Ch. 5: Mechanisms of Neurotransmitter Release

John H. Byrne, Ph.D., Department of Neurobiology and Anatomy, McGovern Medical School

Revised 19 May 2020

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.

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.

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

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.

• First, they occurred in the absence of any stimulus.
• Second, they were small with an average amplitude of about 0.5 mV. The distribution could be fit by a single gaussian function, indicating that the events arose from a common underlying process.
• Third, these events could only be recorded in the vicinity of the synaptic junction.
• Fourth, they were blocked by curare.
• Fifth, they were enhanced by neostigmine.

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.

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.