Author: Ibon Cancio, UPV/EHU Associated Professor in Cell Biology; researcher in the ‘Cell Biology & Environmental Toxicology’ (CBET) research group of the Plentzia Marine Station (PiE-UPV/EHU); Spanish scientific representative in the EMBRC Committee of Nodes

Ricardo Miledi (born Chihuahua, Mexico, 15 September 1927, died Irvine, California, 18 December 2017) was a Mexican neuroscientist known mainly for his work on ‘synaptic noise’ at the frog neuromuscular junction and on the presynaptic role of calcium (Ca2+) in triggering neurotransmitter release. Synapses make nerve stimulus transmission possible, allowing reflexes such as the jet propulsion motion in cephalopods, and creative activities such as the Eureka moment of Watson and Crick on their discovery of the double-helix or the composition of Iggy Pop’s The Passenger. The synapse itself could not be physiologically and biochemically understood without Miledi’s contribution… and without the contribution of the squid Loligo!

Picture of Neuroscience magazine
Front page of the special issue of the journal Neuroscience published in 2020 to honour the life and work of Ricardo Miledi.

Born in a family of seven children emigrated from Lebanon, Miledi graduated in medicine from the National Autonomous University of Mexico. He initiated experimental research on ventricular fibrillation at the National Institute of Cardiology in Mexico City where he would complete his PhD. While there in 1954, he had the opportunity to show his technical skills in delicate microdissections, which would become his hallmark, in front of two visitors from the Marine Biological Laboratory at Woods Hole, Massachusetts. As a result, he was offered a Grass fellowship for early career scientists in neuroscience’ to spend the summer of 1955 in Woods Hole studying lobster stretch receptors. However, it was the synapse in the common squid Loligo pealei that earned his attention during his stay. His early experiments there taught him that forgetting to add enough Ca2+to the experimental medium meant spoiling the experiments. That was his first encounter with the squid’s giant axons and synapse. Both structures in the squid shaped much of 20th century neurophysiological research. Alan Hodgkin and Andrew Huxley discovered their action potential (Nobel Prize, 1963) and Bernard Katz and colleagues identified neurotransmitter synaptic release (Nobel Prize, 1970) using the squid as a model organism. But before that happened, Miledi enjoyed an 18- month fellowship in Canberra, Australia, with John Eccles, the third recipient of the 1963 Nobel Prize.

Picture of Hodgkin, Huxley and Katz
Medicine and physiology Nobel Prize winners (Hodgkin, Huxley and Katz) who worked with the giant axon/synapse of the squid in 1963 and 1970. Loligo is the only one of them who can say that he won two Nobel Prizes. Both Bernard Katz and Loligo were colleagues of Miledi. (Photos and illustrations obtained from Wiki Commons).

In 1958, Miledi met Sir Bernard Katz while the latter was visiting the laboratory in Canberra. Katz offered Miledi a position in his Department of Biophysics at University College London to study how acetylcholine is released in the synapse. That proved to be a very fruitful move. For instance, together they discovered the phenomenon known as spillover, in which neurotransmitters diffuse away and stimulate receptors outside the synapse. Further work on extrasynaptic receptors based on this phenomenon led to the development of the concept of neuromodulation and to the understanding of the functions of glial cells.

The Ca2+ question in neural signalling though remained to be solved. In the early 1960s, Miledi found that absence of Ca2+ in the medium does not stop nerve impulse but completely supresses neurotransmitter (acetylcholine) release. Release was recovered as soon as a little bit of Ca2+ was provided. With Katz, Miledi published a paper with their observations of frogs in 1965, proposing a hypothesis as simple as this one: axon membrane depolarization → Ca2+ influx into the presynaptic neuron → quantal release of acetylcholine into the synaptic space. Textbook neurophysiology today, but just a hypothesis needing further experimental research in that early paper.

Obtaining clear proof required tight control of the action potential at the nerve terminal, and a synapse large enough to be accessed with microelectrodes. The opportunity came in a new visit to Woods Hole to deliver a Forbes talk in 1964. There, Miledi returned to the nearly 1 mm long giant synapse in the stellate ganglion of the squid. First, he repeated the experiments he had ran with Katz on frogs (Miledi and Slater, 1966). So much for comparative neurophysiology! Then, the big experiment came. As the giant axon is so large, Ca2+ can be microinjected right in the presynaptic neuron… However, experiments failed and time in the lab at Woods Hole ran out. Back in London, there were no giant axons nor squids around to work with.

Manipulating the giant axon of the squid under the microscope (Wiki Commons)
Manipulating the giant axon of the squid under the microscope (Wiki Commons)

During the late 1940s, Katz was working on the giant axon at Marine Biological Association (MBA) in Plymouth with the 1963 Nobel Prize winners Hodgkin and Huxley, so the obvious place to go in 1964 was Southern England. However, all efforts were unprofitable because even if squid Loligo forbesi arrived fresh to the lab, they were dead and useless for their experiments. They even tried to carry out the experiments on board of the trawler vessels that collected the specimens in Plymouth Sound.

When one door closes in a marine station another can be opened in different shores, and Stazione ZoologicaAnton Dhorn (SZN) in Naples had experience in working with squids (the discoverer of the giant axon, Sir John Z. Young, had worked with squids in Woods Hole, Plymouth and Naples). The Aquarium at the station could provide live squid on demand. Katz secured a travel grant of the Royal Society for Miledi, and he initiated summer pilgrimages to Naples and fresh squids it provided. Three- to eight-week yearly stays took place between 1965 and 1973. This time, the hero species of the story would have to be the squid Loligo vulgaris. The first paper, a classic, was published in 1967 by Katz and Miledi.

How squids work
The posterior wall of the squid’s muscular mantle is where the giant synapse can be found within the stellate ganglion on each side of the midline. Activation of this synapse triggers the contraction of the mantle musculature and ejection of a jet of water that allows the squid to move rapidly. Three paired giant neurons are organised in sequence relaying the information from the 'squid’s brain' tertiary axons to the contractile muscular mantle. (Modified from source, Wiki Commons)

Definitive proof of the Ca2+ hypothesis required discarding any direct involvement of the axon action potential in acetylcholine release. Miledi thus employed tetrodotoxin extracted from the Japanese poison puffer fish Fugu to inhibit the sodium (Na+) channels in the axon membrane. This, accompanied by a complete blocking of potassium (K+) (completely stopping axon membrane depolarisation to achieve equilibrium potential), provided the means in 1971 and 1972 to completely suppress neurotransmitter release. Then, Miledi restored acetylcholine release by microinjecting Ca2+, providing definitive proof of the hypothesis. Miledi published his results alone in 1973, while Katz obtained his Nobel Prize in 1970 for all the scientific achievements during his career regarding ‘the humoral transmitters in the nerve terminals and the mechanisms for their storage, release and inactivation. Yes, the neural process that allows you to read these lines (and hopefully smile as the information reaches your brain cortex) occurs thanks to this fine-tuned movement of ions and neurotransmitters across neuron membranes that Hodgkin, Huxley, Miledi and Katz helped to unravel in the squid Loligo!

Administration at SZN changed after 1973 and this, together with his new interest in mechanisms in the postsynaptic membranes and compartments, meant that Miledi lost interest both in Naples and the research models. He still returned to Naples once in 1977 and continued to publish his squid Neapolitan results up until 1986. However, the relationship ‘researcher-model organism’ had ended while new neurophysiological techniques were implemented that did not require such large neurons for research.

Miledi joined the faculty of the University of California, Irvine, in the early 1980s developing techniques of microtransplantation to study brain receptors in frog oocytes for drug development. He did not forget his mother country and from the 1990s until his death he was Distinguished Professor at UNAM's Institute of Neurobiology, in Querétaro, Mexico. Miledi received many important awards but one is special within EMBRC. Miledi became ‘Doctor honoris causa’ of the University of the Basque Country (UPV/EHU) in 1992, exactly 20 years earlier than its marine biological station, the Plentzia Marine Station, was opened in 2012. Many inspirational constellations need to converge to see the birth of a research institution like a marine station. Maybe Miledi sowed his seed while visiting the UPV/EHU, exactly as when he micromanipulated Ca2+ into the neurons of his lab alter ego, the squid.

Family portrait
Ricardo Miledi with family and friends after the Doctor honoris causa ceremony at the University of the Basque Country (UPV/EHU) in 1992. Graduates in medicine in Spain wear yellow academic dress, as it was the case of Miledi in the centre of the photograph. Graduates in sciences instead are dressed in blue. (Photo kindly provided by UPV/EHU press office)


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