Neuromuscular Junction

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Neuromuscular Junction

Introduction

The neuromuscular junctions play the important role of transferring action potentials to the skeletal muscle fibers. Fibers of the skeletal muscles are innervated by large, myelinated nerve fibers that originate in the large motor neurons in the anterior horn of the spinal cord. All of these fibers branch out to innervate anywhere from between a few to several hundred muscle fibers.

The neuromuscular junctions act as the point of interaction between the neurons and the muscle fibers. These neuromuscular junctions allow the action potentials to travel from the neurons to the muscle fibers where they travel towards both ends of the fibers. In about 98 percent of the muscle fibers there is only one neuromuscular junction supplying them. 

Thus, it is important to understand the normal anatomy and physiology, the different toxins, pathologies that affect them and the drugs that act on the neuromuscular junction in order for us to fully understand the overall functioning of the skeletal muscles. 

Development

In order for it to develop, the neuro muscular junction requires signals from both the nerve fiber terminals and the central region of the skeletal muscle fibers. Through a process called pre-patterning, the skeletal muscle fibers accumulate their acetylcholine receptors (Ach-R) in their central regions. This process of accumulation is facilitated by the presence of “MuSK kinase” and a Heparin proteoglycan called “Agrin”. MuSK being a receptor Tyrosine kinase induces signals by phosphorylating Tyrosine in self regions and in other cytoplasmic target regions. Upon binding to its ligand, the activated MuSK releases “Rapsyn” and “Dok7”. These two proteins induce the huddling of the ACh-R. Further, the developing nerve fibers release ACh into the synaptic cleft which induces potentials in the post synaptic membrane. This further stabilizes the neuromuscular junction and contributes to its development. Although most of these findings were observed in research conducted on rodents and other organisms, in 2015 scientists were able to create an all human neuro muscular junction in Vitro.

Anatomy

This section describes the anatomy of the neuromuscular junction in order make it easier to understand its overall functioning. The complex structure of the neuromuscular junction allows it to link the nervous system to the skeletal muscles allowing them to work together. 

Motor End Plate

Overall, the structure of the neuro muscular junction can be divided into the terminal button, the presynaptic membrane, the synaptic cleft, and the post synaptic membrane. The myelinated nerve fibers upon reaching their terminal branch outwards to form a system of branching nerve terminals. These branching nerve terminals invaginate into the muscle fibers however they lie entirely separated and outside of plasma membrane of the muscle fibers. This invagination of the nerve terminal into the muscle fibers is known as the synaptic trough or the synaptic gutter. This entire structure is known as the motor end plate. This motor end plate is completely surrounded by one or more Schwan cells that insulate it from the fluids that surround it. 

Synaptic Cleft

On electro micrographic examination, a clear gap is visible between the neuronal terminal membrane and the muscle fiber membrane, known as the synaptic cleft. The synaptic cleft is 20 to 30 nanometers wide and is occupied by thin spongy layers of the reticular fibers. These reticular fibers are actually basal lamina, and they allow the diffusion of extra cellular fluid. 

Sub-Neural Clefts

In order to increase neurotransmission, the muscular membranes display numerous small folds known as Sub-neural clefts these greatly increase the surface are for the synaptic transmission to occur. 

Axon Terminal

On close examination the axon terminals are found to contain numerous mitochondria. These supply the energy for the synthesis of acetylcholine, the excitatory transmitter. Upon their synthesis in the terminal axonal cytoplasm the acetylcholine molecules are immediately packed in small synaptic vesicles and shifted towards the presynaptic membrane. on average three hundred thousand of such vesicles are found in nerve terminals. ACh also directly affect vasculature by binding to muscarinic receptors on the endothelium which causes the release of nitric oxide causing vasodilation Further the acetylcholinesterase enzymes are found in large quantities in the basement membranes, cause the degradation of acetylcholine in the synaptic cleft.

Acetylcholine 

Acetylcholine (ACh) is the neurotransmitter used in the neuro muscular junction.  Chemically, it is an ester bond between acetic acid and choline. Parts of the body making use of or being affected by ACh are known as Cholinergic. The major sites where ACh is used are  the neuro muscular junctions where it stimulates muscle fibers and the Autonomic system where it acts in both the sympathetic and the  parasympathetic ganglia. It also acts on the parasympathetic target organ receptors as well as in the stimulation of the adrenal gland. There are two types of ACh receptors muscarinic receptors that occur in the receptors in the target organ receptors in parasympathetic system and the Nicotinic receptors that occur in both the sympathetic and parasympathetic ganglia and in the target organ receptors in the adrenal gland and the somatic stimulation i.e. the Neuromuscular junction. Further according to pharmacologic evidence, ACh also plays a key role in the formation of memory thus a disruption in its transmission can have adverse implications.

Synthesis

Acetylcholine is formed when a molecule choline is attached to acetyl coenzyme A under the presence of the Choline acyl-transferase enzyme. In relevance to the topic, Choline is absorbed in the axonal nerve terminals from the surroundings via high affinity choline transporter, this can be blocked by Hemicholinium. Furthermore, ACh can be  broken down by the enzyme Acetylcholinesterase into Acetic acid and Choline.

Physiology

On reaching the neuromuscular junction the nerve impulses stimulate the release of about 300 ACh vesicles from the nerve terminals into the synaptic trough. This section describes the normal physiology of the neuromuscular junction. 

On close examination several linear dense bars are present on the surface of the axonal terminal. These are believed to be the voltage gated Calcium channels. When the action potentials reach the axonal terminals, these channels allow calcium ions to enter into the terminals in large quantities. The Calcium ions having an attractive influence on the Ach containing vesicles attracts them towards the presynaptic membrane. Eventually the vesicles fuse with the membranes thus releasing ACh into the synaptic trough through the process of Exocytosis. Although much of the information stated above is still speculative, it is widely agreed that the influx of calcium ions is the most effective stimulus for the ACh release. This is further supported by the fact that the ACh vesicles are mainly emptied near the bar bodies.                                                 

On the post synaptic membrane lie many ACh gated ion channels. These lie towards the mouths of the sub-neural clefts right below the dense bars where the ACH is released into the troughs. These receptors are large protein complexes with total molecular weights of around 275,000. These complexes are composed of 5 subunit proteins and they penetrate deep into the membranes lying side by side in circles forming a tubular channel. Until ACh attaches to one of the subunits, these  channels remain constricted but once ACh molecules attach to them, these channels undergo a conformational change that opens the channels. These channels have a diameter of around 0.5 nm and are large enough to allow the passage of all the important positive ions namely sodium, calcium and potassium whereas the negative ions such as chloride are unable to pass due to the presence of strong negative ions in the mouths of the channels. 

Further, it has been found that in practice sodium ions pass through these channels in greater amounts than any other ions for mainly two reasons; firstly there are only two ions in concentrations large enough to be considered significant, Sodium in the extra cellular fluid and potassium in the intracellular fluid. Secondly the presence of a large negative potential of between -80 to -90mV is able to effectively pull sodium into the membranes while simultaneously preventing the efflux of potassium ions. 

The overall effect of the ACH entering the post synaptic membranes is that they allow a large number of sodium ions to enter inside the muscle fibers. This causes a large increase in intracellular positive ions which in turn causes a local potential. This local potential is called the end-plate potential and it is this potential that initiates the action potential in the muscle fibers.

Degradation of Acetylcholine

Once Ach  gets released into the synaptic trough, it continuously stimulates the Ach receptors for as long as it is present. Thus, it is important to remove it. There are two main mechanisms to remove ACh from the synaptic trough; Firstly As described earlier the enzyme acetylcholinesterase breaks it down into choline and acetic acid, secondly Small quantities of ACh are able to diffuse out of the synaptic trough thus they are unable to stimulate the muscle fibers. Even though ACh remains in the trough for just a few milliseconds it is able to sufficiently stimulate the muscle fibers, it is then removed from the trough thus preventing the fibers from being re-exited after the first action potential.

End plate potential(EPP)

The end plate potential can depolarize the muscle fiber up to +50 to +75 mV. This is way higher than the normal threshold of between 15 to 30 mV. This explains why ACh is  able to sufficiently depolarize the muscle fibers in the small time when it is present in order to create an action potential.

Toxins affecting the Neuromuscular Junction

Curare is a drug that acts as a competitive inhibitor of ACh by attaching to the ACh receptors on the post synaptic membrane. Another toxin that acts on the neuromuscular junction is Botulinum however acts on the nerve terminal preventing the release of ACH.

Diseases

There are several diseases that affect the neuromuscular junction and there are visible variations in their severity as well as their mortality. Further they may be inherited or acquired. Most of them are due to autoimmune disorders or mutations. 

Auto immune disorders

  1. Myasthenia Gravis: It is an autoimmune disorder where anti bodies are produced against either post synaptic muscle-specific kinase or the ACh-receptors igG1. In the seronegative type IgG1 targets low density lipoprotein receptor-related protein competitively inhibiting its ligand from binging to its receptors. There is a lack of solid evidence to whether the seronegative Myasthenia Gravis responds well to standard therapies. Some of the symptoms of myasthenia gravis are Dysphagia, ptosis, Diplopia, muscular weakness, facial paralysis, and difficulty in breathing. Commonly the drug given to myasthenia gravis patients are Cholinesterase inhibitors such as pyridostigmine and neostigmine which prevent ACH breakdown, increasing stimulation. Secondly immuno-suppressants are given in order to decrease the antibody production.
  1. Lambert Eaton Myasthenic Syndrome(LEMS): It is another auto-immune disorder, this however acts on the pre-synaptic membrane. This produces autoantibodies against the calcium channels thus preventing ACh release. It is characterised by a unique triad of symptoms: weakness of the proximal muscles, Areflexia and Autonomic dysfunction which can lead to dry mouth, constipation etc. Over half of the LEMS cases are associated with a tumor usually a small-cell lung carcinoma and often times it co-exists with Myasthenia Gravis. The first measure treatment is 3,4-diaminopyridine which increases the duration where the voltage gated calcium channels are open and blocks the potassium channels which increases the compound muscular action potential and muscle strength. If 3,4-diaminopyridine does not work prednisone and azathioprine are prescribed.

Genetic Disorders

  1. Duchenne muscular dystrophy: It is an X-linked recessive disorder that affects 1 out of 3,600–6,000 males. The affected die by the age of 30. It due to lack of production of  Dystrophin which is a structural protein. It presents with the following symptoms: elevated Creatine kinase, calf hypertrophy etc. If untreated, it may lead to respiratory distress causing death. There is only symptomatic treatment including the use of steroids.
  1. Congenital Myasthenic Syndromes(CMS): It is very similar to MG and LEMS. These are autosomal recessive disorders that affect  synaptic, presynaptic, and postsynaptic proteins in the neuromuscular junction. These syndromes present symptoms at different ages. During fetal period these can cause fetal Akinesia, during the perinatal period they are known to cause ptosis, arthrogryposis, ophthalmoplegia, hypotonia and breathing or feeding difficulties. These syndromes can also get activated during adolescence or adulthood for example, slow channel syndrome. Their treatment is similar to other neuromuscular disorders with . 3,4-Diaminopyridine being the first line of treatment

References

  1. Lodish, H; Berk, A; Kaiser, C; Krieger, M; Bretscher, A; Ploegh, H; Amon, A (2000). Molecular Cell Biology (7th ed.). New York, NY: W. H. Freeman and Company. p. 695.
  2. Marieb, E. N., & Hoehn, K. (2014). Human anatomy & physiology. San Francisco, CA: Pearson Education Inc.