Lower Motor Neurons and Neuromuscular Junction

Lower Motor Neurons and the Neuromuscular Junction

• Muscle spindle fibers, Golgi tendon organs, and upper motor neurons regulate muscle contraction, including gamma co-activation.
• Major factors regulate acetylcholine release and metabolism at the NMJ.
• The sequence of events occur in the sarcolemma in response to an action potential at the NMJ.
• Calcium's role at the NMJ (pre and postsynaptic).
• Common problems associated with the NMJ and their primary mechanism.

Lower Motor Neurons

• Alpha motor neurons innervate extrafusal muscle fibers, causing muscle contraction.
• Gamma motor neurons innervate intrafusal muscle fibers (muscle spindle fibers), regulating the spindle's sensitivity to muscle stretching.
• Alpha and gamma motor neurons are in the ventral horn of the spinal cord; for facial muscles, they are in the cranial nuclei.

Muscle Contraction

• Contraction results from activating alpha motor neurons.
• Activation can be initiated at the spinal level via incoming sensory afferents.
• Activation can also be initiated by descending fibers (UMN).
• Sensory afferents include fibers originating in muscle spindles (1a) or golgi tendon organs (1b).
• Muscle spindle fibers relay information about the length and speed of contraction.
• Golgi tendon organs relay information about the force on the tendon.

Activation of Alpha and Gamma Motor Neurons

• Co-activation of alpha and gamma motor neurons is key to muscle tone (i.e., resistance to stretch).
• LMN damage decreases muscle tone, potentially leading to flaccid paralysis.
• Increased muscle tone occurs when gamma motor neurons are overactive due to increased sensitivity of muscle spindle fibers (spasticity).
• UMN provide inhibitory inputs to gamma motor neurons.
• UMN lesions can cause hyperreflexia due to the lack of inhibition on gamma motor neurons, increasing the sensitivity of muscle spindle fibers.

Motor Unit

• Motor neuron axons travel in the peripheral nerve to supply muscle fibers in a single muscle.
• The motor neuron and the dozens to hundreds of muscle fibers it innervates are referred to collectively as the "motor unit."
• Collaterals of a single aLMN may be spread out across the muscle.
• More motor units are recruited as more contraction is needed.
• Usually, slow fibers are recruited first, then fast fatigable fibers.
• Typically, all available motor units are hardly ever recruited; some remain in reserve.

Neuromuscular Junction

• An action potential (AP) reaches the synapse.
• Calcium (Ca2+) enters the terminal.
• Acetylcholine is released.
• Acetylcholine binds to the nicotinic receptor at the motor end plate.
• Sodium (Na+) enters the motor end plate.
• An AP is generated in the muscle sarcolemma, leading to contraction (power stroke).
• Acetylcholinesterase breaks down acetylcholine, and choline is taken back into the presynaptic cell for recycling.

Acetylcholine Metabolism in the Presynaptic Terminal

• Choline, an essential nutrient usually found complexed with B-vitamins, is key.
• The acetyl group is donated by Acetyl-CoA from mitochondria.
• Choline acetyltransferase is the enzyme responsible for catalyzing this process.
• Increased neuronal activity increases availability of acetyl-CoA and choline uptake from the synaptic cleft.
• Calcium (Ca2+) is involved in both regulatory mechanisms.

Storage of Acetylcholine

• Most ACh is found in vesicles (100 um).
• A small amount of ACh is free in the cytosol.
• Vesicle-bound ACh is not accessible to degradation by acetylcholinesterase (AChE).
• Uptake of ACh made in the terminal into storage vesicles occurs via the vesicular ACh transporter (VACHT).
• VACHT is an antiporter, acidifying the inside of the vesicle by pumping in hydrogen ions to pull in acetylcholine.
• Acidosis can make packaging vesicles less efficient due to the pH gradient changes.

Calcium and Vesicular Release

• Intracellular calcium (Ca2+) promotes the exocytosis of synaptic vesicles from the presynaptic terminal.
• The amount of intracellular calcium determines the number of vesicles that can be released.
• Sufficient extracellular calcium is needed to release about 100 vesicles, producing an endplate potential of approximately 50 mV under normal conditions.
• The amount of calcium that comes in determines the number of vesicles released.
• With repeated firing, calcium builds up and more acetylcholine is released.

Magnesium and Vesicular Release

• Extracellular magnesium acts opposite of calcium (Ca2+).
• Increased extracellular magnesium decreases the number of vesicles released by the AP.
• This effect is likely due to the inhibition of calcium entry into the terminal.
• High extracellular magnesium impairs neuromuscular transmission.
• Decreasing extracellular magnesium can increase the number of vesicles released, resulting in neuromuscular hyperactivity.
• Magnesium competes with calcium for entry into the terminal.
• Low magnesium can cause fasciculations (spontaneous muscle contractions).

Acetylcholine Metabolism in the Synapse

• ACh is rapidly hydrolyzed by the enzyme acetylcholinesterase (AChE).
• AChE has one of the highest catalysis rates known in biology.
• AChE is synthesized in the cell body and distributed throughout the neuron by axoplasmic transport.
• There are multiple isoforms of AChE.
• At the NMJ, AChE comprises four subunits coupled to a collagen tail that anchors the enzyme to the cell membrane of the postsynaptic cell.
• AChE in the presynaptic cell is freely soluble in the cytoplasm.
• Inhibition of ACh breakdown at the synapse significantly alters cholinergic neurotransmission.

AChE inhibitors

• AChE inhibitors can be reversible or irreversible.
• They increase ACh at the synapse, which continues to bind postsynaptic receptors.
• Side effects are due to parasympathetic activation (e.g., bradycardia, hypotension, bronchoconstriction, GI tract hypermotility) and activity at the NMJ.
• Common clinical signs include SLUDGE BAM (parasympathetic) plus muscle spasms (NMJ).
• Examples of reversible inhibitors include Neostigmine, Edrophonium, and Pyridostigmine, used to treat myasthenia gravis, glaucoma, and reverse non-depolarizing neuromuscular blocking agents.
• Examples of irreversible inhibitors are Organophosphate Insecticides (Malathion, Diazinon).
• Organophosphate Insecticides are rapidly inactivated in mammals and birds compared to insects.
• Inhibiting acetylcholine breakdown causes over-excitation of the muscle.

Motor End Plate – NMJ Postsynaptic Side

• Efficient processes occur at the motor end plate.
• T-tubules descend into the muscle for spreading activation and the power stroke.

Nicotinic Receptor

• The NMJ nicotinic ACh receptor consists of 5 subunits: two alpha (α) subunits and one each of beta (β), delta (δ), and gamma (γ).
• These subunits are found in different combinations in different types of nicotinic AChRs.
• ACh binds to the alpha (α) subunits.
• Binding and opening of Na+ channels leads to depolarization.
• Nicotinic subunits contain recognition sites for agonists (e.g., suxamethonium), reversible antagonists (e.g., vecuronium), and α-toxins (cobra α-toxin and α-bungarotoxin).
• Nicotinic receptors at the motor end plate are the target of peripheral muscle relaxants used during surgery.
• The subunits in the nicotinic receptor affect sensitivity and drug response, leading to different sensitivities in the parasympathetic system and the NMJ.
• Non-depolarizing muscle relaxants block alpha subunits to prevent acetylcholine binding.
• Other drugs cause a depolarization block, activating the muscle to the point where it cannot fire anymore.
• Toxins can act as reversible but long-acting antagonists, preventing muscle contractions.

End Plate Potentials and Spreading Activation

• Sodium (Na+) diffuses into the muscle cell.
• Sodium depolarizes the postsynaptic membrane to threshold, eliciting an action potential (AP).
• The AP spreads into the T-tubules.
• Spreading depolarization activates voltage-sensitive calcium channels (DHP).
• Activation of DHP receptors results in a conformation change in Ryanodine receptors (RyR1), which allows the release of Ca2+ from the sarcoplasmic reticulum.

Potassium and Chloride during T-tubule spreading activation

• As the AP propagates down the T-tubule, extracellular potassium (K+) builds up.
• Chloride channels in the T-tubules and sarcolemma reduce the "length constant," preventing transmission of T-tubular depolarization to the surface of the membrane.
• Chloride channels maintain/restore the resting conductance of muscle fibers, preventing sarcolemma over-excitability in response to T-tubular potassium accumulation.
• Without appropriate chloride conductance, the muscle will be hyperexcitable.
• Chloride ions help maintain membrane potentials and decrease excessive spreading activation.

Sarcomere

• Sarcomeres are the contractile units of a muscle.
• The Z disc marks the ends of one sarcomere and the beginning of the next.
• Sarcomeres are composed of thick (myosin) and thin (actin) filaments.
• Thin filaments are attached at one end to a Z disc and extend toward the center of the sarcomere.

Sliding Filament Model

Excitation-Contraction Coupling

• An action potential occurs across the sarcolemma and down T-tubules.
• Calcium (Ca2+) is released from the sarcoplasmic reticulum.
• Myosin and actin interact.
• Muscle contraction occurs.
• Calcium is removed.
• Relaxation occurs.
• Calcium is important on the presynaptic and postsynaptic sides and critical for the power stroke.

Problems at the Neuromuscular Junction

Hypocalcemic tetany

• Hypocalcemic tetany (milk fever; milk tetany) is the involuntary contraction of skeletal muscles that occurs when the extracellular calcium (Ca2+) falls to about 40% of its normal value.
• It can happen when there is a sudden demand for high calcium levels that outpaces the ability to mobilize calcium from bone, such as during lactation.
• There are various effects initially exerted on the plasma membrane that lead to increased spontaneous firing.
• Ion channel gating disruptions (channels may open spontaneously, stay open longer, and close more slowly after activation).
• Threshold potential changes (easier to get to the threshold for action potential).
• Sodium channel function opening more easily and at more negative thresholds.
• There can be impaired calcium shield.
• Over time, if calcium falls too low, paresis will occur as vesicular release will be impaired.
• Calcium forms a shield around membrane channels.
• Low calcium causes increased excitability and easier membrane firing, leading to muscle tremors and fasciculations, eventually leading to paresis.

Synaptic fatigue

• Synaptic fatigue happens when, under intensive stimulation, reuptake, synthesis, and transport mechanisms may be unable to keep pace with neurotransmitter demand.
• During synaptic fatigue, the synapse remains inactive until ACh has been replenished.
• It is primarily observed in fast fatigable muscles (e.g., muscles used for brief forceful movements like jumping).
• It may be observed following myoclonic seizures.

Denervation Hypersensitivity

• In response to an injury, nicotinic receptors spread out over a larger part of the motor end plate.
• Results in an increased sensitivity of the target muscle fiber in the presence of ACh.
• It occurs following spinal cord injury, stroke, burns, prolonged immobility, prolonged exposure to neuromuscular blockers.
• Associated with conditions such as acute canine polyradiculoneuritis (similar to Guillain-Barre syndrome observed in humans) and other polyneuropathies.
• Denervation causes the muscle to add nicotinic receptors, trying to find a signal.

Myotonia and Chloride channelopathies

• Myotonia can be caused by an over excitability of the muscle fiber.
• Myotonia exhibits decreased chloride conductance.
• In myotonic muscle, "after-depolarization" occurs after a series of action potentials.
• This prevents the muscle from relaxing following stimulation.
• Myotonia congenita is caused by an inherited disorder of a chloride channel in the muscles of the skeleton (skeletal muscle chloride channel 1, CLCN1).
• Decreased chloride conductance leads to easier muscle firing and muscle contractions.
• Fainting goats fall over due to chloride channels not being able to repolarize the membrane.

Botulinum toxin

• Interferes with ACh release by disrupting the ability of vesicles to fuse to the presynaptic membrane.
• Cleaves SNARE proteins critical for vesicular docking.

Myasthenia Gravis

• Results in decreased nicotinic receptors on the motor end plate.
• It can be acquired (immune-mediated) or congenital.
• Acquired myasthenia gravis involves the generation of antibodies that bind nicotinic receptors.
• The most common symptom is muscle weakness that worsens with exercise but improves with rest.
• It is often tested with edrophonium (short-acting AChE inhibitor).
• Edrophonium allows acetylcholine to activate the remaining nicotinic receptors more effectively.