PHYSIOLOGY NEWS

summer 2011 | number 83

Decline in physiology teaching in the UK Endurance training–overtraining: a fi ne balance How we learn to walk Life in the womb and the effects on later cardiovascular risk Keep students learning in and out of the lab

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UK physiology ad/penny.indd 1 3/6/10 2:04:41 PM PHYSIOLOGY NEWS Editorial 3 The Society’s dog. ‘Rudolf Magnus Meetings gave me to Charles Sherrington, who Epithelia & Membrane Trasport Themed Meeting 4 gave me to Henry Dale, who gave Networking at Meetings may help your career Keith Siew 5 me to The Physiological Society in Cellular & Integrative Neuroscience Themed Meeting October 1942’ Trevor Smith 6 Opinion Published quarterly by The Physiological Society The decline of physiology teaching in British universities Contributions and queries Richard Naftalin 8 Senior Production Editor Soapbox Jill Berriman Abstracts of Communications: people were faster than Editorial Administrator computers Roger Thomas 12 Maev Fitzpatrick Science News and Views The Physiological Society Publications Office PO Box 502, Cambridge CB1 0AL, UK Do peripheral chemoreceptors in the carotid body serve as sites of glucose sensing? Erica Wehrwein, Michael Joyner 13 Tel: +44 (0)1223 400180 Fax: +44 (0)1223 246858 A key role of orexin (hypocretin) neurons in the fight-or-flight Email: [email protected] response Tomoyuki Kuwaki 15 Website: www.physoc.org Endurance training–overtraining: a fine-tuned balance that 2+ Magazine Editorial Board depends on Ca Niklas Ivarsson, Joseph Bruton, Håkan Westerblad 18 Editor The way we learn to walk Tue Hvass Petersen, Simon Farmer, Austin Elliott Jens Bo Nielsen 20 University of Manchester, Manchester, UK CO2 emissions of locomotion: innovative automobiles do Deputy Editor better than humans Alberto Minetti, Gaspare Pavei 24 Patricia de Winter University College London, London, UK A novel feature of epithelial acetylcholine in intestinal luminal sensing Takaji Yajima, Ryo Inoue, Megumi Matsumoto, Masako Members Yajima 26 Angus Brown University of Nottingham, Nottingham, UK Surviving life in the womb and the implications for vascular Sarah Hall health in adulthood Marianne Tare, Marc Mazzuca, Helena Cardiff University, Cardiff, UK Parkington, Nicoleta Dragomir, Mary Wlodek 29 Munir Hussain Techniques University of Bradford, Bradford, UK Western blotting Patricia Leoni 31 John Lee Noticeboard 34 Rotherham General Hospital, Rotherham, UK Unbelievable! 35 Thelma Lovick History University of Birmingham, Birmingham, UK More on Starling and Bayliss during the 1914–18 War Samantha Passey William Van der Kloot 36 University of Bedfordshire, Bedford, UK Death of a diuretic Prakasa Rao 38 Foreign Correspondents From the archives Austin Elliott 39 John Hanrahan McGill University, Montreal, Canada Book reviews 40 John Morley Society pages University of Western Sydney, NSW, Australia Paton Prize Photographic Competition 42 Ask a Physiologist! 44 © 2010 The Physiological Society Education ISSN 1476-7996 (Print) Meet the Physiologists 45 ISSN 2041-6512 (Online) Undergraduate Vacation Studentship Scheme 47 The Physiological Society is registered in England as a Lab_13 49 company limited by guarantee: No 323575. Registered office: Peer House, Verulam Street, London Affiliate news WC1X 8LZ. Lab Profiles: Welcome to the Muscle Cellular and Molecular Registered Charity: No 211585. Physiology Research Group Samantha Passey 50 Printed by The Lavenham Press Ltd The Society’s journals New Journal Editors 2011 52 Experimental Biology 2011 Giovanni Mann 54 Paul McLoughlin, the new Editor-in-Chief for Experimental Physiology 55 Early Career Author’s Prize 2010 Winners 55 Obituaries Alison Brading Tom Bolton, Richard Vaughan-Jones 56 James (Jim) Edward Pascoe Tim Biscoe, Ann Silver 60

Cover image: Surviving life in the womb Sidney Montague Hilton John Coote, Mike Spyer, Will Redfern 62 and the implications for vascular health in Advancing the science of life adulthood, from p. 29. John Archibald Browne Gray Bruce Lynn 64 PHYSIOLOGY NEWS Action points Guidelines for contributors In this issue Grants These guidelines are intended to assist Welcome to the Summer 2011 The Society offers funding authors in writing their contributions and to reduce the subsequent editing Physiology News. through the following grant process. The Editorial Board of schemes: Travel Grants, Non- Physiology News tries to ensure that all This issue has, completely Society Symposia Grants, articles are written in a journalistic style serendipitously, somewhat of a Outreach Grants, International so that they will have an immediate historical flavour. William Van der Teaching and Research Grants interest value for a wide readership and Kloot tells us about the WW1-era and the Vacation Studentship and will be readable and comprehensible to non-experts. Scientific articles should activities of famous physiological Departmental Seminar Schemes. give a good overview of a field rather brothers-in-law Bayliss and Starling For full information, please visit: than focus entirely on the authors’ own on p. 36, J Prakasa Rao gives us a www.physoc.org/grants research. snippet of diuretic history on p. 38, Format of articles and even my editorial has had a Membership applications The main message or question posed history outbreak. Meanwhile, the Applications for membership should be introduced in the first winning entrants for the Paton Prize to The Physiological Society are paragraph. The background for the Photographic competition (p. 42) considered on a rolling basis, topic should then be established, show that in history, as in other leading up to the final conclusion. and a decision is normally made things, a picture can be worth an within 15 working days. For full Length of articles awful lot of words. information, please visit: This will be determined by the subject www.physoc.org/membership matter and agreed with the Senior One of the things Physiology News Production Editor. seeks to do is air views – and not Is your membership information Submission of articles just views from the magazine correct? Authors should submit articles as a editorial staff, or from The Society's Please check and update your Word document attached to an email. Council or officers, but from you – details at www.physoc.org, under Illustrations should be sent as separate The Society's Members. This issue attachments (see below) and not ‘My Physoc Profile’. embedded in the text. we have two Members expressing strong opinions – Roger Thomas Illustrations and authors’ photographs is on his soapbox (should I say Authors are encouraged to submit 'again'?) on p. 12, while Richard Physiology News diagrams, drawings, photographs or Naftalin explains how and why Deadlines other artwork with their articles and a photograph of the author(s) should he thinks physiology teaching Letters and articles and all other accompany submissions. Illustrations has been in retreat on p. 8. And contributions for inclusion in the and photographs may be colour or black another thing magazines try to Autumn 2011 issue, No. 84, and white, and preferably TIFF, JPEG, PNG, PDF or AI files with a minimum do is encourage debate, so please should reach the Publications resolution of 300 dpi. do write in and tell us if you agree Office ([email protected]) by – or disagree – with either of our References 7 July 2011. Short news items and soapbox-ers. Authors are requested to keep the letters are encouraged, and can number of references to a minimum – Moving to our regular features, usually be included as late preferably no more than two or three. copy if space permits. Please cite all references in the style of this issue has a wide-ranging The Journal of Physiology (see selection of Science News and Suggestions for articles Information and Guidance for Authors at Views that takes us from the womb Suggestions for future articles are http://jp.physoc.org). to adulthood (p. 29), appropriately welcome. Please contact either taking in learning to walk (p. The Physiological Society permits the the Editorial Administrator or a single copying of individual articles for 20), 'fight or flight' (p. 15) and member of the Editorial Board private study or research. For permission endurance (p. 18). We can all agree of Physiology News (see contents to copy or reproduce for any other that modern scientists need plenty page for details). purpose contact [email protected]. of the last quality, and probably the Opinions expressed in articles and letters one before that too. Physiology News online submitted by, or commissioned from, Finally, we say farewell to, and Physiology News online: Members, Affiliates or outside bodies are not necessarily those of The Physiological salute the life and work of, four www.physoc.org Society. stalwarts of UK physiology in our obituaries. Austin Elliott Editor EDITORIAL PN 3

Then and now a genius. By microdissection, he There was a fixed income to the reaches anatomical structures laboratory with no questions asked Pondering a suitable topic for this difficult of access, uses advanced as to how it was spent. I still think Editorial, I used the internet to ask tele-technique for stimulation that this is the basis of every big some of my colleagues and friends and recording, and employs discovery.” [1] what might make a good subject. high magnification stroboscopic A range of ideas were offered, but microscopy for making apparent So to the list of things that there was a common theme. ‘Not complex membrane movements, the underpinned von Békésy’s more gloom’. ‘People’, another amplitudes of which are measured in discoveries we could add good suggested ‘need cheering up’. thousandths of a millimeter…” research facilities; a secure job; and Among the positive suggestions for minimal interference or need to how to be more upbeat, the one that Von Békésy’s work thus had many of justify his research. And thus, in turn, caught my eye was ‘There must be the typical hallmarks of the scientific time to try things – and fail – on the an anniversary of some important process: identifying a problem to way to success. discovery – talk about that’. address; working out a strategy to attack it; devising and refining the Apart from the central place of They had a point, I conceded, about methods to do it; making the most the scientific method, what other the gloom. When one editorializes accurate observations possible; and ‘contemporary resonances’ can we about the issues facing scientists in finding the appropriate theoretical find in von Békésy’s story? One is 2011, it can be hard not to sound framework to interpret them. Von the key role of physical sciences, gloomy. The grim economic climate, Békésy’s background in physics was and numeracy, as a prerequisite for grant success rates at historic also key, and his being able to obtain much physiological and biophysical lows, rocketing university tuition tissue from obliging pathologists investigation. It is a regular fees, rising student expectation, useful, so perhaps we could also complaint of today’s university governments that don’t listen to add ‘interdisciplinarity’. A final and scientists that numeracy among scientists – I could go on. But there perhaps less obvious ingredient, to students is ever-declining – though is a danger that, in talking about which I will return, was also present: this has been a regular complaint for these topics, one ends up repeating the necessary time to work all this at least the last half-century. oneself. Gloomily. out. Another point that comes across So I have decided to take my friends’ Von Békésy’s life took a decidedly strongly is the necessity for time, suggestions, and tell you about ‘non-standard’ route to scientific and a free hand, and a supportive an anniversary. Though if I find greatness. He must surely be the environment – even if mainly contemporary messages there too, only Nobel Laureate in physiology supportive by lack of interference you will have to forgive me. Old to have done his research whilst – for scientific discovery. Though habits. working (for over two decades) for there is far more science, and many the national telephone company. more scientists, now than in von Among the many changes in science Having completed a physics PhD Békésy’s day, many of them can in the modern era, ‘one-person in Hungary in the 1920s he initially be heard saying they are ‘too busy discoveries’ have become struggled to find a scientific job. chasing funding’, ‘too busy doing increasingly rare. This year marks a administration’ or just ‘too busy significant anniversary for a famous “After a certain time I decided to think’. This situation, more and past one, however; it is 50 years to look around systematically more evident in the last few years, since the 1961 Nobel Prize for and find out which was the best has been masked by the funding that Physiology or Medicine was awarded equipped laboratory in Budapest. allows scientists to employ more to Professor Georg von Békésy I found that it was the laboratory junior scientists (or ‘scientists in (1899–1972) ‘for his discoveries of the government controlling the training’) to do the hands-on work. of the physical mechanism of research in long distance telephones, The danger now is that, as funding stimulation within the cochlea’. For telegraphy, and radio stations. becomes ever scarcer, there will be those unfamiliar with von Békésy Hungary was in the middle of Europe fewer of these pairs of hands to do and his work, a quote from the Nobel and therefore communication science, but still just as much other presentation speech by Professor was a very important feature. The stuff to distract scientists’ attention. Bernhard of the Karolinska Institute government was forced by peace If we are to see more von Békésys, gives a good summary [1]: treaties to spend a certain amount I would suggest, we need to strive of money to keep the transmission much harder to keep scientists doing “Von Békésy’s distinction is to lines in good shape. To do so, they science – not filling in forms. have recorded events in a fragile constructed a laboratory and gave biological miniature system. the laboratory a certain amount Austin Elliott Authorities in this field evaluate of money to buy the necessary 1. Von Békésy G (1974). Some biophysical the elaborate technique which equipment. It was this financial experiments from fifty years ago. Annu Rev he developed as being worthy of support which started my research. Physiol 36, 1–16.

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Epithelia & Membrane translational research. This approach Invited speakers is emphasised by combining Transport Themed presentations from basic scientists Frances Ashcroft and clinicians for every one of the Meeting main topics covered. University of Oxford, UK Royal Free Campus, University Jonathan Ashmore College London, UK http://www.physoc.org/em2011 University College London, UK 1–3 September 2011 Topics covered September’s Themed Meeting is Detlef Bockenhauer •Calcium and magnesium an event for all those working in Great Ormond Street Hospital, transport and paracellular Epithelia & Membrane Transport. It London, UK permeability is hosted by Anselm Zdebik, Robert Ted Debnam Unwin and Ted Debnam (University •Luminal sensing in the kidney and College London) at the Royal Free gastrointestinal tract University College London, UK Campus, University College London. Themed Meetings bring together •Novel aspects of phosphate Annette Dolphin basic scientists and clinicians homeostasis University College London, UK working in the Theme to share the latest ideas and cutting-edge •Potassium transport, sodium Dominique Eladari knowledge. Submit your research, transport and cross-talk – from Centre de Recherche des Cordeliers from any topic within the Theme, disease to structure/function de Jussieu, France when abstract submission opens studies Ian Forster on 23 June as there are many •Ion transport protein trafficking opportunities to present your work. and modulation University of Zurich, Switzerland The focused symposium ‘Ion Pascal Houillier channels: Insights from disease and National Institutes of Health, USA animal models’ will explore recent Young Physiologists’ new developments in transport Symposium 2011 Christian Huebner physiology that have surfaced Medizinisch Theoretische Institute with the recent analysis of newly Royal Veterinary College London, Universitätsklinikum Jena, Germany identified genetic diseases and UK animal models. Insights arising Edith Hummler 31 August 2011 from this approach integrate into University of Lausanne, Switzerland a wider perspective in many newly Epithelial physiology across species: developing fields. These include can other species inform your Martin Konrad TRP channels and epithelial polarity, epithelial research? University of Münster, Germany but also their role in calcium and magnesium handling by epithelia. A one-day Young Physiologists’ Michael Pusch Advances in the understanding of Symposium (YPS) is designed National Research Council, Italy genetic diseases has also changed specifically for early career traditional views on potassium researchers in the field of epithelial Bernard Rossier transport, sodium transport and physiology to meet and exchange University of Lausanne, Switzerland has identified diseases where these ideas. Explore the differences and two processes show considerable similarities in epithelial physiology Blanche Schwappach cross-talk. across species, with specific focus University of Manchester, UK on epithelial transport, sensory By tying in speakers from related, epithelium, naturally occurring Victor Sorribas but less traditional fields, such as animal models to facilitate University of Zaragoza, Spain ion transport regulation, inner ear epithelial research, and epithelium transport and even developmental in health and disease. Robert Unwin biology, this opens the epithelial field to other physiologists with University College London, UK Early career researchers can present an interest in ion transport. The the same abstract at the YPS and Oliver Wrong understanding of genetic disease and the Themed Meeting. their analysis using animal models University College London, UK will be of a broad interest, not only http://www.rvc.ac.uk/Meetings/ Anselm Zdebik to physiologists in the epithelial field, YPS/Index.cfm but also to others with an interest in University College London, UK

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Networking at Meetings may help your career been awarded funding by the British Heart Foundation for a three year My Physiological Society journey we launched into the week’s many PhD studentship at the University of began in my hometown of Dublin talks, presentations and socials. Cambridge, starting this October. If at Physiology 2009, just before there is one piece of advice I can give starting my final year of my joint BSc On the night of the Society Dinner to those of you attending this year’s (Hons) Physiology & Pharmacology at Manchester United, our former Meeting in Oxford, it is to never be degree at University College Dublin lecturer Stuart Bund advised us to afraid to ask a question and always (UCD). A last minute decision, some take advantage of this opportunity be prepared for when opportunity classmates and I decided to attend and network, not only with our comes a-knocking. the Meeting in support of our peers but other senior scientists and lecturers who were giving talks at perhaps meet a potential supervisor. Keith Siew the various symposia. It was here our So with this in mind on the last day University College Dublin, of the Meeting, I chose to attend a interest was sparked, and not too Republic of Ireland long after we had all signed up as symposium on my favourite subject Undergraduate Associate Members and passion, renal physiology. It was of The Society. here I met Kevin O’Shaughnessy, University of Cambridge, and his Annual Public Lecture The following year, with the financial talk on renal salt transport and of The Physiological support of bursaries from the Young blood pressure really caught my Society Physiologists’ Bursary Scheme imagination and I decided to hang (YPBS), I encouraged even more of about after the session to ask him a Light, Clocks and Sleep our class to attend Physiology 2010 few questions and get some career Russell Foster (University of in Manchester. This time round, my advice for this area of research. classmate, Naadiya Carrim, and I Oxford, UK) had been accepted to present our I figured it was now or never and 18.00 Tuesday 12 July decided to chance my arm, dropping research at the Young Physiologists’ The Sheldonian Theatre, Symposium YPS), much to our into conversation my earlier success surprise. So you can only imagine at the YPS and my recently obtained Oxford, UK my utter shock when I discovered I first class honours UCD degree. As An internal 24 hour biological had been placed second in the oral luck would have it Dr O’Shaughnessy clock (circadian clock) controls, communications section despite was in search of a new PhD student modulates and fine-tunes our sleep being the only undergraduate to recruit to his lab. Fast forward patterns, alertness, mood, physical presenting a talk. With a great start several emails, phone conversations strength, blood pressure, and every to the Meeting and new YPS friends, and trips to Cambridge and I’ve since other aspect of our physiology and behaviour. Russell’s lecture will look at how circadian rhythms are generated, regulated by light and why we can’t ignore our internal time in both medical treatments and in the way we organise our 24/7 society.

If you wish to participate and are NOT attending Physiology 2011 in Oxford, please email Sarah Bundock at The Society ([email protected])

Physiology 2011 – Early Career Social Monday 11 July from 19.30

A chance for all Early Career scientists to make new friends before Physiology 2011 kicks off. It is free to sign up, so please email UCD Physiology Class of 2010 from left to right: Louise Cully, Naadiya Carrim, Keith Sarah Bundock Siew, Philip Lewis and Naoise O’Ciardha. ([email protected])

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Cellular & Integrative gratitude from the various speakers for everyone who attended their Neuroscience Themed talk, rather than sitting in the sun. Meeting After the talks on the first and 6–8 April 2011 King’s College second days, there were two poster London, UK sessions. These were incredibly useful, as they were held in an As this was my first Cellular & informal setting and everyone was Integrative Neuroscience Themed able to walk around and engage Meeting, I wasn’t entirely sure of in light conversation over research what to expect. In addition, I had they were interested in. All of the never visited the Guy’s Campus of posters were of a high quality and King’s College London and I was one that particularly stood out to left extremely impressed by its me was Jean-Pierre Lin’s (King’s facilities, as the room layouts, of College London), as he had brought both the lecture theatre and the his computer along and had some poster presentation room, worked videos to supplement his poster on incredibly well. The organisers of the how potent a treatment of deep Meeting, Jon Robbins, Pat Doherty brain stimulation (DBS) can be to and Jeremy Ward (King’s College treat dystonia. Walking around the London), had clearly researched Left to right: Jeremy Ward, Roger Nicoll posters, it was quite amazing to see everything thoroughly and the and David Wyllie. Roger delivered the how far some of the research groups result was a meeting that ran Hodgkin–Huxley–Katz Prize Lecture at had travelled from. Just to name a smoothly from start to finish. To be the Meeting. few of the locations: Porto, Portugal; honest, having read through the Brescia, Italy; Cairo, Egypt and programme a few times, I wasn’t Dunedin, New Zealand. I think a lot sure how relevant some of the All the talks were of a very high of those names also appear on my various speakers would be to me, as standard and were very well holiday wish-list, so maybe there will there was a wide breadth of subjects attended, which was extremely covered, and none of the symposia impressive given that the weather be some new post-doc positions in a were on chronic pain, which is my could not have been any nicer, as year’s time. there was glorious sunshine for specific research interest. However, On Thursday evening, The Society my fears were unfounded because, three days straight. This provided thoroughly enjoyable breaks sitting Dinner was held at the Little Ship as the Meeting progressed, the Club and although I wasn’t able to quality of the invited speakers in the grassy quad outside New Hunt’s House and quite a lot of attend myself, I heard great things and selected oral communication the next day. There were fabulous presentations shone through. One views over the River Thames, as the speaker that I was particularly sun set after belting out heat all impressed with was Chris Anacker, day long. In addition, the food and a PhD student from King’s College opportunity for networking were London. He presented a poster and, both top notch. at extremely short notice, stepped in for his ill supervisor, Carmine Overall, the Meeting did exactly Pariante (King’s College London), what every scientific meeting should to give a 30 minute extended do, as novel results were presented, presentation. The presentation, friendships were formed, and ideas on exploring the role of the gluco­ were discussed. For budding and corticoid receptor in depression experienced neuroscientists, the and hippocampal neurogenesis, three-day meeting was beneficial to was given so well that I never would all who attended. Let’s hope in the have guessed he filled in to give future, the next Cellular & Integrative the presentation at the last minute. Neuroscience Themed Meeting I also particularly enjoyed James can bring the same high calibre of Fawcett’s (University of Cambridge, scientific research and maybe, if UK) thought-provoking presentation we’re lucky, some sunny weather. on how to increase the regenerative ability of axons by providing the Trevor Smith right micro-environment for the axons. Participants discussing a poster. University of Liverpool, UK

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13–15 July 2011 Robinson College, Cambridge, UK

A Biochemical Society Focused Meeting DEADLINES: Abstract submission Nuclear envelope disease 11 MAY 2011 and chromatin organization Earlybird registration 13 JUNE 2011

Organizers: Juliet Ellis Sue Shackleton

Overview: Laminopathies are a diverse group of diseases associated with nuclear envelope proteins, ranging from muscle

Image kindly supplied by Sue Shackleton (University of Leicester, UK) Image kindly Sue Shackleton supplied by of Leicester, (University wasting to premature aging disorders. This meeting brings together researchers and clinicians working on diverse aspects of nuclear structure and associated diseases, to review recent progress and define strategies for future research.

Topics: * Nuclear envelope proteins * Disease models and mechanisms * Nuclear envelope protein-dependent stem cell aging * Chromatin interactions * Nucleo-cytoskeletal connections and mechanics

For a full programme please visit www.biochemistry.org

Sponsored by:

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The decline of physiology teaching in British universities Physiology teaching in most British 1870s. The Physiological Society medical schools has decreased over was founded in 1876. One of its the last quarter century. Precise raison d’êtres then, as now, was as a figures on the amount of physiology lobby against the Anti-Vivisectionist now taught are hard to obtain as it movement, which was as vociferous, is no longer as clearly demarcated although rather less menacing then as it once was. However, first year (Sharpey-Schafer, 1927). physiology teaching for medical Richard Naftalin students at King’s College London Experimental physiology in Britain has been cut by more than fifty was initially practised by a very small per cent, as Table 1 below shows. chair in Physiology at Breslau (now number, whose financial support was During the same period medical Wroclaw) from 1823 to 1850 and sourced mostly from their income student numbers have doubled. there opened the first independent from their own clinical work and Currently eight thousand students Physiology Department in 1839 and occasional anatomy and histology annually enrol in UK medical schools. was the first to introduce a course teaching. Most British physiologists King’s College London is the largest of experimental physiology. His gained their research training from medical school in Britain, but is teaching was much influenced by the visits to the established laboratories fairly representative of other UK city ideas of the Swiss educationalist and of eminent German physiologist– universities e.g. those in the rest of social reformer, Johann Pestalozzi physicians or physicists; for example, London, Glasgow and Newcastle. (1746–1827), which were based Carl Ludwig (1816–1895), discoverer This decline in the amount of on the concept of ‘Anschauung’ – of glomerular filtration; his student physiology teaching along with the direct concrete observation from Otto Frank (1865–1944), who bloating of student numbers reflects experiment – ‘A man learns by action worked on the mechanics of muscle; a general trend in the UK. ... have done with mere words’. Rudolf Virchow (1821–1902), the Pestalozzi’s main concerns were to populariser of the cell theory – ‘every Where medical teaching practice maintain an equilibrium between cell originates from another’ – and leads, biology teaching in general head, hands and heart, and with ‘father’ of modern pathology; soon follows. The reduction in what was ‘right’ as opposed to and Hermann von Helmholtz medical physiology practical ‘correct’. Purkinje is remembered (1821–1894), a physician and teaching is symptomatic of a decline today by physiologists mainly scientist, who made seminally in practical teaching extending to because of his innovative use of important contributions to both other disciplines, like pharmacology, the microtome in preparation thermodynamics and optics, and biochemistry and microbiology, of histological specimens which invented the ophthalmoscope. where practical teaching once had a enabled him to discover the large No doubt Helmholtz would take a preponderant role. nerve cells in the cerebellum and the very jaundiced view of the current conducting fibres within the heart, vogue for ‘Translational Medicine’. So what? All academic subjects both of which are now named as He wrote, “Whoever in the pursuit wax and wane according to Purkinje cells. of science, seeks after immediate contemporary needs and vagaries. practical utility may rest assured that Why bewail physiology’s relative In Britain the first chairs in he seeks in vain”. decline any more than that of Physiology were founded in 1836 Ozymandias? at both University College London The life, scientific works and and King’s College London, then philosophy of the French physician What is so important about in Cambridge and Oxford in the and physiologist, Claude Bernard physiology that it needs to occupy Table 1 the ascendant position in the time and thoughts of our ostensibly Year Med School Physiology lecture Tutorial hours Practical hours Total hours (numbers hours hardworking British medical (group size) (group size) attending) students? 1990 KCL 72 (120) 20 (6–8) 36 (2–4) 128

History of academic physiology 1992 Glasgow 62 34 The foundations of the physical– 2011 KCL 46 (450) 8 (8–10) 6 (6–8) 62 chemical approach to physiology 2011 Glasgow 15 — 9 were laid in Germany and France (PBL plenary in the early nineteenth century lectures on and it developed in parallel with physiological physical and organic chemistry. Jan topics) Purkinje (1787–1869) held the first PBL, Problem-Based Learning.

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(1813–1878) were one of the other by the most facts are the best, but at all levels – molecular, subcellular important formative influences even then they are never final, never organelles, cells, tissues and body on the ethos of twentieth to be absolutely believed’. So we see organ systems. From its beginnings century physiology research and that Bernard’s scientific philosophy experimental physiologists teaching. Bernard insisted that all was far ahead of his time and closely have tried to eliminate as many science must be based on ‘facts’ aligned with the views of Karl Popper extraneous factors as possible from established by rigorously controlled (1902–1994). the processes under investigation experiments. ‘Active’ experimental and so improve the precision of physiology was differentiated from Bernard was sceptical about the their conclusions. This reductionist ‘passive’ observation of unvarying Darwinian theory of evolution, trend has meant, wherever possible, phenomena, e.g. histological viewing it as speculation that physiological processes are appearances. ‘An ‘experimenter’ unsupported by proper experimental examined in isolation – after observes the changes in phenomena, evidence. He was, as was Darwin, removal of the living tissues from whilst applying a controlled variable, unaware of Gregor Mendel’s the animal. In most cases this thereby uncovering an otherwise (1822–1884) work, which remained requires acquisition of skills and unrecognised until its rediscovery hidden process’. He conceded, development of expensive and and translation into English (Bateson, nevertheless, that one could sensitive instrumentation. This 1902). take advantage of opportunities atomization of physiological function presented in medical practice, as in has led to growth of sub-specialities A modern criticism of Bernard’s and to a growing divide between the case of Alexis St Martin, who as views and by extension to all a result of a shooting accident had the immediate research interests of contemporary physiology, is that physiologists and the essential topics a gastric fistula. Fortuitously, he he ignored species differences became the personal servant to Dr required for the training of future when applying results from animal clinicians and most biologists. William Beaumont (1785–1853), a models to human clinical conditions. US army surgeon. Beaumont put St The main differences between Clinicians work mainly at the level Martin’s gastric fistula to good use. the physiologies of mice and men of interrelations between the were assumed to be quantitative various body systems and their Claude Bernard asserted that rather than qualitative. We know disorders, e.g. the cardiovascular, whilst Beaumont’s observation and now that genetic variations in respiratory, gastrointestinal, genito- description of the condition of St protein structure can alter enzyme urinary, reproductive and nervous Martin’s gastric mucosa were merely specificities and activities, so both systems, rather than in the cellular acts of passive inspection, examining qualitative and quantitative intra- and subcellular domains, where of the process of digestion by and interspecies differences exist reductionist studies have led. insertion and removal of digestible (LaFollette & Shanks, 1994). These materials via the fistula was the act It is claimed that physiology “lost its differences must make us cautious of an experimentalist – an altogether position as the exclusive paradigm of about extrapolation from animal nobler endeavour. biology ... when behavioural biology, to human models of physiological ecology, population biology, and other and pathological processes. The accident that befell St Martin branches of modern biology developed, However, these real differences do and his subsequent fortuitous it became even more evident how not invalidate, as anti-vivisectionist employment by Beaumont is an unsuitable mechanics was as the propaganda would like us to believe, exceedingly rare coincidence as ‘it paradigm of biological science... In the tried and tested corpus of results would be immoral to experiment on contrast, clinical medicine -- based based on animal experimentation. humans’, and since physiological on observation and comparison (as science must be based on Because Purkinje’s and Bernard’s opposed to the determinists’ controlled experiments on living animals, views were strongly influential on experiments) -- is emerging as a Bernard reasoned that animal medical and biological thought genuine science, and epidemiological vivisection was a necessity for throughout the twentieth century, studies, while irredeemably statistical both advancement of physiological physiology teaching became the in nature, have provided much knowledge and teaching (Bernard, core topic from which medical and valuable information about human 1865). Mme Claude Bernard, biological sciences stemmed. pathology” (LaFollette & Shanks, amongst others, vociferously 1994). disagreed. What is physiology? This argument is analogous to Bernard (Claude) considered that Physiology is the study of functional the statement that the classical experimental hypothesis must relationships of living tissues with mechanical paradigm has been always be based on prior observation each other, using the methods and overturned by the discoveries of – ‘Theories of how systems function precepts of physics and chemistry. quantum mechanics. Although are only hypotheses, verified by more It attempts to understand the quantum physics overrules or less numerous facts. Those verified integration between living states Newtonian physics in the particle

Physiology News | No. 83 | Summer 2011 | www.physoc.org 10 PN OPINION physics domain, this is both untrue These changes might have occurred Threats to physiology from loss of and misleading when applied in in any case, as the rise in student academic departmental structures macroscopic terrestrial domains, numbers coincided with adoption Heads of university Physiology where Newtonian mechanics still in the late 1990s of a new Problem- departments, as with other rules okay. Based Learning (PBL) teaching model scientific disciplines, were imported from North America. It traditionally the regulators of Distrust of statistics and of was evangelised in the UK, at first in new appointments, promotions, ‘empirical’ clinical studies were Manchester, with the avowed aim of ingrained in Bernard’s philosophy allocators of financial resource for encouraging the students towards shared equipment and teaching that dominated early twentieth ‘self-directed learning’, but had the century physiological thinking. These and the ones answerable to higher added advantage that it was seen university authorities. Peer pressure aversions stemmed from Bernard’s as a means of re-distributing the view that trustworthy facts can from national and international increasing teaching load to ‘specialist academic organizations, such as only be legitimately derived from teaching’ staff and relieved the experimental data. In fairness, The Physiological Society and increased burden on research staff. International Union of Physiologists modern statistical methods were PBL introduced students to an not fully developed until the 1920s generally ensured a progressive drift integrated or ‘holistic’ approach in research and teaching. (Fisher, 1925), about forty years to medicine from the outset. As after Bernard’s death. However, another of the aims of PBL is to break The years of faltering economic the canard that contemporary down the ‘artificial barriers between growth in the UK (1975–90 and physiology rejects the statistical the -ologies’, widespread adoption of 2008–2015) with reduction in approach to biology is completely PBL has had a major impact on the financial support from central false and misleading. For example, academic disciplines that have been government for academic research the ‘modern’ theory of chemical co-opted into this venture. and consequent ‘greying of transmission of signals between academia’ together have eroded nerves or nerve and muscle is Encroachment of PBL into the the traditional pyramidal structure based on a statistical theory of early medical teaching curriculum of academic departmental neurotransmitter release (Fatt & has meant that clinicians are now authority. General adoption of Katz, 1952). integrated within these early information technology throughout years and have a major say in early universities in the 1990s meant that Molecular biology, including training. However, only a few it became feasible for the central specifically molecular genetics and scientists are expected to take genetic modification, has made an administrations to take closer charge part in the converse, that is of immense impact into understanding of departmental finances and of clinical teaching. Consequently, any of physiological control mechanisms student progression. Resource perceived need to make good losses of growth and development at the allocation within the pre-clinical from natural attrition in pre-clinical cellular and subcellular levels. These schools, formerly controlled at staff numbers has been overridden studies have brought huge dividends the pre-clinical faculty level, was by the higher priority that the to the understanding of many transferred to central committees Medical School Administration gives genetic diseases and physiological composed of administrators and to the ‘health’ needs of patients – processes. Physiology has always senior clinicians, whose perspectives and for clinical duties. been totally promiscuous in were based on the global needs of adopting any and every method that PBL is more appropriately suited to teaching and research in the whole is appropriate towards elucidation of medical teaching in North America, medical school and university rather living processes – it is not a scientific where every medical student already than the local needs of pre-clinical discipline relying on a specific set of has a degree in biological sciences science departments. Naturally in a methodologies. So these discoveries before entering medical school. hospital environment in which most are not seen as a threat, but as an British students mainly do not have medical schools are based, these important adjunct to physiology equivalent experience in physiology, perceived needs militate against (Noble, 2011). cell biology, biochemistry or science in general and physiology pharmacology before coming to in particular. Physiology was seen The threats to physiology from medical school. Realistically UK as being old-fashioned, based on changes in the medical curriculum students can only be expected to outdated methods – antagonistic As a consequence of the integrate knowledge and solve to clinical science and engaged disproportionate increase in student problems once they have acquired a in expensive and controversial numbers relative to the available firm basis in medical sciences. This vivisection, which required an teaching laboratory accommodation requires that physiology should be expensive protective umbrella of and staff, the shape and design taught and examined prior to the security precautions. PBL was viewed of the medical curriculum has students applying this knowledge to as a cheaper, cleaner and more undergone a fairly radical change. problem solution. contemporary option and had the

Physiology News | No. 83 | Summer 2011 | www.physoc.org OPINION PN 11 added advantage that it could be is both a time-consuming and labour- Jan Purkinje and Claude Bernard’s controlled by clinicians. intensive process – its omission may precepts will be rediscovered. prove to be even more costly. British Perhaps the most important science graduates will become Richard Naftalin consequence of physiology’s loss less competitive and attractive to References of autonomy has been the virtual employers than more thoroughly Bateson W (1902). Mendel’s Principles of abolition of practical physiology trained European competitors, all of Heredity, a Defense, London. Cambridge teaching (see Table 1). Whilst whom still take laboratory teaching University Press. physiology and indeed clinical seriously. Bernard C (1927; reprinted in 1949). An medicine can be illustrated and Introduction to the Study of Experimental Medicine, 1865. First English translation by illuminated by using well-chosen It is not simply because of elitism Henry Copley Greene, Macmillan & Co., Ltd. clinical problems, the premature or conservatism that Oxford and Fatt P & Katz BJ (1952). Spontaneous use of the PBL approach leads to Cambridge have not yet completely subthreshold activity at motor nerve endings. exclusion of important aspects of complied with the trend towards J Physiol 117, 109–128. physiology and loss of the kind integration of departmental Fisher RA (1925). Statistical Methods for of understanding for which, as structures and teaching. For Research Workers. Pestalozzi’s teaching demonstrated, example, in Oxford medical Helmholtz H von (1885). 1821–1894 Popular lectures in Science. Appleton, New York. no substitute exists. students in their first two years undertake both human and animal LaFollette H & Shanks N (1994). Animal Experimentation: the Legacy of Claude The main goal of practical experimental work, carried out in Bernard, pp. 195–210. International Studies in physiology classes – giving the relatively smaller groups (e.g. of 48 the Philosophy of Science, Routledge. student direct personal experience students working in 12 separate Noble D (2011). Neo-Darwinism, the Modern of how deductive reasoning is groups with four staff members Synthesis and selfish genes: are they of use in based on experimental observation present); and in their third years, all physiology? J Physiol 589, 1007–1015. – has been abandoned. Practical work in a laboratory (usually joining Sharpey-Schafer E (1927). History of The Physiological Society 1876–1926, Part demonstrations on key methods in an experimental group taking part in 1 & Part 2. J Physiol 64 (Suppl.), 1–76 and clinical physiology, such as blood ‘cutting edge’ experimental work). 77–181. pressure and electrocardiograph As a result these students enter Acknowledgements and lung function, and widespread their clinical training with at least The author gratefully acknowledges Richard adoption of computer-aided learning some of the mental concept of the Boyd, Michael Lucas and Otto Hutter for their (CAL) programs used as surrogates scientific method and of data at the perceptive comments and discussions on this work. for animal experimentation, centre of the intellectual challenge. although better than nothing, Preservation of pre-clinical science cannot replace experience gained departmental autonomy appears The Physiological from real experiments on living necessary for maintaining their Society Oxford tissue. presence within the curriculum and in conserving the rational approach Debate – is The lessons that Bernard and to medicine which the GMC physiology relevant? Purkinje taught so successfully have prescriptive document ‘Tomorrow’s been abandoned and forgotten. Doctors’ are required to follow. If you enjoyed this article, there's Practical classes are now viewed as more to come – Richard Naftalin an optional and expensive ‘extra’ When, as now, medical education will be arguing in The Society for medical and basic medical and National Health are the debate at the upcoming Oxford science courses. Human or animal playthings of politicians, it is meeting in July (Monday 11th at experimentation, like nothing else, particularly important that clinicians 16.30), where he and Giovanni imprints the need for precision in should be well trained in rational Mann (KCL) will be opposing the preparation, accurate observation, thinking and know what ‘evidence’ motion: recording, collation, analysis and actually means. They should be eventually reporting the precious trained to examine with suspicion 'Physiology as a separate discipline data. Inadequacy of any of these all claims from authority, including is no longer relevant to modern essential elements will lead to those that suggest that particular science.' wasted time, money and in some measures will ‘save money’ in the cases life. This is exactly the kind long term by offering short-term The proposers of the motion will of lesson which is needed by all financial ‘inducements’ to some. be Maynard Case (Manchester) intending clinicians and animal and Ian McGrath (Glasgow), and researchers. But fashions also recur, Ozymandias the debate will be chaired by David (aka Rameses II) arguably has more Eisner (Manchester) and Godfrey Although this ‘old fashioned enduring posthumous celebrity Smith (Glasgow). Come along to approach’ of incorporating extensive than anyone. Certainly his works will hear the views, to give your own laboratory courses in physiology into remain Egypt’s biggest attraction for and to ask questions – or just to medical and medical science courses awhile longer; maybe in a future age see the fur fly.

Physiology News | No. 83 | Summer 2011 | www.physoc.org 12 PN SOAPBOX

Abstracts of Communications: people were faster than computers It is almost 35 years since the of card and labelled it by hand as submission time and paucity of Centenary Meeting of The Society. shown here, in part, on the left of Meetings. The authors have no This leads me to compare the way Fig 1. I wrote the abstract text in chance to correct minor errors, printed abstracts were dealt with long hand, had it typed up by a which used to happen after the then and now. Forty-odd years ago departmental secretary, and sent it abstract was accepted by the abstracts could be submitted up off with the original fi gure by post to Meeting for publication. to 7–9 weeks before a meeting; the Meetings Secretary (D. Noble). nowadays the window closes My submission was accepted and The Oxford Meeting of 2011 is 14 weeks before. The Centenary sent with the rest of the abstracts bigger than the Centenary Meeting, Meeting in 1976 was the largest to Cambridge University Press for but much of the programme was ordinary meeting of The Society to fi gure labelling and printing. The fi xed many months ago. The space date. It was held in Cambridge with fi nished book of abstracts and the for free communications may be much celebration, and fi ve or six programme were posted to all somewhat larger than in 1976, and parallel sessions. It ran from the 1st Members a week or so before the there will be many posters, but there to the 3 July, and the window for meeting. I reproduce part of the must be other factors dictating the long submission time. Ethics scrutiny submitting abstracts was, I recall, fi gure as printed. and checking by Special Interest 3–14 May. I remember the fi rst of After 35 years of amazing Group convenors only takes a week, these dates because I managed to do developments in computers and the and the submitted material simply an important experiment just in the internet, we now have to upload needs sorting into suitable sessions. nick of time. abstracts for the Main Meeting (in What takes so long? The Society In those days there were no posters. Oxford) 14–18 weeks in advance, has a much larger administration with the text and any fi gures ready If the number of submissions than 35 years ago, and many for printing as submitted. (The actual exceeded the time available the powerful computers, but perhaps submission process often requires, Meetings Secretary operated a too few people are involved with the in my experience, much wrestling fi rst-come fi rst-accepted policy. programme preparation. Is it simply with the ScholarOne computer As a result, one aimed to post that people can do things much somewhere in the USA.) the abstract to arrive on the day more effi ciently if computers are not acceptance opened. I managed It used to be that one attended involved? a complete experiment late on a Meeting to learn of the latest Roger Thomas Thursday 29 April, showing that advances. Now most of the intracellular pH regulation by a programme is full of symposia I thank Ann Silver for comments and snail neurone was inhibited by the covering work often done some corrections. + + removal of Na but not K . On the years ago, and even the voluntarily + Friday I cut up my pen recording, Thomas RC (1976). Comparison of the Na submitted material is staler than and H+ pumps in a snail neurone. J Physiol assembled the fi gure on a sheet it used to be because of the long 263, 212P–213P.

Figure 1. Left: part of my fi gure as submitted, though now rather faded. Right: the same part of the published version re-labelled by the printers (Thomas, 1976).

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Do peripheral chemoreceptors in the carotid body serve as sites of glucose sensing? The peripheral chemoreceptors in the carotid body are well known for their role in oxygen sensing and control of respiration. Recently, cellular evidence has shown that these cells are also responsive to glucose. Our recent work is supportive of carotid body involvement in glucose sensing in humans. The brain lives on oxygen, were also required for an appropriate adequate blood pressure and neuroendocrine response to exercise glucose. If any one of these three and carotid body-resected dogs essential elements for brain did not show the normal exercise- function falls below a critical level, induced increase in glucagon, unconsciousness and even death noradrenaline (norepinephrine) or can occur. The carotid arteries cortisol (Koyama et al. 2001). Given are located bilaterally in the neck these compelling findings and the and deliver oxygenated blood and Erica Wehrwein and Michael Joyner strategic location of the carotid glucose to the brain. They contain reflex so that oxygen consumption bodies, we sought to address the strategically located sensory organs is temporarily slowed and the role of the carotid bodies in glucose sensing in conscious humans. that monitor the blood oxygen level brain and heart are protected from (carotid chemoreceptors) before hypoxic damage. In humans, the Our recent work in The Journal it enters the brain and also blood carotid bodies play an important of Physiology entitled ‘Hyperoxia pressure (carotid baroreceptors). If role in oxygen sensing as evidenced blunts counterregulation during the carotid chemoreceptors sense by the loss of hypoxic ventilatory hypoglycaemia in humans: possible a drop in oxygen level, ventilation is response in carotid body-denervated role for the carotid bodies?’ focused stimulated. If a fall in blood pressure patients (Timmers et al. 2003a,b). on the interactions of oxygen and is detected by the baroreceptors, The latter two are great examples glucose sensing in the carotid there are adjustments in the of the role the carotid bodies serve bodies (Wehrwein et al. 2010). heart and blood vessels to raise in an integrative physiological Hyperoxia was used to ‘desensitize’ it. The teleological argument response of the neural, respiratory the carotid bodies since exposure is that the carotid chemo- and and vascular systems, and underpin to 100% oxygen results in a baroreceptors serve to ensure that the notion that the carotid bodies depression of minute ventilation blood is delivered to the brain at an are important homeostatic control (Downes & Lambertsen, 1966) and appropriate pressure with enough points for key physiological immediately drops chemoreceptor oxygen for neuronal survival. Do parameters. activity in the carotid bodies to zero the chemoreceptors in the carotid and initiates a brief apnoea in cats Recently cellular evidence has bodies also function to ensure an (Lahiri & DeLaney, 1975). When emerged that Type I glomus cells adequate supply of glucose to the we inactivated the carotid body in the carotid bodies are in fact brain? chemoreceptors, neuro-hormonal sensors for glucose in addition to The carotid body structure is counterregulation to hypoglycaemia their roles in sensing oxygen and was blunted (Fig. 1) suggesting: (1) conserved among vertebrates and carbon dioxide. There is also animal has also been studied extensively that the carotid bodies play a role work in support of this notion. in glucose sensing and regulation for its role in oxygen sensing. In fish, In dogs, carotid body resection in humans, and (2) there is an there are ‘glomus-like’ cells in the drastically impairs the animal’s interaction between glucose and first gill arch that serve as an oxygen ability to sense and respond to oxygen in the carotid bodies. These sensor between the ‘inhaled’ water hypoglycaemia (Koyama et al. 2000). observations are also consistent with and blood flow. However, in the case During a hypoglycaemic clamp the studies from isolated carotid glomus of fish these cells sense the external glucose infusion rate was higher in cells showing an additive effect environment and are unlikely to denervated dogs, consistent with of oxygen and glucose signalling respond to hypoxaemia and serve a the idea that endogenous glucose such that the glomus cells respond homeostatic mechanism similar to production was blunted. There to hypoglycaemia and that this that described above. In diving birds, were also blunted glucagon and response can be enhanced with there is evidence that the carotid cortisol responses to hypoglycaemia concurrent exposure to hypoxia bodies and their sensing of oxygen (Koyama et al. 2000), suggesting (Pardal & Lopez-Barneo, 2002). is related to dive duration (Milsom that the carotid bodies are integral & Burleson, 2007). Furthermore, to the neuro-hormonal response Before our study there was in diving ducks the carotid bodies to hypoglycaemia. In a study in evidence in healthy humans and mediate at least part of the dive exercising dogs, intact carotid bodies in several patient groups for a

Physiology News | No. 83 | Summer 2011 | www.physoc.org 14 PN SCIENCE NEWS AND VIEWS Figure 1

Hormone Average Area Under Curve diabetes mellitus and hypertension. Arch Med Res 37, 991–997.

100 Czupryniak L, Loba J, Pawlowski M, Nowak Hyperoxia D & Bialasiewicz P (2005). Treatment with continuous positive airway pressure may 80 affect blood glucose levels in nondiabetic ** patients with obstructive sleep apnea 60 * * syndrome. Sleep 28, 601–603. * Dorkova Z, Petrasova D, Molcanyiova A, Popovnakova M & Tkacova R (2008). Effects of 40 CPAP on cardiovascular risk profile in patients with severe obstructive sleep apnea and 20 metabolic syndrome. Chest 134, 686–692. hyperoxia vs normoxia hyperoxia Downes JJ & Lambertsen CJ (1966). Dynamic 0 characteristics of ventilatory depression in Percent hormone response during during response hormone Percent NA ADR Cortisol GH Glucagon man on abrupt administration of O. J Appl Physiol 21, 447–453. Figure 1. Counterregulatory hormone response to hypoglycaemia was blunted during carotid body deactivation under hyperoxic conditions. Area under the curve Hjalmarsen A, Aasebo U, Birkeland K, Sager G & Jorde R (1996). Impaired glucose tolerance for counterregulatory hormones was calculated during the entire 3 h hypoglycaemic in patients with chronic hypoxic pulmonary clamp: noradrenaline (NA), adrenaline (ADR), cortisol, growth hormone (GH) and disease. Diabetes Metab 22, 37–42. glucagon. Area under the curve during normoxia (carotid body active; bars not shown) was normalized to 100% and hyperoxia (carotid body deactivated; grey bars) Jakobsson P & Jorfeldt L (2006). Oxygen supplementation increases glucose tolerance is shown as a per cent of normoxia. Significance denoted as *P < 0.05 for differences during euglycaemic hyperinsulinaemic between normoxia and hyperoxia. All hormone profiles are significantly reduced glucose clamp procedure in patients with under hyperoxic conditions (carotid body desensitized). N = 7. severe COPD and chronic hypoxaemia. Clin Physiol Funct Imaging 26, 271–274. fundamental link between oxygen hypoglycaemia during treatment Koyama Y, Coker RH, Denny JC, Lacy DB, and glucose sensing. Although (Al-Waili et al. 2006). Taken together Jabbour K, Williams PE & Wasserman DH these examples have not been these observations are consistent (2001). Role of carotid bodies in control of linked to the carotid bodies, it is with the concept that oxygen and the neuroendocrine response to exercise. Am J tempting to speculate that multi- glucose homeostasis are linked, and Physiol Endocrinol Metab 281, E742–E748. modal sensing in the carotid bodies our recent publication supports the Koyama Y, Coker RH, Stone EE, Lacy DB, may be involved. Some examples notion that one site of interaction Jabbour K, Williams PE & Wasserman DH (2000). Evidence that carotid bodies play include: (1) Acute reductions is the carotid bodies. These an important role in glucoregulation in vivo. in oxygen saturation in healthy observations along with our findings Diabetes 49, 1434–1442. subjects causes glucose intolerance also have implications that extend Lahiri S & DeLaney RG (1975). Relationship (Oltmanns et al. 2004); (2) In chronic into a variety of diseases in which between carotid chemoreceptor activity obstructive pulmonary disease there is an apparent interaction of and ventilation in the cat. Respir Physiol 24, (COPD) patients, chronic hypoxia is oxygen and glucose homeostasis. 267–286. associated with impaired glucose Milsom WK & Burleson ML (2007). Peripheral tolerance while COPD patients with In summary, our findings support the arterial chemoreceptors and the evolution of cellular, animal and anecdotal clinical the carotid body. Respir Physiol Neurobiol 157, more normoxic blood gases have 4–11. normal response to oral glucose evidence that the cells of carotid Oltmanns KM, Gehring H, Rudolf S, Schultes B, tolerance testing (Hjalmarsen et al. bodies function as glucose sensors. We have shown an interaction Rook S, Schweiger U, Born J, Fehm HL & Peters 1996; Jakobsson & Jorfeldt, 2006); A (2004). Hypoxia causes glucose intolerance (3) Hypoxic COPD patients acutely between oxygen and glucose sensing in humans. Am J Respir Crit Care Med 169, placed on supplemental oxygen in the carotid bodies with systemic 1231–1237. have an immediate improvement implications. The anatomical Pardal R & Lopez-Barneo J (2002). Low in glucose tolerance and insulin location of the carotid bodies makes glucose-sensing cells in the carotid body. Nat sensitivity during euglycaemic them ideal for the strategic control Neurosci 5, 197–198. glucose clamps (Jakobsson & of what the brain needs to function Timmers HJ, Karemaker JM, Wieling W, Marres in humans and other vertebrates. HA, Folgering HT & Lenders JW (2003a). Jorfeldt, 2006); (4) Two days of Baroreflex and chemoreflex function after treatment with continuous positive bilateral carotid body tumor resection. J airway pressure improves glucose Erica A. Wehrwein and Hypertens 21, 591–599. tolerance and insulin resistance in Michael J. Joyner Timmers HJ, Wieling W, Karemaker JM & metabolic syndrome patients with Department of Anesthesiology, Lenders JW (2003b). Denervation of carotid sleep apnoea (Czupryniak et al. Mayo Clinic, Rochester, MN, USA baro- and chemoreceptors in humans. J Physiol 2005; Dorkova et al. 2008); and (5) 553, 3–11. Diabetic patients receiving insulin References Wehrwein EA, Basu R, Basu A, Curry TB, Rizza and exposed to hyperbaric oxygen Al-Waili NS, Butler GJ, Beale J, Abdullah MS, RA & Joyner MJ (2010). Hyperoxia blunts counterregulation during hypoglycaemia in for treatment of diabetic ulcers Finkelstein M, Merrow M, Rivera R, Petrillo R, Carrey Z, Lee B & Allen M (2006). Influences of humans: possible role for the carotid bodies? J frequently experience unexpected hyperbaric oxygen on blood pressure, heart Physiol 588, 4593–4601. reductions in blood glucose and/ or rate and blood glucose levels in patients with http://jp.physoc.org/content/588/22/4593.long

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A key role of orexin (hypocretin) neurons in the fight-or-flight response Stress increases cardiac function, ventilation and body temperature. These changes increase metabolic rate, oxygen supply and conduction velocity of nerve impulses and prepare the body for a fight-or-flight response. A part of the hypothalamus, called the ‘defense area’, has long been known to play a key role but the precise mechanisms are largely unknown. Our recent research suggests that orexin (hypocretin) neurons act as a master switch of the fight-or-flight response.

Research on neural mechanisms of bodies in the perifornical area (PFA) state-dependent adjustments of and dorsomedial hypothalamus central autonomic regulation has (DMH), which overlap the ‘defense been sparse, despite the importance area’, prompted us to investigate the of this event from the perspective possible role of orexin in the defense response against stressors. of quality of life. In addition to Tomoyuki Kuwaki calm and resting states, our daily Orexins (orexin-A and orexin-B), also life involves many perturbations report on the molecular basis of the known as hypocretins (hypocretin 1 that induce active conditions defense response underlying the and hypocretin 2, respectively), are such as locomotion, eating and multi-faceted nature of simultaneous recently discovered hypothalamic communication. During such active and coordinated changes in the neuropeptides (de Lecea et al. periods, cardiovascular, respiratory cardiovascular, respiratory, sensory, 1998; Sakurai et al. 1998). Although and body temperature regulation thermal and behavioural parameters. orexins were first described as needs to be adjusted according to Localization of orexin-containing cell hypothalamic neuropeptides the situational demands, which differ from those during resting states, by modulating or resetting homeostatic points (Kumada et al. 1990). One of Sleep/wake & Emotional Stress Control CardioRespiratory Homeostatic Reflex Pathway the neural substrates regulating such Biological Sleep Stressor CO2 O2 Blood adjustment appears to be located clock substance Pressure in the hypothalamus because SCN VLPO AMG/ BNST stimulation of the so-called ‘defense MR RTN LC NTS area’ in the dorsal hypothalamus LC, Orexin elicits behavioural ‘rage’ along with TMN, Neurons the specific autonomic responses; DR PBN, PBC this was termed the ‘defense response’ (Hess, 1954).

Several neurotransmitters have MLR PAG PVN Phrenic Hypoglossal RVLM been proposed to be involved in the Motoneuron Motoneuron modulation of the efferent pathways of defense responses against Arousal Locomotion Analgesia Stress BAT Diaphragm Pharyngeal Blood vessels stressors (for review see Kuwaki Hormones Dilator & Heart & Zhang, 2010). For example, the activation of serotonin (5HT)-1A Figure 1. Pivotal role of orexin neurons in linking the state-dependent behavioural receptors in the medullary raphe regulation system and cardiorespiratory homeostatic reflex pathways. Among known reduces cardiovascular changes, and connections from/to orexin neurons in the hypothalamus, selected brain nuclei that the inhibition of 5HT-3 receptors in are relevant to our study are indicated by thick lines. Many nuclei located at both the nucleus tractus solitarii prevents input (MR, RTN, LC, NTS) and output (cardiorespiratory motor neurons) interfaces in the homeostatic cardiorespiratory reflex pathway receive projections from the the baroreflex bradycardia inhibition orexin neurons (right half). Simultaneously, orexinergic connections are engaged in during the defense response. sleep–wake regulation and emotional stress-induced behavioural changes (left half). Micro­injections of adenosine into Thus, orexin can modulate cardiorespiratory homeostasis in a state-dependent and the rostral ventrolateral medulla feedforward manner. Arrows indicate a probable excitatory connection, and circles augment the increase in blood indicate an inhibitory connection. Connections shown with thin lines are either direct pressure induced by electrical or indirect. Abbreviations: AMG, amygdala; BAT, brown adipose tissue; BNST, bed stimulation of the hypothalamic nucleus of the stria terminalis; DR, dorsal raphe; LC, locus coeruleus; MLR, medullary defense area. The pros and cons locomotor region; MR, medullary raphe; NTS, nucleus tractus solitarii; PAG, periaqueductal grey; PBC, pre-Bötzinger complex; PBN, parabrachial nucleus; PVN, of glutamate participation in the paraventricular nucleus; RTN, retrotrapezoid nucleus; RVLM, rostral ventrolateral cardiovascular component of the medulla where sympathetic cardiovascular premotor neurons are located; SCN, defense response have been a topic suprachiasmatic nucleus; TMN, tuberomammillary nucleus; VLPO, ventrolateral of debate. However, there is no preoptic nucleus. From Kuwaki & Zhang (2010).

Physiology News | No. 83 | Summer 2011 | www.physoc.org 16 PN SCIENCE NEWS AND VIEWS that influenced appetite and reward process, the pain process, neuroendocrine systems (Kuwaki consciousness, it was later found and autonomic regulation of the & Zhang, 2010). Orexin-containing that orexins also modulate the cardiovascular, respiratory and cell bodies are restricted to the A

B C

Figure 2. A, effect of repeated handling stress on rectal temperature of mice of 4 genotypes. The temperature measurement (insertion of thermistor probe into the animal’s rectum) itself was used as a stressor and repeatedly applied at 10 min intervals for 2 h. Data are presented as mean ± SEM of orexin knockout mice (ORX-KO, n = 37), orexin neuron-ablated mice (ORX-AB, n = 32),

and their corresponding wild-type littermates (WTKO, n = 22 and WTAB, n = 21). B, expression of uncoupling protein (UCP)-1 in the brown adipose tissue (BAT) of stressed or naïve mice. BAT was dissected from stressed mice (4 rectal temperature measurements at 10 min intervals) and from naïve (unstressed) mice of the same genotypes. The total RNA extracted from the BAT and cDNA was reverse transcribed. UCP-1 mRNA was determined by quantitative real-time PCR in triplicate and normalized with β-actin mRNA. Data are presented as means ± SEM of 7–9 animals. Note that handling stress increased the expression of UCP-1 in wild-type and ORX-KO mice but not in ORX-AB mice. C, BAT function test. Change of BAT temperature in response to a β3- agonist, CL316243, was examined in chloralose- (75 mg kg–1, i.p.) and urethane- (750 mg kg–1, i.p.) anesthetized mice. The animals received intraperitoneal injection of saline (dashed lines) or CL316243 (1 mg kg–1, continuous lines). Data are presented as means ± SEM of the peak values during the observation period of 120 min (n = 5 for each group). These results indicate that blunted stress-induced hyperthermia in ORX-AB was caused by the loss of orexin neurons and abnormal BAT regulation and not due to an abnormality of BAT per se. From Zhang et al. (2010). *P < 0.05, **P < 0.01.

Physiology News | No. 83 | Summer 2011 | www.physoc.org SCIENCE NEWS AND VIEWS PN 17 lateral hypothalamic area (LHA), stress in orexin knockout mice a direct pharmacological stimulation PFA and DMH. On the other hand, (ORX-KO) and orexin neuron-ablated (Fig. 2C). These abnormalities orexin-containing nerve terminals mice (ORX-AB) (Kuwaki & Zhang, in ORX-AB were not observed in and receptors are widely distributed 2010). The results of the study ORX-KO, in which orexin peptide in the hypothalamus, thalamus, are summarized as follows. (1) In is defi cient but the neurons are cerebral cortex, circumventricular response to stress, both ORX-KO preserved. Therefore, the integrity organs, brainstem, cerebellum and and ORX-AB exhibited an attenuated (orexin and other co-existing spinal cord, suggesting that the defense response, including increase neurotransmitter/modulators) of orexin neurons have widespread in respiration and blood pressure the orexin neurons is indispensable connections with other regions and stress-induced analgesia. (2) for the complete expression of in the brain. Specifi cally, the Stimulation to the amygdala or multiple facets of the fi ght-or- following cardiorespiratory- BNST, both of which are implicated fl ight response (Fig. 3). Although related areas receive orexinergic in the stress-induced autonomic the concept of co-transmitters innervation: nucleus tractus responses, induced long-lasting in one neuron is well known and solitarii; pre-Bötzinger cardiorespiratory excitation in the related anatomical evidence complex; periaqueductal grey; wild-type mice but not in ORX-AB. has been frequently reported, rostral ventrolateral medulla; On the basis of these results, we the synergic/comprehensive role intermediolateral cell column of the proposed that orexin serves as a of the co-transmitters has been spinal cord; and the retrotrapezoid, central node between the state- scarcely reported. From this point hypoglossal, medullary raphe, dependent behavioural regulation of view, our report is, I believe, an parabrachial/Kölliker–Fuse and system and the cardiorespiratory exceptional one. phrenic nuclei (Fig. 1). Moreover, homeostatic refl ex pathways (Fig. 1). orexin neurons receive inputs Tomoyuki Kuwaki from the regulatory sites of the In line with the above-mentioned Department of Physiology, Graduate circadian clock, sleep–awake cycle, hypothesis, we thought that stress- School of Medical and Dental and emotional stress, such as the induced hyperthermia would also Sciences, Kagoshima University, ventrolateral preoptic area, locus be infl uenced by orexin. On the Kagoshima, Japan coeruleus, dorsal raphe, amygdala, contrary, we found that ORX-AB bed nucleus of stria terminalis but not ORX-KO mice had blunted References (BNST), and suprachiasmatic and stress-induced hyperthermia (Zhang tuberomammillary nuclei. et al. 2010) (Fig. 2A). The brown de Lecea L, Kilduff TS, Peyron C, Gao XB, adipose tissue, which is a major Foye PE, Danielson PE et al. (1998). The hypocretins: hypothalamus-specifi c peptides We have previously shown that thermogenic organ in rodents, with neuroexcitatory activity. Proc Natl Acad orexin plays an important role in did not respond to handling stress Sci U S A 95, 322–327. cardiorespiratory excitation during (Fig. 2B), although it did respond to Hess WR (1954). The Diencephalon: Autonomic and Extrapyramidal Functions. Grune & Stratton, New York. stressor Stressor Kumada M, Terui N & Kuwaki T (1990). Arterial baroreceptor refl ex: its central and peripheral neural mechanisms. Prog Neurobiol (unconscious) 35, 331–361. (conscious) Amygdala, BNST Orexin neuron Kuwaki T & Zhang W (2010). Orexin neurons ORX as arousal-associated modulators of central Cerebral cortex cardiorespiratory regulation. Resp Physiol Orexin Neurons Co-transmitters Neurobiol 174, 43–54. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Emotion Chemelli RM, Tanaka H et al. (1998). Orexins CV Resp BTPain and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled Body temperature receptors that regulate feeding behavior. Cell Fight-or-flight (=defense response) 92, 573–585. Cardiovascular, Zhang W, Sunanaga J, Takahashi Y, Mori T, Respiratory, Sakurai T, Kanmura Y & Kuwaki T (2010). Orexin neurons are indispensable for stress- Analgesic induced thermogenesis in mice. J Physiol 588, responses 4117–4129. http://jp.physoc.org/content/588/21/4117.long Figure 3. Hypothetical diagram showing the key role of orexin neurons in the fi ght-or-fl ight response. When animals confront a stressor, orexin neurons mediate Acknowledgements cardiovascular (CV), respiratory (Resp) and analgesic (Pain) responses by using orexin The work from the authors was supported by as a transmitter. At the same time, orexin neurons regulate body temperature (BT) the Grants-in Aid for Scientifi c Research from by using co-transmitters other than orexin. These responses are collectively called the Ministry of Education, Science, Culture the ‘defense response’ that prepares the body for fi ght-or-fl ight behaviour. and Sports, Japan.

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Endurance training–overtraining: a fi ne-tuned balance that depends on Ca2+ The razor-thin margin between benefi cial effects of endurance training and the deleterious effects of overtraining is a central dilemma in endurance sports. We show here that modest changes in muscle cell Ca2+ homeostasis can cause both these effects. Endurance training is of fundamental importance in all types of endurance sport activities. Moreover, endurance exercise is probably the best way to counteract the large and rapidly growing health problem referred to as the metabolic syndrome: the From left: Joseph combination of central obesity, Bruton, Håkan increased blood lipids and glucose, Westerblad and and increased blood pressure, which Niklas Ivarsson often develop into type 2 diabetes and cardiovascular disease. It is then of kinase cascades and transcription Recent studies in our laboratory natural that there are an increasing factors. Relatively less attention has were performed on cold-acclimated number of studies into mechanisms been paid to primary effectors, i.e. mice. Serendipitously, we observed by which endurance exercise factors that are directly affected by adaptations in their muscles mediates its benefi cial effects. endurance exercise and subsequently resembling both the benefi cial activate signalling pathways and effects observed with endurance Endurance exercise results in trans cription factors. One tentative training and the impaired muscle numerous adaptations in skeletal primary effector is increased free Ca2+ function which can be the result of 2+ muscle, including increased aerobic concentration in the cytosol ([Ca ] i), excessive training, i.e. overtraining 2+ capacity and improved metabolism because [Ca ]i must increase to (Aydin et al. 2008; Bruton et al. with increases in insulin sensitivity drive contractions during exercise, 2010). Both adaptations could be and fat oxidation. Studies addressing and in vitro experiments have shown explained by changes in the cellular 2+ 2+ mechanisms underlying these that increased [Ca ]i can increase Ca handling due to modifi cation bene fi cial adaptations have to a mito chondrial biogenesis in adult of the sarcoplasmic reticulum (SR) large extent been focused on cellular skeletal muscle fi bres (Wright et al. Ca2+ release channel complex, i.e. signalling events, such as activation 2007). the ryanodine receptor 1 (RyR1)

Figure 1. A, the ryanodine receptor (RyR1) complex and one of its associated proteins, FKBP12, can be modifi ed by various stressors. For instance, RyR1 can be phosphorylated (P) and this, possibly together with other modifi cations, can lead to release of FKBP12. 2+ B, minor changes of this sort are accompanied by increased [Ca ]i during tetanic stimulation in fl exor digitorum brevis (FDB) fi bres of cold-acclimated mice (mean ± SEM, n = 10–11). C, conversely, major changes in the RyR1 protein complex are accompanied by 2+ decreased tetanic [Ca ]i in shivering soleus fi bres of cold-acclimated UCP1-KO mice (n = 3–5); these mice cannot generate heat in their brown adipose tissue because they lack uncoupling protein-1 and therefore they are constantly shivering in the cold. B and C adapted from Bruton et al. (2010) and Aydin et al. (2008), respectively.

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expression of the mitochondrial transcription factor A (Tfam, required for transcription and replication of the mitochondrial genome); (iii) the cellular mitochondrial content; (iv) the capacity for fatty acid oxidation. Furthermore, FDB fibres of cold-acclimated mice were more fatigue resistant and maintained much higher forces during fatigue induced by repeated tetanic stimulation than control fibres of mice kept at room temperature (Fig. 2). Interestingly, there was no change in fibre type (i.e. myosin heavy chain isoform) composition between FDB fibres from cold-acclimated and room-temperature mice. This is consistent with results from 2+ Figure 2. The slightly increased SR Ca leak in FDB fibres of cold-acclimated mice was endurance training studies where accompanied by increased fatigue resistance. Continuous force records from fatigue major increases in endurance are induced by repeated tetanic contractions of a fibre from a room-temperature (A) and 2+ accompanied by little or no fibre a cold-acclimated (B) mouse, respectively. C and D show [Ca ]i (upper) and force (lower) from the first, tenth and last (50th) contractions of the fatigue runs. From type changes, especially not between Bruton et al. (2010). slow-twitch type I and fast-twitch type II fibres. To sum up, FDB fibres protein complex (Fig. 1). The RyR1 protein complex with some increase of cold-acclimated mice show 2+ protein complex can be modified by in the RyR1 phosphorylation and increased [Ca ]i and adaptations a wide range of stressors (Bellinger depletion of FKBP12. These changes similar to those observed with et al. 2008a), including endurance were found to be associated endurance training. This occurs exercise and exposure to a cold with increased SR Ca2+ leak and despite them performing no obvious 2+ environment. A critical component of increased [Ca ]i both at rest and endurance exercise and hence the the RyR1 modification is the release during tetanic contractions. An results indicate a key causative role 2+ of the 12 kDa FK506 binding protein increased mitochondrial biogenesis of increased [Ca ]i. (FKBP12, also called calstabin 1), has previously been shown when which results in a destabilized RyR1 caffeine was used to increase Genetically engineered mice 2+ lacking uncoupling protein-1 channel complex and increased SR [Ca ]i in muscle fibres in vitro Ca2+ leak. (Wright et al. 2007). Accordingly, (UCP1-KO) cannot generate heat FDB muscles of cold-acclimated by increasing the metabolism in Toe muscles (flexor digitorum mice displayed increases in: brown adipose tissue. When kept brevis, FDB) do not participate in (i) the protein expression of in the cold, UCP1-KO mice shiver to the shivering response induced by peroxisome proliferator-activated maintain their body temperature. exposure to a cold environment. receptor-γ coactivator-1α (PGC-1α, Soleus muscles, which participate FDB fibres of cold-acclimated mice a key regulator of mitochondrial in the shivering response, of show minor changes in the RyR1 biogenesis); (ii) the mRNA cold-acclimated UCP1-KO mice show major changes in the RyR1 protein complex, with hyperphosphorylation and a major FKBP12 depletion (Aydin et al. 2008). These changes were 2+ associated with decreased [Ca ] i during contractions (see Fig. 1C) and hence also lower forces (Fig. 3). Thus, the combination of shivering and the general stress induced by exposure to a cold environment resulted in severe changes in the Figure 3. The marked modifications of the RyR1 channel complex in soleus muscles RyR1 protein complex and impaired of cold-acclimated UCP1-KO mice were accompanied by decreased force production. Original force records from tetanic contractions (100 Hz, 1 s duration) of soleus contractile function. This resembles muscles from a cold-acclimated wildtype (A) and a UCP1-KO (B) mouse, respectively. the contractile dysfunction observed C, the force–frequency relationship in soleus muscles of cold-acclimated mice with overtraining, which is a (mean ± SEM, n = 6–7). Adapted from Aydin et al. (2008). frequent problem for the elite in

Physiology News | No. 83 | Summer 2011 | www.physoc.org 20 PN SCIENCE NEWS AND VIEWS many endurance-type sports. This The way we learn to walk notion is supported by the fact that similar changes in the RyR1 protein Learning to walk is a milestone in children’s motor skill complex accompanied by contractile development. Newly published research suggests that dysfunction have been observed emergence of oscillatory drive in the corticospinal pathway after a period of intense exercise plays an important role in developing and shaping this motor (Bellinger et al. 2008b). Furthermore, similar changes may also occur in skill. patients with disorders afflicting The world of walking muscle function where muscles have to be used closer to their maximal Parents regard the first few steps capacity even in everyday activities. taken by their child as a huge milestone in their child’s motor Another interesting aspect related to skill development. Learning to walk exercise-induced modifications of the independently usually occurs at RyR1 channel complex and increased around 1 year of age with some SR Ca2+ leak is that this would lead degree of variance. However, to accelerated energy-dependent children can be taught to walk at Jens Bo Nielsen, Ca2+ reuptake into the SR and hence a slightly earlier age by means of Tue Hvass increased heat production. Thus, motor practice. One of the most Petersen and this is likely to contribute to the striking examples comes from an Simon Farmer increase in basal metabolism and the anthropological study by Super (from left top sensation of being hot that remain (1976). He studied children from a clockwise) for prolonged periods after many particular tribe in Kenya and found types of endurance exercise. that they learned to walk slightly by African mothers – simply place earlier than American children and the legs on a surface and perhaps Niklas Ivarsson, Joseph this was likely to be due to the fact jump the infant gently up and down. D. Bruton and Håkan that these Kenyan mothers spent a You would be likely to experience Westerblad remarkable amount of time teaching the legs performing stepping-like their children how to walk by Department of Physiology and movements. These movements means of jumping-like movements Pharmacology, Karolinska Institutet, occur if the infant is younger than and supported walking practice. 171 77 Stockholm, Sweden 2–3 month of age, for reasons we Furthermore, he reported that these will touch upon later. Scientists have References children seemed to spend less of wondered which part of the nervous Aydin J, Shabalina IG, Place N, Reiken S, Zhang their waking time lying down and system generates these movements SJ, Bellinger AM, Nedergaard J, Cannon B, hence learned to crawl later than Marks AR, Bruton JD & Westerblad H (2008). since cerebral projections and hence their American counterparts (this Nonshivering thermogenesis protects against the brain’s control of the leg muscles defective calcium handling in muscle. FASEB J runs contrary to the notion of a child has not fully developed at this stage. 22, 3919–3924. needing to learn to crawl before Bellinger AM, Mongillo M & Marks AR (2008a). it learns to walk). The final motor Stressed out: the skeletal muscle ryanodine Work done by Hans Forssberg receptor as a target of stress. J Clin Invest 118, outcome of teaching children to walk (1985) showed that when very 445–453. independently a few weeks earlier young infants (0–18 months of age) Bellinger AM, Reiken S, Dura M, Murphy PW, than normal is unclear. However, perform stepping-like movements, Deng SX, Landry DW, Nieman D, Lehnart understanding the maturation and they place the foot on its forepart SE, Samaru M, Lacampagne A & Marks AR plasticity processes of the underlying (2008b). Remodeling of ryanodine receptor straight under the body. This is complex causes “leaky” channels: a molecular neural networks responsible for unlike adult human walking which mechanism for decreased exercise capacity. generating the characteristic is characterised by the foot being Proc Natl Acad Sci U S A 105, 2198–2202. pattern of gait will prove helpful in placed in front of the body with a Bruton JD, Aydin J, Yamada T, Shabalina developing rehabilitation strategies IG, Ivarsson N, Zhang SJ, Wada M, Tavi P, clear initial heel strike. In adults, the Nedergaard J, Katz A & Westerblad H (2010). for adults and children learning to soleus and gastrocnemius muscles Increased fatigue resistance linked to Ca2+- walk following damage of the central act on the ankle joint in the later stimulated mitochondrial biogenesis in muscle nervous system. parts of the stance phase to propel fibres of cold-acclimated mice. J Physiol 588, 4275–4288. the body forward and the tibialis http://jp.physoc.org/content/588/21/4275.long Infant stepping – the first few anterior (TA) muscle is responsible steps Wright DC, Geiger PC, Han DH, Jones TE & for lifting the forefoot during the Holloszy JO (2007). Calcium induces increases If you happen to have an infant in swing phase of gait to prevent it in peroxisome proliferator-activated receptor your near vicinity while reading from striking the ground. However, gamma coactivator-1α and mitochondrial this, now would be the right time biogenesis by a pathway leading to p38 when Forssberg investigated the mitogen-activated protein kinase activation. J to lift him or her and perform one individual muscle activation patterns Biol Chem 282, 18793–18799. of the regular exercises performed of the infants he found that the

Physiology News | No. 83 | Summer 2011 | www.physoc.org SCIENCE NEWS AND VIEWS PN 21 electromyographic (EMG) activity pattern generators is provided by it is acknowledged that there exists displayed less modulation than in studies such as that of Yang et al. significant spinal organisation adults through the gait cycle and (2004). In this experiment infants for walking which is increasingly that the different muscles were often stepped on a treadmill during modulated by cortical circuitry as activated in parallel rather than in which reciprocal organisation of the child grows and develops. The a sequence. He argued that these muscle activity between the two process of this ‘corticalization’ of premature stepping movements are legs was demonstrated and this lower limb control and the role of generated by pattern generators activation pattern was modulated the pattern of descending neural located in the spinal cord. Such by the speed of the treadmill, drive is only just beginning to be pattern generators can produce suggesting the presence of a spinal understood. alternating muscle activity in flexor central pattern generator that is and extensor muscles in the same modulated by proprioceptive input. Maturation of gait – the role of leg and even coordinated activity Whilst in babies there are important motor network oscillations between flexor and extensor biomechanical constraints on the Although children are able to walk muscles of both legs. PooledIn other TA-TA human EMG actual coherence output of during spinal walkingpattern and staticindependently contraction after the first few studies, indirect evidence of spinal generators during reflex stepping, years of life, the adult pattern of

Walking 4-6 yrs Static contraction A E 0.5 0.5

Pooled coherence 0 Pooled coherence 0 0 20 40 60 0 20 40 60 Frequency (Hz) Frequency (Hz) 7-9 yrs B F 0.5 0.5 Pooled coherence Pooled coherence 0 0 0 20 40 60 0 20 40 60 Frequency (Hz) Frequency (Hz) 10-12 yrs C G 0.5 0.5 Pooled coherence Pooled coherence 0 0 0 20 40 60 0 20 40 60 Frequency (Hz) Frequency (Hz) 13-15 yrs D H 0.5 0.5 Pooled coherence Pooled coherence 0 0 0 20 40 60 0 20 40 60 Frequency (Hz) Frequency (Hz)

Figure 1. Pooled coherence for the 4 groups of children during walking and static contraction. A–D, pooled TA–TA EMG coherence at frequencies from 1 to 75 Hz for the 4–6 years age group (A, n = 11), 7–9 years age group (B, n = 11), 10–12 years age group (C, n = 10) and 13–15 years age group (D, n = 10) during walking. E–H, pooled TA–TA EMG coherence at frequencies from 1 to 75 Hz for the 4–6 years age group (E, n = 9), 7–9 years age group (F, n = 7), 10–12 years age group (G, n = 8) and 13–15 years age group (H, n = 9) during static contraction. The dashed horizontal lines denoted the upper 95% confidence levels.

Physiology News | No. 83 | Summer 2011 | www.physoc.org 22 PN SCIENCE NEWS AND VIEWS gait may not be fully established corticospinal tract either within stroke, is clinically evident in the until 12 to 15 years of age. The likely the brain or the spinal cord display upper motoneurone ‘dropped foot reason for this is that different neural varying degrees of gait impairment syndrome’ where the forefoot pathways mature at different times depending on the extent and is lifted poorly during the swing during development. One important location of the lesion. The severity of phase. Activity in TA and the role part of this is the development of gait impairment seen in spinal cord of corticospinal drive is easy to the corticospinal pathway. White damage in adults suggests that once study both through examination of matter density changes in this tract connections from brain to the spinal the TMS evoked response during continue until the age of 17 and cord have developed the functional static contraction and walking and the neurophysiological properties relevance of a central pattern through study of the common drive follow the same trajectory of generator is reduced because the to TA motoneurones. Individual development i.e. there is maturation system now relies on complex motoneurones fire at frequencies of lower extremity muscle response interactions between cortical and from 8 to 13 Hz; however, their to transcranial magnetic brain spinal activity. activity is commonly modulated stimulation (TMS) (Muller et al. 1991; by the cortical oscillations at a Eluvathingal et al. 2007). The most The TA muscle motoneurones frequency of around 20 Hz (beta striking evidence of the importance receive prominent drive from the frequencies). When looking at of the corticospinal tract is the fact corticospinal pathway and damage signals from individual pools of that patients with damage to the to this system, for example, in motoneurones it is possible to

Individual coherence during walking Individual coherence during static contraction A C 0.5 r=0.26 0.7 r=0.48**

0.6 0.4 0.5

0.3 0.4

0.3 0.2

0.2 0.1 Peak beta band coherence Peak beta band coherence 0.1

0.0 0.0 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 Age (yrs) Age (yrs)

B 0.5 D 0.6 r=0.54*** r=0.56***

0.4 0.5

0.3 0.4

0.2 0.3

0.1 0.2

0.0 0.1 Peak gamma band coherence Peak gamma band coherence 0.0 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 Age (yrs) Age (yrs)

Figure 2. Individual peak coherence estimates plotted against age. Individual subjects’ peak beta and gamma band coherence in the walking experiment (A and B, respectively) and the static contraction experiment (C and D, respectively) plotted against their age. Statistical analysis showed a significant correlation between age and beta band coherence estimates for the static contraction experiment (r = 0.48, P < 0.01) but not for the walking experiment (r = 0.26, P = 0.097). Statistical analysis showed a significant correlation between age and peak gamma band coherence estimates in both the walking experiment (r = 0.54, P < 0.001) and in the static contraction experiment (r = 0.56, P < 0.001).

Physiology News | No. 83 | Summer 2011 | www.physoc.org SCIENCE NEWS AND VIEWS PN 23 calculate how the signals are band coherence increase was most References correlated in the frequency domain. obvious when comparing the Barthelemy D, Willerslev-Olsen M, Lundell H, Coherence analysis provides an youngest group of children (aged Conway BA, Knudsen H, Biering-Sorensen F estimate of the magnitude of 4–6) with children aged between 7 & Nielsen JB (2010). Impaired transmission common input to the different pools and 15 years. Interestingly, individual in the corticospinal tract and gait disability of motoneurones and during static values of peak coherence in the beta in spinal cord injured persons. J Neurophysiol 104, 1167–1176. muscle contraction coherence is and gamma bands display a linear present in the ~20 Hz beta band. correlation with age (see Fig. 2). In Eluvathingal TJ, Hasan KM, Kramer L, Fletcher JM & Ewing-Cobbs L (2007). Quantitative Beta rhythms are coherent between addition we found a relationship diffusion tensor tractography of association the EEG/MEG and the EMG, and between the strength of gamma and projection fibers in normally developing damage to the corticospinal tract band coherence and a measure of children and adolescents. Cereb Cortex 17, (see, for example, Barthelemy et the efficacy of TA activation during 2760–2768. al. 2010) reduces or abolishes the walking, suggesting that oscillations Farmer SF, Gibbs J, Halliday DM, Harrison LM, ~20 Hz coherence between pairs in this frequency range are helpful James LM, Mayston MJ & Stephens JA (2007). of motoneurones and between in stabilizing the ankle during the Changes in EMG coherence between long and the EMGs of synergistic muscles swing phase of gait. This behavioural short thumb abductor muscles during human development. J Physiol 579, 389–402. indicating that beta band coherence measure matures with increasing within and between the EMGs of age. We argued that this age-related Forssberg H (1985). Ontogeny of human locomotor control. I. Infant stepping muscles reflects the oscillatory increase in oscillations is related supported locomotion and transition to modulation of corticospinal drive to to functional maturation of the independent locomotion. Exp Brain Res 57, spinal motoneurones corticospinal network involved in 480–493. controlling the muscles during gait. We have recently asked whether James LM, Halliday DM, Stephens JA & Farmer The functional role of development SF (2008). On the development of human the ‘corticalization’ of muscle of beta and gamma oscillations still corticospinal oscillations: age-related changes control during development requires investigation, and direct in EEG–EMG coherence and cumulant. Eur J can be reflected as an increase recordings of EEG–EMG coherence Neurosci 27, 3369–3379. in the strength of the ~20 Hz during the development of gait Muller K, Homberg V & Lenard HG (1991). common drive to motoneurones. A remain to be performed. Magnetic stimulation of motor cortex and developmental increase was recently nerve roots in children. Maturation of cortico- demonstrated for EMG–EMG and Gamma band coherent oscillations motoneuronal projections. Electroencephalogr Clin Neurophysiol 81, 63–70. EEG–EMG coherence in upper are important in processes requiring limb muscles (Farmer et al. 2007; attention and prediction. Beta band Petersen TH, Kliim-Due M, Farmer SF & James et al. 2008). In a new set of coherence oscillations are important Nielsen JB (2010). Childhood development of common drive to a human leg muscle during experiments (Petersen et al. 2010), for maintenance of a motor state. ankle dorsiflexion and gait. J Physiol 588, we investigated the development The findings that coherent oscillatory 4387–4400. of oscillations in the corticospinal drive to TA muscle in beta and http://jp.physoc.org/content/588/22/4387.long pathway during human treadmill gamma ranges changes significantly Super CE (1976). Environmental effects on walking in forty-two children of across childhood leads us to motor development: The case of African infant different ages. Firstly we showed speculate that these frequencies play precocity. Dev Med Child Neurol 18, 561–567. an age-dependent increase in the a crucial role in motor control. Their Yang JF, LamT, Pang MY, Lamont E, ~20 Hz oscillatory input to different emergence correlates with the onset Musselman K & Seinen E (2004). Infant pools of TA motoneurones during of the adult pattern of walking in stepping: a window to the behaviour of the steady muscle contraction (a result which the interaction between spinal human pattern generator for walking. Can J Physiol Pharmacol 82, 662–674. analogous to that described for the and cortical circuits becomes more upper limb) (see Fig. 1E–H). More fixed. Seeing how this oscillatory importantly we found strong age drive changes more precisely at the Physiology News effects for activation of TA muscle crucial transition points of motor during treadmill walking. These development and their relation to If you have enjoyed this issue of effects were detected for oscillatory learning and plasticity will be an Physiology News please don’t throw drive at higher frequencies – the interesting topic for further research. it away. Put it in your coffee room 35-40 Hz gamma band which in 1,2 so that others may see it too. a number of studies has been Tue Hvass Petersen , Simon associated with important motor F. Farmer2 and Jens Bo We are always looking for cortex network functions and which Nielsen1 interesting features, meeting can also modulate motoneurone 1Institute of Exercise & Sports reports, news items and firing. In contrast to static muscle Sciences, University of Copenhagen, photographs. contraction in which beta band Copenhagen, Denmark coherence dominates, during Contact us ([email protected]) walking the gamma band coherence 2Institute of Neurology, University with your suggestions. dominates (Fig. 1A–D). The gamma College London, UK

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CO2 emissions of locomotion: innovative automobiles do better than humans Four male friends plan to support it to power the next take-off, again their favourite soccer team at the reducing the work muscles have to stadium located at the opposite provide (Fig. 1B). side of the town. Would they emit Thus, both walking and running more CO2 to reach their destination by jogging or by boarding a hybrid dynamics incorporate a sort of car and driving there? Despite the regenerative braking, the same strategy of the most modern hybrid character of both human and Alberto Minetti and Gaspare Pavei innovative automobile locomotion, fuel-saving cars. modern vehicle technology allows single-step kinetic energy increases Further analogies include the transportation of four passengers (in and decreases, respectively (Fig. 1A), efficiency of muscles (Woledge et town) with lower CO emissions than 2 which can occur at no extra cost al. 1985) and internal combustion if they were running. with respect to the same ‘constant’ engines (Vogel, 1998), about speed, equal-strides walking (Minetti 25–28%, which makes the Hybrid locomotion in biology and et al. 2001 ). This seems to happen production of heat and the related in mechanical engineering for controlled oscillating cycles of water vapour (the most relevant Hybrid vehicles use different energy 6 seconds, up to ±40% sinusoidal determinant of the greenhouse forms to move around. Petrol-based change of walking speed. effect) similar in the two ‘motors’. cars accelerate mainly by converting Human running (and legged Tendons and electric motors, namely fuel combustion into mechanical the hybrid parts in the two ‘vehicles’, work. During the inevitable terrestrial animals trotting/ galloping) uses another strategy to also share similar efficiencies (95 and braking frequently occurring in 88%, respectively). town, though, most of the car keep CO2 emission low. During the kinetic speed is not ‘traditionally’ first half of the contact phase, part of From the metabolic cost of dissipated as heat, but it enters the inevitable mechanical potential transport to CO emission the battery after being converted and kinetic energy decrease is 2 by an electric motor. That motor temporarily stored into elastic The carbon dioxide emission of is successively used to convert structures, tendons, which release a ‘standard’ male runner can be electrical energy back into motion thus, human locomotion and hybrid A car technology have something in common.

During walking at constant average speed, humans (and other bipeds/ quadrupeds) behave like inverted pendula. The inherent constraint with the ground, causing inevitable B raising–lowering and acceleration– deceleration of the body centre of mass within every stride, makes the pendulum an effective strategy to continuously exchange (part of the) body potential energy with kinetic energy. This maintains almost 60% of mechanical energy inside the body, and leaves the remaining 40% to be supplied by the propelling muscles Figure 1. A, a single walking stride is here represented as a raise followed by a (Cavagna et al. 1963). decrease of the total mechanical energy of the body centre of mass. In a series of Also, the walking cost of transport consecutive ‘constant’ speed strides the total energy changes are mechanically (and metabolically) equivalent to longer accelerative/decelerative sequences, as illustrated shows low sensitivity to greater by adding all the positive and the negative changes together. B, in running, part of inter-strides speed changes. The the mechanical energy of the body centre of mass (on the ordinate) during landing mechanics of more ample, multi- is stored, (almost) free of charge, into the elastic proteins of tendons (dashed curve), stride accelerative–decelerative for it to be released as kinetic + potential energy before taking off. This corresponds oscillations can be seen as the to a sort of regenerative braking of human locomotion, very similarly to modern cumulative, separate sums of hybrid cars.

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calculated by knowing his metabolic CO2 emission per unit distance in conclude that running was still cost of transport. Experimental fuel-saving automobiles marginally less polluting than driving –1 measures (di Prampero, 1986) The most fuel-effi cient car available (he used the value of 150 g CO2 km and theoretical models (Minetti, at present produces about 87 g for cars moving at 100 km h–1 on a 2004) analysing world records –1 motorway). It seems a ‘physiological’ CO2 km , but the reported value in the distance range of 1 km to refers: (1) to a ‘combined cycle’, coincidence that one of the EU’s the marathon (42.2 km), usually which is less expensive than the goals in terms of containment of achieved by athletes of about 65 kg ‘urban cycle’, and (2) to transporting pollution is to have automobiles mass, lead to a value of about 270 J a load of 180 kg, corresponding to producing just below ECO2R (95 g –1 per metre travelled, independent 2.4 passengers. When those data CO2 km ) by the year 2020. of the running speed. This has are corrected according to ‘urban’ been inferred (Minetti, 2004) by cycle and 4 passengers, only hybrid Two other interesting limits could be approached in the future, namely assuming a maximum aerobic car emission is lower than ECO2R metabolic power of 1.9 kW and (i.e. the 4 runners, see Fig. 2). Their four walkers and four cyclists, who realistic values of anaerobic energy superior economy with respect to are expected to aerobically produce –1 –1 stores (21 kJ). When we correct competitors (e.g. diesel vehicles) is 50 g CO2 km (ECO2W, at 5.5 km h ) –1 the cost of running according to a based on the very low correction and 25 g CO2 km (ECO2B, at –1 mass of 75 kg, with a respiratory factor for the ‘urban’ cycle, since 30 km h on race bikes), respectively quotient of 0.9 (corresponding to the regenerative brakes and the (Fig. 2). It is likely that at least 80% of the maximum metabolic ‘Stop&Go’ expedient make hybrid ECO2W is within reach by considering power sustainable for 2–3 hours) car economy less sensitive to the that, at the Shell Eco-Marathon and, consequently, a unit conversion frequent speed changes occurring 2010 (www.shell.com/home/ content/ecomarathon/europe/), of 20.6 J = 1 ml O2, the production in town. Today there are about a of carbon dioxide is calculated as dozen models on the market and for an UrbanConcept petrol-driven car –1 13.7 ml CO2 m . By considering that the most fuel saving of these, the designed at the Lycee Louis Delage, –1 Cognac, France, reached a distance 1 mol CO2 (= 44 g) occupies 24,056 emission is about 95 g CO2 km . ml at standard barometric pressure of 303 km with 1 litre of fuel (31 g –1 and at an ambient temperature of Humans vs. automobiles CO2 km when calculated for 4 20ºC, the emission of a ‘standard’ It seems that we have hit a wall: four one-passenger vehicles). In the same –1 male runner is 25.1 g CO2 km . In passengers in a hybrid car produce event the Polyjoule team from Polytech Nantes University achieved order to compare human to car less CO2 per unit distance travelled in emission we need to consider four town than if they all were running. an economy record of 4,896 km –1 runners, a total of 100 g CO2 km (a A similar rationale brought David travelled with (the equivalent energy reference value we named ECO2R). Miller (2008) in this magazine to from their hydrogen fuel cell) 1 litre of gasoline, corresponding to about CO emissions –1 2 2.8 g CO2 km (calculated for 4 one-passenger vehicles).

Despite all the strategies mentioned to contain the metabolic cost of human locomotion, wheels are wheels are wheels. Our mode of

–1 being in contact with the ground km

2 forces continuous raise–descent, acceleration–deceleration sequences g CO of the body centre of mass, inherently requiring a signifi cant amount of work, and thus quite a high metabolic energy. However, even after having invented and refi ned the bicycle, which avoids all those troubles, we are now beaten by (prototypal) technology 9 to 1.

We have mentioned only males, so far, for a reason. The lower average Figure 2. Bars represent CO2 emission per unit distance of the most fuel-saving cars in each engine category, during urban use and full occupancy (4 passengers). body mass (~10 kg) in women Horizontal lines show the emission of 4 runners (at whichever aerobic speed), decreases the load factor of cars 4 walkers (at 5.5 km h–1) and 4 cyclists on race bikes (at the constant speed of less than the metabolic cost of 30 km h–1). 52 commercial vehicles have been included in the analysis. running. Four ‘standard’ females

Physiology News | No. 83 | Summer 2011 | www.physoc.org 26 PN SCIENCE NEWS AND VIEWS still produce during running less A novel feature of epithelial acetylcholine in CO2 than a four-passenger car, independently of the automotive intestinal luminal sensing technology. However, the same Textbooks describe acetylcholine (ACh) as a neurotransmitter ‘green’ attitude of four males in a hybrid car is obtained by extending that is released from cholinergic motor nerves in response to the female group to fi ve (passengers luminal stimuli to control gut motility and ion transport. In and runners). a new approach we demonstrated that ACh is synthesised in colonic epithelial cells and released on the basolateral side by Conclusions luminal chemo-stimulation, which causes chloride secretion. Vehicle science is currently facing a turning point where automobiles, despite their large size, reaching a transport economy challenging our natural gaits (electric-only vehicles already beat ECO2W, although probably due to the lower cost of producing electricity). Arguments against these considerations include Ryo Inoue air pollution and the non-renewable From left, Megumi Matsumoto, Takaji Yajima and Masako Yajima From left: Megumi character of fossil fuels, when Matsumoto, Takaji compared to foodstuff. However, Yajima, Masako Yajima the cost of processing and delivering and Ryo Inoue. foodstuff should also be considered: for the same amount of provided energy, olive oil, for example, ends From left, Megumi Matsumoto, Takaji Yajima and Masako Yajima Ryo Inoue up being much more expensive Acetylcholine (ACh) is the best mechanical and chemical signals (200–300%) than automotive fuels. characterised neurotransmitter from the luminal side of the

1 and is synthesised and stored in intestine (Raybould et al. 2004). Alberto E. Minetti and cholinergic neurons in the brain and Stimulating muscarinic ACh Gaspare Pavei2 gut wall. ACh synthesis is catalysed receptors induces mucosal hydration 1Department of Human Physiology, by choline acetyltransferase (ChAT) due to chloride secretion from the University of Milano, Milan, Italy via the enzymatic conversion of crypt cells and inhibition of sodium acetyl-CoA and choline to ACh and absorption from the apical cells, and 2Faculty of Sport Science, University CoA. Choline, the limiting substrate stimulates mucus secretion from of Milano, Milan, Italy for ACh synthesis, is taken up by the goblet cells. Thus, intestinal neurons by the high-affi nity choline cholinergic regulation plays a role References transporter 1 (CHT1). Synthesised in providing surface lubrication to Cavagna GA, Saibene F & Margaria R ACh is stored in synaptic vesicles propel luminal contents aborally or (1963). External work in walking. J Appl by the vesicular acetylcholine for fl ushing out harmful microbes Physiol 18, 1–9. transporter, and is released from and toxins. di Prampero PE (1986). The energy cost nerve terminals by membrane of human locomotion on land and in action potentials in response to It is also noteworthy that ACh is water. Int J Sports Med 7, 55–72. physiological and pharmacological synthesised and stored in a broad Miller D (2008). Jogging and global stimuli. variety of non-neuronal cells, warming, or the Nike-Adidas CO2 particularly in surface epithelia of footprint. Physiology News 70, 53. The gut wall is richly innervated the airway, bladder, placenta and Minetti AE (2004). Passive tools for with cholinergic neurons, and ACh skin of rodents and humans (Wessler enhancing muscle-driven motion and is a predominant neurotransmitter & Kirkpatrick, 2008). Synthesis and locomotion. J Exp Biol 207, 1265–1272. of the enteric nervous system storage of ACh in non-neuronal cells Minetti AE, Ardigò LP, Capodaglio (Harrington et al. 2010). When has been confi rmed not only by EM & Saibene F (2001). Energetics the cholinergic motor neurons are positive anti-ChAT immunoreactivity and mechanics of human walking at activated via an enteric refl ex by but also by direct measurements oscillating speeds. Am Zool 41, 205–210. luminal stimuli (Fig. 1A), ACh is with high performance liquid Vogel S (1998). Cats’ Paws and Catapults. released at the neuro-enterocyte chromatography combined with Penguin Books Ltd, London. junction. Epithelial cells express a post column enzyme reactor Woledge RC, Curtin NA & Homsher many subtypes of muscarinic ACh and an electrochemical detector. E (1985). Energetic Aspects of Muscle receptors, which are involved in ACh is not stored in vesicles of Contraction. Academic Press, London. the refl ex secretory response to non-neuronal cells and its release

Physiology News | No. 83 | Summer 2011 | www.physoc.org Figure 1 SCIENCE NEWS AND VIEWS PN 27

A B Chemo-stimulation Chemo-stimulation Cl- Cl- Cl- Cl-

ACh x x x M M M x M M ACh ACh ACh Ｘ ACh

M Muscarinic ACh receptor

Submucosal plexus

Figure 1. Schematic diagram showing the neuronal and non-neuronal mechanisms of the cholinergic control of chloride secretion. A, in the neuronal mechanism acetylcholine (ACh) is released from cholinergic motor nerve terminals projecting beneath epithelial cells in response to luminal chemical stimulation via mucosal sensing and a signal substance (X) from an endocrine cell followed by a neuronal enteric refl ex. The X, otherwise, depolarises the nerve terminal and releases ACh. B, novel cholinergic control of chloride secretion is mediated by ACh release from epithelial cells in response to luminal chemical stimulation. ACh released at the basolateral side stimulates muscarinic receptors on neighbouring cells through a paracrine action of ACh.

is mediated by organic cation However, little progress has been (SCFAs) are major colonic microbial transporters (OCTs). Therefore, ACh made regarding the physiological fermentation products and stimulate Figure 2 release from non-neuronal cells is signifi cance of the synthesis and chloride secretion in the rat distal much slower than that from nerve release of non-neuronal ACh into the colon in vitro (Yajima, 1988). terminals. Non-neuronal ACh also intestine. SCFA-stimulated chloride secretion occurs in the epithelial cells of the is induced even under a nerve block small and large intestines of rats Previous studies have demonstrated by tetrodotoxin but is inhibited and humans (Klapproth et al. 1997). that luminal short chain fatty acids by atropine, a strong cholinergic receptor antagonist. Therefore, ACh may be released from cholinergic 3001200 ) 1200 -1 nerve terminals depolarised by an

) unknown signal substance (Fig. 1A) -2 200800 or released non-neuronally from 800 colonic epithelial cells (Yajima, A cm µ

( 1988; Hubel & Russ, 1993). In a 100400 5 min tissue sc I 400 -1 recent study reported in The Journal ACh release ACh of Physiology, we addressed the 00 involvement of non-neuronal ACh

0 0 (pmol g 5 in the secretory response to SCFA in 10 15 rat colon (Yajima et al. 2011). Using 20 25 epithelial sheets mounted on Ussing chambers that did not include the Figure 2. Acetylcholine (ACh) release induced by the luminal short chain fatty acid, enteric nerve plexuses, we showed propionate, in epithelial sheets, without the enteric nerves, mounted on Ussing that the luminal stimulation by chambers in the presence of tetrodotoxin and the acetylcholinesterase inhibitor, the SCFA, propionate, caused ACh eserine. Red line shows the secretory response, which is shown by the changes in release into the serosal side (Fig. 2). short-circuit current (Isc) plotted as mean value per minute. Blue bars indicate ACh release into the serosal fl uid of the Ussing chambers, which is plotted as mean value A signifi cant linear relationship was per 5 min. Propionate (1 mM) was added to the mucosal fl uid 10 min after starting observed between ACh release and the experiment. chloride secretion, as indicated by

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as local signalling for the muscarinic 2.5 receptors of neighbouring epithelial cells following chloride secretion as )

-1 2 well as mucin secretion (Fig. 1B). However, many questions arise, such as what kind of cells store ACh, how 1.5 is the chemo-signal received, how is

tissue 5 min ACh released from the stored cells,

-1 1 and what other physiological roles g ACh release ACh does non-neuronally released ACh 0.5 r = 0.698 play in addition to the secretory ( nmol p<0.003 response? We expect future studies 0 to answer these questions. 0 50 100 150 200 250 300 350 400 1 2 -2 Takaji Yajima , Ryo Inoue , ∆Isc (µA cm ) Megumi Matsumoto1 and 1 Figure 3. Relationship between acetylcholine (ACh) release and secretory response Masako Yajima

(indicated by changes in short-circuit current (Isc) induced by luminal short-chain fatty acid, 1 mM propionate. A linear regression analysis was performed based on the 1Meiji Dairies Research Chair, difference between the amount of ACh released in 5 min and the maximal increase in Creative Research Institution, Isc induced by luminal propionate. Hokkaido University, Sapporo, Japan

2Laboratory of Animal Science, Kyoto changes in short-circuit current the first time in human placenta. (Fig. 3), suggesting that the release Interestingly, the amount of ACh Prefectural University, Kyoto, Japan of ACh occurred non-neuronally and released from human placenta then caused chloride secretion by is comparable to that observed References stimulating epithelial muscarinic during propionate-induced ACh Harrington AM, Hutson JM & Southwell BR receptors. These results were release from rat colonic epithelial (2010). Cholinergic neurotransmission and confirmed in an experiment cells. However, ACh release muscarinic receptors in the enteric nervous showing that a muscarinic receptor occurred spontaneously in the system. Prog Histochem Cytochem 44, antagonist and an inhibitor of placenta epithelial cells without 173–202. chloride secretion inhibited chloride any stimulation, which was not Hubel KA & Russ L (1993). Mechanisms of the secretion but not ACh release. the case in colonic epithelial cells. secretory response to luminal propionate in To address the physiological role rat descending colon in vitro. J Auton Nerv Syst To confirm the non-neuronal 43, 219–229. of non-neuronal ACh release from origin of ACh release in response epithelium, it is important to Klapproth H, Reinheimer T, Metzen J, Munch to luminal propionate stimulation, understand whether ACh is released M, Bittinger F, Kirkpatrick CJ, Hohle KD, we employed a method of crypt Schemann M, Racke K & Wessler I (1997). into the luminal or serosal side. isolation from colonic segments to Non-neuronal acetylcholine, a signalling However, previous studies have not obtain neural components free of molecule synthezised by surface cells of revealed the direction of ACh release rat and man. Naunyn Schmiedebergs Arch epithelial cells. We demonstrated from epithelial cells of the placenta Pharmacol 355, 515–523. the ChAT gene mRNA expression as or bladder. Our study clearly Raybould HE, Cooke HJ & Christofi FL well as the storage of a considerable demonstrated that the ACh release (2004). Sensory mechanisms: transmitters, amount of ACh compared to residual coupled with luminal propionate modulators and reflexes. Neurogastroenterol muscle tissues including the enteric Motil 16 (Suppl 1), 60–63. stimulation occurred on the serosal nerves. Gene expression of OCTs Wessler I & Kirkpatrick CJ (2008). but not CHT1 indicated that colonic side, but not the luminal side in the rat colon. Acetylcholine beyond neurons: the non- epithelial cells use OCTs rather than neuronal cholinergic system in humans. Br J CHT1 to take up choline. Although Pharmacol 154, 1558–1571. Non-neuronal ACh is expected to colonic epithelial cells store ACh, act as a local signalling or trophic Yajima T (1988). Luminal propionate-induced the amount is approximately three secretory response in the rat distal colon in molecule (Klapproth et al. 1997). orders of magnitude less than that vitro. J Physiol 403, 559–575. Our study demonstrated that in neuronal tissues, rat brain and the cholinergic colonic ion transport is Yajima T, Inoue R, Matsumoto M & Yajima myenteric plexus of the distal colon M (2011). Non-neuronal release of ACh regulated not only by neuronal but of guinea-pig. plays a key role in secretory response to also by non-neuronal mechanisms. luminal propionate in rat colon. J Physiol 589, ACh release from non-neuronal Non-neuronally released ACh in 953–962. epithelial cells was reported for response to luminal stimulation acts http://jp.physoc.org/content/589/4/953.long

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Surviving life in the womb and the implications for vascular health in adulthood The early environment of the fetus can shape its risk for cardiovascular disease in adulthood. Females, by and large, fare better than males following these early life insults. However, changes in the function of the uterine vessels may predispose to pregnancy complications and perpetuation of cardiovascular disease risk in the next generation.

The womb is not necessarily a cosy and nurturing environment, as one might expect. Although the fetus may be endowed with genes bestowing physiological fortitude and resilience, the environment in the womb can sculpt the physiological phenotype and ultimately infl uence the risk of disease. The early studies by McCance and Widdowson in the 1960s–1970s laid the scientifi c foundations that life in utero can infl uence, for better or for worse, the ability of the offspring to achieve its full genetic potential after birth (McCance & Widdowson, 1974; Bateson, 2001). In the 1980s the seminal work of Barker and colleagues linked slowed human fetal growth to an increased risk of cardiovascular and metabolic disease From top left clockwise: Marianne Tare, Marc Mazzuca, Helena Parkington, Nicoleta in adulthood (Barker, 1995), and Dragomir and Mary Wlodek. this was the catalyst for an intensive research focus into what is now known as the ‘developmental origins quantity of nutrition and oxygen, maternal nutrition, hormonal status of health and disease’ hypothesis. together with internal cues arising of the mother, stress, placental from the mother such as hormones, function and oxygen delivery to Life in the womb can be a struggle, nutrients and the function of the the fetus have implications for especially when faced with an placenta, can induce developmental vascular structure and function and inhospitable environment. In and functional adaptations in the blood pressure in the offspring. response to adverse conditions, fetus. Myriad epidemiological and The wealth of data emerging from the fetus is able to modify its experimental studies indicate that experimental studies indicates that, development to optimise its survival events occurring around the time of more often than not, there are sex in the womb. This developmental conception, through to pregnancy differences in the expression and plasticity of the fetus enables and early postnatal life, can sculpt severity of vascular dysfunction preservation of life in the short the phenotype of the offspring. and blood pressure (Grigore et al. term, but may have unfavourable 2008). Female offspring tend to consequences for long-term health Cardiovascular disease is one of the fare better than males in response in postnatal life. During fetal life, leading causes of death worldwide. to an early life insult. Furthermore, the organs, vasculature and body Apart from genetic predisposition the raised blood pressure in females systems undergo ‘critical periods’ of and traditional risk factors such seen in some models is resolved development. Disturbances to the as obesity, high blood pressure after puberty. Whether there are environment during these sensitive and smoking, it is now recognised differences in the ‘critical periods’ periods can induce permanent that the quality of very early life is of development in utero or the changes in the structure, function an important factor that can also ‘sensitivity’ to the insult for males and physiological set-points of infl uence cardiovascular disease risk. and females has yet to be elucidated. organs and body systems. External Research into the developmental However, sex hormones appear environmental cues sensed by the origins of health and disease has to modulate the expression of the mother and transduced to the fetus demonstrated that factors such cardiovascular phenotype developed via the placenta, such as quality and as the quality and quantity of in response to early life insults.

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A Weight: postnatal day 1 mediated by endothelium-derived hyperpolarizing factor (EDHF) 4 was impaired (Fig. 2) (Mazzuca et al. 2010). EDHF is an important 3 vasodilator mechanism in small g 2 arteries and arterioles. The stiffness of the uterine artery wall was also 1 Male offspring Control increased in growth-restricted 0 Restricted females and this was associated with an increased proportion B Mean arterial pressure Female offspring Control of thick, less compliant collagen 150 Restricted fibres (Fig. 3). Importantly, vascular dysfunction was localised to the 100 uterine artery and was not evident in the major resistance beds as in the mmHg 50 males, and this probably contributes to the lack of hypertension in the 0 females.

Figure 1. Effect of late gestation uteroplacental insufficiency on offspring weight at Upregulation of endothelial postnatal day 1 (A) and mean arterial pressure (B) in male and female rat offspring. vasodilator factors in the uterine A, body weight (g) at postnatal day 1 in male (n = 9 per group) and female (n = 10 artery, including EDHF, is part of per group) control and restricted offspring. B, mean arterial pressure in 5-month- the natural adaptation that takes old males (n = 7 per group) and 18-month-old females (n = 5 per group). Values are place in this vascular bed in response mean ± SEM. *P < 0.05 between control and restricted offspring. to pregnancy, to ensure adequate perfusion of the fetus. Inappropriate Uteroplacental insufficiency rather stiffening of the arteries, risk adaptation of the uterine vasculature than maternal undernutrition is factors for cardiovascular disease in pregnancy is associated with a common cause of intrauterine (Norman, 2008). In our recent study compromised uteroplacental growth restriction in Western we used a model of late gestation societies. Endothelial and smooth uteroplacental insufficiency by muscle function and arterial bilateral uterine artery and vein A Control Restricted mechanical properties are important ligation in rats. This reduces the regulators of vascular function. supply of nutrients and oxygen Humans of low birth weight tend to the fetus. This model results in to have impaired endothelial fetal growth restriction of 10–15% vasodilator function and increased (Fig. 1A) without the confounding influence of maternal hypertension. B 100 Uterine artery We examined the effect on blood Control Total pressure and vascular function in 75 Restricted 0 Control Restricted adult female offspring. Previous 50

EDHF studies of this model in our 25 50 Control

laboratory demonstrated that adult (%) proportion Restricted 0 collagenadventitia

% Relaxation % male growth-restricted offspring

100 had raised blood pressure, reduced red orange yellow green 10 10 -9 10 -8 10 -7 10 -6 10 -5 nephron endowment, reduced thick fibres thin fibres ACh [M] vasodilator capacity and increased Figure 3. Thick and thin collagen fibres Figure 2. Dysfunction of endothelium- arterial stiffness. The adult growth- are more abundant in the adventitia derived relaxing factor (EDHF)-mediated restricted females also had a reduced of uterine arteries from adult growth- relaxation in uterine arteries of adult nephron endowment, but were restricted females. A, sections of growth-restricted females. Total: found to be normotensive (Fig. 1B) uterine artery stained with picrosirius endothelium-dependent relaxation and had normal vascular function red and viewed under cross-polarized evoked by acetylcholine (ACh) in the in the major resistance beds. The light to reveal birefringent collagen absence of blockers. The response to low nephron number alone was fibre thickness. B, proportion of thick EDHF was revealed after blockade of not sufficient to cause an elevation (red, orange, yellow) and thin (green) nitric oxide and prostanoid production. fibres in the adventitia of arteries from Values are mean ± SEM, n = 5 per group. in blood pressure. However, closer control and restricted female offspring. *P < 0.05 between control and restricted investigation revealed that there Values are mean ± SEM, n = 4 per group. offspring for maximal EDHF-mediated was dysfunction in the uterine *P < 0.05 between control and restricted relaxation. (Taken from Mazzuca et al. artery of growth-restricted females. offspring. Scale bar, 100 µm. (Taken 2010.) Endothelial vasodilator function from Mazzuca et al. 2010.)

Physiology News | No. 83 | Summer 2011 | www.physoc.org SCIENCE NEWS AND VIEWS/TECHNIQUES PN 31 blood flow, growth restriction and Western blotting. pregnancy complications including maternal hypertension. Indeed, Part III emerging evidence in the literature In the last of this series, Patricia in humans and from our own Leoni discusses the process of laboratory studies in rats indicates transferring electropheresed that females born with low birth proteins to a membrane (the weights may have a higher risk of Patricia Leoni complications during pregnancy. 'blot') and the use of antibodies to identify proteins of interest Research in the developmental from the many that present on adapted by Towbin et al. (1) from the origins of health and disease area the membrane. technique developed by Southern (2) has drawn attention to the critical The separation of proteins by for the transfer of DNA (Table 1). role of early life environments in electrophoresis on polyacrylamide determining the cardiovascular gels does not provide any It should be noted that while for and metabolic phenotype. It has information on their identity. SDS-PAGE electrophoresis standard also highlighted the complexity The proteins can be recovered conditions recommended by of the cardiovascular phenotype, from the gel for chemical equipment manufacturers are quite and the reality that truly effective characterization using a combination adequate for most protein extracts, interventions in adults may need to of liquid chromatography and Western blotting can require be tailored according to the nature mass spectrometry. However, careful optimization. For proteins of the early life insult. this is a costly and laborious of medium to high abundance technique useful mostly when and not too low molecular weight, 1 Marianne Tare , Marc trying to identify proteins that are almost any condition will work, Q. Mazzuca2, Helena C. completely unknown or for accurate while proteins of low abundance Parkington1, Nicoleta quantification. and/or low or very high molecular 3,4 weight require much more rigorous Dragomir and Mary E. The use of antibodies provides a empirical testing. Wlodek2 very sensitive tool to identify the presence and relative proportion of Equipment 1Department of Physiology, Monash specific proteins but the detection of A wide variety of electroblotting University, Victoria, Australia proteins directly in the gel presents equipment is commercially 2Department of Physiology and several problems due to diffusion. available, but there are only two 3School of Physics, The University of In order to overcome these, basic designs. In both, the transfer Melbourne, Victoria, Australia proteins can be electrophoretically occurs by applying an electric field transferred from the gel to a perpendicular to the plane of the gel. 4School of Biomedical and Health thin membrane, where they are The main difference between them Sciences, Victoria University, immobilized. This procedure, known is the cost of the equipment and the Victoria, Australia as Western blotting, has been time required for the transfer.

Table 1 References Bateson P (2001). Fetal experience and good Steps involved in Western blotting Conditions to be optimised adult design. Int J Epidem 30, 928–934. Polyacrylamide gel with separated Barker DJP (1995). Fetal origins of coronary proteins heart disease. BMJ 311, 171–174. Equilibration of gel Choice of transfer buffer, methanol and Grigore D, Ojeda NB & Alexander BT (2008). SDS content Sex differences in the fetal programming of hypertension. Gend Med 5 (Suppl A), S121– Membrane staining Choice of reversible stain S132. Electrophoretic transfer to membrane Type of equipment and membrane, McCance RA & Widdowson EM (1974). The transfer conditions determinants of growth and form. Proc R Soc Lond B Biol Sci 185, 1–17. Blocking of free binding sites Choice of blocking agent, concentration of Tween-20 Mazzuca MQ, Wlodek ME, Dragomir NM, Parkington HC, Tare M (2010). Uteroplacental Incubation with primary antibody Antibody dilution, time and insufficiency programs regional vascular temperature of binding dysfunction and alters arterial stiffness in Washes Number and length of washes female offspring. J Physiol 588, 1997–2010. http://jp.physoc.org/content/588/11/1997.long Incubation with secondary antibody Manufacturer’s recommendation Norman M (2008). Low birth weight and the Washes Number and length of washes developing vascular tree: a systematic review. Acta Paediatr 97, 1165–1172. Detection Choice of substrate

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electrodes the membrane. Traditionally they + consist of a buffer of pH higher than the isoelectric point of most of the proteins in the extract, and gel methanol. The most common membrane formulations are: 25 mM Tris, 192 mM glycine, methanol 10–20% filter paper v/v, pH 8.3 and 48 mM Tris, 39 mM glycine, methanol 10–20% v/v, pH 9.2. Transfer cassettecasette Methanol in a concentration of 10 to 20% stabilizes the dimension of the gel and strips SDS from the protein molecules. Figure 1. Diagram of a vertical buffer tank. Gels swell during the run and this Vertical buffer tanks nitrocellulose and polyvinylidene could affect the resolution of the The polyacrylamide gel containing fluoride (PVDF). Both membranes proteins. As swelling depends on the separated proteins is placed are available with pore sizes of 0.2 polyacrylamide concentration, next to a membrane in a cassette and 0.45 μm. The larger pore size is gradient gels expand unequally, and suspended in a tank containing used for most transfers, while the acquiring a trapezoid shape. buffer between two electrodes 0.2 μm pore is more efficient for low (Fig. 1). molecular weight proteins. On the other hand, proteins of high molecular weight can have As the electrodes are several Proteins are thought to attach to limited solubility in the presence of centimetres apart the voltage the membranes by a combination methanol. If the protein of interest is gradient that can be applied should of hydrophobic and electrostatic more than 100 kD, the concentration not exceed 5 V cm–1 to avoid interaction. The binding capacity of methanol should be lowered to 5% overheating; refrigeration is needed of nitrocellulose is 80–100 μg cm2 or even removed completely. when applying currents of 200 mA while PVDF membranes can bind or more. 100–200 μg cm2 and have superior For routine blotting, excess SDS mechanical strength, chemical should be removed from the gel Semi-dry blotting systems resistance and protein detection. by equilibrating it with the transfer These use buffer-wetted filter paper buffer for 5–10 minutes. SDS As a drawback, PDVF membranes are instead of buffer, in close proximity interferes with the ability of the hydrophobic and will not wet-out in with flat-plate electrodes (Fig. 2). protein to bind to the membrane; aqueous buffers. They must be wet this affects particularly low In this system, much higher field first in a solution of 50% or more of molecular weight proteins. Also, strengths can be achieved with lower ethanol, methanol or isopropanol, SDS bound to the protein causes current and cooling is not required. then rinsed in water and finally it to migrate faster through the The maximum applied current is equilibrated with buffer. membrane, not allowing enough 0.8 mA cm–2 and the transfer takes contact time for the protein to 1–2 hours. Transfer buffers adsorb to it. However, when trying Transfer buffers must be conductive to transfer very hydrophobic Membranes and maintain the solubility of the proteins, like protein from cell The most commonly used proteins without interfering with membranes, it is worth considering membranes for protein transfer are the adsorption of the proteins to adding a very small amount of SDS (0.05%) as these proteins may precipitate in the gel in the absence of SDS.

Staining Once the transfer has finished it is important to stain the gel with coomassie brilliant blue to make sure that the transfer has been complete. The fact that your pre-stained molecular weight markers have transferred to the membrane Figure 2. Diagram of a semi-dry system. does not necessarily mean that all

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protein of interest is the primary antibody. Once the primary antibody has been allowed to bind to the target protein, the membrane is washed and incubated with a secondary antibody conjugated to an enzyme that will pinpoint the location of the protein. The 2 secondary antibody is raised against immunoglobulins of the animal species used to raise the primary antibody. Antibodies are diluted in blocking solution to avoid unspecific binding to the membrane. The presence of Tween-20 in the diluent prevents aggregation of the antibodies. It is important that the Figure 3. Mechanism of protein detection using chemoluminescence. concentration of Tween-20 does not exceed 0.05% (v/v) as it has the the proteins in your extract have, should have ionic strength and pH potential to remove a proportion of particularly the high molecular as close as possible to physiological the protein from the membrane. weight ones. In order to monitor the conditions; phosphate-buffered (PBS) quality and efficiency of the transfer, or Tris-buffered (TBS) saline are the The dilutions of primary and the membrane should also be most commonly used. secondary antibody must be stained. There are several reversible optimised for each target protein. A stains that are compatible with Using the wrong blocking agent non-specific signal can be avoided immunodetection: Ponceau S red, or wrong blocking conditions can with higher dilution of the primary Fast green FC and 3,4',4'',4-copper obscure the protein of interest; antibody; high background can be phthalocyanine tetrasulfonic acid some contain products that cause a minimized by higher dilution of the and tetrasodium salt (CPTS) are not high background. For instance, milk secondary antibody. very sensitive (1–5 mg protein per contains biotin and glycoproteins; as band) but are very fast and perfectly a consequence neither biotinylated Washing of the membrane is adequate for checking transfer nor lectin-bound antibodies can be necessary to remove unbound homogeneity and loading. Spyro used. antibodies: too little washing will Ruby and Spyro Rose (Invitrogen) lead to a high background and are highly sensitive fluorescent Blocking buffers of proprietary too much washing can elute the stains (1–2 ng per band). Reversible formulations are commercially antibodies. Washes are done in membrane protein staining kits of available, like Casein blocking PBS or TBS containing 0.02–0.1% proprietary formulation are available buffer (Sigma and Thermo Tween‑20. The right number from Thermo Scientific (Pierce) and Scientific), SuperBlock (Pierce), and length of washes must be Invitrogen Pierce Protein-Free blocking buffer determined experimentally, starting (Pierce) and ECL blocking agent (GE with 3 washes of 5 minutes. Blocking Healthcare). PhosphoBLOCKER was After the transfer of proteins from specifically developed for blocking Detection membranes prior to detection of the gel to the membrane it is The most sensitive detection phosphoproteins by Cell Biolabs, essential to block the unoccupied methods are based on an enzymatic to enhance phosphoprotein signal surface in order to avoid unspecific reaction, using secondary antibodies without increasing background. binding of the antibodies used bound to enzymes like horseradish However, even manufactures show for protein detection. Blocking peroxidise (HRP) or alkaline contradictory results when extolling can be achieved using a variety of phosphatase (AP), β-galactosidase the virtues of their blocking agents, proteins and non-ionic detergent and glucose oxidase. The activity of so it is important to determine solutions, but no blocking agent these enzymes can be detected with empirically which one is the best for is ideal for every occasion, so chromogenic, chemoluminescent the protein of interest. more than one should be tested and fluorescent substrates. to obtain optimum results. The most common blocking agents are Protein detection In chromogenic detection the bovine serum albumin, non-fat milk, Detection of a specific protein enzyme’s reaction results in an casein, gelatin and dilute solutions immobilized on a membrane with an insoluble coloured precipitate. of Tween-20 (0.05–0.1%). Buffers antibody is called immunodetection. WesterBreeze Chromogenic used to prepare blocking solutions The antibody that binds to the (Invitrogen), Amplified AP ( Bio-Rad)

Physiology News | No. 83 | Summer 2011 | www.physoc.org 34 PN TECHNIQUES and Immun-Bolt BCIP/NBT (Bio-Rad) membrane can be partially removed Society Noticeboard are some of the kits commercially so it is not always successful. available for chromogenic detection. Scientific Meetings – 2011 However, this type of detection is The most common methods for Physiology 2011 not very sensitive, requiring 1–2 ng stripping are: 2% SDS, 100 mM The Physiological Society Main Meeting per protein per band. β-mercaptoethanol, 50 mM Tris, pH 6.8. The membrane is incubated at University of Oxford, UK, 11–14 July In chemoluminescent detection 50ºC for 15–30 minutes and rinsed Epithelia & Membrane Transport (Fig. 3) the enzyme catalyses several times in TBS. Themed Meeting a reaction that results in the University College London, UK production of light which is detected A milder stripping buffer is 0.1 M 1–3 September on a film; the sensitivity is at least 10 glycine HCl (pH 2.5–3.0). This buffer Abstract submission and registration open 23 June times higher than the chromogenic will dissociate most antibody– method, making it the method of antigen interactions in less than Vascular & Smooth Muscle Physiology choice. 30 minutes at room temperature or Themed Meeting 37ºC. Edinburgh, UK, 6–8 December Several chemoluminescent detection Abstract submission and registration A simpler and very effective products are available from Thermo open 20 September approach is to re-probe the Scientific, Bio Rad and GE Healthcare, membrane without stripping; all equally suitable. However, there Scientific Meetings – 2012 two or three antibodies can be are products that claim to detect used sequentially, starting with Human & Exercise Physiology Themed proteins in the low femtogram level the one that gives the cleanest Meeting, The Biomedical Basis of Elite that are probably very effective, but background. Ideally, the proteins Performance tend to give high backgrounds and to be detected using this method The Queen Elizabeth II Conference require laborious optimization in should have relatively different Centre, London, UK, 19–21 March order to get the right noise-to-signal molecular weights but there are ratio. ECL and ECL plus from GE Physiology 2012 instances where two proteins with Healthcare give very good signals, Main Meeting, The Edinburgh very similar molecular weights can adequate for most circumstances. International Conference Centre, UK. be detected sequentially providing 3–5 July The only drawback of this method one of them gives a stronger signal. Registration opens 1 January is that the signal is not permanent For instance, connective tissue and fades after a few minutes. The growth factor (CTGF, MW 38,000) reaction can be repeated if needs The Journal of Physiology and glyceraldehyde-3-phosphate be, but the background tends to Symposia 2011 dehydrogenase (MW 36,000) can be increase. detected in this way as long as the UCL Neuroscience Symposium antibody against CTGF, which gives a Institute of Education, London, 1 July Fluorescence detection uses a 2011 Wednesday 13 July fluorogenic substrate that fluoresces weaker signal, is used first. at the site of the enzyme. This Human hand function: the limitations of brain and brawn method is less sensitive than the Storage chemoluminescent but it has Membranes can be stored for use Physiology 2011 Wednesday 13 July the advantage that the signal is at a later date. It is recommended Synaptic Mechanisms in the CNS - stable indefinitely so blots can be that they are stored dry, between Symposium to honour Roger A. Nicoll re-imaged. Invitrogen offers two two sheets of Whatman 3MM, Silverado Resort, Napa Valley, CA, USA products for fluorescent staining: protected by cardboard and inside 18–20 September DyeChrome which has two different a plastic bag. They can be stored fluorescent products, one yellow and at 4ºC or –20ºC for up to 2 months Society-sponsored meetings or at –70ºC for long-term storage. one red, which allow the detection – 2011 of two proteins at the same time I have successfully re-probed a and Ampex Gold, which produces membrane that had been stuck in Advances in the cellular and bright yellow spots. They can be my note book, wrapped in cling film molecular biology of angiogenesis detected and recorded with a UV for several months. Sometimes it is a University of Birmingham, UK, 27–29 June epi-illuminator. matter of luck. Motoneurons, their inputs and Stripping Patricia Leoni outputs Warsaw, Poland, 5–7 September Once one of the proteins has been Imperial College London identified, the membrane can be References stripped and re-probed with a Towbin H, Staehelin T & Gordon J (1979). Proc Travel Grants different antibody. However, during Natl Acad Sci U S A 76, 4350–4354. www.physoc.org/grants stripping some of the proteins on the Southern EM (1975). J Molec Biol 98, 503–517. www.physoc.org/international

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Why the medieval Doctrine sign upon the various means of curing disease which he provided.” of Signatures will become The seeds of the skullcap plant, the dominant biomedical for example, resemble small skulls research tool of the 21st and were thought to be effective in the treatment of head injuries and century there is an outrageously phallically As a scientist suffering from an shaped fungus, the stinkhorn (Phallus externally imposed career hiatus (the impudicus) that was believed to bastards), I have begun to wonder be effective for the treatment of “What’s the point of an individual impotence. The more I read about the Doctrine of Signatures the more I scientific technique, why do some their medieval equivalents. I propose become fashionable and others fall am convinced that it has a number of key advantages over a range of modern a return of “the vapours” as a term by the wayside?” Very often it’s encompassing all forms of psychiatric something of a historical mystery. molecular biology techniques which I have summarised below. and cognitive dysfunction. Consider Appearing transformational is easy for the following modern ailments and some newly emerging technologies – Nine reasons why the medieval their medieval equivalents – asthma questions can be formulated around Doctrine of Signatures is a superior induced by pollution versus “miasmas”, their potential and their limitations technology to modern molecular cervical tuberculus lymphadenopathy are not understood. Some scientists biology techniques. versus “the scrofula” and the winter even manage to arrange entire careers vomiting virus versus “the bloody flux” by hopping from one fashionable (1) The Doctrine of Signatures is – what sounds more imposing and fun? technology to another but nobody ever underpinned by destiny, sin and astral seems to notice that they are often influences. These are likely to be far (6) Explaining the science behind doing the same experiment again and more reliable sources of research molecular biology causes unnecessary again. One good career-enhancing funding than the British government distress to people who believe move seems to be to deploy a given for the foreseeable future. in homeopathy; empathetically technology to answer a question, explaining the science behind the just when the technology has first (2) Using post hoc attribution to discern Doctrine of Signatures to them will appeared, just before everybody the utility of a medicinal object is far make them feel wanted, included and actually realises that the technology less intellectually tiring than employing validated. has been oversold and cannot answer a priori reasoning and statistics. every biomedical question on planet (7) Adopting the Doctrine of Earth. (3) The philosophy behind the Signatures as a mainstream biomedical Doctrine makes far more sense to technology could encourage religious In order to revive my career, I have members of the general public than diversity by attracting pagans, wiccans decided that I need access to the molecular biology. Try explaining deep and satanists to your lab. next generation of transformational sequencing technology to someone and dynamic biomedical technology, with a media studies degree, then (8) When preparing a CV writing in the one that will keep me occupied until explain how the shape and crinkled key skills section “Extensive expertise retirement. I thought about becoming surface on walnuts resemble the brain in the medieval Doctrine of Signatures a fake expert in the emerging and can therefore be used to treat including the use of liverwort, technology of deep sequencing (but attention deficit disorder. They will snakeroot, lungwort, bloodroot, it’s a crowded market and there definitely agree to fund the work on toothwort, wormwood and mandrake are lots of them about). But due to walnuts. to modulate gene expression in stress, I have recently developed an transgenic mice” is likely to lead to irrational fear of rationalism, and as (4) The medieval Doctrine of Signatures interesting job interviews. employs copious quantities of black a consequence have developed a (9) And finally including “a proven bile, phlegm, blood and yellow bile – cunning plan. I plan to revive my career ability to locate and extract mandrake ordering these from Sigma will be far by resurrecting the ancient medieval roots from hallowed ground under a more fun than ordering monoclonal medical technology of the Doctrine of full moon” in the essential skills section antibodies. Signatures. of a job description is an excellent The Doctrine of Signatures was an (5) Once the Doctrine of Signatures is way of excluding uncommitted job all-purpose medical technology fashionable it could lead to the revival applicants. developed by Paracelsus and Jakob of medieval medical terminology. Bohme in the 17th century. They Have you noticed how boring modern Dr Keith Cormorant suggested that God or the divine terminology is? Take psychiatry – there PS. The 17th century botanist and creator left signatures or marks on is a semantically turgid, disorder for herbalist William Coles (1626–1662), plants and animals that could be used every day of the week. Classifying a author of The Art of Simpling and Adam to discern their use in medicine. A disorder as schizoaffective, narcissistic in Eden, stated that walnuts were good theological justification was made for personality or bipolar is clinical for curing head ailments because in this philosophy – “It was reasoned and boring. These disorders need his opinion “they have the perfect that the Almighty must have set his declassifying and renaming with signatures of the head”.

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More on Starling and and Allied Conditions. Bright young army physicians were measuring Bayliss during the blood pressures, which were seldom done at the time; few physicians 1914–18 War kept a sphygmomanometer in their Ann Silver recently wrote in these kit. Patients with wound shock had pages (PN 82, p. 17) about these low pressures – 90 mmHg systolic great physiologists at war, taking or lower. If pressure fell further they their stories up to 1917. This was died. No treatment was known: they before Bayliss’s treatment for were triaged and left to die. In the wound shock was tested in the field spring of 1916, Bayliss had reported and before Starling demonstrated to The Physiological Society that that he had stopped hating all anaesthetized cats deprived of things German. As she wrote, the half their blood had low pressures William Van der Kloot with his Senegal choleric Starling was so outraged and soon died, obeying Starling’s parrot. by the German invasion of neutral law of the heart. Replacing the Belgium that he swore never to lost blood with saline raised blood Cannon and Bayliss injected acid speak another word of German, pressure only transitorily, because into several etherized stray cats; even as lyrics in the Lieder he loved the added salt and fluid were their blood pressure fell and they to sing, and announced that he pushed into the interstitial fluid. A died. Therefore the gum must be would enlist as a rifleman. Friends sustained rise in blood pressure was dissolved in bicarbonate solution. convinced him that he would deal produced by an infusion of saline the Huns a harder blow in the containing 5% gum acacia, a large Cannon’s prestige was such that Royal Army Medical Corps (RAMC). molecule whose colloid osmotic weeks passed while they failed Bayliss, six years older, held the fort pressure held fluid in the plasma. to get gum dissolved in sterile at UCL. The cats survived, demonstrating bicarbonate solutions. Finally the the Starling mechanism. (Frank Captain Starling’s first post was as a acid hypothesis was tested properly P. Knowlton had introduced gum pathologist at the Herbert Hospital when Cannon, Bayliss and Henry acacia, obtained from certain plant in Woolwich — presumably the brass H. Dale, the committee’s secretary, exudates, as a substitute for plasma feared that after years in the lab he injected acid into a healthy cat using proteins in 1911, when he was at was a danger to patients. After the a local anaesthetic. The subject Cambridge on an astonishingly German chlorine gas attack in April played with Dale and strolled about productive sabbatical from Syracuse 1915 he was re-assigned to direct panting for an hour or so. There University.) training of Kitchener’s army in the were no other adverse effects. Dale use of the new protective helmet, had done little to push gum solution Gum acacia solutions were finally because he was sure that shock breathing in through a chemically tested on moribund soldiers in impregnated flannel bag covering was produced by the release of a shock in November 1917. Blood histamine-like molecule. Their goal the head and exhaling from the pressure rose and was sustained. mouth through a rubber valve. should be to identify this molecule It was like watching Lazarus rising and find a drug that stopped it When the familiar box respirator from the grave. By then Starling binding to its receptor. was introduced in 1916 he was had resigned the chair and seldom transferred to anti-gas duties in attended; Bayliss took his place. Gum solutions finally were used Salonica. The Allies had invaded that Even after this dazzling success, during the last months of the war. part of neutral Greece to use it as the committee dragged its feet in Astonishing to us, no tally was a base to keep the Central Powers getting gum solutions to the front. kept of the number treated, their from overwhelming their Serbian The eminent Harvard physiologist state when treated, or the results. ally. They failed. As the Allies sat in Walter B. Cannon, now a captain in Medics were not instructed about their 110­-km-long entrenchments the American Army, had discovered the volume to be infused, when it in Greece, Starling’s martial ardour that the blood of shocked men was should be given, or the scientific cooled. He resigned his commission low in bicarbonate. He argued that rationale for the treatment. Blood as a Lieutenant Colonel on the acid caused shock and treated a was also transfused into shocked grounds that he was now more than handful of cases in early shock with wounded. This was feasible after 50 years old. bicarbonate. They survived. Ever it was discovered that refrigerated the enthusiast, he persuaded the blood could be stored without Back in London in August 1917 he RAMC to stipulate that bicarbonate clotting by chelating calcium with chaired the first meeting of the was to be stirred into tea provided citrate. Again no records were MRC’s Special Committee on Shock for the wounded. In London, compiled. Post-war assessment of

Physiology News | No. 83 | Summer 2011 | www.physoc.org HISTORY PN 37 these treatments was anecdotal and which set daily rations in calories that the British had won the war by swayed by physicians’ perceptions and projected future food needs for starving women and children was of blood as ‘natural’ while a solution the Allies. not popular. made with a substance utilized in chewing gum and glue for postage He wrote an article lambasting the Starling began the war hoping to kill stamps was obviously ‘un-natural’. lack of understanding of science as a warrior; he ended as a civilian Some patients had been killed by that led to so many wartime gaffes helping to prevent starvation. dodgy gum solutions made in the by the establishment and prescribed Bayliss discovered an effective field using tap water. There were no educational reforms including less replacement for lost blood, but his problems with solutions prepared in emphasis on dead languages and role was largely forgotten. Gum the RAMC’s central laboratory. better salaries for teachers. was replaced by dextran, but whole blood or plasma was preferred. He was sent on a secret trip to Starling was doing less in the MRC Now that we realize that infusing Germany in June 1919 to evaluate committee because he was so another’s blood or plasma is a their feeding – surely then he busy chairing the Royal Society transplant that evokes unwelcome spoke German with old friends. He Food [War] Committee. On top of immunological responses, there is wrote a detailed paper about how that he was sent briefly to Italy to renewed interest in large molecules the Germans fed during the war start anti-gas training with the box that can do the osmotic work of and in the months following the respirator, accompanied by Captain plasma proteins. Even in wartime armistice during which the Allies Charles Lovatt Evans who later the brothers-in-law showed what kept blockading food and banned wrote a jolly, irreverent account of physiologists could accomplish. their mission. The Italians purchased saltwater fishing. Many urban dwellers had starved: body weights the respirators because their own William Van der Kloot devices had failed abysmally when were down 15–20%. There were the Austro-German army broke only half of the predicted numbers SUNY at Stony Brook, Stony Brook, through their front at Caporetto late of children in the age groups from New York, USA 1–3 and from 10–15. He thought in 1917. (The site of the gas attack References there was selected by Captain Otto that the Germans might have had sufficient foodstuffs despite the Henderson J (2005). A Life of Ernest Starling. Hahn, the eminent chemist who Oxford University Press. later discovered uranium fission.) blockade, but the shortfall was enhanced by inept rationing, failure Van der Kloot W (2003). Ernest Starling’s analysis of the energy balance of the German Back home, Starling became the to stop farmers from using feed to people during the blockade, 1914-1919. Notes principal scientific advisor to the maintain livestock, and diversion Rec R Soc Lond 57, 185–193. Ministry of Food, and then one of into the black market. British food Van der Kloot W (2010). William Maddock the two British representatives on administrators had averted these Bayliss’s therapy for wound shock. Notes Rec R the Inter-Allied Food Commission, problems. Starling’s implication Soc Lond 64, 271–286.

Hazards for the Committee Talking to current members of The Society Council I get the impression that they may be missing some of the distractions that spiced up Committee service in the ’70s and ’80. In those days the Committee often met the day before a Society Meeting, ending with a Dinner, and overnight accommodation in the same place as the rest of the Members. This had the advantage, at least for the latter, that they could harangue the Committee about any contentious issues over breakfast. But it wasn’t only Members who did the haranguing. I remember breakfasting in an Oxford College where each place was set with a bowl of cereal. The then Treasurer, a porridge-only Scot, pushed his cereal away but had it pushed back by a fierce waitress. Just after that contretemps, a late arrival sat down at the next table; this spurred the waitress into pushing him back on to our table with the words ‘Who’s in charge here?’ Someone indicated the Meetings Secretary; for some reason this provoked her into saying ‘I’m not joking.’

A more alarming episode concerned a winter Committee Meeting in Sheffield. We were dining in Buxton some 18 miles away and the coach took the ‘scenic’ route over the Pennines. It wasn’t all that scenic because it was dark and we came to a halt on what seemed to be a foggy cliff top (but probably wasn’t). Those lucky enough to have known the somewhat eccentric Pat Merton will probably agree that he was not the ideal man for the job but nevertheless he got out of the coach to direct the reversing driver. Later, in the safety of the restaurant, we compared notes on our thoughts during that manoeuvre. Those truly dedicated to The Society had comforted themselves with the thought that there were sufficient absentees to ensure a Committee could be reconstituted but the more pragmatic confessed to hoping their life insurance was up to date. Ann Silver

Physiology News | No. 83 | Summer 2011 | www.physoc.org 38 PN HISTORY

Death of a diuretic considered the golden standard on Mersalyl seems to interact with the the subject. However, there is no thiol groups of cysteine in the pore “Don’t let the patient run to the mention of mersalyl from the sixth of the water channel and interfere bathroom frequently in the night. edition onwards. Failing to locate the with its function. Prolonged use Give it sufficiently early”. That was earlier editions, I turned to the net, of this diuretic was found to cause what the resident used to advise the greatest source of information kidney failure due to mercury us, the interns. ‘It’ of course refers and misinformation. toxicity and signalled its death and to ‘mersalyl’ the mercurial diuretic, deletion from clinical use but its much in vogue those days. That The earliest study using the stop resurgence as a tool of molecular is nearly five decades ago. Soon it flow technique indicates that the biology seems to have begun. The was replaced by the loop diuretic proximal tubule is the site of action question that daunts me is: if Stokes furosemide, and mersalyl just faded where mersalyl stops reabsorption had the modern tools, would he away. But in its day it was the drug of of sodium and water (White & Rolf, have stumbled on the aquaporins. choice for patients with congestive 1963). Cafruny’s review (1968) on Yes, of course. heart failure to provide relief and a the mercurials raises more questions good night’s sleep. The knowledge than it answers, regarding the site of J. Prakasa Rao of its diuretic property I assumed action. A later study (Burg & Green, Department of Physiology, College came from patients with syphilis, 1973) mentions that at least part of Medicine, American University of who were treated with mercury in of the action might be due to the Antigua, Antigua the pre-antibiotic era. Remember the inhibition of chloride transport in the References old saying ‘A moment with Venus thick ascending limb of the loop of and a life time with Mercury’. But Benga G (2009). Water channel proteins (later Henle. However, this inhibition may called aquaporins) and relatives: past present it seems that an Irish man by the be a milder one since furosemide, and future. IUBMB Life 61,112–133. name of Stokes (of ‘Stokes–Adams known to act in a similar fashion, Burg M & Green N (1973). Effect of mersalyl Syndrome’ fame) had foreseen on the thick ascending limb of Henle’s loop. causes profuse diuresis. But all these Kidney Int 4, 245–251. the diuretic properties of mercury reports belong to the pre-porin era. Cafruny EJ (1968). The site and mechanism of much earlier (Ventura et al. 2001). action of mercurial diuretics. Pharmacol Rev Why was mersalyl given up then? The earliest publication 20, 89–116. Possibly because of toxicity of (Whittembury et al. 1984) regarding Jung JS, Preston GM, Smith BL, Guggino WB mercury salts and the fact that it the porins that I could locate, & Agre P (1994). Molecular structure of the water channel through aquaporin CHIP, the was less effective when compared concluded that cell membranes of hourglass model. J Biol Chem 269,14648– with the other diuretics. However, proximal convoluted tubules are 14654. it is still around on the shelves pierced by aqueous pores that are Ventura HO, Mehra MR & Young JB (2001). Treatment of heart failure according to of laboratories as a thiol binding reversibly shut by the mercurial William Stokes: the enchanted mercury. J Card agent. What is the site of action of diuretics. Agre, the Nobel awardee Fail 7, 277–282. mersalyl? For enlightenment on the for aquaporins, himself used salts of White HL & Rolf D (1963). An analysis of meralluride and ouabain on sodium excretion subject, I looked for the ‘classics’ mercury to inhibit water transport by stop flow and slow flow techniques. J like ‘Diuretics for Dummies’ and ‘All through these membranous pores Pharmacol Exp Ther 141, 326–332. you wanted to know about diuretics (Jung et al. 1994). Curiously Benga Whittembury G, Carpi-Medina P, Gonzalez but were afraid to ask’. Finding none (2009), much earlier in Romania, E & Linares H (1984). Effect of para- on the shelves, I turned to two men experimenting with erythrocytes, chloromercuribenzene sulfonic acid and temperature on cell water permeability of of repute – Goodman and Gilman, found water channel proteins that proximal straight tubules. Biochim Biophys Acta for their text on pharmacology is were closed by mercurial diuretics. 775,365–373.

Prize for Meetings report With the introduction of focused scientific meetings, the Meetings Secretary no longer reads the minutes of the previous meeting at the next Society Dinner. Of course a formal record is kept of the numbers attending, the titles of the Symposia, Prize Lecturers and the like, but diners are not now entertained by the Meetings Secretary’s account of highlights or lowlights – both scientific and not so scientific (such as the evacuation of the dining room at Cork in 2004 when the overcooked beef set off the fire alarm).

To continue the tradition that The Physiological Society has room for humour in its records, the History & Archives Committee, in conjunction with the Meetings Secretary, is inviting younger Members to submit a light-hearted report when they have attended a Society Meeting. If suitable, these may be published in Physiology News and/or online. An annual prize of £100 will be awarded for the best submission.

All entries should be submitted to Jill Berriman at [email protected] by the end of July 2011.

Physiology News | No. 83 | Summer 2011 | www.physoc.org FROM THE ARCHIVES PN 39

100 years ago in J Physiol discusses how the effects of these the outside of the cells, or to a change solutions might thus be mediated, in in the previously existing potential The action of tri-valent ions part, directly by H+, whose inhibitory difference. It is conceivable that such an on living cells and on colloidal action on the heart had been famously alteration in potential difference might systems: II. Simple and complex described some 30 years before by cause a change in the surface of the kations. George Ralph Mines Gaskell. Mines compared the actions of cells… modifying their shape…. it can (1911). J Physiol 42, 309–331 the “simple” (non-hydrolysed) Mg2+ and hardly be doubted that the electrical the hydrolysed Be2+. This in turn led him change indicated must play some part This paper represents the first ‘repeat to search for “simple” (non-hydrolysed) in the mechanism of excitation or of appearance’ in From the Archives, being trivalent cations to compare with Al3+, contraction.” the work of the brilliant but ill-fated and thus to the rare earth elements. George Ralph Mines (1886–1914). More “Now the potential difference could on Mines’ tragically short life, and his Obtaining rare earth cations was not be modified in two distinct ways by work, can be found in the Summer 2008 a trivial matter a century ago. Mines substances applied outside the cells, issue (PN71, p. 63 ). The current paper, notes: “I have [previously] shown that (1) by the addition to the surrounding only 3 years on, is a far more mature salts of [several of] the rare earths… fluid of some rapidly moving ion which, work. It shows the clear biophysical agree precisely in their action on the by diffusing, into the cells, would understanding, well ahead of its time, heart of the frog. Through the generosity introduce its own electric charge, (2) that was a feature of Mines’ later work. of some of those chemists who have by the addition to the fluid surrounding isolated them I have been able to the cells of substances capable of The paper is best read together with experiment with several other of these altering the relative permeability of the a report from the previous year that it precious substances.” surface of the cells to positive and to follows up (Mines, 1910). The papers negative ions. The alteration of potential report the effects, mainly on frog heart, The 1911 paper also makes use of difference produced in the first way will of micromolar concentrations of trivalent complex trivalent cations such as quickly disappear when the excess of lanthanide cations, including La3+ and 3+ [Cr(NH3)6] , showing that they are far rapidly moving ion is removed from the Gd3+. Mines shows that doses as low as less potent at inhibiting the heart than environing fluid. ...it is to be expected 1 M produce complete inhibition of the μ the simple trivalent lanthanides. Mines that the alteration of potential difference heartbeat (Fig. 1) then discusses the actions of the ions in induced by the second method will terms of what they might imply about Mines’ interest in rare earth cations persist for a considerable time after the binding sites of trivalent cations in stemmed from his search for appropriate removal from the surrounding fluid of the heart. Noting how slowly reversible congeners to probe the effects of the the agent which induced the change.” the effects of the lanthanide cations physiological divalent cation magnesium. are, he contrasts this with arrest of Nowadays we know that the lanthanides In the 1910 paper Mines explains: the heartbeat by elevated potassium bind tightly to, and block, voltage- 2+ “The study of the reactions of living concentrations, where “restoration of the gated Ca channels. Given that Mines tissues towards [non-physiological] beat takes place promptly on changing was writing some 40 years before the inorganic salts... is of value for several the perfusion fluid to Ringer without the seminal work of Hodgkin, Huxley and reasons. We have a simplification of excess of potassium”. The most prescient their collaborators in the late 40s and knowledge if we can state that some passage of all in the paper is where early 50s predicting the existence physiological property is characteristic Mines uses these facts to distinguish the of ion channels, the insight in the + of a group of substances, otherwise potential mechanisms by which K and above passage is striking. It is worth defined by their chemical or physical the trivalent lanthanides could have their considering how contemporary it would resemblances, rather than of one of actions on the heartbeat: sound if one substituted the words its members. In such classification lies “ion channels” for “surface of cells” and “If... there arises at any stage in the cycle a method of attacking the problem of “gating” or “conformation” for “shape”. of chemical events concerned in a heart how the normal saline constituents of beat a difference in the concentration Gaskell WH (1880). On the tonicity of the the tissue fluids exert their action... of diffusible electrolytes inside and heart and blood vessels. J Physiol 3, 48–92. Particular interest attaches to any case outside the cells, this difference of where a clear parallel exists between the Mines GR (1910). The action of beryllium, concentration will lead to a difference of lanthanum, yttrium and cerium on the frog’s action of salts on a living organ and on electric potential between the inside and heart. J Physiol 40, 327–346. a colloid system… [e.g. with] the alkali metals. Such discoveries help to bring the mechanism of the cell within range of the methods of physical chemistry.”

This statement would have had obvious resonance for later investigators of the ionic basis of membrane excitability, and could still stand today.

Mines began with the divalent cation Be2+ and the trivalent Al3+. However, both hydrolysed noticeably in solution, producing complex forms like [Al(OH)]2+ and acidifying the saline. In another example of his clear logic, Mines Figure 1 from Mines (1911), showing rapid inhibition of cardiac contraction by 10 μM Nd3+.

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Nerve and Muscle properties, such as resistance and words that is hard to resist, making capacitance, influence generation of for an inspiring and engaging read. By Richard D. Keynes, David J. action potentials. Aidley and Christopher L.-H. The content is divided into chapters Huang The chapters on synaptic entitled: ‘Early history – from Galen transmission and neuromuscular to Golgi’, ‘The birth of modern Cambridge University junction review the evidence that neuroscience: Santiago Ramon Press, 4th edition: £24.99, established the role of acetylcholine Y Cajal’, ‘After Cajal: from black 194 pages, paperback as a neurotransmitter in frog skeletal and white to colour’, ‘Breaking muscle, and demonstrate the five the diffraction barrier: from cells ISBN-10: 0521737427 criteria required for a compound to to molecules’, ‘Electricity in the ISBN-13: 978-0521737425 be labelled as a neurotransmitter, brain’, ‘The brain as a circuit’ and although the discovery of ATP ‘From brain structure to brain First published in 1981, this book and nitric oxide as putative function’. Each chapter opens has evolved to emphasise key neurotransmitters over the last with an introduction written by an principles in electrophysiology, 20 years has relaxed these criteria. expert in the field, and provides an stressing concepts rather than The remainder of the book concerns enlightening report of the relevant facts to aid comprehension of most muscle. Most textbooks in physiology background information. The undergraduates undertaking related fail to address the intimate extraordinary images are all more courses in biomedicine. In this relationship of a neuron with its than adequately captioned for the edition, the authors have dedicated muscle fibre and simply discuss general reader and pleasingly so for two extra chapters to cardiac and muscles as an isolated system. Again the more knowledgeable reader, smooth muscle, compared to the using evidence, the authors compare delivering detail on how the images previous editions where the topics and contrast the three major types were obtained and what they were very briefly explored under the of muscle fibre as well as the subtle represent. heading ‘non-skeletal muscle’. The differences involved in their signal true essence of this book, however, transduction, from arrival of action A particular strength of the book is is enmeshed within the review potential to the linking of actin and that through its well-constructed of classical electrophysiological myosin. framework it delivers succinct experiments, which soared in information on a variety of the early 1950s to explain the This updated version of a classic methodologies including microscopy, fundamentals of neuroscience book should be required reading electrophysiology, staining and that we take for granted today. for all undergraduate Neuroscience neuroimaging techniques, with Nonetheless, recent findings are students and is a wonderful model of attention paid to the development also included and, taken as a whole, a concise, clearly written textbook. and applications of such techniques. provide a comprehensive overview of The many references to integrated physiology in neurones and muscles. Angus Brown and Sina Tavakol approaches accurately reflect the growing importance of such methods The book begins with the basic in current and future research. morphology and properties of neural Portraits of the Mind cells. The experimental evidence by Carl Schoonover One of the most exciting methods reviewed in this chapter is enough discussed in this publication is to provide a basic understanding Harry N. Abrams, Inc., New the rather elegant and recently of threshold, excitation, York, £22.50, 240 pages, developed ‘Brainbow’ staining propagation and refractoriness of hardcover technique. This particular technique action potentials in myelinated is presented through some of and unmyelinated neurons. The ISBN-10: 0810990334 the most striking and intricately discussion on the generation of ISBN-13: 978-0810990333 worked images in the book. resting potentials uses key examples Another commendable quality to illustrate the implementation of This fascinating work presents of the writing is the relative ease the Nernst and Goldman–Hodgkin– powerful visualisations from the with which challenging concepts Katz equations, and how different past, present and future directions underlying neuroimaging techniques ionic distributions within and of neuroscientific research. are conveyed to the reader briefly outside can be perceived as a single Written so that both general and and effectively but without voltage across the cell membrane. knowledgeable readerships alike can oversimplification. The book has a section dedicated to take enjoyment from the content cable theory and describes relevant and appreciate the meaning behind However, it is the images that equivalent circuits that mediate the images, this book is wonderfully linger, and the most memorable passive currents or action potentials. accessible. Schoonover and the ones include: on pp. 84–85 axons But most importantly, there is an various contributing authors exhibit a emerging from selected retinal in-depth analysis of how these contagious enthusiasm through their ganglion cells converge on the

Physiology News | No. 83 | Summer 2011 | www.physoc.org BOOK REVIEWS PN 41 optic disc then disappear like water anatomy text, you can have a break six perilous years of deprivation down a plug hole, the scanning EM and colour them in using this book, and enormous risk taking? Against of a neurone on p. 124 looks like not only getting the small thrill that all the odds you have survived the an octopus, on p. 210 the images all well-educated people will get from horrors of the Second World War of white matter tracts in the brain being allowed to deface a book, but but your immediate family has been obtained by MRI resemble strips of reinforcing your anatomy learning killed and you have been deprived coloured plasticine; and on p. 216 at the same time. In fact, in order to of an education since the age of 12, blood vessels penetrating deep into maximise the effect, I recommend all for the crime of being born to a the brain parenchyma. that you sing (or at least hum) the Jewish mother. Despite everything, appropriate anatomy mnemonic there is a flame burning inside In short, Portraits of the Mind takes while colouring (but not in the you – for Science. Me neither. But the reader on a marvellous journey library of course, which is mainly this is where Gerta Vrbová’s latest of discovery you should not be useful for dozing through other autobiographical memoir begins. disappointed with. people’s whispered conversations). The preface gives some useful hints In Betrayed Generation, Gerta Angus Brown and Helen Smith on colouring, so that if you want describes her life first as a student, to you can make your finished then as a medical doctor and finally The Anatomy Colouring products look like genuine anatomy as a research physiologist, wife, and mother of two in communist Book text examples. On the other hand, if you are in colouring iconoclast Czechoslovakia between 1945 By C.R. Constant, C. Brassett & mode, the authors encourage you and 1960. The immediacy of her M. Spear, with illustrations by to let rip – “there is no correct or story comes from the details and anecdotes of everyday life J. Berrangé incorrect colour coding”. You want a blue liver or a green heart, you in the lab and at home, and her New Holland Publishers, 2011, can have one. Unlike some similar candid descriptions of tight-knit relationships with colleagues, friends, £12.99, pp. 240 books, each figure comes with brief but helpful words to explain what family and lovers. The naivety and ISBN-10: 1847738699 you are seeing, and comes with idealism that drove her to embrace ISBN-13: 978-1847738691 numerical labelling (numbers on the the communist ideal and ultimately end of thin lines with a key below), to be let down by it seems incredible It has always seemed to me that ensuring that the diagrams will still today. there is a fairly straightforward be clearly labelled and useful even Life in post-war Czechoslovakia was approach to learning about most after you have finished with your no party but it was infinitely better issues in human biology: what it is, crayons. I think that the figures than the life she had experienced how it works, how it goes wrong. In have been well chosen for first time during the war. However, a other words anatomy, physiology, anatomy students and are neither glimpse of the West, plus falling pathology. But while physiology too complicated nor too simple: they head-over-heels in love with a British and pathology have some powerful serve the purpose of the book well. physiologist, led Gerta once again to unifying principles which often allow Also important, the price is very risk her life and that of her children, you to work things out rather than reasonable. I heartily recommend this time to reach the UK. At this simply remembering them, getting this text to anyone embarking on a point new heroes enter her story, to grips with anatomy requires course in human anatomy. Not only for her escape would not have been some pretty heavy lifting in the rote will it help you learn, you may even possible without the selfless help and learning department, especially discover some unexpected new skills. at the beginning. This is where personal risk-taking of colleagues any available help is welcome, and John A. Lee within the international scientific where this book scores highly. It fits community, many of whom are directly into the educational research Betrayed Generation – Physiological Society Members. tradition that shows that when British physiology, and indeed the you are learning something new, Shattered Hopes and British scientific community at large, multi-modality learning helps it go in Disillusion in Post War owe an enormous debt to these and other unsung heroes. Who today better and makes it easier to retain. Czechoslovakia This nicely produced book, with would risk their career, let alone their judiciously chosen, uncoloured, clear By Gerta Vrbová life, for a colleague? and simple drawings on one side only Zuza Books, £9.99, paperback Gerta Vrbová’s story is the triumph of a double page, can thus be used as of unfailing optimism, hope and a supplement to entry level anatomy ISBN-10: 1907890106 ISBN-13: 978-1907890109 steely determination over experience. learning. When you have had enough Read her book – it will put your own of trying to memorise the lumbar Can you imagine yourself destitute, life into perspective. plexus or the mediastinal relations aged 19, arriving back at your of the lung from your standard childhood home in Slovakia after Thelma Lovick

Physiology News | No. 83 | Summer 2011 | www.physoc.org 42 PN SOCIETY

Paton Prize Bursary Photographic Competition We are pleased to announce the winners of the photographic competition for equipment in use or within a laboratory setting. The two entries submitted were both awarded a prize: Piers Nye and Michael Gardner. Here are the winning photos plus an accompanying explanation of them. Piers Nye These two photographs are from Haldane’s expedition to Pikes Peak, Colorado in 1911 – 100 years ago in July.

During their five week sojourn on the peak at 14,100 ft (4,300 m) Haldane, Schneider, Henderson and Douglas (left to right) gave the first good description of ventilatory and cardiovascular acclimatisation to high altitude while Mabel Fitzgerald (between Haldane and Schneider in the photograph) travelled around mining camps sampling alveolar gases and taking blood samples from fully acclimatised miners. The photograph was taken by Douglas using a shutter-release delay. In the background one can see part of the cog railway that carried their equipment to the summit. (The original of the photograph was bought from Douglas’ effects by John Widdicombe.)

This photograph, which appears in the full publication of the expedition (1; Fig. 3 on p. 188, reproduced with permission of the Royal Society), shows the laboratory they set up for studying alveolar and blood gases. It is lit by an electric light (at 14,000 ft in 1911!) and warmed by the large wood-burning stove. Note the Douglas bag on the left with tubes that largely obscure three sets of Haldane alveolar gas analysis equipment. Behind the stove is a galvanometer and, to its right, is a circular Fuller’s slide rule equivalent to a 25.3 m straight slide rule. Directly in front of the stove, sitting on a water bath heated from below by a Primus lamp, is a saturator for equilibrating blood with expired gas, required for Haldane’s complicated method of measurement of arterial oxygen levels. In front of the window are measuring burettes and, at the right, a kymograph for recording events on smoked paper. On top of the box at the right is a reservoir that supplied controlled drips of water that displaced pure carbon monoxide from a cylinder. Beside and beneath this box are 1- and 10-litre gasometers to measure respiratory gas volumes. The tall white

cylinder on the floor contains ‘oxylith’ (sodium peroxide, Na2O2), to which water was added for the generation of pure oxygen. (This photograph was given to me by Bob Torrance who may have acquired it at the same time as John Widdicombe.) I was given most of the above information when I took the original photograph to Brian Lloyd in 1999.

Reference 1. Douglas CG, Haldane JS, Henderson Y & Schneider EC (1913). Physiological observations made on Pike’s Peak, Colorado, with special reference to adaptation to low barometric pressures. Phil Trans R Soc Lond B 203, 185–318.

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Michael Gardner

David Fisher setting up a demonstration on absorption across the small intestine at a meeting of the European Intestinal Transport Group in The late R.B. (David) Fisher beside a Southampton in 1980. The apparatus consists of 3 identical sets of Fisher & Fisher & Parsons intestinal perfusion Parsons perfusion apparatus (modified from the original by removal of the apparatus being used to study copper serosal bathing fluid). The demonstration showed that: (1) when glucose was transport across rat small intestine. The present in the luminal perfusate, fluid was absorbed but phenol red hardly photograph was taken in about 1972 in crossed the intestine at all; (2) when glucose was omitted from the perfusate, Fisher’s laboratory in the Department of there was no significant water absorption and intestinal barrier function soon Biochemistry, University of Edinburgh failed; and (3) when methyl red was present in the perfusate, instead of phenol Medical School. red, it readily crossed the intestine and appeared to be concentrated during transport, suggesting active transport against a concentration gradient.

The equipment shown on the left is a Fisher & Parsons apparatus for perfusion of animal small intestine, especially used for investigation of intestinal absorption (Fisher RB & Parsons DS (1949). A preparation of surviving rat small intestine for the study of absorption. J Physiol 110, 36–46). This, using bespoke organ chambers and other components made originally by Harold Vincent, Oxford’s ‘ace’ glassblower, was the first successful method for studying intestinal absorption in vitro; a key feature was that the animal’s circulation was maintained under anaesthesia during the setting-up procedure until perfusion with well-oxygenated media was established, since brief anoxia would seriously compromise tissue viability. It provided the earliest evidence that glucose absorption was by carrier-mediated active transport, and that glucose absorption was normally essential for fluid to be absorbed. Arising from the success of this methodology and the underlying principles, a number of other approaches – other perfusion methods including vascular perfusion, everted sacs, tissue accumulation slices, mucosal sheets, isolated cells, etc. – were developed which duly established the basic mechanisms of intestinal transport and inter alia enabled investigation of effects of drugs on intestinal function. Fisher strongly warned that maintenance of physiological viability was de rigeur and that experiments in vitro needed to account quantitatively for rates and activities seen in vivo.

Fisher (1907–1986), originally a student of Sir Rudolph Peters in Oxford, was a University Demonstrator in Oxford (1933–1959) before taking the chair of biochemistry in the University of Edinburgh in 1959. Though his appointment in Edinburgh was in biochemistry, he was primarily a physiologist or physiological biochemist, with interests predominantly in whole-organ function. After retirement in 1976, he returned to Oxford where he continued to perform research on intestinal absorption, including demonstrating active transport of salicylates and zenobiotics. Horace Davenport credited Fisher for having steered him in the direction of elucidating the mechanism of gastric acid secretion when he had spent a Rhodes Scholarship in Fisher’s laboratory in 1937. Fisher’s other work included creatine metabolism, mechanisms of action of insulin, enzyme kinetics, analytical paper chromatography, and many aspects of intestinal absorption. He loved inventing (and using) laboratory equipment and gadgets, and his statistical and numerical abilities were well renowned. His outstanding analytical and statistical skills led to him being seconded to the RE8 (Research & Experimental) Group of Bomber Command during World War II. His personal tutorials were noteworthy, and many of his postgraduate and postdoctoral workers have achieved academic distinctions; most would attribute their scientific successes to his strong influence and intellectual drive.

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Ask a physiologist! Why do men have beards but PS. Beards grow faster than any other women do not? (Lewis, age 11) Why hairs. Without shaving, a man’s beard How many times do you blink in do we have armpit hair? (Chris, age might reach 30 feet in his old age. an hour? Why do we blink? (Alex, 14) Why do men have facial hair? age11) (Oliver, age 14) In most Biology text books it states that a moist surface is required for Dr Patricia de Winter of University Dr Kirsten Poore, University of effi cient gas exchange at respiratory College London replies: Southampton, replies: surfaces yet I seem to remember an This is a very good question but Men and women do sometimes examiner stating that this is not the it does not have a simple answer look quite different don’t they? case and that diffusion would take because blink rate in humans varies The fi rst answer to this question is place more rapidly without a layer tremendously – from 2 to 50 blinks about the development of ‘girls and of moisture. Could you, please, boys’ into ‘women and men’. And it per minute depending on the clarify? (Lesley Thompson, Teacher, can be explained by what goes on circumstances. For example, if you are Spalding High School) around the time of puberty, in the concentrating very hard on reading early ‘teen’ years. In both boys and something, such as this answer, you Although we usually answer questions girls, a part of the brain called the posed by pupils, we thought this was will probably blink less frequently hypothalamus sends more signals to a quite a good one, so asked Dr Glenn than if you were sitting chatting with gland underneath the brain called the Baggott of Birkbeck, University of your friends (not to be recommended pituitary. This gland releases signals London to reply: in class). There are three types that are able to ‘kick into action’ the They are both wrong and right! of blink: spontaneous, refl ex and ovaries (in girls) or testes (in boys). voluntary. The ovaries make the hormone First, it depends on what is meant by Spontaneous blinks are those that we oestrogen and the testes make effi cient gas exchange. For example, don’t notice (unless we start thinking the hormone testosterone. These if there were not a moist surface on hormones are what make children about it) and serve to keep the the alveolus of the lung, by whatever start to look more like adults after eyeball clean and moist. However, it defi nition you use the word effi cient, puberty and help us to tell adult males has been noted that the spontaneous it would be bad. The thin layer of and females apart. The features that blink rate is more than that needed water is needed to contain the develop are called the secondary sex to simply prevent the eyeball from surfactant which ensures adequate characteristics. For girls, these include drying out – so why do we blink more infl ation and defl ation of the alveolus. the development of breasts and the than required? One answer is that Without that, gas exchange will appearance of hair under the arms – indeed be ineffi cient. spontaneous blinking is affected by but not usually on the face. In boys, a substance called dopamine in the there is a deepening of the voice, On the other hand, the proposition brain which affects particular nerve growth in height and strength and that diffusion would be perfect cells – some studies have shown that the appearance of hair on the face, without the moisture depends on giving drugs that affect dopamine as well as on other parts of the body. the gas species. The permeation of levels changes the blink rate, Interestingly though, testosterone is a liquid by oxygen is slower than although how exactly this works is yet also responsible for slowing and even carbon dioxide. The term permeation to be explained. It is also known that stopping hair growth in older men, here refers to the rate of transfer of people with Parkinson’s disease, who leading to baldness. oxygen through the layer. This is not have lower dopamaine levels in the the same thing as diffusion, which brain, blink less frequently. The second answer is actually another question. We could also ask why is depends on diffusion properties of Refl ex blinks are a response to an it that males and females appear so the gas species, the driving force for unexpected event, for example, if different. Why do men have beards, the diffusion and the solubility of the an object approaches your eye (such for example, at all? Perhaps beards gas species in liquid. Carbon dioxide as when someone tries to put eye help to protect against the cold? But in the liquid layer is much less of a drops into your eye when you have this can’t be necessary for survival barrier. For example, in the chicken an infection). Loud noises or any since women don’t need beards. In egg the ability to lose carbon dioxide other startling event can also cause other animals, when there are big to the atmosphere is always greater refl exive blinking. This type of blink differences in how adult males and than the uptake of oxygen in early lasts a fraction of a second longer females look, it is usually to make one development, but as the membranes than spontaneous blinks and is a of them more attractive to the other inside the shell dry, as water is protective mechanism to prevent sex, or more powerful to potential removed from them by osmosis damage to the eyeball from potential rivals. Think about the manes of (during the fi rst week of incubation) threats. male lions or the tail feathers in male the ability of oxygen to permeate the peacocks. This is known as ‘sexual shell and membranes, so reaching the Finally, voluntary blinks are selection’. Not that many centuries circulation, increases substantially. In those which you do consciously ago, most men still had beards, consequence of this, the permeability (deliberately). These also last but these days most men shave off of the shell and its membranes fractionally longer than spontaneous their beards. Has our idea of what is reaches a maximum so allowing the blinks. attractive changed? chick embryo to grow.

Physiology News | No. 83 | Summer 2011 | www.physoc.org EDUCATION PN 45

Meet the Physiologists demonstrations for families and people of all ages to engage with. In partnership with the Manchester For more information, please visit Museum of Science and Industry www.mosi.org.uk (MOSI), The Physiological Society hosted a ‘Meet the Physiologists’ The Society funded three exhibits day at the museum on Saturday 6 at ‘Meet the Physiologists’. Each November 2010. was hosted by Members based at Manchester Metropolitan University The day formed part of MOSI’s or the University of Manchester and monthly series of events at which was funded through our Outreach by all those involved. The following members of the public can meet Grant scheme. are summaries of reports written by scientists and engineers. The events the organisers of the exhibits; full are free and provide a range of The event attracted over 1500 reports can be found on our website exciting hands-on activities and visitors and was thoroughly enjoyed at www.physoc.org/schools Report 1. Manchester meets its physiologists A group of us from the Faculty of Life Sciences and Cardiovascular Research Group at the University of Manchester hosted an event to introduce the field of cardiorespiratory physiology to the public, with a particular focus on the heart, kidney, circulation, blood and lungs. The whole day was very interactive, with hands-on activities designed to suit a wide range of age groups.

The event was held in the Power Hall - a large and popular area of the museum that attracted over 1500 visitors throughout the day. Quiz sheets were handed out at the entrance to encourage older children and parents to visit each station, gather information and find out answers to questions such as “how many chambers does the heart have?”

Posters and plastic models of organs were placed around the stations, providing further information; however, heart and kidney from sheep and pig provided a more realistic demonstration and certainly provoked a variety of interesting reactions from the crowd. The younger children enjoyed the play area, which included a scientist dress-up box, interactive posters, physiological pictures for colouring in and clay for constructing models.

The event provided us with an opportunity to interact with many different people and to hear their views on science. In fact, many people we spoke with expressed the necessity for science and commented on how important research is, which was very gratifying to hear. The event has shown how vital it is to communicate science to the local community and to make the public aware of some of the cutting-edge research that is being carried out in their neighbourhood.

A young physiologist! The organisers (from left to right): Fiona Lynch, Tristan Pocock, Paolo Tammaro, Liz Sheader, Tracy Speake and Alison Gurney. Rebecca Brookfield Comments from visitors on the day: “I thought it was really interesting and I learnt a lot in a fun way!” Child, age 11 “A great learning opportunity. The experiments were great for engaging the kids.” Parents

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Report 2. What do you need to make you move? Members of the School of Healthcare Science at Manchester Metropolitan University developed a group of interactive demonstrations entitled ‘What do you need to make you move?,’ which had been designed to illustrate how different parts of the body contribute to our ability to move.

Activities included: ultrasound imaging, which enabled visitors to see the muscles in their arm; a wireless EMG electrode, held over biceps and linked to a speaker, allowing participants to hear the activity in their muscle when it was activated; and the role of vision in motor control was also demonstrated with a ball catching challenge in which visitors wore stereoscopic glasses to affect their perception of depth. In an additional activity, we asked whether people really stand ‘still’, by inviting volunteers to stand on a pressure mat (kindly loaned from Biosense Medical Ltd) to show that they actually sway and that the amount of sway is affected by whether eyes are open or closed. Younger visitors were encouraged to collect stickers from each stand they visited in order to complete a printed sheet we provided.

The museum received a large number of visitors and it seemed as though we did not stop talking to people from when the doors opened until the end of the day. We were amazed by the range of people who visited and the wonder that was created by showing them simple aspects of their body using tools that have become part of our everyday work routines. The majority of our visitors were families with young children and our hands-on approach really encouraged participation. The parents seemed as impressed as their children and we hope they left with the impression that visiting similar events in the future could be engaging and educational for the whole family. We also found the day rewarding and left tired, but with great plans for future events. Linda Tersteeg, a PhD student, preparing to reveal the muscles in the forearm of a young visitor using B-mode Emma Hodson-Tole ultrasound imaging.

Report 3. A heart to heart talk The event was very busy, with over 1500 visitors of all ages. Our objective was to enthuse people about science, and offer them a glimpse of research and clinical practice related to the heart in particular. We presented posters, plastic models, PowerPoint shows, and The Physiological Society’s DVD on the use of animals in physiological research – quite a few visitors wanted to know more about this subject and the DVD helped. The highlight of our exhibit was an oximeter activity where visitors measured their heart rates. We also had related quizzes, handouts and giveaways.

We learnt a lot about how to make an exhibit look appealing and exciting to young people. We found that hands-on activities like the oximeter were better appreciated than passive presentations and posters, but the posters provided information for the more inquisitive visitors about ongoing research in our research group. We noticed that the most curious visitors were aged 5–11 years.

Support from The Physiological Society was critical in our successful delivery of this exhibit, which has given us our first experience of preparing, organising and delivering an outreach event. Staff at the museum were also very helpful.

Sanjay Kharche

Outreach Grants The Outreach Grant scheme is open to all Ordinary Members, Affiliates and Associates of The Society who would like to communicate the excitement of physiology to young scientists and the wider community. For more information, please visit our website www.physoc.org/grants or email [email protected]

Physiology News | No. 83 | Summer 2011 | www.physoc.org EDUCATION PN 47

Undergraduate Vacation Studentship Scheme

Established in 1994, the Vacation work, attention to detail, organisation “During many moments of the Studentship Scheme provides funds and perseverance. I experienced placement, I was fairly sure that a for undergraduates wishing to gain the fascination this job offers and career in research couldn’t possibly be experience of laboratory-based the frustration of not obtaining the worth the annoyance of inexplicably research. Projects are carried out expected results. I could suddenly dropping glass micropipettes like over a period of eight weeks during apply what I had learned in the books they’re going out of fashion, or the summer, under the supervision for the past two years, and learned to software crashing again. But as I write of an Ordinary Member of The efficiently read scientific papers. This this, in between playing with my Excel Society. placement allowed me to realise that spreadsheets of results and drafting this career path was exactly what I had my abstract, those frustrations seem In 2010, The Society awarded been looking for.” fairly insignificant and suddenly Vacation Studentships to thirty- research doesn’t seem so bad at all. In seven undergraduates who were Ita describes her experience as a fact, all the data in the spreadsheets asked to provide a summary of supervisor: are mine. I obtained them. And that’s their project and overall experience “Having Laura in the lab through pretty cool, actually.” afterwards. Of these reports, the The Physiological Society Vacation James Selvey, student at Cardiff Education and Outreach Committee Studentship was a really positive University was particularly impressed by experience for both the lab and Laura. one undergraduate, Laura Bella, It has given Laura the opportunity to who worked under the direction experience a research environment and of Dr Ita O’Kelly at the University has really spurred her on to aspire to a We are extremely pleased to receive of Southampton (see Vacation career in research.” such excellent feedback this year Studentship Report on next page). and plan to continue running the Feedback from other students and Vacation Studentship Scheme for Laura describes how her placement their supervisors was also positive: the foreseeable future. We wish all changed her perceptions of research “I am immensely grateful to The the awardees the best of luck in their and plans for the future: Physiological Society for funding this studies and future career. “While I found the initial training studentship. Will has been a real asset period quite difficult and demanding, to my laboratory and I sincerely believe For more information about the once I was able to perform the that this funding and the opportunity Undergraduate Vacation Studentship various techniques required for the it allowed will have a long-term Scheme, please visit studentship, I began to enjoy my influence on Will.” experience more. I learned about the Dr James Fisher, Will's supervisor at www.physoc.org/vs importance of factors such as team the University of Birmingham or contact [email protected] Live-Cell Imaging University of East Anglia, 14–16 June 2011

The Physiological Society is supporting a three-day, hands-on workshop for Members and non-members to learn a range of imaging techniques. The workshop has been extended to include a day of image analysis software demonstrations.

For more information, visit www.physoc.org/education or contact [email protected]

This course is organised by Dr Paul Thomas.

Physiology News | No. 83 | Summer 2011 | www.physoc.org 48 PN EDUCATION

Vacation Studentship Report Laura Bella was awarded a Vacation Studentship in 2010 to undertake a lab-based project at the University of Southampton under the supervision of Dr Ita O’Kelly. Here is her report: Investigation on the internalisation and recycling mechanisms of the K2P channel subunits The aim of this project was to investigate the endocytic pathway(s) of the two-pore domain potassium channel, K2P3.1 (TASK1). K2P3.1 shows widespread tissue distribution and is expressed in neuronal, cardiac, pulmonary, genitourinary and gastrointestinal tissues (Duprat et al. 2007). When K2P3.1 is on the plasma membrane, it is active and allows potassium efflux, which changes the electrochemical potential of cells to a more negative state and so regulates cell function. As, therefore, the protein is a regulator of the cell’s function, it is of paramount importance to understand the regulation of the channel itself, in particular the control of surface expression. Surface expression is maintained by balancing the forward transport of membrane proteins with their retrieval from the cell surface (endocytosis). The team I joined have previously identified mechanisms of forward transport for the protein (O’Kelly et al. 2002) and so my aim of identifying mechanisms of internalisation would provide a more extensive knowledge regarding the regulation of surface expression of this protein.

There are numerous protein internalisation pathways, including clathrin- mediated or caveolae-dependent endocytosis. By disrupting proteins that function specifically in these different pathways, the mechanism of internalisation can be deduced. To determine the rate of turnover of the channel at the cell surface, I transfected cells (HEK293) with a tagged channel (tagged with green fluorescent protein on the N-terminus and an external haemaglutinin tag). Using flow cytometry (FACS) and immunocytochemistry (ICC), I monitored the cell surface expression and cell localisation of the channel. In this experiment, cells were treated with Brefeldin A (BFA) for different time periods. BFA prevents protein transport from the endoplasmic reticulum and so prevents forward transport of the channel, thus allowing us to determine the rate of channel retrieval from the cell surface. This revealed that K2P3.1 had a turnover rate in excess of 5 hours. Dr Ita O’Kelly (left) and Laura Bella. The role of phosphorylation in channel internalisation was also investigated with the tagged channel. HEK293 cells expressing the channel were treated with protein kinase A (PKA) activator (8 Br cAMP) or protein kinase C (PKC) inhibitor (Gö6976). Cell surface expression of the channel was examined by ICC and FACS. In both cases, the amount of channel on the cell surface (detected by an antibody to the external tag) was compared to the amount of total channel (detected by green fluorescence protein) in the control and treated cells. I found that activation of PKA or inhibition of PKC resulted in increased surface expression of the channel. This will require further studies but suggests that inhibition of PKC stops channel retrieval from the cell surface as less channel was visualised in the endosomes.

Finally, the role of dynamin in channel internalisation was examined. Dynamin is critical to enable clathrin-mediated endocytosis and caveosome internalisation, and I found cells incubated with dynamin inhibitor (dynasore) showed more channel on the surface of the cell (by FACS and ICC). This indicates that it is likely that K2P3.1 is internalised via a dynamin-dependent pathway. Duprat F et al. (2007). The TASK background K2P channel: chemo- and nutrient sensors. Trends Neurosci 30, 573–580. O’Kelly I et al. (2002). Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals. Cell 111, 577–588.

Annual General Meeting 2011 The AGM of The Physiological Society will be held at the University of Oxford on Wednesday 13 July at 12.30 pm. All Members have the right to attend but only Ordinary and Honorary Members may vote at the AGM. Please note that you do not have to register for Physiology 2011 to attend the AGM. Matters for discussion As well as the formal business, time has been allowed for discussion of items raised by Members. Anyone wishing to raise any substantive matter are invited to indicate this in advance. Note that this is not a requirement. Members remain free to raise any matter they wish during the AGM, although items that have been pre-notified will receive preference if time is short. If you wish to submit a question in advance please contact Orly Burgess ([email protected]).

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Lab_13 has passed it is clear that what they really want is a safe place to do it. I Ignition* is a programme of believe that from this will grow the creative approaches to STEM scientists of the future. (science, technology, engineering, mathematics) subjects in the Tom Carter East Midlands, involving teachers, Tom is Scientist-in-Residence at students, parents, artists, scientists, Crown Hills Community College in inventors, designers, engineers, project to emphasise the importance Leicester. The college has also taken film-makers, writers and journalists. of maths in various professions. The part in other strands of the Ignition* There are six integrated programmes students have to program a robot programme, including Come Alive to promote creativity in the teaching to perform a number of challenges. With Science where students and and learning of these subjects, one These navigational skills will no teachers worked with an architect of which is Lab_13. doubt be valuable when careers in and a media professional to develop Lab_13 is an exploratory space space exploration take off in the creative STEM activities during within a school or college, run by future. National Science and Engineering young people for young people, Week in order to engage the wider After a busy morning I am back in with the guidance of a scientist, school and feeder primaries. As part my lab where I am greeted by a engineer or inventor in residence. of the project they designed and group of eager students who have One scientist-in-residence shares his constructed an installation in which given up their lunch hour to perform typical day… students were able to showcase their own research. The students various forms of media to the rest of are keen to identify which of their the school and visiting primaries. The favourite energy drinks is the best students took leading roles in the and have persuaded me to run on design of the installation, as well as the treadmill after consuming one of in the script writing and film-making their horrible drinks. This is actually process. the third test in a trial of three where I have been asked to run to fatigue The project at Crown Hills to settle the debate. The students Community College was one of over were disappointed to find that the 30 creative STEM collaborations, flavoured water worked best. involving schools, artists and scientists, taking place across the As a Lab_13 Scientist-in-Residence, As a Scientist-in-Residence I am able East Midlands as part of Come Alive I hold a unique and privileged to act as a mediator between STEM with Science. position within our school. My role subjects which can only dream of is to facilitate STEM-based subjects cross-collaboration within the school Ignition* is delivered by Ignite! and to provide opportunities for our timetable and I am able to provide in collaboration with The Mighty students to learn through discovery. an opportunity for students to be Creatives, and funded by the East creative and to explore science on My day begins by supporting a Midlands Development Agency their own terms. It is common for Life Skills class where students are (EMDA). students to want to blow things up learning about sustainability and the and set fire to things but after this environment. My aim is to provide a For further details see: desire to test the limits of science scientific investigation to reinforce www.ignitefutures.org.uk the learning objectives of the lesson. Today we are assessing the energy consumption of household gadgets and students have to conduct an energy survey using plug-in energy monitors. The students were surprised to find that the humble kettle consumed more than 40 times the energy of a standard light bulb. We manage to draw on some numeracy skills as the students calculate the cost of the energy consumed by each item. Lab_13 does Genoa. Crown Hills Lab_13, along with Tom Carter, their Scientist-in- I then head over to the maths Residence, headed off to Genoa for the Genoa Science Festival, to show how creative department where we are trialling a science should be done.

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Lab Profiles: Welcome using a culture force monitor (CFM) that incorporates a force transducer, to the Muscle Cellular and the constructs can also be ‘exercised’ using a piece of bespoke and Molecular equipment called the tensioning Physiology Research culture force monitor (tCFM) to stretch the constructs according Group to pre-programmed regimes Recently I was talking with Sam (Cheema et al. 2005). The associated colleagues and marvelling at Passey molecular and structural changes the incredible range of research can be measured following this questions that are being asked by expertise and equipment, including ‘exercise’ using confocal microscopy, physiology researchers and the an environmental chamber that and Q-PCR. equally large variety of techniques allows for controlled changes in In collaboration with Keith Baar and approaches that are being environmental conditions such as at UC Davis in California we have used to answer them. We thought temperature and humidity during recently started using an alternative it would be interesting to profile exercise, and other physiological in vitro model where the muscle some labs from around the world testing equipment to look at in Physiology News to highlight the exhaled gases, blood lactate and constructs develop in a fibrin matrix range of exciting research that body composition. (Huang et al. 2005). Cells are seeded Phys Soc Members are involved in. on a fibrin gel layer that is anchored However, the major focus of the in place using small pins and suture So here I am to get the ball rolling by MCMPRG is on studying cellular introducing our lab and the research material. As the cells grow and and molecular physiology, and differentiate, the tension formed we are undertaking to further this is where our research group understand muscle physiology. from the anchor points causes really comes into its own. Before them to roll up around the anchors Since March last year I have had moving to Bedford and forming and form a cigar-shaped muscle the great pleasure of working in the MCMPRG in its current guise, construct containing myo­tubes the Muscle Cellular and Molecular Prof Lewis spent many years with aligned along the lines of tension. Physiology Research Group colleagues and collaborators These muscle constructs can be (MCMPRG) led by Mark Lewis. developing a 3-dimensional in vitro electrically stimulated to contract The research group is part of the model of skeletal muscle and it and the force they produce can be Institute of Sports and Physical is this 3D model that is the main measured using a sensitive force Activity Research (ISPAR) Bedford at tool used by current MCMPRG transducer, allowing us to probe the University of Bedfordshire and researchers. muscle structure, function and since its arrival here in September In vivo, skeletal muscle consists molecular makeup in response to 2009 it has been growing and of bundles of aligned contractile different treatments and conditions. developing at an amazing rate. I myofibres surrounded by extra­ These are the tools we use in our am pleased to be able to write this cellular matrix, and these 3D culture lab – so what are we doing with profile of the lab for many reasons, models aim to produce a muscle them? Well, there are a number but particularly because it has construct that mimics the in vivo of exciting ongoing projects that changed so much since I came here structure and behaviour of skeletal range from exercise physiology and now, 1 year on, it is pleasing to muscle. look back and see how much we’ve to neuroscience. PhD students all progressed since I arrived. The 3D collagen model involves Darren Player and Neil Martin are seeding cells within a collagen gel investigating the molecular and As the name suggests, the group is that is tethered at opposite ends by cellular adaptive responses of interested in muscle research and attachment to mesh floatation bars. the in vitro muscle constructs to uses a variety of physiology, cell This tethering provides the tensional mechanical stimulation, aiming to biology and molecular techniques cues required by the skeletal muscle model both endurance exercise and to study muscle physiology and cells in order to properly align with resistance training using the 3D the response of muscle to exercise, each other, fuse and differentiate to models. David Hughes, who is in the ageing, changing environmental form myotubes. Once formed, these first year of his PhD, is undertaking a conditions and in different disease 3D constructs show many structural detailed study on the mechanism of states. and molecular similarities with in action of testosterone in promoting vivo skeletal muscle (Mudera et al. muscle hypertrophy. Due to our convenient location 2010). within the Department of Sport and In addition to the exercise Exercise Science, we have access The tension generated by the physiology projects that are to a range of human physiology muscle constructs can be measured going on in the lab that have clear

Physiology News | No. 83 | Summer 2011 | www.physoc.org AFFILIATE NEWS PN 51 relevance to sports performance, excitement. We have maintained other projects aim to investigate and developed numerous health-related issues that are collaborative projects and working prominent in this country today. In relationships with researchers particular, the lab is interested in the at other institutions including muscular changes that accompany UCL Institute of Orthopaedics ageing, and research by postdoc and Musculo­skeletal Science Adam Sharples is attempting to at Stanmore, the UCL Institute unravel the complex effects of of Neurology, Loughborough ageing on the muscle function University, Cranfield University and and adaptation, and the ability UC Davis in California. So as I enter of skeletal muscle to repair and the second year of my research in regenerate as we age. the MCMPRG, I look forward to more exciting developments, revelations There are also interesting projects and new collaborations in all of our I live in the East End of London with underway that lie at the interface research projects, and am thrilled to my English husband, who I married between in vitro and in vivo exercise be able to contribute to the growth in 2009 – it was a very busy year for physiology. For example, Paul and development of muscle research me! Davies, a second year PhD student, here in Bedford. Living and working in London is is using ultrasound scanning important as I love going to lectures to investigate changes in core Samantha Passey muscle activation with ageing, and exhibitions throughout the year and combining this with molecular Affiliate Representative for and enjoy the history and culture studies of ageing muscle in the 3D The Physiological Society and that the UK has to offer. models. Similarly, James Tuttle is Postdoctoral Researcher at ISPAR, combining traditional in vivo exercise University of Bedfordshire, Bedford Clare Kingston physiology studies with molecular studies in the cellular models to References investigate the involvement of heat Cheema U et al. (2005). Mechanical signals shock proteins in muscle damage and IGF-I gene splicing in vitro in relation to following downhill running and heat development of skeletal muscle. J Cell Physiol stress for his PhD project. 202, 67–75. Huang YC et al. (2005). Rapid formation of The research I have been doing functional muscle in vitro using fibrin gels. J since moving to the MCMPRG is Appl Physiol 98, 706–713. slightly different to many other Mudera V et al. (2010). The effect of cell current lab projects. I am working density on the maturation and contractile jointly with Alec Smith, a final ability of muscle derived cells in a 3D tissue- engineered skeletal muscle model and year PhD student based at the determination of the cellular and mechanical Institute of Neurology at UCL. stimuli required for the synthesis of a postural Our work is aimed at engineering phenotype. J Cell Physiol 225, 646–653. a neuromuscular junction by I joined The Society as Head co-culturing skeletal muscle cells New Society staff and primary moto­neurons in the of Media Communications for in vitro models – very challenging. members 12 months, providing maternity cover for Mary Arbuthnot. In Alec has put in a lot of work during Orly Burgess his PhD to optimise the co-culture addition to maintaining The I was born in Jerusalem and moved Society’s current activities, I will of the primary muscle cells and Clare Kingston joins The Society as Head of Media Communications for 12 months, providing moto­neurons, and the project is to the UK in 2004 after I finished maternitybe conducting cover for Mary Arbuthnot. a review In addition of to media maintaining the Society’s current comms activities, Clareand will becommunications conducting a review of media – and producing communications – producing a new strategy for producing some promising results studying for my first degree in Physoc. Previously, Clare has worked as Press Secretary for the Science Minister and Government History at the Hebrew University. Chiefa newScientific strategy Adviser, as senior for press The officer Society. at the Royal Society and has most recently been although there is still much to do. making science documentaries with the BBC series, Horizon. I spent five years working at the Previously, I have worked as Press The lab has grown and developed Israeli Embassy while studying Secretary for the Science Minister dramatically since I joined in March at Birkbeck College, completing and Government Chief Scientific last year, both in terms of the my Masters Degree in Medieval Adviser, as senior press officer at people working within the research History in 2009. I joined The the Royal Society and have most group and also the lab itself which Physiological Society in January recently been making science has gone from an empty room 2011 as PA to the Chief Executive documentaries with the BBC series, to a bustling hive of activity and and Office Administrator. Horizon.

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Cardiac Biomedical Science and Engineering Center at UCSD. I served as Vice Chair of the Bioengineering Department from 2002 to 2005 and one example, I underwent complete New Journal Editors 2011 Chair from 2005 to 2008. I am the paralysis of the body with curare Director of the HHMI-NIBIB Interfaces Simon Gandevia while awake to determine the Graduate Training Program neural mechanisms which generate and the accompanying UCSD the feeling of breathlessness when Interdisciplinary PhD Specialization carbon dioxide levels rise (Gandevia in Multi-Scale Biology. et al. 1993, J Physiol 470, 85–107). I serve on a number of editorial I was educated at the University boards. Of note was my initial of Auckland, New Zealand in role on the board of The Journal of Engineering Science and Physiology Physiology (1993–2000). I was one receiving my PhD in 1986. I was an I am an NHMRC Senior Principal of the first Australians to contribute NSF Presidential Young Investigator Research Fellow and Deputy Director in this way. I have published more and am a Fellow of the American of Neuroscience Research Australia. than 80 papers in The Journal on Institute for Medical and Biological I began as a medical student in 1972 a wide-range of topics in human Engineering. I served on the Board and subsequently received a number physiology, including the behaviour of Directors of the Bio-Medical of Bachelor and post-doctoral of human muscle spindles, the Engineering Society, and am degrees from the University of New operation of the muscle–tendon currently Associate Editor of Medical South Wales (BSc Med, MBBS, PhD, unit, proprioceptive sensations, and Biological Engineering and MD, DSc). My passion for medical maximal muscle performance and Computing and PLoS Computational research developed during my central fatigue, neural control of Biology and co-Editor-in-Chief of Drug medical course when I stopped to the hand, and human respiratory Discovery Today: Disease Models. I complete a PhD in physiology. muscle performance. I have also am on the editorial boards of the helped develop concepts about the American Journal of Physiology: After my medical training, I ethics of experimental studies in Heart and Circulatory Physiology, subsequently conducted research humans. I was elected a Fellow of Computer Methods in Biomechanics in clinical neurophysiology at the the Australian Academy of Science in and Biomedical Engineering and Prince Henry Hospital. My research 1998. Cellular and Molecular Bioengineering. continues to focus on the way in I gave the Konrad Witzig which the human brain controls Andrew McCulloch Memorial Lecture and the Donald movement, with emphasis on three Wassenberg Memorial Lecture. major areas: the neural mechanisms Recently, I was elected a Fellow of underlying proprioception; the the Cardiovascular Section of the sensory and motor mechanisms American Physiological Society. involved in the control of human I also chair the Physiome and movement and posture; and the Systems Biology Committee of the central control of human breathing. International Union of Physiological With my colleagues, David Burke, Sciences. Ian McCloskey and Erica Potter, I am Professor of Bioengineering My lab uses experimental and I established an independent and Medicine and Jacobs School computational models to investigate medical research institute which Distinguished Scholar at the the relationships between the was named the ‘Prince of Wales University of California San Diego cellular and extracellular structure of Medical Research Institute’, and later (UCSD), where I joined the faculty cardiac muscle and the electrical and renamed ‘Neuroscience Research in 1987. I am a member of the mechanical function of the whole Australia’. I am the only founder still UCSD Institute for Engineering heart during ventricular remodelling, working at the Institute. in Medicine, the California heart failure and arrhythmia. Institute for Telecommunications My work has provided insights into Current interests include developing and Information Technology, a pathophysiological mechanisms multi-scale models of myocyte Senior Fellow of the San Diego in several branches of medicine excitation–contraction coupling Supercomputer Center, and a including neurology, rehabilitation mechanisms and their regulation by member of the UCSD Center for and cardiorespiratory medicine. PKA and CaMKII. My group has also Research on Biological Systems. I One hallmark of this approach scaled cellular level models of these am a Principal Investigator of the has been to use incisive human processes up to the tissue and organ National Biomedical Computation experiments to expose underlying scales to investigate mechanisms Resource and Co-Director of the neurophysiological principles. As of arrhythmias and ventricular

Physiology News | No. 83 | Summer 2011 | www.physoc.org THE SOCIETY’S JOURNALS PN 53 dysfunction associated with targeted I have published more than 170 (1968) from Virginia Polytechnic gene defects and congestive heart original research papers and 45 Institute, and a PhD in Physiology failure. Genetically engineered mice reviews/book chapters. My main (1977) from the School of Medicine, are an important model system for research interests are in neurogenic State University of New York developing and validating these hypertension and interactions with (Buffalo). I am currently a Professor computational models. Important respiratory and immune systems in of Physiology at the University phenotyping techniques in the animal models, and more recently, of Utah School of Medicine and mouse include optical electrical humans. I won the Sharpey–Schafer Director of the Nora Eccles Harrison mapping, isolated muscle mechanics Prize of The Physiological Society Cardiovascular Research and Training testing and magnetic resonance (1998) and delivered the Carl Ludwig Institute (http://www.cvrti.utah. imaging. A second major area of Prize Lecture of the American edu/) at the same institution. My research in the lab has been the Physiological Society (2005). I hold research focuses on heart cells to role of cytoskeletal and membrane a Royal Society Wolfson Research include intracellular pH regulation, proteins in cardiac myocyte Merit Award. In 2002, I was excitation–contraction coupling and mechanotransduction mechanisms appointed Honorary Professor of electrophysiology. I have served on and how defects in costameric the University of Belgrade, Serbia. the editorial board of the American and z-disk protein complexes can In 2005 I presented the inaugural Journal of Physiology: Heart and alter mechanotransduction and Public Lecture of The Physiological Circulatory Physiology and currently lead to dilated cardiomyopathy. Society on hypertension. I have sat serve on the editorial board of the The effects of stretch on myocyte on the British Heart Foundation Journal of Molecular and Cellular membrane configuration and Project grant committee and am the Cardiology. electrical conduction are also under current Deputy Editor-in-Chief for investigation. Finally, we have Experimental Physiology (2001–2011), Irving Zucker been using Drosophila as a model and will remain on their Board system to explore hypoxia tolerance ex-officio. I served as an Editor for and susceptibility mechanisms in Neuropharmacology (2002–2007), conjunction with metabolomics and Autonomic Neuroscience: Basic to metabolic network modeling. Clinical (since 1997) and Archives Italiennes de Biologie (since Julian Paton 2009). I am Vice-President of the International Society for Autonomic Neuroscience (ISAN) and Chair the International Programming Committee for the inaugural joint I am the Theodore F. Hubbard meeting between ISAN and the Professor of Cardiovascular Research American Autonomic Society and Chairman of the Department of (Buzios, Brazil, 2011). I sit on Cellular and Integrative Physiology at the International Programming the University of Nebraska Medical Committee for the International Center in Omaha, Nebraska, USA. I have been Chairman since 1989. I was educated at Taunton School, Union of Physiological Sciences I received my PhD in physiology Somerset, and obtained my meeting (Birmingham, UK, 2012) from New York Medical College in first degree at the University of and the International Society for 1972. I continued my post-doctoral Birmingham in 1984 in Biological Hypertension (Sydney, Australia, training at the University of Sciences (Physiology). In 1987 I was 2012). Nebraska Medical Center where awarded my PhD from the University I became a faculty member in of London. My post-doctoral training Kenneth W. Spitzer 1973. My research involves studies included periods at the Department related to the neural regulation of of Physiology, Royal Free Hospital, cardiovascular function. My studies London (1987–1989), as a Visiting have revolved around cardiovascular Scientist at E.I. DuPont de Nemours reflex control of sympathetic Inc., Wilmington, Delaware, USA nerve activity in animal models (1989–1992) and as an Alexander of chronic heart failure. These von Humboldt Fellow at the investigations focus on the role of University of Göttingen, Germany central mediators of sympathetic (1992–1994). Subsequently, I was nerve activity such as angiotensin II, awarded a British Heart Foundation nitric oxide and reactive oxidant Lectureship (1994–2004) and I obtained a BA degree in Biology (1966) from Virginia Military stress. More recently, I have obtained a Chair of Physiology at carried out experiments designed the University of Bristol in 2001. Institute, an MS degree in Zoology

Physiology News | No. 83 | Summer 2011 | www.physoc.org 54 PN THE SOCIETY’S JOURNALS to understand the regulation of angiotensin II receptors in the brain. I have published over 200 papers in this field and this work has been continuously funded by the National Institutes of Health and the American Heart Association. I am the Principle Investigator of a Program Project currently in its 12th year of funding. I have been the recipient of a MERIT Award from the NHLBI, an Established Investigatorship from the American Heart Association and a recipient of the Wiggers Award from the Cardiovascular Section of the American Physiological Society. I currently serve on the David Paterson looking through The Journal of Physiology, his ‘new' journal, at EB. editorial boards of 10 journals. Note the special JP- and EP-branded bottles of water on the Wiley-Blackwell stand. All In addition to my research, I am 1,100+ bottles of water were taken during the meeting. active in administrative activities UK. The failure of bench-to-bedside of the neurovascular unit in for the American Physiological translation highlights the importance stroke and featured lectures by Society and the American Heart of understanding the mechanisms Constantino Iadecola (Division Association. I was a member of the underlying damage to the brain and of Neurobiology, Department National Research Committee of the the processes involved in its repair. of Neurology and Neuroscience, American Heart Association. I am Tissue plasminogen activator (tPA) Weill Cornell Medical College, USA) currently an Associate Editor of the is the only approved treatment for on ‘Neurovascular protection by American Journal of Physiology. I am a stroke but its beneficial thrombolytic ischemic tolerance: roles of nitric past-President of the Association of actions are counterbalanced by oxide and reactive oxygen species’, Chairs of Departments of Physiology the limited window of efficacy Alastair Buchan (Experimental and a past-President of the American following the onset of symptoms Medicine Division, John Radcliffe Physiological Society. I currently sit and its neurotoxicity. Ischaemia– Hospital, University of Oxford, UK) on the Finance Committee of the reperfusion (I/R) injury after stroke on ‘Cerebral blood flow alteration American Physiological Society. In leads to disruption of the blood– in neuroprotection following my spare time, I enjoy travelling, brain barrier and potentially fatal cerebral ischaemia’, Giovanni E. cooking and photography. I am cerebral oedema and an increased Mann (Cardiovascular Division, happy to be a member of the incidence of haemorrhage. The initial BHF Centre of Research Excellence, editorial team at The Journal of rapid loss of viable brain tissue in School of Medicine, King’s College Physiology. the ischaemic core region is often London, UK) on ‘Improving followed by subsequent damage to outcome after stroke by targeting Experimental Biology the surrounding penumbra. Rescuing the Nrf2-Keap1 defence pathway the penumbra is a key objective for neurovascular protection’ and 2011 for stroke research, and targeting Ulrich Dirnagl (Department of Washington, DC, USA endogenous defence mechanisms Experimental Neurology, Center 10 April 2011 by which the brain protects itself for Stroke Research Berlin, Charité and recovers from ischaemic – Universitätsmedizin Berlin, The Journal of Physiology sponsored damage may provide novel insights Germany) on ‘Pre-, per-, post- and a research symposium entitled for effective treatment strategies. remote conditioning of the brain: ‘Molecular mechanisms underlying Although ischaemic preconditioning expressway or roadblock?’. neurovascular protection in stroke’ at (‘tolerance to ischaemia’) has been the Experimental Biology meeting employed to improve endogenous The Symposium review articles held in Washington DC, USA on antioxidant brain defences, the will be published in a special 10 April 2011. The symposium cellular mechanisms underlying Neuroscience issue of The Journal of was organised by Giovanni Mann protection of the neurovascular unit Physiology and should be of interest (Department of Physiology, King’s remain to be elucidated. to physiologists and researchers College London) on behalf of The in the field of neuroscience and Journal of Physiology. The Symposium provided an vascular biology. up-to-date overview of basic Stroke is associated with one of and clinical research focused Giovanni E. Mann the highest rates of mortality, with on characterising the cellular ~150,000 cases each year in the mechanisms underlying protection King’s College London

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The runner-up is Thomas Seifert for their paper ‘Glycopyrrolate abolishes the exercise-induced increase in cerebral perfusion in humans’ Paul McLoughlin, the of Experimental Physiology. The journal has made great advances new Editor-in-Chief for under the editorship of David Experimental Physiology Paterson and it is ideally poised to become a leading journal in the field of integrative and translational physiological research. I’m really looking forward to developing the journal over the coming years.’

Under his five-year tenure, Paul Thomas Seifert, James P. Fisher, plans to maintain the journal’s Colin N. Young, Doreen Hartwich, focus on original, integrative Shigehiko Ogoh, Peter B. Raven, Paul physiological research and reviews, J. Fadel & Niels H. Secher (Exp Physiol whilst developing new areas such (2010) 95, 1016–1025). as genomics, bioinformatics and Both these papers are available free imaging – which offer novel insights online: http://ep.physoc.org/ The Physiological Society is pleased into physiological mechanisms. to announce the appointment of The Prizes will be presented on Paul McLoughlin as new Editor-in- He commented: “It is a particularly Tuesday 12 July at Physiology 2011 Chief of Experimental Physiology. exciting time for the journal. in Oxford UK. Whole genome sequencing, rapid Following a long association with the developments in bioinformatics, For details of the 2011 prize and journal and having previously served advances in in vivo genetic entry requirements, see Early Career on the Editorial Board from 2002 to manipulation and powerful novel Author’s Prize on the website. 2010, Paul took up his post on 10 imaging technologies mark a April 2011. new era in physiological and New cover for Experimental pathophysiological research. Paul is currently Professor of Physiology from June 2011 Experimental Physiology is extremely Physiology and Head of Biomedical well placed to publish research from Sciences at the School of Medicine this ‘post-genomic’ era and really and Medical Sciences, University advance the field of integrative College Dublin. He obtained his physiology.’ degree in medicine from University College Dublin in 1983 and undertook his doctoral studies at St Early Career Author’s Thomas’s Hospital London, gaining Prize 2010 Winners his PhD from the University of London in 1993. The winner of the 2010 Experimental Physiology Early Career Author’s Prize Paul’s special interest is the vascular is Thomas D. O’Brien, for the paper biology of the lung. His research entitled ‘In vivo measurements of focuses on the key mechanisms in muscle specific tension in adults and the development and progression children’. of lung diseases such as adult Erratum respiratory distress syndrome, It has been brought to our chronic obstructive pulmonary attention that the article by Richard diseases and cystic fibrosis. His Boyd (Physiology News 82, 24) group are working to identify novel contained a couple of errors: therapies through the exploration of these mechanisms, and their work Richard Keynes was born in 1919 on the pathogenesis of pulmonary and died art the age of 90 (not 93). hypertension is recognised internationally. Also, Alan Hodgkin lived from 1914 Thomas D. O’Brien, Neil D. Reeves, to 1998. Thus, he was 5 years older Paul commented on his Vasilios Baltzopoulos, David A. Jones than Richard Keynes and therefore appointment: ‘It is a great honour to & Constantinos N. Maganaris (Exp the senior author of the cited take up the position of Editor-in-Chief Physiol (2010) 95, 202–210). articles.

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Alison Brading Alison was born in 1939. Her father convalescence, she was informed was an army officer and so, as that she would not be permitted to 1939–2011 was common in such cases, she enter the medical course, because attended boarding school while of her disability. Her subsequent life her father was posted abroad. As then involved an ongoing fight to well as being academically gifted remain independent and mobile. In at school, she was also a superb her early years, with considerable athlete, winning the Victor Ludorum will-power, she was able to walk at Maynard School, Exeter, for her distances using crutches (her athletic achievements. During ‘sticks’, as she called them) which the long vacations she would visit placed considerable demand on her her parents in Nigeria. She made forearms and elbows. As she got three such trips, usually with her older, however, walking became brother Roy but tragically on the increasingly difficult, and she was last she contracted polio, which forced to resort more to a wheel- caused severe disease. She was Alison Brading (photo supplied by Anant chair. Nevertheless she was supreme Parekh, source unknown). hospitalised in an iron lung, first in captain of this transport, issuing firm Africa and then, after an emergency and precise commands to whosoever flight home, in England. She was Alison Brading, who died on 7 was pushing at the time. Alison was fortunate to recover, but suffered January 2011, was a well-known also undeterred by the prospect of permanent and considerable loss of and much loved figure in The international travel. She frequently motor function. She was unable to Physiological Society who will went abroad, whenever possible walk unaided and had restricted use be sorely missed. To those with friends or relatives. Thus, she of her respiratory muscles, which who worked with her she was was able to visit most international caused her particular discomfort a source of inspiration, a vast scientific meetings to which she with bronchial infections and even fund of enthusiasm and scientific was invited. In 1976, for example, the common cold. All this was a knowledge, and a loyal and she travelled to Leningrad, Moscow considerable blow to someone who, supportive friend. Her services to and Kiev with Edith Bülbring and until then, had been outstandingly fit The Society were extensive. She Tom Bolton, visiting palaces and and able. There followed a prolonged was elected in1970, at a time when places of local interest, and finally period of convalescence, which membership was strictly limited, contributing to the Smooth Muscle involved operations to improve the peer-reviewed in competition Symposium in Kiev organised by motor function of her hands. with others, and largely male. She Mykhailo Shuba. Alison also travelled served on Council, was an editor Before Alison’s last trip to Africa extensively in Europe, the United of The Journal of Physiology, and she had applied for, and been States and the Far East, including Chairman of the editorial board accepted, to read medicine at Taiwan and Japan, where she had of Physiological News between Oxford. However, after two years of good friends and contacts. Even in 1990 and 1992. She was elected the final year before her death she to Honorary Membership in 2008. travelled to a scientific meeting in Although stricken with polio in Japan. her late teens, she was fiercely independent and determined to lead Not discouraged by her early a normal academic life. She showed rejection from Oxford, Alison indomitable spirit in dealing with applied to read for a BSc in Zoology her disability. Her early training at Bristol and was accepted. She was in Zoology at Bristol University. obtained a first-class degree and She came to the Department of then chose to study for a PhD with Pharmacology, Oxford, to work Peter Caldwell, an expert membrane with Professor Edith Bülbring of biophysicist, investigating the whom she became a life-long friend, somatic muscles of the nematode establishing a trust to support young roundworm Ascaris lubricoides. scientists when Edith died. She was a She qualified in 1965, but still held familiar and respected personality at Oxford in her sights. It succumbed Physiological Society Meetings, Lady when she joined Professor Edith Margaret Hall and the Pharmacology Bülbring at the Oxford Department Department. Throughout her life, of Pharmacology, as a post-doctoral she made substantial contributions Alison Brading (right) with Edith research assistant, a position which to our understanding of smooth Bülbring (photo supplied by Anant continued until 1971 when she muscle function. Parekh, source unknown). was appointed as a Departmental

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Demonstrator, although she retained with transverse dimensions not summary of smooth muscle a close association with Edith. She greatly different in size from those research, with Edith (and T. Tomita was elected Fellow and Tutor in of the local extracellular space; and A. W. Jones) in 1970 and a later Physiology at Lady Margaret Hall thus it can be difficult to identify edition of this book in 1981. Alison’s in 1967 and University Lecturer ion fluxes specifically across the early pioneering flux work was in 1972; she became Professor of plasmalemma, especially when ion summarised in her paper, presented Pharmacology in 1996 and Emeritus diffusion is slowed by binding to in a symposium at the Royal Society Fellow at Lady Margaret Hall in 2005. extracellular sites. Measurements of (Brading, 1973). Another brief intracellular ion concentrations also publication from this period was Although Alison’s PhD training was required elaborate correction for the with Tadao Tomita. They reported, on nematode somatic muscle, all her extracellular space. at a meeting of The Physiological subsequent work was in the field of Society and published as a refereed mammalian smooth muscle. While in Alison’s meticulous experimental abstract, that smooth muscle can Edith Bülbring’s research group, she approach set the scene for a more generate action potentials in low began to elucidate mechanisms that rigorous application, particularly of sodium solution; indeed the rate generate ionic asymmetry across the the radiotracer technique. Although of rise and overshoot was greater cell membrane. In smooth muscle initially part of the Bülbring group, when extracellular Na+ concentration this was a largely unexplored and she first published her work was reduced (Fig. 1). This was a open field. Although the Bülbring independently with Tadao Tomita crucial step in the discovery that research group was internationally from Nagoya, Johan Setekleiv smooth muscles generally have pre-eminent for the study of smooth from Oslo, and Alan Jones from calcium-based rather than sodium- muscle receptors and contractility, Philadelphia who were visiting based action potentials. A full paper and for the first electrophysiological workers at the time. Edith did not followed (Brading et al. 1969). The studies of smooth muscle using have the now popular habit (and report was thus the forerunner of sharp microelectrodes, little was funding necessity) of group leaders a major branch of smooth muscle known about carrier-mediated ion adding their name to publications physiology and pharmacology, transport. Alison’s work initially from individual members. As a including the clinical use of calcium involved using radioactive tracers result, Alison published only a antagonist drugs that influence and flame photometry. The task couple of original papers with Edith smooth muscle contraction. proved difficult as smooth muscle although she did co-edit a book, cells are generally long and thin ‘Smooth Muscle’, a comprehensive The analysis of ion fluxes and intracellular concentration continued to be a principl focus when Alison established her own laboratory in the Pharmacology Department, following her tenured appointment. She was joined by her first PhD student, Jonathan Widdicombe, and together they studied Na+-dependent ion transport across the plasma membrane. These included Na+/ Na+, Na+/K+ and Na+/Ca2+ transport mechanisms.

The problems associated with radiotracer and flame photometric techniques continued to dog the smooth muscle field. Another technique, however, for recording local ion concentration was coming on-stream through the work of Roger Thomas in Bristol. He had developed the use of ion-selective microelectrodes (ISMs) for monitoring intracellular Na+, Cl– and pH directly inside snail

+ neurones. Alison was joined for a Figure 1. a and d, action potentials, spontaneous and evoked, in normal [Na ]o (Krebs + period by a post-doctoral assistant, solution) and b, c and e, in 10 mM [Na ]o (sucrose substitution). Note the increase in + rate of rise and overshoot in low [Na ]o indicating that the action potential is calcium Richard Vaughan-Jones, from the based. Taken from Brading et al. (1969). Thomas laboratory. Vaughan-Jones

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started to use ISMs initially to types, of the ICRAC (capacitative entry) colleagues she studied the effects of measure intracellular Cl– and H+ ion channel. partial outflow obstruction on the concentrations (with Tom Bolton) in pig bladder. skeletal muscle, and later in cardiac The 1980s saw Alison being joined by muscle. Alison was joined a few many visiting scientists from abroad, While collaborating on her clinical years later by a further post-doctoral and her interest moved increasingly studies, Alison also investigated the assistant, Claire Aickin, again from taenia coli to other smooth fundamental cellular properties of from the Thomas stable. Claire muscles such as vas deferens and detrusor muscles, thus helping to extended the ISM technique to ureter. She formed collaborations fuse more effectively both basic and include mammalian smooth muscle. with surgeons and clinical urologists, clinical science. This work involved Applying the technique was difficult such as Jacek Mostwin, Gary Sibley electrophysiological investigations enough in tissues with relatively and Mark Speakman, and later by doctoral, postdoctoral and with others at the John Radcliffe large cells, such as those of skeletal visiting academics such as Inoue, Hospital in Oxford. This shift into muscle and cardiac Purkinje fibres. Burdyga, Teramoto, Nakayama, more clinically oriented research Adapting the technique to the small Parekh, Bramich and many others. began with investigations of the contractile cells of smooth muscle Investigations were also extended overactive bladder and the resulting required true perseverance, and not to smooth muscles from a variety of incontinence it causes. The quest, inconsiderable serendipity. But with other organs, and from a widening an excellent example of a successful Alison’s enthusiasm and support, range of species, including pigs and translational research axis for a and Claire’s expertise, it was even humans. basic scientist (and long before achieved. This led to a major series of it was fashionable or politically In addition to her research on the studies on the membrane transport encouraged) would occupy Alison bladder, Alison in later years studied of Cl–, H+ and Na+ ions, including the for the next twenty-five years. Her the properties and responses to first description in smooth muscle research involved examination drugs of anorectal smooth muscles. of the Cl–/HCO – exchanger and 3 of bladder innervation and the As with much of her earlier work, its participation in the process of effects of potentially useful drugs this was a largely unexplored area, intracellular pH regulation. Further to relax the detrusor muscle. In ripe for development. Her interest + ISM recordings of intracellular Na addition, with the help of her clinical focused on two areas: to what led to important functional work on the Na+/Ca2+ exchanger (Fig. 2; Brading et al. 1987), a transporter whose expression in smooth muscle had previously been questioned. This ISM work in the 1980s (and reviewed in Brading & Aickin, 1990), produced several milestone publications on the fundamental physiology of smooth muscles.

Alison’s work on Na+/Ca2+ exchange was paralleled by her studies of smooth muscle contractile activity, particularly the role of the sarcoplasmic reticulum as a receptor-operated store of releasable Ca2+. Indeed, she was among the first to moot for smooth muscle the possibility of capacitative entry of extracellular Ca2+ directly into intracellular stores as a means of rapid refilling, a phenomenon that to the present day is still being researched and debated. Indeed, a later doctoral student,

Anant Parekh, now Professor of 2+ Figure 2. Sodium–calcium exchange in ureter of guinea-pig. Raising [Ca ]o Physiology at Oxford, cut his teeth hyperpolarizes the membrane, causes contraction due to the increase in calcium + + + 2+ in Alison’s laboratory on studies of entry, and a reciprocal reduction in [Na ]i. Lowering [Na ]o allows the Na /Ca Ca2+ regulation, and has gone on to exchanger to hyperpolarize the membrane, while the reduction in calcium extrusion specialise in the study, in various cell results in contraction (Fig. 1 from Brading et al. 1987).

Physiology News | No. 83 | Summer 2011 | www.physoc.org OBITUARIES PN 59 extent derangement of bladder expertise. For her, it was a lifelong (detrusor) function was associated love affair with science, and with the with alterations in anorectal teaching of science. smooth muscle function, and whether diseases such as irritable In the end, Alison was defeated bowel syndrome were associated physically by her disability. She with changes in the properties of contracted pneumonia and, with anorectal smooth muscle. Many of her respiratory difficulties, this was her studies were again collaborative, hard to fight for a prolonged period. notably with Mortensen, a But Alison’s spirit, even during this consultant gastro-intestinal surgeon last illness, was never dimmed. In at the John Radcliffe Hospital. all, she should be celebrated for Most recently Alison studied her tenacity in the face of early the innervation and function of misfortune, for her energy and the anal sphincter and rectum indomitable enthusiasm, for her in collaboration with Radomirov loyalty and support of colleagues and Ivancheva of the Institute of and friends, and for her broad and Neurobiology, Bulgarian Academy of Alison Brading at the tiller of her narrow far reaching scientific and academic Sciences. boat (photo courtesy of Roger Thomas). achievements. There is no doubt The Physiological Society has been the In her determination to be mobile better for her. teacher, who talked with, rather than after her convalescence from polio, at, her students. She did not tolerate Alison learned to drive a car in 1 fools gladly, but she was genuinely Tom Bolton and Richard which the controls were modified. 2 caring about students’ welfare and Vaughan-Jones Although this could sometimes academic development. And she be rather scary for an unwary 1Biomedical Sciences, St George’s was passionate about enthusing passenger, it enabled Alison to lead University of London, London, UK students with an understanding and an essentially independent life, respect for science. That was also 2 eventually commuting between Burdon Sanderson Cardiac Science true of her approach to her own her Department in Oxford and Centre, Department of Physiology, scientific learning. In her early years her cosy cottage in the outlying Anatomy & Genetics, University in the Department of Pharmacology village of Thrupp. In later years, her Laboratory of Physiology, Parks at Oxford it was not uncommon for relatives moved in next door, so Road, Oxford, UK Alison to appear suddenly on her that family members were gathered ‘sticks’ in one’s office, enthusing around her. The house bordered the References about a marvellous paper she had Oxford canal, and so she bought just read in the most recent edition Brading AF (1973). Ion distribution and a narrow-boat and enjoyed trips the role of calcium in cellular function. Ion of The Journal of Physiology, often in the company of friends and distribution and ion movements in smooth in an area far outside her own muscle. Philos Trans R Soc Lond B Biol Sci 265, students. Just as she was captain of 35–46. her wheel-chair, she was captain of her boat, issuing firm commands Brading AF & Aickin CC (1990). Ions, transporters, exchangers and pumps in from the wheel-house, with crew smooth muscle membranes. Prog Clin Biol Res members dispatched regularly onto 327, 323–343. the towpath to operate lock-gates Brading AF, Aickin CC & Walmsley D (1987). and wave her through. For those An investigation of sodium–calcium exchange involved, the trips were immensely in the smooth muscle of guinea pig ureter. J sociable, and always memorable. Physiol 391, 325–346. Brading AF, Bülbring E & Tomita T (1969). The The job of a Tutorial Fellow at Oxford effect of sodium and calcium on the action requires maintaining a research potential of the smooth muscle of the guinea- group, but also admitting, teaching pig taenia coli. J Physiol 200, 637–654. and mentoring undergraduate Brading AF & Tomita T (1968). The action students, in Alison’s case in Medicine potential of the guinea-pig taenia coli in low sodium solution. J Physiol 197, 30P–31P. and Physiological Sciences at Lady Margaret Hall. Her dedication to her Bülbring E, Brading AF, Jones AW & Tomita T (1970). Smooth Muscle. Edward Arnold, research group was matched by her London. commitment to her College and her Bülbring E, Brading AF, Jones AW & Tomita undergraduate charges. She was T (1981). Smooth Muscle: an Assessment of an extremely gifted and vigorous Photo kindly supplied by Karen Brading. Current Knowledge. Edward Arnold, London.

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James (Jim) Edward We also did some experiments together and he wrote the software Pascoe for an early laboratory computer, 1924–2011 the Z80. He would stand in the lab dictating the machine code while Jim Pascoe was a Reader in Physiology Michael Duchen and I would listen at University College London. He was in awe and write it down. And the born in Falmouth and at the age of program worked, enabling us to 12 moved to Penzance. Thence at measure changes in sensitivity of age 16 he worked at his mother’s primary afferent nerve terminals in behest in an apprenticeship at Boots, the spinal cord in real time. transferring to Plymouth College to start his studies for the Diploma in He had a great fund of stories about Pharmacy which he completed at the the previous heads of department School of Pharmacy, London where he had known including G.L. Brown, he then became a Junior Lecturer Andrew Huxley and Doug Wilkie – and where he met Margaret, a Photo with kind permission of Alice Pascoe. stories always told with great good pharmaceutical chemist who became humour and affection. He had the his wife. He studied Physiology ‘sod them’, and he never really got greatest respect for the College and whilst working nights in Boots, round to publishing much after that its secular traditions, and for the Piccadilly and so came to UCL at the rejection. Such is the untoward power Department of Physiology with a long invitation of G.L. Brown, the head of of editors. history going back to the foundation department, and then on to the staff of the College in the 1820s. where he was known for his absolute He was a sailor, and Ann Silver integrity and devotion to the subject. describes one of his trips: We worked most closely during the In 1954 he was elected a Member of “He sailed his boat along the south 1980s when there was considerable The Physiological Society. coast and round to East Anglia, then activity in the Palace of Westminster up the River Deben to Woodbridge. over the issue of animal experiments. In the ’60s and ’70s the Annual This involved his successful The anti-vivisectionists were very General Meeting of The Physiological negotiation of the tricky Horse active at this time and increasingly Society was always at UCL. There shoal at the mouth of the river. I violent, for example, attempting would be Jim Pascoe listening to was very impressed by this but what to set fire to the house of a Nobel the Communications in the lecture impressed me even more was that Laureate and attaching a bomb which theatre, often alongside G.L. Brown, when I drove over from Cambridge fell off and detonated under the car and clearly a close friend. Sometimes to show him some of north Suffolk, of a Bristol physiologist. The Act of I thought they appeared very similar he made sandwiches for us without Parliament under which we all did except that Jim was thinner. He had benefit of breadboard, slicing the loaf experiments supervised by the Home that intent questioning look that held vertically between his knees”. Office Inspectorate had been passed did not allow you to get away with Furthermore she describes on this in 1876. It was clear that with the any nonsense. At the same time he trip how “We visited Orford Castle changes in biological science there was wonderfully jolly and positive if and several splendid churches. Jim was a need to update the Act. Thus he judged you to be serious about couldn’t resist the urge, left over we became involved with the political advancing the subject. At that time I from his choirboy years, to test the process in The Lords and the House did not know him at all well – he was acoustics by bursting forth into of Commons. There was a Private to me a figure in The Society. Always hymns and psalms – occasionally Member’s Bill in the Lords and in the something of a stirrer, he enjoyed startling some poor lady engrossed in Commons, more seriously, there tweaking the tail of The Physiological arranging the flowers.” was a Private Member’s Bill and a Society Committee. After joining the Government Bill succeeding it. There Committee in 1979, he remained the I came to know him well after I went were a number of MPs who were blunt-speaking champion of many to work at UCL. He was a marvellous positive supporters – Sir Nigel Fisher, causes. ally who would drift in to see me Mr Ray Mawby and some others, from time to time to discuss the but the most outstanding one was He quite naturally, perhaps inevitably, department and check out how I was Tam Dalyell. As ever he was brave, had something of a falling out with getting along running it. We would articulate and hugely supportive of The Journal of Physiology where the talk about all manner of problems the scientific endeavour. editors rejected a paper on the and he was always absolutely frank grounds, so he was told, that there with me, telling me if he thought During discussion of the Private was too much pharmacology in it. I was wrong about some problem Member’s Bill in the Committee He was of course in good company and also if I was correct. Such Stage there was an accusation since the great Nobel Prize winner straightforward advice from someone that experiments were being done Eccles had a paper rejected. Jim’s of absolute integrity was invaluable involving cutting off the feet of reaction in the demotic argot was to me. rabbits, and this procedure should

Physiology News | No. 83 | Summer 2011 | www.physoc.org OBITUARIES PN 61 be stopped. It turned out that there ‘blackwork embroidery’. He took were no such experiments in the the photographs of embroideries UK and if there were such things and materials that she needed for done in any country they were on her book, a technology that he had anaesthetised mice. Tam Dalyell learned as a physiologist and in suggested that Jim and I bring to the which he was expert. They travelled House a rabbit and a mouse which abroad with Jim as the significant he could then produce during the other, a reversal of earlier roles debate to show to our opponents the which he greatly enjoyed. Such was difference between these species. his expertise that he then went on This we did and were, all four of to photograph the work of others us, smuggled into the Palace of in the Embroiderers Guild as well as Westminster through a back door by the historical collection of the Guild Tam Dalyell and so to the Committee housed at Hampton Court Palace. room. Naturally the display of these animals caused embarrassment to After Margaret died in 1995 he the other side and even some mirth. stayed on in Molesey and eventually The Chairman of the Committee, Mr in 2004/5 went to live in Chacewater Ted Leadbitter, did not demur. Not in Cornwall and latterly in Truro. long after this the Bill was withdrawn. He was cremated at the Penmount There followed the long period Crematorium a place where my father of argument and debate over the used to take services and was also Government Bill in which Tam Dalyell cremated, a touch of continuity that was steadfast and Jim was continually Jim would have greeted with a merry involved along with a small group laugh. He also laughed heartily when of other physiologists including Ann A cartoon of Jim, drawn by Ralph Sallon long ago I told him that my father Silver, Cecil Kidd, Tony Angel and (with kind permission of Alice Pascoe). had made an insurance claim for his David Whitteridge. Robert Comline cassock which caught fire while he the Treasurer of The Society insured without him we would all have been was waiting to take a service at the that whatever funds were needed much less effective. same crematorium. were available. He retired from UCL Physiology and He was a great colleague and a dear Through this long period, perhaps then, as he said, was a bag or case friend to many of us; he lived life and 4 or 5 years, Jim retained his sense carrier for his beloved wife Margaret was much loved. of humour and moral support and who had become an authority on Tim Biscoe

An aside from Ann Silver The oldest Members of The Society will have started their animal experiments under legislation enacted in 1876. Since this was long out-of-date by the middle of the 20th Century, it is understandable that from about 1965 onwards several moves were afoot to modernise the legislation. The Physiological Society and the Research Defence Society (now ‘Understanding Animal Research’) were at the forefront of those keen to weed out any new, ill-conceived regulations that could hinder research with no proportionate benefit to animals. Tim Biscoe indicates above that Jim was particularly active in these efforts. As well as priming Tam Dalyell at Parliamentary Subcommittees, he spent many a Saturday in Leeds with Cecil Kidd, Bernard Ginsborg and me, discussing the latest government proposals and formulating The Society’s response. In addition, he once came to Cambridge for a memorable meeting in St Catharine’s College at the invitation of Robert Comline, then the Society’s Treasurer. Although productive, it was memorable for the wrong reasons. A cold, overcast day, Robert took us into Hall for a self-service lunch before we began our discussion. The lighting was rather poor and the most recognisable offerings were the salads but Robert insisted that we needed something hot. He hustled us along the counter to a rather tempting steak and kidney pudding with a thick suet crust; to this Cecil and I added a good helping of cabbage. Jim, invoking his choirboy days, smugly said that as it was Friday he would have the fish. As soon as Cecil and I started to eat we realised our mistake – our pudding was not full of steak and kidney but sweet, stewed, plums that went none too well with the cabbage. To add to the problem, I had a stone – plum not kidney – and not wanting to disturb Robert, I stored it, hamster like, in my cheek. Had he been aware of our dilemma, the ever-hospitable Robert would have insisted Cecil and I started again. When Jim and I went to fetch the coffee he said ‘What the hell is the matter with you two?’ Inevitably there was much scarcely suppressed mirth whenever we caught each other’s eyes during our afternoon deliberations. A year or so later, The Committee met (I think in Leeds) ready for the Scientific Meeting beginning the following afternoon. When it came to the question of the pre-Meeting lunch, Robert said, indicating Jim, Cecil and me, ‘You can’t trust this lot not to disgrace us.’ Apparently our failure to identify puddings had been revealed to Robert, during a convivial gathering the previous evening, after I had gone off to bed.

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Sidney Montague cardiovascular disease. At the end of the 1950s his attention began to Hilton move to the regulation of the heart 17 March 1921–28 January 2011 and vascular system by the brain, and it was here that arguably he made his Sidney Montague Hilton who died biggest contribution. He had become recently had been Bowman Professor fascinated by the hypothesis of and Head of the Department of Cannon concerning the fright–flight– Physiology at the Medical School fight response (defence response) in Birmingham from 1965 to so fundamental to survival in wild 1983. He was a distinguished animals but when over-expressed physiologist who made leading in humans can lead to chronic contributions to understanding hypertension. At the time little was brain control of cardiovascular known as to how the brain initiated changes accompanying behavioural the defence response. Hilton realized reactions associated with sudden that a key feature of this response is a emergencies like the fear–flight–fight selective vasodilatation in the skeletal reaction. These have relevance to muscle vascular bed and this could psychosomatic disease and to chronic Sidney Hilton on his arrival in possibly be used as a marker to locate hypertension. Hilton was in the Birmingham in 1963. relevant brain regions in experimental vanguard of thinkers who provided animals. To study this he needed experimental evidence against (MRC) at Mill Hill, London, where to gain experience of behavioural the long-held view that there is a he worked in the division led by methods and for this there was no single brain centre controlling blood another great figure in biomedical better place than the laboratory of pressure. He was an excellent lecturer science, Professor Feldberg, who had Professor Kornoski (a former pupil and travelled widely. made major discoveries concerning chemical transmission between of Pavlov) at the renowned Nencki Sidney Hilton was educated at St nerves. Hilton’s major interest at the Institute of Experimental Biology Paul’s School, London from where time was the local mechanical and of the Polish Academy of Sciences he won a place to study medicine chemical control of blood vessels. He in Warsaw. Thus, he returned to at Cambridge having gained an found that the widespread dilatation the land of his grandfather, a Polish exhibition at Jesus College. During the in the large arteries supplying Jew who had emigrated to London second World War he was allowed skeletal muscles during contraction from Lodz in the early part of the time to take the Part II Honours depended on transmission by 20th century. It was at the Institute Tripos course in Physiology following smooth muscle conducting elements in Warsaw that he was fortunate which he did his clinical training at in the walls of the arteries. This to team up with Andrzej Zbrożyna, Guy’s Hospital, London where he conducting system turned out a brilliant young behavioural subsequently held several house to be functionally important for physiologist who later joined Hilton at appointments. He then was required ensuring that a local stimulus caused the MRC Institute in England. The two to do National Service and for this he a widespread opening up of the were then joined by Viv Abrahams served in the RAF at the Institute of arteries supplying an organ. This and together they used the muscle Aviation Medicine, Farnborough. Here was probably the earliest evidence vasodilatation in anaesthetized he assisted in developmental work pointing to a chemical mediator that animals as an identifying feature on the design of flying equipment we now know is nitric oxide, a major when electrically stimulating at sites and with Professor J Beattie at the dilator in all vascular beds although throughout the brain. Subsequently Royal College of Surgeons carried Hilton concluded at the time it was repeating these experiments in out studies on ways to accurately a mechanically driven mechanism. conscious animals it was shown these measure oxygen levels in the blood, There followed studies on blood flow were the same regions that evoked non-invasively. On leaving the RAF in and secretion in salivary glands which defence responses. These studies 1950, Sidney returned to Cambridge in collaboration with Dr Graham provided the first description of brain to the lab of Professor Adrian (a Lewis provided the first evidence regions that are essential for initiating Nobel Laureate, later to become that the increase in blood flow the typical pattern of cardiovascular, Lord) who had done ground-breaking essential for secretion was not solely respiratory and skeletal muscle work on the generation of vasomotor dependent on nerve-mediated release changes that comprise defensive nerve activity by the brain, a key of acetylcholine but also on the behaviour. More recently modern element in the control of blood simultaneous release of polypeptides. brain imaging techniques and deep pressure. It was here that he decided Subsequently the polypeptides brain stimulation, which is primarily on a career in physiological research became recognized by others as used to treat a variety of brain- and possibly the seeds were sown important agents for ensuring blood based disorders, has confirmed that for his later major contributions. flow in organs such as the heart and similar brain regions are involved He then joined the scientific staff other vascular beds, and were later in aggressive behaviour in humans. of the Medical Research Council targeted in clinical treatments for From these studies it was clear to

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Hilton that no longer could a single with a special relationship to the cardiovascular and respiratory localized area in the hind brain be University of Birmingham. The early recordings were collected on a considered responsible for blood days of Hilton’s time in Birmingham multi-channel chart recorder, all pressure regulation, a concept were very happy ones for budding analysis was manual, manuscripts that had stood for 50 years and physiologists who not only enjoyed were first written out in long-hand, appeared in every textbook. It was the intense 24/7 research activity and there was no PubMed. He had an now necessary to define a model but also the social events of parties office adjoining our lab, and would based around discrete reflex control in Gerta Vbová and Hilton’s home as pop in enthusiastically, puffing on of parallel pattern generators well as other social occasions. During a cigar, inadvertently triggering for the cardiovascular responses this time Hilton served as Secretary a nasopharyngeal reflex in the accompanying various types of of The Physiological Society where anaesthetised rat with the smoke. behaviour. These studies which his irreverent sense of humour was were started at the MRC Research not always enjoyed although his I had read most of his published work Institute, continued with Andrzej contributions to scholarly debate at on central cardiovascular control Zbrożyna, who moved with Hilton scientific meetings of The Society during the course of my PhD, and it on his appointment to the Bowman were often a highlight and much had captured my imagination. His Chair of Physiology in Birmingham, missed on his retirement. In 2004 he writing style reflected his personality, a university where his younger was awarded the distinction, given particularly in his reviews: in person brother Peter, a highly distinguished to only a few, of being elected as an he was engaging and witty, with a mathematician, had recently Honorary Member of The Society. formidable intellect. Where most occupied the chair of mathematics. physiologists grappled with ‘single Interestingly Dr Zbrożyna had He retired in 1984 and moved with issue’ topics, he quite happily took also played a significant part in his third wife Mary to what up to on multi-functional, integrated helping Hilton’s future second wife then had been her family holiday responses that were fundamental Gerta Vbová and her two children home, a lovely rural but somewhat to survival of the organism. He was to accomplish a remarkable and remote setting on a mountain side, very territorial in ‘owning’ this area, harrowing epic escape from Soviet- above Lanelltyd near Dolgellau in and like a gamekeeper apprehending occupied Czechoslovakia described West Wales. Despite being quite cut a poacher, would tackle head-on in Professor Vbová’s recent book off from the scholarly environment anyone who wasn’t going about it ‘Betrayed Generation’ (for a review of he appeared to be very happy, much correctly! Before I joined his group this book, see p. 41). enjoying the attention of Mary’s I saw him doing this on more than children and grandchildren. Recently one occasion at scientific meetings Hilton was a complex, perhaps he had renal failure but was looking – it wasn’t just what he said, but egotistic, person whose measure forward to reaching his 90th birthday the subtly disparaging inflections of mischief in scientific and in March of this year but this sadly in his voice – which later left me administrative confrontations often was not to be. He is survived by his apprehensive ahead of my PhD led to acrimony and this perhaps third wife Mary and a number of viva, and always somewhat in awe resulted in his not receiving the full children from previous marriages. of him. Fortunately I avoided ever credit for his scientific contribution. being on the receiving end of this; However, the move to Birmingham John Coote and Mike Spyer on the contrary, he was always very provided an opportunity to build encouraging and supportive. Sidney up a department that had lost Will Redfern (former postdoc of operated in a golden age of ‘holistic’ most of its staff. Hilton built up SMH) continues: systems physiology (not to be its teaching and its research to a Sidney Hilton was the external confused with the current trend for state where it became not only one examiner for my PhD in 1982, and ‘systems biology’) in the UK. He and of the strongest departments in after the viva he invited me to join his his numerous collaborators have left the University but also in the UK, group as a postdoc. I was honoured a lasting legacy in our understanding whilst its research quality became to be asked, and am still proud to of centrally co-ordinated patterns of recognized internationally. It was have been a part of the last research responses to internal and external also a happy department and during group he had before he retired. I challenges and perturbations. I joined this time it nurtured many ‘prima was funded jointly by the MRC and the pharmaceutical industry after donnas’ providing three Heads of ICI, and worked with him from my postdoc at Birmingham, and Departments at other universities 1982 to 1985 in the Department of eventually moved to AstraZeneca, as well as a Director of the Army Physiology at Birmingham. The bulk Alderley Park (formerly ICI), which Personnel Research Establishment of my project involved a comparison I suppose could be considered a in Farnborough. During this time of electrical stimulation of brain return on their investment. I last saw Hilton also helped to establish the stem ‘defence areas’ (hypothalamus Sidney at his 80th birthday ‘do’ in Physiology Department in the new and periaqueductal grey) in the rat Birmingham in 2001, where he was in Medical School in Salisbury (Harare), with pharmacological stimulation fine form, and I’m so sorry he didn’t Southern Rhodesia (now Zimbabwe) using an excitatory amino acid. Back make it to his 90th. It wasn’t like Sid that had been founded in 1963 then, everything was analogue, to miss a party.

Physiology News | No. 83 | Summer 2011 | www.physoc.org 64 PN OBITUARIES

John Archibald his employment for some years as a member of the MRC External Browne Gray FRS Staff. This enabled him to move to 1918–2011 his home area in Devon and base himself at the Marine Biological Died Jan 4th 2011, age 92 Association Labs in Plymouth. From Pacinian corpuscles to the There he worked with his old friend MRC Eric Denton on the physiology of John Gray was one of an amazing the lateral line in fish, publishing generation of physiologists who two papers in Nature in 1979 and contributed so much to our subject 1982, as well as a number of other in the years immediately following papers through to 1991, mostly in the Second World War. the Journal of the Marine Biological Association. After studying at Clare College, Cambridge and UCL Medical School, For me, John Gray was an John Gray graduated in medicine inspirational teacher and supervisor. in 1942. He then did wartime As mentioned at the start, he physiological research, much of it at belonged to the post-war cohort sea with the Pacific Fleet. Post war John Gray from his days at the NIMR, Mill whose memorial is surely that their he worked at the MRC’s National Hill. research provided the foundation Institute for Medical Research (NIMR) that fostered the development of at Mill Hill with J.L. Malcolm and G.L. 122, 610–636) was a technical physiological science over the last Brown (always known as G.L.). tour-de-force and significantly 70 years. pushed forward our understanding The topic was sensory transduction of mechanoreception. Bruce Lynn of touch and pressure and whether this involved direct mechano- John Gray played an active part in planning and delivering the teaching The Society also notes with regret electrical transduction or occurred the death of Francis (Frank) Beswick, via an intermediate chemical programme at UCL. A big project was trying to get more integration who died peacefully on 11th January step. The parallel was with the 2011 and was elected a Member in then great controversy about across departments within the biomedical degree programmes. 1960, and Carlos Chagas Filho, who synaptic transmission – chemical was elected a Member in 1952 or electrical? G.L. was firmly in He also insisted that all physiology the chemical camp on synaptic students get serious university-level transmission, but one of the main teaching in physics and chemistry. The Journal of Physiology achievements of John Gray was to His Inaugural Lecture (he became Symposia 2012 show that certainly for mammalian a professor in 1959) was entitled mechanoreceptors, chemical The Place of Physiology in University Neuromuscular function, muscle intermediates were not involved. Education and he argued for the phenotype and mass regulation When G.L. moved to head Physiology value of physiology as a broad-based Biomedical Basis of Elite at UCL he soon recruited John Gray education in life sciences. John Gray Performance became Dean of the Science Faculty as a Reader. John Gray started his Queen Elizabeth Hall,London, UK, at UCL in the early 1960s and found work on Pacinian corpuscles at 20 March Mill Hill and continued it at UCL. himself much distracted by academic Speakers: Simon Gandevia, Peter In a series of papers from 1950 to administration. Zammit, Stefano Schiaffino, Jan 1957 in The Journal of Physiology he In 1966 he was offered the post Lexell and Marco Narici established the rapidly adapting of second secretary at the MRC, nature of this mechanoreceptor, with the prospect of soon taking Exercise Metabolism demonstrated its remarkable over as Secretary (in practice Chief Biomedical Basis of Elite sensitivity to rapid movements Executive). In the end he decided Performance (threshold less than 1 µm) and to desert research and ran the MRC established the importance of ion Queen Elizabeth Hall, London, UK, until 1977. He explained part of his 20 March gradients for the receptor potential. thinking to me as follows: “If I am The experiments were mostly in vitro to become an administrator, better Speakers: Michael J. Rennie, Bente and were carried out with a series to do this in an organisation that Kiens, Erik Richter and Martin of students and postdocs including understands administration”. Gibala Peter Matthews, David Inman, Jack Diamond and notably M. Sato. The When he finished as MRC Secretary (For 2011 Symposia see Gray and Sato paper in 1953 (J Physiol he arranged a deal that ensured Noticeboard on p. 34)

Physiology News | No. 83 | Summer 2011 | www.physoc.org The Biomedical Basis of Elite Performance 19–21 March 2012 Queen Elizabeth II Conference Centre London, UK

• Cardiac, respiratory and vascular aspects of performance • Neuromuscular function, muscle phenotype and mass regulation • Drugs in sport Published in: • Exercise metabolism The Journal of Physiology Experimental Physiology British Journal of • Thermoregulation Pharmacology The Scandinavian Journal • Sport and exercise medicine of Medicine and Science in Sports • Genomics and exercise

www.bbep2012.org

PN_190411.indd 1 4/19/2011 5:19:47 PM stressor Stressor

(unconscious) (conscious) Amygdala, BNST Orexin neuron ORX Cerebral cortex Orexin Neurons Co-transmitters

Emotion CV Resp BTPain

Body temperature Fight-or-flight (=defense response) Cardiovascular, Respiratory, Analgesic responses

Hypothetical diagram showing the key role of orexin neurons in the fi ght-or-fl ight response (p. 15).

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