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NEUROMUSCULAR JUNCTION
Dr. Mohammad Alqudah, Ph.D.
Department of Physiology and Biochemistry
School of Medicine , M2 5th floor
[email protected]
1
Synapse:
a functional connection between a neuron
Axon of motor neuron
and another cell
 Two types of synapses
1. Electrical synapse
2. Chemical synapse, i.e. neuromuscular junction
evidences for the intermediate step (Chemical
synapse):
1. Synaptic delay of .2 msec
2. Unidirectional stimulation
3. Curare blocks the transmission even though the
muscle and the nerve still independently capable
of firing.
Neuromuscular junction
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
Motor neurons and motor units
Motor unit 1
As axon approaches muscle , it divides into
many terminal branches and loses its myelin Motor unit 2
sheath
Each of these axon terminal forms special
junction ,a neuromuscular junction with one
of the many cells that form the whole
muscle
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)
Presynaptic terminal
contains:
1. Mitochondria
2. Synaptic vesicles
storing Ach about
10,000 Ach molecule
per vesicle. Released
into the cleft by
exocytosis
Choline
acetyltranferase
Physiological anatomy of the neuromuscular junction
1. Terminal button
2. Motor end plate
Sequence of events at the neuromuscular junction
Sequence of events at the neuromuscular junction
1. Arrival of the action potential:
causes depolarization of the
terminal that opens voltage gated
calcium channels allowing calcium
influx.
2. Calcium influx
1
Action potential
2
Ca2+
Presynaptic
terminal
Voltage-gated
Ca2+ channel
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
Ca2+
Synaptic
vesicle
Acetylcholine
Ca2+ diffuse into the cell and cause synaptic vesicl
acetylcholine, a neurotransmitter molecule.
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
Action potential
Ca2+
1
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
Synaptic
vesicle
Voltage-gated
Ca2+ channel
Presynaptic
terminal
Synaptic cleft
2
3
Acetylcholine
Postsynaptic
membrane
Na+
Acetylcholine bound
to receptor site opens
ligand-gated Na+
44
channel
Acetylcholine Opens Na+ Channel
Sequence of events at the neuromuscular junction
Na+
Acetylcholine bound
to receptor site opens
ligand-gated Na+
channel
Motor end plate
Acetylcholine molecules combine with their receptor sites and
cause ligand-gated Na+ channels to open.causing local depolarization at
the motor end plate called end
plate potential EPP
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.
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
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
Drugs that affect the transmission at the neuromuscular junction
Ach release :
1. Ca+2
2. Mg+ and Mn+
3. Botulin toxin
Bind to the receptors
1. D – tubocurare ( curare) inhibits transmission
2. Carbachole
3. Methacholine
Ach like effect
4. Nicotine
Cholinesterase inhibitors:
1. Irreversible – nerve gas ( diisopropyl flluorophosphate) and insecticides
Respiratory muscles
2. Reversible - neostigmine and physiostigmine
Ach release :
Action potential
Ca2+
1. Ca+2 increases Ach release and thus prolonged its effect.
2. Mg+ and Mn+ decreases Calcium influx thus decreases AchVoltage-gated
Ca channel
release
2+
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 .
Presynapt
terminal
Bind to the receptors
1. D – tubocurare ( curare) inhibits transmission: competes with Ach by binding
to Ach receptor sites; blocks neuromuscular transmission and thus paralyze
the skeletal muscle
2. Carbachole
3. Methacholine
4. Nicotine
Ach like effect but they are not deactivated by AChE
Cholinesterase inhibitors:
1. Irreversible – nerve gas ( diisopropyl flluorophosphate) and insecticides
2. Reversible - neostigmine and physiostigmine
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?...
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
http://media.pearsoncmg.com/bc/bc_0media
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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
Triad Structure
Excitation –Contraction Coupling
- Links action potential to contraction
1. 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
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
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
NMJ of Smooth Muscle
29
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
30
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
Intracellular Structures of Smooth Muscle
Contractile filaments are anchored diagonally, causing smooth
muscle to contract in a corkscrew manner.
Smooth Muscle Contraction
Mechanisms of Smooth Muscle Contraction
MLC
Phosphatase
Kinase
MLC phosphatase (PP-1cd)
MLC kinase (Ca2+/CaM)
MLC-p
Contraction
MLC
MLCK
MLC
MLCPase
MLC-p
Contraction
MLCK
MLCPase
MLC-p
Relaxation
Contraction is biphasic and has different
mechansims mediating contraction
Agonist
Initial/Ca dependent Sustained/Ca independent
G proteins, effector enzymes and second
messengers in smooth muscle contraction
Receptor
DAG
PIP2
PLC-b1
PKC
IP3
IP3R-I IP3
SR
Ca2+
Ca2+/CaM
Gaq
Depolarization
VO channel
Ca2+ Influx
[Ca2+]i
Ca2+/CaM
+
MLC
RyR
SR
[Ca2+]
MLCK
MLCPase
MLC-p
i
Ca2+/CaM
+
Contraction
Smooth muscle contraction
m3 receptors
a13.GTP
g
b
p115RhoGEF
RhoA.GDP
inactive
RhoA.GTP
active
Rho kinase
m3
m3
40
30
Gaq
Ga13
20
10
PLC-b1
RhoA
0
0
100
200
300
400
500
600
Seconds
IP3
ROCK
DAG
p-MYPT1
MLC
Ca2+/CaM
+
MLCK
MLC-Pase
MLC-p
CPI-17
Contraction
Murthy et al. Biochem J. 374: 145, 2003
PLD
PA
DAG
PKC
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)
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
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
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?
http://people.fmarion.edu/tbarbeau/physio_muscle_supplements.htm
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:
1. Recruit more M.U.s
2. Increase freq.
(force –frequency)
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
Summation of Calcium transients produces tetanus
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.
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
Isometric/isotonic contractions
• Isometric: muscle
contraction without
movement  no
muscle shortening
• Isotonic: muscle
contraction with
movement  muscle
shortens
Three Potential Actions During Muscle Contraction:
• shortening
Biceps muscle shortens
during contraction
(Isotonic: shortening against fixed
load, speed dependent on M·ATPase
activity and load)
• isometric
• lengthening
Biceps muscle lengthens
during contraction
Most likely to cause
muscle injury
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
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 1.95.
• 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
The velocity of muscle contraction varies inversely with the afterload
• Concentric contraction – shortening of the muscle
• Eccentric contraction lengthening
Isometric contraction
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.
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
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
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
 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.
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
 Muscles can be classified based on their metabolic properties (Peter and coworker):
1. Slow oxidative (SO)
2. Fast glycolytic (FG)
3. 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
• Burke classified muscle fiber based of their mechanical properties into:
1. 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
• 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
ATP hydrolysis is the source of energy for mechanical work
(Cross-bridges formation)
How? Myosin ATPase
H2O
ATP  ADP + Pi + Energy
57 KJ
ATP is also needed for other reactions in muscle :
1. Calcium reuptake into SR
2. Sodium-Potassium ATPase to maintain the ionic composition of the two side of the
cell membrane
3. 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
Metabolism regenerates ATP in different time scales and capacities
1. Cytoplasmic ATP (5 mM) can support full contraction for about 1-2 second at most.
2. 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
3. Glycolysis rapid but low capacity supply of ATP for fast twitch fibers
(Glycogen or blood) 1 glucose…….2 ATP
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
1. 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
During intense exercise there will be:
1. short rest periods between contractions.
2. More fast glycotic fibers are recruited over the oxidative fibers, causing more lactic
acid release
3. 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.
Fuel sources
2. Fat
3. Protein
Fuel type vary with the type, intensity and duration of exercise
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
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)
 Exercise increases glucose transporter (GLUT4) in the muscle sarcolemma
 Resistance training hypertrophies muscle, increases muscle fiber size not number
 Signals that control muscle mass:
1.
2.
3.
4.
5.
6.
Stretch
Hypoxia
Androgens
glucocorticoids
Ca 2+
Myostatin negative regulator
 Hypertrophy takes place through recruitment of satellite cells
Myostatin knock-out
Exercise and force velocity relation ship
Muscle Atrophy
– Lack of muscle
activity
• Reduces muscle
size, tone, and
power
Steroid Hormones
• Stimulate muscle growth and hypertrophy
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