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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.

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. Through mechanisms that are not fully understood, calcium in the presynaptic terminal leads to the release of the chemical transmitter substance.

Calcium Hypotheses for Chemical Synaptic Transmission

Quantal Nature of Transmitter Release

(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 releaseable pool. Newly synthesized vesicles are found in the storage pool. The process by which a vesicle migrates from the storage pool to the 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.

Figure 5.4

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.

Figure 5.6

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Figure 5.6, above, illustrates a summary of the steps involved in the process of synaptic transmission at the neuromuscular junction.

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