The toxin hunters were an adventurous bunch. Some spent a month each year gathering cone snails in the Red Sea, alternately diving for snails and extracting the venom; it might sound like a holiday, but it was actually hard work, to say nothing of the added frisson provided by the need for eternal vigilance to avoid being stung. Other intrepid investigators travelled to North Africa, where they would venture out into the desert at night. Under a vast sky that glittered with stars, the ground appeared a deep inky black, free from danger. Switch on an ultraviolet lamp, however, and the many scorpions swarming around their feet leapt into view, for scorpions fluoresce brightly in ultraviolet light. Thousands were carefully packed into large milk churns and taken back to France, and a whole week each month was devoted to milking them for their venom. This operation requires extreme care, but an experienced researcher can avoid being stung by grabbing the scorpion close to the tip of its tail. The scorpion reacts by secreting a drop of venom from its sting, which is then carefully collected. It is a very time-consuming process because venom from as many as 150,000 scorpions is needed to isolate enough toxin for experiments.
The cornucopia of sodium channel poisons illustrates just how crucial sodium channels are for nerve and muscle function. Their special properties of voltage sensitivity and selective permeability to sodium ions are essential for the generation and conduction of nerve impulses, and the transmission of information along nerve axons, such as those that travel along motor axons from your brain and spinal cord to your muscles. What happens when those impulses reach the nerve terminals and how they then excite the muscle fibre is considered next.
4
Mind the Gap
the leap
thought makes at the synaptic gap.
Brian Turner, ‘Here, Bullet’
Botox is the latest tool in the armoury of the cosmetic surgeon and is used by film stars and ordinary people alike to smooth out the wrinkles etched on our faces by age. But it is actually a virulent poison called botulinum toxin, and in my youth it was far more famous for causing fatal food poisoning. In those days, tinned corned beef was a staple food, but if the cans were not correctly sealed they could become infected with the bacterium Clostridium botulinum. The toxin the bacteria produced led to the death of anyone who unwittingly ate the contaminated corned beef.1 Other meat products can also harbour the toxin: indeed, the word ‘botulinum’ derives from the Latin name for sausage (botulus).
Botulinum toxin is one of the most potent natural poisons known. An amount sufficient to cover the head of a pin is more than enough to kill an adult and it is estimated as little as a gram would kill a million people. It is alleged to have been used by the Czech resistance to assassinate the notorious SS-Obergruppenführer Reinhard Heydrich, a high-ranking Nazi whom Hitler considered as a possible successor to himself. Heydrich was attacked in Prague in the spring of 1942 by two British-trained Czech patriots, who lobbed a grenade, reputedly impregnated with botulinum toxin, into his car. Although Heydrich’s injuries were not thought to be life-threatening, and the surgery he received was successful, he died eight days later from complications. Whether botulinum toxin actually caused his death remains highly controversial. Nevertheless, several countries have explored its potential as an assassination agent; the CIA, for example, laced some of Fidel Castro’s favourite cigars with it (they were never used).
Botulinum toxin acts by preventing muscle contraction. When ingested, it gradually relaxes the respiratory muscles until they stop functioning, causing paralysis and death from asphyxiation. In the last decade or two, however, it was realized that if a minute quantity of the toxin is injected under the skin, it will paralyse the muscles in a highly localized way. At first this was used to treat people with an unfortunate condition in which their neck or shoulder muscles become permanently frozen, so that their head is twisted to one side. But it was soon recognized that botox had another effect – it ironed out the furrows produced by years of frowning and the crinkles due to smiling. As the Swiss physician Paracelsus once said, ‘The right dose differentiates a poison and a remedy’.
The virtue of botox is that it binds so tightly to its target that it is only slowly washed away and the muscles remain relaxed for many months. But every six months or so, the procedure must be repeated. The downside is that the toxin also blocks contraction of the muscles used in facial expressions such as smiling, and thus tends to produce a smooth expressionless sphinx-like stare. Worse still, if too much is used it can cause local paralysis, resulting in droopy eyelids or a downturned mouth.
A Nobel Dream
Botox causes paralysis by blocking the transmission of electrical impulses from nerve to muscle. These two types of cell are not physically connected and the impulse cannot leap the gap that separates them. Instead, a chemical messenger is used to send signals from one cell to another. Transmission of information from nerve cell to muscle cell (or another nerve cell) takes place at specialized junctions called synapses, where the gap between the two cells is very tiny – less than one hundredth-millionth of a metre (about thirty nanometres). For obvious reasons, the upstream cell that releases the transmitter (in this case the nerve cell) is known as the pre-synaptic cell and its target as the post-synaptic cell.
The tip of the nerve fibre is densely packed with small membrane-bound vesicles filled with a chemical transmitter. At the synapse between nerve and muscle the transmitter is acetylcholine, but many other chemicals are used to signal information between different types of nerve cells in the brain. When an electrical impulse arrives at the nerve ending it causes the vesicles to release their contents into the gap between the two cells. The transmitter that is liberated diffuses across the gap and attaches to a receptor on the surface of the post-synaptic cell, triggering an electrical impulse. In a muscle cell this electrical impulse causes contraction.
The use of chemicals to transmit information from nerve to muscle was discovered by Otto Loewi, a brilliant scientist who worked for much of his life in the Austrian city of Graz. Born in Frankfurt in 1873, he was initially more interested in painting, music and philosophy than in science and desired to become an art historian. Dutifully, however, he bowed to family pressure and went to medical school instead. There, he quickly became captivated by science. Loewi had an irrepressible zest for life, an enthusiasm for science and a sense of humour that lasted throughout his life: even during his last years, he maintained that ‘excitement was good for him’.
In 1921, Loewi showed that chemicals are involved in transmission of electrical impulses between nerve and muscle cells. He attributed this breakthrough to an inspiration that came to him during a dream. He wrote, ‘The night before Easter Sunday of that year I awoke, turned on the light, and jotted down a few notes on a tiny slip of thin paper. Then I fell asleep again. It occurred to me at six o’clock in the morning that during the night I had written down something most important, but I was unable to decipher the scrawl. The next night, at three o’clock, the idea returned. It was the design of an experiment to determine whether or not the hypothesis of chemical transmission that I had uttered seventeen years ago was correct. I got up immediately, went to the laboratory, and performed a simple experiment on a frog heart according to the nocturnal design.’
Loewi knew that if an electric shock were applied to the nerve innervating a frog’s heart, the heart rate would slow. If, as he suspected, this was caused by a chemical transmitter released from the nerve endings, then the chemical should pass into the fluid bathing the heart. First in his dream, and then in reality, Loewi showed that if this solution were collected and then flowed over a second heart, the rate at which the second heart beat would also slow. Ergo, the first heart secreted a soluble chemical messenger that then acted upon the second.
Loewi called his chemical messenger Vagus-stoff because it was released by the vagus nerve that supplies the heart. Today, we know it to be acetylcholine, as indeed Loewi himself conjectured. Too cautious to publish a speculation that migh
t prove premature, it was only after a considerable number of further experiments that he tentatively concluded, in 1926, that it might be acetylcholine. Loewi was lucky, because the nerve that supplies the frog’s heart contains two sets of fibres: one set releases a chemical that causes slowing of the heart rate (acetylcholine) and the other secretes a substance (noradrenaline) that speeds it up. The frequency at which Loewi stimulated the nerve to the heart must have been exactly right to ensure that the effects of acetylcholine dominated. Once again, serendipity played a key role in scientific discovery.
In 1936, Loewi (together with his lifelong friend, the British scientist Sir Henry Dale) was awarded the Nobel Prize for his work. A mere two years later, on 12 March 1938, Germany invaded Austria. Loewi was informed late that afternoon that the Nazis had taken over the country, but being preoccupied with his research he ignored the significance of the news. He found out his mistake at three o’clock the following morning when a dozen armed storm troopers burst into his bedroom and dragged him off to prison, along with many other Jews. Uncertain of his future, and expecting to be murdered at any time, he scribbled his latest results on a postcard, addressed it to a scientific journal (Die Naturwissenshaften) and persuaded a prison guard to stick it in the post. He felt indescribable relief that his results would not be lost.
Two months later Loewi was released from jail and allowed to leave the country – but only after he had given all his assets to the Nazis, which included instructing a Swedish bank in Stockholm to transfer his Nobel Prize money to a Nazi-controlled bank as a ransom for his life. He escaped to England without a penny.
After spending time with Dale in London, Loewi held temporary appointments in Brussels and Oxford, before sailing to the United States in 1939 to take up a research professorship in pharmacology at New York University Medical School. He arrived in New York in June 1940, aged sixty-seven, armed with a visa and a doctor’s certificate. Glancing at the latter while waiting to see the immigration officer he was horrified to discover it proclaimed ‘Senility, not able to earn his living’. Fortunately the officer chose to ignore this questionable handicap, and Loewi was allowed into the United States. He was never bitter about the tumultuous upheaval to his life. In fact, he considered fate had been kind to him, as in the USA he was able to pursue his scientific endeavours at an age at which in Austria he would have been forced to retire. For another twenty-one years, he continued to inspire successive generations of students and spend his summers in ever-animated discussion at the Marine Biological Laboratories at Wood’s Hole.
Hitler’s Gift2
Sir Henry Dale was both a great scientist and a wise, influential and authoritative spokesman for science, who was held in affectionate veneration by his all colleagues. A tall, warm-hearted man, with a capacious memory, he played an important role behind the scenes in rescuing Jewish biologists, including Loewi, from Nazi Germany. He was also intimately involved in the discovery of chemical transmission. Like Loewi, Dale stressed the role of ‘fortunate accidents’ in his research. Equally important, however, was his perseverance. Dale’s involvement in the story began in 1913 when he received an extract of ergot (a fungus) for routine testing. It had a potent and unexpected physiological action that aroused his interest. Using rigorous classical chemical methods, his colleague, the chemist Arthur Ewins, succeeded in identifying the active principle, which turned out to be acetylcholine. The physiological effects of acetylcholine mimicked those produced by stimulating certain nerves and led Dale to comment that if there was any evidence for the presence of acetylcholine in animal tissues it would be a good candidate for a neurotransmitter. World War I then intervened and Dale was occupied with other duties, but years later he succeeded in demonstrating that acetylcholine is indeed found naturally in animals and in isolating it from the spleen of a horse.
Dale’s interest in acetylcholine was rekindled when he learnt of Loewi’s dream-inspired experiment and he set out to see whether acetylcholine was secreted from nerve terminals at the nerve–muscle junction. This was not an experiment for the faint-hearted, for only tiny amounts of acetylcholine are released and, as Dale commented, it had an extraordinarily evanescent action. What Dale needed was a highly sensitive assay. Providence, in the form of the Nazis, provided it for him.
In 1933, shortly after coming to power, Hitler ordered that all Jews employed by state institutions should be sacked. Almost overnight, large numbers of academics were out of a job. William Beveridge, Director of the London School of Economics, inspired British academics not only to devise a rescue plan but to support it financially themselves. He also persuaded the Rockefeller Foundation to set up a special fund for Jewish scientists to enable them to move to American universities. Many Jews were helped to flee to the United States and Britain. They were Hitler’s great gift to the Allies. Hitler was seemingly unaware of their value, reputedly stating that, ‘If the dismissal of Jewish scientists means the annihilation of contemporary German science, then we shall do without science for a few years.’ It proved to be a suicidal policy.
But it was to be of considerable value to Dale. Wilhelm Feldberg, who had developed a sensitive assay for acetylcholine, relates how one day in 1933 he was summarily dismissed from his post at the Physiological Institute in Berlin because he was a Jew. A few weeks later he sought help from a representative of the Rockefeller Foundation of New York who was visiting the city with the aim of trying to rescue the brightest Jewish scientists. While the man was sympathetic, he said, ‘You must understand, Feldberg, so many famous scientists have been dismissed whom we must help that it would not be fair to raise any hope of finding a position for a young person like you. But at least let me take down your name. One never knows.’ On learning Feldberg’s name he hesitated, and riffling through his papers exclaimed with delight, ‘Here it is. I have a message for you from Sir Henry Dale. [...] Sir Henry told me, if by chance I should meet Feldberg in Berlin, and if he has been dismissed, tell him I want him to come to London to work with me.’3 Feldberg left Germany at once.
Feldberg’s technique provided a highly sensitive bioassay that not only showed that acetylcholine was indeed released on stimulation of the nerve innervating a muscle, but also allowed the amount of the transmitter to be measured. This was achieved by flowing a solution over the nerve when it was stimulated, collecting the solution, applying it to a leech muscle and measuring the strength of the contraction it produced. The secret to the success of the leech test was the use of a chemical (eserine) that prevented the breakdown of acetylcholine by endogenous enzymes, thus prolonging its ‘evanescent’ action. It was the finding that acetylcholine was the transmitter at the nerve–muscle synapses which led Dale to share the Nobel Prize with Loewi in 1936. In his acceptance speech, Dale tentatively suggested that chemical transmission might not be confined to the neuromuscular junction but also take place in the central nervous system. His words were prophetic.
The War of Soups and Sparks
While Dale favoured chemical transmission, the flamboyant Australian neurophysiologist John Carew Eccles was equally certain that communication was electrical, believing that transmission at nerve–nerve synapses was too fast to be chemical. A long-running debate known as the ‘war of the soups and sparks’ began that greatly enlivened the previously rather staid scientific meetings of the Physiological Society. Young, excitable, domineering and extremely energetic, Eccles presented his views with characteristic forcefulness. Dale, a member of the Establishment, and by now both a Fellow of the Royal Society and a Nobel Laureate, adopted a calm magisterial air. Yet Dale and Eccles were essentially shadow boxing. Although their public debates could be tense, highly charged, and, to some observers, quite astonishingly adversarial, their personal relationship was far from acrimonious, as they exchanged friendly letters and shared their results prior to publication. Furthermore, their scientific disagreement provided a valuable incentive for them to seek far more evidence to support their ideas than might otherwise have b
een the case.
Eccles was impressed by the long time it took for the heart to slow down when the vagal nerve was stimulated. Since this was known from Loewi’s work to involve a chemical transmitter, he inferred that the much faster transmission that took place at the junction between nerve and skeletal muscle could not be chemical and thus must necessarily be electrical. He was outraged at Dale and Feldberg’s suggestion that acetylcholine mediated transmission at the nerve–muscle junction. By 1949, however, sufficient evidence had accumulated for Eccles to concede that transmission at the neuromuscular junction was indeed chemical.
He reserved judgement, however, about what happened at nerve–nerve synapses in the spinal cord and brain, remaining convinced that here, at least, electrical transmission might prevail. The crucial experiment that resolved the debate was carried out late one night in mid-August 1951 in Dunedin, New Zealand, by Eccles and his colleagues Jack Coombs and Lawrence Brock. Eccles claimed it was inspired by his conversations with the philosopher Karl Popper who argued that nothing could be proved in science, only disproved. So Eccles set out to prove that neurotransmission in the central nervous system was not electrical and – to his immense surprise – he succeeded. The breakthrough came because the team used fine glass micropipettes, which they inserted into neurones of the spinal cord to pick up the electrical signals in the post-synaptic cells when the nerve was stimulated. Coombs, an electrical engineer, designed, built and operated the specialist apparatus needed to stimulate and record from the neurones. Eccles had arranged the experiment so that the potential trace would go down if transmission were chemical and would go up if it were electrical. It went down, and Eccles was momentarily stunned – the electrical transmission hypothesis was thereby falsified. It was a dramatic night in other ways too. Coombs’s wife gave birth, her baby girl being delivered by his co-worker Brock (a medic) while Eccles continued to experiment into the early hours of the morning.
The Spark of Life: Electricity in the Human Body Page 9