Synaptic Transmission and Quantal Release

Put an electrode inside a resting frog muscle fibre at the point where the nerve ends, stimulate nothing, and you still see small depolarizations arriving at random, each about the same size and each far too small to fire the fibre.

Paul Fatt and Bernard Katz reported these miniature endplate potentials in 1952. They turned out to be the bricks from which every nerve-evoked response at that junction is built. This lesson follows a signal across a chemical synapse, shows how del Castillo and Katz counted the bricks, and uses the count to explain why two different diseases make muscles weak.

From spike to fusion

A synapse is a site where one neuron passes a signal to another cell. At a chemical synapse the presynaptic terminal releases a transmitter that crosses a gap, the synaptic cleft, and acts on receptors of the postsynaptic cell. The vertebrate neuromuscular junction, where a motor axon meets a skeletal muscle fibre, is the best-studied example. How it is built during development belongs to the neuromuscular junction in Developmental Neuroscience, along with central synapses and synapse elimination.

The sequence runs like a relay. The action potential invades the terminal and opens voltage-gated calcium channels. Calcium flows in down a steep gradient. Synaptic vesicles near the membrane have already been docked and primed, made ready to fuse. Calcium binds a sensor, synaptotagmin-1 in the neuronal machinery, and three SNARE proteins (synaptobrevin on the vesicle, syntaxin-1 and SNAP-25 on the plasma membrane) zip together and pull the two membranes into one. The molecular mechanics of fusion are taught in vesicle traffic, and calcium handling in calcium signaling.

At the neuromuscular junction the transmitter is acetylcholine. It diffuses across the cleft and opens nicotinic acetylcholine receptors, whose gating belongs to ion channels as receptors. The channel passes both sodium and potassium, so its reversal potential sits near 0 mV, between the two ions’ equilibrium potentials. At a frog muscle fibre’s resting potential, far more negative than the reversal potential, the driving force (the difference between the membrane potential and the reversal potential) is large and inward, and the fibre depolarizes. This depolarization is the endplate potential (EPP), the muscle’s version of an excitatory postsynaptic potential (EPSP), a depolarization caused by transmitter. Acetylcholinesterase in the cleft hydrolyzes acetylcholine and ends the signal. Vesicle membrane is then retrieved: Heuser and Reese followed an extracellular tracer from the frog terminal’s surface into internal cisternae and then into new vesicles, a story continued in endocytosis.

Not every synapse uses a transmitter. At an electrical synapse, channels called gap junctions join the two cells’ cytoplasm, and current passes directly with no transmitter step. Such junctions are often drawn as symmetric pipes, but some rectify, passing current more easily one way. Shui and colleagues (2020) found that junctions built from the worm innexins UNC-7b and UNC-9, expressed in Xenopus oocytes, strongly favour current from the UNC-9 cell to the UNC-7b cell.

Packets: the miniature and the quantum

Fatt and Katz noticed that the spontaneous miniatures looked like scaled-down copies of the evoked endplate potential, and were largest at the endplate. They attributed them to spontaneous release of acetylcholine from the terminal. Del Castillo and Katz then asked whether the evoked response was made of the same units.

They bathed the frog junction in a solution with less calcium and more magnesium, which cut release to a trickle. Now a nerve impulse often produced nothing at all, a failure. When it did produce a response, the amplitudes clustered at the size of one miniature, at twice that, and at three times that. The evoked response came in packets. Each packet is a quantum, and its postsynaptic effect, the miniature amplitude, is the quantal size, q. A quantum is a multimolecular packet, later identified with the contents of one vesicle; it is not one molecule of acetylcholine.

Think of coins dropped into a jar on a scale. You cannot see the coins, but if every reading is a multiple of one coin’s weight, you can count them.

Two cautions keep this honest. The size of a miniature depends on how much transmitter a vesicle holds and on how many receptors face it, so a small miniature does not by itself say which side is at fault. And the frog endplate is only one kind of synapse: in cultured rat hippocampal boutons (small presynaptic terminals), Maschi and Klyachko (2020) saw two vesicles fuse at the same bouton after a single action potential.

Counting quanta: two estimates and their assumptions

The mean quantal content, m, is the average number of quanta one impulse releases. You can estimate it two ways, and the two must agree if the model is right.

Direct method: m = mean EPP (including failures) / mean miniature

Failure method (Poisson): m = −ln(number of failures / number of trials)

The second comes from the Poisson distribution, P(k) = exp(−m) × m^k / k!, which gives the chance of releasing exactly k quanta. For k = 0, P(0) = exp(−m), so m = −ln P(0). The model assumes many independent release sites, each with a small and equal chance of releasing, and, for the direct method, a stable quantal size and quanta that add linearly; the failure method needs only the Poisson assumption and failures you can reliably detect. Poisson statistics describe release well only when the release probability at each site is small, as in low calcium.

Worked problem. In this made-up recording, a frog junction sits in low calcium and high magnesium. You stimulate the nerve 200 times. The mean miniature is q = 0.40 mV. The mean EPP, counting failures as zero, is 0.40 mV. Seventy-four stimuli give no response.

Step 1: direct method. m = 0.40 mV / 0.40 mV = 1.00 quantum per stimulus.

Step 2: failure method. P(0) = 74 / 200 = 0.37. m = −ln(0.37) = 0.99. The two estimates agree, which supports the Poisson model for this recording.

Step 3: predict the rest. With m = 1.0, P(1) = exp(−1) × 1 = 0.368, so about 200 × 0.368 = 74 trials should show one quantum. P(2) = 0.368 / 2 = 0.184, so about 37 trials should show two. If the amplitude histogram has peaks near 0.40 and 0.80 mV with roughly those counts, the data are consistent with the model.

Step 4: the safety factor in normal calcium. Now restore normal calcium and suppose m = 60, q = 0.40 mV, and, as an illustration, that the fibre fires when the EPP reaches 12 mV. Assuming linear summation, EPP = 60 × 0.40 = 24 mV. The safety factor, the ratio of the EPP to the depolarization needed to fire, is 24 / 12 = 2.0. Linear summation is a simplification: as a large EPP depolarizes the fibre, the driving force falls and quanta add less than linearly.

Calcium and the steep dependence

Release needs external calcium, and magnesium opposes it. F. A. Dodge and Rami Rahamimoff (1967) measured how strongly. At the frog neuromuscular junction they varied calcium and plotted EPP amplitude against calcium concentration on logarithmic axes. At low calcium the slope was 3.78 (SD 0.2, 28 experiments), and it became shallower as calcium approached its normal level.

A slope of about 4 means that release rises roughly as the fourth power of calcium over that range. Double calcium on the steep part and release grows by 2^3.78, about 14 times. That steepness is why a modest loss of calcium entry can cut release sharply.

The slope fits a model in which several calcium ions act together to trigger release. It does not count binding sites. In Dodge and Rahamimoff’s data, adding magnesium lowered the EPP without changing the slope; a fitted slope can still be shaped by saturation and by how calcium spreads in the terminal, and it flattens toward normal calcium. Treat “four calcium ions” as a reading of one curve in one preparation, not a molecular fact.

When the safety factor fails

In myasthenia gravis, antibodies in most patients target the muscle’s acetylcholine receptors; others have antibodies against MuSK, a protein needed at the junction. Antibodies to the receptor can cross-link receptors and speed their removal. Fewer receptors means a smaller q. Toyka and colleagues (1975) transferred an immunoglobulin fraction from patients’ serum into mice, which then had smaller miniatures and fewer junctional receptors.

Return to the worked example. Cut q from 0.40 to 0.24 mV. With m = 60 the EPP is 14.4 mV, which still fires. But release runs down during repeated impulses. Suppose m falls by 25 percent, to 45. The normal EPP is 18 mV and still fires; the myasthenic one is 45 × 0.24 = 10.8 mV, below 12 mV, and that fibre fails. This is the fatigable weakness of myasthenia: muscles that work at first and weaken with use. Acetylcholinesterase inhibitors act by letting each quantum’s acetylcholine last longer.

In Lambert-Eaton syndrome the fault is presynaptic. Antibodies against the terminal’s voltage-gated calcium channels reduce calcium entry, so m is low while q is normal. Because release depends steeply on calcium, repeated stimulation, which lets calcium build up in the terminal, raises release.

Design problem

Two made-up muscle biopsies arrive for intracellular recording. In A, the miniature amplitude is reduced and the quantal content is normal. In B, the miniature amplitude is normal, the quantal content is low, and it rises during 20 Hz stimulation. Where is each defect, and what experiment would test your answer?

Next, transmitter systems asks where the brain’s other transmitters are made and cleared, and synaptic plasticity asks how release changes from one impulse to the next.