Did these studies then lead to the postulation of the radial-unit hypothesis?Yes! The cortex enlarges its surface by adding more ontogenetic columns. However, humans have a larger pool of neural stem cells than, for example, mice even before the first neurons are generated. Initially the stem cells divide symmetrically, so that after each division the number of progenitors and radial glial cells is duplicated, leading to an exponential increase in the number of cortical columns. At a certain point, which is different in each species, cells begin to divide asymmetrically, each division producing one postmitotic neuron that migrates up along the radial glia and another that remains near the ventricle to divide again. Now the growth is linear.

A larger number of columns therefore provides a more complex behavioural repertoire?That is correct. Importantly, ontogenetic columns in the various cytoarchitectonic areas are different in size, cell composition, synaptic organization and expression of signalling molecules, which can explain both the differential expansion of some areas and the introduction of new areas during evolution, as formulated in the protomap hypothesis, which was based on simple ablation experiments. This concept has now been confirmed with the more sophisticated methods of molecular genetics in my and many other laboratories. Some of these studies provide insight into how the cortex evolves by adding more radial units to enlarge some functional areas and to introduce new areas.

When did you postulate your radial-unit hypothesis, and was that hypothesis widely accepted at the time? It certainly has been very influential for more recent neuroscience.The idea originated in a paper I published in 1972, but a more developed hypothesis was formulated in my 1988 article. Initially it did not receive much attention, as most neuroscientists focus on a single structure in a single species. By contrast, I have always studied the development of the cortex simultaneously in multiple species, to learn something from the differences. I am glad that the principle of glia-guided migration

the highest cognitive functions, including perception and language. The question of how such a precise map is generated intrigued me to the extent that I abandoned neurosurgery to obtain a Ph.D. in Developmental Biology and Genetics in order to study how this occurs at the molecular level.

When did you postulate the role of radial glial cells in neuronal migration?In the early 1970s I began to use a new method of electron microscopy to analyse migrating neurons in the embryonic primate brain. This was before the era of computers, and we had to reconstruct the migratory pathways from serial sections and thousands of photomicrographs pasted with silotype (Supplementary information S1 (figure)). I observed that the migrating neurons faithfully followed elongated glial shafts that spanned the fetal cerebrum from the ventricular to the pial surface, and I suggested that these shafts guided migrating neurons to their proper destinations. I named this mode of migration ‘glio-philic’, to emphasize that moving neurons are attracted to and selectively follow the surface of the radial glia. This observation led to the idea of differential cell adhesion mediated by heterotypic adhesion molecules that can distinguish between glial and neuronal surfaces. Later we used glia-specific markers to prove that the radial cells are indeed glia, as had been suspected from classical anatomical studies. Thus, neurons in primates migrate a very long distance across the fetal white matter and bypass earlier-born cells to form distinct radial units, which I call ‘ontogenetic’ cortical columns.

Pasko Rakic, Yale University

Initially you studied medicine. How did

you get into neuroscience and into studying cortical development?Yes, I received my M.D. at Belgrade University, where I also started a residency in neurosurgery. I was lucky to receive an International Clinical and Research Fellowship to go to Harvard Medical School. There I was exposed to the new methods for studying brain development, and after my return to Belgrade I labelled fresh human post-mortem embryonic brain tissue with DNA-synthesis markers to label dividing cells. I observed that dividing neuronal progenitors were present only around the cerebral ventricles — not in the cortex itself.

Did that result surprise you?The finding was not a total surprise as Wilhelm His had suspected it in the nineteenth century, based on the distribution of mitotic figures in histological preparations, and it had also been shown in mice. However, I found it fascinating that none of the neurons that make up the largest part of the human brain are formed locally. Furthermore, as the human cortex is not only approximately 1,000 times larger than that of the mouse, but also convoluted, postmitotic neurons have to migrate longer distances and take more complex routes to their final destinations in the regions that subserve

The first ever Kavli prizes, in astrophysics, nanoscience and neuroscience, have been awarded this year. The million-dollar-prize winners in neuroscience are Pasko Rakic, Professor of Neurobiology and Neurology at Yale University School of Medicine, USA; Sten Grillner at the Department of Neuroscience, Karolinska Institute, Sweden; and Thomas Jessell at the Department of Biochemistry and Molecular Biophysics, Columbia University, USA. The Kavli prizes in neuroscience recognize the scientists’ influential contributions to our understanding of neuronal circuits in the developing and adult brain and spinal cord. Claudia Wiedemann talked to the prize winners about their scientific careers and their outlook on the future of neuroscience.

AN INTeRvIeW WITh…

The Kavli prize winners

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has been confirmed and further elaborated on by many developmental neuroscientists, and that it has been accepted in the field of evolutionary biology and even by the general public.

Is there a key anecdote or experience that influenced your scientific career?one, relevant to my choice of research subject, occurred when I applied for a grant to study the development of the primate cortex. I reasoned that, although the basic principles of cortical development are similar in all mammals, changes in gene expression and modifications of developmental events during millions of years of evolution produce not only quantitative changes (for example, in the number and size of neurons, and in the timing and sequence of cellular events), but also qualitative changes (for example, the elaboration and introduction of new neuronal types, additions of functionally specialized cortical areas as well as different patterns of synaptic connections). For example, radial glial cells are born much earlier, mature much earlier and are more stable in humans than in rodents. one referee of my grant application stated that “since the basic developmental principles are the same, there is no reason to waste money on studying primates.” Interestingly, this same person rejected a study on mice because the authors used different strains in experimental and control groups, saying: “there is a huge difference between mouse strains”. In spite of such difficulties, I was generally well funded, and I feel that studying the cerebral cortex, the site of human uniqueness, is both intellectually appealing and scientifically exciting.

Is there a fundamental question that you would like to see resolved before you can retire?I think that we will understand the development of the brain before we understand how it works. At the moment, there are methods available for identifying genes and studying in-depth molecular mechanisms of development. We are studying right now which factors determine the transition from symmetrical to asymmetrical divisions, which can explain how we got more

radial units. of course, this will not explain all cortical differences, but it is the first essential step. In addition, if migration is disturbed by gene mutations or environmental factors, the cortex will be abnormal and can provide insight into causes of congenital disorders of higher brain functions. Finally, studying cortical development may give us insight into the evolution of human uniqueness.

Adult neurogenesis in the human brain is one of the most heated topics of discussion in modern neuroscience. What is your view on that?For the cerebral cortex, the structure that we have been talking about, there is no doubt that there is no addition or turnover of neurons in the adult — we and others could not see it in monkeys, and 14C dating in humans shows convincingly that all human cortical neurons are generated by the time of birth. Inexplicably, some people seem to be disappointed by this finding, as if it is a bad thing. I find it fascinating that our cortical neurons are as old, or even a little older, than we are, and that during our prolonged lifespan we always use the same cells. This is not the case for other organs and tissues, or even for the brain of many other vertebrates. For example, fish, amphibians and reptiles maintain neurogenesis all their lives and can regenerate optic nerves or make an entirely new spinal cord. There has to be an evolutionary reason why this is not the case for humans, with our highly evolved neurons. In 1985 I proposed that during evolution we lost the ability to regenerate in order to gain the ability to retain complex learned behaviours in the permanent sets of neurons. So far I have not seen a better explanation for the fascinating durability of the most precious elements of our body. This is not my research interest, but I would put my money on investigations into how to preserve my old and experienced neurons, rather than on introducing new and naive neurons that would probably wreak havoc in my brain.

Where do you think neuroscience is heading?Neuroscience these days has spectacular possibilities. We have recently had a decade of the brain, and there is a sense that this will be a century of neuroscience. At the end of the nineteenth century William Hiss could ask the same questions that we can ask today, but he could not get the answers as the appropriate methods were not available. Now, young students who are entering the field of neuroscience have the opportunity to apply new methods to old questions, such as: what exactly are the structural and

developmental differences in our brain that make us human?

Do you have any recommendations or advice for students entering neuroscience?I think that they should identify the question that they want to ask and then look for methods that can be used to answer it, rather than — and some make this mistake — identify methods that they want to use and then look for the question. It is the question that should drive them, as the most suitable methods can easily be learnt.

Sten Grillner, Karolinska Institute

How did you get into neuroscience?

When I was studying medicine I became interested in how the body functions. I thought that the most interesting aspects were the control systems of the brain, and that is why I started to study these in the Physiology Department at the University of Gothenburg.

In the mid 1960s, I started to look at the synaptic connectivity between reticulospinal and supraspinal pathways — that was the state of the art at the time. I soon realized that even if you knew the connectivity and all the pathways, you still would not understand their role in the control of behaviour. At that point I started to think about the need to study the active nervous system in order to understand the neural basis of behaviour.

Initially you studied motor behaviour in the cat and then later you switched to the lamprey. What was the rationale behind that initial choice?In short, I studied cat models in the beginning but later looked for a simpler preparation in which I could study the neuronal components participating in active behaviour. In the mid 1960s it had been shown that well-coordinated locomotor behaviour could be induced in cats in which the forebrain was not present. So essentially you could have an active ‘behaving’ preparation in which you could apply all the different neurophysiological techniques available, and that stimulated me.

There was evidence from the earlier literature that there were central networks, but there was very little detail. We established that mammals have networks that can produce the detailed motor pattern that underlies locomotion — a central pattern

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I find it fascinating that our cortical neurons are as old, or even a little older, than we are, and that during our prolonged lifespan we always use the same cells.

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generator in the spinal cord — and later that other vertebrates also have this network, meaning that in the spinal cord we have a network that can be activated from the brainstem and that can then turn on all the different muscles in the appropriate sequence.

once we had established the central pattern, we could show that sensory input also plays an important part in the control system. on the one hand there is a very clever central network, and on the other hand you have superimposed on that a sensory network that can adapt the movements. As an example, when you walk there are perturbations that interrupt a regular rhythm, and these need to be compensated for at the spinal level. Having established the basic organization of the spinal control system with both central and sensory components, our next question was how do these networks operate — what is their intrinsic funtion in terms of interacting neurons and synapses?

Was that when you started to work on the lamprey?We initially started to look in the cat spinal cord for different types of neurons and their connectivity, but soon realized that, owing to the complexity of the mammalian system, it would be very difficult to draw up a connectivity map. I therefore started to look for a simpler model system and, after working with the dogfish for a while, we started on the lamprey in the late 1970s. Although the lamprey has a very long gestation time (7 years), the main reason that we used the lamprey was that the preparation was much simpler and it has all the basic parts of a vertebrate nervous system but with fewer neurons. Another plus was that there was a fair bit of comparative anatomy on the lamprey: owing to its phylogenetic position, the lamprey had been of interest to comparative anatomists more than 100 years ago — including Sigmund Freud. In those days, comparative anatomy was the thing to do as a young neurologist, and Freud published a good paper on the lamprey that showed that one type of dorsal cell was sensory. People did not believe it at the time, and it took another 70 years before they did. of course, Freud went on to do other things afterwards.

We therefore started to gradually build up our knowledge about the lamprey, including cell types, connectivity, synaptic properties and the membrane properties of the neurons. In the late 1980s we understood the basic principle of how the network that underlies the swimming behaviour operates, including how it is activated, the sensory control, its transmitters and its membrane properties, although much detail was later added. For all of that work, modelling has been extremely important for testing different hypotheses, and I have had a long-standing and fruitful collaboration with Anders Lansner since the late 1980s.

At that point we had a fair understanding of the spinal pattern-generating network. Now subsequent neural-control problems could be addressed, such as how this network is normally activated and how the body orientation — or posture — is controlled. Since then we have gained a solid understanding of the basic principles that underlie steering control, control of body orientation and control of propulsion, and also a little bit about eye-movement control; however, there is always more to learn.

What is the fundamental question you would like to see resolved?one main problem that we are trying to understand at the moment is the neural mechanisms that underlie selection of behaviour. How does the lamprey brain decide that the animal likes to swim, to turn, or to do something different? In that network, the basal ganglia, the striatum and the pallidum are very important players.

We have shown, to our pleasure but also somewhat to our surprise, that the striatum of the lamprey is in many respects almost identical in basic design to the human striatum: it has the same type of inputs, similar membrane properties and similar symptoms on dopamine deprivation. The lamprey can be a good model system, as it has a very limited behavioural repertoire — in contrast to primates and mammals — from which it should be easier to deduce basic principles for modi of operation.

I feel that understanding the basal ganglia is a fundamental next step, as a number of different motor programmes that are embedded in the brain stem are under the control of the basal ganglia, and they are more or less responsible for selecting which programme is to be active. For the control of most patterns of behaviour it may be that the striatum is rather more important than the cortex. This may be perceived as a rather radical standpoint in the cortico-centric view of current neuroscience.

What types of techniques did you apply most for your studies?We have always used all available and applicable techniques; it is not feasible, however, to use transgenic techniques in the lamprey as the gestation time is so long. However, some of the more modern tools can probably be applied, such as transfection with channelrhodopsin and halorhodopsin to control neuronal activity by light.

Where do you think neuroscience is going?I feel very enthused by the fact that neuroscience has moved from having a very large focus on the cellular and molecular levels to now having a focus on the circuit and behavioural levels. A variety of new techniques will make important contributions, including the molecular techniques with which you can transiently inactivate different components. I am convinced that we will gain a deeper understanding of the networks that control sensory perception, memory and different aspects of movement control. I think that we will also gain further insight into the function of the subcortical structures, such as the basal ganglia and the other forebrain structures, and that this will be extremely important in understanding the neural basis of behaviour. My expectation is that in the not-too-distant future we will be able to understand the neural bases of many patterns of behaviour. If you engage in the questions regarding the thought process or even consciousness, however, I believe that it will take a few more years.

Thomas Jessell, Columbia University

What prompted your interest in neuroscience at the beginning of your career?I first became interested in the entire field of brain circuitry and function as an undergraduate at the University of London in Chelsea College, where a very persuasive lecturer, John Bevan, introduced me to neurotransmitter systems, how they function in the CNS and how they control different aspects of behaviour.

At that point in time, drug interactions with neurotransmitter receptors were beginning to be understood. I think that my general interest in manipulating synapses and circuits within the CNS stems from that period.

My expectation is that in the not-too-distant future we will be able to understand the neural bases of many patterns of behaviour.

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…what intrigues me now is how spinal circuits extract sensory signals from the outside world and use this information to coordinate motor output.

I was fortunate to have the opportunity to train with Leslie Iversen at the Neurochemical Pharmacology Unit in Cambridge, which at the time was by far the best environment for analysing brain neurochemistry. With Leslie’s guidance I started working on a number of transmitter systems, and very soon began to focus on a novel class of transmitters, the neuropeptides, which had gained attention through the pioneering work of Susan Leeman, Thomas Hokfelt and Sol Snyder. The realization that substance P, the best-characterized neuropeptide at the time, was expressed by primary sensory neurons prompted me to examine the release of substance P from sensory terminals, as well as its interactions with other peptidergic systems, in particular the opioid system. These studies demanded an understanding of pain circuits in the spinal cord, and from this came my continuing interest in spinal cord circuitry and function.

After a brief postdoctoral stay in Japan working with Masanori otsuka, who provided early physiological evidence of a role for substance P as a sensory transmitter in the spinal cord, I joined Gerald Fishbach’s laboratory in the Pharmacology Department at Harvard Medical School. There I began working at a more cell-biological level on the question of how motor neurons organize synaptic connections at the neuromuscular junctions. This focus maintained my interest in spinal circuits but switched the emphasis from sensory to motor systems and to a cell-biological analysis of the development of circuits.

What kind of methodology did you apply back then?We were using a number of cell-biological and physiological methods; Fischbach had pioneered the use of co-cultures of nerve and muscle cells, and this permitted him to monitor the formation of synapses between motor neurons and skeletal muscle cells using physiological and receptor- binding techniques. α-Bungarotoxin, an essentially irreversible ligand for the muscle acetylcholine receptor (AChR), was available

and enabled us to examine the influence of motor neurons on the organization of AChRs. These studies provided support for the idea that synapses do not form at pre-existing AChR clusters; rather, clusters are induced by the ingrowing motor neuron.

The hypothesis at the time was that motor neurons release factors that organize the postsynaptic membrane, a process essential for achieving a mature and functional synapse. The biochemical search for the nerve-induced AChR-organizing factors began. Today we recognize agrin and neuregulin as two molecules that influence different aspects of peripheral nerve–muscle synapses. In those days, molecular strategies that are routinely used today were not available, and so we began a set of somewhat crude biochemical purifications based on receptor clustering and induction assays.

For me this was an important period, because Fischbach introduced me to key ideas in neural development that set the stage for studies on other aspects of circuit organization in the spinal cord.

In 1981 I took a Faculty position in the Neurobiology Department at Harvard Medical School. Influenced by what had happened in the Fischbach laboratory, but also by my continued interest in sensory systems, I set out to study the way in which sensory–motor systems in the spinal cord are organized and assembled. It was that period at Harvard in the early 1980s, together with the advent of using monoclonal antibody strategies, that got us interested in how to recognize different neuronal types by virtue of their molecular character and then use that information to piece together how sensory circuits are formed. Much of the work in the laboratory since that period is really an extension of ideas that emerged during that period, although obviously the methodologies have changed, and in some respect the nature of the questions has also changed.

What were the milestones that led to the identification of the key players in spinal cord circuit assembly?Classical anatomy had told us that there must be a high degree of specificity in terms of synaptic connections in the spinal cord. The spinal cord was a system in which there was reason to think that the organization of certain circuits was hard-wired — as opposed to, for example, the situation in the visual cortex, where classic work had established that sensory experience has an important role in establishing the final pattern and visual connections. In the spinal cord,

eric Frank’s work had shown that changing patterns of activity does not obviously change monosynaptic sensory–motor connectivity patterns. Together with findings from Corey Goodman and Lynn Landmesser, these studies strongly suggested that there is a molecular basis for synaptic connectivity in the spinal cord. The key breakthrough in testing these ideas came from a set of developmental studies in which we found that the floor plate provided signals that guided spinal axons and induced spinal neurons. We then began a series of collaborative studies with Thomas edlund’s group in Sweden. Thomas, in a completely different set of experiments, had identified a transcription factor, ISL1, that is involved in regulating insulin gene expression in pancreatic β-cells. edlund observed that this transcription factor was not restricted to pancreatic endocrine cells: it was also expressed in a group of ventral neurons in the spinal cord. He faxed me a picture of the ISL1 expression pattern, and very quickly it became clear that this nuclear protein was a long-sought-after marker for spinal motor neurons. This observation gave us a molecular entry point into understanding the specification of motor neurons and, eventually, of spinal cord circuits.

Did you realize the importance of this finding at the time?It did not take long to realize that perhaps the general logic of spinal cord neuronal identity, as well as later connectivity, might have its basis in transcriptional programmes that these neurons expressed. So it was in this very active and brief period around the late 1980s and early 1990s that the chemoattractant and induction properties of the floor plate, as well as some of the molecular players, revealed themselves.

A more systematic study of the way in which floor-plate-derived signals both pattern neuronal cell types and guide axons followed. The more we knew about the cell types, the more it became clear that this was of relevance not just to general patterning strategies but also to the way in which circuits that control motor output are organized. each of the functional attributes of spinal motor neurons, of which there are approximately 100 different classes, has its basis in these transcriptional codes, which in turn are set up by the environmental patterning signals. Together with the advent of mouse genomics, these developments permitted us to tackle some of the problems in a genetically rigorous way that just had not been possible before.

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How had developmental studies on Drosophila patterning influenced your thinking at these times?Fly genetics had provided — not in the context of the nervous system, but in the context of embryonic patterning in general — a way of thinking about how one could approach developmental problems. We have always tried to tread the slightly wobbly line between developmental biology and systems neuroscience. Therefore, by using insights from developmental mechanisms, we progressively went back to some of the original problems of circuitry and function and readdressed them at a physiological and anatomical level.

What is the fundamental question you would like to see resolved before you allow yourself to retire?Retirement is not an option. But what intrigues me now is how spinal circuits extract sensory signals from the outside world and use this information to coordinate motor output. A hundred years of physiology

and anatomy have given us a reasonably clear picture of what motor systems can do — from simple locomotor tasks to learned motor behaviours. Yet we still do not understand the core principles of motor circuit function. The neurons that contribute to these circuits can be identified and we are beginning to have insight into the identity of unique markers of neuronal subtypes. We hope to use this knowledge, with the help of genetics, to try to understand the organization of these circuits, how selective connections are formed and how motor systems use sensory and local circuitry to direct motor output.

Do you see your line of research converging with that of Sten Grillner?Yes, they are converging in a very substantive way. We have begun to work with Sten to identify core features of circuitry at a molecular level. A lot of what we are doing in mice is influenced by the principles of motor-system organization that Sten has described in the lamprey. There is a close

parallel in terms of the types of questions that can be asked in these divergent vertebrate spinal motor systems.

Do you have any career advice for young generations of neuroscientists?Without sounding too avuncular, what I have always done is follow my passion and intuition. The important thing is to choose a problem that you think is important and representative of a general set of issues in the field. There will be many moments of failure and sober contemplation, but the initial choice of problem and the determination to stick with it have sustained me over the years.

FURTHER INFORMATIONThe Kavli Prize: http://www.kavliprize.no/Pasko rakic’s homepage: http://rakiclab.med.yale.edu/sten Grillner’s homepage: http://ki.se/ki/jsp/polopoly.jsp?d=21984&a=54779&l=enThomas Jessell’s homepage: http://sklad.cumc.columbia.edu/jessell/ SUPPLEMENTARY INFORMATIONsee online article: S1 (figure)

All linKs Are AcTive in The online Pdf

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