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Chapter 5: Mechanisms of Neurotransmitter Release

John H. Byrne, Ph.D., Department of Neurobiology and Anatomy, The UT Medical School at Houston


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5.1 Role of Calcium in Transmitter Release

Calcium is a key ion involved in the release of chemical transmitter substances. Bernard Katz and his colleagues examined its role using the skeletal nerve muscle synapse. Electrodes were placed near the presynaptic terminal to initiate an action potential in the terminal (Figure 5.1). The preparation was perfused with a solution free of calcium. In order to precisely control the delivery of calcium, another microelectrode was filled with calcium. Since Ca2+ is positively charged, it can be delivered to the vicinity of the synaptic terminal by briefly closing a switch connected to a battery in such a way that the positive pole forces minute amounts of calcium out of the electrode. In the absence of Ca2+ ejection, stimulation of the motor neuron produced no EPSP.

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Just before the presynaptic axon was stimulated a second time, the switch was briefly closed to eject a small amount of calcium in the vicinity of the presynaptic terminal. A normal EPSP was recorded. The experiment was repeated a third time, but now the calcium ejection occurred after the presynaptic axon was stimulated. There was no EPSP. This experiment demonstrates that calcium must be present before or during the action potential in the presynaptic terminal. Based on this experiment and others like it, Katz and colleagues proposed the calcium hypothesis for chemical synaptic transmission.

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Figure 5.2

The figure above illustrates some of the key features of the calcium hypothesis for chemical synaptic transmission at the neuromuscular junction, but this hypothesis is generally applicable to all chemical synapses in the nervous system. There are two parts to this hypothesis. First, the depolarization of the presynaptic terminal leads to an increase in Ca2+ permeability. Just as there are voltage-dependent Na+ and K+ channels, there are also voltage-dependent Ca2+ channels. The structure of the voltage-dependent channels is very similar to the structure of the voltage-dependent sodium channels. Indeed, just a few amino acids can make the difference between a channel being selectively permeable to calcium and one that is selectively permeable to sodium. The Ca2+ channel is normally closed, but if there is a depolarization of the membrane (caused by a presynaptic action potential), the channel opens and the opening of the channel allows calcium influx. The second part of the calcium hypothesis for chemical synaptic transmission involves the consequences of the Ca2+ influx. The opening of the Ca2+ channel allows for calcium to flow down its concentration gradient from the outside to the inside of the synaptic terminal. This influx leads to an increase in the concentration of the Ca2+ in the presynaptic terminal, which by interacting with proteins associated with synaptic vesicles leads to the release of the chemical transmitter substance.

5.2 Calcium Hypotheses for Chemical Synaptic Transmission

Quantal Nature of Transmitter Release

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Figure 5.3

How does the increase in the intracellular concentration of Ca2+ cause transmitter release? The answer to this question came from an experiment which initially seems unrelated to the issue. Using high amplification of the electrical recording system, Katz noticed small deflections that occurred spontaneously and randomly at a rate of about once every 50 msec (Panel A of the figure to the right).

These small deflections had interesting properties.

Based on these considerations, Katz called these events miniature endplate potentials or MEPPs. They appeared very similar to endplate potentials, but they were only about 0.5 mV in amplitude compared to the 50 mV amplitude of the normal EPP. Katz suggested that MEPPs were due to the spontaneous and random release of ACh. This idea intuitively makes good sense. If there is an abundance of ACh in the presynaptic terminal, perhaps some will leak out and diffuse across the cleft, bind to ACh receptors, and produce a small potential change. ACh is likely to be spontaneously released occasionally because there is a basal level of calcium in the presynaptic terminal. Each vesicle actually contains enough transmitter to open about 1,000 individual ACh-sensitive channels. Therefore, because the MEPP is about 0.5 mV in amplitude, the opening of a single channel produces a potential of about 0.5 μV.

(The designation MEPP has a very specific meaning. It refers to those small endplate potentials that occur randomly in the absence of any stimulation. For example, small endplate potentials (EPPs) can be recorded in the presence of curare or low levels of extracellular Ca2+ , but they are not MEPPs.)

Katz suggested, as a result of the experiment illustrated in Figure 5.3, that the normal EPP is due to the summation effects of many vesicles being released at the same time. One vesicle produces a potential of about 0.5 mV. The release of 100 of those vesicles at the same time could produce a potential which is 100 times as great (50 mV).

The illustration below (Figure 5.4) shows one of these vesicles in the process of fusing with the membrane and releasing its contents into the synaptic cleft through a process called exocytosis. For illustrative purposes, each synaptic vesicle is shown to contain three molecules of transmitter. In reality, each vesicle contains about 10,000 molecules of transmitter. Vesicles ready to be released are found in a region near the presynaptic terminal membrane called the readily releaseable pool. Newly synthesized vesicles are found in the storage or reserve pool. The process by which a vesicle migrates from the reserve pool to the readily releaseable pool is called mobilization. After fusing with the membrane and releasing its contents, the membrane is recycled to form new synaptic vesicles. This process is called recycling. Additional details of this process are found in Chapter 10.

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Figure 5.4

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Figure 5.5

An experiment by Katz that further supported the quantal hypothesis for chemical synaptic transmission is shown above. The extracellular concentration of calcium was lowered to reduce the size of the evoked endplate potential. Because less Ca2+ is in the extracellular medium, less Ca2+ will be available to enter through the voltage-dependent Ca2+ channels. At the arrow, the electrical shock was delivered to the motor axon. Eight successive stimuli were delivered to the presynaptic terminal. EPSPs with stars are the miniature endplate potentials (MEPPs). Note that they are uncorrelated with the stimulus. The evoked endplate potentials are small and highly variable. Sometimes the EPP was 1.6 mV in amplitude; sometimes there was no EPP at all. Sometimes the EPP was 0.4 mV. Katz noticed that these amplitudes showed a specific kind of distribution. The smallest evoked responses were 0.4 mV. He called these responses "units". Other times he recorded EPPs that were about 0.8 mV and called such responses "doubles" because they were twice the unit, and sometimes responses were 1.6 mV. Figure 5.5 is a plot of the number of times an EPP of various amplitudes was observed. Katz noticed that the amplitude of the smallest EPP that could be evoked was the same amplitude (0.5 mV) as the amplitude of the MEPP.

Based on these results Katz proposed the quantal hypothesis for chemical synaptic transmission. An action potential in the presynaptic cell produces an influx of Ca2+which promotes the exocytosis of synaptic vesicles from the presynaptic terminal. There is a statistical variability in the amount of vesicles that can be released. When the extracellular calcium concentration is low, sometimes there is not enough calcium to release any vesicles. At other times, there is enough calcium to cause the release of one vesicle and other times two vesicles, or three vesicles, and so forth. Each peak is therefore an integral multiple of the next, indicating that these vesicles are released in a quantized fashion. With normal levels of calcium, there is sufficient influx of Ca2+to release about 100 vesicles, which produce an endplate potential (EPP) of about 50 mV.

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Figure 5.6
Summary of the steps involved in the process of synaptic transmission at the neuromuscular junction.

 

Test Your Knowledge

  • Question 1
  • A
  • B
  • C
  • D
  • E

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1

B. 100

C. 10,000

D. 100,000

E. 10,000,000

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1 This answer is INCORRECT.

B. 100

C. 10,000

D. 100,000

E. 10,000,000

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1

B. 100 This answer is INCORRECT.

C. 10,000

D. 100,000

E. 10,000,000

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1

B. 100

C. 10,000 This answer is INCORRECT.

D. 100,000

E. 10,000,000

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1

B. 100

C. 10,000

D. 100,000 This answer is CORRECT!

The opening of a single ACh-sensitive channel produces a potential change of about 0.5 microvolts. Therefore, it takes about 1,000 ACh-sensitive channels to produce a potential change equal to the size of a miniature end-plate potential (MEPP), which has a value of about 0.5 mV. A normal end-plate potential is about 50 mV. Therefore, one needs 100 times the number of channels (1,000) that are needed to produce a MEPP to produce a normal end-plate potential.

E. 10,000,000

In a normal, healthy individual the number of ACh-sensitive channels in a skeletal muscle cell opened as a result of a single action potential in a motor axon would be approximately.

A. 1

B. 100

C. 10,000

D. 100,000

E. 10,000,000 This answer is INCORRECT.

 

 

 

 

 

 

 

 

  • Question 2
  • A
  • B
  • C
  • D

An isolated nerve muscle preparation treated with a moderate dose of curare would be associated with a decrease in the (NOTE: there is more than one correct anwer.)

A. Frequency of MEPPs

B. Amplitude of MEPPs

C. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

D. Amplitude of the EPP

An isolated nerve muscle preparation treated with a moderate dose of curare would be associated with a decrease in the (NOTE: there is more than one correct anwer.)

A. Frequency of MEPPs This answer is INCORRECT.

The frequency of MEPPs would not be affected. The frequency of MEPPs is determined strictly by the levels of basal calcium in the presynaptic terminal. Curare is a competitive antagonist of ACh, hence it would affect the amplitude of the miniature end-plate potential, but not the frequency.

B. Amplitude of MEPPs

C. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

D. Amplitude of the EPP

An isolated nerve muscle preparation treated with a moderate dose of curare would be associated with a decrease in the (NOTE: there is more than one correct anwer.)

A. Frequency of MEPPs

B. Amplitude of MEPPs This answer is CORRECT!

Curare is a competitive inhibitor of ACh, hence it will inhibit or reduce the amplitude of the miniature end-plate potential.

C. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

D. Amplitude of the EPP

An isolated nerve muscle preparation treated with a moderate dose of curare would be associated with a decrease in the (NOTE: there is more than one correct anwer.)

A. Frequency of MEPPs

B. Amplitude of MEPPs

C. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential This answer is INCORRECT.

Curare effects the ACh receptors on the postsynaptic side of the synapse. Hence, it will have no effect on the release of transmitter from the presynaptic terminal.

D. Amplitude of the EPP

An isolated nerve muscle preparation treated with a moderate dose of curare would be associated with a decrease in the (NOTE: there is more than one correct anwer.)

A. Frequency of MEPPs

B. Amplitude of MEPPs

C. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

D. Amplitude of the EPP This answer is CORRECT!

Since curare affects the amplitude of the miniature end-plate potential, it would also affect the amplitude of the end-plate potential through the same mechanisms, competitive inhibition of ACh for its receptor.

 

 

 

 

 

 

 

 

  • Question 3
  • A
  • B
  • C
  • D

An isolated nerve muscle preparation exposed to a low concentration of extracellular Ca2+ would be associated with a decrease in the (NOTE: there is more than one correct answer.)

A. Amplitude of the EPP

B. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

C. Influx of Ca2+ into the presynaptic terminal

D. Amplitude of MEPPs

An isolated nerve muscle preparation exposed to a low concentration of extracellular Ca2+ would be associated with a decrease in the (NOTE: there is more than one correct answer.)

A. Amplitude of the EPP This answer is CORRECT!

The decreased extracellular concentration of calcium results in less calcium entering the presynaptic terminal during an action potential. Less transmitter would be released, and there would be a smaller end-plate potential.

B. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

C. Influx of Ca2+ into the presynaptic terminal

D. Amplitude of MEPPs

An isolated nerve muscle preparation exposed to a low concentration of extracellular Ca2+ would be associated with a decrease in the (NOTE: there is more than one correct answer.)

A. Amplitude of the EPP

B. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential This answer is CORRECT!

The reduced concentration of extracellular calcium would result in decreased influx of calcium into the presynaptic terminal with an action potential in the presynaptic terminal. The reduced level of intracellular Ca2+ would lead to decreased release of transmitter.

C. Influx of Ca2+ into the presynaptic terminal

D. Amplitude of MEPPs

An isolated nerve muscle preparation exposed to a low concentration of extracellular Ca2+ would be associated with a decrease in the (NOTE: there is more than one correct answer.)

A. Amplitude of the EPP

B. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

C. Influx of Ca2+ into the presynaptic terminal This answer is CORRECT!

Decreased extracellular calcium would lead to decreased influx of calcium through voltage-dependent calcium channels that are opened as a result of a presynaptic action potential.

D. Amplitude of MEPPs

An isolated nerve muscle preparation exposed to a low concentration of extracellular Ca2+ would be associated with a decrease in the (NOTE: there is more than one correct answer.)

A. Amplitude of the EPP

B. Release of transmitter from the terminal of the motor axon triggered by a presynaptic action potential

C. Influx of Ca2+ into the presynaptic terminal

D. Amplitude of MEPPs This answer is INCORRECT.

There would be no change in the amplitude of the miniature end-plate potential. The amplitude of the miniature end-plate potential is dependent upon the amount of transmitter contained in an individual vesicle. This would be independent of the Ca influx that occurred during an action potential and independent of the concentration of extracellular calcium.

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