Neurotransmitter Release Case File

Neurotransmitter Release Case File

EUGENE C.TOY, MD, RAHUL JANDIAL, MD, PhD, EVAN YALE SNYDER, MD, PhD, MARTIN T. PAUKERT, MD

CASE 8

A very distraught 22-year-old mother brings her 9-month-old baby boy to the emergency room in the early evening. She states that he started to have difficulty eating this afternoon and that he has been “drooling” more. He has also been much less active and his crying is weaker today. She did not have to change his diaper since morning. He had his normal diet since last night, but was given a special treat for his good behavior. He has been meeting all of his developmental milestones previously. On physical examination, the baby is diffusely weak with poor head control. He has pooling of saliva in his mouth. He appears lethargic with bilateral ptosis. During examination he moves only minimally and gives a quiet cry. Based on the clinical presentation, the baby is diagnosed with botulism, a disease that interferes with neurotransmission and the neuromuscular junction.

• What ion is involved in the normal release of neurotransmitter?
• What neurotransmitter is involved at neuromuscular junctions?
• How would the child have most likely acquired botulism?

ANSWERS TO CASE 8: NEUROTRANSMITTER RELEASE

Summary: A 9-month-old baby boy presents to the emergency room with poor feeding, drooling, weakness, and constipation.

• Ions involved in neurotransmitter release: Ca2+ions are usually required for the release of neurotransmitter.
• Neurotransmitter at the neuromuscular junction: Acetylcholine.
• Source of botulism toxin: Eating honey.

CLINICAL CORRELATION

Infant botulism is a rare disease resulting from the ingestion of the bacteria Clostridium botulinum in contaminated soil, home-canned goods, and honey. Estimates place the incidence at approximately 250 cases per year in the United States with more than half of all cases occurring in California. Most of the cases involve infants between the ages of 6 weeks to 9 months. Symptoms, including constipation, cranial nerve abnormalities, hypotonia, and respiratory difficulties, classically appear 12-36 hours following ingestion of the contaminated food. Parents will complain that breast-feeding babies will have poor suction and weak cries. The flaccid paralysis usually descends as the disease progresses. In severe cases, the respiratory muscles may be affected. Following ingestion of contaminated food or soil, the spores germinate and colonize in the infant’s gastrointestinal tract. Once the bacteria are established, they begin to produce the botulinum exotoxin which is absorbed throughout the intestinal tract. The exotoxin makes its way to the presynaptic neurons of the neuromuscular junction, where they bind irreversibly to the presynaptic cholinergic receptors and enter the cell by endocytosis. Once inside the cell, the toxin functions as a protease and cleaves integral membrane proteins of the acetylcholine-containing synaptic vesicles. This prevents fusion of the vesicles to the presynaptic membrane and, ultimately, exocytosis of the neurotransmitter. The decreased levels of acetylcholine at the neuromuscular junction produce the weakness that is the hallmark of botulism poisoning.

APPROACH TO NEUROTRANSMITTER RELEASE

Objectives

1. Describe the role of Ca2+ ions in the release of neurotransmitter.
2. Know what constitutes a quantum of neurotransmitter.
3. Identify the proteins involved in the fusion of synaptic vesicle to the presynaptic membrane.
4. Describe how vesicles are recovered following exocytosis.

Definitions

Presynaptic cholinergic receptors: Receptors for the neurotransmitter acetylcholine found on the presynaptic terminal of the nerve.

Active zone: The region of the axon terminal with high concentrations of synaptic vesicles.

Quantum: A term for the amount of neurotransmitters contained in one synaptic vesicle.

Quantal synaptic potential: The postsynaptic potential generated by the release of neurotransmitters from one synaptic vesicle.

DISCUSSION

Stated simply, the release of neurotransmitter at the neural synapse requires the depolarization of the presynaptic terminal by the action potential. This in turn leads to the binding of vesicles to the membrane at the active zone and the release of neurotransmitter into the synaptic cleft.

The voltage-gated Na+ and K+ channels in the presynaptic terminal depolarize the surrounding membrane in response to the action potential. In addition to the Na+ and K+ channels, the presynaptic terminal has a large concentration of voltage-gated Ca2+ channels clustered around the active zone of the presynaptic terminal. Although they are slower to open than voltage-gated Na+ and K+ channels, their relative proximity to the site of neurotransmitter release allows for rapid opening of the channels. It is the influx of Ca2+ (and not Na+ or K+) as a result of membrane depolarization that is responsible for vesicle release of neurotransmitter into the synaptic cleft. The amount of neurotransmitter released is directly proportional to the influx of Ca2+ ions.

Experiments have shown that neurotransmitters are released in a discrete package called a quantum. Each synaptic vesicle carries one quantum of neurotransmitter. A single quantum results in a fixed quantal synaptic potential in the postsynaptic cell. The total postsynaptic potential is comprised of multiple quantal synaptic potentials. In the central nervous system, one action potential can result in the release of 1-10 synaptic vesicles. At the neuromuscular junction, a single action potential can result in the release of up to 150 vesicles. Furthermore, the release of neurotransmitters from the vesicles is an all-or-none phenomenon. If the vesicle binds to the presynaptic membrane, the entire quantum of neurotransmitter is released.

The synaptic vesicles are found clustered in the active zone of the presynaptic terminal. Synaptic vesicles in the cytosol are anchored in place to cytoskeletal filaments by proteins called synapsins. These vesicles cannot release their contents until they move adjacent to the presynaptic membrane. With influx of Ca2+ ions, the synapsins are phosphorylated and freed from their anchors and move toward the active zone. Membrane-bound proteins, Rab3A and Rab3B, are thought to be important for the targeting of the vesicles to the target zone. These proteins bind and hydrolyze GTP into GDP and inorganic phosphate.

One hypothesis for vesicle docking relies on the interaction of a group of integral SNARE proteins. Integral SNARE proteins in the vesicle bind to their counterpart on the presynaptic membrane and hold the vesicle directly adjacent to the membrane. Neurotransmitter release relies on specialized transmembrane proteins which serve as fusion pore. These pores are likely preassembled hemichannels and may resemble gap junction channels. As Ca2+ ions enter the terminal, the fusion pore opens and allows for the exocytosis of neurotransmitter into the synaptic cleft in a mechanism that is not completely understood at this time. The membrane of the vesicle is incorporated into the presynaptic membrane following release of the neurotransmitter.

Calcium ions must be cleared from the terminal to prevent the exhaustion in supply of the synaptic vesicles. Cytosolic proteins rapidly bind and sequester the Ca2+ ions and prevent further vesicle release. The Ca2+ ions are also actively transported into storage cisterns in the terminal. Finally, Na+/Ca2+ exchange transporters use the concentration gradient of Na+ ions to pump Ca2+ out of the terminal and into the extracellular space.

In order to replenish the supply of vesicles, the vesicle membrane must be recovered from the presynaptic membrane. The recovered vesicles are then transported to the membrane-bound organelles in the presynaptic terminal and readied for future synaptic vesicle formation. There are several methods detailed for the retrieval of the synaptic membrane. The classical pathway relies on endocytosis of the synaptic membrane by means of clathrin-coated pits. Clathrin is a cytosolic protein which coats the invaginating membrane and helps to form the new synaptic vesicle. In the kiss-and-run pathway, the

vesicle does not completely fuse with the presynaptic membrane. Once the fusion pore opens, neurotransmitter can be released from the vesicle. As soon as all of the neurotransmitter is released, the pore closes and the vesicle can be recycled. Finally, in the bulk endocytosis pathway recovery of excess membrane occurs without the use of clathrin-coated pits.

COMPREHENSION QUESTIONS

[8.1] A 63-year-old male with a known history of lung cancer complains of progressive weakness. You perform a thorough history and physical examination of the patient and order additional ancillary studies. Based upon your findings, you diagnose him with Eaton-Lambert syndrome, an autoimmune disease which causes destruction of the presynaptic voltage-gated Ca2+ channels. Which of the following events involved in normal neurotransmission would most likely be disrupted by the destruction of these channels?

A. Propagation of an action potential to the presynaptic terminal

B. Release of neurotransmitter in response to nerve terminal depolarization

C. Response of the postsynaptic cell to neurotransmitter

D. Removal of neurotransmitter from the synaptic cleft

[8.2] A 27-year-old man is brought into the emergency room with complaints of severe nausea and vomiting as well as diplopia and weakness. His friend reports he cans his own vegetables and had eaten some the previous evening. Your initial suspicion is that this man has botulism, and you immediately administer botulinum antitoxin. Which of the following processes involved in neurotransmission is inhibited by botulinum toxin?

A. Anterograde axonal transport which delivers neurotransmitter precursors to the synaptic terminal

B. Synthesis of neurotransmitter in the presynaptic terminal

C. Packaging of neurotransmitter into synaptic vesicles

D. Fusion of the synaptic vesicles with the neuronal membrane resulting in neurotransmitter release

[8.3] Which of following best describes the kiss-and-run model of synaptic vesicle recovery?

A. Synaptic vesicles completely fuse with the presynaptic membrane, releasing their contents into the synaptic cleft. The vesicle components are then recovered by clathrin-mediated endocytosis.

B. Synaptic vesicles completely fuse with the presynaptic membrane, releasing their contents into the synaptic cleft. The vesicle components are then recovered by endocytosis without clathrin-coated pits.

C. Synaptic vesicles approach the presynaptic membrane but do not completely fuse with it. They open pores with the membrane, releasing their contents into the synaptic cleft, and then separate from the membrane intact.

D. Synaptic vesicles fuse with the presynaptic membrane, releasing their contents into the synaptic cleft. The vesicle components are not recovered, but rather new components are synthesized in the soma and transported to the presynaptic terminal.

Answers

[8.1] B. Because Eaton-Lambert is a disorder that stems from destruction of the presynaptic calcium channels, it follows that those steps in neurotransmission that directly involve calcium channels, specifically release of neurotransmitter in response to nerve terminal depolarization, would be disrupted. Recall that opening of voltage-gated calcium channels results in calcium influx, which results in fusion of secretory vesicles with the presynaptic membrane and release of neurotransmitter into the cleft. Action potentials depend on sodium and potassium channels, so destruction of calcium channels will not prevent them. In Eaton-Lambert syndrome, there is a deficient response of the postsynaptic cell, but this is not caused by a disruption in response to neurotransmitter, it is because there is insufficient neurotransmitter released. The ability of the postsynaptic cell to respond remains intact; there is simply nothing to respond to. Removal of neurotransmitter from the synaptic cleft is unaffected by the loss of calcium channels.

[8.2] D. Botulinum toxin binds to and inactivates the acetylcholine synaptic vesicle docking complex, which normally facilitates the fusion of the synaptic vesicles with the neuronal membrane allowing neurotransmitter release. This prevents fusion of the vesicle with the presynaptic membrane and release into the synaptic cleft. The other steps listed in the question are not inhibited by botulinum toxin. Packaging of norepinephrine into vesicles is inhibited by the drug reserpine.

[8.3] C. Answers A and B describe the classical pathway for vesicle recycling and the bulk endocytosis pathways respectively. Answer D is not a described method for vesicle recycling.

NEUROSCIENCE PEARLS ❖ Ca2+ (the influx of which is triggered by membrane depolarization) is necessary for the fusion of the synaptic vesicles with the neuronal membrane and the release of neurotransmitters into the synaptic cleft. ❖ The amount of neurotransmitter released is directly proportional to the influx of Ca2+ ions. ❖ Synaptic vesicles in the cytosol are anchored in place to cytoskeletal filaments by proteins called synapsins. ❖ The three known pathways by which synaptic membranes are retrieved include: the classical pathway, the kiss-and-run pathway, and the bulk endocytosis pathway. ❖ The classical pathway describes retrieval by endocytosis of the synaptic membrane by means of clathrin-coated pits. ❖ The kiss-and-run pathway states that instead of the vesicle completely fusing with the presynaptic membrane, the vesicle’s fusion pore opens, releasing the neurotransmitter, and then closes so the vesicle can be recycled. ❖ The bulk endocytosis pathway describes retrieval of excess membrane without the use of clathrin-coated pits.

REFERENCES

Bear MF, Connors B, Paradiso M. Neuroscience: Exploring the Brain. 3rd ed. Baltimore, MD: Lippincott Williams & Wilkins; 2006. 

Purves D, Augustine GJ, Fitzpatrick D, et al, eds. Neuroscience. 3rd ed. Sunderland, MA: Sinauer Associates, Inc.; 2004. 

Zigmond MJ, Squire LR, Bloom FE, Landis SC, Roberts JL, eds. Fundamental Neuroscience. 2nd ed. San Diego, CA: Academic Press; 1999.

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