Difference between revisions of "2011 Group 6 Project"

From CellBiology
Line 329: Line 329:
- several Tm’s isoforms
- several Tm’s isoforms
Actin/Myosin Movie 1 [http://www.youtube.com/watch?v=VQ4OMSi6qAg&feature=related]
Actin/Myosin Movie 2 [http://www.youtube.com/watch?v=pt53JOXP-GE&feature=related]
=Development of the neuromuscular junction=
=Development of the neuromuscular junction=

Revision as of 13:22, 28 April 2011


Neuromuscular Junctions


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 on how this junction works, which may lead to a skeletal muscle contraction, followed by a description for each of its engines involved during this process. Additionally, the NMJ is the site of some well-known diseases which will be briefly described in a table of NMJ’s disorders. Currently, the NMJ is a research topic in many areas such as pharmacology, pathology, physiology and medicine.


Thomas Willis

Historic researchers in NMJ:

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 the observed paralysis was mediated via the NMJ, although the precise mechanism was at that time unknown. [3]

In 1914, Sir Henry Dale, (in 1936, he was warded the Nobel Prize for Medicine and Physiology), investigating the pharmacological properties of 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 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.[5] [6]

Mechanism of action

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. These steps are:

  • Nerve impulse reaches the motor nerve terminal[7]
  • Specialized proteins forming ion channels in its cell membrane open quickly[8]
  • Calcium enters into the axon terminal[9]
  • Synaptic vesicles are filled with ACh[10]
  • Ca causes some of the vesicle membranes to fuse with the nerve terminal membrane[11]
  • ACh content is released into synaptic cleft[12]
  • ACh diffuses rapidly across the gap and binds to the ACh receptors (AChRs)[13]
  • When this binding occur, small positively charged sodium (Na) ions enter the muscle[14]
  • This lead to the depolarization across membrane[15]
  • End-plate potential in turn opens the voltage-sensitive Na channels at the synaptic fold[16]
  • An “all or nothing” action potential starts which propagates along the muscle fibre in each direction[17]
  • Initiation of a muscle contraction occur[18]
  • Then, the AChR pore closes[19]
  • ACh unbinds and broken down[20]

To understand where this happens and the steps involved, please watch this video

Motor neuron

Motor Neuron
  • Efferent neurons
  • Originate in the ventral horn of the spinal cord[21]
  • Synapse with muscle fibres[22]
  • Carry information from the central nervous system to muscles[23]
  • Facilitate muscle contraction
  • Somatic once are directly involved in the contraction of skeletal muscles[24]
  • Excitatory
  • Influenced by input descending from the brain[25]
  • Affected by a class of diseases known as motor neuron diseases[26]
  • 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.[27]

Acetylcholine (ACh)

  • Cholinergic neurons[28]
  • Skeletal muscle movement
  • Regulation of smooth muscle
  • One of the principle neurotransmitters of the peripheral nervous system
  • Released by a motor neuron at the NMJ[29]
  • Bind and activate a receptor protein[30]
  • A very effective deliverer of sodium ions, which stimulate muscle contractions and excites nerves[31]
Acetylcholine Nicotine.jpg
  • The enzyme acetycholinesterase (AChE) hydrolyzes acetylcholine into acetic acid and choline[32]
  • Choline travels back to be recycled into acetylcholine and start the process over again[33]
  • Concentration of ACh remains higher if the AChE is inhibited[34]
  • AChE inhibitors delay the degradation of acetylcholine[35]
  • This inhibitors are used to reverse muscle relaxants and sometimes to treat Alzheimer's disease[36]

Acetylcholine Receptors (AChR's)

Nicotinic and Muscarinic receptors are receptors for acetylcholine. The important difference between the two is their mode of action

  • Nicotinic receptors (nAChRs)

-Controls skeletal muscle contraction [37]

-Ionotrophic receptors

-located at synapses between two neurons and at synapses between neurons and skeletal muscle cells

  • Muscarinic receptors (mAChRs)

-Controls smooth muscle contraction[38]

-Metabotropic receptors (G-protein coupled receptors)

-located at the synapses of nerves with smooth or cardiac muscle

-Trigger a chain of chemical events referred to as signal transduction

-Excitation and inhibition response

-Response of mAChRs is slower

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

Motor End Plate

  • Specialised region of the sarcolemma
  • Highly folded
  • Holds a high concentration of AChRs [39]
  • Also called Myoneural Junction [40]
  • Receive neurotransmitters in order to propagate an Action Potential [41]
  • Responsible for the terminal tree like branching of a motor axon on a muscle fibre [42]
  • Maintains muscle tone through stretch reflex [43]

Important Structural Components

Important structural components of the NMJ
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 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 Presynaptic membrane.jpg
Synaptic cleft The space between the presynaptic membrane (axon terminal) and the postsynaptic membrane (motor end plate) 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. [44] Synaptic cleft.jpg
Synaptic vesicles Found within the axon terminals of the neuron 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.[45]
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. Ion channels.jpg
Postsynaptic membrane Separated from presynaptic membrane by synaptic cleft 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. [46] Postsynaptic membrane.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. 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.

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. 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.

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
Transmission Electron Micrograph

Differences between scanning EM and transmission EM:

  • Scanning 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

  • Transmission EM

-Passes a beam of electrons THROUGH a specimen

-Can view inside objects

-The TEM can magnify up to 1,000,000x

Cellular organization of skeletal muscle:

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

Muscle fibers:

  • Characterized by the presence of several nuclei
  • These nuclei are located below the sarcolema
  • Contain a semifluid cytoplasm called sarcoplasm
  • Sarcoplasm is filled with mitochondria and myofibrils
  • Filaments (thin and thick) lie parallel to the long axis of the myofibrils
  • Sarcoplasmic reticulum ( SR- a saclike membranous network) surrounds each of the myofibrils
  • SR is associated with transverse tubules ( T tubules) which are connected with the sarcolema
  • T tubules help to transmit signals from the sarcolemma to the myofibrils

Proteins of muscle filaments:

  • Actin

- 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

  • Myosin

- large complex

- dissociated into two identical heavy chains and two pairs of light chain

- 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

- the light chains are associated with the head

  • Troponin (Tn)

- Tn complex bind to tropomyosin’s surface

- complex of three subunits

1. TnT attaches to tropomyosin

2. TnC binds Ca ions

3. TnI inhibits the actin-myosin interaction

  • Tropomyosin (Tm)

- long thin molecule

- contains two polypeptide chains

- Tm molecules are bound head to tail

- forming a polymer that run over the actin subunits

- binds to actin and acts as a molecular barrier

- blocks myosin-binding sites in relaxed muscle

- thus, prevents the crossbridge cycle from occurring

- myosin-binding sites are exposed when Ca ions are released

- several Tm’s isoforms

Actin/Myosin Movie 1 [1]

Actin/Myosin Movie 2 [2]

Development of the neuromuscular junction

During week 9, i.e. the beginning of the fetal period, the first neuromuscular junctions appear on the newly created myotubes (muscle fibers form from the fusion of myoblasts into multi-nucleated fibers).

The development of NMJ requires reciprocal signals from nerve and muscle. It can be divided into the following stages:

- Formation of clusters of acetylcholine (ACh) concentrated in the central regions of the myofibers. This is induced by kinase releasing activity of the muscle specific MuSK protein.

- ACh released by branching nerve endings regulates and refines the localization and stabilization of nerve-muscle contact sites.

- Synaptic contacts are stabilized by agrin released from the branching nerve. Agrin enhances the MuSK activity and ACh accumulation.

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

Common neuromuscular junction disorders

Neuromuscular junction disorders are due to impaired transmission of impulses at the neuromuscular junction. This may result from disorders that affect receptor function, pre- or postsynaptic membrane function, or acetylcholinesterase activity. The majority of diseases in this category are associated with autoimmune, toxic, or inherited conditions.

Neuromuscular junction 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. [47] 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. [48] 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 [49] Myasthenia.jpg
Lambert–Eaton Myasthenic Syndrome (LEMS) Autoimmune, presynaptic disorder which involves impaired release of acetylcholine. [50] 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. [51] 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. [52]
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. 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). 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. For infants, Botulism Immune Globulin Intravenous-Human (BIG-IV) also known as BabyBIG. Antitoxins (Trivalent, Heptavalent)
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. [53] 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. [54] Treatments vary depending on the type of CMS. Cholinesterase inhibitors, quinine, and fluoxetine are some effective clinical treatments. [55] Ptosis.jpg
Black widow spider venom The black widow spider produces a protein venom that causes a complete depletion of acetylcholine. [56] 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 [57] 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. [58] 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. [59] Redbackspider.jpg
Cobratoxins Cobratoxins bind specifically to the acetylcholine receptor. [60] 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. [61] 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. [62] Indiancobra.jpg
Aminoglycosides and excess Magnesium These interfere with calcium-mediated release of quanta. Symptoms include nausea and vomiting, impaired breathing, hypotension, hypocalcemia, arrhythmia and asystole [63] 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 [64] Usually treatment is to simply stop the cause of the overdose. If severe symptoms are present, sometimes dialysis and an IV drip are required [65] Magnesium.jpg

Current associated research

Future research

Potential Application of Induced Pluripotent Stem Cells in Cell Replacement Therapy for Parkinson's Disease.

PMID: 21495962

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. [66]

External links


  • Ionotrophic receptors in the NMJ are receptors that allow ions to pass through them when they bind to acetylcholine
  • Signal Transduction is the process by which an extracellular signaling molecule activates a membrane receptor that in turn alters intracellular molecules creating a response


  1. Farrugia M.E. 2002, "Myasthenia Gravis", JR Coll Physicians Edinb, vol 34, pp. 14-18.
  2. Leake, C. D. An Historical Account of Pharmacology to the Twentieth Century; Charles C. Thomas: Springfield, IL, 1975.
  3. Sneader, W. Drug Discovery: The Evolution of Modern Medicines; Wiley: New York, 1985
  4. Feldberg WS. Henry Hallett Dale, 1875-1968. Biogr Mem Fellows R Soc. 1970;16:77–174.
  5. Fatt P & Katz B (1951). An analysis of the end-plate potential recorded with an intra-cellular electrode. J Physiol 115, 320–370
  6. del Castillo J & Katz B (1954). Quantal components of the end-plate potential. J Physiol 124, 560–573
  7. <pubmed>6152289</pubmed>
  8. <pubmed>19820706</pubmed>
  9. <pubmed>4296699</pubmed>
  10. <pubmed>6476821</pubmed>
  11. <pubmed>4301843</pubmed>
  12. <pubmed>6476821</pubmed>
  13. <pubmed>6476821</pubmed>
  14. <pubmed>4301843</pubmed>
  15. <pubmed>8034713</pubmed>
  16. <pubmed>4301843</pubmed>
  17. <pubmed>8034713</pubmed>
  18. <pubmed>8034713</pubmed>
  19. <pubmed>6476821</pubmed>
  20. <pubmed>6476821</pubmed>
  21. <pubmed>19321640</pubmed>
  22. <pubmed>20215342</pubmed>
  23. <pubmed>4107829</pubmed>
  24. <pubmed>20215342</pubmed>
  25. <pubmed>4107829</pubmed>
  26. <pubmed>7925318</pubmed>
  27. <pubmed>20215342</pubmed>
  28. <pubmed>19209176</pubmed>
  29. <pubmed>5963300</pubmed>
  30. <pubmed>5327039</pubmed>
  31. <pubmed>5919565</pubmed>
  32. <pubmed>5963300</pubmed>
  33. <pubmed>5327039</pubmed>
  34. <pubmed>19209176</pubmed>
  35. <pubmed>5919565</pubmed>
  36. <pubmed>9396010</pubmed>
  37. Guyton AC, Hall JE: Textbook of Medical Physiology (2006) Elsevier Saunders, Philadelphia. P.87
  38. Guyton AC, Hall JE: Textbook of Medical Physiology (2006) Elsevier Saunders, Philadelphia. P.87
  39. <pubmed>5786980</pubmed>
  40. Junqueira C.L. & Caraneiro J., 2005. Basic Histology, text & atlas, 11th edition. McGraw-Hill Companies.
  41. <pubmed>5811099</pubmed>
  42. <pubmed>4653422</pubmed>
  43. <pubmed>4669373</pubmed>
  44. <pubmed>21470699</pubmed>
  45. <pubmed>205437</pubmed>
  46. <pubmed>1009425</pubmed>
  47. <pubmed>20130418</pubmed>
  48. <pubmed>16150851</pubmed>
  49. <pubmed>18567867</pubmed>
  50. <pubmed>15257511</pubmed>
  51. <pubmed>16805718</pubmed>
  52. <pubmed>15146039</pubmed>
  53. <pubmed>15367858</pubmed>
  54. <pubmed>15269664</pubmed>
  55. <pubmed>21365067</pubmed>
  56. Martin, Louise (1988). Black Widow Spiders. Rourke Enterprises, Inc.. pp. 18–20.
  57. Preston-Malfham, Ken (1998). Spiders. Edison, New Jersey: Chartwell Books. p. 40.
  58. Insects and Spiders. New York: St. Remy Media Inc. / Discovery Books. 2000. p. 35.
  59. Martin, Louise (1988). Black Widow Spiders. Rourke Enterprises, Inc.. pp. 18–20.
  60. <pubmed>14285499</pubmed>
  61. <pubmed>17555709</pubmed>
  62. <pubmed>17710455</pubmed>
  63. Pritchard JA. The use of the magnesium ion in the management of eclamptogenic toxemias. Surg Gynecol Obstet. 1955; 100:131–140
  64. Lu JF,Nightingale CH. Magnesium sulfate in eclampsia and pre-eclampsia. Clin Pharmacokinet. 2000; 38:305–314
  65. Pritchard JA. The use of the magnesium ion in the management of eclamptogenic toxemias. Surg Gynecol Obstet. 1955; 100:131–140
  66. Stem cell-derived neurotrophic support for the neuromuscular junction in spinal muscular atrophy PMID20955113

Coordinator Comment to all Groups

I will add a general comment that will be the same to all groups under this heading.

Referencing Extension Problem

--Mark Hill 13:16, 3 May 2011 (EST) As mentioned in the lecture, I am aware of the referencing extension problem on your project pages. I have the following temporary solution, of removing the extension, so that groups can continue to add content to their project pages. I am also giving everyone a 1 week extension before the peer assessment.

This should only be done if your project page is not allowing you to save changes!

A. The Easy Way....

The following 4 steps can be done on the webpage or select all content in edit mode, copy and paste into a text editor. All steps must be completed before you attempt to save.

  1. In page edit mode, find all <pubmed> reference tags.
  2. Replace this tag with [http://www.ncbi.nlm.nih.gov/pubmed/ Note, there should be no spaces between the internet address and the pmid number.
  3. Now find all </pubmed> reference tags.
  4. Replace this second tag with ]

This will generate a numbered reference list that we can later fix up.

B. The Better Looking Result....

Whatever is between the <ref> </ref></pubmed> tags is what will appear in your reference list, so you can format the reference and link to appear in your reference list.

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