Neuromuscular Physiology

Prof. Vajira WeerasingheDepartment of Physiology

Faculty of MedicineUniversity of Peradeniya

• Nerve conduction

• NMJ

• Muscle contraction

Nerve conduction

• Electrochemical basis• concentration gradient, membrane permeability

ionic channels

• Resting membrane potential (RMP)• K+ efflux, Na/K pump• Leaky channels

• Action potential (AP)• depolarisation, repolarisation• Voltage-gated channels

• Propagation of AP• Local current flow

Membrane potential

• A potential difference exists across all cell membranes– This is referred to as “Resting Membrane Potential” (RMP)– Inside is negative with respect to the outside

What are excitable tissues?

• They are capable of generating electrochemical impulses and transmitting them along the membrane

• Excitability of a tissue depends on its membrane potential– Excitable tissues have more negative RMP

( - 70 to - 90 mV)– Non-excitable tissues have less negative RMP

( - 40 mV)

excitable Non-excitable

Red cellGIT

neuron

muscle

Resting Membrane Potential

• This depends on following factors– Ionic distribution across the membrane– Membrane permeability – Other factors

• Na+/K+ pump

Factors contributing to RMP

• One of the main factors is K+ efflux (Nernst Potential: -94mV)

• Contribution of Na influx is little (Nernst Potential: +61mV)

• Na/K pump causes more negativity inside the membrane

• Negatively charged protein remaining inside due to impermeability contributes to the negativity

• Net result: -90 mV inside

Ionic channels

• Leaky channels (K-Na leak channel)– More permeable to K– Allows free flow of ions

K+

Na+

Na/K pump

• Active transport system for Na-K exchange using energy

• It is an electrogenic pump since 3 Na influx coupled with 2 K efflux

• Net effect of causing negative charge inside the membrane

3 Na+

2 K+

ATP ADP

Action Potential (A.P.)

• When an impulse is generated– Inside becomes positive – Causes depolarisation– Nerve impulses are transmitted as AP

Dep

olar

isat

ion R

epolarisation-90

+35

RMP

Hyperpolarisation

Physiological basis of AP

• When the threshold level is reached– Voltage-gated Na channels open up– Since Na conc outside is more than the inside– Na influx will occur– Positive ion coming inside increases the positivity of the membrane

potential and causes depolarisation

– When it reaches +35, Na channels closes– Then Voltage-gated K channels open up– K efflux occurs– Positive ion leaving the inside causes more negativity inside the

membrane– Repolarisation occurs

Physiological basis of AP

• Since Na has come in and K has gone out

• Membrane has become negative

• But ionic distribution has become unequal

• Na/K pump restores Na and K conc slowly– By pumping 3 Na ions outward and 2 K ions inward

• At rest: the activation gate is closed• At threshold level: activation gate opens

– Na influx will occur– Na permeability increases to 500 fold

• when reaching +35, inactivation gate closes– Na influx stops\

• Inactivation gate will not reopen until resting membrane potential is reached

outside

inside

outside

inside

-90 Threshold level +35Na+ Na+

outside

inside

Na+m gate

h gate

– At rest: K channel is closed– At +35

• K channel open up slowly• This slow activation causes K efflux

– After reaching the resting still slow K channels may remain open: causing further hyperpolarisation

outside

inside

outside

inside

-90 At +35

K+ K+

n gate

Propagation of AP

• When one area is depolarised

• A potential difference exists between that site and the adjacent membrane

• A local current flow is initiated

• Local circuit is completed by extra cellular fluid

Propagation of AP

• This local current flow will cause opening of voltage-gated Na channel in the adjacent membrane

• Na influx will occur

• Membrane is deloparised

Propagation of AP

• Then the previous area become repolarised

• This process continue to work

• Resulting in propagation of AP

AP propagation along myelinated nerves

• Na channels are conc around nodes

• Therefore depolarisation mainly occurs at nodes

AP propagation along myelinated nerves

• Local current will flow one node to another

• Thus propagation of A.P. is faster. Conduction through myelinated fibres also faster.

• Known as Saltatory Conduction

Membrane stabilisers• Membrane stabilisers (these decrease excitability)

• Increased serum Ca++– Hypocalcaemia causes membrane instability and spontaneous activation of nerve membrane– Reduced Ca level facilitates Na entry– Spontaneous activation

• Decreased serum K+• Local anaesthetics• Acidosis• Hypoxia

• Membrane destabilisers (these increase excitability)• Decreased serum Ca++• Increased serum K+• Alkalosis• Caffeine• strychnine

NMJ function

• Pre-synaptic membrane• Ca channels• Acetycholine release

• Postsynaptic membrane• Acetylcholine receptors• Ligand-gated channels

• Synaptic cleft• cholinesterase

Synapse

• A gap between two neurons

• More commonly chemical

• Rarely they could be electrical (with gap junctions)

Although an axon conducts bothways, conduction through synapse is oneway

presynaptic neuron to postsynaptic neuron

A neurone receives more than 10000 synapses. Postsynaptic activity is an integrated function

Presynaptic terminal (terminal knob, boutons, end-feet or synaptic knobs) Terminal has synaptic vesicles and mitochondria Mitochondria (ATP) are present inside the presynaptic

terminal

Vesicles containing neurotransmitter (Ach)

Presynaptic terminal (terminal knob, boutons, end-feet or synaptic knobs) Presynaptic membrane contain voltage-gated Ca

channels The quantity of neurotransmitter released is

proportional to the number of Ca entering the terminal Ca ions binds to the protein molecules on the inner

surface of the synaptic membrane called release sites Neurotransmitter binds to these sites and exocytosis

occur

Ca2+ Ca2+

• Postsynaptic membrane contain receptors for the neurotransmitter released

• eg: Acetylcholine receptor

AchNa+

•This receptor is Ach-gated Na+ channel•When Ach binds to this, Na+ channel opens up•Na+ influx occurs

• Na+ influx causes depolarisation of the membrane– End Plate Potential (EPP)

• This is a graded potential• Once this reaches the threshold level• AP is generated at the postsynaptic membrane

Ach release

• An average human end plate contains 15-40 million Ach receptors• Each nerve impulse release 60 Ach vesicles• Each vesicle contains about 10,000 molecules of Ach

• Ach is released in quanta (small packets)• Even at rest small quanta are released • Which creates a minute depolarising spike called Miniature End

Plate Potential (MEPP) • When an impulse arrives at the NMJ quanta released are

increased in several times causing EPP

Ach vesicle docking

• With the help of Ca entering the presynaptic terminal• Docking of Ach vesicles occur• Docking:

– Vesicles move toward & interact with membrane of presynaptic terminal

• There are many proteins necessary for this purpose• These are called SNARE proteins• eg. Syntaxin, synaptobrevin etc

Axoplasmic transport

• The process by which mitochondria, synaptic vesicles and other cytoplasmic constituents travel to and from the cell body

• axon contains 100 times the volume of the cell body

• proteins for neurotransmitters and membrane repair are constantly transported anterogradely – to maintain normal axonal function

• If the axon is deprived of proteins because it is severed or crushed– the segment that is distal to the injury cannot support itself

and will degenerate

NMJ blocking

• Useful in general anaesthesia to facilitate inserting tubes

• Muscle paralysis is useful in performing surgery

Earliest known NMJ blocker - Curare• Curare has long been used in South America

as an extremely potent arrow poison

• Darts were tipped with curare and then accurately fired through blowguns made of bamboo

• Death for birds would take one to two minutes, small mammals up to ten minutes, and large mammals up to 20 minutes

• NMJ blocker used in patients is tubocurarine

• Atracurium is now used

Neuromuscular blocking agents• Non-depolarising type (competitive)

– Act by competing with Ach for the Ach receptors– Binds to Ach receptors and blocks– Prevent Ach from attaching to its receptors– No depolarisation– Prolonged action (30 min)– Ach can compete & the effect overcomes by an excess Ach– Anticholinesterases can reverse the action– eg.

• Curare• Tubocurarine• Gallamine• Atracurium

Neuromuscular blocking agents• Depolarising type (non-competitive)

– Act like Ach, but resistant to AchE action– Bind to motor end plate and once depolarises – Persistent depolarisation leads to a block

• Due to inactivation of Na channels– Two phases

• Phase I – depolarisation phase – fasciculations • Phase II – paralysis phase

– Ach cannot compete– Quick action (30 sec), short duration (10 min)– Anticholinesterases cannot reverse the action– eg.

• suxamethonium• Ach in large doses• nicotine

Neuromuscular blocking agents• AchE inhibitors

– Inhibit AchE so that Ach accumulates and causes depolarising block

• Reversible– Competitive inhibitors of AChE– Block can be overcome by curare

• physostigmine, neostigmine, edrophonium

• Irreversible– Binds to AChE irreversibly

• , insecticides, nerve gases

NMJ disorders

• Myasthenia gravis – Antibodies to Ach receptors– Post synaptic disorder

• Lambert Eaton Syndrome (myasthenic syndrome)– Presynaptic disorder (antibodies against Ca channels)

• Botulism– Presynaptic disorder– Binds to the presynatic region and prevent release of Ach

NMJ disorders

• Snake venom (Presynaptic or postsynaptic disorder)– Krait (bungarotoxin)

• Postsynaptic disorder

– Cobra• Postsynaptic disorder

– Russell’s viper• Presynaptic disorder

Botulinum toxin • Most potent neurotoxin known

• Produced by bacterium Clostridium botulinum

• Causes severe diarrhoeal disease called botulism

• Action: – enters into the presynaptic terminal – cleaves proteins (syntaxin, synaptobrevin) necessary for Ach vesicle release with

Ca2+

• Chemical extract is useful for reducing muscle spasms, muscle spasticity and even removing wrinkles (in plastic surgery)

Video clip

Organophosphates

• Phosphates used as insecticides

• Action– AchE inhibitors– Therefore there is an excess Ach

accumulation– Depolarising type of postsynaptic block

• Used as a suicidal poison • Causes muscle paralysis and death

• Nerve gas (sarin)

Snake venom

• Common Krait (bungarus caeruleus)– Produces neurotoxin known as

bungarotoxin – Very potent

• Causes muscle paralysis and death if not treated

• Cobra– venom contain neurotoxin

Myasthenia gravis• Serious neuromuscular disease

• Antibodies form against acetylcholine nicotinic postsynaptic receptors at the NMJ

• Characteristic pattern of progressively reduced muscle strength with repeated use of the muscle and recovery of muscle strength following a period of rest

• Present with ptosis, fatiguability, speech difficulty, respiratory difficulty

• Treated with cholinesterase inhibitors

Muscle contraction

• T tubules

• Voltage-gated Ca channels

• Actin-myosin cross bridges

• Muscle contraction

• ATP release

• Reversal of the process

Muscle contraction

• Depolarisation of the muscle membrane spreads through the muscle

• Causes muscle contraction

Muscle contraction

• Excitation - contraction coupling

– Excitation : electrical event– Contraction : mechanical event

Ca++Ca++

• AP spreads through t tubule into the muscle tissue

• Close to the sarcoplasmic reticulum, AP activates a receptor (votage-gated Ca++ channel)

• Ca flows to the myoplasm in the vicinity of actin & myosin

• Ca++ binds to troponin• Troponin shifts tropomyosin• Myosin binding sites in actin filament

uncovered• Myosin head binds with actin• Cross bridges form• Filaments slide with ATP being broken down• Muscle shortens

• New ATP occupies myosin head• Myosin head detaches• Filaments slide back• Cycling continues as long as Ca is available

TroponinActin

Myosin

Tropomyosin

ATP

Ca2+TroponinActin

Myosin

Tropomyosin

Ca2+Myosin binding sites

Ca2+Ca2+

Detachment Sliding

Binding

• Relaxation– This occurs when Ca++ is removed from myoplasm

by Ca++ pump located in the sarcoplasmic reticulum

– When Ca++ conc is decreased– Troponin returns to original state– Trpomyosin covers myosin binding sites– Cross-bridge cycling stops

slow & fast fibres

• Slow twitch fibre (type I fibre)

• Fast twitch fibre (type II fibre)

Slow twitch fibre (type I fibre)– Slow cross-bridge cycling– slow rate of shortening (eg. soleus muscle in calf)– high resistance to fatigue– high myoglobin content– high capillary density– many mitochondria– low glycolytic enzyme content– They are red muscle fibres

Fast twitch fibre (type II fibre)– rapid cross-bridge cycling,– rapid rate of shortening (eg. extra-ocular

muscles)– low resistance to fatigue– low myoglobin content– low capillary density– few mitochondria– high glycolytic enzyme content– fast twitch fibers use anaerobic metabolism

to create fuel, they are much better at generating short bursts of strength or speed than slow muscles