2011 Group 6 Project

From CellBiology
NMJunction.jpg

The Neuromuscular Junction

Introduction

A Neuromuscular junction (NMJ) is a connection between an axon of a motor neuron and the motor end plates on a muscle fibre. The NMJ can be found in both smooth and skeletal muscle fibres, with different purpose and mechanisms which may involve different receptors. Starting with historic researches, this group project establishes detailed information for this junction. Additionally, the NMJ is an important carrier of orders commanded from the brain and also an important transmitter for the skeletal muscle which execute the final movement. The neurotransmitter contained and released from vesicles in NMJ is elaborated parallel with the overall NMJ’s functional mechanism. This research will also discuss the early formation of NMJ that begins in fetal development. As motor neurons develop, several proteins are made which stimulate the formation of a muscle specific kinase. Furthermore, main receptors for the neurotransmitter are discussed in details followed by their role.

This research elaborates the cell components of the NMJ as well as proteins involved supported by scientific research articles which are synthesised within the text. However, NMJ abnormalities lead to some well-known diseases which will be briefly described in a table of NMJ’s disorders followed by their clinical manifestations and treatments. Currently, the NMJ is a research topic in many areas such as pharmacology, pathology, physiology and medicine.Some current findings from different research articles are elucidated followed with the future study areas in NMJ.

History

Thomas Willis


In 1672, Myasthenia gravis (MG) was first described by Thomas Willis. It is an acquired autoimmune disease with antibodies against the nicotinic acetylcholine receptor (AChR) at the NMJ. [1]

In 1842, Claude Bernard concluded that the arrow poison 'curare' acts at the NMJ to interrupt the stimulation of muscle by nerve impulses. [2]

In 1850, Claude Bernard demonstrated that paralysis was mediated via the NMJ, although the precise mechanism remained unknown. [3]

In 1914, Sir Henry Dale, (in 1936, he was awarded the Nobel Prize for Medicine and Physiology), investigating the pharmacological properties of acetylcholine (ACh), distinguished two actions that were reproduced by the alkaloids, muscarine and nicotine. As the effects of muscarine mimicked the parasympathetic nervous system, he termed the receptors muscarinic, whereas those in autonomic ganglia and at the skeletal NMJ were termed nicotinic. [4]

In 1936, The release of ACh into the NMJ has been demonstrated at vertebrate motor nerve endings when stimulated. [5]

In 1949, At the Paris conference Jack C. Eccles was convinced of the part played by acetylcholine at the neuromuscular junction but still thought of electric currents as being responsible for excitation and inhibition in the central nervous system. [6]

In 1952 - 1954, the name end-plate potential is used since Fatt&Katz (1952) and Del Castillo & Katz (1954) studied the endplate potential (epp) at NMJ. They concluded that the ACh molecules were released in packets containing several thousand ACh molecules revealing the quantal nature of synaptic transmission.[7] [8]

In 1978, it has been demonstrated in frog muscles that nerve terminals persist at synaptic sites on basal lamina sheaths in frogs following degeneration of muscle fibers. The conclusion was that factors that guide axon's growth are present at synaptic sites outside of myofiber. Axons can differentiate into nerve terminals even when the postsynaptic myofibers are absent.[9]

In 1981, it was first recognized that the formation of NMJ involve complex signalling between nerve and muscle cells. [10]

In 1986, M. John Anderson experiments with Xenopus laevis embryos and observes that proteolytic enzyme systems used in tissue remodeling may also contribute to nerve and muscle cell interaction during synaptogenesis. [11]

In 1990, Agrin has been first identified to cause AChR cluster in cultured myotubes. [12] It has since been described to play a crucial role in the development of neuromuscular junction.

In 2000, Discovery of the Rabies virus entry at the neuromuscular junction in nerve-muscle cocultures. [13]

In 2004, ACh released from developing neurons was shown to have a developmental role in axon pathfinding in Drosophila visual system. [14]

In 2005, in vivo experiment in mice provided evidence on the antisynaptogenic effect of ACh on developing skeletal neuromuscular junction. The same study also proved the physiological role of agrin in counteracting this inhibitory effect of ACh. [15]

Mechanism of action

The NMJ establishes the communication between a nerve fibre and a muscle cell[16]. However, a motor nerve fibre loses its myelin sheath when it reaches a striated muscle fibre, and also is divided in terminal branches which each of them lies in a groove of the muscle, forming the NMJ [17]. Regarding its location, the NMJs are highly distributed in skeletal muscles[18]. Nevertheless, the nerve terminates at the pre-synaptic membrane, which is separated by a synaptic cleft from the post synaptic membrane of the muscle [19]. The mechanism of action of NMJ involves many steps and engines which harmonically follow each other in which the ability of a muscle to contract or relax depend on. The steps that lead to muscle contaction are as follow:

For a visual representation of these steps, see this video

1. Nerve impulse reaches the motor nerve terminal[20]

2. Specialized proteins forming ion channels in its cell membrane open quickly[21]

3. Calcium enters into the axon terminal[22]

4. Synaptic vesicles are filled with ACh[23]

5. Ca causes some of the vesicle membranes to fuse with the nerve terminal membrane[24]

6. ACh content is released into synaptic cleft[25]

7. ACh diffuses rapidly across the gap and binds to the ACh receptors (AChRs)[26]

8. When this binding occur, small positively charged sodium (Na) ions enter the muscle[27]

9. This lead to the depolarization across membrane[28]

10. End-plate potential in turn opens the voltage-sensitive Na channels at the synaptic fold[29]

11. An “all or nothing” action potential starts which propagates along the muscle fibre in each direction[30]

12. Initiation of a muscle contraction occur[31]

13. Then, the AChR pore closes[32]

14. ACh unbinds and broken down [33]


Motor neurons are efferent neurons that carry impulses away from the Central Nervous System which innervate skeletal muscle to cause movement. They originate in the ventral horn of the spinal cord [34] and synapse with muscle fibres[35] and carry information from the central nervous system to muscles[36], facilitating muscle contraction.

Motor Neuron
  • Somatic once are directly involved in the contraction of skeletal muscles[37]
  • Excitatory
  • Influenced by input descending from the brain[38]

However, they are affected by a class of diseases[39]. Motor neurone disease is a chronic degenerative disorder that affects both lower motor neurones and the descending tracts to the spinal cord. Degeneration of ventral horn cells causes weakness, wasting, hypotonia and fasciculation of the limb muscles known as progressive muscular atrophy. Additionally, Amyotrophic lateral sclerosis is characterized by loss of lower neurons in spinal cord and brain stem and upper motor neurons that project in corticospinal tracts. Skeletal muscles innervated by the degenerated lower motor neurons show neurogenic atrophy.[40]


Acetylcholine (ACh) is one of the principle neurotransmitters of the peripheral nervous system and produced by cholinergic neurons[41] which are motor neurons at the NMJ[42]. ACh binds and activates a receptor protein[43] and it is a very effective deliverer of sodium ions, which stimulate muscle contractions and excites nerves[44]

Acetylcholine Nicotine.jpg

However, the enzyme acetycholinesterase (AChE) hydrolyzes acetylcholine into acetic acid and choline[45] where choline travels back to be recycled into acetylcholine and start the process over again[46] Additionally, concentration of ACh remains higher if the AChE is inhibited[47] The AChE inhibitors delay the degradation of acetylcholine[48] and these inhibitors are used to reverse muscle relaxants and sometimes to treat Alzheimer's disease[49]


There are two main types of Acetylcholine Receptors (AChR's): Nicotinic AChRs (nAChRs) and Muscarinic AChRs (mAChRs). Nevertheless, the nAChRs control skeletal muscle contraction [50] and known as Ionotrophic receptors. They are located at synapses between two neurons and at synapses between neurons and skeletal muscle cells, while the mAChRs control smooth muscle contraction and they are metabotropic receptors (G-protein coupled receptors)[51]. They are located at the synapses of nerves with smooth or cardiac muscle and trigger a chain of chemical events referred to as signal transduction: Excitation and Inhibition response. However, the response of mAChRs is slower.

For more information on nAChRs and mAChRs see: Nicotinic and Muscarinic Acetylcholine Receptors


The Motor End-plate is a specialized region of the sarcolemma, highly folded for increased surface area and contains a high concentration of AChRs[52]. Also called Myoneural Junction [53]. The Motor End-plate receives neurotransmitters in order to propagate an Action Potential [54] Additionally, it is responsible for the terminal tree like branching of a motor axon on a muscle fibre [55] and maintains muscle tone through stretch reflex [56]

Important Structural Components

Component Location Description Picture
Presynaptic membrane The presynaptic membrane is the membrane of the neuron that faces the membrane of the muscle. In between these two membranes is the synaptic cleft[57] In a NMJ, the presynaptic membrane is the axon terminal. This is where the nerve axon terminates. The axon terminal contains many ion channels for the intake of Ca+ and also is responsible for exocytosis of acetylcholine vesicles into the synaptic cleft[58]
Presynaptic membrane.jpg
Synaptic cleft The space between the presynaptic membrane (axon terminal) and the postsynaptic membrane (motor end plate)[59] Separates the presynaptic from the post synaptic cells, the electrical signal cannot cross this space from the neuron, but activates a chain of reactions that causes the muscle to contract. [60]
Synaptic cleft.jpg
Synaptic vesicles Found within the axon terminals of the neuron[61] The synaptic vesicles are membranous organelles that contain the neurotransmitter Acetylcholine (Ach). They are released from the presynaptic membrane through exocytosis. Ach is released when it fuses with the postsynaptic membrane.[62]
Synaptic vesicles.jpg
Ion gated channels and receptors Ion gated channels and receptors are located on the presynaptic membrane (axon terminal) and on the postsynaptic membrane (motor end plate) The depolarisation/repolarisation of the NMJ membrane is controlled by sodium, potassium, calcium, and chlorine ion channels. These channels are either opened or closed in response to certain chemical or voltage changes. The release of ions into the presynaptic membrane, synaptic cleft and postsynaptic membrane controls the depolarisation of the membrane and thus an action potential occurring.[63]
Ion channels.jpg
Postsynaptic membrane Separated from presynaptic membrane by synaptic cleft[64] In the NMJ, the postsynaptic membrane is the motor end plate. Myonuclei are located underneath this membrane where they locally synthesise AChRs and other postsynaptic proteins. MuSK provides a structural scaffold necessary to initiate aggregates of postsynaptic molecules. Characterised by the accumulation of nAChRs and voltage-gated sodium channels. [65]
Postsynaptic membrane.jpg
Schwann Cell Schwann cells wrap around axons of neurons to form the myelin sheath and help in nerve impulse conduction. A single segment of an axon’s myelin sheath is made up by a single Schwann cell which arranges themselves in a series of cylinders that serves as a guide for sprouts of regenerating axons.[66]
Schwann cell.jpg
Mitochondria Mitochondria are concentrated within the presynaptic nerve terminal at both peripheral and central synapses. They are presumably related to the transmitter release machinery.[67] The key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves.[68]
562px-Animal mitochondrion diagram en (edit).svg.png
Basal lamina (BL) Surrounds the Schwann cells of the axon and muscle fibre, Projects into folds that enclose the postsynaptic membrane Layer of extracellular matrix, Synaptic cleft is filled with dense BL, Contain ACh-esterase, Lamina densa comprising the intermediate part of BL, procollagen type 4 is the most important component [69]
Basal lamina.jpg

Light microscopy and electron microscopy pictures of the Neuromuscular Junction

Light Micrograph of a NMJ

This is a light microscopy image of a neuromuscular junction. You can see the striated muscle fiber on the left hand side with the neuron that innervates this muscle fiber on the right. Light microscopy was invented around the 1600's and was the first type of microscope used by scientists. [70] The image from an optical microscope can be captured by a normal camera to generate a micrograph. Digital microscopes are available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eyepieces. The main limitations with light microscopy is that the maximum normal magnification only reach around 1000x normal. [71]

Benefits of light microscopy:

  • Cheap
  • Simple to use
  • Gives good detail to very small objects
  • Can view living specimens (as opposed to the electron microscope where specimens need to be fixed)
  • Can view specimens in colour (as opposed to the electron microscope where specimens are in black and white)
Scanning Electron Micrograph of a NMJ

This is a scanning electron micrograph of a neuromuscular junction. This type of microscope was invented in the 1930's. The scanning electron microscope uses electrons to scan a sample which can be magnified from about 10 times to more than 500,000 times, which is about 250 times the highest magnification of light microscopes. [72] In the following two pictures you can see the highly folded region of the post synaptic membrane (motor end plate) where the neuron synapses with the muscle fiber. These folds increase the surface area of the junction and thus the number of available acetylcholine receptors. The increased number of AchR's allows the membrane to depolarise more quickly so the muscle fiber is activated to contract in a smaller period of time. More AChR's are also needed for the membrane potential to reach threshold so an action potential can propagate. [73]

Advantages of Electron microscopy:

  • Higher resolution and magnification
  • Capability to observe inside of samples
  • Greater depth of field, creating more of a 3D image
  • Finer magnification control, to be able to view a wider range of magnifications instead of a set 100x to 400x

[74]

Transmission Electron Micrograph

Differences between scanning EM and transmission EM:

-Scans a beam of electrons across the object (does not penetrate the object)

-Measures energy from the electrons to create a three-dimensional picture of the surface of an object

-Able to produce images of larger objects (eg an ant)

-The SEM can magnify up to 200,000x [75]

-Passes a beam of electrons THROUGH a specimen

-Can view inside objects

-The TEM can magnify up to 1,000,000x [76]

Cellular organization of skeletal muscle:

Skeletalmuscle.jpg


The NMJ innervates muscle to contract, the following points outline the components of skeletal muscle:

  • Epimysium, an external sheath of dense connective tissue surrounds the entire skeletal muscle
  • Muscle fibers are arranged in regular bundles
  • The connective tissue, perimysium surrounds each bundle
  • Each fiber is surround by a delicate connective tissue called endomysium which is mainly composed of basal lamina and reticular fibers [77]
  • Skeletal muscle must continuously make ATP to provide the energy for muscle contraction. There are three principal ways where a muscle fibre forms ATP:

1. ATP produced by phosphorylation of ADP by creatine phosphate which its phosphate in can be transferred to ADP to form ATP in a process called substrate phosphorylation.

2. ATP produced by glycolytic processes.

3. ATP produced through Krebs cycle and oxidative processes in the mitochondria.[78]


Muscle fibers are characterized by the presence of multiple nuclei located below the sarcolemma. The fibers contain sarcoplasm, a semifluid cytoplasm filled with mitochondria and myofibrils. Filaments lie parallel to the long axis of the myofibrils. Each myofibril is surrounded by the sarcoplasmic reticulum which is a saclike membranous network associated with transverse tubules which are connected with the sarcolemma. Transverse tubules, or T-tubules, help to transmit signals from the sarcolemma to the myofibrils. [79]

Myofibrils contain the fiber's contractile machinery and are composed of a fundamental unit called a sarcomere[80]. Studies with the electron microscope reveal that the sarcomere pattern is characterised by the presence of thin and thick filaments. These two filaments are made up of actin and myosin proteins which are refered as contractile proteins because they constitute the machinery that generates contractile force. However, the thin filaments contain also regulatory proteins: tropomyosin and troponin[81].

To watch the sliding filament theory of muscle contraction video click here


Actin: Its function depends on a dynamic equilibrium between the actin filaments and the pool of actin monomers. Many actin filaments are unstable, but by associating with other proteins they can form stable structures in cells such as the contractile apparatus of muscles. They can form permanent structures such as microvilli and also form temporary structures such as contractile rings that pinch the cytoplasm in two when the cell divides. Additionally many protein bind to actin and modify its properties, regulate actin polymerization and create different structures. [82]

Actin/Myosin Movie 1
Actin/Myosin Movie 2

The structure of actin is as follows:

- long filamentous polymers

- consists of two strands of globular monumers

- diameter 5.6nm

- twisted around each other

- double helical formation

- each monomer contain a binding site for myosin [83]

Myosin: There are two types of the myosin: myosin I and myosin II. Myosin I is found in all cells and it is one headed domain while myosin II is found in muscle cells and it is a dimer (two heads) that bind to each other to form myosin filaments. Aditionally, myosin bind to and hydrolise ATP which provides energy for their movement along actin filaments. The dissociation of the myosin results into two idental heavy chains and two pairs of light chains. The ATP-binding sites are located in the heads of heavy chains, enabling ATPase activity and actin binding. Myosin plays a vital role in muscle contraction and cell motility. [84]

The structure of the myosin is as follows:

- large complex

- heavy chains are thin, rod-like molecules which are twisted together

- the heads of heavy chains have ATP-binding sites, ATPase activity and the ability to bind to actin [85]

- the light chains are associated with the head


Troponin (Tn) binds to the surface of tropomyosin (Tm). The Tn complex consists of three subunits:

1. TnT: attaches to tropomyosin

2. TnC: binds Ca ions

3. TnI: inhibits the actin-myosin interaction [86]

Tropomyosin (Tm): Its principal function is to deliberate the cooperativity upon the Tn, so that Calcium switching of one Tn can control the activity of many actins. The Tm's structure is characterised by a long thin molecule consisting of two polypeptide chains. Tm molecules are bound head to tail forming a polymer that runs over the actin subunits. [87] Additionally, Tm bind to actin and acts as a molecular barrier which blocks the myosin-binding sites on actin in relaxed muscle and prevents crossbridge cycling. In the presence of calcium ions, Tm changes formation and exposes myosin-binding sites. [88]

Embryonic Development of the neuromuscular junction

Human embryo

During week 9, i.e. beginning of the fetal period, the first neuromuscular junctions appear on the newly created myotubes (muscle fibers formed from the fusion of myoblasts into multi-nucleated fibers).[89]Formation of the neuromuscular junction during embryonic period is only partially understood. It is a multistep process requiring coordinated interactions between nerve terminals and muscle. The formation of a subsynaptic apparatus at the neuromuscular junction is an example of extreme subcellular differentiation induced by the motor neuron in a small segment of the muscle fiber. Four key components are involved in this process: agrin, MuSK, acetylcholine receptors and rapsyn.

Stage 1. Nerve-independent establishment of AChR clusters within central regions of muscle

- The growing end of motor neuron axons secret a protein called agrin.[90]

- MuSK is a receptor tyrosine kinase on muscle cell. Once activated by a signal protein (agrin in this case), the kinase domain of MuSK transfers a phosphate group from ATP to selected tyrosine side chains on itself. This process is called autophosphorylation.[91]

- Upon activation by agrin, MuSK signals via proteins (CK 2[92], Dok-7[93]and rapsyn) to induce clustering of acetocholine receptors (AChR).[94]

- Formation of clusters of AChR occur and are concentrated in the central regions of the myofibers.

Stage 2. Nerve-dependent refinement and regulation of AChR clusters

- ACh released by branching nerve endings causes AChR-induced postsynaptic potentials that appear to determine endplate localization. Thus ACh regulates and refines the localization of developing synaptic contacts.[95]

- The active ACh/AChR contact sites may prevent AChRs clustering in non-contacted regions.[96]

Stage 3

Motor axons branch onto specific regions (endplates) of individual muscle fibers.

Stage 4

Presynaptic specialization of motor nerve terminals occurs. [97]

Stage 5

Postsynaptic stabilization of innervated AChR clusters occurs. [98]


Novel Mouse Model Reveals Distinct Activity-Dependent and –Independent Contributions to Synapse Development

The precise mechanisms that control AChR aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. A study proposed that the muscle AChR is central to this process. The following outlines this study and the original paper can be found here

During early embryonic period, AChRs are composed of α2βγδ subunits (AChRγ) and are replaced by adult AChRs composed of α2βεδ subunits (AChRε) during later embryonic stages and early postnatal development.[99]

Genetic mutation of genes coding for subunits of the AChR can greatly affect its activity. The mutation of ε subunit can reduce AChR’s affinity for ACh. This mutation was used to generate γ/ε-fc mice, in which a mutated ε subunit cDNA fragment is fused into the γ subunit gene by homologous recombination to replace AChRγ with functionally “silent” receptors (AChRγ/ε-fc) during embryonic development. Mice diaphragms (a thin sheet of muscle under the lungs) were dissected and prepared for the study. [100]

The study findings show that AChRγ/ε-fc is expressed, clustered in endplate-like structures, and developmentally regulated like AChRγ in wild type (WT) mice. However, the lack of AChR-mediated activity leads to a series of synaptic abnormalities during embryonic development. [101]

The γ/ε-fc mice exhibit abnormal endplate distribution, axonal growth, muscle innervation, NMJ formation and distribution, as well as endplate morphology. Thus, the γ/ε-fc mouse line demonstrates the contribution of activity-dependent and -independent AChR-mediated processes to the formation of NMJs.


- Endplates distribution

Endplates distribution

In the images on the right, the endplates in γ/ε-fc diaphragms are spread out (B), not as centrally located as in WT diaphragms (A). Endplate band of γ/ε-fc diaphragm is more dispersed (C). The percentage of diaphragm witdth (%DW) occupied by endplate band in embryonic week 18 (E18) is reduced comparing to that in E16 in WT (p=0.022) (D). This is due to the lateral growth of myotubes without affecting endplate distribution. In γ/ε-fc diaphragm, there is no significant change (p>0.6) in %DW between E18 and E16. This implies that new endplates are continuously being formed in myotubes. [102]

Method: Endplates were imaged using a Zeiss Axioplan2 microscope, visualized with Alexa-488-labeled α-bungarotoxin.


- Innervation of individual endplates

The innervations pattern of γ/ε-fc diaphragms is disorganised where aberrantly located endplates attract nerves otherwise spreading directionless.

Innervation of individual endplates

In WT diaphragms (A), nerve branches (green) terminate on endplates (red). In γ/ε-fc diaphragms (B), longer branches are formed to contact the wider spread out endplates and they continue to grow beyond the endplate, contacting additional endplates. Close-up of a single γ/ε-fc endplate (C) shows additional nerve sprouting after endplate innervations.

Method: Images of endplates were taken using a Leica TCS NT confocal microscope. Neurofilament and endplates are GFP- and r-bgt (red fluorescent AChR stain)-labeled.


- Presence of Multiple Endplates

Presumably, the effect of ACh prevents formation of additional NMJs on the same myofiber through unknown, possibly activity-regulated retrograde signals. If lack of activity impairs retrograde signalling, formation of multiple synapses on the same myofiber could be expected, i.e. the fiber could present several fully innervated endplates. Slow muscle fibers were used to test this hypothesis as they are present in smaller numbers than fast fibers in the developing diaphragm.

Confocal images showing presence of multiple endplates on muscle fibers Presence of multiple endplates illustrated in graphs

(A) and (B) are confocal images showing muscle fibers (blue) and endplates (green) in WT and γ/ε-fc mice respectively. Asterisks mark location of endplates on individual muscle fibers. The γ/ε-fc diaphragm presents multiple endplates on single muscle fibers (B). (C) illustrates a higher percentage of muscle fibers of γ/ε-fc embryo display more than one endplates per fiber when compared to WT embryo during different embryonic stage. (D) shows that average number of endplates per muscle fiber in E18 WT (1,12±0,03, white) and γ/ε-fc (1,95±0,07, gray) embryos (p<0,001).

Method: Muscle fibers are stained with anti-slow myosin antibody and endplates by Alexa488-labeled α-bungarotoxin. Confocal pictures were taken using a Leica TCS NT (Leica Microsystems) confocal microscope.

Common disorders

Disorder Description Clinical Manifestation Treatment Picture
Myasthenia gravis Autoimmune neuromuscular disorder caused when antibodies block post-synaptic acetylcholine receptors in neuromuscular junctions. When the passage acetylcholine across the synapse is inhibited, muscles are unable to function normally and as a result they are quick to fatigue. [103] There are two forms of clinical manifestations of myasthenia gravis: ocular and generalized.In about 10-40% cases, weakness is restricted to the ocular muscles. The rest experience a fluctuating degree and variable combination of weakness in ocular, bulbar, limb, and respiratory muscles. Common symptoms include breathing difficulty, chewing or swallowing difficulty, fatigue, facial paralysis, double vision and eyelid drooping. [104] There is no cure, but long-term remission is possible. Some medications, such as neostigmine, improve the communication between the nerve and the muscle. Immunosuppressants may be used if symptoms are severe and other medications don't work well enough [105]
Eyelid drooping due to muscle weakness
Lambert–Eaton Myasthenic Syndrome (LEMS) Autoimmune, presynaptic disorder which involves impaired release of acetylcholine. [106] Symptoms include impaired proximal muscle function, eg hip and shoulder movement. Caused by antibody blockage voltage-gated calcium channels (VGCCs) in the presynaptic cell, resulting in decreased release of acetylcholine at the neuromuscular junction. Most occurrences follow cancer although LEMS may be diagnosed first. [107] Symptoms resemble those of Myasthenia gravis, most notably muscle weakness in limbs Azathioprene, steroids, and/or immunoglobin as immune system suppressants. Pyridostigmine, dyaminopyridine to enhance acetylcholine release. [108]
X-ray of a small cell lung cancer (SCLC) that often precedes LEMS
Botulism A disease caused by the excrement of the bacteria Clostridium Botulinum. This excrement, known as Botulinum toxin, is an extremely lethal neurotoxin. It works by blocking the release of acetylcholine at neuromuscular junctions, resulting paralysis and ultimately respiratory failure. The bacteria may enter the body through wounds, or improperly canned or preserved food. [109]

Infant botulism occurs when living bacteria or its spores are eaten and grow within the baby's gastrointestinal tract. However, recently developed medical techniques use the toxin beneficially (botox). [110]

Clinical manifestation of muscle weakness without fever is characteristic of botulism. It starts with facial muscles weakness, which then spreads to the arms and legs. Symptoms include diplopia or ptosis, dysphagia and dyspnoea. In addition, patients may experience dry mouth and GI related symptoms e.g. vomiting and constipation especially in infants. [111] For infants, Botulism Immune Globulin Intravenous-Human (BIG-IV) also known as BabyBIG. Antitoxins (Trivalent, Heptavalent) [112]
This 14 year old boy was fully conscious at the time of this photograph
Congenital myasthenic syndrome (CMS) Inherited disorder where not enough acetylcholine crosses the neuromuscular synapse with onset at or shortly after birth or in early childhood. [113] CMS can be pre-synaptic, where not enough acetycholine is produced or released in the first place, or post-synaptic, where acetylcholine receptors malfunction and do not stay open long enough. Symptoms are similar to those expressed through Myasthenia gravis. [114] Treatments vary depending on the type of CMS. Cholinesterase inhibitors, quinine, and fluoxetine are some effective clinical treatments. [115]
Muscle weakness causing eyelid droop, much like MS
Black widow spider venom The black widow spider produces a protein venom that causes a complete depletion of acetylcholine. [116] Local pain from the spider bite may be followed by severe muscle cramps, abdominal pain, weakness and tremors. In worse cases symptoms include nausea, vomiting, fainting, dizziness, chest pain, and respiratory difficulties [117] Alpha-latrotoxin causes the opening of cation channels in the presynaptic membrane. This channel opening causes increased release of neurotransmitters which over-stimulates motor endplates, causing pain, muscle cramps, tremors etc. [118] Most bites can be treated with opioid analgesics. Antivenom is usually only used in severe cases as it has a high incidence of allergic reactions. [119]
A Black Widow Spider
Cobratoxins Cobratoxins bind specifically to the acetylcholine receptor. [120] Cobratoxin causes paralysis by preventing the binding of acetylcholine to the nicotinic receptor (nicotinic acetylcholine receptor antagonist). Acetylcholine causes muscles to contract when activated, so when these receptors are blocked by cobratoxin, it results in muscle paralysis. [121] There seems to be no current cure for cobratoxin, but cobratoxin is controversially used as an analgesic due to its ability to block the connection between muscle and neuron. [122]
Indian Cobra
Aminoglycosides and excess Magnesium These interfere with calcium-mediated release of quanta. Symptoms include nausea and vomiting, impaired breathing, hypotension, hypocalcemia, arrhythmia and asystole [123] Increased magnesium decreases impulse transmission across the neuromuscular junction. This results in loss of deep tendon reflexes, and muscle paralysis. Since this also affects smooth muscle function, excess magnesium can cause decreased or stopped respiration [124] Usually treatment is to simply stop the cause of the overdose. If severe symptoms are present, sometimes dialysis and an IV drip are required [125]
Magnesium block

Current associated research

Aging MCK-UCP1 mice show progressive NMJ deterioration.


Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons (2009): Dupuis et al looked at abnormalities of the mitochondrial energy metabolism. Their findings proved that this defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases. Furthermore they suggested that muscle selective alterations in mitochondrial function might initiate NMJ pathology and participate in triggering motor neuron degeneration in ALS. Biochemical measurements were carried out followed by electromyography recordings and PCR analysis. [126]

Overall, this study elaborated the impact of the mitochondrial uncoupling in skeletal muscle. This leads to muscle weakness but preserves muscle structure, affects the integrity of Ach clustering. Additionally, there was an abnormal muscle electrical activity. Muscle mitochondrial uncoupling triggers spinal cord astrocytosis and mild motor neuron degeneration. These findings strongly suggested that chronic muscle-restricted mitochondrial uncoupling is able to induce a mild but clear motor neuron pathology, likely as a result of the progressive degenerative process initiated at the NMJ. [127]


Immuno electron micrographs from embryos showing neurexin protein immunoreactivity in both presynaptic terminals (A), and in postsynaptic muscle (B,C).


Neurexin in Embryonic Drosophila Neuromuscular Junctions (2010): In this study, Chen et al unfolded the role of the neurexin in Drosophila glutamate receptor abundance. Their results suggested that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. During the research several methods were used. Starting with the hybridization where embryos were manually staged and dissected to examine neurexin expression. Dissections were performed as for confocal imaging and electrophysiology. For electron microscopy, genotyped Drosophila eggs were dechorionated with bleach. [128]

This study explored the role and the structure of the Neurexin which is a single pass transmembrane protein with a short cytoplasmic C-terminus and a long extracellular N-terminal part. Neurexin form heterologous cell contacts with post-synaptic cell surface proteins at synaptic connections. There are two types of neurexin; alpha (α) and beta (β). Furthermore, the study suggested that Neurexin serves as a receptor for the black widow toxin α-latrotoxin, a black widow spider venom component that causes massive neurotransmitter release, and also it clearly plays an important role in nervous system development and function.However, the most controversial suggestion provided by this research mentioned in the article is the possibility that neurexin in Drosophila NMJs might be present in postsynaptic muscle, where it appears to contribute to formation of proper glutamate receptor clusters in embryos along with presynaptic neurexin


Loss of TBPH function affects synaptic growth and boutons shape.

TDP-43 Regulates Drosophila Neuromuscular Junctions Growth by Modulating Futsch/MAP1B levels and Synaptic Microtubules Organization (2010): Godena et al, unfolded that TDP-43 is an RNA binding protein and plays numerous roles in mRNA metabolism such us transcription, pre-mRNA splicing, mRNA stability, microRNA biogenesis, transport and translation. However, in pathological conditions such as ALS and FTLD, TDP-43 appears as a large insoluble protein aggregates which is redistributed within the cytoplasm. They concluded that the changes observed at the level of NMJs and synaptic boutons formation can be explained by defects at the cytoskeleton level. [129]


This research based on other similar researches displayed several TDP-43’s characteristics:

-TDP-43 is RNA binding protein of 43 kDa

-It belongs to the hnRNP family

-It plays numerous roles in mRNA metabolism such us transcription, pre-mRNA splicing, mRNA stability, microRNA biogenesis, transport and translation

-TDP-43 is very well conserved during the evolution, especially with regards to the two RNA-recognition motifs (RRMs), the first (RRM1) being responsible for the binding of TDP-43 with UG rich RNA

-TDP-43 prevalently resides in the cell nucleus where it co-localizes with other members of the RNA processing machinery

-TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions

-Pathologically, TDP-43 appears in the form of large insoluble protein aggregates redistributed within the cytoplasm in diseases such as ALS and frontotemporal lobar degeneration (FTLD)

-However, Godena et al highlighted that it is not clear how these alterations may lead to neurodegeneration. In theory, the cytosolic accumulation of TDP-43 may induce a toxic, gain of function effect on motoneurons whilst the exclusion of TDP-43 from the cell nucleus may lead to neurodegeneration due to a loss of function mechanism.

Future research

Clostridium botulinum
C. elegans, adult hermaphrodite
Undifferentiated stem cells
synaptic vessicles crossing a gap junction


  • Botulinum neurotoxin works at the neuromuscular junction, but many questions remain about the way it works. What proteins are involved in the transport the neurotoxin into the cholinergic nerve terminal? What factors control how long the toxin acts? New biotechnology has made further research feasible. Answers to these questions would restrict the toxin’s appeal as a tool for bioterrorists and increase its effectiveness as a therapeautic agent for disorders of the neuromuscular junction. [130]
  • Analysis of neuromuscular development and function in C. elegans. Future studies involving organisms such as C. elegans will identify the molecular machinery that is required for the development and function of the neuromuscular junction [131]
  • Synaptic vesicle dynamics within neuromuscular junctions of synapto-phluorin expressing mice.Relationships between synaptic structure and function at the mammalian neuromuscular junction. Current experiments involving the expression of various genes such as synapto-pHlaurin (spH) in transgenic lines of mice reveal reasons for changes in synaptic strength and structure. These experiments lay the groundwork for future studies that will look into the processes underlying the plasticity of the nervous system. [132]
  • Potential Application of Induced Pluripotent Stem Cells in Cell Replacement Therapy for Parkinson's Disease. Current research has shown that the use of embryonic stem cells to replace damaged tissue can be beneficial in various neurodegenerative diseases. These stem cells integrate and form connections with host cells. The problem with some stem cell treatments is the capability of these cells to form long axons from the spinal cord to the muscle cell and forming a junction with the muscle. Further research into the signaling mechanisms of the NMJ may find additional mechanisms by which transplanted cells may be of therapeutic benefit. [133]

External links

Glossary

  • acetylcholine (ACh): A chemical neurotransmitter in the brain and peripheral nervous system; the dominant neurotransmitter in the peripheral nervous system, released at neuromuscular junctions and synapses of the parasympathetic division.
  • acetylcholinesterase (AChE): An enzyme found in the synaptic cleft, bound to the postsynaptic membrane, and in tissue fluids; breaks down and inactivates acetylcholine molecules.
  • actin: The protein component of microfilaments that forms thin filaments in skeletal muscles and produces contractions of all muscles through interaction with thick (myosin) filaments
  • action potential: A conducted change in the transmembrane potential of excitable cells, initiated by a change in the membrane permeability to sodium ions; see also nerve impulse.
  • axon: The elongate extension of a neuron that conducts an action potential.
  • axon hillock: In a multipolar neuron, the portion of the cell body adjacent to the initial segment.
  • choline: A breakdown product or precursor of acetylcholine.
  • cholinergic synapse: A synapse where the presynaptic membrane releases acetylcholine on stimulation.
  • cholinesterase: The enzyme that breaks down and inactivates acetylcholine.
  • depolarization: A change in the transmembrane potential from a negative value toward 0 mV.
  • electrical coupling: A connection between adjacent cells that permits the movement of ions and the transfer of graded or conducted changes in the transmembrane potential from cell to cell.
  • gap junctions: Connections between cells that permit electrical coupling.
  • innervation: The distribution of sensory and motor nerves to a specific region or organ.
  • ion: An atom or molecule bearing a positive or negative charge due to the donation or acceptance, respectively, of an electron.
  • ionotrophic receptors in the NMJ are receptors that allow ions to pass through them when they bind to acetylcholine
  • muscarinic receptors: Membrane receptors sensitive to acetylcholine and to muscarine, a toxin produced by certain mushrooms; located at all parasympathetic neuromuscular and neuroglandular junctions and at a few sympathetic neuromuscular and neuroglandular junctions.
  • myasthenia gravis: A muscular weakness due to a reduction in the number of acetylcholine receptor sites on the sarcolemmal surface; suspected to be an autoimmune disorder.
  • myoblast: A type of embryonic progenitor cell that gives rise to a muscle cell
  • myofiber: Another name for muscle fiber
  • nerve impulse: An action potential in a neuron cell membrane.
  • neurofilaments: Microfilaments in the cytoplasm of a neuron.
  • neuromuscular junction: A synapse between a neuron and a muscle cell.
  • nicotinic receptors: Acetylcholine receptors on the surfaces of sympathetic and parasympathetic ganglion cells; respond to the compound nicotine.
  • peripheral nervous system (PNS): All neural tissue outside the central nervous system.
  • rapsyn: A peripheral membrane component required for the aggregation of the AChRs in the muscle membrane.
  • repolarization: The movement of the transmembrane potential away from a positive value and toward the resting potential.
  • resting potential: The transmembrane potential of a normal cell under homeostatic conditions.
  • sarcolemma: The cell membrane of a muscle cell.
  • sarcomere: The smallest contractile unit of a striated muscle cell.
  • sarcoplasm: The cytoplasm of a muscle cell.
  • signal Transduction is the process by which an extracellular signaling molecule activates a membrane receptor that in turn alters intracellular molecules creating a response
  • sliding filament theory: The concept that a sarcomere shortens as the thick and thin filaments slide past one another.
  • synapse: The site of communication between a nerve cell and some other cell; if the other cell is not a neuron, the term neuromuscular or neuroglandular junction is often used.
  • thick filament: A cytoskeletal filament in a skeletal or cardiac muscle cell; composed of myosin, with a core of titin.
  • thin filament: A cytoskeletal filament in a skeletal or cardiac muscle cell; consists of actin, troponin, and tropomyosin.
  • threshold: The transmembrane potential at which an action potential begins.
  • tropomyosin: A protein on thin filaments that covers the active sites in the absence of free calcium ions.
  • troponin: A protein on thin filaments that binds to tropomyosin and to G actin.

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2011 Projects: Synaptic Junctions | Gap Junctions | Tight Junctions | Desmosomes | Adherens Junctions | Neuromuscular Junction