Neuromuscular Transmission and Blockade

What is the motor unit composed of?

the motor neuron and all the muscle fibres it innervates

What is the motor neuron?

• where is it located?

• how does it innervate the muscle fibres?

• single cell body located in the ventral horn of the spinal cord

• axon extends peripherally to innervate the muscle fibres

• one neurone can innervate many muscle fibres

How many neurons can innervate one muscle fibre?

one

What does the ratio of neuron-to-fibre vary by?

the function of the muscle

Do fine control muscle fibres have a higher or lower ratio of neuron-to-fibre?

lower

What is the NMJ?

• what is it the site of?

The NMJ is a specialized synapse where a motor neuron communicates with a skeletal muscle fibre.

It is the site at which neuromuscular transmission occurs and where many drugs and toxins exert their effects.

Define: Presynaptic Terminal

axon terminal of motor neurone; contains synaptic vesicles loaded with ACh

Define: Motor Endplate

specialised postsynaptic membrane with junctional folds and nicotinic AChRs

Define: Junctional Folds

Deep infoldings of the postsynaptic membrane; increase the surface area for AChRs

Define: Synaptic Cleft

gap (~50nm) between the pre- and postsynpatic membranes; contains AChE

Define: Synaptic Vesicles

Membrane bound vesicles (~50nm); each contains ~10 000 ACh molecules (1 quantum)

Define: AChE

acetylcholinesterase; rapidly degrades ACh in the cleft; bound to ECM

What are the 6 events at the synapse?

1. Action Potential: AP arrives at the presynaptic terminal of motor neuron

2. Ca²⁺ influx: voltage gated Ca²⁺ channels open; Ca²⁺ enters the terminal

3. Transmitter Release: Ca²⁺ triggers exocytosis of ACh from synaptic vesicles

4. Receptor Binding: ACh diffuses across the cleft and binds nicotinic AChRs

5. Ion Channel Opening: Na+ influx & K+ efflux; generates endplate potential (EPP)

6. ACh Degradation & Recycling: AChE degrades ACh; choline is recycled back into the terminal

What are the 2 precursors for the synthesis of ACh?

• where do they come from?

1. choline- from the phospholipid membrane

   1. acetyl-CoA- metabolic intermediate linking glycolysis to the citric acid cycle

What is the synthesis enzyme (rate-limiting enzyme) of acetylcholine?

• where is it found?

choline acetyltransferase (CAT)- located in the axon terminal

Where is the ACh packaged and what facilitates the packaging?

ACh is packaged into synaptic vesicles; vesiclular ACh transporter (VAChT) facilitates this packaging

What enzyme degrades ACh?

• where is it found?

• how does it degrade ACh?

AChE; located in the ECM and the postsynaptic cell

• rapidly hydrolyses ACh → acetate + choline

What happens to the choline after degradation of ACh?

choline is transported back into the axon terminal via a high-affinity transporter (HAChT) and reused to synthesize new ACh

What are SNARE proteins?

SNARE (Snap receptor) proteins are a large superfamily (60+ members) whose primary role is to mediate fusion of vesicles with the target cell membrane

What are V-SNAREs?

• what are they incorporated into?

• give 2 examples

• what do they interact with?

• incorporated into vesicle membranes

• e.g. synaptobrevin/VAMP

• interact with t-SNAREs on target membrane

What are t-SNAREs?

• where are they located

• give 2 examples

• what do they form

• located in the target (plasma membrane)

• e.g. syntaxin, SNAP-25

  • form tight junctions with v-SNAREs

What is Ca²⁺ sensor: Synaptotagmin

• vesicle membrane protein

  • Rising Ca²⁺ binds to Synaptotagmin

• triggers fast membrane fusion & exocytosis

What are the 4 steps of Vesicle Release?

1. Mobilisation: only mature vesicles reach the presynaptic membrane (actin/myosin dependent)

2. Docking: vesicles attach via sec6/8 and Rab proteins (reversible)

3. Priming: SNARE complex formation readies the vesicle for fusion

   1. Fusion: Ca²⁺ binds → Synaptotagmin → irreversible fusion → ACh released into the cleft

What is Quantum?

the amount of NT contained within one vesicle

What is an mEPP?

• what produces it?

• what does it demonstrate?

• produced by spontaneous release of a single quantum

• ~1/100 - 1/200 the amplitude of a normal EPP

• occurs even without nerve stimulation

  • demonstrates quantal (not continuous) ACh release

What is an EPP?

• how is it generated?

• how does it trigger a muscle AP?

• what type of AP does it produce?

• generated by synchronous release of many vesicles

• normally suprathreshold → triggers muscle AP

• Na+ influx and K+ efflux through nAChR ion channels

• all-or-nothing action potential if threshold reached

How many nAChRs does the NMJ endplate contain?

up to 5 million

Describe the structure of the nAChR.

• structure

• arrangement

  • type of opening

• pentameric: 2α, 1β, 1γ, 1ε

• arranged in a rosette forming a central ion channel

• both α subunits must be occupied by ACh for channel opening

  • all-or-nothing opening (not graded)

For an ion current and EPP to occur, what 2 ions are in influx and efflux?

Open channel: Na+ influx and K+ efflux

How does an EPP occur?

net depolarisation

How does summation occur?

action potential if threshold reached

How many channels needs to be open for an AP?

only 5-10%

What is a receptor reserve?

• Where has the greatest reserve>

• Where has the least reserve?

Receptor reserve: proportion of AChRs that can be blocked before any reduction in muscle contraction is observed; 250 000-500 000 channels (5-10%) must be open to trigger an AP

Greatest reserve: respiratory and large limb muscles

Least reserve: faciala nd extraocular muscles

What is botulism?

• what toxin produces it?

  • where does this toxin come from?

Botulism: rare but life threatening illness caused by botulinum toxin; produced by Clostridium botulinum; one of the most potent neurotoxins known

What are the 5 types of botulism and what are there sources?

1. Foodborne: home-canned/low acid foods

2. Wound: infected injuries (IV drug use)

3. Infant: intestinal colonisation (honey)

4. Latrogenic: excess therapeutic toxin

5. Adult intestinal toxaemia

What is the mechanism of action of botulinum toxin?

• toxin cleaves SNARE proteins (synaptobrevin, syntaxin, SNAP-25)

• prevents vesicle fusion with presynaptic membrane

• blocks ACh release → flaccid paralysis

• descending pattern: cranial nerves → limbs → diaphragm

  • autonomic involvement: dry mouth, ileus/constipation, urinary retention Wha

What are 5 key sources of botulinum toxin?

1. home-canned/preserved foods

2. honey

3. infected wounds

4. improperly stored meats & fish

5. fermented foods

List 7 clinical symptoms of botulism.

1. double/blurred vision (diplopia)

2. drooping eyelids (ptosis) - early sign

3. difficulty swallowing (dysphagia)

4. slurred speech (dysarthria)

5. descending flaccid muscle weakness

6. respiratory failure (life threatening)

7. autonomic: dry mouth, constipation, urinary retention

List 6 ways of diagnosing botulism.

1. Clinical: classic descending paralysis pattern

2. Mouse bioassay: gold standard for toxin detection

3. Stool/wound/serum cultures

4. EMG: characteristic of nerve conduction changes

5. Tensilon test negative

6. Electrophysiology

How to treat botulism?

• heptavalent antitoxin (HBAT)

  • wound debridement + penicillin

What is not used in the treatment of botulism?

Cholinesterase inhibitors

What is tetanus?

• what is the name of the toxin?

• what bacteria produces it?

• how does one get a tetanus infection?

Tetanus toxin (tetanospasmin) is produced by Clostridium tetani from contaminated wounds

What type of paralysis does tetanus produce?

spastic

What is the mechanism of action of the tetanospasmin?

• toxin enters peripheral motor neurons at the NMJ

• transported retrogradely to spinal cord/brainstem

• cleaves synaptobrevin (VAMP) in inhibitory interneurons

• blocks GABA and glycine (inhibitory NTs)

• loss of inhibition → uncontrolled motor neuron firing (spastic paralysis)

What are 4 clincal features of tetanus?

1. trismus (lockjaw)

2. risus sardonicus (facial spasm)

3. opisthotonus (severe spinal arching)

4. generalised muscle rigidity and spasm

What is the incubation period of contaminated wound for tetanus?

3-21 days

What are 3 methods in the management of tetanus?

1. tetanus immunoglobulin (TIG)

2. wound debridement + metronidazole

3. benzodiazepines for spasms