PHYSIOLOGY NEWS

spring 2011 | number 82

Bob Edwards’ pioneering research into fertility The cost of breathing in heart failure A tribute to Richard Keynes’ legacy What makes some axons more excitable? PUBLISH PERISH

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UK • GERMANY • USA • BRAZIL • CHILE • INDIA • JAPAN • CHINA • MALAYSIA • NEW ZEALAND • AUSTRALIA PHYSIOLOGY NEWS Editorial Patricia de Winter 3 The Society’s dog. ‘Rudolf Magnus Meetings gave me to Charles Sherrington, who Physiology 2011 – University of Oxford 4 gave me to Henry Dale, who gave Epithelia & Membrane Transport, UCL 5 me to The Physiological Society in Cross Themed Meeting Stephen Kocher 6 October 1942’ The GL Brown Prize Lecture reunites with his descendants Published quarterly by The Physiological Society Helen Brown 7 The run-up to IUPS Thelma Lovick, Bridget Lumb 8 Contributions and queries Senior Production Editor My 10 key papers John Coote 10 Jill Berriman Opinion Editorial Administrator Peter Wilmshurst, a Consultant Cardiologist at the Royal Maev Fitzpatrick Shrewsbury Hospital, gives his personal experience of the The Physiological Society Publications Office PO Box 502, Cambridge CB1 0AL, UK effect of the English libel laws on scientific debate 16 Tel: +44 (0)1223 400180 Living History Fax: +44 (0)1223 246858 Bob Edwards – 2010 Nobel Laureate in Physiology or Medicine Email: [email protected] Richard Gardner 18 Website: www.physoc.org Letter from Portland, Oregon Magazine Editorial Board Rebecca St George 23 Editor History Austin Elliott R.D. Keynes (1913–2010): the legacy from two papers University of Manchester, Manchester, UK Richard Boyd 24 Deputy Editor Science News and Views Patricia de Winter Two arms as one: position and movement sense in an elbow- University College London, London, UK matching task Masahiko Izumizaki, Uwe Proske 26 Members A novel way to test human motoneurone behaviour during Angus Brown muscle fatigue Chris McNeil, Peter Martin, Simon Gandevia, University of Nottingham, Nottingham, UK Janet Taylor 29 Sarah Hall Cardiff University, Cardiff, UK What makes some axons more excitable than others? Munir Hussain Louise Trevillion, James Howells, Susan Tomlinson, Hugh Bostock, University of Bradford, Bradford, UK David Burke 32 John Lee NOS1, S-nitrosylation and cardiac contraction Rotherham General Hospital, Rotherham, UK Honglan Wang, Mark Ziolo 36 Thelma Lovick NO is pivotal in motor pathologies, but is it harmful or University of Birmingham, Birmingham, UK protective? Bernardo Moreno-López 39 Samantha Passey The ‘cost’ of breathing in heart failure Bristol Heart Institute, Bristol, UK Thomas Olson, Bruce Johnson 42 Foreign Correspondents John Hanrahan Techniques McGill University, Montreal, Canada Western blottintg. Part II Patricia Leoni 44 John Morley Noticeboard 46 University of Western Sydney, NSW, Australia Unbelievable! 47 © 2011 The Physiological Society Book reviews 48 ISSN 1476-7996 (Print) From the archives Austin Elliott 49 ISSN 2041-6512 (Online) Society pages The Physiological Society is registered in England as a Ask a physiologist! 50 company limited by guarantee: No 323575. Registered office: Peer House, Verulam Street, London The Society’s journals WC1X 8LZ. David Paterson, the new Editor-in-Chief for The Journal 51 Registered Charity: No 211585. An interview with William Large, the outgoing Editor-in-Chief 51 Printed by The Lavenham Press Ltd Optogenetics themed issue 53 Statistics: Little and often, softly softly, don’t scare the horses, oaks and acorns Gordon Drummond 54 Affiliate news Life as a Physiological Society Affiliate representative Samantha Passey 55 Dearest Affiliate Federico Formenti 55

Cover image: background, guinea pig corpus luteum (from Dr Dhia Mukhtar, University of Southampton; large photo of Bob Edwards Advancing the science of life (courtesy of Ruth Edwards): top to bottom of 3 images on left: female reproductive system (from SPL); rabbit blastocyst; and a 12 week fetus. p. 18. PHYSIOLOGY NEWS Action points Guidelines for contributors In this issue Grants These guidelines are intended to assist Welcome to the Spring 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 The centrepiece of this issue is Society Symposia Grants, articles are written in a journalistic style obvious – Sir Richard Gardner’s Outreach Grants, International so that they will have an immediate riveting personal account of Bob Teaching and Research Grants interest value for a wide readership and Edward’s Nobel Prize winning work and the Vacation Studentship and will be readable and comprehensible to non-experts. Scientific articles should (p. 18), which Richard himself was a Departmental Seminar Schemes. give a good overview of a field rather part of in the 1960s and 70s. With For full information, please visit: than focus entirely on the authors’ own some wonderful hitherto-unseen www.physoc.org/grants research. contemporary photographs, we Format of articles hope it is a fitting tribute to Bob Membership applications The main message or question posed Edwards, a real visionary and a Applications for membership should be introduced in the first worthy (and long overdue) Nobel of The Physiological Society are paragraph. The background for the Laureate. considered on a rolling basis, topic should then be established, and a decision is normally made leading up to the final conclusion. In some ways this Spring 2011 within 15 working days. For full Length of articles issue is a kind of ‘serendipitous’ information, please visit: This will be determined by the subject special one for the history of www.physoc.org/membership matter and agreed with the Senior late 20th century physiology. Production Editor. Thus, Richard Boyd comments Is your membership information Submission of articles on the lasting legacy of another correct? Authors should submit articles as a Cambridge physiologist, Richard Please check and update your Word document attached to an email. Keynes (p. 24), while John Coote details at www.physoc.org, under Illustrations should be sent as separate gives us his ten key papers on the attachments (see below) and not ‘My Physoc Profile’. embedded in the text. brain–heart connection (p. 10), which span the years 1960–2001. Illustrations and authors’ photographs Meanwhile, nerves and muscles Authors are encouraged to submit – including heart – also form a Physiology News diagrams, drawings, photographs or (wholly spontaneous) theme in our Deadlines other artwork with their articles and a photograph of the author(s) should Science News and Views articles. Letters and articles and all other accompany submissions. Illustrations contributions for inclusion in the and photographs may be colour In Society matters, we welcome Summer 2011 issue, No. 83, or black and white, and preferably David Paterson as the new should reach the Publications TIFF, JPEG, PNG, PDF or AI files with a Editor in Chief of The Journal of minimum resolution of 300 dpi. Office ([email protected]) by Physiology (p. 51), and interview 7 April 2011. Short news items References his predecessor, William Large, and letters are encouraged, and Authors are requested to keep the now departed for retirement in number of references to a minimum – can usually be included as late the sunnier climes of Southern preferably no more than two or three. California. Though sadly we don’t copy if space permits. Please cite all references in the style of find out if William is going to be Suggestions for articles The Journal of Physiology (see Information and Guidance for Authors at surfing. Suggestions for future articles are http://jp.physoc.org). welcome. Please contact either Finally, even though 2011 is not the Editorial Administrator or a The Physiological Society permits the long underway, meeting organisers single copying of individual articles for are already gearing up for this member of the Editorial Board private study or research. For permission of Physiology News (see contents to copy or reproduce for any other Summer’s meetings (pp. 4–5) page for details). purpose contact [email protected]. and indeed for some a couple of years beyond that (p. 8). Start Opinions expressed in articles and letters planning now ... given the fairly Physiology News online submitted by, or commissioned from, grim news on UK science funding Physiology News online: Members, Affiliates or outside bodies are (see Editorial), I dare say we will be www.physoc.org not necessarily those of The Physiological Society. needing a get-together and a bit of mutual support. Austin Elliott Editor EDITORIAL PN 3

All change please… onerous, on the face of it, but the devil, several critical decisions to be taken as ever, is in the detail. The weekly before universities can plan properly government funding repayment rises to £34.61 per week for for the future, including: 1) the those earning £41,000, for example. amount of funding that will remain for terminates here And the £21,000 earnings threshold laboratory and clinical education; 2) The result of the votes on higher refers to earning some years in the what level of funding might continue education (HE) funding and student future; comparisons to current earnings for postgraduate taught programmes, finance in the House of Commons suggest most graduates will start loan where there is no underpinning loans on 9th December 2010 will change repayments as soon as they get their package; 3) the conditions under the face of English higher education first full-time job. Overall, the minimum which a university may be permitted to entirely. Anyone hoping that the debt a student will incur via fees is charge a fee higher than £6,000; and 4) House of Lords would rescind the £18,000, to which the cost of student whether the government will maintain coalition government’s victory was loans for living and other expenses will a cap on student numbers at each sadly disappointed, as the Lords’ votes need to be added. For the fees loan, university”. Professor Grant is quite on 14th December supported the those earning above the repayment correct – the proposals, and votes, proposals by a clearer majority than threshold will be charged interest at have clearly been rushed through for the earlier 21-vote margin in the Lower a variable rate reaching a maximum political reasons, and do not really House (Table). of the retail price index (RPI) plus 3%. seem to have been thought through. In November 2010 the RPI was 4.7%1, Quite apart from the funding Had the eight Liberal Democrat MPs which means that someone earning who either abstained or were absent rearrangements, the new structure £41,000 would be charged a minimum turns university applicants and voted against the motions to increase of £1386 interest per annum. Clearly it tuition fees, the government would students very much into consumers; will be advantageous to pay your debt have still won. Had the 27 Liberal ‘power in the hands of the student’, off as quickly as possible, if you earn Democrats who voted with the as the government proudly proclaims. enough to do so. government rebelled, and stuck to Student choices, we are told, will shape higher education. Be very their pre-election promise not to raise Whilst the media focus on loan afraid. What undergraduate students tuition fees, the government would repayments, more disturbing to generally want – maximum outcome have been defeated. Amongst the academics is the change to university for minimum effort – is not conducive Lords who voted for the motions to funding that accompanies the new to producing a fit-for-purpose graduate, increase fees were academics Baroness structure. Currently the money an that is, one equipped with the critical Blackstone, former Master of Birkbeck, English university receives from the thinking skills for lifelong learning, and University of London, and currently public purse for teaching is ring-fenced educated to a level that is beneficial to Vice-Chancellor of the University of for that purpose. The charging of fees, subsequent employers. Greenwich, and Lord Krebs, son of Hans to the disappointment of academics, and currently Principal of Jesus College, will replace government funding It is not difficult to understand the Oxford. Surprisingly, the vast majority rather than complement it (with the government’s reasoning, of course. of Contents (>160) were Labour Peers, exception of clinical and laboratory If you wish to educate more than the and most Not-Contents Conservative. science, which will have government top, say, 15–20% of the population Such is politics. The changes to tuition funding drastically reduced rather to degree level, the pre-1998 system fees will little affect those who actually than abolished). This means, however, of tax-payer-funded HE in the form of voted, except perhaps for the 27 that income derived from fees need grants is not financially viable. So how ‘turn-coat’ Lib-Dems – who may find no longer be spent exclusively on can England (university funding in their electorate sadly disillusioned teaching. With reductions in research Scotland and Wales is independent of (and ready to exact penalty) at the funding, it may be tempting for many the English vote and that of Northern next trip to the polling booth. The universities to divert fee income away Ireland is as yet undecided) retain changes resulting from the politicians’ from pedagogy. a competitive edge in the global actions, however, will fundamentally market? Give it a decade or two, many alter HE when they take effect on Although the charging of fees is now commentators pedict, and we will 1st September 2012. effectively written in stone, it remains probably go back to restricting, not unclear to universities exactly how The issue runs deeper than simply the widening, participation. After all: what much income they can expect from fees students will pay, but I will start goes around, comes around. 2012–13. According to UCL Provost, there. The government’s argument is Malcolm Grant, in his Newsletter of Patricia de Winter that nobody will have to pay fees up 13th December 2010: “There are still 1. http://www.statistics.gov.uk/ front, a compelling point. Furthermore, they need not pay back a penny until Table. How they voted – the results from the Lower and Upper Houses as recorded in they earn over £21,000, and then only the Business Papers for each House at a rate of 9% of earnings above this amount, so for example, a graduate Motion House Ayes/Contents Noes/Not-contents earning £25,000 will repay their loan Basic amount increase to £6k Commons 323 302 at a rate of £6.92 per week. Finally, Higher amount increase to £9k Commons 323 302 after 30 years, any outstanding debt Basic amount increase to £6k Lords 283 215 is written off. This does not sound too Higher amount increase to £9k Lords 273 200

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Physiology 2011 – & Respiratory Physiology, Cellular & architectural commission, and the Integrative Neuroscience, Epithelia Oxford Museum of Natural History University of Oxford, & Membrane Transport, Human & are just two of the historic venues in Exercise Physiology, Metabolism the University that we are using for UK & Endocrinology and Vascular & the Meeting. However, Oxford has 11–14 July 2011 Smooth Muscle Physiology – give so much to offer, and key highlights The dreaming spires of Oxford will you the opportunity to explore the include the Bodleian Library, which play host to the largest physiology advances in your field as well as is one of the oldest libraries in meeting in the world this summer. learning from others. The Society is Europe, and the Radcliffe Camera, From 11 to 14 July, the Department proud that we offer researchers of which functions as a reading room of Physiology, Anatomy and Genetics all levels the opportunity to share for Oxford students. It is well worth at the oldest university in the UK will their work as one of 108 short oral viewing for its grand exterior. Other be the place for global physiologists communications that we have notable highlights include Hertford to meet, learn, collaborate and drive scheduled. Bridge or the Bridge of Sighs, and forward scientific breakthrough. Balliol, University and Merton Colleges, Over 1000 people came to the Main At the heart of the Meeting will which each claim to be the ‘oldest’ in 2 Meeting last year in Manchester, be a 1000 m marquee (one of the the University, with founding dates and The Society expects that we will largest in the country) which will in the 13th century. All are examples see record numbers of delegates at be the home to over 250 poster of the collegiate Gothic architecture Physiology 2011. communications and a 30-strong for which Oxford is renowned. These trade exhibition from key companies are all a stone’s throw away from the What can you expect? Highlights for supplying the scientific community. Meeting, so taking a break from the last year’s Meeting attendees include All catering will be here, and we science is easy and rewarding. ‘Listening to state-of-art lectures expect to serve 5000 cups of from world-renowned scientists and coffee, nearly 5000 sandwiches and Your Physiology 2011 Oxford to-do making new friends and starting 1000 bottles of beer! For further list should definitely include a visit to build relationships with other refreshment, take a trip to one of to at least one museum, visit at least researchers’, ‘I enjoyed all of it, from the city’s many historic pubs. The one college and – if possible – hear oral communications to socials, a Bear is the oldest pub in Oxford, one of the world-class college chapel must for physiology graduates’ and founded in 1492, and probably has choirs. We might also suggest a ‘I had such a wonderful experience the lowest ceilings of any pub in pint or two in a traditional pub with and met so many excellent scientists the city, the Eagle and Child was the friends and colleagues, old and new. in my field’. We also listened to your frequent haunt of the Inklings, a The local Scientific Organisers from advice on how we could improve group of Oxford literary dons that the University of Oxford – Andrew the meetings so this year we are included CS Lewis and JRR Tolkien, Parker, David Paterson and Richard providing all abstracts on a USB and the Turf Tavern, an ancient pub Vaughan-Jones – and all at The stick and are allowing more time for (a favourite with Inspector Morse), Society look forward to seeing you moving between sessions. We have is an unmissable Oxford institution in July. also made more strategic scheduling that many consider to be the best to avoid clashes with symposia. pub in the city. For more information visit the website at: Twenty symposia around The The Sheldonian Theatre, which was Society’s six core Themes – Cardiac Sir Christopher Wren’s first major www.physiology2011.org Prize Lectures 2011

The Physiological Society is delighted to announce that the following Prize Lectures will be delivered in 2011.

Lecture Lecturer Venue Annual Public Russell Foster (University of Oxford) Physiology 2011, Oxford, July Annual Review Carla Shatz (Stanford University) Physiology 2011, Oxford, July GL Brown Lucilla Poston (King’s College London) Various across UK & RoI Hodgkin–Huxley–Katz Roger Nicoll (University of California – San King’s College London, April 2011 Francisco) Michael de Burgh Daly Tobias Wang (Aarhus University) Physiology 2011, Oxford, July Paton John West (University of California – San Diego) Physiology 2011, Oxford, July Sharpey–Schafer Walter Marcotti (University of Sheffield) Physiology 2011, Oxford, July

Physiology News | No. 82 | Spring 2011 | www.physoc.org MEETINGS PN 5 Epithelia & Membrane Transport Royal Free Campus, University College London, UK 1–3 September 2011

Themed Meeting of The Physiological Society including a focused symposium: Ion Transport: Insights from disease and animal models

Abstract submission & registration open 23 June 2011

www.physoc.org/em2011

PhysiologyEM PN82.indd News 1 | No. 82 | Spring 2011 | www.physoc.org 1/18/2011 1:11:30 PM 6 PN MEETINGS

Cross-Themed meeting Durham University, UK 15–17 December 2010 Well ... there are benefits of holding a meeting in the week before Christmas! Being a southerner, it’s not often that I see snow like this. As I look out of the window on the train home from Durham, I look out at the blizzard and reflect on The Left to right: Meera Senthilingam, Henning Franzel, Gary Lewin, Stefan Lechner, Ewan Physiological Society’s Cross-Themed Smith and Kate Poole. meeting. talks: my personal favourite was the The final day took us through In the days approaching the oral communication given by Angus mechano-transduction, where we conference, the North–East had Wann (Queen Mary, University of covered a range of subjects including received some of the heaviest London) as he presented a new primary- and stereo-cilia. Here, I snowfall for years with Durham technique for applying forces to was privileged to present an oral having around a metre: some of the primary cilia in cartilage. In addition communication of my work which effects were still apparent as piles to the high profile speakers, I I felt was well received considering of ploughed snow over two metres really felt that this conference the conversation it stimulated high remained scattered around the attracted exceptionally interesting afterwards! At lunch time, I was still city. Despite this, the attendance research from early-career scientists receiving suggestions for further was very good from the outset. displaying a range of novel experiments that I could try: a The first day of the Meeting was techniques to provide insight into a testament to the benefit of Themed compelling; we were introduced small field. In the evening I attended Physiological Meetings. Bringing to the complex field around The Society Dinner at St Hild & St together academics working in acid-sensing ion channels (ASICs) Bede College. We were treated to a closely related fields allows greater and were posed with a question: is a culinarily unique meal (fillet of beef understanding, the chance to share mechano-sensitive channel the same with a coffee gravy) followed by ideas and knowledge, and therefore as a stretch-sensitive channel? In the entertainment from a local group benefits all. first poster session that followed, playing the Northumbrian pipes, a violin and a harp – The Border The final comments of the Meeting we were able to browse some of returned to our initial question which the research that has recently been Minstrels. We might not have known the songs (except for Pip Garner, we were set – is a mechano-sensitive occurring in the field and discuss channel the same as a stretch- our opinions on this question that University of Leeds, who informed me that one of them was used in a sensitive channel? Personally, I feel had been put to us. A group of it all depends on the experiment. us retired to a local pub next to BirdsEye fish advert) but they were beautiful nonetheless. When we truly start applying forces Durham prison where the drink to channels and not just the near of choice was Gaols (pronounced vicinity, I think we can start to fully Jails) Ghouls. Conversation turned appreciate the need for clarity. My from mechano-sensitive channels to final comment must turn to Stuart our experiences of research in our Wilson (University of Dundee) who respective fields, the effect of the was returning to Durham where dreaded comprehensive spending he studied as an undergraduate. review and finally, the broad Throughout the Meeting, I learned incomprehensible Durham accent that Stuart really appreciates from some inebriated locals. Durham as a beautiful city, and Throughout the week, further it is. But Stuart, I’m sure Durham weather warnings had been issued will appreciate you in your new for heavy snow: I would say that on University of Durham top that you Thursday morning, it delivered. As were wearing so proudly on the final I walked in at 09.00, I was able to day. Fantastic! Now, I just hope this take some beautiful snowy pictures snow doesn’t get any worse. of Durham Cathedral. I was glad to finally make it to the venue where Left to right: Esther Lopez, Pedro Stephen Kocher we were treated to some fantastic Redondo Liberal and Natalia Dionisio. University of Bristol, UK

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The GL Brown Prize Lecture reunites with his descendants

Sir Lindor Brown, GL, our father, father-in-law and grandfather, was a great man. He died relatively young at just 70 years almost 40 years ago. At that time we, his offspring, were busy with our own lives. I only became aware this year that The Physiological Society continued to honour him by arranging the GL Brown Prize Lecture after my son discovered them on the internet. In recent years family and professional responsibilities have made travel Graham McGeown, the recipient of the 2010 GL Brown Prize, presented here by Lucy Donaldson from Bristol University. somewhat difficult for me and this year was the first time I was able to the middle aged and white-haired I feel that it has been a great attend a lecture. elderly, sitting in one row, initially privilege for us to celebrate our The postponement of the second felt somewhat out of place in the father’s life and work almost forty half of the lecture series (given audience of today’s students. I held years after his death. I would like to by Graham McGeown, Queen’s my breath when Professor McGeown thank The Physiological Society very much for the opportunity to attend University Belfast, UK) until the end started with a biography of GL... this Lecture and celebrate GL’s legacy of last year due to the impact of fortunately we had no quarrels with once again. the eruption of Eyjafjallajökull on it and enjoyed the descriptions of his work and personality. We all air travel was fortuitous and meant Helen Brown, MB BS FRCP(c) that a number of us might attend appreciated the lecture; even those one of the sessions. Telephone of us with no medical or scientific Ottawa, Canada background were pleasantly wires hummed and emails crossed On behalf of: the Atlantic in rapid succession to surprised at how much they enjoyed Christopher and Wendy Brown, enable us to make the pilgrimage. it. The most fascinating part was Humphrey Brown, Katherine Brown, We took the opportunity for a family realising how far the investigative David Hartshorne, and Jane Uygur. reunion and managed to gather one sciences have evolved in the past daughter of GL, two grand-daughters decades. Now one works on the (all in the medical profession), two minutiae of a single arterial muscle Measurement of gene sons and two in-laws. cell with the aid of exceedingly expression using real-time accurate and expensive measuring quantitative PCR The Canadian contingent was the equipment. Seventy years ago they first to arrive, a few days before the had to smoke their own drums to 27–28 April 2011, King’s College lecture. We spent time acquainting record the contraction of the whole London ourselves with the city and finding muscle. How things have changed. Affiliates and Members can the location of the lecture hall, and register for free to attend this the hills of Bristol gave our legs a We found it a memorable occasion, workshop, which will provide an fantastic challenge. Other members one that GL would have appreciated. opportunity to gain both practical trickled in from Dublin, Ipswich and He continued throughout his life to and theoretical experience of gene Leighton Buzzard. We gathered have a keen interest in teaching and expression analysis using real-time together to exchange memories and eagerly encouraged those who came RT-qPCR. memorabilia and contemplate the after him. The day was rounded off huge amount of work GL managed by The Society’s invitation to dinner. To register, please email to accomplish. We shared stories of We enjoyed the opportunity to [email protected] his artistic and poetic achievements, have conversations with Professor his sense of humour and his great McGeown and to meet the members Course organised by David Sugden capacity to find the right word at the of the University of Bristol’s and Patricia de Winter right time. Many of his phrases are Department of Physiology. It was passing down the generations! interesting to learn of some of the issues in physiology today and a On the day of the lecture we pleasure to share in an evening of gathered six strong to present good food, wine and humour that GL ourselves at the lecture theatre. We, himself would have enjoyed.

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The run-up to IUPS – Thelma Lovick talks to Bridget Lumb, Chairman of the Organising Committee What goes on behind the scenes to organise a big meeting like the International Union of Physiological Sciences (IUPS)? The next IUPS congress will be held in Birmingham in 2013 so the organisation is now well under way. Thelma Lovick talked to Bridget Lumb, Chair of the IUPS Organising Committee. Thelma Lovick (TL). Most of us go to The Physiological large international meetings without Society, so giving a thought to what goes on I brought behind the scenes to put them on. experience We’ve got IUPS coming to the UK of organising in 2013. Can you tell me a bit about national and how that decision was made? international Bridget Lumb (BL). IUPS organises meetings. So I suppose that a scientific meeting every 4 years Thelma Lovick (left) and Bridget Lumb for which it invites tenders made me the natural choice. from constituent Societies. The TL. How was the Organising Physiological Society put in a bid Committee set up – did you choose TL. How many people make up the about 5 years ago during the IUPS its makeup? Organising Committee? meeting in San Diego in 2005. We BL. No, that was determined by BL. We have five key people, were in competition with three other the Executive of The Society. including a representative from bids. Two were from within Europe – People were approached – usually FEPS, who remain on the committee Austria and the Czech Republic – and members of various subcommittees throughout the 10 years or so the third was from China. or Executive – according to their that it actually takes to organise TL. But surely the thinking behind particular experience and expertise. the meeting. This is to maintain the bid must have started well It’s more than just the obvious things continuity. Then there are seven before that? like sorting out the programme and people who are representatives from arranging the venue. For example, Phys Soc Exec at any point in time. BL. Oh yes, we’d already done a lot What is critical is maintaining that there’s also a lot of outreach activity, of background work to decide on core representation. the venue. We looked at various hosting events for the public, talks places in the UK before we decided about physiology in schools and TL. Do you also have local people on on Birmingham. What really turned things like that. the ground? us on to Birmingham was the TL. How did you get to be Chair of BL. Yes, of course. It’s important conference centre right in the city the Organising Committee? to have people with local centre. It’s such an attractive venue knowledge; so we have a local BL. At the time the committee was on the canal side with lots of bars organising committee as well, set up I was Meetings Secretary of and restaurants very close by. The with representation on the main other really very good thing there Organising Committee. is the capacity to accommodate TL. How much other help do you everyone within walking distance of get? Do you hire professional the conference centre. Birmingham conference organisers? is also incredibly accessible. It has BL. We’re very lucky – we’ve got an international airport very close to Birmingham’s floral trail our own. The Phys Soc office – led the city centre – it’s just the perfect by Nick Boross-Toby – offers us venue. experienced support; they advise Geographically IUPS moves round and lead the academic committee the globe – it’s not only the in many ways. There’s also David science, it’s a question of getting Bennett, who is the International the geographical location right Affairs and Events Manager. They too. You’ve got to remember that bring with them experience in people come to meetings not only hosting national meetings on behalf for the science, but also for a cultural of The Society. Our annual meeting experience – and Birmingham offers in the summer now attracts around fantastic potential as a base for a thousand delegates and covers a exploring the UK. We had to appeal diverse scientific remit, so the Phys scientifically and economically. Birmingham’s floral trail. Soc team is pretty experienced.

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experience. Anything above this will be a bonus. TL. Everything is getting so specialised these days – don’t you think the days of the big general meeting are over? BL. No, I don’t think so – IUPS will be something that cuts across the boundaries of different scientific disciplines. TL. It all sounds a bit like organising the Olympics BL. Very similar!

Birmingham Town Hall.

TL. So essentiallyBirmingham the summer Town Hall a proportion of the cost of funding meeting is a dress rehearsal for IUPS? young physiologists and delegates BL. Yes, I guess you could say that. from developing countries. TL. Will Phys Soc be putting on its TL. Even so, it’s likely to be a lot annual meeting as well as IUPS in compared to going to The Society’s 2013? annual meeting. BL. No. Phys Soc won’t host a main BL. What you’ve got to remember meeting that year, nor will FEPS or is that our national meeting is the Scandinavian societies. They are massively subsidised by The Society. all going to designate IUPS as their What you are asked to pay is meeting that year. nowhere near the real cost. So we in the UK sometimes have a rather TL. What about satellites – who unrealistic view of what these events organises them? cost to put on. The Sea Life centre. BL. Interesting question. Satellites TL. How many people are you are still quite a lively topic of debate! For further information on the IUPS expecting to come? There will be the opportunity for meeting visit The Society website at satellites in the UK and Europe BL. 3000–4000 based on previous www.iups2013.org but their exact format hasn’t been finalised. What I can say at this stage Microelectrode techniques for cell physiology is that a call will be going out. The Marine Biological TL. How are these meetings funded? Association, Plymouth, UK BL. They’re underwritten by the host country’s physiological society. 7–21 September 2011 But the IUPS also raises funds, particularly to support young 28th workshop scientists to attend, and people from developing countries – those who A variety of marine and other preparations will be used to illustrate the would find it very difficult to raise possibilities and limitations of these techniques. The workshop is intended the money to come. mainly for postgraduate students and postdoc workers from any discipline who wish to learn techniques for use in their research. TL. How do you decide on the registration fee? International Closing date for receipt of applications 30 April 2011 meetings are getting very expensive to go to these days Application details from: BL. The fee is a realistic amount that www.mba.ac.uk/microelectrodecourse.php covers the cost of each delegate, plus [email protected]

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My 10 key papers John Coote selects the ten papers which he considers had a major influence on the understanding of heart–brain interactions A topic which is rapidly gaining region of the hypothalamus or of interest from clinicians is the brain the periaqueductal region of the control of the heart and vascular mid-brain areas which integrate system. This area of research some alerting, aggressive display and 40 years ago, when I became flight are also responsible for the involved, would have seemed of autonomic responses accompanying purely academic interest. Now the defence reaction. These facts the importance of the brain in alone imply complex interactions cardiovascular diseases such as within the central nervous system. hypertension, stroke, cardiac failure This and subsequent studies changed and arrhythmias is well recognized ideas about a vasomotor centre and a strong focus of research. in the brainstem. The hypothesis stimulated much of my own work on Interestingly the link between brain John, giving a lecture in China in 2010. patterns of autonomic response. We and heart has been topical since now know that other cardiovascular the time of Aristotle (384–322BC) outflows mediated via different brain response patterns such as although then the emotions and regions in the medulla, mid-brain, those accompanying changes in feelings now known to emanate hypothalamus and cerebral cortex environmental temperature, feeding, from brain structures were thought which act via parallel pathways to water submersion, sleep and to be in the heart. It is a huge topic parasympathetic and sympathetic and I wondered whether I could pick exercise are similarly organized and preganglionic neurones in medulla involve different brain regions. The out just 10 papers from the 100,000 and spinal cord. or so that might be of current studies of the defence reaction also interest to readers of Physiology The following papers are my laid the foundation for numerous News. I decided to take on the rather personal choice of studies that studies suggesting a preservation of daunting task and the chosen papers I consider underpinned the an intense defence response in some are ones that had particular impact subsequent explosion of knowledge individuals could be causally related on my own studies but I believe have which daunts many young to hypertension. played an important part in the main researchers at the present time and 2. Rostral ventrolateral medulla developments in thinking on the may be studies of which they are and the vasomotor centre heart–brain connections. Inevitably unaware. this is a personal choice and has Amendt K, Czachurski J, Dembowsky K & meant leaving out many important 1. The brain elicits patterns of Seller H (1979). Bulbospinal projections autonomic response to the intermediolateral cell column: a contributions. neuroanatomical study. J Auton Nerv Syst 1, Abrahams VC, Hilton SM & Zbrozyna 103–107. My involvement with the brain–heart AW (1960). Active muscle vasodilatation connection started when I was a produced by stimulation of the brain stem: its Up to the early 1970s pressor and student at the Royal Free Hospital significance in the defence reaction. J Physiol depressor regions of the medulla, School of Medicine in the early 154, 491–513. which were thought to constitute 1960s as more and more scientists Autonomic responses rarely appear a vasomotor centre, had been worldwide began to question the in isolation, and are usually seen as localised to the dorsal and medial idea of a vasomotor centre in the part of a pattern of change involving reticular formation. A dramatic medulla. Studies were initially a number of organs and systems change in thinking occurred with Charles Downman who was that participate in a behavioural when Feldberg & Guerzenstein then Professor of Physiology at the response. This was the central (1972, J Physiol 224, 83–103) and Royal Free. The field has expanded idea put forward by Sidney Hilton Guerzenstein & Silver (1974, J Physiol considerably since then with the first at the MRC centre in Mill Hill, 242, 489–503) found that dramatic advent of modern anatomical London and then at the University changes in blood pressure occurred tracing methods and the application of Birmingham. The paper from when drugs were topically applied of new neurophysiological and Hilton’s group showed that sites in to the ventral surface of the rostral pharmacological techniques. the brain that elicited a sympathetic medulla just caudal to the trapezoid Instead of the old concept of cholinergic vasodilatation in bodies. This was not the first such a brainstem centre containing muscles in the cat whilst increasing observation since almost 20 years neurones responsible for cardiac sympathetic activity to other previously Loeschcke & Koepchen or vascular control, current models organs were the same as those (1958, Pfluegers Arch 266, 628–641) of autonomic control are based that elicited the defence reaction. reported that the ventral surface of around discrete reflex control of It was the first study showing that the medulla was a site from which sympathetic and parasympathetic stimulation of the perifornical respiratory and circulatory effects

Physiology News | No. 82 | Spring 2011 | www.physoc.org MY 10 KEY PAPERS PN 11 were obtained by topically applied to other cardiovascular regions of & Barman (1985) was something drugs. The cardiovascular effects the brain and can be affected by of a tour de force, carrying out obtained from this region in the peripheral afferent receptors whose single cell recording of brainstem latter experiments largely went primary role is to influence higher neurones at two sites and also unnoticed because the authors regions of the brain as is evident in recording peripheral sympathetic had been most concerned with blood volume regulation and more nerve activity. It was the first to respiration and chemosensitivity. complex behaviour. provide evidence that assemblies The significance of Feldberg and of interconnected neurones acted Guerzenstein’s results was much 3. Origin of vasomotor tone as multiple oscillating networks, debated because it was argued Gebber GL & Barman SM (1985). Lateral the output of which is synaptically by some that the applied drugs tegmental field neurons of cat medulla: a coupled to bulbospinal vasomotor potential source of basal sympathetic nerve neurones which are suitably phase could have diffused as far as the discharge. J Neurophysiol 54, 1498–1512. pressor region in dorsal–medial related to bursts of activity in areas of the medulla. The question It has long been known that various postganglionic sympathetic nerves was resolved by the Amendt et al. rhythms originating in central to various organs. This accords with study (1979) which used retrograde nervous structures, including results showing that brainstem transport of horseradish peroxidase respiratory rhythm, are reflected pre-sympathetic neurones (Lovick, (HRP) to demonstrate that a in autonomic outflows. The degree 1987, J Physiol 389, 23–35) as well group of neurones located close of neural activity in sympathetic spinal preganglionic neurones to the brain surface in the rostral nerves to blood vessels and the heart (Boczek-Funcke et al. 1993, J ventrolateral medulla, projected is fundamental to the operation Auton Nerv Syst 43, 189–200) to the sympathetic region of the of efficient cardiovascular control are functionally dedicated. The grey matter of the third thoracic and allows the brain to direct significance of the latter data and segment. This was the beginning cardiac output to organs that are studies by Gebber’s group seem to of numerous investigations on the essential in a particular behaviour. have been largely ignored. I believe characteristics and organization of A major quest in cardiovascular the multiple oscillator hypothesis pre-sympathetic neurones, which research has been to identify better explains the data and will contribute to vasomotor tone and how tonic activity originates. This prove to be a major landmark that can be identified by antidromic paper is just one of 12 papers in understanding the origin of activation from the spinal cord. For from Geri Gebber’s lab that by vasomotor tone. I suspect that many it appeared that all that had focusing on the basic rhythms because Gebber used mathematical happened was that the vasomotor of vasomotor activity provide a analyses, such as time series centre had been moved from very plausible explanation of tone and spectral plots to rigorously the dorsal medulla to the rostral generation. It had been thought show significant relationships, ventrolateral medulla. However, that a population of neurones in the many who were less skilled with numerous studies now show that brain, previously thought to be in these techniques were somewhat there are parallel pathways from the vasomotor centre of the dorso- suspicious about the conclusions. other regions of the brain that medial medulla but subsequently I should refer to another school of contribute to vasomotor tone. in the bulbospinal neurones of the thought which was that spontaneous Interestingly, the study of Amendt et ventrolateral medulla, randomly activity in vasomotor nerves al. also showed there are neurones generated discharges which were arises from the intrinsic activity of in the nucleus of the solitary tract synchronized by powerful inputs pacemaker neurones amongst the that send axons which directly end from cardiovascular and respiratory ventrolateral bulbospinal neurones. in the sympathetic intermediolateral sources. Gebber’s group showed So far there is no strong evidence cell column at T3. The latter finding that the oscillations could be for this although it may be that has been largely ignored but the uncoupled from these inputs and some neurones are conditioned to neurones may well represent they still persisted after removal of be pacemakers by specific synaptic the inputs. The paper by Gebber inputs. inhibitory neurones that studies Hypothesis of vasomotor tone generation in my lab have shown to cause a Afferents RVLM Tegmentum 4. Multiple oscillators in autonomic hyperpolarisation of pre-sympathetic control interneurones at T3 of rat (Lewis & Coote, 2008, Neurosci 152, Staras K, Chang HS & Gilbey MP (2001). 534–546). Resetting of sympathetic rhythm by somatic afferents causes post-reflex coordination of sympathetic activity in rat. J Physiol 533, My personal opinion is that the 537–545. main role of the ventral medullary pre-sympathetic neurones is Recordings from sympathetic in cardiovascular–respiratory Oscillating postganglionic nerves to coupling and thus they play a key networks cardiovascular end organs reveals role in matching cardiac output to Skin Muscle that they display a rhythmic metabolism. As a consequence, the Hypothesis of vasomotor tone gener­a­tion. bursting activity which has several region has two-way connections RVL, rostral ventrolateral medulla. periodicities in the firing pattern;

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2–6 Hz, 10 Hz and frequencies visualized under fluorescence At present, the function of brain related to the cardiac and respiratory microscopy. The method was based catecholamine neurones in cycles. Gebber et al. (1994, Am J on that devised by Hillarp and Falk cardiovascular regulation is unclear Physiol 267, R387–R399), provided and described by Falk (1962), Acta even though much is known of persuasive evidence that the Physiol Scand 56, 1–25. Forty-five the electrophysiology and cellular spontaneous activity of vasomotor years later it is difficult to convey actions of each of the amines on nerves was dependent on network the importance of this and the sympathetic neurones in brain and oscillators as I referred to above. later paper by Fuxe (1965, Acta spinal cord. In fact, more specifically, Gebber Physiol Scand 64, 37–85) and the et al. found there is a variability in excitement created. Now for the first 6. Extrahypothalamic pathways to the rhythmic patterns of activity time the brain could be visualized spinal sympathetic neurones measured in the nerves supplying like words on the pages of a book Sawchenko PE & Swanson LW (1982). vascular systems of different instead of a seemingly tangled web Immunohistochemical identification of neurons in the paraventricular nucleus of the organs suggesting there are of various shapes of neurones so meticulously described by the earlier hypothalamus that project to the medulla multiple oscillators. The studies by or to the spinal cord in the rat. J Comp Neurol Gilbey’s group on the innervation neuroanatomists. Furthermore, it 205, 260–272. suggested that catecholamines that of the tail vasculature support this are chemical neurotransmitters in Prior to this paper studies from interpretation since it reveals that the peripheral nervous system might several groups had shown that there the sympathetic nerves display a also be involved in transmission are direct neural projections from characteristic rhythm of discharge, between neurones in the brain and the paraventricular nucleus (PVN) the T rhythm, that does not appear in particular might have a role to to the spinal cord. The importance in sympathetic nerves to the kidney. play in blood pressure regulation. of the work by Sawchenko and They are involved with the vascular This stimulated numerous studies Swanson was that it characterized responses to body temperature and has benefitted understanding the phenotype of some of the changes. Gebber’s group showed of mechanisms involved in neurones in the PVN that projected that the fundamental rhythms can behaviour, endocrine reactions, to the intermediolateral cell be entrained by afferent inputs, arousal and sleep to name but column of the upper thoracic cord. providing the frequency of the a few. It was soon realized that By using a method that allowed input is close to the uncoupled sympathetic preganglionic neurones the concurrent localization of frequency of the discharge, thus in the spinal cord were densely retrogradely transported fluorescent supporting the idea of oscillating contacted by catecholamine dye and an antigen it was shown networks. The paper from Gilbey’s terminals and the search began, that a high proportion of neurones group, reported that stimulation of in many groups worldwide, to contained either vasopressin or afferent fibres in a somatic nerve, determine their functional role in oxytocin. This was a remarkable the radial nerve of the fore leg in rats blood pressure regulation, and is discovery since up to this time these or pinching the paw, can reset the still ongoing. In particular, there peptides had only been thought of is evidence that some spinally oscillatory network responsible for as intrahypothalamic hypophyseal projecting catecholamine neurones the T rhythm in thermoregulatory hormones. Here was evidence for sympathetic vascular nerves of the are located in the ventrolateral the first time, surprisingly indicating tail and transiently synchronize medullary vasomotor region and that the peptide precursors can neurone firing. I consider this study synthesize adrenaline since they are be transported long distances important because it provided a immunohistochemically positive in axons projecting from the robust test of the multiple oscillatory for the enzyme PNMT. This led to forebrain to distant regions of the network hypothesis and the result strong opinions that adrenaline was spinal cord. Subsequent studies lends it further support. a neurotransmitter mediating tonic vasomotor drive from the brainstem. by several groups including my 5. Central catecholamine pathways However, my own studies and those own showed that these peptides and cardiovascular control of others have shown that the are also expressed in nerve terminals surrounding sympathetic Dahlström A & Fuxe K (1965). Evidence for concentration of adrenaline in the the existence of monoamine-containing spinal cord of cat and rat is so low preganglionic neurones. One neurons in the central nervous system. I. it is hardly measureable, whereas study using electronmicrosopy is Demonstration of monoamines in the cell both noradrenaline and dopamine particularly outstanding in showing bodies of brain stem neurons. Acta Physiol oxytocin terminal synapses on Scand 232,1–55. are present in significant amounts. The mystery deepens because at least one population of these This paper describes the location of no evidence so far has shown a sympathetic neurones (Hosoya et discrete groups of catecholamine- contribution of catecholamine al. 1995, Exp Brain Res 107, 9–16) and 5-hydroxytryptamine-containing neurones or their terminals in so providing a strong indication neurones in brain sections that had the spinal cord to tonic activity the peptides could be involved been freeze dried and exposed to in postganglionic cardiovascular in synaptic transmission. These formaldehyde vapour, and then nerves or blood pressure regulation. neuroanatomical discoveries soon

Physiology News | No. 82 | Spring 2011 | www.physoc.org MY 10 KEY PAPERS PN 13 led to physiological experiments is impaired in heart failure and is 8. Cerebral cortex and cardio- by a number of groups, initially possibly one reason for sympathetic vascular control spearheaded by Mike Brody’s lab activity continuing to increase Oppenheimer SM & Cechetto DF (1990). in Iowa but sadly cut short by his in this condition despite greater Cardiac chronotropic organization of the rat untimely fatal heart attack whilst at distension of the caval–atrial region insular cortex. Brain Res 533, 66–72. a conference in Australia. Data from with the inability of the heart to This paper was highly signifi cant Quentin Pittman’s group in Canada respond adequately to venous since it demonstrated that cerebral and my own have documented a return. At the present time, this control of cardiovascular organs predominant excitatory action of is a subject of intense research by may be highly selective. It describes the peptides and a clear function a number of groups. Historically, a study that was the fi rst, of several of their role in combating increases knowledge of the atrial refl ex effects from these authors, to show that and decreases in blood pressure on sympathetic nerves started stimulation of a specifi c region and changes in plasma volume. with Bainbridge’s report (1915, J of the cerebral cortex can affect Furthermore, there is increasing Physiol 50, 65–84) of an increase in changes in heart rate and no evidence that there is a functional heart rate with increased venous other autonomic parameter. The impairment of the action of the fi lling, which was largely forgotten 20th century saw several studies extrahypothalamic peptidergic until revived by Ron Linden and his indicating that autonomic functions system in heart failure (Patel, group in Leeds of which Karim et al. are considerably infl uenced by areas 2000, Heart Fail Rev 5, 73–86) and were a part. The studies reported of the cerebral cortex linked to the salt-sensitive cardiovascular disease from this group were the centre forebrain. There was a problem in (Toney & Stocker, 2010, J Physiol 588, of many acrimonious discussions interpreting the results of these 3375–3384). at Physiological Society meetings studies since the methods were in the ’70s, the main protagonists not suffi ciently precise to conclude 7. Refl ex effects of cardiac atrial being Linden on the one side and that the cardiac or vascular effects receptors Hilton on the other. Sadly we see were primary or secondary to other Karim F, Kidd C, Malpus CM & Penna PE few such eloquently argued view events. (1972). The effects of stimulation of the left points at the meetings these days. atrial receptors on sympathetic efferent nerve There is now much evidence that the This was overcome in the studies activity. J Physiol 227, 243–260. atrial-sympathetic refl ex is pivotal initiated by Cliff Saper in USA and I chose this paper because it to plasma volume regulation and David Smith in Oxford, which is an excellent example of theSummary my own labof has current shown that it isknowledge provided clear ofevidence brain that circuits basic principle of the response of mediated via the paraventricular cerebral control of one parameter sympathetic cardiovascular nervesinvolved nucleus andin involves atrial the extra-receptormay befeedback accomplished not fromjust the to signals from a specifi c group hypothalamic vasopressin and by connections to cardiovascular of receptors in that each evokes oxytocin pathways. heartregions of the brainstem (Yasui et al. a unique pattern of autonomic activation. The cardiac atrial receptors play a signifi cant role in moment-by-moment plasma volume regulation. The experiments on anaesthetized dogs conclusively demonstrated that activation of atrial receptors by distension of small balloons at the pulmonary vein– atrial junctions causes an increase in cardiac sympathetic nerve activity and simultaneously a decrease in renal sympathetic nerve activity. This indicated that vagal afferent receptors situated at a site ideal for detecting changes in venous return, refl exly initiate a selective pattern of sympathetic nerve response that quickly corrects for alterations in Glycine fl uid status. Importantly, the atrial refl ex threshold becomes higher Renal sympathetic nerve in pregnant rats but returns to normal after parturition (Deng & Summary of current knowledge of brain circuits involved in atrial receptor feedback Kaufman, 1995, Am J Physiol 269, from the heart. RVL, rostral ventrolateral medulla; PVN, paraventricular nucleus; R552–R556). Interestingly the refl ex AVP, arginine vasopressin; OT, oxytocin; NTS, nucleus of the solitary tract.

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1991, J Comp Neurol 303, 355–374) sympathetic preganglionic neurones in the Additionally, a much argued but also via direct connections third thoracic segment of the cat. J Auton Nerv question related to inhibitory inputs Syst 13, 201–244. to spinal sympathetic neurones concerning whether they are direct (Bacon & Smith, 1993, Neuroscience The pattern of tonic neural activity or mediated via interneurones or 54, 719–728). Oppenheimer & to heart and blood vessels plays a only mediated by an action on Cechetto (1990), went a step key part in optimizing cardiac output supraspinal neurones. Two earlier further by devising a technique of and blood supply to every organ. A studies by others had only achieved phasic cortical microstimulation leading idea that I have highlighted short time intracellular recordings synchronized with the R wave of earlier, is that input to ventral and so the answer was still uncertain. the ECG that enabled identifi cation medullary vasomotor neurones The importance of this paper by of regions in the insular cortex arises from an oscillatory network Dembowsky et al. is that it describes that elicited only heart rate effects. in the brainstem tegmentum and for the fi rst time, in a beautiful series is entrained by baroreceptor and Subsequent studies showed that of remarkably stable intracellular respiratory inputs which then there is a selective lateralization recordings obtained in anaesthetised drive sympathetic preganglionic of cardiac vagus versus cardiac cats, ongoing synaptic activity neurones in the spinal cord, sympathetic control between the consisting of EPSPs and IPSPS some resulting in synchronized bursts of right and left posterior insular of which occurred monosynaptically activity observed in postganglionic cortices and the neurones receive following stimulation of axons nerves recorded in rodents, cats, baroreceptor input. Oppenheimer (probably of supraspinal afferents) in went on to confi rm that a similar dogs and humans. However, this is only one of many synaptic the dorsolateral funiculus. It was also organization was present in apparent that there were no slow primates, including humans, the inputs since studies have shown membrane potential oscillations and latter by taking advantage of deep that sympathetic preganglionic that synaptic activity was far greater brain stimulation in patients treated neurones are synaptically contacted than inferred from extracellular for epilepsy (Oppenheimer et al. by several types of chemically recordings, thus establishing that 1992, Neurology 42, 1727–1732). coded supraspinal axons forming the preganglionic neurones were These studies have been the parallel pathways that originate acting as integrators rather than forerunners of many others that are from different cell groups in the relay neurones. More recently much leading to new understanding of brainstem, hypothalamus and myocardial injury in patients with cerebral cortex. In the absence of of the fi ndings have been confi rmed stroke and induction of arrhythmias intracellular recordings, speculation for the rat (Lewis & Coote, 2008, and may well help to direct surrounded the possibility that Neuroscience 152, 534–546). These treatment for patients. a single source of synaptic input in vivo studies and a host of others gave rise to slow membrane using in vitro preparations show that 9. Spinal neurones and sympathetic depolarisations in sympathetic sympathetic preganglionic neurones tone generation preganglionic neurones rather have a number of characteristics Dembowsky K, Czachurski J & Seller H (1985). similar to those that were displayed such as increases and decreases An intracellular study of the synaptic input to by spinal respiratory neurones. in fi ring, gain enhancement,

Coronal section through cerebrum of rat showing sites in the insular cortex from which blood pressure and heart rate increases are elicited by electrical stimulation with a microelectrode (This material is reproduced with permission of John Wiley & Sons, Inc. from Yasui et al. 1991, J Comp Neurol 303, 355–374).

Physiology News | No. 82 | Spring 2011 | www.physoc.org Coronal section through cerebrum of rat showing sites in the insular cortex from which blood pressure and heart rate increases are elicited by electrical stimulation with a microelectrode (Yasui et al 19991). MY 10 KEY PAPERS PN 15

215, 789–804) and, as it transpired later, others in Oxford had also realized the advantage of this approach (McCloskey & Mitchell, 1972, J Physiol 224, 173–186). Studies in this area on experimental animals and humans subsequently expanded enormously and continues to this day, and some very clever experiments on human subjects have shown that the cardiovascular response to exercise is both a refl ex and a central command- induced response. This has led John with a group of Chinese colleagues at Nakai University, Tianjin, China. to an understanding of how the arterial baroreceptors function in prolongation of action and burst the circulation to the limbs. The exercise and how cardiac vagal and activity. These properties are paper by Lind et al. (1964), took a sympathetic activity is infl uenced. probably due to modulation of slightly different approach by using Furthermore, we now have an idea intrinsic membrane conductances sustained (isometric) contractions of the nature of the stimuli within by the variety of chemically coded of muscles that had the advantage contracting muscle and some insight inputs acting either directly or via of enabling easy quantifi cation of into the role of the afferent fi bres spinal interneurones. Therefore the stimulus as well as the use of in heart failure as illustrated by a there is great versatility in the different groups of muscles including paper in a recent issue of J Physiol control of tonic vasomotor activity those controlling hand grip or (Wang et al. 2010, 588, 5033–5047). as a consequence of integration even one fi nger. The study showed Also the signifi cance of regular at the spinal cord level, a feature that the pressor and tachycardia exercise in treatment of heart failure that needs to be accounted for responses were proportional to the and hypertension is now better when considering the causes of percentage of maximal voluntary understood. cardiovascular diseases such as contraction and were enhanced hypertension or cardiac arrhythmias. by occluding the blood supply Concluding remarks 10. The refl ex nature of the cardio- to prevent any possible chemical This series of papers provides a vascular response associated with mediator leaving the contracting brief history of one part of my life exercise muscles. Though this work and in medical research, showing the that of others on human subjects startling increase in knowledge Lind AR, Taylor SH, Humphreys PW, Kenelly provided suggestive indirect BM & Donald KW (1964). The circulatory and understanding of brain–heart effects of sustained voluntary contraction. Clin evidence, direct experimental proof control that has occurred over the Sci 27, 229–244. of the possible refl ex nature of the last 40 years. Many of the early cardiovascular effect of muscle papers may have been forgotten Up to the time this paper was contraction was still awaited. The or not even known about by published, the nature of the stimulus importance of this paper was that it younger scientists. Interestingly, giving rise to the cardiovascular indicated a way of testing whether all of the studies I quote are based response to muscular exercise had the exercise pressor response was on whole experimental animals not been identifi ed. It was originally centrally derived or a refl ex in or humans. Modern genetic or postulated that it was due to a experimental animals. I turned my molecular approaches have yet to direct action of the cerebral motor attention to this response in my own provide much new fundamental cortex on circulatory centres in lab because I had wondered what information although I expect/ the brainstem but little conclusive possibly could be the physiological hope this will happen in the future. evidence was forthcoming. An function of the huge population of However, it will be necessary for alternative hypothesis was that it small myelinated and unmyelinated the modern scientist skilled with was a refl ex initiated by afferent afferent fi bres in muscle that the newer techniques, to read the fi bres in the contracting muscles. increased sympathetic activity and older literature to get a better idea Considerable strength for this idea raised blood pressure and heart of what the important questions are came from studies by Alam & Smirk rate (Coote & Perez-Gonzalez, and even to apply them in the whole (1937, J Physiol 89, 372–383; 1938, J 1970, J Physiol 208, 261–278). animal. Physiol 92, 167–177) and Asmusssen From the knowledge that isometric et al. (1943, Acta Physiol Scand 6, contractions were a feasible method John Coote 168–175) using dynamic exercise to mimic exercise it was a small of forearms or legs showing that a step to evoke these by stimulation Professor Emeritus, School of Clinical pressor or heart rate increase was of the motor fi bres in ventral roots and Experimental Medicine, College proportional to work done and this of anaesthetized or decerebrate of Medical and Dental Sciences, was potentiated by prior occlusion of animals (Coote et al. 1971, J Physiol University of Birmingham, UK

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Peter Wilmshurst, a Consultant Cardiologist at the Royal Shrewsbury Hospital, gives his personal experience of the effect of the English libel laws on scientific debate NMT Medical is an American effect the author of virtually the company, which makes the STARFlex entire 2800 word article by Shelley device for closing holes in the heart. Wood and not just the 79 words in It is suing me for both libel and the article that she attributed to me slander in the High Court in London in quotes. after I spoke in October 2007 at Shelley Wood attributed quotes the Transcatheter Cardiovascular to NMT that I believe most people Therapeutics (TCT) conference in Peter would consider defamed me more Washington DC. TCT is the World’s Wilmshurst than what I had said about them. largest interventional cardiology They told Shelley Wood that I had conference with over 10,000 never been a co-principal investigator delegates. My comments to a She also spoke to executives of NMT in the trial, but she also pointed out Canadian journalist were put on an and wrote an article. I made it clear that she had found numerous press American website. The website and that I believed that information releases on NMT’s website that said the journalist are not being sued. I being presented was both inaccurate I was a co-principal investigator. In am not being sued in the USA. and incomplete. Shelley Wood’s addition, in her article Shelley Wood article pointed out my concerns. She stated that NMT executives had The law suit arises out of my also presented and discussed the said that I had been removed from work as co-principal investigator comments of Dr Dowson and NMT the trial because I had committed and principal cardiologist in the executives. MIST (Migraine Intervention with protocol violations. She also wrote STARFlex Technology) Trial. This was On my return to the UK after that NMT had refused to tell her a multicentre UK trial sponsored by TCT, I found that Dr Dowson had what the violations were. In fact, NMT to investigate whether closing a sent me a copy of a paper about if I had committed any protocol type of hole in the heart that allows the trial, which he said had been violations the CRO should have officially informed me of them and a right-to-left shunt of blood (called a accepted for Circulation. Circulation they have not. patent foramen ovale or PFO) using a is the cardiology journal with the highest Impact Factor and is the STARFlex device would cure migraine The MIST paper was published main journal of the American Heart with aura. in Circulation in March 2008. Association. I wrote back to Dr I immediately contacted the I was invited to speak at TCT 2007 Dowson saying that I was unwilling editor about the inaccuracies and about PFO closure. My lecture to be an author because I could not omissions. I supplied hundreds included comments about the MIST vouch for the integrity of the article of pages of documents. Eighteen Trial, which had ended in early 2006. because NMT had refused to allow months later, in September Some data had been presented the steering committee to see all 2009, Circulation published a 700 at scientific meetings, but journal the data – but that even without word correction, a 4 page data publication was awaited. all the data it was clear to me that supplement and a new version of the article contained errors. I also The other co-principal investigator the paper. Despite that vindication officially informed the independent of my view that the paper was in the MIST Trial was Dr Andrew Clinical Research Organisation (CRO) Dowson, who is a headache inaccurate and incomplete, NMT has and the National Research Ethics not withdrawn its action. Indeed, in specialist. When I arrived at TCT in Service that I intended to ask the October 2007, I discovered that Dr October 2010 they sent me a further GMC to investigate the conduct of letter before action threatening Dowson was also speaking at the Dr Dowson. One other doctor on meeting about the MIST Trial. This a new writ for comments that I the steering committee (Dr Simon made on the BBC Radio 4 Today was contrary to the agreement that Nightingale) also refused to be a I would present MIST data at cardiac Programme in November 2009 when co-author for the same reasons. I spoke about NMT’s libel action meetings and that Dr Dowson would against me. That threat was made present at headache meetings. The next day, NMT’s lawyers wrote against me not the BBC. Shelley Wood, a journalist for the threatening legal action, enclosing online cardiology magazine called copies of the email correspondence The libel case has cost me a lot of Heartwire, heard both presentations I had had the previous day with money and an enormous amount and realised that there were the CRO. In the course of the of time. I felt that NMT was bullying disparities. She spoke separately to proceedings it became clear that me, and life has been made very Dr Dowson and to me about these. they were alleging that I was in difficult for my family and me. For

Physiology News | No. 82 | Spring 2011 | www.physoc.org OPINION PN 17 example, a letter was emailed to should this go to trial, my costs are defamation laws are used to stop me with a Claim Form sealed by likely to exceed £2 million. There is scientists speaking out. Doctors the Court ‘by way of notice rather no guarantee that I will recover my and scientists need the protection than service’. It was sent to me as costs even if I win. of a cheap and easy ‘public interest a non-lawyer, without explanation, defence’ so they do not have to at 17.09 h on the last working day The English defamation laws are make the choice between their before Christmas 2007. Because it weighted in favour of the Claimant, wealth and their patients’ health. was sent just after lawyers’ offices and some lawyers act for Claimants Peter Wilmshurst closed for Christmas, I was unable in a libel case on a CFA, getting up to to get legal advice for 2 weeks. For double the actual costs. For lawyers Consultant Cardiologist, Royal 2 weeks, I had no idea that it had no to act for a Defendant in a libel case Shrewsbury Hospital, Senior legal standing until served. In fact, on a CFA is very rare, because of the Lecturer in Medicine, University of NMT had 4 months to serve it. They high risk of losing and ending up Keele served it at the last possible moment with no money after a lot of work. in April 2008 – nearly 4 months of I am therefore very grateful to my E H Starling and W M lawyers, who are acting for me worrying from Christmas to Easter. Bayliss: the 1914–18 How did issuing a Claim Form but because they consider the issues not serving it help NMT’s reputation? here so important for medicine and War science. If they had not agreed to act The first Bayliss–Starling Lecture, Since then the case has dragged on on a CFA, I risked being bankrupted given by Charles Lovatt Evans in very slowly, but it has involved an or fighting the case on my own as a 1963, has already provided the odd enormous amount of paper work. litigant in person. item for Physiology News (76, p. 14). For example, my Defence to NMT’s I believe I have no option but to Here is another: Claim is 95 pages. NMT’s Reply is 55 continue to fight the case, because pages. Sorting out the thousands of there is a moral imperative that Lovatt Evans mentioned how the pages of supporting documents has medical researchers ensure that 1914–18 War affected physiology consumed every weekend and nearly the data are accurately and fully at University College London. every day of annual leave in the published. The reasons are: (1) The foreign visitors went home last 3 years. I took 2 weeks annual The ethical agreement between almost instantly, among them G. V. leave just to re­-check the 95 pages doctors and patients who are Anrep and B. P. Babkin from Russia of my Defence. The latest hearing subjects in the research involves who, thanks to Starling, were in December 2010 was for ‘security an assurance that the data would able to come back after the war for costs’, to ensure that NMT pays be accurately reported; and (2) If as refugees. The war was a great money into the court so my costs there is inaccurate or incomplete shock to Starling who had German can be paid when I win (as I am sure reporting, patients entering future sympathies but, according to Lovatt I should). Otherwise, I might find trials based on our reported findings Evans, he functioned on the ‘all-or- that I win and have difficulty getting may be put at risk. My Defences are nothing principle’. Not only did he the company, whose share price ‘justification’ (that what I said is true) wish to attack the Germans but he has fallen as low as 18 cents, to pay and ‘qualified privilege’ (that what I spurned their Lieder and language. my costs. At that hearing NMT was said was to people who were entitled Instead he learnt French which he ordered to pay £200,000 security for to know). ‘masticated teutonically.’ costs as a first tranche (the time for payment was extended as a result In addition to fighting the case, I Bayliss stayed on running the UCL of NMT’s application for more time, had to spend considerable time lab and working on wound shock and they have paid £61,000 of the getting Circulation to publish a major but Starling was put in charge of £200,000 so far) or the case would correction and data supplement. I the Anti-gas Department at the be struck out with the verdict in my therefore face a costly libel action for Royal Army Medical College in favour. This £200,000 is just the first fulfilling my ethical responsibilities, London; Lovatt Evans worked with instalment, because my costs will be as a doctor and scientist, to ensure him there. Starling’s advice that much higher by the time the case that published research is accurate. β-β-dichloroethyl sulphide (mustard gets to trial. gas) could be used as an offensive From my experience, I believe agent was rejected. When, later on, The case cost me £100,000 before that scientists and doctors are the Germans used it, he protested my solicitor (Mark Lewis) and under threat of a gagging libel vigorously. In response he was barrister (Alastair Wilson QC) action whene­ ver they question the promoted ‘and sent to Salonika kindly agreed to take my case on a messages that device and drug as Army Chemical Adviser, with Conditional Fee Agreement (CFA – manufacturers want put across. nothing particular to do. He did no win, no fee). Since then the costs Medical and scientific advances this very well...’. have escalated to over £250,000. will be delayed, and the public and The independent estimate is that patients will be put at risk while the Ann Silver

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Bob Edwards – 2010 Nobel Laureate in Physiology or Medicine Sir Richard Gardner gives a personal account of Bob Edwards’ pioneering research into fertility which culminated in the establishment of human IVF Pre-Cambridge years included various additional topics Born in Batley in Yorkshire in that reflected the research interests 1925, Bob Edwards completed of other members of staff. We were his schooling in Manchester thus offered a series of lectures and before embarking on a degree practical classes on reproductive in Agriculture at the University physiology given primarily by Bob. College of North Wales in Bangor. His treatment of the subject was Following a four year interruption in impressively wide-ranging, extending his studies for military service, his from endocrinology to the initial interests shifted towards more basic stages of mammalian development, biology which was reflected in his a subject that was just starting to enrolling in Conrad Waddington’s progress from an almost purely laboratory at Edinburgh University Richard Gardner descriptive to an experimental after completing his degree to do phase. Both I and a fellow Part II a Diploma in Genetics and then a building. This was the start of Bob’s student, Martin Johnson, were PhD. After five very productive years association with Cambridge that was completely captivated by Bob during which he both collaborated to endure for the remainder of his Edwards’ extraordinary energy and with, and then married Ruth research career. enthusiasm, and most especially by Fowler, one of Ernest Rutherford’s his treating us like intellectual equals Bob as mentor grand-daughters, Bob obtained in numerous discussions we had with support to spend a year at the I embarked on Part II Physiology in him both during his practical classes California Institute of Technology the third and final year of the Natural and thereafter. In consequence, in Pasadena. Here he developed a Sciences Tripos at Cambridge with and in defiance of advice from friendship with the late Albert Tyler the aim of doing research either some traditional physiologists, we who inspired his enduring interest in biophysics or neurophysiology, elected to become his first research in reproductive immunology. It providing my degree was good students which was made possible was for pursuing research in this enough to allow me to secure the through our both completing Part II area that Bob gained a five year necessary funding. These were the with Firsts. Thus, we joined the contract to work in the Division of two areas in which the Department Marshall Laboratory in the early Experimental Biology at the National had particular research strengths Autumn of 1966, with me sharing Institute for Medical Research at in the 1960s, and which therefore Bob’s small laboratory and Martin Mill Hill. At the end of this stint, dominated the Part II course. This occupying another room nearby. Bob accepted an appointment in course, irreverently referred to as This was for both of us the start of an Glasgow University’s Department of ‘blood and guts and biophysics’, endlessly stimulating and rewarding Biochemistry where, together with the late Drs Paul and Cole, he made the first attempts to derive cell lines from pre-implantation mouse and rabbit embryos. This, of course, later became a major preoccupation of many laboratories in the drive to extend the bounds of regenerative medicine.

After just one year in Glasgow, Bob was recruited to Cambridge in 1963 by his former head of laboratory at Mill Hill, Alan Parkes, through the offer of a Ford Foundation Fellowship in Reproductive Physiology. Parkes, who had been appointed three years earlier to the newly established Marshall Chair of Reproductive Physiology, occupied laboratories on the top floor of the Physiology Bob in lecturing mode in the 1960s (courtesy of Ruth Edwards).

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dye that bound specifically to DNA, but this proved incompatible with further development of the sexed embryos. Hence, we then resorted to biopsy and staining of the excised tissue which did allow continued normal development so long as the operated embryos were well expanded within a minimally damaged investing coat on transfer to the uteri of recipient does. In this way we were able to demonstrate that the sex of all resulting term offspring had been correctly determined at the blastocyst stage. This proof of principle of pre-implantation genetic diagnosis, which was first successfully applied in man somewhat over two decades later, caused a considerable stir at the time. We presented Bob’s small working area within the Marshall Laboratory. Left to right: Barry Bavister this work as an alternative to who, together with the late Patrick Steptoe, co-authored the 1969 Nature paper on post-implantation diagnosis and achieving human fertilization in vitro, the author, Bob and Alan Henderson, one of his abortion for avoiding the birth of collaborators (from Getty Images). males to women who were carriers relationship with one of the most untenable, simply to sharpen our of serious X‑chromosome-linked notable and independently minded critical faculties. Often, the three genetic diseases like Duchenne-type scientists we could ever have hoped of us would repair to the nearby muscular dystrophy. But, so far to encounter. Fountain pub for discussions over as the media and public were lunch, which typically consisted of concerned, the possibility of being Bob’s style of supervision suited us a couple of pernicious cheese and able to choose the sex of one’s both extremely well, and I am sure it onion rolls washed down with a pint offspring and the various issues this enabled us to operate independently or two of Tartan bitter. might raise were foremost in mind. in research rather sooner than Of course, without any means of might otherwise have been the Proof of principle of pre- accessing pre-implantation human case. Thus, having initially discussed implantation genetic diagnosis embryos at that time, this approach with each of us an impressive range was not a realistic prospect clinically, While I was still engaged in of subject areas we might wish to a situation that was to change developing microsurgical techniques dramatically within the next decade. consider, Bob then assigned us the for transplanting cells and tissues task of choosing a project through between pre-implantation embryos, The origins of IVF acquainting ourselves with the Bob invited me to collaborate relevant literature. On coming back on determining the feasibility of By the time Martin and I joined with our choice, we were then undertaking genetic diagnosis on him in the Marshall Laboratory, challenged by him to defend it in such stages. We chose the rabbit for Bob was already thinking along terms of both feasibility and the this purpose because its blastocyst the lines of bringing the emerging likelihood of generating findings attains a diameter of 5 mm before technologies of induced ovulation, that were novel, interesting, and implantation. The aim was simply and in vitro fertilization and embryo substantial enough for a thesis. to see if we could sex the embryos culture in laboratory and domestic Once this was settled, we were very without compromising their mammals to bear on the problem much left to our own devices, but subsequent development. Our of human infertility. In fact, one of on the clear understanding that it assay was the presence or absence his precious human oocytes was was up to us to seek him out if we in cell nuclei of the sex chromatin among the earliest specimens we needed further advice or guidance. body, which represents the one of examined together. However, since This was not a problem since he was the two X chromosomes in females a first degree in agriculture and a invariably very approachable, but that is genetically inactivated to PhD in mouse genetics are hardly proved an exceedingly tough critic equalize the dosage of genes on apposite qualifications for carrying where necessary. He could also be this chromosome between the two out novel procedures on women, a infuriatingly robust in championing sexes. Initially, we attempted this on critical task for Bob was to identify a viewpoint that seemed utterly intact blastocysts using a fluorescent a clinician who was sympathetic

Physiology News | No. 82 | Spring 2011 | www.physoc.org 20 PN LIVING HISTORY to his aims. This proved quite a Purdy, who I shared with Bob, as the successfully for libel on several challenge since even those in the UK pair of them disappeared, often at occasions. Given the present who were prepared to provide him short notice and for very variable almost universal acceptance of with ovarian tissue often failed to intervals of time, travelling back assisted conception in vitro by both appreciate the need for it to arrive and forth between Cambridge and professionals and public, it is very from the operating theatre in a fresh Oldham with their mobile laboratory. difficult to convey the extraordinarily rather than fixed condition. In fact, poisoned atmosphere Bob and he had to cross the Atlantic to find While, with hindsight, Martin Patrick had to endure during the the first obstetricians who were and I recognize that we were early years of their work. To a really attuned to his ideas, namely extraordinarily privileged to witness humble research student, it was Howard and Gorgeanna Jones, the origins of IVF from ring-side quite an ‘eye-opener’ to see how who were then at Johns Hopkins seats, our view of this endeavour was visceral rather than cerebral the University in Baltimore. Back in the rather different at the time. Bob felt reactions were of many for whom UK Bob’s search took longer but was it was vitally important to inform objectivity was supposed to be the eventually richly rewarded by his and engage the public about what stock in trade. Even today, I find encountering Patrick Steptoe whose he was trying to achieve. To this end, people refer to Bob as ‘controversial’, particular skills in laparoscopic he seized every opportunity to write which misses the point that he could surgery were vitally relevant for popular accounts of his work in both never have achieved what he did retrieving human oocytes. Thus broadsheet and tabloid newspapers. in the climate that then prevailed began a long and close collaboration This had a number of consequences. without being so. between Bob in Cambridge and First, it encouraged the view among Patrick in Oldham. Despite the the mainstream physiologists in the Prominent scientists were among invasiveness of the procedure for building that the Marshall Laboratory the more vociferous critics of IVF, obtaining pre-ovulatory oocytes, was inhabited by noisy self-publicists including two Nobel Laureates, which required general anaesthesia, of dubious scientific merit. Second, JD Watson and MF Perutz. Rather than being cowed by such eminent inflating the abdomen with CO2, and it meant that when Bob was away then probing it with instruments from the laboratory, Martin and I persons, Bob always rose to the inserted though the navel, there had to deal with nosey and often challenge, and almost delighted in seemed to be no shortage of extremely devious reporters. Third, delivering robust responses. This volunteers. This was despite these it fuelled widespread criticism and is illustrated particularly clearly by unsung heroines being clearly given condemnation of Bob and, perhaps the following exchange in which to understand that the chances of surprisingly, extraordinary hostility I was the unwitting instrument alleviation of their own infertility from the medical profession, in providing Bob with critical were slim. For me, the collaboration most notably the British Medical ammunition. Publication on 15 meant the inconvenience of Association. This organization February 1969 of the paper in Nature repeatedly being denied the services branded Bob ‘Dr Frankenstein’, in which Bob and his colleagues of a wonderful technician, Jean thereby prompting him to sue reported having obtained the early stages of human fertilization in vitro provoked a very prompt response from Victor Rothschild in the House of Lords. Rothschild, who had earlier worked on fertilization in sea urchins, questioned in rather patronizing terms whether Edwards et al. really had demonstrated the early stages of fertilization in vitro. Entirely coincidentally, at precisely that time, I had purchased a copy of Rothschild’s book entitled ‘Fertilization’ from Heffer’s Bookshop for 21 shillings. Though devoted entirely to studies on this phenomenon in the sea urchin, it unaccountably had a mouse egg and associated sperm as its frontispiece. The nub of the exchange that appeared in the correspondence section of the March 8 1969 issue of Nature under the title of ‘Did Bob with Patrick Steptoe (courtesy of Ruth Edwards). fertilization occur?’ read as follows:

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Rothschild …“Without wishing to engage in semantic hair-splitting, one must observe that the ‘early stages’ of fertilization may be and, in the note by Edwards et al., are so early as to raise the question whether fertilization, if the word is to have any meaning, occurred at all.”

Edwards, Bavister & Steptoe ….“It should also be noted that conclusions in many papers reporting fertilization in progress have been based on the evidence of pronuclear stages … Indeed, Rothschild is hoist with his own petard, for the frontispiece of his Bob in discussion with the distinguished geneticist Mary Lyon, who spent a sabbatical year in the Marshall laboratory (courtesy of Ruth Edwards). book on fertilization shows a mouse egg with a spermatozoan in the weighing 5lb 12oz. The fact that cases are ‘idiopathic’ and, contrary perivitelline space and labelled “”A Louise Brown was a normal healthy to the view persisting from Tudor live fertilized mouse egg showing girl caused much of the hostility times that problems typically lay the whole spermatozoan in the towards their work to evaporate with the female partner, male factor cytoplasm””. This illustration shows almost overnight, and some of their infertility has emerged as a major fertilization in the same early stage harshest critics within the medical consideration. as in our figure 4B which presents profession wasted little time in a human egg with a perivitelline jumping on the bandwagon and Interestingly, particularly early in sperm.” developing careers within the field their long collaboration, Steptoe’s of assisted conception in vitro. This, name almost invariably preceded In the early days Martin and I could however, was not the end of difficult that of Edwards whenever they were not understand why a highly original times for Bob because Patrick’s referred to by the media. However, creative scientist of Bob’s stature retirement from Oldham meant that Patrick always openly acknowledged was allowing himself to be diverted they had to find a base in Cambridge that Bob provided the intellectual from fascinating and challenging in order to capitalize on their impetus for their work. This was basic scientific problems in the success. What the University had to certainly clear to all the staff at field of mammalian reproduction offer proved entirely inadequate, so Bourn Hall by whom they were and development by what we then that their work was suspended for known as ‘Steppy’ and the ‘Boss’. regarded as a rather pedestrian three years while they sought private piece of applied research. To obtain Other interests funding to establish Bourn Hall clinic. reproducible in vitro fertilization During this time, the ever energetic Bob was throughout these years not and pre-implantation development Bob wrote a massive tome entitled only engaged in other research, but in the mouse was the result of ‘Conception in the Human Female’, also very active in the international years of work in many laboratories, which was described by one of his arena, notably in the founding of requiring the use of literally millions former scientific critics as the best the European Society for Human of oocytes and embryos. To achieve book on obstetrics published in the Reproduction and Embryology this in man, where the numbers of 20th Century. and the editing of its journals. He oocytes would preclude systematic, disengaged from such editorial work controlled, exploration of the myriad Once Bourn Hall was established, on failing to persuade colleagues of potentially relevant variables work soon progressed rapidly with to embrace state-of-art publication seemed to us an intellectually more consistent success in IVF being practices which he then adopted unrewarding and utterly achieved so that the birth of the first in a new journal he founded called daunting exercise. Nonetheless, 1000 babies was celebrated in 1987, Reproductive Medicine Online. This despite numerous reverses and sadly, the year when Patrick died. quickly gained success for its disappointments, Bob and Patrick Most interestingly, medical opinion speed of publication and emphasis pursued this goal doggedly for nine in the early years that IVF would on promoting lively debate of years following publication of their only be of value in treating infertility contentious matters. An issue of this 1969 Nature paper before they were due to blockage of the fallopian journal is currently being prepared finally rewarded by the birth at tubes proved to be entirely wrong. to celebrate Bob’s editing of it for 11.47 pm on July 25 1978 of a baby Most of the successfully treated the first decade of its existence

Physiology News | No. 82 | Spring 2011 | www.physoc.org 22 PN LIVING HISTORY and, after the project was started, often began with “How are you old also the excellent news of his Bean?”, and were invariably spliced Winner of the being awarded the Nobel Prize. It with his infectious enthusiasm and Physiology News contains selected editorials, articles wonderful sense of humour. and other contributions he made annual prize I feel very privileged to have to the Journal during that period, Every year, Physiology News awards had such an original and prefaced by commentaries prepared a prize of £200 for the best article visionary scientist as my mentor, by me together with its new editor, written or co-writen by either an collaborator and friend. Bob not Martin Johnson. What the selection Affiliate Member of The Society only revolutionized the treatment serves to show is the extraordinary or someone within two years of of infertility, thereby making the breadth of Bob’s interests and his having obtained their PhD. deep knowledge of the literature early stages of our own embryonic within the very diverse fields he development accessible to study, chose to explore. Of particular note but also introduced the concepts of is his very early engagement with pre-implantation genetic diagnosis the ethical implications of his work and use of surplus IVF embryos for regenerative medicine. For which featured in a seminal paper this astounding legacy, he richly published in 1971. Nowadays, deserved to be honoured with with the existence of countless the Nobel Prize in Physiology or committees, chairs and, indeed, Medicine. The only shame is that the entire university departments award was not made earlier when devoted to bioethics, it is difficult to he was in good enough health to be recall that the subject hardly existed able to collect it in person. A clue The 2010 prize has been awared at that time. as to why it was so long coming to Peter Bayguinov for the article I ceased to have regular contact might be gleaned from the Vatican’s ‘Generation of complex neuronal with Bob following my migration to bioethics spokesman’s response to behaviour in a mammalian nervous Oxford in 1973, but we continued the announcement as “totally out of system’, which was published in to meet at conferences at which, order”. Physiology News 80, pp. 29–32. particularly in latter years, he was an avid attendee. Before his recent Sir Richard Gardner, KB, FSB, illness we often chatted on the Hon FIAT, FRS Get involved and phone about common research Formerly Edward Penley Abraham write an article for interest and other matters. I greatly Research Professor of the Royal miss these conversations, which he Society in the University of Oxford Physiology News Have you done something in Professor John Conrad Waterlow FRS remembered your studies you would like to – with fondness recommend to other young scientists, attended an amazing I read with sadness obituaries to Professor Waterlow in the press and training course or got an issue listened with equal sadness to the tribute on Radio 4’s Last Word. Both you’d like to get off your chest? marked his passing on October 19 2010 aged 94. If you enjoy writing then why not Some twenty five years or so ago at a Society scientific meeting, I was contribute to Physiology News? We failing in my attempts to withstand some aggressive questioning about have an annual prize of £200 for a presentation I had just made. I thought the questioning was harsh in the best published article written content but in particular, in tone. I did not have the experience to mount by an Affiliate or young scientist. an effective counter. Just before the vote – remember those? – with some If that isn’t enough incentive, difficulty, a frail figure stood up and with immaculate diction, proceeded contributing to the magazine is a to provide much needed and welcome support. My communiqué was great extra on your CV and a nice accepted. way to tell a broader audience about the things you do. We Afterwards, I went and thanked my supporter whose act of kindness, are always looking for people to modesty and graciousness created and left a lasting impression. I was contribute to the Affiliate pages in informed later that my advocate was Professor Waterlow. the magazine and would love to hear from anyone who would like It seems that what he did was entirely characteristic and my memory to get involved. reflects that of those who had the privilege to work with him. I take this opportunity to add my tribute. Email us for more information or to discuss ideas at Edward M. Winter [email protected]

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A letter from reciprocal relationship, choose the wrong word and confusion results: Portland, Oregon ‘A “stationary” cupboard? When Shortly after the US economy had did the office supplies cabinet ever taken a nose-dive and when Obama move?’ was in the early months of his Studying postural control in presidency, I made my big move to Professor Fay Horak’s lab has been the US. I was thrilled to be starting great. Fay is a trail-blazer who my post-doc position at the Oregon has forged an impressive career Health & Sciences University (OHSU). in the field of balance disorders I had spent very little time in through her ‘can do’ attitude, America but growing up in Australia Rebecca and her husband along the Columbia River Gorge. strong collaborations and stellar I was served the standard dose of grant writing skills. I have been Americana, so felt I understood the on my walk to the train station. busy investigating how deep brain basics – I would just need to apply stimulation affects balance in people adjectives such as ‘bigger’ and I have become more of an outdoorsy person since moving here. People with Parkinson’s disease. It is still ‘louder’ to most things. But Portland, poorly understood why delivering I can report, is a special case. assume that as an Australian -- and one from the country no less -- I high-frequency current to parts of Portland is a town where riding a must already be an avid bushwalker, the basal ganglia should improve unicycle carries more caché than or rather ‘hiker’. Not so. In Australia tremor and muscle stiffness, but driving a Hummer. In fact, it is only those with a disposition for the improvements are remarkable. rare to spot the huge gas-guzzlers enduring long hours in the heat Unfortunately, I am finding that amongst the sensibly sized hybrids. while being pestered by flys ever balance control is not as responsive. Thanks to some thoughtful civic get involved in hiking. In Portland In fact, in some cases balance planning, including light rail and everyone is a hiker. Even pale nerds may be worse after deep brain bike lanes, I haven’t needed to buy pull on a polo-fleece and can be seen stimulation surgery. a car which is remarkable for a small walking along the Columbia River As a post-doc I am just beginning the US city of around 500,000 people. Gorge on weekends (see photo). daunting task of applying for grants. The Portlanders I’ve met also do not There are many places to explore. In America there are numerous fit my American stereotype. Their Mount Hood stands to the East of opportunities to apply for federal ambitions are not for fame and the city and to the North looms funding throughout the year, while plastic surgery, rather they aspire Mount St Helens, which is close non-profit and private funding is on to secure a plot in the community enough to have dusted volcanic ash a scale that doesn’t exist in Australia. garden, brew their own beer over Portland during its eruption in It is possible to be perpetually and be on a first-name basis with 1980. I have peered into the gaping writing grant applications! But there the local chicken farmer. Living wound left by that massive side-ways is also intense competition for funds more simply and sustainably in blow-out, and the rising steam hints and I see the familiar shake of the Portland, as far as I can tell, is not she still has more to come. head and shrug of the shoulders in a reaction to recent environmental the lunchroom when people discuss doomsday-speak. Rather, it seems I am also exploring a new language. I have found there is an efficiency their futures. The struggle remains a combination of the ideals from much the same. the hippies that moved North from and directness in American English San Francisco once the 60s petered – qualities that would benefit my I am very fortunate to have become out, and a genuine reverence for the scientific writing. Try ordering a jug colleagues with the emeritus Russian astonishing physical beauty of the of beer and you will be met with Professor, Victor Gurfinkel. The man region. a chuckle, ‘did you mean a pitcher is a treasure chest full of tales about of beer?’ A jug is a tiny version of his seminal scientific discoveries ‘Green’ does not just describe the a pitcher – who knew? You can’t and varied life experiences. At 89 attitude of the inhabitants, it is expect to find beer or wine in a he remains active in the lab with also the dominant colour here. liquor store, there are distilled liquids many ideas yet to test. He stresses This is not the hazy grey–green of only. Also, you don’t hire a car, you that accepted knowledge is often the dry Australian landscape, but would hire a person to drive you based on poor experimentation and a crisp, deep-green produced by about in a car that you have rented. a narrow point of view. The same steady drizzle and rich soil. I’m lucky I can generally find the appropriate could be said of my assumptions enough to work at the OHSU West American terminology: lift instead about America, which have been Campus which is on the site of an old of elevator, trash can instead of challenged by living in this vibrant conifer forest and I still whip out my rubbish bin etc., so watching years of pocket of the country. camera when I spot a deer, squirrel, American television does have some hummingbird or other foreign critter educational value. But this is not a Rebecca St George

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R.D. Keynes (1913– It is widely appreciated that the in aqueous solution); and the gating concept of the ‘ion channel’ of translocation (e.g. by voltage). 2010): the legacy arose from work carried out by And for potassium channels they also Hodgkin and his colleagues both in include another distinct functional from two papers Plymouth (at the Marine Biological characteristic, the ‘long-pore effect’ R.D. Keynes, who died at the Laboratory) and in Cambridge (at of Hodgkin & Keynes. How was this age of 93 in June last year, was a the Physiological Laboratory) during property discovered? Figure 1 from distinguished, indeed central figure the decade 1945–55. This central the relevant 1955 Hodgkin & Keynes in British physiology of the second idea arose from the functional paper shows the key experimental half of the 20th century. In adding characterization of ionic flow finding, and the attached equation to what has already been written, (measured both as current and by what their interpretation of this was. including the delightful piece by radiochemical fluxes) across the Christopher Huang (Physiology plasma membrane of the giant The X-ray crystallographic News 80, 51) and will doubtless yet axon of squid. The 2003 Nobel observations made 50 years later be written (more systematically) Prize for Physiology or Medicine (see Fig. 2) spectacularly prove these concerning his scientific was awarded to R. MacKinnon for postulates. As MacKinnon wrote in contributions, here I want simply his group’s spectacular structural his Nobel lecture: ‘As early as 1955 to draw attention to two seminal work that laid open the exquisite experimental evidence for channel experimental papers that Keynes atomic detail that underpinned mediated ion flow was obtained wrote early in his career. These the functional properties of ion when Hodgkin and Keynes measured contributions, published (jointly and channels, specifically of potassium the directional flow of +K ions across significantly with A.L. Hodgkin) in channels. But what, for such axon membranes using the isotope The Journal of Physiology in 1955, led potassium channels, were these 42K (Hodgkin and Keynes, 1955). rather directly to, indeed could be properties? They include not only the They observed that K+ flow in one seen to under-pin, no less than two specificity of ion translocation (e.g. direction across the membrane different Nobel prizes, awarded to of K+ but not Na+); the high velocity depends on flow in the opposite other very distinguished scientists of ion translocation across the direction, and suggested that ‘the some half-century later. membrane (near-diffusion limitation ions should be constrained to move in single file and that there should, on average, be several ions in a channel at any moment’… After … our crystals improved dramatically, and we were able to solve an initial structure at a resolution of 3.2 Å (Doyle et al., 1998). We could not clearly see K+ in the pore at this resolution, but my years of work on K+ channel function told me that Rb+ and Cs+ should be valuable electron dense substitutes for K+, and they were. Rubidium and Cs+ difference Fourier maps showed these ions lined up in the pore, as Hodgkin and Keynes might have imagined in 1955’.

n' It is also appreciated, though Influx [K]  = O eEF/ RT  perhaps less widely, that in a quite Efflux [K]f  separate study also published in Figure 1. Passive potassium ion movements across the axon membrane observed by 1955, Hodgkin & Keynes provided Hodgkin and Keynes showing the relationship between the measured driving force, the evidence that showed how in

Em–EK (abscissa) and the ratio observed for potassium fluxes (influx/efflux) (ordinate). nerve the process of action potential Each data point is from an individual determination. Note that the slope of the generation (by ionic current flow (continuous) line fitted to the data is steeper than that predicted (dashed line) by a through gated channels) was ‘simple’ Nernst relationship. The equation shown below the data is from the same experimentally distinguishable paper, and suggests an explanation for this discrepancy, namely that the relationship from the subsequent reactions observed is that predicted were potassium ions to flow through a ‘long pore’ that restored (by ion pumping) containing on average more than a single ion, where n' describes the mean number of potassium ions present (‘the occupancy’) in these channels. From their data, n' has the pre-existing ion gradients a value of about 2.5. (‘recovery’). For example, in this

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energy phosphate ester I thought that it could be the substrate [for my enzyme]’.

Both of the Hodgkin & Keynes, 1955, papers are beautifully written studies which draw attention to intrinsic experimental limitations. There is balance in consideration of competing plausible theoretical underpinnings, then the incisive use of quantitative reasoning and finally a set of unambiguous conclusions. Out of this arose insights that are very much alive and kicking 60 years later. What, in our experimental discipline, could be better examples Figure 2. The atomic structure of a potassium channel with ion-binding sites in the than these two papers, for any permeation pathway (of the bacterial potassium channel KcsA). Two diagonally undergraduate or graduate to look opposed subunits of the tetrameric channel are shown. The selectivity filter is at, to read and to study today if they coloured in red. K+ binding sites along the permeation pathway are shown as spheres are to find what is meant by deep with the water molecules surrounding them shown as a blue mesh (modified from rather than ephemeral science? Zhou & MacKinnon, 2003). paper it was shown that these two than that of the (pumped) K influx Richard Boyd processes differed in: their rates (as indicated in the figure). Department of Physiology, Anatomy of ion flow (rapid for the action & Genetics, University of Oxford potential; slow for recovery); their All this preceded the discovery of + + dependence (using dinitrophenol) the Na +K ATPase (Skou, 1957); its stoichiometry (Sen & Post, 1964); References on metabolism (absolute for and its electrogenicity (see Thomas, Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo recovery; not required for the action 1972). It was for his 1957 discovery A, Gulbis JM, Cohen SL, Chait BT & MacKinnon potential ‘spike’); their temperature + + R (1998). The structure of the potassium of the Na +K ATPase that much later + dependence (high Q for recovery; channel: molecular basis of K conduction and 10 Skou was awarded his Nobel prize. In low Q for the action potential). selectivity. Science 280, 69–77. 10 his Nobel lecture Skou acknowledges In this seminal paper (Fig. 3) they Hodgkin AL & Keynes RD (1955). The the critical importance of this potassium permeability of a giant nerve fibre. showed that the ‘recovery’ events Hodgkin & Keynes (1955), J Physiol 128, 61–88. causing the restoration of the publication: ‘I had little knowledge Hodgkin AL & Keynes RD (1955). Active ionic gradients themselves had about the active transport of Na or transport of cations in giant axons from sepia characteristic properties including: K … the closest that I came across and loligo. J Physiol 128, 28–60. mutual interdependence (‘coupling’) [in the literature] was Hodgkin & MacKinnon R (2004). Potassium channels and of Na and K fluxes; metabolic energy Keynes 1955 who had shown that the atomic basis of selective ion conduction. requirement; and the (relative) The Nobel Prizes 2003, ed. Frängsmyr T. Nobel poisoning giant axons with DNP, Foundation, Stockholm, 2004. voltage independence of these cyanide or azide decreased the active fluxes. An additional feature was that Sen AK & Post RL (1964). Stoichiometry transport of sodium suggesting that and localization of adenosine triphosphate- the size of the (pumped) Na efflux high energy phosphate esters are dependent sodium and potassium transport in was slightly but significantly larger the substrate, and as ATP is a high the erythrocyte. J Biol Chem 239, 345–352. Skou JC (2003). The identification of the sodium potassium pump. Nobel Lectures, Chemistry 1996–2000, ed. Grenthe I. World Scientific Publishing Co., Singapore. Skou JC (1957). The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim Biophys Acta 23, 394–401. Thomas RC (1972). Electrogenic sodium pump in nerve and muscle cells. Physiol Rev 52, 563–594. Zhou Y & MacKinnon R (2003). The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a Figure 3. The Hodgkin and Keynes scheme for ion transport in the axon membrane, protein conformational change underlie high distinguishing events involved in action potential generation from those in recovery. conduction rates. J Mol Biol 333, 965–975.

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Two arms as one: position and movement sense in an elbow- matching task Masahiko Izumizaki and Uwe Proske have found that when subjects match the position of one unseen arm by placement of the other, sensory information coming from both arms is used to achieve the observed matching accuracy.

This story is about the kinaesthetic is a largely selective stimulus for the senses. They are part of a group of primary endings of muscle spindles. senses, the proprioceptive senses, The extra impulses generated by concerned with signalling the body’s the vibration lead to an illusion own actions. The kinaesthetic of limb movement and displaced senses comprise the sense of limb position (Goodwin et al. 1972). We position and the sense of limb wanted to know whether altering movement (Proske & Gandevia, the conditions of the indicator arm 2009). Other proprioceptive senses affected matching accuracy when Masahiko Izumizaki (left) and Uwe include the senses of effort, tension the reference arm lay relaxed and Proske and balance. The proprioceptive when it was being vibrated, that senses are somewhat mysterious and are able to match positions with is, in the presence of a large, false because we are largely unaware of errors of 2 deg or less. If, instead of proprioceptive signal coming from them. When we look at an object using a matching process, subjects the reference arm. or touch it we experience a specific, are asked to indicate the position of identifiable sensation. By contrast, one arm with a pointer, they are less The manipulations of the indicator we know precisely where our limbs accurate. It has led us to conclude arm were of four different kinds. In are when we are not looking at that arm matching is a cooperative all trials the reference arm lay hidden them, but that awareness is not process in which both arms play a from the subject’s view. In the first accompanied by any identifiable contributory role. In order to further series of matches the experiment sensation. It presumably means that explore this idea we have done some was done in the traditional way, the sensory information concerned simple experimental manipulations with the subject blindfolded so that with signalling the position and of one arm to see what effect it had they could see neither arm. In the movement of our limbs is processed on matching accuracy (Izumizaki second series subjects could see largely unconsciously. et al. 2010). We carried out these their indicator arm; in the third the matches under two conditions, indicator was placed out of view and The present day view is that the one where the reference arm lay subjects were required to match the principal kinaesthetic sensors are relaxed on a support and the second, perceived position of the reference the muscle spindles. At some joints, when its elbow flexor muscles were arm by placement of a dummy arm. particularly the finger joints, skin and vibrated at 80 Hz. Muscle vibration Finally, a mirror was slipped into joint receptors also contribute to the the partition between the two arms senses of position and movement. (Fig. 1). The position of the mirror At the elbow joint skin stretch was arranged so that the reflected receptors are likely to supplement image of the indicator arm appeared the main kinaesthetic signal coming to occupy a position coincident from spindles in elbow muscles. with that of the reference arm. Here The primary endings of muscle subjects were instructed to look only spindles are thought to contribute at the mirror image and not at the to both the sense of limb position actual indicator. Example records of and the sense of limb movement matching trials are shown in Fig. 2 while secondary endings of spindles and the group results are shown in contribute to the sense of position Figure 1. Subjects sat in front of a Fig. 3. (McCloskey, 1973). two-compartment box. The partition between the compartments was a The first result was that for the We have been studying position mirror that showed an image of the left, matching series where the reference sense at the forearm in a simple indicator arm. Position of the image was arm was not being vibrated, subjects matching task. One arm, designated arranged to coincide with the position managed to achieve comparable the reference arm, is placed in of the right, reference arm. The mirror matching accuracy no matter what could be covered up. Subjects’ arms a particular position and the the condition of the indicator. Errors were taped to paddles which hinged at a blindfolded subject is asked to match point close to the elbow joint. Joint angle were similar (Fig. 3) when subjects it with their other, indicator arm. was measured with potentiometers were blindfolded and could not see Subjects are normally quite accurate coincident with the elbow joint. either arm, or they could see the

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Figure 2. Forearm position matching. Each Blindfolded Arm panel represents two matching trials. The reference arm, which was always hidden from view, was placed at 30 deg to the horizontal and several seconds later the subject matched its position. The blue trace is the position of the indicator during matching without vibration, the red trace during vibration of the reference arm. The end of each trace was the point where the subject declared Dummy Mirror they had achieved a satisfactory match. The four conditions were matching blindfolded, matching while watching the indicator arm, using a dummy indicator arm for matching and looking at the mirror image of the indicator during matching. Notice during vibration that matching angles are small, that is, the indicator is more extended than the reference. indicator or its mirror image, or they able to see their indicator arm In a simple interpretation, based on used the dummy to match positions. (Fig. 3). However, the errors were experience, vision of a particular arm This last result is interesting in that it significantly smaller, 8.6 deg, when posture is calibrated in terms of the implies subjects were equally able to subjects used a dummy to match normally expected proprioceptive match position using proprioceptive and 8.4 deg when they looked at signal. The discrepancy between the information only (blindfolded) or a mirror image of their indicator expected position indicated by sight visual information only (dummy). arm during matching. With the of the dummy and that indicated by However, significant differences dummy, subjects were using a the proprioceptive signals coming between conditions emerged when visual cue to match the position of a from the reference arm led to a the reference arm was vibrated. proprioceptively perceived position. reduction in the position errors

Vibration of the reference arm elicited a strong illusion that the arm was moving into extension and that its position was more extended than really was the case (Figs 2 and 3). The brain was falsely interpreting the muscle spindle signal from vibration as an elongating muscle, that is, an extending forearm. The size of the measured illusion was largest when the subject was blindfolded and they relied entirely on proprioceptive signals for matching. Here, presumably, the subject was trying to match the spindle signal coming from the non-vibrated arm with the much larger vibration-evoked signal coming from the reference arm. They did this by adopting an extended position with their indicator, in the process stretching its flexor muscles and raising the Figure 3. Position matching errors for the group. Matching angle differences for the flexor spindle signal to a level group of 15 subjects shown as means (± S.E.M.), without vibration (blue) and with vibration of the reference arm (red). The four conditions were as in Fig. 2. Angle somewhere near that coming from difference was calculated as the position of the reference arm minus the position of the reference arm. The observed the indicator arm. 0 deg represented a perfect match. Positive errors were where error was 12 deg (Fig. 3). the indicator arm had adopted a more extended position relative to the reference; negative errors were where the indicator had adopted a more flexed position. Interestingly the error was only Asterisks indicate significant differences (P < 0.05) from the blindfolded condition for slightly smaller, 11 deg, when the vibration trials and from the condition where the subject could see their indicator subjects matched while being arm for the non-vibration trials.

Physiology News | No. 82 | Spring 2011 | www.physoc.org 28 PN SCIENCE NEWS AND VIEWS produced by the vibration. We think The main conclusion from this All of the observations described something similar was occurring first set of experiments was that so far were concerned with the when subjects looked at the mirror the condition of the indicator arm sense of forearm position. What image of the indicator arm. Here an clearly played an important role about the sense of movement? additional factor was likely to be that in determining the accuracy of The way we explored this was as subjects were unsure of which arm forearm position matching. Under follows: a vibration illusion was they were looking at. In the trials normal circumstances, vision generated in one arm as before, where subjects simply looked at and proprioception can be used but this time subjects were asked the indicator arm during matching, interchangeably during matching. to track the perceived movement vibration errors were only slightly However, if a false proprioceptive with the vibrated arm itself. So now smaller than in the blindfolded signal is introduced in one arm with during vibration the arm is slowly condition as here both visual and vibration, this creates a conflict extending according to what the proprioceptive signals coming from between the information provided subject felt. We measured the speed the indicator would be available and, by the proprioceptive and visual of the movement on a computer. very probably, the proprioceptive inputs from the two arms to reduce Throughout this time the other arm signal would be prioritised. the effect of the vibration. was held still at a particular angle. Then the experimenter began to move the non-vibrated arm in either the direction of extension or flexion. When the movement was in the direction of extension, subjects were seen to slow the speed of their tracking movement with the vibrated arm. When the movement was into flexion, movement of the vibrated arm speeded up (Fig. 4). Remarkably, when subjects moved their non-vibrated arm up and down in a continuous motion, they reported a ‘flutter’ sensation of slowing and speeding-up of their vibrated arm, even when this was not moving at all. There can be no more dramatic example of an interaction between proprioceptive signals coming from the two arms than this change in the quality of the sensation evoked by vibration in one arm that depended entirely on what the other arm was doing.

Our interpretation of this result is that what is felt during vibration is based, at least in part, on a difference signal derived from the inputs from the two arms. When the Figure 4. Movement sensations in a vibrated arm during movement of the other arm. non-vibrated arm was moving into Upper panel, perceived movement of the vibrated arm with the other arm stationary extension its elbow flexor muscles and then moving in the direction of extension (red trace) or the direction of flexion would be stretched and flexor (blue trace). The arrows indicate the point at which the non-vibrated arm began to spindles would therefore raise their move. The bar at the top of the figure indicates the period of vibration. Notice the level of discharge, thereby reducing illusion of forearm extension in the vibrated arm took about 3 s to develop and that the difference in proprioceptive in the two trials shown its speed before the other arm was moved was not quite signal between it and the vibrated the same. However, as soon as the non-vibrated arm was moved into extension, arm. It led to slowing of the tracking perceived movement speed of the vibrated arm slowed and when the non-vibrated movement by the vibrated arm. arm was moved into flexion it sped up. Lower panel, the pooled data from seven subjects. Mean (± S.E.M.) values for the movement illusion speed (deg s-1) when the Conversely, if the non-vibrated arm non-vibrated arm was held still, when it was moved in the direction of extension (red) was flexed, its spindles would be and when it was moved in the direction of flexion (blue). Asterisks indicate significant slackened by the muscle shortening differences in speed between when the arm was still and when it was moving (both and their discharge would be slowed, P < 0.05). increasing the difference in signal

Physiology News | No. 82 | Spring 2011 | www.physoc.org SCIENCE NEWS AND VIEWS PN 29 between it and the vibrated arm. A novel way to test human motoneurone That led to an increase in speed of the tracking movement. behaviour during muscle fatigue The present study has shown that Descending voluntary drive during a sustained contraction both the sense of limb position makes it difficult to test the true effect of fatigue on the and the sense of limb movement excitability of spinal motoneurones. Transient, artificially of one arm are dependent to induced interruption to voluntary drive enables assessment of some extent on the information motoneurone behaviour during muscle fatigue. This technique coming from the other arm and that this information can be either reveals a profound reduction in motoneurone responsiveness visual or proprioceptive. Such a not seen during intact voluntary drive. conclusion should be kept in mind Fatigue is one of the most common in experiments on proprioception symptoms reported to general allegedly involving only one arm. practitioners. Although it may be Input from the other arm may not difficult to separate general malaise be entirely irrelevant. When the from muscle fatigue in some two arms are involved in a motor circumstances, muscle fatigue is an task where they are required to unavoidable consequence of life. act cooperatively, for example, This is true across the full spectrum fine manipulations that require of activity, from simple daily tasks both hands close together, the such as standing or climbing stairs central comparator is influenced to voluntary exercise or training. From top left clockwise: by information coming from both A commonly accepted definition Janet Taylor, Chris arms, be it proprioceptive, visual or of muscle fatigue is ‘any exercise- McNeil, Simon Gandevia both. At the same time the identity and Peter Martin induced reduction in the ability of each arm is preserved. So in some to exert muscle force or power circumstances the two arms can be regardless of whether or not the task considered to be acting as one, a contractions (Taylor & Gandevia, can be sustained’ (Bigland-Ritchie single instrument, in the execution 2008). Despite this significant & Woods, 1984). It is well known of certain skilled tasks. contribution, the proportion of that muscle fatigue has both central central fatigue which resides in the 1 and peripheral components which Masahiko Izumizaki and Uwe motor cortex versus spinal cord occur in the central nervous system Proske2 remains unclear. and working muscle, respectively. 1Department of Physiology, Showa Changes within the muscle usually One method to test for fatigue- University School of Medicine, have the greater contribution to related changes at the level of the Tokyo, Japan muscle fatigue and as a consequence spinal cord in humans is to stimulate 2 Department of Physiology, Monash much more research has been the corticospinal tract non-invasively University, Clayton, Victoria, devoted to peripheral rather than at the cervicomedullary junction. Australia central fatigue. However, it has This form of stimulation is usually been estimated that as much as 25% References accomplished by fixing electrodes of force loss can be attributed to behind the ears to the mastoid Goodwin GM, McCloskey DI & Matthews PBC (1972). The contribution of muscle afferents nervous system changes during a processes and passing an electrical to kinaesthesia shown by vibration induced sustained 2 min maximal voluntary current pulse between the illusions of movement and by the effects of contraction and this proportion is electrodes. The stimulus produces paralysing joint afferents. Brain 95, 705–748. even greater for weaker sustained a short-latency cervicomedullary Izumizaki M, Tsuge M, Akai L, Proske U & Homma I (2010). The illusion of changed position and movement from vibrating one arm is altered by vision or movement of the other arm. J Physiol 588, 2789–2800. http://jp.physoc.org/content/588/15/2789.long McCloskey DI (1973). Differences between the senses of movement and position shown by the effects of loading and vibration of muscles in man. Brain Res 63, 119–131. Proske U & Gandevia SC (2009). The kinaesthetic senses. J Physiol 587, 4139–4146. Figure 1. Silent period induced by high-intensity transcranial magnetic stimulation. Acknowledgement The responses obtained during two brief (2–3 s) maximal efforts are overlaid. The We would like to thank Professor Ikuo Homma shaded area identifies the period of electrical silence which follows the motor cortical for his insight and encouragement during the stimulus. Note, in this study and many others, the onset of the silent period is project. actually measured from the time of the motor cortical stimulus.

Physiology News | No. 82 | Spring 2011 | www.physoc.org 30 PN SCIENCE NEWS AND VIEWS motor-evoked potential (CMEP) in intrinsic motoneuronal properties protocol in an attempt to accomplish the electrical signal recorded from caused by their repetitive discharge this (McNeil et al. 2009). the target muscle. As the stimulus during the sustained maximal is delivered below the level of the effort; and (2) disfacilitation of the Transcranial magnetic stimulation motor cortex, a CMEP can be used motoneurones caused by a fatigue- is a non-invasive means to activate to assess motoneurone excitability related reduction of excitatory input neurones within the motor cortex. without the need to consider cortical from muscle afferents. However, the A stimulating coil that produces a changes induced by fatigue. question of mechanism is difficult magnetic field is held against the to address because motoneurone appropriate region of the scalp. Using this method of stimulation, responsiveness is strongly affected When a strong stimulus is delivered we have demonstrated that by the presence and magnitude of during a voluntary contraction, CMEP size decreases during the descending voluntary drive from the discharge of the coil affects last 30 s of a sustained 2 min the brain and it is uncertain if the cortical output cells and transiently maximal voluntary contraction level of this drive that reaches the interrupts descending voluntary due to a reduced excitability of motoneurones increases, decreases drive. This stops output from the the motoneurone pool (Butler et or has no net change with fatigue. motoneurones and causes a period al. 2003). What mechanisms are Hence, we wanted to assess of silence in the electrical activity responsible for this fatigue-related motoneurone excitability without, recorded at the target muscle reduction in excitability of the or with minimal influence from, the (Fig. 1). If a corticospinal stimulus motoneurones during a sustained confounding factor of an unknown were delivered in this silent period, maximal effort? We proposed level of descending voluntary drive. motoneuronal properties could be two candidates: (1) a change to We recently used a paired-stimulus tested during the development of fatigue without the complication of unknown levels of descending drive. Thus, we paired a corticospinal stimulus with a strong transcranial magnetic stimulus delivered 100 ms earlier. This pair of stimuli was given at regular intervals throughout a sustained 2 min maximal effort to assess the effect of fatigue on motoneurone excitability during the silent period (Fig. 2A). Raw traces showing CMEPs during the silent period are displayed for a single subject in Fig. 2B.

In the absence of descending voluntary drive from the brain, there is a rapid and profound decrease of motoneurone excitability during a sustained maximal effort (Fig. 3, filled symbols). This result is in stark contrast to the previous and current finding that motoneurone excitability (normalised to the increase of the peripheral potential; Fig. 3, red line) shows no change for the first 90 s of maximal effort when assessed in the presence of Figure 2. Individual traces of biceps electrical activity recorded from a single subject descending voluntary drive (Fig. 3, in brief control maximal efforts and a sustained 2 min maximal effort. A, schematic open symbols). A comparison of diagram of maximal contractions performed and the timing of paired motor cortical– fatigue-related changes to CMEPs corticospinal stimuli. Red arrows identify the paired stimuli for which electrical recorded during the silent period responses are shown in panel B. B, electrical responses obtained after paired motor to CMEPs recorded during ongoing cortical–corticospinal stimuli during three brief control maximal efforts are overlaid voluntary drive (Fig. 3, filled and in the top trace. The bottom traces are two of the responses recorded during the open symbols) demonstrates the sustained 2 min maximal effort. The dashed box surrounds the CMEPs evoked in the silent period following the conditioning motor cortical stimulus. In this subject, potency of descending voluntary the CMEP in the silent period is abolished within 30 s of contraction. Modified from input to the motoneurones. Namely, McNeil et al. 2009. the facilitatory effects of this input

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spindle input has only a limited contribution to the profound fatigue- related decrease of motoneurone excitability.

In summary, a test of motoneurone responsiveness after interruption of descending voluntary drive shows that the facilitatory effect of this input to the motoneurones masks a profound and rapid decrease of motoneurone excitability during a sustained maximal effort. We believe that a stimulus to the corticospinal tract paired with a preceding strong stimulus to the motor cortex is a promising technique to explore Figure 3. Normalised CMEP responses during a sustained 2 min maximal effort. spinal contributions to central N = 8. Data are mean values (± S.E.M.) for CMEPs in the presence (○) and absence (●) of descending voluntary drive and are expressed as a percentage of values obtained fatigue in humans. during brief control maximal efforts. The red line represents an approximation of the typical growth of the compound muscle action potential (Mmax) during a 2 min Chris J. McNeil, Peter G. maximal effort, which is a consequence of changes in the muscle fibre membrane. Martin, Simon C. Gandevia The area of CMEPs in the presence of voluntary drive increases during the first 90 s and Janet L. Taylor of contraction but this is largely the result of an increase in the area of Mmax. The decrease in the area of these CMEPs during the final 20 s of contraction (data points Neuroscience Research Australia denoted by arrows) represents a reduction in excitability of the motoneurones. In and University of New South Wales, contrast, the area of CMEPs in the absence of voluntary drive decreases rapidly and Randwick, NSW, Australia reaches a nadir within 30 s of contraction. Modified from McNeilet al. (2009). References delay any manifestation of reduced in a human, but intracellular Bigland-Ritchie B & Woods JJ (1984). Changes in muscle contractile properties and neural motoneurone responsiveness recordings from animals show control during human muscular fatigue. until 90 s of maximal effort have that motoneurones adapt to a Muscle Nerve 7, 691–699. elapsed. Further, the decrease in constant-current input and discharge Butler JE, Taylor JL & Gandevia SC (2003). responsiveness seen after 90 s is still at a reduced rate due to altered Responses of human motoneurons to markedly attenuated compared to conductances and a lower input– corticospinal stimulation during maximal voluntary contractions and ischemia. J the reduced excitability seen in the output gain of the motoneurone Neurosci 23, 10224–10230. silent period less than 20 s into a pool (see Powers & Binder, 2001 McNeil CJ, Martin PG, Gandevia SC & Taylor sustained maximal contraction. for review). Such processes, and no JL (2009). The response to paired motor doubt others, are at the heart of cortical stimuli is abolished at a spinal level Despite the ability to provide novel the generic term changes to intrinsic during human muscle fatigue. J Physiol 587, insight into the effects of fatigue properties of motoneurones. The 5601–5612. on motoneurone excitability, limits to probing these changes in http://jp.physoc.org/content/587/23/5601.long the combined use of transcranial humans prompted us to examine Powers RK & Binder MD (2001). Input-output magnetic stimulation and functions of mammalian motoneurons. Rev the role of the reduced excitatory Physiol Biochem Pharmacol 143, 137–263. corticospinal tract stimulation is input from muscle afferents. We necessarily non-invasive and hence Taylor JL & Gandevia SC (2008). A comparison tweaked our previous protocol (seen of central aspects of fatigue in submaximal it remains difficult to identify the in Fig. 2A) to include intermittent and maximal voluntary contractions. J Appl mechanisms responsible for the tendon vibration to increase muscle Physiol 104, 542–550. marked fatigue-related reduction afferent input to the motoneurones of motoneuronal excitability. during the silent period. Tendon Physiology News Recall that our earlier experiment vibration strongly excites muscle with intact descending voluntary If you have enjoyed this issue of spindle afferents which should Physiology News please don’t throw it drive (Butler et al. 2003) led to two provide excitatory input to the away. Put it in your coffee room so that proposed mechanisms, a change to motoneurones innervating the others may see it too. intrinsic motoneuronal properties muscle in which the afferents and a reduction of excitatory input are located. Preliminary findings We are always looking for interesting from muscle afferents on to the indicate that vibration during a features, meeting reports, news motoneurones. It is unacceptably sustained maximal contraction does items and photographs. Contact us ([email protected]) with your invasive to obtain a direct recording not change CMEP size. A possible suggestions. of intrinsic motoneurone properties interpretation is that reduced muscle

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What makes some axons more excitable than others? Have you ever wondered why some motor axons may be activated before all others in a muscle as an electrical stimulus is increased? It could just be that the low threshold represents a quirk of anatomy and current flow, but it could also be that the excitability of the axon is greater than that of other axons innervating the muscle. The latter seems to be the case and a key factor seems to be the hyperpolarisation-activated current (Ih) The excitability properties of human axons can be studied non-invasively by tracking the changes in the strength of the electrical stimulus required to generate an action potential. This technique is known as threshold tracking.

We teach that the largest nerve fibres not only conduct faster but are also easier to excite electrically than smaller axons. Small axons arising from small motoneurones are recruited first by voluntary effort or by reflex inputs (according to Henneman’s size principle; Henneman et al. 1965). On the Left to right: James Howells, Susan Tomlinson, David Burke, Louise Trevillion and other hand, sensory axons in Hugh Bostock. human peripheral nerve are excited 100 electrically at lower threshold than motor axons due, in part, to a Depolarization difference in sodium channels. 0 Now a study using the threshold H tracking of human motor axons y p has shown that the first motor -100 e axons to be excited electrically r p are those with greater activity of o the hyperpolarization-activated l -200 a current, Ih. Ih is a depolarizing r current that plays a pacemaker role i z in the heart and CNS and is due to a hyperpolarization-activated cyclic %) reduction, (threshold Excitability -300 t i nucleotide-gated (HCN) channels. o n This previously unsuspected role -400 of HCN channels in controlling 0 50 100 150 200 250 300 350 the resting excitability of axons Delay (ms) may be linked to their role in the hyperexcitability of the nociceptive Figure 1. Excitability changes in sensory and motor axons of the median nerve in afferents responsible for neuropathic response to subthreshold polarizing currents (unpublished mean data for 3 subjects). Threshold electrotonus was recorded by tracking compound sensory nerve action pain. potentials (dashed lines; recorded from digit 2) and compound muscle action potentials (continuous lines; recorded from abductor pollicis brevis). The injected Sensory and motor axons depolarizing current was set to 40% of the 1 ms threshold for the target potential and Differences in membrane was 100 ms in duration (black). The injected hyperpolarizing current was set to –40% conductances between sensory (red), –70% (green) and –100% (blue) of the threshold for the target potential and and motor axons have been was 100 ms, 200 ms or 300 ms in duration, respectively (duration of conditioning demonstrated in human peripheral current indicated by horizontal lines just above the X-axis). Note that the threshold changes induced by hyperpolarization were less for sensory axons than motor nerves. There is a significant axons, reflecting greater activity of Ih in sensory axons. Note also that the threshold difference in the accommodation overshoot when the polarizing current was switched off (110–175 ms, red traces; to hyperpolarization between 210–300 ms, green traces) was greater for sensory axons. The accommodation has a sensory and motor axons (shown relatively slow onset and, as indicated for sensory axons by arrowed lines, it increases as changes in excitability in Fig. 1). with the degree of hyperpolarization.

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Sensory axons appear to have a strength of an externally applied muscle fibres innervated by a single greater activity of Ih (Fig. 1; Bostock electrical stimulus required to motor axon) and then painstakingly et al. 1994). It is believed that generate an action potential. adjusted stimulus voltage by hand the role of the current is to limit (Typically adhesive ECG electrodes to track changes in the threshold activity-dependent hyperpolarization are used with the cathode positioned of the axon. He made meticulous following the conduction of trains of over the nerve and the anode placed and methodical recordings from impulses. Sensory axons also have a remotely on electrically less excitable single motor units in order to study greater persistent Na+ conductance tissue nearby.) The stimulus is recovery from single and repetitive (Bostock & Rothwell, 1997), reflected submaximal, and is adjusted by activation and the influence of in a longer strength–duration time computer to activate a preset ischaemia, electrical polarization constant (Panizza et al. 1992). submaximal nerve volley (usually and temperature on these This Na+ conductance is active 40–50% of maximum). The nerve processes. From these non-invasive, at threshold and contributes to volley generates a compound muscle in vivo studies, Bergmans was resting membrane potential. These action potential (CMAP), recorded able to describe the mechanisms differences have been suggested using surface electrodes placed responsible for the different phases to render sensory axons more over the belly of the innervated of post-tetanic hyperpolarization. susceptible to ectopic activity than muscle, and a compound sensory motor axons, while making them nerve action potential (CSNAP or, The development of an automated less susceptible to conduction block. more simply, SNAP) recorded with technique for tracking the ring electrodes placed around the threshold of a compound sensory In addition to differences between innervated digit. If axons become or muscle action potential (Bostock sensory and motor axons, there are depolarized (for example, during et al. 1998) gave researchers a differences within an axon type. It is ischaemia induced by inflation of remarkable tool that allowed well documented that motor units of a pressure cuff), they are more them to rapidly assess axonal a muscle differ in their motoneurone excitable and a smaller current excitability. In a matter of minutes, properties and the mechanical and will be required to produce the the stimulus–response relationship, histochemical properties of the target potential. If axons become strength–duration properties, innervated muscle fibres. Motor hyperpolarized (for example, after accommodation to subthreshold units are remarkably consistent conducting trains of impulses during changes in membrane potential in their recruitment thresholds, a sustained muscle contraction), and recovery after activation can be whether they be in voluntary or they are less excitable and more measured, providing information reflex contractions on the one current is required. This technique about both nodal and internodal hand, or in response to electrical of threshold tracking provides conductances. The measurements stimululation on the other. All measures of excitability which are made non-invasively and in things being equal, voluntary effort are determined by the electrical real time, allowing the time course should recruit motoneurones with properties of the axonal membrane of interventions such as drug smaller, more slowly conducting at the point of stimulation. The administration and renal dialysis to axons first (Henneman et al. 1965), procedure differs from nerve be followed. while electrical stimulation should conduction studies in which a activate larger axons first. There Many studies have been conducted supramaximal stimulus is used to are, however, a number of factors using this tool and there is now ensure that all axons within the which need to be taken into account. a considerable body of literature nerve are excited. In studies in human subjects, describing findings in normal nerves stimulation is percutaneous, at some The field of nerve excitability owes and in numerous pathophysiological distance from the closest nodes of a debt to Joseph Bergmans who, in conditions ranging from nerve Ranvier. Do proximity to the stimulus 1970, documented the excitability disorders, such as channelopathies and anatomical factors determine properties of single human motor and demyelinating conditions, which axons are first recruited from axons. He found that single motor to metabolic disorders, such as any one site rather than variations axons could be selectively activated renal failure and diabetes. Further, in intrinsic excitability due to over a large range of stimulation differences between the excitability differences in their biophysical strengths and that the same motor properties of axons in the upper properties? If low threshold is a units could be activated by threshold and lower limbs and also between reflection of increased excitability, stimuli on repeated occasions when the proximal and distal segments what are the determinants? the stimulating electrodes were within the same nerves have been placed in the same location. He found. The technique has also been Nerve excitability and threshold defined threshold as the lowest successfully applied to the study of tracking stimulation strength that would elicit animal nerves (and single axons), The excitability properties of human three to five consecutive motor unit giving rise to a body of literature on axons can be studied non-invasively potentials (a motor unit potential the changes in axonal excitability by tracking the changes in the is the potential generated by the with maturation.

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Excitability properties of single of differentiation between the may have a greater activity of motor axons axons was found to be in the Ih and may, therefore, be more Automated threshold tracking has accommodation to hyperpolarizing secure against the development recently been applied to investigate currents (Fig. 2). Low-threshold of conduction block in disease the excitability of selectively axons were found to have greater processes. The flipside is that the activated single motor units (a accommodation to hyperpolarizing increase in excitability makes motor unit being the motoneurone, changes in membrane potential, these axons more prone to ectopic its motor axon and the muscle fibres presumably due to a greater activity, such as fasciculation, and it innervates) to determine whether hyperpolarization-activated perhaps to motoneurone loss in the reason that they have a low inwardly rectifying conductance, amyotrophic lateral sclerosis. A threshold to electrical recruitment is Ih. This conductance has been corollary of this is that axons from due to intrinsic properties (Trevillion shown to have a pacemaker role in smaller motoneurones, recruited et al. 2010). Threshold tracking spontaneously firing neurones and more readily into voluntary enabled comparisons to be made the sinoatrial node. HCN channels contractions, may be more likely between the excitability properties mediate the inwardly rectifying to develop conduction block when of the single motor axons with those conductance and are known to exist hyperpolarized during activity. of higher threshold motor axons in four isoforms. All have been found This has implications for conditions recruited in compound potentials in the mammalian nervous system, such as multiple sclerosis, chronic recorded in the same experimental although channel expression in the inflammatory demyelinating session. human peripheral nervous system is polyneuropathy and multifocal yet to be characterised. The findings motor neuropathy, where these Based on earlier published findings for single motor axons suggest that conduction abnormalities occur. on the ionic determinants of HCN channels provide a depolarizing threshold (Bostock & Rothwell, current at rest and therefore play a The stimulus–response curve for 1997), it was anticipated that a role in setting membrane potential. motor axons is relatively steep, difference in the persistent Na+ reflecting minimal variation in conductance between the low- and Physiological implications threshold. However, the above findings demonstrate a significant high-threshold axons would be The above findings suggest that the variation in the intrinsic properties revealed. However, this proved motor axons that are less readily of different motor axons. This may not to be the case. The key point recruited by voluntary activation have implications for some motor unit number estimation (MUNE) techniques that are based on the ‘mean’ motor unit measured using surface electromyography.

Spontaneous discharges from injured peripheral nerves are believed to be responsible for the induction and maintenance of neuropathic pain syndromes. Upregulation of

Ih in injured nerves is believed to be a factor in the spontaneous action potentials (Chaplan et al. 2003), making HCN channels the targets for pharmacological treatments for neuropathic pain.

Just as Ih expression varies between motor axons, resulting in threshold

variation, it may be that Ih expression Figure 2. Threshold changes in motor axons during threshold electrotonus. varies considerably between sensory Subthreshold currents, both depolarizing (40% threshold for the target potential) axons rendering some more prone to and hyperpolarizing (–40% threshold for the target potential), were applied for generating the spontaneous activity 100 ms (as indicated by bar just above the X-axis) and the threshold of the single seen in neuropathic pain syndromes. motor units (filled circles) and compound potentials (40% of maximal amplitude; open circles) were tracked before, during and after the currents. Note the divergence Computer-controlled threshold in the threshold changes to hyperpolarization between the low-threshold axons tracking provides a reliable method of the single motor units and the high-threshold axons recruited in the compound potential. Hyperpolarization produces a lesser threshold change for the single motor for assessing Ih expression in units, much as for sensory axons shown in Fig. 1 (see the responses to 100 ms human motor and sensory axons, hyperpolarization). This and other findings support the conclusion that there is non-invasively by tracking compound greater inward rectification, due to greater Ih, on the low-threshold single motor action potentials (Tomlinson et al. axons. Data presented as mean ± S.E.M. (n = 22). 2010). In fact, threshold tracking

Physiology News | No. 82 | Spring 2011 | www.physoc.org SCIENCE NEWS AND VIEWS PN 35 could be used to assess the efficacy http://jp.physoc.org/content/588/13/2503.long of HCN channel blockers in animal Paton Prize Bursary Photographic models of neuropathic pain and to Competition monitor the effects of these agents on axonal function. 1 The Paton Prize fund promotes interest in an historical perspective Louise Trevillion , James on physiological research. Under the auspices of the History & 1 1 Howells , Susan Tomlinson , Archives Committee, a £100 prize is offered for the best photograph 2 Hugh Bostock and David showing equipment in use or within a laboratory setting. Burke1 1Institute of Clinical Neurosciences, Suitable photographs should show equipment with modern or Royal Prince Alfred Hospital and historical significance, either by dint of association with renowned The University of Sydney, Sydney, experimentalists or as items that are fascinating technically. Australia Where relevant, multiple images or a brief movie are acceptable. A 2Sobell Department of photograph that is itself historic would be welcome, but one that Neurophysiology, Institute of shows experimental equipment and its users could be ideal. Neurology, Queen Square, London, UK Hugh Bostock and colleagues run occasional 3-day, hands-on nerve excitability workshops, which provide a detailed introduction to the theory and practice of nerve excitability testing. Those interested are invited to email h.bostock@ion. ucl.ac.uk for further information. References Bergmans J (1970). The Physiology of Single Human Nerve Fibres. Vander, University of Louvain, Belgium. Bostock H, Burke D & Hales JP (1994). Differences in behaviour of sensory and motor axons following release of ischaemia. Brain 117, 225–234. Bostock H, Cikurel K & Burke D (1998). Threshold tracking techniques in the study of human peripheral nerve. Muscle Nerve 21, 137–158. Bostock H & Rothwell JC (1997). Latent addition in motor and sensory fibres of human peripheral nerve. J Physiol 498, 277–294. Chaplan SR, Guo H-Q, Lee DH, Luo L, Liu C, Kuei C, Velumian AA, Butler MP, Brown SM & Dubin AE (2003). Neuronal hyperpolarization- activated pacemaker channels drive neuropathic pain. J Neurosci 23, 1169–1178. An early ECG recording system Henneman E, Somjen G & Carpenter DO (Reproduced from the Ministry for Social Policy, Health, the Elderly and Community Care, (1965). Excitability and inhibitability of Malta) motoneurons of different sizes. J Neurophysiol 28, 599–620. Panizza M, Nilsson J, Roth BJ, Basser PJ & The best examples offered, as well as the prize-winning entry, will Hallett M (1992). Relevance of stimulus appear in Physiology News and on The Society’s website. duration for activation of motor and sensory fibers: implications for the study of H-reflexes and magnetic stimulation. Electroencephalogr Submissions: Please provide a jpg file or equivalent. (Where the Clin Neurophysiol 85, 22–29. photograph itself is a venerable object, please arrange for scanning Tomlinson SE, Burke D, Hanna M, Koltzenburg and enter it as an electronic file.) M & Bostock H (2010). In vivo assessment of HCN channel current (Ih) in human motor axons. Muscle Nerve 41, 247–256. Photographs need to be supported by a brief statement to explain Trevillion L, Howells J, Bostock H & Burke D the equipment and to highlight its claim to fame. (2010). Properties of low-threshold motor axons in the human median nerve. J Physiol 588, 2503–2515. Address your entry to Simon Kellas: [email protected] http://jp.physoc.org/content/588/13/2503.long

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NOS1, S-nitrosylation and cardiac contraction Nitric oxide (NO) is a major regulator of cardiac contraction in both healthy and diseased hearts. NO is produced via enzymes termed nitric oxide synthase (NOS). The neuronal NOS (NOS1) isoform alters key cardiac proteins via post-translational modifications resulting in enhanced contractile function. Therefore, it provides a potential therapeutic target for clinic treatment and new drug development.

Contraction of cardiac myocytes modification related to cellular signal occurs by a process termed transduction (Ziolo, 2008). RyR2 excitation–contraction coupling contains sulfhydryl residues which (ECC) (Bers, 2002). The initiating can be modified by NO through event is the opening of L-type Ca2+ S-nitrosylation (Xu et al. 1998). channels (ICa), which leads to a Our recent study demonstrated release of Ca2+ from the sarcoplasmic that there were decreased RyR2 reticulum (SR) via the SR Ca2+ release Honglan Wang and Mark Ziolo S-nitrosylation levels in NOS1 channel (ryanodine receptor, RyR2) knockout (NOS1–/–) hearts (Wang (i.e. Ca2+ transient). Upon RyR2 NOS3 signalling ultimately decreases et al. 2010) (Fig. 1). Along with the opening, the large efflux of Ca2+ the functional response to β-AR deceased RyR2 S-nitrosylation levels, will diffuse and then activate the stimulation and protects against NOS1–/– myocytes also displayed myofilaments to contract. During arrhythmias (Champion et al. 2004; reduced RyR2 activity. We employed relaxation, the majority of Ca2+ is Massion et al. 2004; Wang et al. various techniques to measure RyR2 resequestered into the SR by the 2008b). activity ranging from direct single SR Ca2+-ATPase–phospholamban RyR2 protein activity to indirect (SERCA–PLB) complex while the Unlike NOS3, NOS1 is localized measurements in intact cardiac remaining Ca2+ is removed from the to the SR along with RyR2. NOS1 myocytes. cell by the Na+–Ca2+ exchanger. signalling in the myocyte leads to enhanced contraction and Data from single RyR2 channel Nitric oxide and cardiac function accelerated relaxation. Another recordings showed that the Within cardiac myoctes, nitric difference compared to NOS3 is knockout of NOS1 resulted in a oxide (NO) is endogenously that NOS1 targets RyR2 (Gonzalez 3-fold reduction in RyR2 channel et al. 2007; Wang et al. 2010) and produced via two constitutively open probability (Po) compared to expressed enzymes: endothelial NO PLB (Wang et al. 2008a) through a RyR2 channels from wild-type (WT) synthase (NOS3) and neuronal NO cGMP-independent pathway. hearts (Fig. 2A). In the physiological synthase (NOS1). Another isoform Ca2+ range (pCa 7 to pCa 3), NOS1 (inducible NO synthase, NOS2) is RyR2 S-nitrosylation and activity in knockout resulted in decreased not constitutively expressed, but NOS1 knockout hearts [3H] ryanodine binding to RyR2 its expression is induced during an S-nitrosylation has emerged as an (Fig. 2B) in SR vesicle preparations, inflammatory response. NO has been important protein post-translational indicative of decreased Po. In intact shown to play an important role in mouse myocytes, NOS1 knockout both physiological and pathological RyR2RyR2 S-NitrosylationS-nitrosylation levels Levels or acute inhibition significantly states. Depending on its source of reduced Ca2+ spark frequency and SNO RyR2 production, NO may be beneficial amplitude (Fig. 2C). Finally, in a more or detrimental to cardiac function 2.0 Total RyR2 physiologically relevant approach, and remodelling. Although NO is a NOS1–/– myocytes and WT myocytes highly diffusible gas, signalling via 1.5 with acute NOS1 inhibition (SMLT) the two constitutively expressed * had a significant rightward shift in isozymes (NOS1 and NOS3) is 1.0 the SR Ca2+ leak–load relationship compartmentalized and each compared to control WT myocytes isoform modulates cardiac function 0.5 (Fig. 2D). Thus, in four distinct differently (Barouch et al. 2002; Ziolo (A.U.) RyR2 SNO/Total methods to measure RyR2 activity, et al. 2008). we consistently found impaired RyR2 0.0 -/- WT NOS1 NOS1–/– activity when NOS1 was absent or NOS3 is localized to the acutely inhibited. plasmalemmal caveolae. It has Figure 1. RyR2 from NOS1 knockout hearts (NOS1–/–) have decreased RyR2 been shown that NOS3 signalling –/– S-nitrosylation levels measured by the NOS1 hearts and myocytes exhibit decreases the β-adrenergic receptor DyLight Switch method. Representative a blunted force–frequency response (AR)-stimulated ICa via activation of blots show S-nitrothiols (SNO) (top) and and decreased contraction (Barouch the cGMP-dependent protein kinase. total RyR2 (bottom). *P < 0.05 vs WT. et al. 2002; Wang et al. 2008a).

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We suggest that decreased RyR2 A RyR2 PPo –/– 0 B WT NOS1-/- A –/–-/- B activity via reduced S-nitrosylation WT NOS1 NOS1 0.07 RyR2 –/– openopen levels observed in NOS1 hearts 0.06 contribute to the depressed closeclose 0.05 contraction. If this concept is correct, 0.04 then addition of a NO donor (or 0.03

S-nitrosylating agent) should be able pmoles/RyR2 0.02 Ryanodine] Binding Ryanodine]

to reverse the reduced RyR2 activity 3 [ 0.01 by increasing S-nitrosylation leading 0.00 to increased myocyte contraction. 10 pA 8 7 6 5 4 3 200 ms pCa Can a NO donor increase NOS1–/– C Ca2+ spark RyR2 activity? WT NOS1–/– WT + SMLT SNAP (S-nitroso-N-acetyl-DL- penicillamine), a NO donor and S-nitrosylating agent, was used to test our hypothesis. In NOS1–/– mouse hearts, SNAP was able 20 μm to increase RyR2 activity in our distinctive methods. That is, SNAP 1 2 400 ms F/F0 significantly increased RyR2 Po, Leak–load relationship enhanced both Ca2+ spark frequency D and amplitude, and caused a leftward shift in the SR Ca2+ leak– load relationship. There was little effect of SNAP in WT hearts. More importantly, SNAP now normalized RyR2 activity in the NOS1–/– hearts to WT levels. Taken together, SNAP is able to reverse the blunted RyR2 activity.

Can the increased RyR2 activity Figure 2. Decreased RyR2 activity with NOS1 knockout or acute inhibition. A, representative traces from WT (left) and NOS1–/– (right) single RyR2 channels by SNAP reverse the depressed showing decreased open probabillity (P ). B, WT and NOS1–/– [3H]ryanodine binding –/– o contraction observed in NOS1 at different Ca2+ concentrations. C, representative images of Ca2+ sparks from WT, myocytes? NOS1–/– and WT+SMLT (NOS1 selective inhibitor) myocytes. D, left, representative Contraction in myocytes was trace of the SR Ca2+ leak–load protocol from a WT myocyte. Right, NOS1 knockout or determined by simultaneous acute inhibition results in a significant rightward shift. measurement of Ca2+ transients and cell shortening. SNAP resulted Shortening WT NOS1–/– WT + SMLT in an increase in Ca2+ transient and cell shortening amplitudes in WT myocytes, NOS1–/– myocytes and WT 2% RCL myocytes with acute NOS1 inhibition 500 ms (Fig. 3). However, SNAP had a much 2+ larger effect in myocytes with NOS1 Ca transient knockout or acute inhibition. More importantly, SNAP now normalized the Ca2+ transient and cell shortening amplitudes in these myocytes. Thus, SNAP is indeed able to reverse ΔF/F0 the depressed contraction in 0.2 NOS1–/– myocytes.

–/– Is RyR2 S-nitrosylation involved in Figure 3. The NO donor SNAP reversed the depressed cardiac function in NOS1 and SMLT (NOS1 selective inhibitor)-treated myocytes. Representative shortening the effects mediated by SNAP? (top) and Ca2+ transient (bottom) traces. Dotted black line highlights decreased In NOS1–/– mouse hearts, SNAP Ca2+ transient amplitudes with NOS1 knockout or acute inhibition. Dotted red line increased RyR2 S-nitrosylation levels. highlights normalized Ca2+ transient amplitudes after treatment with SNAP.

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As with contraction, SNAP had a Wang H, Viatchenko-Karpinski S, Sun J, Gyorke channel (ryanodine receptor) by poly-S- much larger effect on NOS1–/– RyR2 I, Benkusky NA, Kohr MJ, Valdivia HH, Murphy nitrosylation. Science 279, 234–237. E, Gyorke S & Ziolo MT (2010). Regulation Ziolo MT (2008). The fork in the nitric oxide S-nitrosylation compared to WT of myocyte contraction via neuronal nitric RyR2. Further, in the presence of road: cyclic GMP or nitrosylation? Nitric Oxide oxide synthase: role of ryanodine receptor 18, 153–156. SNAP, RyR2 S-nitrosylation levels S-nitrosylation. J Physiol 588, 2905–2917. were now normalized. These data http://jp.physoc.org/content/588/15/2905.long Ziolo MT, Kohr MJ & Wang H (2008). Nitric oxide signaling and the regulation of suggest that the effect of SNAP to Xu L, Eu JP, Meissner G & Stamler JS (1998). myocardial function. J Mol Cell Cardiol 45, reverse the contraction is, in part, via Activation of the cardiac calcium release 625–632. increased RyR2 S-nitrosylation. In conclusion, NOS1 signalling results Paton Prize Bursary in S-nitrosylation of RyR2, increasing its activity that ultimately leads to The Paton Prize should not be confused with the Paton Lecture. The Prize enhanced contraction. Therefore, the was established in 1994 and takes the form of a bursary of up to £1000 to NOS1-mediated post-translational cover travel and incidental expenses. It is open to all Members and Affi liates modifi cation of RyR2 is an important of The Society, as well as established scientists. modulator of cardiac myocyte Professor Sir William Paton originated the Prize to encourage the historical function and may be a useful study of major ideas and concepts that have shaped modern physiology. target for clinical treatment of The History & Archives Committee administers the Prize and would be cardiomyopathies. delighted to receive applications describing a proposed piece of historical Honglan Wang and Mark T. research which could lead to a published paper, a booklet or an article for Ziolo Physiology News. Department of Physiology and The Paton Prize was awarded in 2007 to David Miller for research into the Cell Biology, Davis Heart and Lung life and work of Sydney Ringer. Research Institute, The Ohio State University, Columbus, OH 43210, USA

References Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA et al. (2002). Nitric oxide regulates the heart by spatial confi nement of nitric oxide synthase isoforms. Nature 416, 337–339. Bers DM (2002). Cardiac excitation- contraction coupling. Nature 415, 198–205. Champion HC, Georgakopoulos D, Takimoto E, Isoda T, Wang Y & Kass DA (2004). Modulation of in vivo cardiac function by myocyte-specifi c nitric oxide synthase-3. Circ Res 94, 657–663. Gonzalez DR, Beigi F, Treuer AV & Hare JM (2007). Defi cient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes. Proc Natl Acad Sci U S A 104, 20612–20617. Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C et al. (2004). Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates β-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation 110, 2666–2672. A solution for the heart: the life of Sydney Ringer (1836–1910) Wang H, Kohr MJ, Traynham CJ, Wheeler DG, Janssen PM & Ziolo MT (2008a). Neuronal David Miller (2007) (available on The Society website at: nitric oxide synthase signaling within cardiac www.physoc.org/site/cms/contentviewarticle.asp?article=759) myocytes targets phospholamban. Am J Physiol Cell Physiol 294, C1566–C1575. Applications, in the form of an outline of the proposed work on one side of Wang H, Kohr MJ, Wheeler DG & Ziolo MT (2008b). Endothelial nitric oxide synthase A4, should be submitted to Simon Kellas ([email protected]) by the last decreases β-adrenergic responsiveness via day of March 2011 for consideration by the committee in April. Anyone inhibition of the L-type Ca2+ current. Am J wanting more details or to talk through an idea is invited to contact the Physiol Heart Circ Physiol 294, H1473–H1480. committee chair, Dafydd Walters ([email protected]).

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NO is pivotal in motor pathologies, but is it harmful or protective? Dysregulation of the expression of nitric oxide synthase (NOS) is a hallmark of many neuropathological conditions. Using a model of peripheral motor neuropathy, we demonstrated that NOS up-regulation delays nerve repair and evokes many of the changes that occur in damaged motoneurons. Therefore, NOS might be a pivotal target for the development of therapeutic tools for diverse neuropathies.

Traumatic injury of a This type of injury up-regulates the peripheral motor nerve evokes three major isoforms of NOS in the physiopathological changes in affected nerve: nNOS accumulates the damaged motoneurons that in the growing motor axons, eNOS are common to a broad array of is over-expressed in vasa nervorum neuropathological states, including in the distal stump and around the dysregulation of protein expression, Bernardo injury site, and iNOS is expressed function and/or aggregation. One of Moreno-López by the recruited macrophages and the proteins involved is nitric oxide phagocytic Schwann cells (Fig. 1). synthase (NOS), which synthesizes It is also noteworthy that NOS nitric oxide (NO), a highly reactive Although the pivotal role of NO is expression is up-regulated in gas. First identified in biological evident, whether it is harmful or motoneurons and reactive astrocytes systems as the endothelial-derived protective is less clear. In nNOS or in amyotrophic lateral sclerosis (ALS) relaxing factor (EDRF), NO is iNOS knockout mice, peripheral and in neurons and/or glial cells in an intercellular messenger that recovery is impaired due to multiple sclerosis, HIV dementia, freely crosses plasma membrane, delayed Wallerian degeneration, and Alzheimer’s, Parkinson’s and with multiple functions in the axon breakdown and Schwann cell Huntington’s diseases, to cite cardiovascular, immunological reaction after nerve injury (Zochodne the most prevalent neurological and nervous systems. The relative & Levy, 2005); this supports a pathologies. Up-regulation of ubiquity of NO actions suggests beneficial role of NO. Paradoxically, this enzyme also occurs after that this molecule is important for chronic systemic inhibition of NOS peripheral and central traumatic physiological and pathological states accelerates the onset of functional lesions of the nervous system. and confers upon NOS important muscle reinnervation, indicating As a result, unmasking the role therapeutic possibilities for a variety a harmful impact on nerve repair of NOS imbalance in neuron of neuropathies. because NO slows down axonal physiopathology is central to the regrowth (Sunico et al. 2008). There are three major isoforms of development of feasible therapeutic This apparent controversy was NOS, coded by different genes and tools. Over the past decade, our resolved by the finding that chronic, differing in localization, regulation, team has focused on investigating catalytic properties and inhibitor the role of NO in nerve repair and systemic, specific inhibition of eNOS sensitivity (Alderton et al. 2001). motoneuron pathology after the – but not specific inhibition of n/ The isoform predominantly found traumatic lesion of a nerve. We iNOS – accelerated neuromuscular in the neuronal tissue (nNOS) was selected the hypoglossal motor reconnection. To avoid unwanted the first to be purified and cloned. system of the adult rat as an easily side-effects due to the systemic The inducible isoform (iNOS) occurs accessible system and crushing as administration of drugs and to in a wide range of cells and tissues, the method of nerve injury. improve specificity, an adenoviral including activated macrophages, vector was used to locally express and in pathological states of the Physical injury to a nerve is the a dominant negative of eNOS central nervous system, in astroglia most frequent cause of acquired within the injured nerve. A single and microglia. Finally, the primary peripheral neuropathy, which is injection of this adenovirus on the isoform found in vascular endothelial responsible for loss of motor, sensory day of nerve lesion increased axonal cells is known as eNOS. At the and/or autonomic functions. Injured regeneration and significantly low tissue concentrations of NO axons in the peripheral nervous accelerated functional recovery of generated by nNOS and eNOS, NO system maintain their capacity neuromuscular junction (Sunico acts as a molecular messenger in to regenerate in adult mammals. et al. 2008). This suggests that NO multiple physiological transduction Therefore, the identification of endothelial origin slows down pathways; at high concentrations, of molecules that regulate muscle reinnervation by detrimental such as those produced by iNOS, degenerative and regenerative actions on axonal regeneration NO is cytotoxic. Remarkably, processes is indispensable in after peripheral nerve injury. These over-expression of eNOS or nNOS developing therapeutic tools to experiments identified eNOS as in pathological conditions can also accelerate and improve functional a potential therapeutic target yield toxic NO levels. recovery following nerve injury. for treatment of traumatic nerve

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up-regulate nNOS in the hypoglossal motoneurons (HMNs), we used two different approaches. Crushing of the XIIth (hypoglossal) nerve provokes axonal transection of motoneurons. The alternative procedure was to transduce motoneurons by injecting an adenoviral vector, directing the expression of nNOS, into the hypoglossal nucleus (Fig. 2). The XIIth nerve crushing induces de novo nNOS expression in the perikarya of motoneurons, which normally lack this enzyme.

At the central level as well, NO Figure 1. Diagram at the nerve level of the sources and actions of NO after motor has both harmful and beneficial nerve injury. The three isoforms of NOS are up-regulated: eNOS in vasa nervorum, roles for motoneuron survival nNOS in fibres and iNOS in infiltrated macrophages and reactivated Schwann cells. after nerve injury. The gas is a NO from different origins mediates pro- (+) and anti- (–) regenerative processes. pro-apoptotic molecule affecting NO favours vasodilatation, inflammatory cell infiltration, angiogenesis, Wallerian motoneuron survival after motor degeneration (WD), myelin breakdown and clearance, all of which are expected to be beneficial for axonal regeneration. On the other hand, NO could exert anti- nerve transection: nNOS deficiency proliferative and toxic effects on Schwann and endothelial cells, as well as axonal or inhibition effectively reduced growth cone collapse. Figure modified from Moreno-López, 2010, with permission motoneuronal apoptosis induced from Wiley-Blackwell (for a more detailed description, see that paper). by avulsion of the sciatic nerve in adult mice. Motoneuron apoptosis injuries, highlighting the potential In a recent study reported in The mediated by NO was accompanied value of gene therapy using viral Journal of Physiology, we analysed the by an increase in the NO subproduct vectors to suppress the activity of role of induced NO in motoneuron peroxynitrite (ONOO–), a potent eNOS. pathology (Montero et al. 2010). To oxidant, and in protein nitration (Martin et al. 2005) (Fig. 3). In addition, chronic NO synthesis strongly increases intrinsic AVV-eGFP motoneuron excitability by inhibiting + GFAP background potassium currents, suggesting that induced nNOS sensitizes injured motoneurons to apoptosis by mediating the hyperexcitability experienced by the motoneuron following axonal injury (Fig. 3). This NO-mediated action may sensitize motoneurons to excitotoxic damage (Gonzalez-Forero et al. 2007), with detrimental effects on motoneuron viability after axonal damage. On the contrary, however, nNOS up-regulation mediates the loss of excitatory synaptic inputs on injured HMNs (Fig. 3). This appears to be an NO-mediated protective action against excitotoxic stimuli. The activation of regrowth programs in adult motoneurons following axotomy may require a reversion to an immature electrical phenotype. Figure 2. Specificity of viral transfection. Confocal photomicrograph of a coronal Whether these apparently opposing section, obtained 7 days after adenoviral (AVV) administration in the hypoglossal impacts of NO on motoneuron nucleus, at 300 μm rostral to the injection site, showing a broad number of eGFP- expressing hypoglossal neurons (green). Immunohistochemistry against the glial survival form part of a motoneuron fibrillary acidic protein (GFAP) illustrates the absence of infected astroglia (red). Scale dedifferentiation process is a topic bar, 40 μm. that merits attention.

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and Fas death receptor linked by DNA damage and p53 activation. J Neurosci 25, 6449–6459. Montero F, Sunico CR, Liu B, Paton JF, Kasparov S & Moreno-Lopez B (2010). Transgenic neuronal nitric oxide synthase expression induces axotomy-like changes in adult motoneurons. J Physiol 588, 3425–3443. http://jp.physoc.org/content/588/18/3425.long Moreno-Lopez B (2010). Local isoform-specific NOS inhibition: a promising approach to promote motor function recovery after nerve injury. J Neurosci Res 88, 1846–1857. Sunico CR, Gonzalez-Forero D, Dominguez G, Garcia-Verdugo JM & Moreno-Lopez B (2010). Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinase-dependent mechanism preceded by myosin light chain phosphorylation. J Neurosci 30, 973–984. Sunico CR, Portillo F, Gonzalez-Forero D, Figure 3. Diagram at the soma level of the sources and actions of NO after motor Kasparov S & Moreno-Lopez B (2008). nerve injury. After nerve injury, nNOS appears de novo at the motoneuron. The nNOS- Evidence for a detrimental role of nitric derived NO reacts with different targets, compromising motoneuron survival. NO oxide synthesized by endothelial nitric – can react with the anion superoxide (O2 ) to form the potent oxidant peroxynitrite oxide synthase after peripheral nerve injury. (ONOO–), with the subsequent increase in protein nitration that precedes Neuroscience 157, 40–51. motoneuron apoptosis. Furthermore, through inhibition of background potassium Sunico CR, Portillo F, Gonzalez-Forero D & currents, NO increases excitability. This might sensitize motoneurons to excitotoxic Moreno-Lopez B (2005). Nitric oxide-directed damage. In contrast, NO mediates a protective mechanism against excitotoxic synaptic remodeling in the adult mammal stimuli such as excitatory synaptic withdrawal from injured motoneurons. Figure CNS. J Neurosci 25, 1448–1458. modified from Moreno-López, 2010, with permission from Wiley-Blackwell. Zochodne DW & Levy D (2005). Nitric oxide in damage, disease and repair of the peripheral Disruption of the trophic These results point to nNOS as a nervous system. Cell Mol Biol (Noisy-le-grand) 51, 255–267. communication between moto­­ pivotal target for the development of neurons and their target myocytes therapeutic tools for the treatment Acknowledgements causes a well-characterized range of of peripheral neuropathies and This work was supported by grants from functional and synaptic impairments neuro­degenerative disorders Spain’s Ministerio de Ciencia e Innovación (SAF2008-01415) and Consejería de in insulted motoneurons. The characteristically accompanied by Innovación, Ciencia y Empresa of the Junta NO-mediated disturbances involve nNOS up-regulation. This finding de Andalucía (PAI2007-CTS-02606). I thank changes in intrinsic membrane also opens a line of research to Elaine Lilly, PhD (Writer’s First Aid), for English properties and anatomical synaptic establish a method for studying language revision of this manuscript. deterioration that suggest a major the role of dysregulated proteins pathological role of nNOS (Sunico in the neuronal impairment that Physiology News et al. 2005, 2010; Gonzalez-Forero occurs during the course of so many archives now et al. 2007). However, nNOS is neuropathological conditions. only one of the numerous proteins available dysregulated after nerve damage. Bernardo Moreno-López Thus, the actual role for nNOS is less Have you ever wanted to search clear within the complex synergistic Grupo de Neurodegeneración y through the back issues of and/or antagonistic actions of Neuroreparación (GRUNEDERE), Área Physiology News? Until now, this has multiple dysregulated proteins. We de Fisiología, Facultad de Medicina, meant laborious searching of each further scrutinized the effects of Universidad de Cádiz, Cádiz, Spain issue separately. We are pleased to virally induced de novo expression announce, therefore, that we have References of nNOS in motoneurons, together now made available an archive of with complementary attempts to Alderton WK, Cooper CE & Knowles RG all Physiology News issues, plus the (2001). Nitric oxide synthases: structure, forerunners – Physiology Magazine down-regulate nNOS expression in function and inhibition. Biochem J 357, damaged hypoglossal motoneurons 593–615. (July 1992 to Autumn 2001) and the Newsletter (February 1984 to using virally mediated gene Gonzalez-Forero D, Portillo F, Gomez L, knock-down (Montero et al. 2010). Montero F, Kasparov S & Moreno-Lopez May 1992). Our results demonstrate that nNOS B (2007). Inhibition of resting potassium These are available from the over-expression in motoneurons is a conductances by long-term activation of the NO/cGMP/protein kinase G pathway: A new Physiology News website, and sufficient signal to evoke many of the mechanism regulating neuronal excitability. J means you can now search the electrophysiological and anatomical Neurosci 27, 6302–6312. whole archive of Physiology News in changes associated with axonal Martin LJ, Chen K & Liu Z (2005). Adult motor one easy step. damage. neuron apoptosis is mediated by nitric oxide

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The ‘cost’ of breathing in heart failure Heart failure patients develop a number of pulmonary abnormalities which increase the work and oxygen cost of breathing. Demands of the respiratory muscles are further accentuated during activities of daily living that require moderate exercise. In the face of limited cardiac reserve, heavily recruited respiratory muscles preferentially ‘steal’ blood flow from the working skeletal muscles. This phenomenon may be one mechanism that contributes to the enhanced perception of locomotor muscle fatigue commonly encountered in patients with heart failure. The two most common symptoms manifestations from the failing heart failure (HF) patients suffer heart, the work and oxygen cost of from include shortness of breath breathing for a given level of oxygen (dyspnoea) and fatigue. As such, consumption will be accentuated exercise intolerance is a hallmark in HF and requires greater relative of symptomatic HF. Initially, blood flow than that seen in healthy investigators sought to determine individuals. This increase becomes which measure of cardiac function particularly exacerbated during (left ventricular ejection fraction, Thomas Olson (left) and Bruce Johnson increasing intensities of exercise; cardiac output, filling volumes, however, it is possible that even etc.) best related to an individual’s is particularly concerning when low levels of physical activity in HF exercise tolerance. Unfortunately, coupled with a severely blunted patients (e.g. representing typical these studies failed to find a clear ability to increase cardiac output due activities of daily living) may result in link between resting measures to the disease pathology. a redistribution of blood flow away of cardiac function and exercise Exercise elicits an increased from the working skeletal muscles to tolerance in patients with HF. need for blood flow to both the respiratory muscles in an effort to compensate for the respiratory the respiratory and working While altered cardiac function muscle work in the setting of limited skeletal muscles. Under normal clearly plays an initiating role, HF cardiac reserve. If true, a respiratory circumstances, during exercise in becomes a systemic illness which muscle blood flow ‘steal’ from the healthy individuals, a combination impacts on multiple organ systems. locomotor muscles may be one of localized vasoconstriction (renal, One system particularly influenced mechanism which contributes splanchnic, etc.) and an increase is the pulmonary system. This to heightened perceptions of in cardiac output combine to system is intimately linked with fatigue that are prevalent in this meet the demands of both the the cardiovascular system both population. Consistent with this respiratory and locomotor muscles. anatomically and haemodynamically premise, previous work in animal in that the lungs lie in series with This appears to be the case up to models of HF have suggested that the heart, share a common surface approximately 75–80% of maximal the diaphragm (a primary muscle area within the thoracic cavity, and intensities. However, at higher of respiration) will take blood flow are exposed to similar intrathoracic intensities, the respiratory muscles from working skeletal muscles pressure changes. Heart failure begin to preferentially recruit blood during physical activity. In addition, patients often develop mild flow at the expense of the working O’Donnell and colleagues have pulmonary function impairments skeletal muscle. In fact, Harms shown that providing human HF including restrictive and to a and colleagues suggested, during patients with inspiratory pressure lesser extent obstructive changes, maximal cycle exercise in healthy support using a ventilator resulted reduced lung diffusing capacity individuals, reducing respiratory in longer submaximal steady-state (surface area for gas exchange), muscle work by ~60-65% resulted in exercise duration and reduced and reduced respiratory muscle ~4–5% increase in blood flow to the leg discomfort (O’Donnell et al. strength. They also require greater legs whereas increasing respiratory 1999). Further, Borghi-Silva et al. levels of ventilation for a given muscle work by ~25–30% led to (2008) have demonstrated that HF metabolic demand, due to a more ~7% reduction of blood flow to the patients undergoing constant load rapid–shallow breathing pattern, legs (Harms et al. 1997). From these exercise at 70–80% of peak work altered matching of ventilation findings, the authors concluded that while using a proportional assist to perfusion, and chronic, mild the work of breathing exhibited ventilator increased skeletal muscle hyperventilation. The end result of during heavy exercise results in oxygenation and an estimate of leg these pulmonary manifestations a reflex vasoconstriction of the blood volume. of HF is an elevated work and cost locomotor muscle vascular beds of breathing, particularly during contributing to reduced leg blood Accordingly, we sought to determine activities of daily living that require flow. the relationship between respiratory moderate exercise intensities (Olson muscle work and leg blood flow et al. 2006). This increase in the work As noted previously, due to a during moderate-intensity exercise and cost of breathing during activity number of pulmonary system in patients with HF (Olson et al.

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A. B B. A 15 A. Control 15 Heart Failure Baseline B. Baseline

O) 15 10 Control O) 15 Heart failure 2 Control 10

Unloading 2 Heart Failure BaselineBaseline Baseline UnloadingBaseline O) O) O) 10 2

2 10 O) 5 UnloadingUnloading 2 5 Unloading

2 Unloading 05 05 -50 -50 -10-5 -10-5 -15-10 -15-10 -20-15 -20-15 -25-20 -20

Esophageal Pressure (cm H (cm Pressure Esophageal -25 Esophageal Pressure (cm H (cm Pressure Esophageal -25 Esophageal Pressure (cm H (cm Pressure Esophageal -30 -25 Esophageal Pressure (cm H (cm Pressure Esophageal -30 Oesophageal pressure (cmH

0.30.60.91.21.51.82.12.42.73.03.3 Oesophageal pressure (cmH 0.30.60.91.21.51.82.12.42.73.03.3 -30 -30 3.3 3.0 2.7 2.4 2.1Volume1.8 (L)1.5 1.2 0.9 0.6 0.3 3.3 3.0 2.7 2.4 2.1Volume1.8 (L)1.5 1.2 0.9 0.6 0.3 Volume (l) Volume (l) C Volume (L) D Volume (L) C. D. 15 ControlControl 15 Heart failureFailure C. Baseline D. Baseline O) O) 15 O) 15 O)

10 2 10

2 Control 2 Heart Failure 2 UnloadingLoadingBaseline Unloading LoadingBaseline

O) 10 O) 10 2 5 Loading 2 5 Loading 05 05 -50 -50 -10-5 -10-5 -15-10 -15-10 -20-15 -20-15 -25-20 -25-20 Esophageal Pressure (cm H (cm Pressure Esophageal H (cm Pressure Esophageal Oesophageal pressure (cmH Oesophageal pressure (cmH -25 -25 Esophageal Pressure (cm H (cm Pressure Esophageal -30 H (cm Pressure Esophageal -30 3.3 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 3.3 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 -30 -30 Volume (L)(l) 0.30.60.91.21.51.82.12.42.73.03.3 VolumeVolume (L) (l) 0.30.60.91.21.51.82.12.42.73.03.3 Figure 1. Influence of inspiratoryVolume (L)pressure unloading and inspiratory pressure resistance Volumeon the (L)pleural pressure–volume response during steady-state exercise. A, inspiratory unloading in healthy control participants. B, Inspiratory unloading in heart failure patients. C, inspiratory resistance in control participants. D, inspiratory resistance in heart failure patients.

2010). During this study, HF patients muscle unloading via a ventilator, or using an oesophageal balloon to exercised at a constant submaximal with inspiratory muscle resistance quantify intrathoracic pressure rate of 60% peak work while (Fig. 1). During these exercise bouts (a measure of work) as well as breathing room air, with inspiratory we measured the work of breathing leg blood flow by thermodilution  using catheters in the femoral vein. %%D LBF/QLBF/Q We observed that reducing the -25 0 25 50 75 100 normal inspiratory muscle work via 25 inspiratory pressure support during r = -0.88 exercise resulted in a significant 0 p < 0.001 increase in leg blood flow in HF patients, but not in healthy control participants (Fig. 2). In contrast, -25 increasing the work of breathing by adding inspiratory resistance did not alter leg blood flow or cardiac output -50 in HF patients, but increased cardiac

Ppl Insp Mean output in the healthy controls. D Ppl Insp mean D

% -75 These results suggest that the HF

% patients were able to redistribute blood flow to the locomotor muscles -100 when respiratory muscle work was reduced, but were unable to reduce locomotor blood flow beyond that -125 observed during normal breathing Figure 2. Relationship between the per cent change in mean inspiratory pleural when inspiratory muscle work was pressure (%DPpl Insp mean) and the per cent change in the leg blood flow/cardiac output ratio (%DLBF/Q) during the transition from normal room air breathing increased. This was most probably during exercise to inspiratory assistance in heart failure patients. This relationship due to the lack of cardiac reserve and demonstrates the impact of a reduction in the work of breathing on leg blood flow a maximal level of vasoconstriction during exercise in patients with heart failure. in the active locomotor muscles.

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In summary, our research in HF is Western blotting. Part II consistent with that observed in healthy athletic adults and is an In the last issue, Patricia Leoni reviewed protein electrophoresis. example of the intricate balance In this article she examines the different methods for preparing between the demands for blood flow protein extracts from cells, tissues or fluid media. Selecting relative to the available reserves. the method that is suited to your sample is important as it will For the HF patient, very modest amounts of activity appear to provide more efficient protein extraction and better results. challenge the ability to deliver blood Sample preparation for protein flow to both the respiratory muscles electrophoresis and locomotor muscles, while in the The majority of parameters athlete this only occurs near maximal regarding equipment, gel levels of exercise. However, in both preparation and running conditions cases, the respiratory muscles have been optimised by commercial appear to preferentially ‘steal’ blood companies so that there is no longer Patricia Leoni flow at the expense of the locomotor a need to investigate the right muscles. This ‘steal’ phenomenon conditions in the laboratory. What is may result in a mismatch in oxygen still left to the individual researcher supply and demand and be one cocktails of protease inhibitors is the preparation and loading of containing different combinations mechanism which contributes to the sample which is a crucial step the enhanced perception of fatigue and phosphatase inhibitors for a successful separation. The containing combinations that inhibit commonly encountered by patients most important considerations living with HF. most proteases and phosphatases are: complete solubilisation, are commercially available. The most accurate measurements and lack of common protease inhibitors contain Thomas P. Olson and Bruce D. modification of the proteins through different combinations of antipain, Johnson proteolysis and de-phosphorylation bestatin, chymostatin leupeptin, during preparation. These are crucial Division of Cardiovascular Diseases, phosphoramidon, EDTA, aprotinin, to get reproducible results. Mayo Clinic, Rochester, MN 55905, pepstatin, E-64 and 4-(2-aminoethyl) USA benzenesulfonil fluoride. Preparation of protein extracts Phosphatase inhibitor cocktails References from cells in culture include sodium orthovanadate, Borghi-Silva A, Carrascosa C, Oliveira CC, The preparation of cell extracts sodium molybdate, sodium tartrate, Barroco AC, Berton DC, Vilaca D, Lira-Filho EB, depends on the relative amount of sodium fluoride, imidazole, okadaic Ribeiro D, Nery LE & Neder JA (2008). Effects the protein of interest and location acid and calyculin A. Protein of respiratory muscle unloading on leg muscle within the cell. For a protein that concentration can be measured at oxygenation and blood volume during high- this point. intensity exercise in chronic heart failure. Am J is relatively abundant, a whole-cell extract will be adequate. Cells Physiol Heart Circ Physiol 294, H2465–H2472. Proteins of low abundance need must be rinsed quickly with cold Harms CA, Babcock MA, McClaran SR, to be enriched; in order to achieve Pegelow DF, Nickele GA, Nelson WB & phosphate-buffered saline (PBS) in this, as many irrelevant proteins Dempsey JA (1997). Respiratory muscle work the monolayer or after centrifugation as possible need to be removed compromises leg blood flow during maximal if grown in suspension. It is exercise. J Appl Physiol 82, 1573–1583. from the extract. Nuclear proteins important that excess PBS is or proteins that are embedded O’Donnell D, D’Arsigny C, Raj S, Abdollah removed completely using a suction H & Webb K (1999). Ventilatory assistance in the plasma membrane are improves exercise endurance in stable pump. Cells should be lysed with a relatively easy to isolate, eliminating congestive heart failure. Am J Respir Crit Care detergent-containing buffer which the majority of soluble proteins Med 160, 1804–1811. does not interfere with the protein’s and proteins contained in other Olson TP, Joyner MJ, Dietz NM, Eisenach biological or immuno reactivity such sub-cellular structures. Nuclei can JH, Curry TB & Johnson BD (2010). Effects as RIPA. A typical formulation for this of respiratory muscle work on blood flow be isolated by centrifugation for M distribution during exercise in heart failure. J buffer is: 150 m NaCl, 1.0% NP-40, 1 minute in a refrigerated bench top Physiol 588, 2487–2501. 0.5% sodium deoxycholate, 0.1% centrifuge after lysing the cells in a http://jp.physoc.org/content/588/13/2487.long SDS and 50 mM Tris, pH 8.0. Other hypotonic buffer, and membranes Olson TP, Snyder EM & Johnson BD (2006). formulations exclude SDS. Protease can be isolated using linear sucrose Exercise disordered breathing in chronic heart inhibitors must be added to the lysis gradients and centrifugation at high failure. Exerc Sport Sci Rev 34, 194–201. buffer. If the protein sample is to speed (26,000 revs min–1, average of be analysed for phosphoproteins, 90,000 g) for 3 hours in a swing-out Acknowledgements ~ the buffer should also contain rotor. Sucrose gradients allow Dr Olson’s research program is supported by NIH/NCRR CTSA–KL2 RR024151. Dr Johnson’s phosphatase inhibitors. Inhibitors the recovery of cytosolic proteins research program is supported by NIH can be added individually but, as and proteins from all subcellular 5R01HL71478-7. more than one type are necessary, structures.

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There are also commercially The classic method to eliminate a pellet after centrifugation at available protein extraction kits that albumin from serum is to use affinity 35,000 g for 30 minutes, but they allow enrichment if not purification binding to the dye cibachron blue, need to be dialysed in order to of the different compartments. in the right conditions of ionic eliminate excess ammonium sulfate For example, cytoplasmic/nuclear strength and pH. Resins bound to before electrophoresis. Protein preparations, membrane and the dye are available from several pellets can be dissolved in 30 mM hydrophobic protein preparations companies (Pierce blue from Pierce, Tris-HCl pH 7.4–8.0. Any insoluble and cytosolic/membrane/nuclear/ HiTrap blue from GE Healthcare). residue should be removed by cytoskeletal preparations can be This dye also binds other proteins, so centrifugation. Protein concentration achieved using products such as the it can only be used if these proteins should be measured at this stage. ReadyPrep range made by Bio Rad are not being investigated. Also, and the ProteoExtract range from albumin-associated proteins will Trichloroacetic acid Calbiochem/Merck. These greatly be removed from the extract. A The negatively charged reduce labour, although they are more selective method to remove trichloroacetate (TCA) triggers expensive. actin is immunoprecipitation protein unfolding by disrupting using a monoclonal antibody. the electrostatic interactions that Extraction of proteins from tissue Immunoglobulins can be effectively stabilise the native conformation removed using a mixture of Fresh tissue can be dissociated using of proteins. Partial unfolding of protein A and protein G sepharose. a buffer containing a collagenase proteins results in coalescence preparation suitable for the tissue Concentration of diluted samples of protein molecules leading to of interest. This will result in a their precipitation. Proteins are suspension of all the cell types If the sample is too dilute for direct precipitated in ice-cold 10% TCA analysis it will require concentration contained in the tissue. Alternatively solution for 2–3 hours. After tissue should be homogenised in a as the total volume of sample centrifugation for 30 min at 15,000 g detergent-containing buffer like RIPA is limited by the capacity of the at 4°C, the pellet is washed two or or T-Per tissue protein extraction wells. There are several methods to three times with absolute ethanol reagent from PIERCE, in the presence concentrate protein extracts. If the to remove the remaining TCA, as of protease and phosphatase volume of the sample is large, an remaining acidity would impair inhibitors. easy and fast method is by filtration solubilisation. The pellet can be against dry or a concentrated dissolved in 0.1–0.2 N NaOH, Homogenisation can be done solution of polyethylene glycol (PEG; removing any insoluble residue by manually in a Dounce homogenizer, Carbowax M-20). The sample is centrifugation. Protein concentration or automatically in a bead-mill placed in a large-diameter dialysis should be measured, prior to the homogeniser such as the Tissuelyser tube and this is submerged in a addition of Laemmli buffer. (Qiagen) or GentleMACS (Miltenyi concentrated solution of PEG or biotec). Frozen tissue can be covered with dry PEG. The classic Ethanol and acetone precipitation pulverised in a mortar and pestle, rotatory vacuum evaporator like adding liquid nitrogen during the Buchi, is a fast and gentle method to Upon dehydration by organic grinding. The resulting powder can concentrate samples of any volume. solvents, protein molecules will be extracted with an appropriate Ultrafiltration membranes are attract each other by van der Waal’s buffer. disposable, centrifugal devices with forces strongly enough to become membranes that filter out water and insoluble in the ethanol–water Proteins from plasma, serum or ions (Amicon from Millipore, Pierce and acetone–water mixtures. conditioned medium protein concentrators from Pierce). Ethanol precipitation (66% final Extraction of proteins from liquid They are very effective and fast, but concentration), and acetone sources presents two problems: the there is some protein loss during the precipitation (85%) require several proteins of interest might be too process. hours, typically overnight, at –20°C. dilute and need to be concentrated Acetone precipitation is particularly and the presence of other proteins Precipitation useful when retrieving proteins that are present in large quantities Most protein will aggregate in the from interfaces after nucleic acid (albumin, immunoglobulins). Such presence of high salt concentation. extraction. After precipitation highly abundant proteins may need Because of its high solubility the proteins are recovered by to be eliminated prior to the run, as (NH4)2SO4 is commonly used to centrifugation for 30 minutes at they would otherwise constitute the increase ionic strength. Different 15,000 g at 4°C. The pellet can be greater part of the sample, thereby proteins precipitate at different dissolved in a suitable buffer, like reducing the sensitivity for detecting ammonium sulfate concentrations. PBS. Any insoluble residue should proteins present in low quantities, as This could be used to selectively be removed by centrifugation, prior the total amount of protein that can precipitate the proteins of interest. to the measurement of protein be loaded onto a gel is limitied. The proteins are recovered in concentration.

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Sample loading blue binds directly to the protein Society Noticeboard in the gel and has a detection Full solubilization and accurate Scientific Meetings – 2011 measurement of protein sensitivity of approximately 1 μg. concentration are essential With stains containing metal salts, Cellular & Integrative Neuroscience for high-quality informative usually silver chloride, the grains of Themed Meeting electrophoretic runs. The amount metal precipitate on the protein. King’s College London, UK, 6–8 April of protein loaded on the gel varies The detection sensitivity is in the Physiology 2011 according to the thickness of the nanogram order of magnitude. The Physiological Society Main Meeting gel, the volume of the well and Staining with copper or zinc chloride University of Oxford, UK, 11–14 July the detection method. Though is a fast and sensitive method to Abstract submission 1–31 March, early 50–200 mg is usually sufficient in detect proteins; they show as clear registration now open, ends 1 June bands in an opaque background. This the most common models, when Epithelia & Membrane Transport method takes only a few minutes, stained with Coomassie blue, it is Themed Meeting does not affect the proteins and can better to determine this empirically University College London, UK by running different quantities of be removed quickly by rinsing. After 1–3 September extract and staining the bands. Prior photographing the gel, the stain Abstract submission and registration to the electrophoretic run, sample can be rinsed and the proteins can open 23 June be electro-eluted or blotted. This is buffer ( 2–8x) should be added Vascular & Smooth Muscle Physiology according to the dilution of the not possible with the other stains Themed Meeting sample. DNA in the sample distorts because the proteins are fixed on the Edinburgh, UK, 6–8 December the run and can be eliminated gel during staining. Zinc or copper Abstract submission and registration either by incubating the sample staining can detect 10–100 ng of open 20 September with DNAse (5 units ml–1) or by protein in a band. shearing it using a Hamilton syringe, Scientific Meetings – 2012 taking care not to introduce air Fluorescent staining of proteins Human & Exercise Physiology Themed bubbles into the sample. If the Fluorescent dyes provide a fast Meeting, The Biomedical Basis of Elite buffer contains a reducing agent and very sensitive method to stain Performance and/or SDS, the sample should be proteins in acrylamide gels. These The Queen Elizabeth II Conference boiled for 5 minutes in order to dyes fluoresce when bound to Centre, London, UK, 19–21 March proteins and are compatible with achieve complete binding of SDS Physiology 2012 UV base imagers. Some can be and disulfide bond disruption. Main Meeting, The Edinburgh The most commonly used sample visualised with a 302 UV box and International Conference Centre, buffer was described by Laemmli in photographed with a Polaroid black Edinburgh, UK, 3–5 July 1970 (1); it contains 2% SDS, 10% and white film. The most commonly glycerol, 5% 2-mercaptoethanol, used are Sypro orange, Flamingo The Journal of Physiology 0.002% bromo phenol blue and orange, Oriole and Sypro ruby from Symposia 2011 0.065 M Tris–HCl buffer pH 6.9 in Bio Rad and FITC and fluorescein Physiology, pharmacology, and its final concentration. Glycerol can dyes from Pierce. pathology of tissue fluid exchange be replaced by solid sucrose in order Satellite Symposium at the 31st to achieve up to 8x concentration; These dyes are relatively photo stable and can be photographed International Symposium on Intensive this is often useful to avoid diluting Care and Emergency Medicine several times; they are also the protein extract too much. After Brussels, Belgium, Tuesday 22 March heating, it is advisable to centrifuge compatible with electro-eluting the sample for 5 minutes to remove and enzyme digestion prior to mass Molecular mechanisms underlying neurovascular protection in stroke any insoluble proteins, as this spectrometry. This method can detect 1–10 ng of protein in a band. Experimental Biology 2011, would cause streaky bands. Other Washington, DC, USA, Sunday 10 April factors affecting band pattern and Patricia Leoni separation are protein overloading, Human hand function: the limitations uneven gel polymerization, Imperial College London of brain and brawn temperature gradients within the Physiology 2011 Wednesday 13 July Reference gel, uneven well surface and the 1. Laemmli UK (1970). Nature 227, 680–685. Non-Society meetings presence of small air bubbles in the YLS 2011, Autonomic control in health gel. In the third and final article in this and disease series of Techniques articles, the University of Birmingham, 26 May 2011 Gel staining process of transferring proteins to a The most common method to membrane and the use of antibodies Travel Grants visualise proteins is staining. The to detect specific proteins will be www.physoc.org/grants soluble stain Coomassie brilliant discussed. www.physoc.org/international

Physiology News | No. 82 | Spring 2011 | www.physoc.org UNBELIEVABLE! PN 47

How I learned to love How does the unique physiology of an individual my aura (not really – it organism interact with the cost me £60) universe to transmit an energy signature that can be received I realised that I might be suffering by the ‘sensitive’? What are the from a personality disorder or at hidden physiological processes, the very least be missing a chunk of the biomedical benefits and my frontal cortex when I became the possible applications in erotically obsessed with Sarah Palin. famine-stricken countries that During daylight hours I couldn’t stop exploring the mysteries of the thinking about her and every night aura might uncover? inappropriate obsessions. I don’t have she would stalk my sub-conscious a biomedical background, so you will mind. I don’t really know what it Actually, its bullshit! I am being have to explain this simply. How are is about that woman, but I have narcissistic, the real reason I am auras generated by living things?’ the same vivid dream every night. interested in auras is not to provide I see her slowly running across the me with any unique insights into What I discovered was fascinating. Alaskan wilderness wearing a white physiology, but because a close ‘Auras are the electromagnetic fields negligee accompanied by stirring relative claims he can see them. He that surround living things, in your Wagnerian music and carrying one is an emotionally balanced man – he case the field will vibrate at different of those really neat military assault thought for 20 years or so that L frequencies depending on your rifles. It’s the way she caresses the Ron Hubbard was the Messiah and state of mind’. ‘Scientists (you evil rifle that makes the dream seem so that a recurrent shoulder injury scum) are unable to measure such vivid and when she finally points the was caused by the trauma of being subtle physiological effects’. ‘Most rifle at a critically endangered but struck by an axe during one of his of this knowledge is suppressed as unbelievably cute looking piece of previous lives. So when he claims that it is cheap and it would affect drug Alaskan wildlife and shoots, I wake auras can be used to divine a wide company profits’ and this little gem ‘the technology behind this stretches up feeling exhausted and drenched in range of physiological dysfunctions all the way back to Atlantis’. sweat. and diseases I believe him. Another sensitive relative (who unfortunately When I casually mentioned this ‘What is the primary focus of your is a physiotherapist and so allowed to a psychologist, he suggested I obsessions?’ he then asked almost near patients in a hospital) then paid seek professional help. He said I as an afterthought. ‘Mainly an erotic £50 to have her aura read. Dr Aura might be suffering from a form of interest in Sarah Palin’ I replied. He told her she was a caring person obsessive compulsive disorder with didn’t react much, but put my hand and a healer. Being an insensitive the strangely alluring Ms Palin as its on a plate and produced a picture of disturbed focus. It was at this point misanthrope I do remember saying my hand surrounded by an energy I decided to act like a responsible ‘You have just given him £50 for field. It looked quite pretty and he adult and so I looked up treatment 15 minutes work – he’s not going suggested that a number of things options for obsessive disorders on to call you a bastard is he?’. (N.B. were wrong, that my life lacked the internet. There weren’t many, this particular paragraph is entirely balance and that I might be suffering mainly cognitive behavioural therapy accurate). from stress. I asked him about my or drugs that would interfere with my obsessions but he was a bit evasive So I made an appointment with an serotonin system. but did suggest I was a caring person aura consultant (best not say who, and a potential healer. Eventually So I obsessed for several days and or where in case he decides to sue, after an hour, I paid him his £60 fee then decided to take a course we have really strange libel laws in and left. of action that would change my England). He was a friendly man and life. I decided to have my aura when I told him I was a social worker After paying £60 and having my read! Although I have a PhD in (as I thought it might be the type energy fields probed by a technology neuroscience, I have always been of profession that was sensitive to developed in Atlantis, am I any interested in the quirkier side of electromagnetic fields), he conducted the wiser? Not really! But I have physiology, neuroscience and a detailed clinical analysis: ‘What discovered I am a caring person and the mind: spontaneous human path are you choosing in life?’ he a healer (it’s news to me) and I have combustion (I tried it once on a asked, followed by ‘what would developed an extreme sensitivity to neighbour – the fire went out and you describe as your basic health electromagnetic fields. Now when they just smouldered a bit – I think problem?’. This might possibly have I dream about Sarah Palin walking it was the rain), the use of colonic activated my mischievous streak so I across the Alaskan wilderness she is irrigation to influence an animal’s explained ‘I have a range of problems. surrounded by an aura. physiological state (I tried it on a I can’t sleep or concentrate, I am fruit fly, it died) and above all the drinking too much and I think that I Dr Keith Cormorant mysteries of the aura. might be suffering from a range of PS Some parts of this article are true.

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You are what you hear – like fMRI, are referenced at the more glial cells compared to ‘normal’ bottom of each page. I quite like people in the left parietal cortex, How music and territory this format because it is interesting a region believed to be involved in make us who we are to know where all the diverse and language, mathematics and spatial By Harry Witchell obscure facts were gleaned from, learning. In the last few chapters without having to keep turning to a of the book, Koob postulates the Algora Publishing, 2010, bibliography at the end. various roles astrocytes could play £14.30, 232 pages, paperback in dreaming, affective disorders, I read this book by dipping into drug abuse as well as degenerative ISBN-10: 0875868045 it a chapter at a time, sometimes disorders, trauma and cancer. ISBN-13: 978-0875868042 accompanied by music but often in silence in my ‘safe’ home territory. The main thesis, repeated numerous Music is organised sound, created to I also read it on trains and buses to times throughout the text, is elicit responses in the listeners. So the accompaniment of noise. Did the that glia play vital roles in various why is music so different from noise music make any difference? I tried neurophysiological functions; in or even talking? Harry Witchell’s Chapter 1 to the Eagles and then a particular, they serve a role in thesis is that humans have music Malian band called Tamikrest. I tried thought processing, suggesting that to establish and reinforce social some later chapters with Vivaldi or glia rather than neurons are the territory. a bit of Mozart. I don’t think any of substrate of the root of thought. them made a difference. Did I feel At points Koob even goes so far By the author’s own admission, safer or more territorial with the there are not as much controlled as to argue that ‘Neurons have no music on? Not really. But I did quite experimental data to support the reason to exist except to support enjoy reading the book. hypothesis as one would normally astrocytes’, acting simply as the ‘roads’ to the ‘glial cities’ but offers like to see, as an academic. However, Thelma Lovick Harry Witchel has read widely. He insufficient evidence to support this, or many of his other statements provides examples ranging from the The root of thought behaviour of songbirds and the Third throughout the text. However, Reich to modern day shopkeepers, By Andrew Koob whilst much of the content is merely speculative, the evidence detailed is to illustrate how music is used Financial Times/ Prentice Hall, very interesting and does illustrate to manipulate the behaviour of 2009 populations. the many complex functions of £17.99, 192 pages, hardback glial cells. Although it is tempting The 11 chapters each pose a to believe that astrocytes could be question related to a different aspect ISBN-10: 0137151713 involved in intelligence, the reader of music. Why does music make sex ISBN-13: 978-0137151714 is advised to take the information in better? Can music cure? Does violent In his first book ‘The root of this book with a pinch of salt. music lead to violent behaviour? thought’, author Andrew Koob aims A further criticism of the book Although packed full of interesting to enlighten the reader about the is that the level that the book facts and anecdotes related to music, under-appreciated glial cells that has been pitched at seems to be the thesis of music and territory comprise 90% of the cells in our oversimplified for the neuroscience does seem to get overlooked. In brains, cells that were previously Chapter 9, for example, on whether thought to be simply the glue that aficionado yet too complex to be music makes the brain grow larger, holds neurones together. appreciated by a general readership. there is an intriguing story about Despite the fact that this book the fate of Haydn’s brain, which Koob opens with a brief overview of consists largely of metaphors, relates how, after being removed exactly why we should pay attention intertwined with biased personal from his coffin by bodysnatching to glia, including such compelling opinion and numerous references phrenologists, his skull passed facts as ‘the ratio of glia to neurons to popular culture, it does present through the circles of Viennese increases with our definition of an interesting hypothesis as well as society for the next 145 years before intelligence’, and that glia can highlighting the error of considering finally being reunited with the rest of communicate through networks glial cells as uninteresting, but which his body. There’s another fascinating via increases in intracellular calcium are in fact complex, multifunctional story about the fate of Einstein’s levels. The reader is then taken on cells, in a similar way that nucleic brain. The mathematical genius was a journey though the history of acids were initially overlooked as also an accomplished violinist. neuroscience and neurotransmission before being introduced to glial cells, the material of heredity due to their The book aims to be accessible to and learning how they communicate, simple structure. a wide audience. It is an easy read about their potential roles in and source material, together with development and memory, and how Angus Brown and Lucy explanations of technical procedures, Einstein’s brain possessed many Brooks

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The physiological action of and scientific birthright for a mess of β-iminazolylethylamine pottage”. H.H. Dale and P.P. Laidlaw (1910) Utimately, the presence at Wellcome J Physiol (December 31) 41, 318–344 of organic chemists, and commercial There is a natural tendency to picture production facilities, played a key role eminent scientists as their ‘mature’ in the WPRL’s success. Indeed, the selves. This is obvious in the choice of WPRL offers an early exemplar of many photographs to accompany valedictory of the things that would characterise Wellcome Images or memorial articles, images which later successful pharmaceutical industry Sir Henry Dale in turn tend to become the ‘received’ drug discovery programmes. Thus, Dale’s operation was well-resourced, version of the person. Thus it is with Sir guinea-pig correspond in a suggestive with trained scientific personnel already Henry Hallett Dale OBE (1875–1968), manner with those described ... as the within the company, and easy access Nobel Laureate, President of the Royal effects of poisoning in that animal by ... to analytical and synthetic chemists Society, Chairman of the Wellcome serum or other protein in the sensitised and to the company’s chemical and Trust, scientific adviser to Churchill’s War guinea-pig (“anaphylactic shock”).” Cabinet, Member of the Order of Merit, biological materials. Apart from drawing and first director of the MRC’s National Several years later, a chance observation on the expertise of his Wellcome Institute of Medical Research (NIMR). If relating to animal sensitization to serum colleagues, Dale could and did remain any image of Dale is familiar to readers, (which was produced by the Wellcome in close contact with the academic labs it is almost certainly that of the eminent Laboratories and tested at the WPRL) elsewhere in London and in Cambridge, elder that accompanied his obituaries. was to lead Dale to return to sensitized as well as further afield. smooth muscle (especially uterine George Barger (1878–1939) and Patrick One has to stop a moment to recall muscle) and the spasm produced by Laidlaw (1881–1940), Dale’s co-workers, that many of the physiological greats of antigen re-challenge. By simple and both became eminent FRSes. The Dale’s generation did their pioneering elegant experiments Dale showed that Anglo-Dutch Barger was a chemist, a experimental work comparatively the antigen must be localized in the cells Cambridge acquaintance of Dale’s who young, at the stages we now call PhD and tissues, rather than circulating in the was already at Wellcome when Dale student, postdoc/research fellow, and blood (Dale, 1913). junior lecturer. It is for this reason that it arrived. He went on to be Professor of seems appropriate to include the other Together, this body of work amounted Medical Chemistry in Edinburgh. Patrick picture of a much younger Henry Dale, to a key pioneering contribution Laidlaw was some years younger than taken in 1904 when he was around 30. to understanding anaphylaxis. It is Dale, and had been taught by Dale This was the time when Dale took up his described in depth in a fascinating article when a 16­­-year-old pupil at Dale’s old first substantive post at the Wellcome by science historian and Physiological school in Cambridge – Dale, like many Physiological Research Laboratories Society archivist Tilli Tansey (Tansey, an impoverished student, was tutoring (WPRL), then situated in a large house 2003). Tansey, who has written to supplement his allowance. Laidlaw (since disappeared) in South East extensively about Dale, his work and his became a distinguished pathologist London’s Brockwell Park. milieu, relates that Dale was always keen and virologist, later re-joining Dale to credit both serendipity, and the work at the MRC and becoming Deputy The paper is from this period of Dale’s and insights of colleagues, as key to his NIMR Director to Dale’s Director. He is career, part of a series in 1910–11 own discoveries. Of Dale’s arrival at the remembered particularly for his work in which Dale and his co-workers WPRL she tells us: on isolating the influenza virus, and on George Barger and Patrick Laidlaw developing a canine distemper vaccine. describe the identification in ergot “[Dale] had ... no immediate research Though Laidlaw’s role in the histamine fungus, and the biological actions of project of his own in mind, and very story is less well-known, Dale saw it β-iminazolylethylamine – or histamine, as readily followed Henry Wellcome’s as critical, writing in Laidlaw’s British it shortly afterwards came to be known. personal suggestion that he investigate Medical Journal obituary that “The first the pharmacology of ergot, a fungus detailed studies on histamine were his Barger & Dale (1910) had reported some known to contaminate cereal crops work as much as mine.” months earlier that histamine was a such as rye ... [and] often used as an component of ergot fungus. Dale and abortificant … Ergot has been described Though Dale remains best known for his Laidlaw describe the actions of histamine as ‘a treasure house for drugs’, and many Nobel-winning work on acetylcholine, injections in intact animals and in a range of those treasures were discovered by several earlier nominations for the of perfused and/or isolated organs. Inter Dale and his colleagues at the WPRL. Prize specifically mention his histamine alia, they show intravenous histamine Indeed, much of [Dale’s] research career work. He retained a life-long interest in causes a profound fall in blood pressure. stems directly from his investigations of histamine and anaphylaxis, publishing This effect is independent of actions on ergot and its constituents.” and giving lectures on the topic, and the heart, and reflects dilatation in the even being elected President for Life of vascular beds of many organs. Dale always said his major motivation the Histamine Club formed in the late in taking the Wellcome post was the 1940s. Perhaps most striking, they note that in desire to do research unencumbered vivo histamine injection causes “acute by other commitments. At the time, Austin Elliott obstruction of air-entry ... this must be though, the move was unprecedented. Barger G & Dale HH (1910). Proc Physiol Soc 40 attributed primarily to bronchial spasm.” Dale’s later colleague and obituarist (Suppl), xxxviii–xl (abstract). Wilhelm Feldberg noted: “[Dale took Dale HH (1913). J Pharmacol Exp Ther 4, They continue: the job] against the advice of nearly 167–223. “It may be noted ... that the symptoms all his scientific advisers, who told him Tansey EM (2003). Stud Hist Phil Biol & Biomed and post-mortem condition in the that he would be selling his medical Sci 34, 455–472.

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Ask a physiologist! coordinate automatically to make the Finally, no cell in the brain would be action happen faster. able to work at all without a highly I know that our brain tells our body effi cient fuel supply, provided by the what to do, but how does the brain Finally, perhaps you were actually oxygen and glucose delivered in the know? (George, age 11) asking how our brain ‘makes’ our blood by an intricate network of blood consciousness. That is an even vessels. Ultimately it is this blood Austin Elliott, University of bigger question, and one that lots of which ‘powers’ the brain to function in Manchester, replies: scientists would like to solve! such an awesome fashion. There are lots of answers to this How come our brains are so question! Why do we have bogeys? (Philip, age powerful? (Anna and Connor, age 11) 11) First of all, many of the nerves in Dr Stuart Allan, University of Sam Wilson, Shoreham Academy, your body outside your brain carry Manchester, replies: replies: information about what is going on back to the brain. There are your The power of the human brain stems In the air that we breathe there are ‘special senses’ (vision, hearing etc.); from its incredible complexity. Indeed, many small dust particles and millions there is sensing of things like touch, the human brain is probably the most of microbes, which are not visible pressure, temperature and balance; complex thing on planet Earth. Within to us; however, they could cause us and in a typical nerve in the body that the 1.4 kg adult human brain there are problems if we just inhaled them deep runs to one of your muscles, as many 100 billion neurones. These neurones into our lungs. So our nostrils are as 80% of the nerve fi bres (axons) interconnect with their neighbouring designed to fi lter out all of those small cells to form local networks, and these are bringing information back to the particles and microbes that are present networks can in turn send messages brain about how hard the muscle is in the air. Lining our nostrils are small to other networks of neurones in a contracting, the length of the muscle, bones which are crinkled and uneven, different brain region, and so on, until its position, the position of the limb, a bit like the inside walls of a cave; and so on. Finally, the body has many, whole systems of communication within the brain are established. These they are covered in a slimy liquid called many other ‘internal sensors’ to tell it ‘mucus’, otherwise known as ‘snot’. about what is going on inside it – like complicated interconnecting networks of neurones are what allow the brain So imagine a cave covered in slime, how much oxygen is in your blood, for with jagged and uneven walls which instance. to perform diverse functions, such as the control of breathing, recognition are so close together that any air being The next answer is that all this of faces, sleep regulation and the sucked into the cave crashes into the information is ‘received’ by centres in formation of new memories, to name walls, and any small particles in the air the brain (mostly) and decisions then but a few. get stuck to the slime. made – sometimes by special cells, Now small hairs called ‘cilia’ brush that sometimes by ‘networks’ of many cells Each specialised function occurs in slime carrying the dust and pathogens working together – about what signals specifi c regions of the brain, and many (microbes) down towards the pharynx to send out to control the body. A lot of these functional areas are shared (throat), where we swallow it; of these are automatic, like controlling between humans and other species. However, it is the outermost layer of fortunately we are not very aware that your heartbeat. Some things you do the human brain, the cerebral cortex, we are doing this! The mucus carrying are voluntary – like standing up, if that is most advanced in humans, the dust and pathogens is then you decide to! – but need the brain to standing us apart from other animals destroyed by our very strong stomach ‘automatically’ make a lot of different and ultimately making our brains so things happen. You choose to stand acid. However, the mucus near to the powerful. up, but the brain’s processing then entrance to the nose dries out quite tells all the various muscles that help quickly and can’t be moved by the Neurones pass messages to each cilia; it is this dried mucus carrying you stand up to coordinate their other using electrical and chemical actions to make it happen. the dust and microbes which forms a signals in a complex process known as bogie. synaptic transmission. At any one time Some things require thinking at millions of neurones in your brain will fi rst, but as you do them over and Now more about the slime. The mucus be fi ring off messages to each other to in the nostrils can be different to the over again get progressively more allow you to function in everday life. automatic – like learning to stand and mucus in the trachea (windpipe) and Amazingly, the billions of connections different again to the mucus lower walk as a baby, for instance, or learning that exist in the human brain are not to catch a ball. Catching a ball involves down in the ‘respiratory’ tract. If fi xed and neurones have the ability to you have an infection, such as a cold your eyes tracking the ball, your brain change how they respond over time. your respiratory tract produces more predicting its fl ight, and your body, This ‘plasticity’ of the brain is a key mucus, in an attempt to trap more arm and hand moving to catch it, but contributor to its amazing power. this may all happen so fast – think microbes and particles; the mucus a catch in the slips in cricket – that However, neurones are not alone in often becomes thicker and more sticky much of it is semi-automatic without making the brain function. They also you having to consciously think ‘Must rely on glial cells such as astrocytes, A note of caution: catch ball – thinks – must move arm.’ micro glia and oligodendrocytes to This is because all the practise has allow them to work properly, and we While we are continually ‘re-wired’ your brain’s circuitry to still have much to learn about how sub consciously swallowing our snot, allow the relevant bits – eyes, tracking, these glial cells contribute to brain scientists are not convinced that eating prediction, move to catch ball – to function. bogeys is a good thing!

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David Paterson, the new Editor-in-Chief for The Journal ‘Personally I am delighted that internationally and is funded by the David has been appointed as the British Heart Foundation, Wellcome Editor-in-Chief of The Journal of Trust and BBSRC. David has a long Physiology. He will bring energy, association with The Physiological commitment and innovation to the Society. In addition to his five years role. The Society views The Journal as Editor-in-Chief with Experimental as a beacon for physiology and is Physiology, he delivered The Society’s committed to its development to GL Brown Prize Lecture in 2000. maintain its international reputation for excellence.’ David says: ‘It is a great honour to be given the opportunity by The David is a Professor of Physiology Physiological Society to lead The at Oxford University and a Fellow Journal of Physiology into its next of Merton College, positions he phase. I am of the opinion it is our has held since 2002 and 1994, job as editors to publish the best respectively. He completed his science presented to us so we can The Physiological Society is delighted doctoral studies in physiological maintain and grow the international to announce that David Paterson sciences at Oxford as a scholarship standing of The Journal, and the has been appointed the new Editor- winner from the University of discipline of physiology.’ in-Chief of The Journal of Physiology. Western Australia. He was made a David, who is the current Editor- Fellow of the Institute of Biology in He continued, ‘This is a very exciting in-Chief of Experimental Physiology, 2003 (now the Society of Biology) time to be in physiology. The Journal will take up his new appointment in and the Royal Society of Medicine in is well positioned to become the April. David will take over the reins 2005. opinion leader in the physiological from William Large, who has served sciences that bridge many of as Editor-in-Chief for five years. David’s research is in the area of the exciting discoveries made cardiac neurobiology. His team’s in the post-genomic area. There Mike Spyer, President of The Society, work looking into the neural causes will be challenges, but also great commented on the appointment: of cardiac arrhythmia is recognized opportunity.’

An interview with William Large, the outgoing Editor-in-Chief

William Large has been the Editor- contributing to the impact factor we in-Chief of The Journal of Physiology were publishing about 100 papers since 2005, during a time of (about 10%) that had not been uncertainty and change in the world cited. I felt that the overall quality of scientific publishing. To mark his of The Journal would be increased retirement in April we asked William by raising the bar for acceptance to about the challenges he has faced eliminate the least influential papers. over the past 5 years. In addition, I wished to increase the variety of articles to broaden 1. What were the ‘big issues’ for The the interest of The Journal and to Journal of Physiology when you took encourage discussion of published over as Editor-in-Chief? articles by more junior members of When I took up the role of Chair the research community (leading of the Editorial Board in 2005 I had to the introduction of Journal Club been a member of the Board for articles). Also, I wished to take this 6 years and my major concern was theme further to make The Journal that whereas we published some of limited influence. We embarked (and physiology) of relevance very strong papers, we were also on citation analysis and found, outside our core readership of publishing many papers that were for example, that in the 2 years research physiologists. A first

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enactment of policies that was very difficult in the past.

4. How have the issues changed during your time as EiC and how has this affected The Journal?

Many of the issues are the same, i.e. trying to improve the overall quality of The Journal and make it more accessible to a wider readership. When I started in 2005 the issue of ‘open access’ was a major concern but this seems to have faded over the last few years. I think areas of increasing importance concern the different types of access to The Journal via the web, mobile phones, Facebook, Twitter etc.

5. What have been the highlights William, with his wife Kathy, before they set off for California. and low points of your time as EiC? step was to introduce Clinical Journal which coincided with the I gained most satisfaction from Perspectives to attract clinicians who establishment of the Publications working with members of the Board might not otherwise read The Journal Committee by The Physiological and the staff in the Publications regularly. More recently we have Society who decided to appoint the Office to bring about so many started publishing non-technical Editor-in-Chief instead of an election changes, which I believe will have a summaries, which we hope will be by the Board. lasting positive effect on The Journal. of interest not only to high school It was a great pleasure to work with students and undergraduates but Inevitably these changes were so many wise and conscientious also to members of the general resisted by some members of the individuals who were committed to public who are interested in science Board because some power was improving The Journal. The greatest and health issues. removed from the Board and also headaches were caused by ethical scientists are most conservative problems and I was amazed that so 2. How did you tackle these and often do not like change. A many scientists seem to be unaware issues and what problems did you major difficulty was persuading of legal aspects of publication encounter? reviewing editors to raise the bar for ethics. During my time a much acceptance and reject more papers. closer relationship with The Society These were major challenges and It seemed that some editors have developed which brought some I thought that there needed to a fixed view of the type of paper obvious benefits, but I also think the be fundamental changes in the that is published in The Journal and erosion of the independence of the organisational structure of the were reluctant to alter that standard Board has had some downsides that Board to allow editorial policies to and become much stricter. I believe were detrimental to The Journal. be formulated and then enacted. the appointment of Senior Editors I felt that these policies should be helped to overcome that difficulty. 6. What makes The Journal of developed by an Editor-in-Chief (not Physiology unique from other similar Chair of the Board) together with an 3. What have you given to The journals? Executive Committee consisting of Journal during your time/what senior members of the Board plus impact have you had on The Journal? The Journal has a remarkable history representatives of our publishers in publishing many of the most and The Society. Moreover, we I am not sure that I am the right influential papers in physiology appointed a team of Senior Editors, person to answer this question. I since 1878 – I think it is fair to say experts in their respective fields and think we made many changes from that this is unequalled in the field who were loosely connected to the being much stricter on acceptance, of physiology. Although it has Table of Contents subdivisions. The introducing new types of articles – suffered from the rise of specialist Senior Editors have the final say on especially those that reach out to journals (e.g. in neuroscience), which acceptance and therefore control the individuals that would have not read have attracted many authors, The quality of published papers. All of The Journal in the past – to more Journal still publishes outstanding these changes required substantial pragmatic issues such as altering papers across the full spectrum of changes in the governance of The the governance, hence allowing physiology.

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7. What trends in the physiological universities!) – none of us is always Contrary to popular belief, community are you most excited correct. Thirdly, develop achievable optogenetics is not all about using about? Any predictions? aims that can, preferably, be light-sensitive ion channels, such quantified. Finally, enjoy the role – it as channelrhodopsin, although I am not sure how wise a comment is a great honour to act as Editor-in- this particular method has led to a I can make or whether I can say Chief of The Journal of Physiology. flood of high-profile publications. anything useful but a simple Strictly speaking, optogenetics observation is that several years 9. What will you be doing in means experimentation which ago the rapid advances in molecular retirement? involves a combination of genetic techniques and knowledge pulled manipulation and optics. Therefore, At present I am adjusting to life in many physiologists into these perhaps, its history should date back the US after relocationg with my areas and physiology seemed to the first days when scientists American wife, Kathy, to southern old-fashioned to many scientists. started experimenting with green California. In the next few months However, it is now evident we still fluorescent protein or even earlier. I will be kept busy renovating a know very little about the function Genetic engineering enables condominium which we have bought of many gene products and how targeted expression, in mammalian in West Hollywood, LA. After that I they interact in terms of integrative cells, of various constructs, which will have to find something to keep physiology (is this what some can be subdivided into reporters and me out of trouble since drinking people call systems biology?) and unsurprisingly, this approach has margaritas in the sunshine is far too disease. This is obviously where been swiftly adopted particularly by attractive. However, I have no firm physiologists will play an enormous neuroscientists. The brain represents ideas and I am happy to see what role in answering these fundamental a particularly difficult experimental develops. problems – in the end it will come target; there, the numerous to the whole body. Associated cell types are intermingled and with these aims, I believe some interconnected, they are hard to physiologists are becoming overly visualize and almost impossible to ambitious in their conclusions control selectively other than by in papers, often published in using patch clamp on a cell-by-cell high-impact journals. Often data Optogenetics Themed basis. Therefore, understanding the obtained from series of experiments Issue complex behaviours of neuronal using several diverse techniques may networks is a formidable task. The be consistent with a physiological recent appreciation of the active role or pathophysiological function. of astroglia has added yet another However, in many instances, at least dimension to it. At the same time, in my field of ion channels in smooth optogenetics deserves a wider muscle, several of these conclusions application than only neuroscience, have been shown to be at least so and we hope to excite our diverse incomplete that they are of little readership with the possibilities it value and quite often to be actually offers. incorrect. My guess is this happens more now and in higher impact journals than in the past.

8. What advice would you give David Paterson as he takes over?

It would be presumptuous of me to advise David who has acted in this role for Experimental Physiology but I can make a few suggestions that I found to be valuable. Firstly, use http://ep.physoc.org/content/96/1.toc the great wisdom and experience of all members of the Editorial The January issue of Experimental Board and the Publications Office Physiology contains a set of papers when developing journal policies. that summarize experiences of a Secondly, listen carefully to those number of internationally recognized arguments that go counter to laboratories working with the The issue was organised by Editor ones’ own idea (not something technology currently known as Sergey Kasparov (pictured above) at that happens currently in British ‘optogenetics’. Bristol University.

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Statistics: Little and of ‘no hypothesis’ if one sends in a statistician who is my co-author a paper that states ‘we studied…’ and we conducted our own, rather often, softly softly, rather than overtly parroting ‘we depressing, survey of recent articles, hypothesised that…’. confirming yet again that things don’t scare the ‘could do with improvement’. We horses, oaks and In physiology, it was soon clear to me suggested that we would write a that things were different. No-one series to see if we could stimulate acorns recognised my quotation “Absence change. It’s been done before, of I came to The Physiological Society of evidence is not evidence of course: for example, the American from a medical background and absence” which to me was the most Physiological Society did so in 2004 editorship in a clinical journal, and striking faux pas in many papers. (Curran-Everett & Benos, 2004) and I had always been in awe of The Everything was dynamite plunger triggered some debate, but less Society’s ways and its prestige. plots, you could search high and low change (Curran-Everett & Benos, When I became an Editor, and later to find how many individuals were 2007). Perhaps it’s presumptuous joined the board of The Journal of in a study, and as for suspecting that of us to think we can do better, Physiology, I remained in awe of the data could be skewed… I started to but the approach we have taken standards and rigour of the science, feel very uneasy. The old aphorism of is more gentle: a light touch, little but … I had a nagging feeling that in the lamp post for a drunkard, more and often, nothing too threatening, clinical medical publications we had support than illumination, came to just enough for a coffee break at least been doing one thing better. mind. Thus, when Peter Cahusac and no need for an icepack to get That was the statistics! launched his broadside against the to the end. We shall try to avoid equations, and use diagrams Essentially concurrent with to illustrate key messages. my research career, the The Journal Board have been use of statistical analysis very supportive, as have many in medical research has others in other journals we improved greatly. As a have written to, not the least statistical novice, I watched Doug Everett who wrote the the effects of a concerted APS guidelines, and many campaign by well-known others, too numerous to statisticians such as Mosteller, list. Please read the articles, Swinscow, Altman, Bland and let me know what you and others, with the help think of them: feedback of prominent platforms will be gratefully received provided by journals such and incorporated. I hope to as the British Medical Journal encourage contributions in and the New England Journal specialist areas, so volunteers of Medicine. I read their will be valuable. articles with the interest of a budding researcher Gordon Drummond and watched as there was University of Edinburgh, a substantial improvement Edinburgh, UK in the understanding and use of statistics in medical t test and the null hypothesis, in References journals. Of course, these changes Physiology News (Cahusac, 2009) Cahusac P (2009). Statistical methodology were also driven by a variety of it was like the little boy and the and reporting – the case for confidence other factors, not the least being Emperor’s new clothes! These intervals. Physiology News 74, 33-36. the increased regulation of data views would have been considered Curran-Everett D & Benos DJ (2004). collection in association with mainstream and even quite gentle Guidelines for reporting statistics in journals drug licensing. It was a long haul. published by the American Physiological in my personal training arena, the Society. Am J Physiol Heart Circ Physiol 287, Although we’ve come a long way, Anaesthetic Research Society, which H447–H449. one can still occasionally find fault. was at the time not for the faint- Curran-Everett D & Benos DJ (2007). However, most journals are now hearted presenter. In fact, the point Guidelines for reporting statistics in journals far more rigorous and exacting, had been made already (Watters & published by the American Physiological giving comprehensive guides to Goodman, 1999). Society: the sequel. Advances in Physiology contributors, and are never slow to Education 31, 295–298. pick up failings in papers submitted I was galvanised into action and Watters MP & Goodman NW (1999). submitted a paper to the Board Comparison of basic methods in clinical for consideration. Indeed, things studies and in vitro tissue and cell culture may have swung too far: it’s almost of The Journal, outlining some studies reported in three anaesthesia journals. impossible to avoid the criticism suggestions: I collaborated with Br J Anaesth 82, 295–298.

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Life as a Physiological and the different activities that it enthusiastically supports, and also Society Affiliate how hard the committee members, Council trustees and Society staff Representative work to ensure The Society runs well Well, two affiliate representatives and provides useful benefits to its to be more accurate! Members. I would whole-heartedly encourage anyone who is tempted Affiliate reps are the voice of the to put themselves forward for Affiliate Members on The Society nomination in the coming months Council and committees and we as a new Affiliate rep. In addition to are your current representatives – Attending the Council meetings has being enjoyable it is great for the CV. Samantha Passey from the University offered a unique and fascinating of Bedfordshire, and Federico viewpoint on the running of The Samantha Passey Formenti, currently at the University Society but for me, sitting on the University of Bedfordshire and of Oxford but soon to be jetting off Education committee and Editorial Affiliate representative of The to sunny New Zealand to take up a Board of Physiology News has Physiological Society position as Lecturer at the University been much more involved. Time of Auckland. consuming in some instances, yes, Dearest Affiliate In July 2011 it will be time to vote in but hugely rewarding, very enjoyable a new representative for the affiliate and totally worth it. membership. As the existing reps we The Education committee meets would like to highlight the activities about 3 times a year, and its remit that we have been involved in to is to support the learning and encourage other Affiliate Members teaching of physiology both in to step forward in the coming schools and at university level. months to be voted in as a new rep. This includes supporting outreach events at schools, universities and I put myself forward for the role public science festivals, running the of Affiliate rep in Spring 2009 as vacation scholarship scheme for I have been honoured to be your I was keen to get involved in the university students, contributing representative for The Physiological activities of The Society, especially to physiology teaching curricula at Society for a fantastic year and a in their outreach programme, and universities, and also organising half. It has given me the unique also to write for Physiology News. various teaching workshops for opportunity to see how our beloved I have thoroughly enjoyed myself, Society Members to offer instruction Society is run from within, how met some fantastic and inspirational in specific techniques such as PCR, much effort and care is put into people and have learned a great deal microscopy and in vivo techniques. keeping it lively and, we hope, about the huge range of activities The Education committee is hugely helpful to its Members. Most The Society is involved in. enthusiastic and I was excited to activities of The Society require a great deal of long-term planning, As I understood it the major role see the range of activities that The critical debates and consultation of an Affiliate rep was to be the Society supports and encourages. It with Trustees, and close constructive voice of the Affiliate membership was good to have the opportunity to collaboration between the scientific at Society Council meetings. These get involved in this exciting area of experts across the UK and the typically begin with a dinner and The Society’s endeavours. sparkling Society staff. The Society social event the night before the As a member of the Editorial Board as it appears to Members and to the meeting, which provides an excellent for Physiology News, I contribute public represents only a very small opportunity to meet and chat with articles and try to encourage other part of their work. the Council members and Society Affiliate Members to contribute too! staff in an informal setting before It is exciting to have the opportunity During my time as a rep, I sat on the more official Council meeting to play a role in the content and look the Meetings committee, where the following day. As the Affiliate of the magazine. Along these lines, if the planning and organization reps are not full Council members anyone has a burning desire to write of Society meetings takes place. or Society Trustees we do not have an article, we are always interested In addition to participating in voting rights at the meetings, but in hearing from Affiliate Members. discussions on the format and we can participate in discussions locations of the meetings, I also about the running of The Society and So for me, being Affiliate rep has had the opportunity to put forward it provides an opportunity to raise been a hugely rewarding experience ideas on new elements that could be Affiliate issues and the needs of the and has really opened my eyes included. Although I was the most Affiliate membership. to how much The Society does junior member of Council and of the

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Meetings committee, I was always ‘Phys Soc’ so much that I am now given a chance to present my ideas, contemplating the idea of joining Physiology in the which would then be discussed with our Kiwi friends in the Physiological school curriculum other Members. I still think that Society of New Zealand (yes, there is this is a privilege, as it would not one indeed). I think they also have a The Society believes it is essential necessarily happen in my country. dog as a symbol, but it looks white, that physiology is accurately almost tailless, very fluffy and it finds represented and recognised as I was able to contribute to decisions it very difficult to scratch its ears a central component of biology regarding future activities of with its hind feet. education at all stages. We believe The Society. For example, I was that supporting physiology involved in the reviewing process New Zealand has a reputation for education, in schools in particular, of applications for the Young Life being a fantastic country, inhabited will promote awareness of the Scientists Symposia, which will be by very lovely people. In the seven discipline and encourage a high run jointly with the Biochemical years I spent in England, my dad quality entry of school leavers into Society and the Pharmacological visited twice, driving his camper physiology-related careers. Society. They encourage young life van through Europe, because of his scientists (students and postdocs) aeroplane phobia. He has now left In recent months, we have to organize and run a symposium on our hometown Parona di Valpolicella, been working with awarding a topic within the life sciences. This aiming to be in Auckland for Easter organisations, in collaboration with gives them invaluable experience in (2012). I might feel the need of some the Society of Biology and other what it takes to organize a scientific European contact between now and member organisations, to develop meeting. then, so please get in touch if you their GCSE Science specifications plan to visit. (for first teaching in 2011). Our Being a rep gave me the opportunity main focus was to make sure that to meet many authors of papers I Thank you once again for giving me all physiology content was accurate had enjoyed reading, to talk about the opportunity to serve The Society, and to provide recommendations our research, and have a chat about and I wish you all the very best for when appropriate. our lives. It was not only meetings your future, and professional discussions. As yet, none of the GCSE Trustees of The Society give a high Federico Formenti specifications has been priority to human aspects in our accredited: Ofqual concluded University of Oxford and Affiliate work, so there would always be they did not represent a clear and Representative for The Physiological significant improvement over a social component in the plan Society for the day. Overall, I found being current specifications. Awarding your representative a very valuable organisations are now working experience, which will certainly help closely with Ofqual to ensure me in my future. Understanding Life that the new specifications are accredited and ready for first Life now asks me to move on to A new version of Understanding Life teaching in September 2011. a new job, which starts soon in is now available. Understanding Life It is worth noting that the coalition Auckland, New Zealand. I was told is The Society’s main educational government will soon launch a full I have good networking skills, and publication. It provides an review of the National Curriculum clearly this time I have branched introduction to the subject of and The Society expects to make out greatly. Although I will be sad physiology – why we study it and an active contribution to any to leave, it opens up a very exciting how we can apply our knowledge. changes that follow. opportunity for a new Affiliate The booklet gives an insight into Member representative, and he the central role of physiology in The Society has exciting plans to or she might be you. This is an the progress and understanding increase support for the teaching experience that most of you would of biological and clinical sciences, of physiology in schools; together thoroughly enjoy and I most strongly and explains how physiologists with contributing to curriculum recommend putting your name are involved in the detection and changes, we will also be developing forward to work for The Society, as I prevention of disease. resources (including a new am sure that you would find it a very website) to support physiology rewarding and helpful experience. For hard copies, please email teaching. [email protected] Obviously I will keep on following the activities and developments of If you would like to contribute to The Physiological Society, now from Alternatively, you can download a The Society’s activities in these a new, different perspective (from copy at: areas, please email education@ down under). I enjoyed being in the http://www.physoc.org/education physoc.org

Physiology News | No. 82 | Spring 2011 | www.physoc.org In 2013, The International Convention Centre, Birmingham will host the largest physiology meeting in the world

Plenary & Keynote Lecture Nominations

You can now nominate yourself or another individual for one of the keynote lectures, the public lectures, or the following named lectures:

Wallace Fenn Lecture Bayliss-Starling Prize Lecture Ernst Knobil Lecture GSK Prize Lecture TP Feng Lecture Hodgkin-Huxley-Katz Prize Lecture Schmidt-Neilsen Lecture Joan Mott Prize Lecture Robert Pitts Lecture Michael de Burgh Daly Prize Lecture August Krogh Lecture Paton Prize Lecture FEPS Lecture Sharpey-Schafer Prize Lecture Annual Review Prize Lecture

Deadline 1 April 2011

For more information or to make a nomination, please visit: www.iups2013.org/lectures

IUPS PN82.indd 1 1/17/2011 12:59:53 PM AVV-eGFP + GFAP

Confocal photomicrograph after adenoviral administration in the hypoglossal nucleus (p. 39).

A publication of The Physiological Society www.physoc.org