Professor Max Bennett, neurobiologist

Neurobiologist

Professor Max BennettProfessor Max Bennett was born in Melbourne in 1939. He earned a Bachelor of Engineering (Electrical) from the University of Melbourne in 1963. This background, combined with a philosophical interest in how the human mind works, led him to the study of neurophysiology. Continuing at the University of Melbourne, he received an MSc in 1965 and a PhD in 1967. In 1969 Bennett joined the Department of Physiology at the University of Sydney and has remained there ever since. He was Director of the Special Research Centre of Excellence in Neurobiology from 1982 to 1990. Over his career, Bennett has made many significant findings and chief among these was the discovery that nerve terminals on muscles release transmitter molecules other than noradrenaline and acetylcholine, going against the prevailing scientific paradigm.

Teachers' notes to accompany this transcript.
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Interviewed by Dr Max Blythe in 1996.

Contents


A blend of contrasting family influences

Max, you were born in Melbourne, in 1939, to fascinating parents.

My father was a Jew, very dedicated to his family's religion. His parents had come to Australia from Galatz, on the border between Russia and Romania – a part of the world where people of Jewish descent would say either that they were Russian or that they were Romanian, depending on how the pogroms were going.

My mother's parents, however, came from County Cork, Ireland. I really feel like Bloom, in the novel Ulysses, in that I have both those rather interesting heritages which I was taught to live up to, yet found slightly contradictory.

Would you say that one of your parents was a stronger figure than the other?

Not really. Certainly my father was a very strong person. He was fascinated with engineering, although he wasn't able to practise it much himself because he went away to the Second World War and that broke up his career prospects. But he was determined that his son would be an engineer. He was also a man of great philosophical depth and a very spiritual, religious man.

So you were headed toward engineering. What about your brother Richard?

He was very interested in pursuing engineering, but when he was quite young he had a rather bad physical accident which derailed him from any career like that at all.

When I became old enough to go to school, my father was away at the war. So my mother, being of Irish descent, naturally enough sent me to the nearest school in our neighbourhood – a Catholic school. There I was, the only Jew in a Catholic school of several hundred. It was an interesting experience. I kept winning the religious prize and they didn't know quite what to do with me, because it didn't seem appropriate for a Jewish boy to go up on the stage and receive the prize from the Archbishop of Melbourne.

Even after my father returned, my mother demanded that I continue my Catholic education. He rather insisted that I shouldn't, but in the end I stayed on at my school. My mother could be quite strong too.

What kind of a war did your father have?

At first he was stationed in Australia, and then he went to New Guinea, which was a very tough area in the Second World War – many Australians died on the Kokoda Trail and in other such areas. He came back to Melbourne after the war somewhat shattered by his experiences, and perhaps as a consequence he became much more centred in the spiritual life. He started to read a lot of Eastern philosophy and religion, and the final 45 years of his life were spent in isolation, effectively as a Buddhist monk. He died last year at just on 85 years of age.

My father's philosophical bent, plus his interest in engineering, have more or less dominated my own thinking in the last 45 years also.

Where did you go to secondary school?

I went to another nearby Catholic school, run by the Christian Brothers. That is where, when I was about 14, I was lucky enough to have as a teacher Brother Kilmartin, who subsequently became head of Catholic Education Victoria. He had a tremendous influence on me – because he would never accept a facile answer to reasonably penetrating questions, he lit the light of inquiry as to how things operate, and particularly the larger questions of cosmology and so on.

He would give me books to read, and in that short period of about 18 months he instilled in me a real fascination with the world around me. He was the key influence, after my father, in shaping my interests in the first 20 years of my life.

So you began to read quite interesting philosophical works.

Well, I did. The influence of my father on the spiritual side, and Kilmartin on the religious side, for some reason channelled me into the general direction of reading a lot of Plato, especially his early dialogues, and then I went on to read Descartes and Leibniz and some of the other philosophers of the 16th and 17th centuries.

Mind and body philosophy?

Yes. And somehow the mixture of that with engineering set me onto trying to think through an analytical approach to how the brain works in the development of consciousness. By the time I was about 18 that had become a dominating stream in my life, and I have pursued it ever since.

Did you study any biological sciences at school?

No. In those days you didn't do biological sciences at a Catholic school! If you opened up a textbook of biology you might find out something about the reproductive tract, and that might get you asking questions which would embarrass people. So that kind of biology was completely missing from a Catholic education. That was a great shame, because it meant you went down a stream either of law (or sociology), or else of the physical sciences, leaving aside the greatest growth industry in natural philosophy at the end of the 20th century – biology.

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Via electrical engineering and philosophy to neurophysiology

You went off to Melbourne University to do engineering. What happened to your other interests?

Well, I did engineering because of my father's influence, but that didn't divert me from my philosophical interests. Indeed, as an undergraduate I spent more time doing philosophy than engineering. And there I came across Cameron Jackson, who'd worked with Wittgenstein and who introduced me to Wittgenstein's great works, the philosophical Tractatus and Philosophical Investigations. I got heavily involved in those. We formed a group of undergraduate philosophers, the Athenian Society, and every Friday we would get together to read philosophy, mostly from Wittgenstein but also from another, much earlier Cambridge philosopher, Alfred North Whitehead.

Whitehead was an extraordinary figure who became Professor of Mathematics at University College, London, and later Professor of Philosophy at Harvard. He mixed mathematics with philosophical investigations and was the mentor of Bertrand Russell – who was, in turn, Wittgenstein's mentor.

Towards the end of engineering, however, I decided that really the only way to approach the problem of how the mind arises from the brain was not to sit down doing philosophy but to actually tackle questions in neurophysiology. So I went across to the medical school as a vacation student in my fourth year of engineering.

As a consequence, I got to know some of the good neuroscientists on campus. I was lucky enough to discover two people, Mollie Holman and Geoff Burnstock, who were by far the best neuroscientists of their generation in Australia – although I didn't realise that at the time. They gave me some simple experiments to try out which really electrified me: at last I was, with my own hands, investigating nature and getting a buzz out of discovering things that no-one else had ever discovered. This became a major turn-on for me.

So, Max, with no biological education you go to university to do engineering. But you're desperately keen on philosophical issues and somehow with a holiday job you break into a biological arena.

Yes. I have been Professor of Physiology at Sydney University for many years now but I've never in my life done any formal studies in biology – not one subject and certainly no undergraduate degrees in it.

Incredibly, though, just when I had finished my engineering course – in fact, before I went to the graduation ceremony – my mentor Geoff Burnstock said to me, 'Why don't you come on and do a higher degree in biology? I said, 'That would be interesting but I really want to do philosophy.' He then went away on vacation for four or five weeks and meanwhile I started to do some gastrointestinal tract experiments which turned out to be quite new and significant.

I took a piece of the gastrointestinal tract (which can be thought of as a tube) and placed recording electrodes on either side of the muscle of the tract. And then I made a move which had never been made before: I put a series of electrodes for stimulating the intrinsic nerves within the gastrointestinal tract.

Do those nerves form a kind of nerve plexus?

Yes, the nerves within the muscle are a plexus which is responsible for the phenomenon of peristalsis – the rhythmic movement of food down the gut, the gastrointestinal tract. You may remember from Julius Caesar that soothsayers would kill an animal and take out the entrails, and eight hours later the entrails, still moving, were used in making forecasts about the future. The reason why those entrails are moving is the enteric neurons: the plexus is still alive and still causing peristalsis, even though the creature is dead.

And you directly went and stimulated those nerves?

Yes – and it turned out that they had not been stimulated in this way before. People had stimulated the extrinsic nerves coming in to the gastrointestinal tract from the spinal cord, but they hadn't actually stimulated the intrinsic nerves. In doing that for the very first time, I recorded a potential change with a particular shape, called an increase in negative potential.

The standard theory was that if you ever recorded such a potential it would be due to noradrenaline, a substance which is released from nerve terminals. That idea had been around for nearly 100 years, from work by Langley. In fact, the concept that noradrenaline was the transmitter being released from these nerves had won two Nobel Prizes. The first went to Otto Loewi and Sir Henry Dale, who claimed that nerves called sympathetic nerves were releasing adrenaline – which is actually found in the adrenal medulla, above the kidney. Subsequently, von Euler discovered that it was not adrenaline at all but something very close to it, noradrenaline, and he too won the Nobel Prize, together with Bernard Katz and Axelrod.

So when I stimulated these nerves I expected the potential that I recorded to be due to noradrenaline being released. But when I then put on a substance which would block noradrenaline, the potential remained exactly the same. The implication was that the main control system which was producing relaxation of the gastrointestinal tract, and was responsible for the movement of food down the gastrointestinal tract, was not due to noradrenaline at all.

If the major component of the nervous control in the gastrointestinal tract was not noradrenaline, what was it?

That is exactly what I was asking myself! Only one other substance was supposed to be acting to control the internal organs – acetylcholine. But acetylcholine was known to produce the opposite effect to adrenaline; it was supposed to produce a potential change which went up, not down. Nevertheless, I blocked acetylcholine. Again there was no effect on the potential. So we had discovered that there were nerves in there controlling internal organs by a 'new' transmission which involved neither acetylcholine nor noradrenaline.

It has turned out, over the last 30 or 40 years, that this new transmission controls not just the gastrointestinal tract but most of the viscera and vasculature. The transmitter substance which is responsible for this event, and which was not blocked when we blocked noradrenaline or acetylcholine, is a substance which seems to control, for example, the contraction of your urinary bladder, or the contraction of a muscle in the eye called the nictitating membrane, which is found in many animals, or the bronchi of your lungs. So this transmitter is widespread as a major component of the control of your internal organs.

Now, that experiment was done when I was 23, before I graduated in electrical engineering. It was an extraordinary struggle to get my findings accepted.

These were very significant discoveries. Had anything already primed you, as it were, to go in this direction?

Well, I was very lucky that six or eight months earlier I was in Burnstock's office doing some wiring for him (as a technician at that stage, while I was still finishing off my engineering) when in walked Sir John Eccles, who had just finished writing his major treatise on the synapse – the region of apposition between a nerve and a muscle – and how that operates. This became a large book called The Physiology of the Synapse, but at that stage it had not yet gone to press and Eccles gave Geoff a proof copy, about 200 printed pages. Geoff passed it on to me that night, and there I came across the existence, in parts of the nervous system, of potential changes called inhibitory potentials: they inhibited the ongoing activity of the nervous system.

Consequently, on seeing what came up on the oscilloscope screen when I stimulated the intrinsic nerves in the gastrointestinal tract, I realised that I had come across an inhibitory potential – which then proved to be due to neither acetylcholine nor noradrenaline.

I didn't quite take in the significance of all this, but my colleague who was doing a PhD at the same time, Graeme Campbell, had done biology and had taken Exhibitions in biology at Melbourne University. So he realised straight away that this was 'big', in the sense that I had come across something in this recording which was contradictory to the standard paradigm that had been in place for so many decades.

The significance did become apparent to me, however, by the time we had sent the paper off to Nature and it was published, because the pharmacological and physiological community in Great Britain were aghast. They were dominated by the paradigm which Sir Henry Dale had set in place. He had been President of the Royal Society and had won the Nobel Prize in 1936, and was really the father of modern, 20th century pharmacology. He had set it in concrete that these transmitters were the only ones operating to control the internal organs. Most people are in hospital not because there's something wrong between their Adam's apple and the top of their skull – that is, with their brain – but because there's something wrong between their Adam's apple and their pelvis – that is, with their heart or their gastrointestinal tract, or emphysema or something like that – so the discovery of new transmitter substances for the control of the internal organs was really of some significance.

There was still an unanswered question, wasn't there?

Yes: what is the substance which produces these potential changes, and which is causing the control of many of the internal organs outside of the system which releases noradrenaline and acetylcholine?

My laboratory colleague Graeme Campbell, together with our mentor, Geoff Burnstock, took up this question just after my PhD time. And after about four years they found that one of the main substances causing these potentials is adenosine triphosphate (ATP).

It took about 35 years – until only about two or three years ago – for that to be accepted. First, we were overturning such a well-held paradigm, and, secondly, ATP is such a ubiquitous substance. It's found in all cells, it's the main source of all energy in cells, and the idea that a substance which is everywhere could be used specifically as a transmitter was a no-no. But now it's known to be a transmitter in the central nervous system in the brain as well. Working on this has become a real growth industry, strangely enough, nearly 40 years after my first discovery.

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A PhD with Geoff Burnstock

Anyway, you did commit yourself to doing a PhD with Burnstock?

Yes, I went on with Burnstock. And we had to work very hard to convince the British community that we had actually overturned the standard paradigm.

But matters got worse for me, because six months after my transmitter discoveries I learned how to push very fine electrodes inside individual cells. These cells are only three-thousandths of a millimetre in diameter, so it is very difficult to get electrodes into them – plus the fact that in the case of the gastrointestinal tract everything is moving a few centimetres up and down.

How did you get the technique? Was that finicky and time-consuming?

I was meant to learn the technique from Mollie Holman, the most brilliant woman scientist in Australia, who was by then in the Physiology Department at Monash University. She had learned the technique in about 1957, at the time of its invention in Oxford. I went over to Monash to learn the technique but in the six months I worked with her she never succeeded in getting an electrode inside a cell. So I had to go back to Melbourne University and 'reinvent' the technique for myself.

I remember vividly the week that I left Monash: it was the week in 1963 when it was announced that Alan Hodgkin, together with Jack Eccles (who was then in Canberra) had won the Nobel Prize. That was cause for great celebration. Alan Hodgkin had discovered that the influx of sodium ions into a cell was responsible for the rising phase of the action potential, which is the whole basis of communication between one nerve and another. That inspired me, when at last I could put an electrode into these very small cells, to put an electrode inside a smooth muscle cell and record the action potential. I wanted to show that Alan Hodgkin was dead right – that what he had shown for the squid giant axon held for the mammalian autonomic nervous system.

I took all the sodium ions out of the medium surrounding a piece of smooth muscle tissue, to show that the action potential would get smaller and then gradually collapse. But what happened when I took all the sodium ions out was – nothing. The action potential remained perfectly normal. So I had discovered an action potential which was not due to the influx of sodium ions.

The only ion I could change which would greatly modulate this potential was calcium. That was the discovery of the first calcium action potential in the nervous system. So I sent that off also to the British physiological community, only about 12 months after they had copped my previous discoveries, and they were not very pleased.

You were shaking all the established ideas!

Yes, but this was quite accidental. It didn't require any great analytical power at all. In a sense, it just happened because an engineer had come bumbling into the area, without any preconceived ideas, and was doing fairly simple-looking experiments on organs and tissues which hadn't been looked at before. They were technically difficult to deal with, so it was taken for granted that they would act the same way as, for example, Hodgkin had showed the squid giant axon worked – that the action potential was due to an influx of sodium ions. But that wasn't true. It turns out that all the internal organs (with the exception, perhaps, of the heart) work by means of a calcium action potential, not sodium at all.

This block of work had taken me about four or five years altogether, and it was really my first introduction to biology. I was extremely lucky to have stumbled across all this without having done any formal biology at all. So '63 was a great year for me. Other years may have equalled it, but I don't think they've ever been better.

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Do nerves use electrical or chemical transmission?

You mentioned Jack Eccles, who would have had a profound effect on anybody working in this field. Did you have a chance to meet him in those early years?

Yes, I met him on a couple of occasions. But I think the longest conversations I have had with him have been in the last two years, while he was in his 90s. These have been about his early research life and why he was subjected to such cruel derision as a consequence of his idea, back in the early 1930s, that nerves throughout the peripheral nervous system work not by releasing a transmitter substance – such as noradrenaline or acetylcholine – which then acts on a muscle, but by imposing an electrical pulse onto the cell that the nerves end on. This is the concept of electrical transmission.

You see, when Eccles first started to record the electrical signs of what happens when you stimulate nerves to tissues such as the smooth muscle of your micturating bladder or the muscle in the nictitating membrane of the eye, for example, he discovered that he couldn't block those electrical potentials with the standard blocking agents that Sir Henry Dale had said must block them. (Dale had shown independently that the nerves going to the nictitating membrane were releasing noradrenaline and those going to the bladder were releasing acetylcholine.)

Eccles stood up with the logic and said, 'I've recorded electrical pulses which are due to transmission. They're not blocked by the agent which Dale says should block them – by chemical transmission – therefore transmission should be electrical, and not chemical at all.'

Dale's reply to all this was very influential, because at that stage it was known that he would soon win the Nobel Prize, which he did in 1936, five years later. He said, 'That's a lot of nonsense. What is almost certainly happening here is that the nerves are coming down and forming a synapse. There's a muscle on which they're terminating, and when the transmitter – which we might say is noradrenaline – is released, it is immediately at such high concentrations in the region between the nerve terminal and the muscle that the blocking agents you're introducing from outside can't block it in there. What you're recording is an electrical pulse which is due to the action of this transmitter acting on the receptors on that muscle, but it's not blocked by my agents because this substance works mainly by diffusing out from there and acting on parts of the muscle which don't give rise to an electrical pulse at all. Therefore you don't record the effects of the chemical transmission, and you're not getting any block of your chemical transmission because the concentration of the substance is too high for the blocking substances to act.'

Dale, because of his authority, carried the day on that issue. And what Eccles and I have been discussing in the last 18 months is that the real reason is that the transmitter substance wasn't noradrenaline in the case of the nictitating membrane, or acetylcholine in the case of the micturition bladder – it was in fact adenosine triphosphate. So they were both wrong. It was not an electrical transmission, either.

That was a ding-dong debate in the early '30s.

Yes, very acrimonious. It used to lead, I think, to tremendously difficult periods for meetings of the Physiological Society of Great Britain, where Eccles and Dale would enter into quite vitriolic argument and their respective students were pitted against each other.

In fact, the real explanation was certainly that we were dealing with a different transmitter. The paradigm that Dale had in place was not correct, and I don't suppose Eccles' was either. But the nice thing about Eccles' argument is that the logic was true, whereas I think Dale's argument is a sleight of hand. He had to bring in another variable – that the concentration of his transmitter was too high for the blocking agents to act on. He stuck with that argument almost to the end of his life.

I've written about that episode in an introduction to a Nobel Symposium on the mechanism of transmitter release at the synapse.

Max, talking about Eccles leads us to your interest in the history of this field of neuroscience. Eccles went to Oxford to team up with Sherrington, but you have written about an even more exciting story before that.

Well, Eccles went as a Rhodes Scholar to work with Sherrington, who was then in his 70s and remained working actively till he was about 76, when he retired as head of the Department of Physiology at Oxford. Sherrington would be regarded, I think, as the major conceptualising figure of the 20th century on how the central nervous system works. He wrote The Integrative Action of the Nervous System – which I think sets the whole of Eccles' main contributions to science, because Eccles followed the Sherringtonian paradigm.

Sherrington himself was introduced to neuroscience by John Langley, who was the Professor of Physiology at Cambridge, having just taken over from Michael Forster. It was in 1888 that Langley and Sherrington published their first paper together.

For me, Langley was the gigantic figure in the whole story that I've been recounting. He was a real genius, but I don't think he has been quite recognised for his enormous impact. On the one hand, his school was primarily responsible for the idea of chemical transmission occurring at all in the nervous system. Also, he developed a whole new line of research on what we now call the plasticity of the nervous system – the extent to which nerves can grow in a mature person and make new connections. It is now clear that during events such as memorising something, laying down new memories in the hippocampus of your brain, there must be changes to the synapses which are responsible for this.

This general area of the plasticity of nerve connections was begun by Langley in the early 1900s. And it was in reading his work on the autonomic system (the system that I have just been talking about in relation to my own first work) that I stumbled across these great papers of his on the phenomenon of plasticity.

How did Langley's work influence yours?

The great question which Langley seemed to me to have left up in the air was: once a nerve terminal in a mature animal has been lesioned in some way, to what extent can it regrow and find its right connection again? And so, at the end of the 1960s when I had finished my first block of work on the autonomic nervous system, I thought I would set up a laboratory to examine this phenomenon of plasticity. The laboratory that was offered to me was at Sydney University, so I left Melbourne and went there – and I have continued occupying those premises for nearly 30 years, still working in the lab that I set up.

In this next block of work I went not to the autonomic nervous system that controls the internal organs but to the nervous system that controls the muscles which we have locomotory control over, such as in our forelimbs and our hind limbs. These muscles consist of individual muscle fibres, each of which has a nerve coming down and forming a discrete, single ending in a very specialised region which is referred to as the motor end platelet.

So the first question I set myself – one which some people had worked on since Langley, but deriving very controversial opinions – was what happens if you sever the axon. We knew that the part of the axon which was no longer connected to the cell body of the neuron would degenerate, and that a growth cone, a bulbous protrusion, would grow on the axon where it had been cut. The axon would then regrow, but the question was to what extent it could form a connection on the muscle.

The first thing I discovered was that if you don't cut the axon too far back from the muscle, the nerve will regrow and it will form connections, but only in the position on the muscle fibre that it was originally connected to. That site on the muscle fibre must contain some information which stops the growth cone from growing any longer, so that it anchors itself and forms a normal terminal.

So the first point was the concept that there are information molecules specifying where nerves make connections?

That's right. The next question I asked was: is this site already on the muscle fibre during very early embryonic development, when the muscle fibres have not yet seen a nerve? Normally during development you put out your limbs, they gradually develop muscle cells, and then the nerves grow out from your spinal cord and find the limb, grow in there and form connections on the muscle cells. So I asked myself whether those muscle cells already have information about where the nerves connect, before the nerves come down.

And what I discovered was that they don't. If a muscle cell has never seen a nerve terminal in its life, the nerve comes down and forms a synapse just anywhere on the surface of that virgin muscle cell. So there are no information molecules on the surface of the cell at all to delineate where the nerve should connect. But then the nerve imprints onto the surface of that cell the information molecules which, if that nerve is severed later in life, will be used to determine that the nerve, when it regrows, will form a connection only there.

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Practical aspects of regrowing and reconnecting severed nerves

Were the experimental challenges of that work quite daunting when you came to Sydney in 1969?

No, it was not very difficult to do. I suppose if there's any daunting side of it, it's in the way in which you go about doing the dissections. But I had a PhD student working with me, Alan Pettigrew (now the Vice-Chancellor at the University of Queensland) who was superb at doing the actual dissecting and suturing work to set up these experiments. We were using mostly rabbits, rats and other mammals like that, so the experiments themselves were not all that difficult. And we had fairly well established techniques, like the electron microscope, available to us. I think the way we designed the experiments was helpful in establishing how the connections were being made.

The two sets of experiments showed that excitable tissues like neurons and muscle – that is, cells which can give rise to electrical pulses – have on their surface, in mature cases, little patches of information as to where nerves anchor. If they are lesioned, then that is where they can grow back to, and nowhere else. But in early development those sites aren't there and the nerve has to impress the sites into the muscle cells. They are very specifically imprinted.

There are two main problems associated with trying to get nerves to reconnect in a lesioned human being. For example, anyone who watched the Hollywood presentation of the Academy Awards last night would have seen Christopher Reeve, who formerly played Superman, sitting in his wheelchair as presenter. He has a lesion at about C1, cervical level 1, and is unable to move any voluntary muscles at all from there down. The question of how to remedy that condition is twofold.

The first problem is to get the axons to regrow through the lesion. The next is to get them to connect up to the right cells. So the question of identification of the molecules which confer specificity on the nerve connection has to be delineated before we will ever be able to get those connections to 'mend' after a spinal cord lesion.

In neuroscience now there is a tremendous amount of interest in trying to remove inhibitory factors which stop the nerve from regrowing through the lesion, and then, once it has done so, to make sure that the nerve connects up to the right informational molecules on the right cells so that the necessary specificity is recapitulated.

Can only one information site be imprinted on each muscle?

No. Torsten Wiesel and David Hubel had shown that there was plasticity of this kind in the visual cortex, in the occipital lobe at the back of the brain. And they showed that during early development there is tremendous plasticity in which different connections can be made, depending on the kind of visual experience you have in early development.

They were particularly interested, then, in our discovery that during early development of the animal, when this imprinting process was occurring, a muscle cell didn't have just one terminal on it, as it does in the mature animal, but several terminals. That is, we have not just one neuron connecting to a muscle as in the normal mature case, but several terminals coming down and competing with each other for the final connection with the muscle cell. All but one of these terminals are eradicated, however; only one remains.

It turned out that our description of that in muscles also held for the brain. During normal development of your brain there is a tremendous excess of connections between one neuron and another neuron, but gradually they are downloaded and you remove a lot of them. Part of the removal process is conditional on your experiences. Your visual experience of the world, for example, affects the visual cortex such that the removal of terminals is either accelerated or not, and this fashions the extent to which the neural connectivity – in this case, the visual cortex – can mediate your visual experience.

For example, suppose that a baby saw nothing but vertical bars. It is said that aristocratic families in Victorian times liked to put their families into beautiful white nurseries – white walls and curtains, a white crib – where the only thing in colour might be the vertical bars on the crib. The child would perceive only vertical bars, which would give rise a set of connections in the child's visual cortex which favoured seeing the vertical rather than the horizontal. That is, as terminals in the visual cortex became eliminated, the ones left in a strong condition (throughout life) would be those subserving the child's vision of verticals. A glass of water would appear to consist of vertical sides, with no top or bottom at all. The horizontals would have 'gone'.

So the question of the elimination of these terminals as a consequence of experience is a very important one in terms of developing, in this case, the mature visual cortex.

I was asked by Torsten Wiesel to go to Harvard and on to a meeting of the Cold Spring Harbor Symposium in 1975 to talk about this work. It was a delight to do that.

I suppose you then looked at why the 'spare' nerve connections did not survive?

Yes. After we talked to Wiesel and to some people working in those days on the development of these connections in the peripheral nervous system, we decided to examine an idea which had just come up from Levi-Montalcini, a very great neuroscientist who won the Nobel Prize for this work. She showed that, in the peripheral nervous system, the nerves which release noradrenaline will only stay alive during early development if they get a growth factor from the muscles on which they make their connection.

Suppose some nerves make connection onto an internal organ muscle such as the micturating bladder, where they release noradrenaline. Each of those nerves will only stay there if it receives a growth factor from the muscle. If not, the growth factor will not be transported back up the nerve to the cell body which provides this nerve terminal with nutrient, and consequently will not get to the cell body nucleus. The cell body will die.

It is quite normal, as a human being develops sympathetic neurons connecting to the internal organs, for a lot of these neurons to degenerate and be lost as a consequence of the competition between nerve terminals for this growth factor. Some of the nerve terminals get it, and some of them fail to get it.

Is this because an early one, getting on well, forms a sink?

No, not necessarily at all. Even the one that got there first could lose out, perhaps because it has made the wrong connection and doesn't get access to the growth factor as easily as those that have made the right connection. And therefore this mechanism is one of eliminating incorrect connections and keeping the right ones – that is, connections such that a neuron cell body in the spinal cord, for example, receives an appropriate input for the motor cortex and operates a particular muscle in a functionally useful way.

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Do nerves in the brain rely on a growth factor?

You have been talking mostly about the peripheral nervous system. Does the same sort of thing happen in the central nervous system?

That is the next question we asked: does Levi-Montalcini's concept that growth factors are supplied by target organs to nerve terminals – and that, if they're not, the neuron cell body which is supplying those nerve terminals degenerates – work also in the central nervous system? Do connections onto, say, a neuron found in the spinal cord remain intact because they've got a growth factor, not in this case from muscle but now from this neuron?

In our next block of work, then, we set out to try and see whether the brain works on the paradigm that Levi-Montalcini had set up for sympathetic nerves in the periphery. Do nerves stay alive in the brain because they get a growth factor?

The preparation that I chose for this, and which I worked on with a dear, close colleague of mine, Bogdan Dreher, was the neurons in the eye that connect it to the brain. (We chose those neurons because we had worked out a nice technique for isolating them into a culture dish containing the normal nutrients which keep cells alive.) The technique which we developed was to inject an enzyme into the part of the brain that the eye normally projects to. This enzyme is then taken up by the nerve terminals which are projecting from the eye to the brain, and so transported back into the eye. Therefore, the only neurons in the eye which are labelled with this enzyme are the ones which project to the brain. None of the other 20 or 30 other cell types in the eye, such as the photoreceptors that take in the photons, are labelled.

That sounds like an ingenious approach. Was it productive?

Yes. We could now dissociate the retina, even of young animals – foetuses, if you like – and detect which were the ganglion cells connecting the retina to the brain. And that technique enabled us to do two things.

First, we could count the number of neurons in the eye that connect it to the brain during normal development. We found that about half the neurons present in your eye when you were quite young will have degenerated and been wiped out during the normal development of your eye's connections to the brain. It is now known that the same sort of thing happens right throughout the brain. You've now got about half as many neurons in your brain, Max, as you had when you were tiny. That is, you had laid down an excess of neurons.

Second, because we could identify the neurons from the eye in a dish, we were then able to see what would keep them alive. The natural thing to do was to follow Levi-Montalcini's paradigm, that what would keep them alive is a growth factor supplied by the normal cells in the brain that the retina connects to.

So we took out the visual centres of the brain that the eye connects to, mashed them up into their individual molecules and put them into the plate with the neurons we had been able to isolate from the eye, which connect the eye to the brain. And we showed that the only part of the brain that would keep these neurons alive was the part that the neurons normally connected to. Other parts, like the cerebellum, which doesn't get an input from the eye at all, did not contain a growth factor for these neurons.

That even goes beyond the paradigm you were testing, doesn't it?

That opened up the paradigm that there are growth factors right throughout the central nervous system which are specific for certain neuron classes – the ones actually projecting to the part of the nervous system from which you have derived the growth factor. Most of the work I did in the '80s was concerned with trying to isolate the growth factors for these specific parts of the brain, in particular the retinal ganglion cell which connects the retina to the brain.

That work I had the great pleasure of presenting to Levi-Montalcini in 1984 at a meeting of the Pontifical Academy, in Rome. Shortly after that she won the Nobel Prize, because it was realised in the late 1980s that the concept she had developed in the '60s, concerning these sympathetic nerves which released noradrenaline to muscle, held for the entire nervous system. And now a tremendous amount of pharmaceutical work is done in isolating these various growth factors, because they could be implicated in a whole range of diseases.

For example, Parkinson's Disease involves the degeneration of neurons which release dopamine. These neurons are found in the substantia nigra and they might be degenerating because they are not getting their normal growth factor from the regions of the brain they project to. In Alzheimer's Disease there is a loss of neurons in the parts of your brain concerned with memory – old people gradually lose their memory because they have a form of dementia which involves the degeneration of these neurons. The neurons won't degenerate if you give them their normal growth factor.

So growth factors are very important if we want to keep neurons alive.

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A cocktail of transmitters

Are you continuing to work on growth factors?

No, in the last eight or nine years I've gone back to looking at the mechanism by which transmitter substances are released from nerve terminals onto muscle cells. That became possible because we were able to develop special imaging techniques to apply to a cell while it was normally functioning, and so to bring recording electrodes down to specific parts of the nerve terminal at our will.

Consider a nerve terminal abutting on a muscle cell. Each nerve terminal has little bulbous regions in it, and any of these little bulbs can release a packet of transmitter. Because we can now visualise these individual little bulbs – these boutons or varicosities, as they are called – we were able to bring electrodes up and record the release of transmitter from an individual element of the nerve terminal. And what we discovered was that, within a single synaptic arrangement on one nerve terminal, each of the boutons or varicosities has its own individuality. You can't treat a nerve terminal as if it is a homogeneous structure. Each one has quite a distinct capacity to release transmitter on the arrival of a nerve impulse down the axon.

Is it one transmitter or more?

It's more than one transmitter; it's mixtures. And that concept was due to my mentor Geoff Burnstock, who argued – against the establishment – that the packets of transmitter coming out don't just contain the classical transmitters but also lots of other things, such as neuropeptides. It's now known that all release of transmitter involves co-transmitters. For example, at nerve terminals on muscles that you use voluntarily, not only the classical transmitter acetylcholine is released but also substance P and calcitonin gene-related peptide, and adenosine triphosphate.

Our early work with Burnstock and Campbell, back in the 1960s, has led us to become interested in the release of different transmitters at different nerve terminals, and then also in the elaboration of Burnstock's concept that from within a single terminal a whole cocktail of transmitters is coming out, not just one or two transmitters. What is more, our work recently has shown that there is considerable heterogeneity within a single nerve terminal as to its capacity to release transmitter.

Are there many boutons on a nerve terminal?

Yes, there are massive clusters of hundreds of thousands of boutons on a single nerve terminal. And they all behave in ways which are not homogeneous – independently in the sense that they have different properties to release transmitter, but with an ability to interact with each other in complex ways.

The concept that the nerve terminal is inhomogeneous leads on to the fact that mostly it isn't doing anything until you actually bring it into action as a consequence of needing it, such as in the laying down of a memory. If you looked in the brain of a mature human being you would find that most of the nerve terminals there are not doing anything. If you're going to incorporate new information into, for example, the hippocampus (the part of the brain concerned with memory) you have to up-regulate some of these terminals so they become effective, but you can't do that if they're effective already. So there are great reserves.

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The influence of great neuroscientists

It seems that from the 1960s you've actually come full circle, back to transmitters.

Yes. My work has been greatly affected by some great neuroscientists, particularly Bernard Katz but also Eccles and Kuffler, Torsten Wiesel and David Hubel. Stephen Kuffler was a great supporter of bringing new techniques in to open up scientific questions in neuroscience. Particularly at this level of analysis, of the synapse, this would be the way he went. That I found very inspiring, and so we took this track.

Did Katz have a specific influence?

Katz would be regarded, I think, as the synapse genius of the last half-century. He laid down, in the early 1950s, the conceptual framework of the way in which we operate in terms of understanding synapses. And some of the first breaks with that tradition are that the terminals he looked at and treated as if they were homogeneous are inhomogeneous – the subcomponents of a single terminal are different – and also that there are co-transmitters. That is, the terminals are releasing cocktails of transmitter. These are the two main shifts in the paradigm which he put in place.

It's very interesting to me, Max, to be talking to you here in the Royal College of Physicians, in Sydney, because the most famous photograph in the history of brain sciences was taken about 200 metres from where we're sitting now, in Macquarie Street. Taken in 1940, it shows Eccles, Katz and Kuffler, who worked just opposite where we're sitting now, together in the Kanematsu Institute. I have that inspiring photograph on my wall, as do many other neuroscientists throughout the world.

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The philosophy and physiology of consciousness

Max, we've looked at your career in a series of blocks, bringing us to the versatility that is possible within the nerve terminals. Does that take you back to a philosophical approach, to think of memory and mind?

I think the dominating drive that I've had in the last 40 years has been reading philosophy and also trying to get insights through neuroscience into the physical basis of consciousness.

Until eight years or so ago I wouldn't have dared say that to you or to anybody else, because I would have been laughed at and regarded as someone who's gone senile. I would have blushed! But about 10 or 15 years ago Roger Penrose, the Ball Professor of Mathematics at Oxford, and Francis Crick, who left molecular biology to enter neuroscience, began to consider neuroscience questions in the context of their main interest – the physiological basis of consciousness.

As it has turned out, they have come to totally contradictory positions on the matter. But because one was regarded as one of the great mathematicians in the world and the other is commonly regarded as the greatest biologist since Darwin, it made the field respectable. That is, you can now talk about consciousness as much as you like at neuroscience meetings and very few people are at all embarrassed by it.

This subject seemed to me to be best tackled not by getting lost in the wiring diagram of the brain but by coming down to what's happening at the synapse. My whole life has been dominated by an attempt to elucidate synaptic function, either in terms of natural function, of transmitter release, or in terms of plasticity. I think, as do Eccles and certainly Roger Penrose, that the secret of how the brain operates to give rise to memory and consciousness is to be found in the way in which these terminals either increase their efficacy or decrease it, or grow in different ways. That has been the experimental side of my life, coloured by the philosophical needs.

Where is the synapse story headed now? If polypeptides have now entered the field in addition to some of the 'traditional' transmitters and those you looked at earlier on, that could be a very sophisticated story of communication.

There is no doubt that the way the synapse operates is now known to be very complex. There is a cocktail of substances coming out from the terminal – the neuropeptides, like encephalin, substance P and others – and acting on the muscle cell or the neuron on which the terminal impinges. But those substances also act back on the terminal to change its capacity to secrete transmitter. And they change the capacity of the receptor molecules, which grasp the transmitter after it is released, to actually identify and interact with the transmitter. So we've got a very complex machine here, which I think will require quite a number of years of elucidation.

But is a basic model in view, for the formation of a unit of memory?

The theory about how memory is laid down is a rich but fairly straightforward one which involves changes in the capacity of these synapses to operate. It is no longer very mysterious. The debating point, though, is the way in which consciousness arises. As well as the contributions by Penrose and Crick which I have mentioned, Gerry Edelman – who won the Nobel Prize in immunology in 1972 – has become a major theorist on the physiological bases of consciousness.

I've written about the subject for a number of journals in an attempt to present a kaleidoscope of some of the excitement being experienced in philosophy and in neuroscience as we delve deeper into brain structure. We can now use non-invasive imaging techniques such as positron emission tomography and functional electromagnetic resonance imaging to see the brain actually functioning and giving rise to conscious thoughts. And this field is undoubtedly growing at a great pace.

Our unit in Sydney is still centred on the examination of synaptic function, making great use of high-resolution imaging techniques.

If the experimental investigation of consciousness is now focused on synaptic function, what does philosophy have to say about it?

Well, I've just finished writing The Idea of Consciousness, which gives the various views of neuroscientists such as Gerald Edelman, Francis Crick and Roger Penrose about the workings of the nervous system – what they think might be going on to give rise to consciousness. And those opinions tend to be polarised into two camps, with the possibility that neither is right.

On one side we have an idea derived from the work of Schrodinger, who with Heisenberg invented quantum mechanics. Schrodinger was a fellow of Magdalene College at Oxford in the '30s, after he left Nazi-occupied Austria, and he had a large effect on a co-fellow of that College, John Eccles, before Eccles left Oxford to work in the Kanematsu Institute. Eccles developed the idea, under Schrodinger, that there were quantum mechanical phenomena going on between the nerve terminal and the neuron on which it impinges, and that the mysteries of quantum mechanics could enable one to tease out the basis of consciousness.

The greatest proselytiser of that idea at present is Roger Penrose, who has written two major books on it. And Eccles himself, working with a quantum mechanicist from one of the Max-Planck-Institutes in Germany, published a paper three years ago in Proceedings of the National Academy [of Sciences] on the way in which quantum mechanical principles might explain the way in which conscious phenomena could be derived from the workings of the synapse.

The standard argument against the idea is that the brain is too hot to allow the interference phenomena that occur in quantum mechanics to occur. There is certainly one part of the brain, though, where quantum mechanical principles operate: where there is capture of photons by the rods and cones of the eye. Photons are essentially a quantum mechanical particle, and their interaction with the pigments in the eye must be a quantum mechanical phenomenon.

Nobody yet has been driven to use quantum mechanical methodology to try and work out some phenomenon at the synapse. That is, at present all the phenomena that we have been able to derive at the synapse have been capable of being explained on classical ideas – using classical physics, effectively. But as we delve in closer and closer in a totally reductionist way, you never know what we'll come across.

So what is the other side of the coin?

That takes advantage of the fact that nothing quantum mechanical in the way of analyses has ever had to be used to explain any brain phenomenon whatsoever. It is the view which Francis Crick leads off with, particularly in his recent book The Astonishing Hypothesis, and it's generally supported by Gerald Edelman.

Crick, in his typical hard-headed fashion, says, 'It's the wiring that's doing the job.' He says that a phenomenon which the wiring of the brain gives rise to – an ability to use language and to hear the language through the auditory pathways – is itself capable of explaining anything we want to know about the origins of consciousness. And the reason we regard consciousness as mysterious is that we are experiencing such an enormous range of extraordinary phenomena when we go through a stream-of-consciousness event. It is hard for us to grasp how the 1015 synapses of the brain, and their connectivity, could give rise to this sort of phenomenon.

The main popular paradigm there comes out of the work of Wolf Singer. We have known for a long while that there are different modules in the brain subserving different functions – audition, vision, smell. But when we see a beautiful woman pass by, carrying a bunch of roses, we have a holistic experience. The smell and the vision come together as one phenomenon. So how is it that a brain module concerned with language, or with smell, and a separate module concerned with vision, can give rise to a holistic experience in our consciousness?

What Wolf Singer has shown is that the firing patterns of neurons in these disparate parts of the brain come into synchrony. So the neurons which are subserving your experience of an holistic event are in phase and are firing together at the same frequency, whereas the neurons in the rest of the brain – the huge amount of it which is not experiencing or contributing to your holistic experience – may be taking note of a lot of other things going on but are not contributing to consciousness. That is, they are not firing in phase, with the same frequency.

At present, the argument of the Cricks and the Edelmans of this world for the development of consciousness at any moment in your brain is that it is derived from those parts of the brain which are firing in this pattern. It is through the horizontal connections across the brain that they are brought into synchronous firing, and it is that synchrony of these neurons which gives rise to the holistic experience that you're having as you look at me now and listen to me talk.

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Scientific collaboration and the need for research funding

Max, let's turn to how you as a scientist relate to the science environment. You have set up at least one action group, I think.

Yes. I've had an ongoing concern about the way in which research is supported in this country, and as a consequence in the 1980s I chaired a group (under the auspices of the Australian Academy of Science) which set up the Federation of Australian Scientific and Technological Societies. That brought the 80 scientific and technological societies in this country into a common forum, in order to promote appropriate government funding for research. It has been operating very effectively since 1984.

In addition, more recently, I set up the International Society for Autonomic Neuroscience (ISAN), which is concerned with the study of the peripheral nervous system controlling the internal organs. The society is having its main congress next year in Cairns, Queensland. It is the sister organisation, we think, to the International Brain Research Organisation (IBRO), which is the main umbrella group for the study of brain functions. ISAN is for the neck down, and IBRO is for the neck up.

Even more recently we have founded the Sydney Institute of Biomedical Research, the main institute for biomedical research in the Sydney region, at Sydney University. On any objective criteria, its members – the main biomedical researchers at Sydney University – are collectively the most powerful group of medical researchers in New South Wales, and probably one of the two or three most powerful groups in the country. That is moving along quite excitingly, because we get a lot of new multidisciplinary research going on as a consequence of bringing such groups together on the campus.

Have you won progress in government funding of science? In England we have not gone forward; we tend to have gone backward.

Well, in Australia we have a problem which is probably not found in most other developed countries. For various historical reasons, there has been an almost complete lack of business funding for research and development in this country. Virtually ever since the First World War, when CSIRO was founded, the Australian government has been supporting research and development at a level comparable with the average or better in most countries of the Organisation for Economic Co-operation and Development (OECD). But that by itself is not enough, and because of the malaise in our business community against putting money into research and development, Australia has an appalling tradition in funding research and development.

That has turned around very dramatically in the last several years as the government has offered the business community a 150 per cent tax break on research and development. That has moved our research and development ahead quite fast, but the improvement comes off such an extraordinarily low base that the total funding of research and development in Australia is still low.

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A symbiotic partnership

To round off this very pleasant meeting, would you tell me about your family life. Did you marry?

Yes, I have been married for 31 years to a very successful painter of landscape, who does a lot of work concerned with the outback. She goes on trips into very exotic areas in Central Australia such as the Bungle Bungles, and she lives a life which – luckily enough for me – is really independent of mine. (Because I work a seven-day week, my family life is very restricted.) Gillian has been a phenomenal companion. Without her I couldn't have kept up the pace that I felt necessary in order to probe so many questions.

Have I missed asking about anything in your research story?

No, Max. The time you have given me has been very generous. Thank you very much for letting me bash your ears about things which have excited me so much and have been such a great experience for me.

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© 2017 Australian Academy of Science

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