Thomas Huxley once described ‘the slaying of a beautiful hypothesis by an ugly fact’ as the great tragedy of science. But Eccles did not mourn the loss of his idea – he wrote immediately to Dale informing him he was now convinced that neurotransmission must be chemical. Dale replied, congratulating Eccles on the beauty of his observations and wryly commenting that ‘your new-found enthusiasm is certainly not going to cause any of us embarrassment’. He later wrote that Eccles’s conversion to the chemical hypothesis was like that of Saul on the road to Damascus, ‘when the sudden light shone and the scales fell from his eyes’. It is one of the great strengths of the scientific method, and a measure of the quality of a scientist, that when the data show conclusively that a favoured hypothesis is wrong, it is quickly abandoned.
Mind the Gap
When the nerve impulse arrives at the end of the axon, it must somehow cause the release of transmitter from the tiny vesicles in which it is stored. Calcium ions play a crucial role in this process. The concentration of calcium ions is more than ten thousand times less inside our cells than outside it, and it is held at this level by molecular pumps that quickly remove any calcium that enters, either by ejecting it from the cell or by storing it in intracellular compartments. One reason calcium is kept so low is that it functions as an intracellular messenger, conveying information about events at the cell membrane to intracellular proteins and organelles. At the nerve terminal, for example, calcium triggers the synaptic vesicles to release the acetylcholine they contain into the gap between the nerve and the muscle.
When a nerve impulse arrives at the nerve terminal, it causes calcium channels to open, allowing calcium ions to flood into the cell. This triggers synaptic vesicles filled with the neurotransmitter acetylcholine to move to, and fuse with, the cell membrane, releasing their contents into the synaptic gap. Acetylcholine then diffuses across the gap and binds to its receptors in the muscle fibre membrane. Binding of the neurotransmitter opens an intrinisc ion channel in the acetylcholine receptor, enabling sodium ions to enter the cell. The flow of sodium current triggers an electrical impulse in the muscle. In this way, the electrical signal passes from nerve to muscle via a chemical intermediary.
Calcium enters the cell via calcium channels in the pre-synaptic nerve membrane that open in response to the voltage change produced by the arrival of the nerve impulse. It is crucial that these channels open only when an impulse arrives and that they only remain open for a brief time; uncontrolled calcium influx can be dangerous as it triggers prolonged release of transmitter. One of the many toxic ingredients of the venom of the deadly black widow spider is alpha-latrotoxin, which inserts itself into the cell membrane, forming calcium-permeable pores that allow calcium ions to flood into the cell in an unregulated fashion and cause massive transmitter release and muscle spasms.
Similarly, increased transmitter release is produced by genetic mutations that prolong the duration of the nerve impulse and so increase calcium influx. People with such mutations may experience periodic attacks of dizziness, uncontrollable muscle shakes and uncoordinated movements so that they find it difficult to walk and lose their balance. They may also vomit. Attacks are often brought on by emotional stress, such as the excitement of watching your favourite football team play. Given the symptoms, it is not surprising that people with this condition are sometimes castigated for being drunk, a fact which the affected individual may find particularly galling if, as has been known, they happen to be teetotal.
On the other hand, inadequate calcium influx means that too few vesicles are encouraged to liberate their contents so that transmitter release is insufficient to trigger muscle contraction. This happens in Lambert Eaton myasthenic syndrome (LEMS), a condition in which the body produces antibodies against the calcium channels at the neuromuscular junction. These antibodies bind to the calcium channels and cause them to be removed from the nerve membrane so that nerve impulses fail to release any transmitter. The result is muscle weakness or paralysis. Most cases of LEMS are actually due to a tumour (usually a lung cancer) elsewhere in the body that possesses a similar type of calcium channel. The immune system’s response to this cancer is to attack it by producing antibodies and those directed against the cancer cells’ calcium channels cross-react with calcium channels present in the nerve terminals. LEMS is thus a red flag, warning the clinician to search for a possible tumour. It can be a valuable sign, for the sooner the lung cancer is treated the better the outcome for the patient.
All Docked Up and Ready to Go
It is sometimes envisaged that the interior of a cell resembles a pea soup, in which chemicals and organelles mill around in a random fashion. This is very far from the truth. Inside the cell everything has its place and is anchored in its correct position by a highly structured protein network called the cytoskeleton. This is particularly evident at the nerve terminal, where vesicles packed with transmitter are released only at specialized sites known as ‘active zones’. Here several vesicles sit docked with the membrane, pretriggered for immediate release as soon as they get the signal to go. The calcium channels sit adjacent to the docking sites, reducing the distance that calcium has to travel once it enters the cell. This helps make transmission very fast. Within a millisecond (a thousandth of a second) of an electrical impulse reaching a nerve terminal, about 30 million molecules of acetylcholine are released. These quickly diffuse across the gap to their receptors on the muscle membrane, where they only remain bound for a couple of milliseconds, so that everything is over in about 20 milliseconds.
A small number of docked vesicles are ‘trigger happy’ and do not wait for a calcium signal: they are spontaneously released at a low frequency (they are too few to cause muscle contraction). Having a system that is all geared up and ready to go ensures that the arrival of a nerve impulse results in very rapid transmitter release – something you may be grateful for when your brain tells your hand to withdraw from a scalding-hot pan handle.
Complex molecular machinery is needed to overcome the enormous energy barrier that normally prevents the membrane of the synaptic vesicle fusing with that of the cell. This includes the numerous proteins that make up the docking and release complex, which act as molecular midwives, facilitating vesicle docking and membrane fusion. Precisely how binding of calcium ions to these proteins triggers the cascade of conformational changes that causes the vesicle and surface membranes to fuse is still unclear. However, inhibiting midwife protein function blocks neuromuscular transmission. Botulinum toxin, for example, prevents transmitter release and muscle contraction by destroying a specific set of these proteins.
Not all synaptic vesicles are primed for release. Most are stored at some distance from the release sites and must move to the membrane before they can be released; they must also undergo maturation processes that ready them for docking and release. Calcium also serves as a signal for mobilizing these troops of vesicles.
Poison Darts
Once liberated from the nerve terminal, acetylcholine diffuses across the tiny gap to the post-synaptic membrane of the muscle fibre, where it interacts with its receptors. Binding of the transmitter causes a conformational change in the acetylcholine receptor that opens an intrinsic ion channel, allowing a simultaneous influx of sodium and efflux of potassium ions. This decreases the voltage difference across the muscle membrane and (if it is sufficiently large) triggers an electrical impulse in the muscle fibre. In this way, acetylcholine serves to link the action potential in the nerve to one in the muscle, and ultimately to muscle contraction.
A large number of drugs and poisons work by interfering with the action of acetylcholine at its muscle receptor. The most famous is curare – the poison used by South American Indians to tip their arrows and the darts used in their blowpipes. Curare blocks binding of acetylcholine to its receptors in the muscle membrane and so prevents the nerve from stimulating the muscle fibre. Consequently, an animal hit by a dart is completely paralysed and falls out of the tree to the ground,
where it is either slaughtered or dies from respiratory failure. Fortunately, curare is poorly absorbed by the digestive system, so animals killed in this way are safe to eat.
Curare was once also used in warfare and even the slightest nick with a poisoned arrow could be fatal. It was much feared. In his account of the discovery of Guiana (Guyana), Sir Walter Raleigh wrote that ‘the party shot endureth the most insufferable torment in the world, and abideth a most ugly and lamentable death’. Depending on the dose, the victim can be awake, aware and sensitive to pain, but unable to move or breathe: unless given artificial respiration they will eventually die of respiratory failure. Toxins like curare have been used to tip arrows and spears for hundreds of years and the ancient Greek word toxicon, from which the name toxin derives, meant ‘bow’ or ‘arrow poison’.
Curare can be extracted from many different South American plants, but the best known is the climbing pareira vine Chondrodendron tomentosum. The great Prussian explorer Alexander von Humboldt was the first European to describe how it is prepared, in 1800. He noted that the juice from the vine was extracted and mixed with a sticky preparation from another plant to make a thick treacly substance that could be glued to an arrowhead. Some years ago, I asked to view the curare-tipped blow darts owned by the Pitt Rivers Museum in Oxford. I was allowed to see them, but not to handle them or borrow them for a public lecture, for health and safety reasons. I was somewhat indignant, as I was certain the drug must have long since deteriorated. I was wrong: recently, curare that was 112 years old was shown to still be effective. Interestingly, the pure toxin was first isolated from a native preparation of curare held by the British Museum; it had been stored in a bamboo tube and the active alkaloid was hence named tubocurarine (tube-curare).
A disaffected group of conscientious objectors are reputed to have hatched a bizarre plot to kill the British Prime Minister Lloyd George using curare. Mrs Alice Wheeldon, her daughters Winnie and Hettie, and her son-in-law Alfred Mason all belonged to the No Conscription Fellowship that campaigned against compulsory military service (introduced because of very heavy losses on the Western Front) and the punishment and imprisonment of conscientious objectors (COs) during World War I. Alfred, a qualified chemist and a lecturer at Southampton University, obtained the curare. In late December 1916, the group was successfully infiltrated by two secret agents, Alex Gordon and Herbert Booth, who posed as conscientious objectors. Herbert Booth testified that he was given an airgun and pellets tipped with curare and detailed to shoot the Prime Minster as he walked on Walton Heath. There was enough curare to kill several individuals and, despite their protestations that the drug was intended to kill dogs guarding CO internment camps rather than the Prime Minister, and that this course of action had been suggested to them by Booth and Gordon, Alice, Winnie and Alfred were convicted of conspiracy to murder. Whether the assassination attempt could have been successful is unclear. Equally uncertain is if this was a genuine assassination attempt or a government plot to discredit the anti-conscription movement and the anti-war factions – were Booth and Gordon in fact agents provocateurs?
What is more definite is that the US government gave curare to operatives on covert operations, in case they were caught and had to take their own life to avoid being tortured. When Francis Gary Powers flew his U2 spy plane over Russia during the Cold War he carried with him an American silver dollar that had a small straight pin inserted in the side. The pin consisted of an outer sheath covering a fine sharp needle with a grooved tip coated with a brown sticky substance that Powers states he was told was curare. When he was shot down and captured, the Russians found the pin and tested it on a dog, which stopped breathing within a minute of being pricked and was dead thirty seconds later. It is questionable, however, whether curare was the sole ingredient on the pinhead as its action appears to have been unusually fast.
The poison hemlock (Conium maculatum) contains several alkaloids, but one of the most potent, coniine, acts like curare by blocking the action of acetylcholine and paralysing the respiratory muscles. The plant was used as a means of judicial execution in Europe for centuries. Its most famous victim was Socrates, whose death is described in the Phaedo, which details how the paralysis developed, beginning with the feet and moving gradually upwards towards the chest.
Curare-like drugs, such as vecuronium, are often used in operations as muscle relaxants to enable the surgeon to operate more easily and to allow a lower level of anaesthesia to be used. This is especially important during abdominal surgery because contraction of the abdominal muscles might make it difficult for the surgeon to gain access without the intestines being squeezed out of the wound. Although the respiratory muscles are those least affected by curare, patients are usually artificially ventilated to help them breathe. The caveat with the use of curare-like drugs is that if anaesthesia is inadequate, the patient may be awake but unable to move, speak or communicate their distress. Each year, this happens to about 0.1 per cent of people undergoing surgery in the United States – approximately 25,000 people. About a third of them can feel the pain associated with their operation and the remainder have some awareness of what is going on without suffering pain. It is a particular problem during Caesarean sections when a lighter level of anaesthesia may be used to avoid anaesthetizing the unborn child.
Nerve Gas
Clearly, if a muscle is to be able to respond to a second nerve impulse, the first signal must be switched off rapidly. This is achieved in two ways. First, the transmitter remains attached to its receptor for only a short time before it spontaneously detaches. Secondly, the transmitter is rapidly removed. At the nerve–muscle junction, acetylcholine is destroyed within about five milliseconds of its release by an enzyme called acetylcholinesterase that sits in the synaptic gap.
Agents that inhibit the action of acetylcholinesterase are lethal. The most famous is the nerve gas sarin. The Aum Shinrikyo sect came to public notice in 1995 when they released an impure form of sarin in the Tokyo Metro, killing twelve people, seriously injuring fifty and temporarily affecting the sight of almost a thousand more. The terrorist attack was timed to coincide with the morning rush hour. Equally horrendous were the tests conducted forty years earlier by the British government.
In May 1953, a number of young servicemen were asked to participate in trials for a new cure for the common cold. But the volunteers were misled in a brutal and unforgivable fashion, as they were not exposed to the cold virus but to the nerve gas sarin. Twenty-year-old Ronald Maddison died horribly forty-five minutes after the agent was dripped onto his skin, suffering from convulsions so severe it appeared to an eyewitness that he was being electrocuted. His lungs became clogged with mucous and he died of asphyxiation. Maddison was used as a human guinea pig to determine how much of the lethal agent was required to kill the enemy. His death was witnessed by a young ambulance man, Alfred Thornhill, who was traumatized by what he saw and afraid to speak out because the authorities threatened him with prison if he did so. The incident was quickly hushed up and only became known fifty years later when the Wiltshire police finally opened a second inquest into Maddison’s death. The previous verdict of death by misadventure was overturned and replaced by one of unlawful killing. Maddison’s sister said that until the inquest she and her family had never known the truth about how her brother died. Britain was not alone in wishing to test sarin on troops. Extraordinarily, in the 1960s, US military scientists requested permission from the Australian government to test the nerve agent on Australian troops.
Inhibition of acetylcholinesterase is fatal because it leads to a build-up of acetylcholine in the synaptic gap. The consequence is overstimulation of acetylcholine receptors, which results in muscle convulsions. Because acetylcholine is the transmitter at the nerves that innervate the glands, acetylcholinesterase inhibitors also cause excessive salivation, drooling and watering eyes. The symptoms of acute poisoning by sarin and other nerve gases are well described by the mnemonic SLUDGE: salivation, lacrimation, u
rination, diarrhoea, gastrointestinal upset and emesis (nausea and vomiting). The victim can also suffer from dizziness, skin irritation, tightness of the chest and involuntary muscle twitching. In the worst case, they may die by suffocation from convulsive spasms of the chest muscles.
Atropine is used to treat patients who have been poisoned by nerve agents. It acts by blocking acetylcholine receptors and thus reduces the ability of excess acetylcholine to exert its effect. Military personnel carry autoinjectable ‘Combo’ pens, consisting of a spring-loaded syringe containing a needle and a barrel filled with atropine which can be used to self-administer the drug rapidly in an emergency. The top of the pen also contains a Valium tablet to reduce levels of stress (which is probably much needed!). Too much atropine, however, can also incapacitate the soldier because it knocks out acetylcholine action too effectively. In this case, nerve–muscle transmission is prevented, resulting in muscle weakness.
Oximes are also used as antidotes to nerve gases, but are generally given in advance of a possible nerve agent attack. They reactivate acetylcholinesterase by removing the phosphate molecule that is added to the enzyme by the nerve agent.
The Deadly Calabar Bean
Another substance that inhibits the action of acetylcholinesterase is physostigmine, the active ingredient of the Calabar bean, Physostigma venenosum. The Nigerian name of the plant is esere, from which its alternative scientific name, eserine, is derived. It was eserine that enabled Feldberg and Dale to demonstrate that acetylcholine was released from nerve terminals, by preventing its breakdown by endogenous acetylcholinesterases.
The Spark of Life: Electricity in the Human Body Page 10
