhoA.GDP
RhoA.GTP
inactive
active
Rho kinase

40. m3 m3 G13 Gq RhoA PLC-1 ROCK PLD IP3 DAG PA MLC + Ca2+/CaM MLCK
Contraction (%)
RhoA
PLC-1
Seconds
ROCK
PLD
IP DAG
p-MYPT1 PA
MLC +
Ca2+/CaM
MLCK
MLC-Pase
DAG
MLC-p
CPI-17
PKC
Contraction
Murthy et al. Biochem J. 374: 145, 2003

41. Neuromuscular Junction
Components of neuromuscular junction
Motor neuron
End plate region
Presynaptic terminal ( mitochondria and synaptic vesicles
10,000 Ach per vesicle)
Synaptic cleft (or gap) (Cholinesterase)
Postsynaptic membrane ( neurotransmitter’s receptors)
Is the location of neural stimulation
Action potential (electrical signal)
Travels along nerve axon
Ends at synaptic terminal
Synaptic terminal:
releases neurotransmitter (acetylcholine or ACh)
into the synaptic cleft (gap between synaptic terminal and motor end plate)

42. Characteristics of muscle contraction
Single action potential (stimulation) causes single muscle contraction (Twitch)
Twitch three phases : Latent, contraction and relaxation
+ Stimulator
Nerve
Don’t confuse the action potential with the muscle twitch

43. The muscle twitch lasts much longer than the action potential
(The trigger for muscle contraction)
A very short stimulus causes a single muscle contraction (twitch). Force rises then falls, the falling time is longer than the rise time
Figure 12.16

44. Tension Produced by Whole Skeletal Muscles Depends on
Muscle force depends on the number of motor units that are activated
Gradual increase in stimulus strength produces stronger twitch , as progressively increasing stimulus activates more motor neurons , which activates more motor units which leads to more force.
(recruitment)
Motor unit is the motor neuron and all of its innervated muscle fibers
The size principle : Motor units are recruited in order of their size, What do you think the rational behind this phenomenon?
Tension Produced by Whole Skeletal Muscles
Depends on
Internal tension produced by muscle fibers
External tension exerted by muscle fibers on elastic extracellular fibers
Total number of muscle fibers stimulated
Motor units in a skeletal muscle
Contain hundreds of muscle fibers
That contract at the same time
Controlled by a single motor neuron
Recruitment (multiple motor unit summation)
In a whole muscle or group of muscles, smooth motion and increasing tension are produced by slowly increasing the size or number of motor units stimulated
Maximum tension
Achieved when all motor units reach tetanus
Can be sustained only a very short time

45. Recall The Motor Unit:
motor neuron and the muscle fibers it innervates
Spinal
cord
The smallest amount of
muscle that can be activated
voluntarily.
Gradation of force in skeletal
muscle is coordinated largely
by the nervous system.
Recruitment of motor units
is the most important means
of controlling muscle tension.
To increase force:
Recruit more M.U.s
Increase freq.
(force –frequency)

46. Muscle force can be increased by increasing the frequency of motor neuron firing
The action potential is much shorter than the muscle twitch
Thus, the nerve can stimulate the muscle before the muscle has relaxed or even before it reaches its peak tension
The frequency must exceed 1/twitch time (period) in order for summation to take place
Example: twitch time 100 ms summation begins at frequency of 1/.1s =10Hz.
At high frequency the force shows no waviness, this is called Tetanus

47. Summation of Calcium transients produces tetanus

48. Molecular rational behind frequency summation and tantalization
Single action potential causes single Ca2+ transient.
Sequential SR release leads to summation of myoplasmic calcium concentration.
Force development depends on intracellular Ca2+ concentration , so repetitive stimulation causes repetitive Ca2+ transients and hence more force.

49. Effect of consecutive stimuli: Treppe
Treppe: gradual increase in contraction intensity during sequential stimulation
Might be due to calcium ions accumulating in the cytoplasm with each stimulation
Figure 12.15

50. Isometric/isotonic contractions
Isometric: muscle contraction without movement  no muscle shortening
Isotonic: muscle contraction with movement  muscle shortens

51. Three Potential Actions During Muscle Contraction:
Biceps muscle shortens
during contraction
shortening
(Isotonic: shortening against fixed load, speed dependent on M·ATPase activity and load)
isometric
Most likely to cause
muscle injury
lengthening
Biceps muscle lengthens
during contraction

52. Muscle force depends on the length of the muscle
Stretching a muscle produces a passive force
The active tension rises and then falls with the stretch of the muscle
Active tension = Total tension - passive tension
The relationship between active force and muscle length is the Length-tension curve

53. The sliding filament hypothesis predicts that force depends on the overlap of thick and thin filaments
At a sarcomere length of 3.65u there is no force because there is no overlap.
At progressively shorter length the overlap increases and the force increases as well
Until at 2.2 sarcomere length, there is maximal overlap and maximal force.
This force does not decrease until the sarcomere shortens to less that
At shorter length the thin filaments begin to run into each other and the number of cross bridges decrease .
When the thick filaments butt up against the Z-disk the force falls precipitously.
Figure 12.18

54. The velocity of muscle contraction varies inversely with the afterload
Isometric contraction
Concentric contraction – shortening of the muscle
Eccentric contraction lengthening

55. Muscle power Power is the force times the velocity
Muscle power peaks at about one-third of maximal force
Figure from Berne and Levy, Physiology
Mosby—Year Book, Inc., 1993.

56. Skeletal Muscle Tone
Even when muscles are at rest, a certain amount of tautness usually remains.
This is called muscle tone.
Because normal skeletal muscle fibers do not contract without an action potential to stimulate the fibers, skeletal muscle tone results entirely from a low rate of nerve impulses coming from the spinal cord

57. Fiber types and muscle energetics
Various muscles with different Twitch time.
They are all the same active force, they differ in their velocity of shortening
+ Stimulator
Nerve

58. Rate of shortening in a muscle fiber (sarcomere) depends directly on the turnover rate of the cross-bridges
Each cross-bridge cycle slides the thin filament about 10 nm past the thick filament
Rapid cross-bridge cycling means that the thin filament slides the thick filament more quickly.
Thus, the velocity of shortening the muscle (each sarcomere), depends on the turnover rate of the cross-bridges.
The turnover rate of the cross-bridges depends on the ATPase activity of myosin, which depends on the myosin isoforms

59. Myosin isoforms are encoded by separate genes, in the adult there are two basic varieties:
- Slow myosin – slow fibers
- Fast myosin - fast fibers
Myosin isoforms stain differently in histological sections.
Myosin staining is one basis for fiber classification.

60. Brook classification of muscle fiber : depending on myosin staining
Type I ( Slow) and Type II ( Fast) fibers
Type IIb fast glycolytic Type IIa fast oxidative Type I slow
Fiber types characterized using ATPase histochemistry
Note: single muscle contains all isoforms with different ratios

61. Muscles can be classified based on their metabolic properties (Peter and coworker):
Slow oxidative (SO)
Fast glycolytic (FG)
Fast oxidative-glycolytic (FOG)
In general:
Red fibers contains
A lot of mitochondria
A lot of myoglobin
Have large oxidative capacity
They are slower and fatigue resistant

62. Burke classified muscle fiber based of their mechanical properties into:
Slow (S)
2. Fast fatigue resistant (FR)
3. Fast intermediate (FI)
4. Fast fatigable (FF)
Whole muscles in the body are mixtures of muscle fiber types
Single muscle can be predominantly one type or another.
The ratio of a given muscle fiber types in a specific muscle vary between individuals

63. Muscle fiber types differ also in the isoforms of many different proteins for example:
Fast twitch fiber contains SERCA1a & TnC2 while slow twitch fiber contains SERCA2a & TnC1
Muscle fiber types also differ in the relative amount oF organelles:
1. mitochondria
2. SR volume
3. SR calcium pump
4. myoglobin

64. ATP hydrolysis is the source of energy for mechanical work
(Cross-bridges formation)
How? Myosin ATPase
H2O
ATP  ADP + Pi + Energy
57 KJ

65. ATP is also needed for other reactions in muscle :
Calcium reuptake into SR
Sodium-Potassium ATPase to maintain the ionic composition of the two side of the cell membrane
Other functions of the cell such as protein expression
Note that during heavy activity, cross-bridges formation is the main drain on ATP stores in muscle cell.
Rate and amount of ATP consumption varies with the intensity and duration of the exercise

66. Metabolism regenerates ATP in different time scales and capacities
Cytoplasmic ATP (5 mM) can support full contraction for about 1-2 second at most.
Creatine phosphate(CP) regenerates ATP fastest to its normal cytoplasmic concentration
CP + ADP = ATP + Creatine
This source of energy supports maximal muscle contraction for another 5 to 8 seconds
Glycolysis rapid but low capacity supply of ATP for fast twitch fibers
(Glycogen or blood) 1 glucose…….2 ATP

67. Metabolism regenerates ATP in different time scales and capacities
4. Oxidative phosphorylation: slower but high capacity source of ATP:
Electron transport chain
1 glucose……30 ATP
Fuel sources
Carbohydrates: stored as Glycogen which mobilized by glycogenolysis
rapid muscle activity utilizes Glycogenolysis, resorted during rest.
Glycolysis the source of glucose either from glycogen or blood
Glut4 …..Effect of exercise
Glucose converted into ATP, pyruvate and NADH and it doesn’t require oxygen
(anaerobic metabolism)
Mitochondria generates NAD+ in order for glycolysis to continue
Or Lactic dehydrogenase converts pyruvate into lactic acid and generates NAD+
during rapid bursts of glycolysis

68. During intense exercise there will be:
short rest periods between contractions.
More fast glycotic fibers are recruited over the oxidative fibers, causing more lactic acid release
Increase sympathetic innervation leading to more glycogenolysis, meaning more pyruvate and thereby more lactate
Thus, During intense Exercise there is more lactic acid production in the muscle even if it is fully oxygenated.

70. Fuel sources
Fat
Protein
Fuel type vary with the type, intensity and duration of exercise

71. Muscle fatigue
Muscle fatigue is a reduction in developed force resulting from previous muscle activity
maximal force that can be generated from resting muscle, any decrease of this maximal force is called fatigue. Maximal force can be sustained only very short time (only once)
Metabolic fatigue is a reduction in submaximal force after prolonged repetitive stimulation
usually exercise is done at submaximal force from many repetition, then we become tired and be unable to do this submaximal force

72. Fatigue in maximum sustained contraction is not in the brain in humans
Pi and H+ in muscle interfere with force development by actomyosin ATPase
Fast twitch muscle use PC and glycolysis for ATP generation, PC accumulates Pi and Glycolysis accumulates lactic acid. The pH of and exercising muscle falls to pH 6.0
Both Pi and pH reduce developed force at the level of cross-bridges formation which is perceived as fatigue.
Fatigue at submaximal force can be postponed by glycogen supercompensations (carbohydrate loading)

73. Exercise increases glucose transporter (GLUT4) in the muscle sarcolemma
Resistance training hypertrophies muscle, increases muscle fiber size not number
Signals that control muscle mass:
Stretch
Hypoxia
Androgens
glucocorticoids
Ca 2+
Myostatin negative regulator
Hypertrophy takes place through recruitment of satellite cells

74. Myostatin knock-out

76. Exercise and force velocity relation ship

77. Muscle Atrophy Lack of muscle activity
Reduces muscle size, tone, and power

78. Steroid Hormones Stimulate muscle growth and hypertrophy
Growth hormone
Testosterone
Thyroid hormones: elevate rate of energy consumption in resting & active skeletal muscles
Epinephrine: stimulate muscle metabolism and increase the duration of stimulation and force of contraction
stimulate synthesis of contractile proteins & enlargement of skeletal muscles
Growth hormone & testosterone – stimulate synthesis of contractile proteins & enlargement of skeletal muscles
Thyroid hormones – elevate rate of energy consumption in resting & active skeletal muscles
Epinephrine – stimulate muscle metabolism and increase the duration of stimulation and force of contraction