Accueil BIU Santé — BIU Santé, Paris - 16 Neuroscience · 2007-03-29 · Foster, S.G. and...

408

Sir John Eccles (see p. 53), when reflectingin 1977 upon his peripatetic scientificcareer, wrote about his period in the JCSMRfrom 1952 until 1966: ‘Without doubt it wasthe high point of my research career’. Sim-ilar words would be used by many of hisclose collaborators when referring to thetime they spent in Canberra during the sameperiod. When Eccles commenced hisresearch career in 1927, with an investiga-tion of cat spinal reflexes as a pupil of SirCharles Sherrington in Oxford, there was

considerable speculation as to whether exci-tation and inhibition at mammalian centralsynapses were chemical or electricalprocesses. At that time, however, equip-ment and techniques were not available forinvestigating these processes at a cellularlevel in vivo. Accordingly, Eccles studiedsynaptic transmission in autonomic gangliaand neuromuscular junctions in vitro, ini-tially in Oxford and later in Sydney from1937 until 1943. His observations resultedin a firm belief that central synaptic trans-

16Neuroscience

Research in Neurophysiology began in 1951, with the appointment of John Eccles,the oldest (b. 1903) and most distinguished (FRS, 1941) of the founding professors,as Professor and Head of the Department of Physiology. He moved to the temporarylaboratories in Canberra in 1953, and with the help of Jack Coombs, the electronicsengineer who accompanied him from New Zealand, began work on neurotransmis-sion for which he was awarded the Nobel Prize in 1963. From the outset, and espe-cially after the move to the permanent building in 1957, he attracted a succession ofdistinguished Visiting Fellows and bright students. Among the latter was David Cur-tis, who graduated PhD in 1957, set up a subdepartment of Neuropharmacology in1962, was appointed Professor in 1966 and, when space became available, Head ofa new Department of Pharmacology in 1973. In 1967 Eccles was succeeded as Headof the Department of Physiology by Peter Bishop, who established a strong group inVisual Science. In 1984 he was succeeded by Peter Gage, but visual neurosciencewas maintained by William Levick, who was appointed a Professor within theDepartment of Physiology in 1983. Studies in neuroscience were further expandedin 1980, when Robert Porter was appointed Director of the JCSMR. Bringing withhim a large NH&MRC grant, he set up an Experimental Neurology Unit, of whichStephen Redman was appointed Head in 1984. With the introduction of the divi-sional structure in 1989 the groups headed by Curtis, Gage, Levick and Redman werecombined as the Division of Neuroscience.

Life in Eccles’ Laboratory

by David Curtis

mission was an electrical process.From 1944 until 1951 Eccles occupied

the Chair of Physiology in the University ofOtago, and advances in neurophysiologicalequipment and techniques enabled him toresume his interest in the spinal cord in vivo.Results to 1950, including an analysis ofpotentials recorded extracellularly nearspinal motoneurones using insulated metalelectrodes, reinforced his opinion that cen-tral neurotransmission was electrical. InJune 1951, however, he and his colleaguesJack Coombs (see p. 413) and LaurenceBrock pioneered the extremely difficult taskof recording intracellular potentials fromspinal motoneurones in vivo, using elec-trolyte-filled glass microelectrodes. Theinitial results led Eccles to immediately anddramatically reject his very strongly heldelectrical theories and to accept that centralsynaptic transmission was chemical innature. Further experimentation inDunedin, however, was prevented by therequirement for further specialized equip-ment to be designed by Coombs, togetherwith Eccles’ acceptance of the JCSMRChair and his commitment to deliver theWaynflete Lectures in Oxford early in 1952.

In 1950, Coombs, a physicist later aptlyacknowledged by Eccles as a shy genius,began the design of an innovative, versatile,reliable and readily operated electronicstimulating and recording unit, whichbecame widely known as the ‘ESRU’ (seep. 412). This unit, based on the advice ofEccles and Archie McIntyre, Reader inEccles’ Department, regarding the require-ments of neurophysiologists, provided facil-ities which were unmatched in laboratorieselsewhere. Coombs also designed stableand low-noise amplifiers and ‘cathode-fol-lower’ input stages essential for recordingthrough high resistance microelectrodes.Accompanying Eccles to Canberra in 1952,he established an electronics workshop inthe Department and continued to develop alarge range of specialised equipment, inaddition to participating in the animalexperimentation.

In the total absence of appropriate appa-ratus from commercial sources in Australia

and abroad, Eccles also insisted on havingfirst class mechanical workshop facilitiesand personnel in his Department. Tworemarkably innovative and skilled mechani-cal and electrical engineers were appointedas Head Technical Officers, Gerry Wins-bury from 1952 to 1958 and Lionel Daviesfrom 1959. They designed and constructedanimal frames, micromanipulators (see p.62), vertical microelectrode ‘pullers’ andnumerous other specialised devices requiredfor the technically difficult task of recordingintracellularly from central neurones invivo. Many items of equipment developedin the Department were later manufacturedin the School Workshop, some even becameavailable commercially with no benefit tothe School, apart from an occasional ‘Can-berra-type’ label. Eccles was also fortunatein bringing to Canberra Arthur Chapman,who had previously provided skilled techni-cal assistance in his laboratories in Oxford,Sydney and Dunedin.

Thus, when laboratories became avail-able in the temporary JCSMR buildings inMarch 1953, the stage was set for a remark-able and intense period of research whichcontinued for over 13 years. During thistime 74 investigators, including PhD Schol-ars, from 20 different countries, worked inthe Department of Physiology, 41 of whomcollaborated and published with Eccles.Initially only three electrophysiological lab-oratories were available, but from 1957 theadditional space in the School’s permanentbuilding included six large laboratories withelectrically shielded rooms, which enabledexpansion of the research staff and anenhanced interest in neuropharmacologyand neurochemistry.

Given Eccles’ extensive knowledge ofexperimental neurophysiology, particularlyof the spinal cord, his strong motivation toexploit intracellular recording techniques inboth the cord and elsewhere in the centralnervous system, so retaining the lead he hadestablished over other laboratories, and theopportunity he had to attract experiencedand beginning investigators to Canberra, theDepartment rapidly became an exciting anddynamic scientific environment. Initially

409

16. Neuroscience

his investigations centred on the spinal cord,including its neuronal organization (see pp.414 and 416) and the mechanisms of synap-tic excitation and inhibition (see p. 411).From 1961 he concentrated on dorsal col-umn and related thalamic nuclei, and from1962 on the hippocampal and then the cere-bellar cortex (see p. 422).

Eccles had the ability to combine expe-rienced investigators and new recruits toform productive research teams. As teamleader he was demanding, and expected allmembers to cooperate and contribute fully.Groups of 2-4 were essential to maintaincontinuity of effort during in vivo experi-ments lasting as long as 36 hours, generallytwice weekly. These were preceded by thepreparation and filling of glass microelec-trodes, and followed by the task of measur-ing photographic records on ever increasinglengths of 35mm film, calculations usingeither a slide rule or logarithmic tables andthen plotting the results on graph paper.Later, with increasing sophistication ofequipment and the availability of calculat-ing machines, the processing of resultsbecame faster and less tedious, but themajority of illustrations in departmentalpublications continued to be based on pho-tographs of records from oscilloscopes.

Few animal experiments failed to pro-vide new and publishable results. Eccleswas a prolific writer; his bibliography lists568 items, including 19 books of which hewas sole author of 12. His name on a pub-lication always indicated his active partici-pation in all aspects of the investigation, andbased on research in Canberra over the 1953to 1966 period he authored or co-authored196 publications, including 4 books. Thetotal number of papers published by allother members of the Department of Physi-ology over the same period was approxi-mately 224. The Department was wellserved by one Secretary, who typed from avariety of handwritten originals all draftsand final manuscripts using ‘carbon-paper’to prepare multiple copies.

Friday afternoon departmental seminarswere the rule each week, dealing with newresults, drafts of papers being prepared for

publication and critical analyses of otherinvestigator’s papers. One aspect of the lab-oratory appreciated by all members of thestaff and research students, in both the tem-porary and permanent buildings, was itsclose proximity to the Medical Library.This incorporated an accumulating collec-tion of neurophysiological and related disci-pline journals, to become one of the best inthe world for many years, with 24-houravailability every day of the year. In theseearly days, long before journals world-widecould be accessed via the Internet, theSchool’s relative isolation from Europe andUSA was reduced to some extent by thereceipt of correspondence and unsolicitedreprints from colleagues abroad long beforethe journals arrived in Canberra. Latersome journals were received by air-mail, aswas Current Contents. Eccles’ deep interestin the Library was recognized in 1997 bynaming it The Eccles Medical SciencesLibrary.

During long periods of continuousexperimentation, late supper in Eccles’study, frequently involving several teams,provided an opportunity to discuss currentprogress and particularly for him to talkabout his earlier experiences, his travels toscientific conferences, his impressions ofother scientists and colleagues and hisassessment of their scientific achievements.The combination of Eccles’ experimentalexpertise, scientific ambition and stimula-tion, together with the satisfaction of con-tributing to interesting and frequently newscientific achievements, provided experi-ences never to be forgotten by his collabo-rators. Despite the remote location ofCanberra, the reputation of Eccles and thequality of research being carried out pro-vided numerous opportunities for the staffand PhD scholars to meet and attend lec-tures and seminars by numerous distin-guished scientists from abroad.

Apart from its scientific aspects, life inhis laboratory also has to be seen in relationto living in Canberra, then a relatively iso-lated city with a population of only 28,645in 1953 and 96,032 in 1966 (approximately308,400 in 1998). Fortunately, the Univer-

410

JCSMR - The First Fifty Years

411

16. Neuroscience

sity had an excellent housing scheme andprovided accommodation for staff, researchstudents and visitors, albeit often in distantsuburbs amongst the Australian wild life,and University House, a few minutes walkfrom the JCSMR, was available for those

without children. Eccles and his wife weregracious hosts at weekend tennis parties onthe family court and at regular countrydancing sessions; he was an enthusiasticexpert in both forms of exercise.

Further Reading

Eccles, J.C. (1977). My scientific odyssey. Annual Reviews of Physiology, 39, 1-18.Eccles, J.C. (1982). The synapse: from electrical to chemical. Annual Reviews of Neuroscience, 5,

325-339.Foster, S.G. and Varghese, M.M. (1966). The Making of the Australian National University. Allen &

Unwin, Sydney.

Biophysical Properties of Motoneurones and Ionic Mechanism ofCentral Synaptic Transmission

by David Curtis

In June 1951, John Eccles, Jack Coombsand Laurence Brock, working in Dunedinand recording intracellular potentials fromcat spinal motoneurones in vivo for the firsttime, dramatically changed the understand-ing of the mechanism of synaptic transmis-sion in the mammalian central nervoussystem. The recording of depolarizing exci-tatory and hyperpolarizing inhibitory post-synaptic potentials convinced Eccles thathis previously strongly held electrical the-ory of synaptic excitation and inhibition wasincorrect, and provided the basis for furtherstudies of chemical synaptic transmissionusing intracellular recording techniqueswhen he took up his appointment in Can-berra (see p. 26).

Beginning in March 1953, Eccles,Coombs and Paul Fatt used single- and dou-ble-barrel electrolyte-filled microelectrodesto record intracellularly from alphamotoneurones in the lumbar spinal cord of

anaesthetised cats. Fatt, a biophysicist, hadpreviously used intracellular microelec-trodes with Bernard Katz at University Col-lege London to examine the changes inmembrane ion permeability at excitatorysynapses of the neuromuscular junction andat inhibitory synapses on crustacean muscle.In the spinal cord, one barrel of double-bar-rel electrodes was used to record membrane,action and synaptic potentials. The otherenabled current to be passed through theneurone membrane to set the membranepotential above or below the resting level.Additionally, ions could be ejected fromsingle, or either barrel of double, electrodesby suitably directed electrical currents inorder to alter intracellular cation or anionlevels.

The electrical properties of the motoneu-rone membrane were measured, and thecharacteristics studied of the ion fluxes andpumps contributing to resting and action

412


potentials. The observation that inhibitorypostsynaptic potentials, initially recorded ashyperpolarizations, gradually converted todepolarizations, led to the recognition thatthis was the consequence of the leakage ofions from intracellular recording microelec-trodes containing potassium chloride. Elec-trodes were then used containingcombinations of a series of cations andanions of different hydrated ion diameter,from which ions were passed intracellularly.This technique was combined with anexamination of the influence of changes inmembrane resting potential on inhibitoryand excitatory synaptic potentials.

The results indicated that at the synapseson motoneurones of the ‘direct’ and recur-rent spinal inhibitory pathways the activatedpostsynaptic membrane was transiently

converted to a sieve-like structure perme-able to ions smaller than a critical size. Anincreased permeability to chloride andpotassium ions thus accounted for hyperpo-larizing membrane currents and potentials.In contrast, depolarization at excitatorysynapses in the spinal cord was generatedby a non-selective increase in the perme-ability to larger ions including sodium, sotransiently short-circuiting the membranepotential. It was essentially these and laterrelated studies which resulted in the awardof the Nobel Prize to Eccles in 1963.

Coombs, Eccles and Fatt also examinedthe interaction between monosynaptic exci-tatory and ‘direct’ inhibitory potentials interms of the transient nature of the changeof permeability at inhibitory synapses andthe time courses of the excitatory and

The Electronic Stimulating and Recording Unit (ESRU) was designed by Jack Coombs while he was inthe Physiology Department of the University of Otago. It was based on the use of thermionic ‘valves’,and a double-beam oscilloscope having a linear time base and the facility for sweep expansion. In 1951this provided unique facilities for neurophysiological research. Three electrical pulses, independentlycontrolled for intensity, duration and timing along the oscilloscope sweep, were available to stimulate, viaisolation transformers, the same or different nerves. The prototype unit, constructed in Dunedin, waslater used by Eccles in Canberra from 1953 until 1966. The Industrial Development Department ofCanterbury University College in Christchurch undertook the mechanical design and construction of fourunits for the Department of Physiology of JCSMR; two were installed in the Department’s temporaryquarters in 1951–52, and all four in the new School building in 1957. The stimulator portion of one wasin continuous use in the Department of Pharmacology until late 1985, and provided the basis for thedesign and manufacture by A. Saeck of an entirely solid state stimulating unit which was used until 1995.

413

16. Neuroscience

inhibitory postsynaptic potentials. The timecourses of the transient ionic currents atinhibitory and excitatory synapses wereestimated in 1955 by Eccles, Coombs andDavid Curtis (see p. 75) using directlymeasured electrical properties of themotoneurone membrane. Subsequent re-examination between 1959 and 1962 of theionic processes contributing to the resting,action and synaptic potentials of motoneu-rones by Eccles and a number of colleagues,

including Masao Ito, using an extendedseries of anions and cations, essentially con-firmed Eccles’ earlier proposals regardingthe involvement of chloride and potassiumions at activated spinal inhibitory synapses.

An investigation in 1955 by Eccles,Coombs and Curtis led to the description ofthe components of orthodromic, antidromicand direct action potentials of alpha-motoneurones in terms of the morphologyof these cells. In 1956 Eccles, Rosamond

Left John Saxon Coombs (1917–93) received his BSc and MSc degrees from the University of Dunedinand was appointed to a Lectureship in the Department of Physics in that University. His involvement inneurophysiological research began in 1950 when asked by J.C. Eccles, then Professor of Physiology inthe University, to design and supervise the construction of electronic stimulating and recordingequipment having features which were then not provided by commercially available equipment. Thehighly successful production of the ‘ESRU’, and his close and continuing participation in the pioneeringintracellular recording from spinal motoneurones, led to his appointment as a Fellow in the JCSMRDepartment of Physiology in September 1952 (Electronics Engineer, 1961). In Canberra, from 1952 untilhis retirement in 1977, he continued to design and make innovative electronic equipment which providedspecial features of inestimable value to investigators in the Departments of Physiology andPharmacology.Right Masao Ito (1928–). Graduating MD (1958) and PhD (1959) from the University of Tokyo, Ito wasan ANU PhD Scholar from 1959 until 1961, and a Research Fellow until May 1962. He investigated,with Eccles and a number of colleagues, the biophysical properties of motoneurones and the ionicconductance changes underlying synaptic inhibition in the spinal cord. On his return to the Faculty ofMedicine, University of Tokyo, where he was successively Associate Professor (1963), Professor ofPhysiology (1970), and Dean (1986), he and his colleagues demonstrated that cerebellar Purkinje cellswere inhibitory neurones, and proposed that the transmitter released was GABA. From 1989 he was ateam leader, and from 1992, the Director General of the RIKEN Frontier Research Program. In 1997 hewas appointed the Director of the newly established RIKEN Brain Science Institute. Amongst hisnumerous awards are Foreign Membership of the Royal Society (1992) and the Japan Prize (1996).

414


Eccles (see p. 415) and Anders Lundberg(see p. 416) demonstrated that there are twosubclasses of spinal motoneurone, havingaxon conduction velocities and durations ofpost-action potential hyperpolarizationsappropriate to the innervation of fast or slowextensor muscles. Subsequently, in 1958Eccles, Rosamond Eccles and Arthur Bullershowed by section and cross union of the

nerves to fast and slow muscles that the typeof motoneurone innervating a muscle deter-mined its contractile properties. Later stud-ies from 1960 by Russell Close wereconcerned with the neural influence on theexcitation-contraction properties whichcontrol the speed of shortening of fast andslow muscle fibres.

Further Reading

Curtis, D.R. and Andersen, P. (2001). John Carew Eccles, 1903-1997. Historical Records ofAustralian Science, 13(4), 439-473.

Eccles, J.C. (1964). The Physiology of Synapses. Springer-Verlag, Berlin.

Organization of Spinal Excitatory and Inhibitory Pathways

by David Curtis

Two very significant investigations whichbegan in the Department of Physiology in1953 indicated that central neurones hadeither an excitatory or an inhibitory effect onother neurones. John Eccles, Paul Fatt andK. Koketsu investigated the recurrent inhibi-tion of motoneurones by antidromic volleysin the axons of other motoneurones. Thispostsynaptic hyperpolarization, which wasreduced by the convulsant alkaloid strych-nine, was found to involve the prior excita-tion of ventral horn inhibitory interneuronesby acetylcholine at synapses of theintraspinal collateral branches of the axonsof motoneurones. The inhibitory pathwaywas thus disynaptic, and the interneuroneswere called Renshaw cells in honour ofBirdsey Renshaw (1911–1948) who had ear-lier recorded their action potentials at theRockefeller Institute, New York.

This was the first direct demonstrationthat acetylcholine was a neurotransmitter in

the mammalian central nervous system.Furthermore, since the axons of motoneu-rones also release acetylcholine peripherallyat the neuromuscular junction, this studyalso provided support for the proposal in1935 by Sir Henry Dale (1875–1968) thatthe same transmitter is released at all of thesynapses made by the axon of a neurone.Studies of the electrophysiology of Renshawcells and of the distribution of recurrent inhi-bition in the lumbar spinal cord were subse-quently carried out by Eccles, RosamondEccles, Ainsley Iggo, Masao Ito (see p. 413)and Anders Lundberg.

In the initial study by Eccles, Fatt andKoketsu some compounds effective at thenicotinic receptors at the neuromuscularjunction had no action at the synapsesbetween motor axon collaterals and Renshawcells when administered systemically. Whenthese compounds were administered micro-electrophoretically (see p. 425) close to these

415

16. Neuroscience

cells, however, David Curtis and RosamondEccles demonstrated in 1957 that a blood-brain-barrier accounted for these apparentanomalies. A further microelectrophoreticinvestigation of acetylcholine receptors onRenshaw cells by Curtis and Ronald Ryallfrom 1962 indicated the presence of nicotinicand muscarinic sub-types, both of whichwere probably involved in the excitation ofthese neurones by impulses in axon collateralfibres under experimental conditions.

Also in 1953, Eccles, Fatt and SvenLandgren showed that another strychnine-sensitive inhibition in the spinal cord, the‘direct’ short-latency and short-durationhyperpolarizing inhibition of spinalmotoneurones by afferent volleys, regardedat the time to be monosynaptic, involved theprior excitation of an inhibitory interneu-rone located in the spinal intermediatenucleus, and was thus disynaptic. Confir-matory evidence was later provided byEccles, Ito and T. Araki in 1960. Accord-

ingly, the central synapses of primary affer-ent (dorsal root) fibres were established asexcitatory, and the inhibitory effects ofimpulses in such fibres were accepted asfollowing the excitation of relatively shortaxon inhibitory interneurones. In later stud-ies, Eccles and his collaborators identifiedand established the role of other inhibitoryinterneurones in the spinal cord, dorsal col-umn nuclei, thalamus, hippocampus andcerebellum (see p. 422).

An extensive study of the patterns ofdistribution of the excitation and inhibitionof individual spinal neurones, includingcells of origin of ascending pathways, byimpulses in various types of primary affer-ent fibre of muscle and cutaneous origincommenced in 1956, initially involvingEccles, Rosamond Eccles and Lundberg,and later numerous collaborators, includingOlaf Oscarsson, who had a particular inter-est in spino-cerebellar pathways.

Further Reading

Eccles, J.C. (1969). The Inhibitory Pathways of the Central Nervous System. Charles C. Thomas,Springfield, Illinois.

Lundberg, A. (1979). Multisensory control of spinal reflex pathways. Progress in Brain Research,50, 11-28.

Rosamond Margaret (Eccles) Mason (1929–).After graduating MSc at the University of Otago inDunedin, New Zealand, in 1951, RosamondEccles was an ANU Overseas PhD Scholar at theUniversity of Cambridge and joined theDepartment of Physiology in Canberra in 1954 tocomplete her PhD thesis research. As aResearch Fellow from 1955, Fellow 1962, shecontinued her investigation of transmissionthrough mammalian sympathetic ganglia, andwith a number of colleagues studiedinterneurones, including Renshaw cells, andmotoneurones involved in spinal reflexes. WithJ.C. Eccles and A.J. Buller she studied theproperties of motoneurones innervating fast andslow limb muscles, and later contributed to theinvestigation of presynaptic inhibition in the spinalcord. For family reasons she resigned in 1966,but maintained her research interests as a VisitingFellow until 1968. Photograph by David Coward.

416


In the 1950s, John Eccles and his colleaguesshowed that increases in membrane poten-tial (hyperpolarization) and conductanceaccounted for the synaptic inhibition ofspinal neurones (see p. 412). Inhibition inthe mammalian central nervous system thenbecame regarded as essentially a postsynap-tic phenomenon, by which transmittersreleased at inhibitory synapses reduced theexcitability of neurones. In 1957, however,Kay Frank and Michael Fuortes at the USNational Institutes of Health provided evi-dence for another type of spinal inhibition,in which a prolonged reduction of excitatorytransmission at synapses of primary afferent(sensory) fibres on motoneurones occurredin the absence of detectable changes inmotoneurone membrane potential and

excitability. One explanation of thisinhibitory process was a presynaptic reduc-tion of the release of excitatory transmitter,‘presynaptic’ inhibition.

From early 1960 until 1966 many mem-bers of the Department of Physiology, ledby Eccles, became engaged upon an inten-sive investigation of the organization andmechanism of this type of inhibition in thecat lumbar and cervical spinal cord and dor-sal column nuclei. The long latency andprolonged inhibition of monosynapticspinal reflexes by stimulating segmentalafferent fibres was found to be accompaniedby depolarization of the excitatory terminalsof the afferent fibres generating the reflex.This primary afferent depolarization wasreferred to as ‘PAD’. Since changes in the

Anders Lundberg (1920–). Before graduating MDat the University of Lund in 1947 and PhD inMedicine at the University of Stockholm in 1948,Anders Lundberg started his research career inLund in 1945, and moved to Stockholm in 1946 towork with Professor Ragnar Granit. He was aVisiting Investigator at the Rockefeller Institute,New York, from 1949 to 1950, and a SeniorResearch Fellow in the Department of Physiology,JCSMR, from January 1956 until October 1957.Prior to his period in Canberra he had usedintracellular microelectrodes to investigatesalivary glands, transmission in sympatheticganglia and in the dorsal spinocerebellar tract.Returning to Sweden, he was AssociateProfessor of Physiology in Lund 1959–1961,Professor of Physiology in Goteborg 1961–86,and since 1987 has been Professor Emeritus inGoteborg. In Sweden, Lundberg and hiscolleagues extensively investigated the functionalorganization of afferent, ascending anddescending pathways and interneuronesassociated with the reflex activity of themammalian spinal cord.

Presynaptic Inhibition and Transmission in the Somatosensory Pathway

by David Curtis

excitability of the postsynaptic neuronescould not be detected, the inhibitionappeared to be essentially presynaptic innature, and was considered to be an impor-tant negative feed-back control mechanismof the relay of somatosensory informationinto the spinal cord and to supraspinal cen-tres.

The types of muscle and cutaneousafferent fibre receiving and generating PADwere identified, as were the neuronal path-ways associated with both the production ofPAD, and the inhibition of spinal reflexesand of the relay of sensory information tosupraspinal centres. A number of studiesconcerned the mechanism by which PADdepressed transmitter release. In 1961,Eccles, Per Andersen and Thomas Searsdemonstrated that PAD was generated byimpulses in pathways descending fromhigher centres of the nervous system. Inaddition to its role in the lumbar and cervi-cal spinal cord, presynaptic inhibition wasalso shown to control the transmission ofimpulses in primary afferent dorsal columnfibres through the dorsal column nuclei butnot to be significant at the next relay in thesomatosensory pathway through the ven-trobasal thalamus.

Unlike postsynaptic inhibition in thespinal cord (see p. 412), presynaptic inhibi-tion of spinal reflexes and PAD were notreduced by the convulsant alkaloid strych-nine. Both the inhibition and PAD, how-ever, were found in 1961 by Eccles, RobertSchmidt and William Willis to be reducedby intravenous picrotoxin. Picrotoxin, aconvulsant without effect on strychnine-sensitive inhibition, was known to block theinhibitory effect of gamma-aminobutyricacid (GABA) in crustacea. Since this aminoacid had been detected by other investiga-tors in the mammalian brain and spinal cordin the mid-1950s, Eccles, Schmidt andWillis suggested that GABA could be thedepolarizing transmitter generating PAD inthe mammalian spinal cord.

Eccles had proposed in 1961 that PADresulted from the prolonged action of adepolarizing transmitter at synaptic contacts(axoaxonic synapses) on the terminal bou-

tons of primary afferent fibres. The reducedrelease of transmitter from these boutonswas attributed to a reduction of the ampli-tude of presynaptic action potentials, result-ing from both the depolarization and theunderlying increased membrane conduc-tance. In 1962, George Gray reported inNature that axo-axonic synapses occurred inthe mammalian spinal cord, and later stud-ies by others established that the postsynap-tic elements of these complexes were theterminals of primary afferent fibres. Thepresynaptic elements, terminals of spinalinterneurones, were also later shown tosynapse upon neuronal dendrites in a ‘triad’arrangement. The presence of these triadsprovided morphological support for severalsubsequent reports that ‘presynaptic’ inhibi-tion in the spinal cord was often accompa-nied by postsynaptic hyperpolarization ofmotoneurones at predominantly dendriticsynapses. Histochemical evidence alsobecame available that GABA was mostlikely the transmitter synthesised and storedwithin the presynaptic terminals of theseaxo-axonic/axo-dendritic triads.

In 1967, David Curtis and his colleaguesdemonstrated that GABA hyperpolarizedspinal motoneurones, an action not blockedby strychnine. Subsequently, in 1970, therole of this amino acid in spinal inhibitionwas supported by the finding of Curtis, Gra-ham Johnston and their colleagues that bothPAD and presynaptic inhibition of spinalreflexes were blocked by intravenous bicu-culline. This convulsant alkaloid, a specificantagonist of central GABA receptors nowrecognized as being of the GABA-A sub-type, also blocked the hyperpolarization ofcentral neurones by GABA (see p. 429).

It was not until 1976, however, that Cur-tis, with David Lodge and later other col-leagues in the Department of Pharmacology,combined microelectrophoretic (see p. 425)and electro-physiological techniques toinvestigate in vivo the pharmacological andbiophysical properties of single primaryafferent axons within the spinal cord. Directevidence was obtained for the first time thatGABA depolarized unmyelinated ‘termina-tions’ (presynaptic axons and synaptic bou-

417

16. Neuroscience

418


tons) of primary afferent fibres, but had noeffect on intraspinal myelinated axons.Depolarization by GABA was accompaniedby an increased membrane conductance,and was blocked by microelectrophoreticbicuculline, which also reduced synapticallygenerated PAD of terminations.

The actions of a series of GABA ana-logues on afferent terminations indicatedthat the bicuculline-sensitive receptors weresimilar to those at which GABA hyperpo-larized spinal and other central neurones byincreasing the permeability of the postsy-naptic membrane at inhibitory synapses tochloride ions. The higher intracellular con-centration of chloride in primary afferentneurones, and their central terminals, than inneurones having cell bodies within the cen-tral nervous system accounts for the oppo-site actions of GABA on the membranepotentials of these cells. Both a conduc-tance increase and depolarization of afferentterminal decreased the amplitude of presy-naptic action potentials, so reducing theinflux of calcium ions through presynapticvoltage-activated channels which initiatestransmitter release. The effect of smalldepolarization alone is probably minimal.

More recent investigations suggest thatthe role of GABA in spinal presynaptic inhi-bition may be more complex than activationof pre- and post-synaptic GABA-A recep-tors linked to chloride channels. In 1979,baclofen [beta-(4-chlorophenyl)GABA],which had been synthesised by CIBA in1962 as a potentially therapeutically effec-tive GABA analogue which penetrated the

blood-brain barrier, was shown by Curtis,Lodge and their colleagues to selectivelyreduce the release of excitatory transmitterfrom primary afferent terminations in themammalian spinal cord, an effect, however,not accompanied by depolarization and notblocked by bicuculline. In laboratoriesabroad, using tissue preparations in whichthe action of baclofen could be directlycompared with that of GABA in the pres-ence of bicuculline, both baclofen andGABA had been demonstrated to activate abicuculline-insensitive receptor, nowreferred to as the GABA-B sub-type. Later,in 1988, a study in Canberra indicated that areduction of the influx of calcium ionsthrough channels activated by presynapticaction potentials was the most likely expla-nation of the reduction by baclofen of trans-mitter release at primary afferentterminations.

Subsequently, in 1992, following thesynthesis by CIBA-GEIGY of selectiveGABA-B receptor antagonists which passthe blood-brain barrier, Curtis and GaryLacey found that these antagonists consider-ably attenuated the long latency and pro-longed ‘presynaptic’ inhibition of spinalreflexes. In contrast to bicuculline, how-ever, these compounds were not convulsantsand enhanced rather than reduced PAD.These observations raised as yet unan-swered questions about both the role of PADin the presynaptic inhibitory process and thefunctional significance of ‘presynaptic’inhibition in the spinal cord.

Further Reading

Curtis, D.R. and Lacey, G. (1998). Prolonged GABA-B receptor-mediated inhibition in the catspinal cord: an in-vivo study. Experimental Brain Research, 121, 319-333.

Eccles, J.C. (1969). The Inhibitory Pathways of the Central Nervous System. Charles C. Thomas,Springfield, Illinois.

Schmidt, R.F. (1971). Presynaptic inhibition in the vertebrate central nervous system. Ergebnisse derPhysiologie, 63, 20-101.

419

16. Neuroscience

Left Per Oskar Andersen (1930–) graduated MD from the University of Oslo in 1954, PhD in 1960. Heheld academic appointments in the Anatomical Institute of the University from 1951 until 1964, and fromNovember 1961 spent two years as a Rockefeller Visiting Fellow working with John Eccles and hiscolleagues studying presynaptic inhibition, synaptic mechanisms in dorsal column and thalamic nucleiand basket cell inhibition in the hippocampal and cerebellar cortices. From 1964 he was a member ofthe staff of the Neurophysiological Institute in Oslo, Associate Professor 1964–1972, Professor1972–1997, and since 1997 has been Senior Stipendiat in the Physiological Institute. His majorresearch interests include hippocampal neurobiology, the relation between structure and function ofcentral neurones, and the synaptic mechanisms associated with learning and memory. One of Europe’smost distinguished neuroscientists, he is a Member of a number of Academies, including the NorwegianAcademy of Science, Academia Europea, and the Royal Swedish Academy, and is a Foreign Associateof the US National Academy of Sciences.

Right Thomas A. Sears (1928–). From 1961–1963 Sears, a Lecturer in the ElectroencephalographyDepartment at the Institute of Neurology, London, was a Wellcome Fellow in the Department ofPhysiology JCSMR. In addition to pursuing his interests in the electrophysiology and connections ofthoracic respiratory neurones using intracellular recording techniques, he also worked with John Ecclesand Per Andersen on presynaptic inhibition in the spinal cord evoked by stimulating the cerebral cortexand on transmission through ventrobasal thalamic nuclei. This latter study led to the development of atheory to account for the phased discharge of cortical neurones which underlies the generation of theelectroencephalogram. His ANU PhD was awarded in 1964. Returning to the Institute of Neurology hecontinued to investigate the respiratory system and also undertook studies in experimentaldemyelination. In 1973 he was appointed a Professor at the Institute, and in 1975 Head of the newlyestablished Sobell Department of Neurophysiology, a position he held until retirement in 1993. AsProfessor Emeritus he is currently an Honorary Senior Research Fellow at King’s College London.

420


Left Robert Franz Schmidt (1932–) graduated MD from the University of Heidelberg in 1959 and was aPhD Scholar with John Eccles from 1960 to 1962 investigating presynaptic inhibition in the spinal cord.His ANU PhD was awarded in 1963, and following academic positions in the Institute of Physiology inHeidelberg he was appointed as Associate Professor in 1970. From 1971 until 1982 he was Professorand Director of the Institute of Physiology at the University of Kiel. Subsequently, from 1982 until 2000he was Professor and Director of the Institute of Physiology at the University of Wurzburg, and iscurrently a Professor Emeritus of that University and an Associate Investigator at the Institute ofNeuroscience, Univerisdad Miguel Hernandez, San Juan de Alicante in Spain. His research interestshave continued to be concerned primarily with sensory neurophysiology, and his contributions have beenrecognized by the award of Lectureships and Honorary Memberships by many learned societies. In1996 he received a DSc honoris causa from the University of New South Wales.

Right William Darrell Willis, Jr. (1934–) graduated MD from the University of Texas SouthwesternMedical School, Dallas in 1960, and his ANU PhD was awarded in 1963 on the basis of research relatedto presynaptic inhibition carried out with John Eccles from 1960 until 1962. After a postdoctoral year inPisa he returned to Dallas and was appointed Professor and Chairman of the Department of Anatomyfrom 1964–1970. He then moved to the University of Texas Medical Branch in Galveston as Chief,Laboratory of Comparative Neurobiology of the Marine Biological Institute, and has been Director of thatInstitute since 1978. Since 1986 he has also been Chairman of Anatomy and Neurosciences inGalveston. He has continued a research interest in spinal presynaptic inhibition, central pain pathwaysand the processing of nociceptive information.

421

16. Neuroscience

Left David Lodge (1941–) graduated BVSc from Bristol University in 1963, and after seven year’sinvolvement in veterinary clinical practice at the University’s Veterinary Hospital, undertook PhD studiesin the Department of Physiology, University of Bristol, graduating in 1974. In Canberra, from 1974–75as a Postdoctoral Fellow and 1975–79 as a Research Fellow, he collaborated with David Curtis in anumber of projects concerned with the central pharmacology of inhibitory and excitatory amino acids,including the role of GABA in spinal presynaptic inhibition. From 1979–91 he was successively SeniorLecturer, Reader, Professor and Head of the Department of Physiology at the Royal Veterinary College,London, investigating central excitatory amino acid receptors. In 1991 he joined Eli Lilly and Co as aResearch Scientist, and is currently Research Advisor and Director of that company’s Stroke ResearchProgram.

Right Gary Lacey (1960–) graduated BSc(Hons) at Chelsea College, University of London, in 1983, andgained a PhD degree in Pharmacology at the Royal Free Hospital School of Medicine, London, in 1986.He then spent two years at St Bartholomew’s Hospital Medical College, and was a Postdoctoral Fellowin the Division of Neuroscience, JCSMR, in 1989, and a Research Fellow from 1992 until his resignationin 1995. In the JCSMR he collaborated with David Curtis in investigating transmitter releasemechanisms in the mammalian spinal cord, including the mode of action of baclofen and the role ofGABA-B receptors in presynaptic inhibition. Since 1996 he has been a member of the Drug SafetyEvaluation Branch of the Therapeutic Goods Administration in Canberra.

422


Per Andersen’s (see p. 419) previous expe-rience in Oslo of investigating excitatorypathways in the hippocampal cortex led in1962 to a study with John Eccles and YngveLøyning of the large and prolonged chlo-ride-dependent inhibitory hyper-polariza-tions recorded from pyramidal cells in theCA3 region. Intracellular records frompyramidal cells, and extracellular records ofthe action potentials of interneurones, werecombined with a systematic depth analysisof extracellular potentials generated byantidromic and afferent volleys. This latteranalysis took advantage of the laminararrangement of synapses originating fromvarious sources. The study resulted in theidentification of basket cells, the axon ter-minals of which form a dense networkaround the soma of pyramidal cells, asinhibitory interneurones. Such a location ofinhibitory action would be the most effec-tive site for controlling the discharge ofaxonal impulses by pyramidal cells. Subse-quently, this very significant first correla-tion between the inhibitory function of aneurone and the arrangement and locationof its axonal terminations served as a guidefor the identification of other centralinhibitory neurones and their synapses.

The following year, Andersen, Ecclesand Paul Voorhoeve, using the same proce-dure, demonstrated that basket cells in thecerebellar cortex, which also has a laminararrangement of synapses and neurones,inhibited Purkinje cells through synapseslocated on the soma. This was followed byan intensive and systematic study, led byEccles and including Rodolfo Llinas, KazuoSasaki and Piergeorgio Strata, of the organi-zation and properties of all major types ofneurone in the cat cerebellar cortex, includ-ing their connectivity with other regions ofthe central nervous system and particularlyby spino-cerebellar and olivo-cerebellartracts. In this investigation, Eccles was

influenced by the Hungarian neu-roanatomist, Janos Szentagothai, whosedetailed knowledge of cerebellar micro-anatomy was unrivalled, and by the observa-tions of Masao Ito (see p. 413) who hadspent three years in Canberra from 1959.Later, in Tokyo, Ito and his colleagues hadshown that Purkinje cells were inhibitoryneurones, and, on the basis of antagonism byintravenous picrotoxin, proposed thatGABA was probably the inhibitory transmit-ter at synapses made by Purkinje cell axons.

The Canberra group established that inaddition to Purkinje and basket cells, super-ficial stellate and Golgi cells were alsoinhibitory neurones. Stellate cells inhibitedPurkinje cells, and Golgi cells had a stronginhibitory action at mossy fibre-granule cellsynapses of spino-cerebellar tracts to thecerebellar cortex. In contrast, granule cellsand neurones of the inferior olivary nucleuswere excitatory neurones, giving rise to par-allel and climbing fibres respectively, bothexciting Purkinje cells. The considerableconvergence of hindlimb muscle, joint andcutaneous afferent impulses on olivary neu-rones was also demonstrated. This study ofthe mammalian cerebellum was continuedby Eccles in the United States from 1967until his retirement in 1975, and overall, asthe first comprehensive analysis of the modeof operation of the main neurones of thecerebellar cortex, was a major contributionto the understanding of its functional role.

The proposal in 1961 by Eccles,Schmidt and Willis that GABA was likely tobe the transmitter of strychnine-insensitiveprolonged presynaptic inhibition in thespinal cord (see p. 417) led in 1963 to sev-eral investigations in the Department ofPhysiology of the effects of strychnine onthe prolonged postsynaptic hyperpolarizinginhibitions of neurones in the hippocampal,cerebellar and cerebral cortices and in thethalamus. All of these supraspinal inhibi-

Excitation and Inhibition in Hippocampal and Cerebellar Cortices

by David Curtis

423

16. Neuroscience

tions were found to be insensitive to strych-nine, suggesting that either the transmittersor the postsynaptic receptors involved dif-fered from those at strychnine-sensitiveinhibitory synapses in the spinal cord. Onthe basis of antagonism by bicuculline, Cur-tis, Graham Johnston and their colleagues

proposed in 1970 that GABA was the trans-mitter of supraspinal inhibitions not influ-enced by strychnine (see p. 429). Theseincluded basket cell inhibition of hippocam-pal pyramidal cells, Purkinje cell inhibitionof lateral vestibular nucleus neurones andbasket cell inhibition of Purkinje cells.

Further Reading

Andersen, P. and Eccles, J.C. (1965). Locating and identifying postsynaptic inhibitory synapses bythe correlation of physiological and histological data. Symposia Biological Hungarica, 5, 219-242.

Curtis, D.R. and Johnston, G.A.R. (1974). Amino acid transmitters in the mammalian centralnervous system. Ergebnisse der Physiologie, 69, 97-188.

Eccles, J.C., Ito, M. and Szentagothai, J. (1967). The Cerebellum as a Neuronal Machine. SpringerVerlag, New York.

Left Paul E. Voorhoeve (1927–) studied medicine and histology at the University of Amsterdam andphysiology at the University of Leiden (The Netherlands), graduating MD in 1954. After two year’s armymedical service he carried out electro-physiological research on fusimotor neurones with Professor YvesLaporte at Toulouse and received his PhD in 1959. He then spent two postdoctoral years with ProfessorAnders Lundberg in Lund, and was a Visiting Fellow in the Department of Physiology, JCSMR from 1962to 1964 collaborating with John Eccles on the cellular physiology of the cerebellar cortex. From1965–1990 he was Professor of Neurophysiology at the University of Amsterdam.

Right Rodolfo Llinas received his MD from Universidad Javeriana in Bogota, Colombia in 1959, and wasa Research Fellow in the Department of Neurosurgery, Massachusetts General Hospital, HarvardMedical School (1960–61), and in the University of Minnesota (1961–63). From 1963, as an ANUResearch Scholar, he carried out research with John Eccles on the cellular physiology of the cerebellarcortex and was awarded his PhD in 1966. From 1976 he has been Chairman of the Department ofPhysiology and Neuroscience, Thomas and Suzanne Murphy Professor of Neuroscience, at the NewYork University School of Medicine, Manhattan, New York. He was elected to the US National Academyof Sciences in 1986.

424


Left Kazuo Sasaki (1929–) graduated MD in 1954 at the Faculty of Medicine, Kyoto University, andgained his PhD in 1959 from the Graduate School of Medicine of that University. Following appointmentsin the Department of Physiology in Kyoto, he was a Research Fellow in the Department of Physiology,JCSMR, from 1963 until 1966, carrying out research with John Eccles on the cellular physiology of thecerebellar cortex. From 1970–93 he held a Professorship at Kyoto University, within the Institute forBrain Research (1970–90) and the Graduate School of Medicine (1990–93). In 1993 he joined theNational Institute of Physiological Science, in Okasaki, and has been Director-General since 1997. In1998 he was awarded a Prize of the Japan Academy.

Right Piergiorgio Strata (1935–) graduated MD at the University of Pisa in 1960, and commenced hisresearch career in the University’s Department of Physiology with the distinguished neurophysiologistProfessor G. Moruzzi. For a year from August 1965 he was a Visiting Fellow with John Eccles inCanberra, and then for a year in Chicago, investigating excitatory and inhibitory interneurones and theirconnections in the cerebellar cortex. In 1967 he was appointed Associate Professor of Physiology inPisa, and, in 1975 Professor of Physiology. Later he was appointed Professor of Neurophysiology in theMedical Faculty of the University of Turin, and is currently a member of the Department of Neuroscience,Rita Levi Montalcini Center for Brain Research of the University of Turin. President of the ItalianNeuroscience Society (1988–90), Vice-President of the European Neuroscience Society (1988–92), hecontinues to hold senior positions in IBRO and the Human Frontier Science Program, and has beenawarded numerous prizes for his contributions to central nervous system neurophysiology.

425

16. Neuroscience

Considerable difficulties exist in ascribingthe effects on central nervous system neu-rones in vivo of compounds administeredintravenously, intra-arterially, topically orby pressure injection, to the activation ofspecific receptors at synapses on the neu-rones under observation. Hence techniqueswere developed from 1957 by David Curtis(see p. 75) to combine the recording ofextra- or intra-cellular potentials from singleanatomically and physiologically identifiedcentral neurones whilst administering com-pounds of pharmacological interest directly

into their immediate vicinity. This tech-nique was developed as a modification ofthat previously used by others to administeracetylcholine and other agents close to neu-romuscular junctions of isolated musclepreparations. Rather than using pressure toeject pharmacolgically active compoundsfrom solutions within fine glassmicropipettes, electrical currents were usedto control both the leakage and the ejectionof active cations or anions from aqueoussolutions of appropriate salts.

Skilled glass blowers in the School

Microelectrophoresis

by David Curtis

Left Teun Van Arkel, photographed in 1981, fusing six glass tubes around a central tube to produce aseven barrel glass microelectrode blank.Right A seven barrel glass microelectrode blank held in the heating coil of an electrode puller by twochucks, the upper fixed and the lower attached by a vertical rod to the plunger of a solenoid. The meltingglass is initially drawn out slowly over a pre-set distance by gravity, and then more rapidly when thesolenoid is turned on.

426


Workshop, including G. Allen, L. Vande-neer, T. Van Arkel and L. Wells, developedtechniques to fuse as many as six glass tubestogether around a central tube. The result-ant seven-barrel micropipette blank wasthen drawn out using a vertical microelec-trode puller designed and constructed in theSchool. The glass, melted in an electricallyheated coil was initially pulled slowly bygravity, and then more rapidly by a solenoidto produce a long tapered shaft with a tipless than one micrometre in overall diame-ter. After the tip had been ground back to anexternal diameter of 6-8 micrometres, theindividual barrels (orifice diameters 1-2micrometres) were filled from the top withappropriate solutions, and the assembly thencentrifuged to drive the solutions to the ori-fices of every barrel. Since the central bar-rel was used as an extracellular recordingmicroelectrode, the assemblies were calledmultibarrel microelectrodes, and the tech-nique microelectrophoresis (or microion-tophoresis).

The central barrel could also be used asa stimulating microelectrode, using briefcurrent pulses of the order of 1-2 microampsto measure the excitability of central nervefibres, synaptic terminations and neuronesin the immediate vicinity of the electrodetip. For intracellular recording a separatesingle or double barrel glass recordingmicroelectrode was cemented along the sideand parallel to the multibarrel assembly, andprojecting beyond its tip by 40-100micrometres in order to impale a single neu-rone.

Standard anatomical and neurophysio-logical procedures were used to locate andidentify particular types of central neurone,and to record and process extracellular andintracellular potentials. The major signifi-cance of the introduction of seven-barrelmicroelectrodes in Canberra was the oppor-tunity to examine the actions of as many assix different agents on a single neurone. Itthus became possible to mimic, antagonizeor enhance the actions of synapticallyreleased transmitters. Useful informationwas provided for identifying the transmitterreleased by known excitatory or inhibitory

pathways, for determining the nature of theneuronal postsynaptic receptors involved,the changes induced in the postsynapticmembrane, the processes by which releasedtransmitter was inactivated and the mecha-nism of transmitter release.

This new method of investigating thepharmacology of central synapses in vivowas used initially by Curtis and RosamondEccles in 1957 to confirm the cholinergicnature of the excitation of Renshaw cells byimpulses in motor axon collateral fibres (seep. 414). Subsequently, it was used in theinvestigation of other potential central neu-rotransmitters, including amino acids, (seep. 427), and was rapidly taken up by manylaboratories abroad.

The major limitation of this technique isthe inability to determine the extracellularconcentrations of administered compoundswhich are achieved in nervous tissue. Fur-thermore, since administration occurs fromvirtually a point source, concentrations arehighest close to the pipette orifice and lowerfurther away, depending on distance, diffu-sion in the complex extracellular space andthe processes which inactivate a particularsubstance. Thus, when extracellular actionpotentials are being recorded by an elec-trode close to the soma of a neurone, theconcentration of an administered compoundmay be too low to influence receptors ondistal dendrites. This problem may be over-come by using two multibarrelmicropipettes, either firmly attached with apreselected tip spacing or separately manip-ulated (see p. 434). The latter procedure hasalso been used to examine the synaptic con-nections between neurones, onemicropipette being used to record from andexcite one type of cell and the other todetect the subsequent excitation or inhibi-tion of another type. In studies of thisnature, the use of an excitatory amino acidto excite neurones provided a considerableadvantage over electrical excitation, sinceexcitatory amino acids, unlike electricalpulses, do not excite axons passing throughthe region of interest.

427

16. Neuroscience

Elucidation of the nature of the transmitterreleased at identified central excitatory andinhibitory synapses is essential for a fullunderstanding of the transmission process.Additionally, such knowledge enables therational development of therapeutic agentswith specific effects at particular synapsesfor treating neurological disorders associ-ated with defective transmission. Knowl-edge of transmitters is also of value inaccounting for the mode of action of knowndrugs and toxic agents influencing the cen-tral nervous system.

A critical observation influencing thisinvestigation was the earlier finding by JohnEccles and his colleagues that the convul-sant alkaloid, strychnine, blocked the post-synaptic action of the inhibitory transmitterproducing ‘direct’ and recurrent inhibitionof spinal motoneurones, inhibitions associ-ated with membrane hyperpolarization (seep. 412). Other short latency and durationspinal postsynaptic inhibitions studied laterby David Curtis were also reduced bystrychnine.

The appointment in 1958 of JeffreyWatkins, an organic chemist, initiated a pro-longed collaborative investigation whicheventually resulted in general acceptancethat simple amino acids were the majorexcitatory and inhibitory transmitters in themammalian central nervous system. Micro-electrophoretic techniques (see p. 425) wereused to determine the effects of compounds

known to be present in brain and spinal tis-sue on physiologically identified spinal, andlater other central, neurones. This techniquehad demonstrated that acetylcholine, widelyregarded at the time as possibly an impor-tant central transmitter, did not influence theexcitability of spinal neurones other thanRenshaw cells (see p. 415).

Reports of both the inhibitory effect incrustacea of gamma-aminobutyric acid(GABA), and the unique presence of thisamino acid in mammalian brain tissue, ledCurtis, John Phillis and Watkins to examinethe effects of GABA and related aminoacids on neurones in the cat spinal cord.GABA (and glycine) depressed neuroneexcitability, but hyperpolarization was notdetected and the depressant action of GABAwas not reduced by intravenous strychnine.Picrotoxin, a GABA antagonist in crustacea,is not ionized in solution and could not beshown in microelectrophoretic experimentsto reduce the depressant action of GABA onneurones. Later, the reduction of spinal‘presynaptic’ inhibition in the cat by intra-venous picrotoxin suggested a role forGABA in this type of strychnine-insensitiveinhibition (see p. 417).

In marked contrast to GABA, the acidicamino acids aspartic and glutamic, alsopresent in central nervous tissue, depolar-ized and excited neurones. These observa-tions resulted in an intensive investigationof the effects on spinal and other central

Further Reading

Curtis, D.R. (1964). Microelectrophoresis. In Physical Techniques in Biological Research, Vol. 5,(W.L. Nastuk, ed.), Academic Press, New York, pp. 144-190.

Duggan, A.W. (1982). Microiontophoresis and the central nervous system. Journal ofElectrophysiological Techniques, 9, 101-112.

Identification of Central Amino Acid Neurotransmitters

by David Curtis

neurones of numerous structurally relatedbasic and acidic amino acids, some of whichwere synthesised by Watkins, and in pro-posals about the possible nature of centralamino acid receptors. No evidence wasobtained, however, that these naturallyoccurring depressant and excitatory aminoacids were inactivated by extracellularenzymic degradation, as had been demon-strated for acetylcholine. In view of this,and the failure to detect a hyperpolarizingaction of GABA, amino acids were consid-ered by the Canberra group in the early1960s as unlikely to be central transmitters.A particularly significant finding, however,when the structure/activity relationships ofexcitant amino acids were further explored,was the very potent activity of N-methyl-D-aspartic acid (NMDA), synthesised byWatkins, and later to be of considerableimportance in the identification of subtypesof excitatory amino acid receptor.

Late in 1966, Curtis, Graham Johnston,also an organic chemist who was appointedin 1965 to the position previously occupiedby Watkins, and colleagues began a reinves-tigation of the role of amino acids as centraltransmitters. This followed the proposal byan American group that glycine was a spinalinhibitory transmitter, based on the associa-tion of this amino acid with spinal interneu-rones and the observation that glycinehyperpolarized motoneurones. In Canberra,an improved technique was developed tocombine intracellular recording with extra-cellular microelectrophoretic administrationof amino acids. Both glycine and GABAwere shown to hyperpolarize spinalmotoneurones with an increase in membraneconductance similar to that produced byspinal inhibitory transmitters. Furthermore,in contrast to GABA, the inhibitory action ofglycine was selectively and reversibly antag-onized by microelectrophoretic strychnine.On the basis of this antagonism, depressantamino acids were classifed as either glycine-like or GABA-like, and, of the former, theneurochemical evidence strongly favouredglycine as the transmitter at strychnine-sen-sitive inhibitory synapses of spinal ‘glycin-ergic’ interneurones. This convincing

evidence for an inhibitory transmitter func-tion of glycine, together with an increasingacceptance that carrier-mediated transportinto neurones and glial cells was primarilyresponsible for maintaining low extracellu-lar levels of amino acids rather thanenzymic inactivation, further stimulated astudy of central excitatory and inhibitoryamino acid receptors.

This was based on the use of compoundshaving specified stereochemical, conforma-tional and electronic parameters, whichselectively mimicked, antagonized orenhanced synaptic transmission. Informa-tion was thus obtained about the optimalrequirements for interaction with receptorsand inactivation processes at synapses stud-ied in vivo, and about membrane bindingsites, transport systems and metabolizingenzymes studied in vitro, for particularamino acids. Since both GABA and glu-tamic acid are flexible molecules, the find-ing in 1968 that the heterocyclic isoxazolesmuscimol and ibotenic acid had potent andprolonged depressant and excitant effectsrespectively led to the design, synthesis andevaluation of the actions of numerous con-formationally restricted analogues ofGABA and glutamic acid.

From 1968 until his resignation in 1980,Johnston and his colleagues synthesisedmany new compounds for these in vivo andin vitro investigations. Collaboration from1974 until 1992 with Povl Krogsgaard-Larsen and his colleagues at the Royal Dan-ish School of Pharmacy in Copenhagen alsogenerated many novel and pharmacologi-cally significant compounds. New agentswere developed having selective effects onsubtypes of receptors and transporters,many of which became available commer-cially. The discovery by Krogsgaard-Larsen and Johnston of nipecotic acid as aninhibitor of GABA transport resulted in thedevelopment in Europe of a new class ofanticonvulsant drug.

A major breakthrough regarding thepossible structure of GABA receptor antag-onists followed Johnston’s analysis of thestructural features of strychnine and otherglycine antagonists considered to be essen-

428


tial for interaction with glycine receptors atinhibitory synapses. As a consequence, in1970, a number of tetrahydroisoquinolineswere tested, of which bicuculline was themost potent convulsant. When adminis-tered electrophoretically, bicucullinereversibly antagonized the depression ofspinal interneurone firing by GABA-likeamino acids, including muscimol, with littleor no reduction of the actions of glycine-likeamino acids. The first report of this selec-tive antagonism was published in Nature onJune 27 1970, having been submitted onMay 19 by Johnston, Curtis, Arthur Dugganand Dominik Felix. By the end of that year,bicuculline had been shown to block strych-nine-insensitive synaptic inhibitions in thethalamus, the lateral vestibular nucleus, thehippocampal, cerebral and cerebellar cor-tices, the olfactory bulb and ‘presynaptic’inhibition in the spinal cord. In combina-tion with neurochemical studies of the pres-ence of GABA and its synthesising enzyme,glutamic acid decarboxylase, bicucullinethus became a valuable pharmacologicaltool with which to identify central inhibitoryinterneurones which released GABA as atransmitter.

In 1972 Johnston prepared the more sol-uble and stable bicuculline methochloride,still widely used as an antagonist of GABAreceptors now defined as the GABA-A sub-type. Many other convulsants were studiedin Canberra and some, including ben-zylpenicillin, were selective GABA-Aantagonists. Others were glycine antago-nists. Later, two subtypes of bicuculline-insensitive GABA receptor were defined,GABA-B, activated by (-)-baclofen [(-)-beta-(4-chlorophenyl) GABA], for whichspecific antagonists and agonists were sub-sequently developed elsewhere (see belowand p. 418), and GABA-C activated by cis-4-aminocrotonic acid, a conformationallyrestricted analogue of GABA synthesisedby Johnston and his colleagues in 1974.

Numerous GABA analogues and othercompounds were tested in vitro as inhibitorsof high and low affinity GABA transportprocesses. Although some of the mosteffective, including (-)-nipecotic acid,

enhanced and prolonged the inhibition ofneuronal firing by microelectrophoreticGABA in vivo, a direct effect on GABA-mediated synaptic inhibition could not bedemonstrated. Anaesthetic barbiturates,however, which increase and prolongsynaptic inhibitions mediated by GABA,were shown by David Lodge and Curtis in1977 to increase and prolong the inhibitoryeffect of GABA but not that of glycine onspinal neurones. This effect, unrelated toinhibition of GABA transport, was laterestablished to be a direct action of barbitu-rates and other anaesthetics in the vicinity ofGABA-A receptors.

In 1954, Eccles, Curtis and VernonBrooks had found that the convulsant neu-rotoxin of Clostridium tetani blocked shortlatency and duration strychnine-sensitiveinhibitions of motoneurones. Later, in1971, Curtis, Felix, Christopher Game andRoy McCulloch demonstrated that tetanustoxin also blocked the longer latency andduration bicuculline-sensitive ‘presynaptic’inhibition of spinal reflexes and the accom-panying PAD (see p. 416). Furthermore,this toxin also blocked GABA-mediatedbasket cell inhibition of Purkinje cells in thecerebellar cortex. In contrast, however, tothe selective antagonism by strychnine andbicuculline at postsynaptic glycine andGABA receptors respectively, tetanus toxindid not significantly reduce the inhibitoryactions of these amino acids on spinal andPurkinje cells. Accordingly, the convulsantaction of tetanus toxin was proposed toresult from a presynaptic reduction of therelease of glycine and GABA at inhibitorysynapses.

Recognition of the transmitter functionof glycine and GABA, and of the impor-tance of cellular uptake in removing aminoacids from the extracellular medium, alsoled to renewed interest in Canberra in aspar-tic and glutamic acids as central excitatorytransmitters. This was accompanied andmade possible by the synthesis of many newcompounds by Johnston, and later byKrogsgaard-Larsen, and their colleagues. In1971, the ionic mechanism of the depolar-ization of motoneurones by aspartic and

429

16. Neuroscience

glutamic acids was shown to be similar tothat of synaptic excitation. Although sev-eral of a large number of analogues andother compounds tested at this time reducedthe sensitivity of neurones to both aminoacids, a search for a selective antagonist foreither naturally occurring amino acid wasnot successful.

Neurochemical evidence provided byJohnston, Duggan and investigators else-where was consistent with glutamic acidbeing the transmitter of dorsal root primaryafferent (sensory) fibres and aspartic acidthat of intraspinal excitatory interneurones.This proposal was supported by Duggan’ssubsequent finding that spinal interneuronesexcited monosynaptically by afferentimpulses were significantly more sensitiveto L-glutamate than to L-aspartate. In con-trast, the reverse was observed for cellsexcited polysynaptically but not monosy-naptically by afferent impulses. Greater dif-ferences in sensitivity were observed in1973–74 using kainic acid, a conformation-ally restricted analogue of L-glutamic acid,and NMDA, and Johnston proposed thatkainic acid and NMDA were selective ago-nists of ‘glutamate-’ and ‘aspartate-prefer-ring’ receptors respectively. By the late1970s, the existence of multiple excitantamino acid receptors was clearly becomingapparent, receptor classification dependingon activation by NMDA, kainic acid,quisqualic acid or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA), and eventually on the increasingavailability of selective antagonists.AMPA, structurally related to ibotenic acid,had been synthesised by Krogsgaard-Larsenand shown in Canberra in 1980 to be apotent excitant of central neurones.

By this time, many groups of investiga-tors abroad were becoming involved in thiscomplex field of central excitant amino acidreceptors, including Watkins, who played amajor role. Although there was interest indiscovering specific antagonists for deter-mining the nature of excitant amino acidreceptors at particular excitatory synapses,there was the added attraction to pharma-ceutical companies of the potential thera-

peutic use of antagonists, particularly asanticonvulsants and as ‘neuro-protective’agents. Potent excitant amino acids, such askainic acid, had been shown under experi-mental conditions to destroy central neu-rones but not nerve fibres or synapticterminals in vivo. Subsequent in vitro and invivo studies led to proposals that excessiverelease of endogenous glutamic acid, as dur-ing cerebral hypoxia or trauma, or the inges-tion of excitatory amino acids able to crossblood-brain-barriers, resulted in the destruc-tion of central neurones. Such ‘excitotoxic-ity’ may be important as an aetiologicalfactor in some ‘neurodegenerative’ disor-ders and in increasing the amount of neu-ronal damage produced by trauma or astroke. As expected, however, from the par-ticipation of amino acid excitatory transmit-ters widely throughout the central nervoussystem, successful clinical use of antago-nists has yet to be achieved, psy-chotomimetic activity being a major sideeffect.

Following Johnston’s appointment to theChair of Pharmacology in the University ofSydney in 1980, the reduced ‘amino acidgroup’ retained an interest in excitant aminoacid neuropharmacology, and collaboratedwith chemists in Australia and abroad, par-ticularly with Krogsgaard-Larsen. Theexpanding interests internationally in thisfield, however, led to the decision to con-centrate upon the complex role of GABA inspinal ‘presynaptic’ inhibition (see p. 417).In 1980, on the basis of the similar effects ofGABA and the GABA analogue (-)-baclofen on peripheral tissue preparations,Norman Bowery and his colleagues in theUnited Kingdom had classified GABAreceptors as GABA-A, blocked by bicu-culline and not activated by baclofen, andGABA-B, insensitive to bicuculline andactivated by GABA and baclofen. Earlier,in 1973, bicuculline had been shown inCanberra not to diminish two central effectsof baclofen: the reduction of excitability ofspinal and other central neurones, and thereduction of the release of excitatory trans-mitter from the terminals of primary affer-ent fibres in the spinal cord. No selective

430


431

16. Neuroscience

GABA-B receptor antagonists were thenavailable, however, with which to elucidatethe functional significance of these recep-tors.

The demonstration in the late 1970s byWatkins and his colleagues that omega-phosphonic analogues of glutamic acid wereselective NMDA antagonists led in 1986 tothe synthesis by David Kerr, Rolf Pragerand colleagues in Adelaide of the phos-phono derivative of baclofen (phaclofen),and later a sulphonic derivative (2-hydroxy-sulphonic acid). Both were weak baclofenantagonists in the peripheral nervous sys-tem, and when tested in Canberra reversiblyblocked the presynaptic effect of baclofenon transmitter release in the spinal cord.Baclofen, a GABA analogue which pene-trates the blood-brain-barrier, had been ini-tially synthesised in 1962 by CIBA in Basleand had been used clinically for many yearsto alleviate spasticity of spinal origin inhumans. The company had retained aninterest in developing agonists and antago-nists. Hence, reports in the early 1990s bychemists at CIBA-GEIGY of the synthesisof a series of baclofen agonists and antago-nists, which were phosphinic acid analoguesof GABA, were of considerable interest.Curtis and Gary Lacey confirmed the ago-nist or antagonist actions of many of this

series at spinal GABA-B receptors, includ-ing those on the terminals of primary affer-ent fibres (see p. 418). Differences werefound in vivo between spinal andsupraspinal receptors which suggested theexistence of several subtypes of centralGABA-B receptor, the functional signifi-cance of which have yet to be established.

Beginning in 1958, investigators in theDepartments of Physiology and later Phar-macology introduced and developed micro-electrophoretic techniques to study centralamino acid neurotransmission at a cellularlevel in the mammalian central nervous sys-tem in vivo, and pioneered the use of numer-ous compounds, many of which weresynthesised expressly for a specific purpose.Apart from glycine, GABA, strychnine,bicuculline, bicuculline methochloride,aspartic and glutamic acids, key compoundsof invaluable practical neuropharmacologi-cal importance include: AMPA, cis- andtrans-4-aminocrotonic acid, NMDA, threo-3-hydroxy-L-aspartic acid, baclofen andbaclofen antagonists, isoguvacine, D- andL-homo cysteic acid, ibotenic acid, kainicacid, dihydrokainic acid, muscimol,nipecotic acid, quisqualic acid, L-serine-O-sulphate, 3-aminopropanesulphonic acid,piperidine-4-sulphonic acid and 4-amino-tetrolic acid.

Further Reading

Chebib, M and Johnston, G.A.R. (2000). GABA-Activated ligand gated ion channels: Medicinalchemistry and molecular biology. Journal of Medicinal Chemistry, 43, 1427-1447.

Curtis, D.R. and Johnston, G.A.R. (1974). Amino acid transmitters in the mammalian centralnervous system. Ergebnisse der Physiologie, 69, 97-188.

Froestl, W. and Mickel, S.J. (1996). Chemistry of GABA-B modulators. In The GABA Receptors,2nd edition (S.J. Enna and N.G. Bowery, eds.), Humana Press, New Jersey, pp. 271-296.

Meldrum, B. and Garthwaite, J. (1990). Excitatory amino acid toxicity and neurodegenerativedisease. Trends in Pharmacological Sciences, 11, 379-387.

Watkins, J.C. (2000). L-Glutamic acid as a central neuro-transmitter: Looking back. BiochemicalSociety Transactions, 28, 297-310.

432


Left Jeffrey Clifton Watkins (1929–) graduated BSc(Hons) in 1950 from the University of WesternAustralia, and gained a PhD in Organic Chemistry at the University of Cambridge in 1954. Afterpostdoctoral research at Cambridge and Yale Universities, he was appointed to a Research Fellowshipin the Department of Physiology JCSMR in January 1958 (Fellow 1961). Until 1965 he worked withDavid Curtis on the identification of excitatory and inhibitory amino acids as central transmitters,synthesising many new compounds, and then moved to the United Kingdom. At the ARC Institute ofAnimal Physiology he worked on model biological membranes, and from 1968–73 renewed his interestsin central neurotransmission at the MRC Neuropsychiatry Unit at Carshalton. From 1973 until hisretirement in 1999, he took up a series of appointments in the Departments of Physiology andPharmacology in the School of Medical Sciences of the University of Bristol (Honorary Professor 1989)where he concentrated his research on establishing and elucidating the central transmitter role of L-glutamic acid, mainly by developing specific ligands for glutamate receptor sub-types. Elected FRS in1988, he was awarded the Thudichum Medal by the Biochemical Society (UK) in 2000. He continues tomaintain close links with excitatory amino acid research in Bristol.

Right Graham Allan Ross Johnston, AM (1939–) graduated BSc(Hons) in 1959 from the University ofSydney, and gained a PhD in Organic Chemistry at the University of Cambridge in 1964. Afterpostdoctoral research at the University of California in Berkeley he was appointed to a ResearchFellowship in the Department of Physiology JCSMR in 1965 (Fellow 1968, Senior Fellow 1972). Hecollaborated with David Curtis on the neuropharmacology and neurochemistry of central amino acidtransmitters until 1980 when he was appointed to the Chair of Pharmacology at the University of Sydney.There he established the Adrien Albert Laboratory of Medicinal Chemistry and continued chemicalstudies on GABA and glutamic acid as neurotransmitters, developing specific ligands for subtypes ofamino acid receptors and transporters. Elected to Fellowship of the Australian Academy ofTechnological Science and Engineering in 1993, he is the recipient of a number of medals, and in 1998his services to bio-organic chemistry and pharmacology, to scientific organizations and to science policydevelopment was recognized by appointment as a Member in the Order of Australia.

433

16. Neuroscience

From 1973, Arthur Duggan, together withJohn Hall, Max Headley, Bernadette Grier-smith and Steve Johnston, investigated theeffects of morphine on neurones of centralpathways involved in the processing ofnociceptive (pain) information with the aimof understanding how opiates relieve pain.They rapidly confirmed previous work thatintravenous morphine reduced the excita-tion of neurones in the dorsal horn of thelumbar spinal cord of anaesthetised animalsby peripheral stimuli considered to bepainful, but the results of administeringmorphine microelectrophoretically near thesame neurones were puzzling. Under theselatter conditions morphine was a weakdepressant of neuronal firing, but this effect

was not mediated by opiate receptors since,unlike the analgesic effect of intravenousmorphine, it was not reversed by the opiateantagonist naloxone.

When multibarrel micropipettes are usedto both record neuronal activity and admin-ister pharmacologically active compounds,the tips need to be positioned close to thebodies of the neurones of interest (see p.426). Hence Duggan and his colleaguesdecided to explore the possibility that opiatereceptors were located on or near the dor-sally directed distal dendrites in superficialspinal laminae of neurones with cell bodieslocated deeper in laminae IV and V. Twoindependently manipulated multibarrelmicropipettes were used; one positioned to

Povl Krogsgaard-Larsen (1941–) graduated fromthe Royal Danish School of Pharmacy in 1967and received his PhD in 1970, DSc in 1980. Hewas appointed an Associate Professor in theSchool of Pharmacy in 1970, and since 1986 hasbeen Professor of Medicinal Chemistry. Hisinterests in the structure/activity relationships ofneurotransmitter amino acid analogues led to hisappointment as a Visiting Fellow in the JCSMRDepartment of Pharmacology in 1974, 1977, 1981and 1987 and collaboration with Curtis, Johnstonand their colleagues in the testing of a very largenumber of new and novel compoundssynthesised in Copenhagen. He is the recipientof numerous prizes and awards, several HonoraryDoctorates, is a member of a number of learnedAcademies including the Danish Academies ofNatural and Technical Sciences, and has been amember of the Board of Carlsberg A/G and of theBoard of Directors of the Carlsberg Foundationsince 1993. He has a particular interest inencouraging the commercialisation oftherapeutically effective compounds.

Morphine, Neuropeptides and Pain

by Arthur Duggan

record extracellular neuronal action poten-tials in laminae IV and V and the other withits tip more dorsally in a superficial laminaof the spinal cord, the substantia gelatinosa.The neurones were excited alternately byperipheral cutaneous stimulation, noxiousby localized heat and innocuous by mechan-ical deflection of hairs.

These experiments were immediatelysuccessful; morphine administered in thesubstantia gelatinosa powerfully and selec-tively reduced or abolished the excitation ofdeeper cells by the noxious stimuli. Thisaction was reversed by naloxone adminis-tered locally into the substantia gelatinosaor intravenously. Furthermore, naloxoneadministered after morphine often producedhyperexcitability of the neurones, suggest-ing that the process of dependence on mor-phine had commenced. The opioid peptidemethionine enkephalin had the same selec-tive depressant action as morphine. Muchsubsequent work showed that the opioidreceptors in the substantia gelatinosa are

located on small intrinsic neurones of thislamina, which synapse with the dendrites ofneurones in deeper laminae, and on thepresynaptic terminations in this area of cuta-neous nociceptive afferent fibres.

In the same year (1976) that this workwas published in Nature, a publication fromthe laboratory of T.L. Yaksh in the UnitedStates reported that the intrathecal adminis-tration of morphine around the caudal spinalcord of rats produced analgesia restricted tothe hind limbs. Together with the investiga-tion in Canberra, this established a powerfulcase for the clinical use of epidural andintrathecal morphine in humans. This tech-nique soon proved to be very effective, andcontinues to be used for a variety of pur-poses including cancer pain, neuropathicpain and during surgical procedures.Sophisticated catheters and pumps havebeen developed so that patients retainmobility. Relatively high doses can be usedat the spinal level without the more generaladverse side effects which accompany thesystemic administration of high doses ofmorphine.

This study of the action of morphine ledDuggan to investigate other aspects of thephysiology and pharmacology of pain.Although there was good evidence that L-glutamic acid was the transmitter released atthe first central synapse of rapidly conduct-ing primary afferent fibres from skin andmuscle (see p. 430), there were suggestionsthat the transmitter of slowly conductingfibres which convey nociceptive informa-tion was the neuropeptide, Substance P. Atthat time, the techniques used for collectingand identifying transmitters released in thecentral nervous system were relativelycrude. A perfusing solution was eitherapplied to the surface or passed through atube inserted into the brain or spinal cord.An entirely new and sophisticated approachwas developed in 1985 by Duggan, IanHendry (see p. 441) and Robert Morton, inwhich the outer surface of fine glassmicropippetes was coated with antibodies toa neuropeptide of interest using the methodsof enzyme immobilization. When insertedinto the central nervous system a proportion

434


Diagrammatic partial cross section of spinal cordshowing the arrangement of micropipettes torecord the activity of single neurones in dorsalhorn laminae IV and V (A) and to administercompounds microelectrophoretically moredorsally in the substantia gelatinosa (B). Theterminals of a primary afferent fibre are shownsynapsing with the dendrites of the deeperneurone.

435

16. Neuroscience

of synaptically released neuropeptide mole-cules bound at discrete sites on the surfaceof these antibody microprobes. These sites,subsequently detected as deficits in autora-diographs of the probes following incuba-tion in a solution of a radiolabelled form ofthe particular peptide, were readily relatedto specific regions in histological prepara-tions of the tissue under examination. Acomputerized digitizing procedure wasdeveloped to average the autoradiographs ofa number of microprobes which had beeninserted into similar regions of the spinalcord and subjected to the same peripheralphysiological stimuli.

Duggan, together with Zhi-Qi Zhao andWilliam Hutchison, soon showed thatsevere peripheral noxious stimuli releasedSubstance P in the substantia gelatinosa,

and that it was derived from the terminals ofnociceptive afferent fibres. Subsequentinvestigations demonstrated that inflamma-tion of peripheral tissues caused a massiverelease of the neuropeptide in the spinalcord, and other investigators implicated thisrelease to hyperexcitability of spinal neu-rones and the enhanced perception of pain.The available evidence, however, does notfavour a role for Substance P as the excita-tory transmitter released at the central ter-minals of nococeptive fibres, but it appearsto be co-released with glutamic acid mainlywhen inflammation occurs peripherally.

The antibody microprobe technique hasalso been used to study the release of sev-eral other neuropeptides in the brain andspinal cord, including beta-endorphin,galanin, dynorphin and neurokinin A.

Further Reading

Duggan, A.W. (1985). Nociception and antinociception: Physiological studies in the spinal cord. InClinics in Anaesthesiology, (S.F. Biena & S.L. Chapman, eds.), W.B. Saunders & Co., London.pp. 17-40.

Duggan, A.W. (1995). Release of neuropeptides in the spinal cord. In Progress in Brain Research,104, (F. Myberg, H.S. Sharma and Z. Wiesenfeld-Hallin, eds.), Elsevier, Amsterdam, pp. 197-224.

Arthur William Duggan (1932–) graduated MBBSfrom the University of Queensland in 1960, havingreceived a BSc degree in 1958. After seven year’sclinical practice, including two years involvementin Brisbane in a study of traffic accident injuries,for which in 1970 he gained a MD degree, hecommenced his PhD studies in the JCSMRDepartment of Physiology in January 1968. Hisinvolvement in investigating the spinal effects ofinhibitory and excitatory amino acids, of theconvulsants strychnine and bicuculline, and ofmorphine led to the award of his PhD in 1971.From 1971 until 1973 he further developed hisinterests in amino acid transmitters and opiateanalgesics as a C.J. Martin Fellow in theDepartment of Physiology in the University ofBristol. The third year of this Fellowship wasspent in the JCSMR, and in 1974 he wasappointed as a Fellow (Senior Fellow 1978) in theDepartment of Pharmacology, where he continuedhis investigations of pain pathways, the actions ofmorphine and of neuropeptides, includingendogenous opioid peptides. In 1987 he wasappointed Professor of Veterinary Pharmacologyin the Royal Dick Veterinary College of the

University of Edinburgh. Ten years later he returned to Australia and is currently Visiting Professor inthe Department of Anaesthesia and Pain Management at Royal North Shore Hospital, University ofSydney. In 1994 he was elected a Fellow of the Royal Society of Edinburgh.

436


Most of the initial investigations of synaptictransmission carried out in the Departmentof Physiology and subsequently in theDepartment of Pharmacology were con-cerned with excitation and inhibition in themammalian central nervous system. Therewas, however, an early interest in excitationin autonomic ganglia and at the neuromus-cular junction, synapses which previouslyhad been used by numerous neurophysiolo-gists, including John Eccles, as more readilyinvestigated models of transmission mecha-nisms within the central nervous system.

In 1955, Rosamond Eccles (see p. 415)reported the first successful intracellularrecording of resting, action and excitatorysynaptic (junction) potentials from neuronesin rabbit superior cervical ganglia. WilliamLiley (1929–1983) and John Hubbard(1930–1995), both of whom had beenBMedSc students with Eccles in Dunedin,studied excitatory transmission mediated byacetylcholine at neuromuscular junctions inthe rat diaphragm. Liley, a PhD Scholar inthe JCSMR from 1955 to 1957, examinedthe spontaneous and evoked release oftransmitter by recording and analysing‘miniature’ and end-plate potentials. Hub-bard, a PhD Scholar 1958–1960, ResearchFellow 1961, Fellow 1962 and Senior Fel-low 1964–1967, alone and with a number ofcolleagues, investigated the electrophysio-logical properties of motor nerve terminals,the factors which influenced acetylcholinerelease by nerve impulses, post-activationpotentiation of release and the relationbetween release and the intracellular distri-bution of synaptic vesicles.

Following the appointment of DavidHirst in 1981, another aspect of autonomicneurotransmission, transmission at the ter-minals of ganglion neurones on the smoothmuscle cells of arteries, arterioles and thegastrointestinal tract, was intensively inves-tigated. Hirst, in collaboration with Dirk

van Helden, examined the properties ofsmooth muscle cells in arterioles. The rest-ing membrane potential of these cells, hith-erto considered to be regulated by theactivity of a sodium/potassium pump, wasfound to be essentially determined by theactivity of an ion channel selectively perme-able to potassium ions. In collaborationwith Alan Finkel of the Experimental Neu-rology Unit, who had developed a singleelectrode voltage clamp (see p. 453), directmeasurements were made of the time courseand properties of the membrane currentsunderlying excitatory junction potentialsproduced by stimulation of the perivascularsympathetic nerves. This was a very signif-icant step forward in understanding thetransmission process at sympathetic nerveterminals.

Mutual interests in sympathetic innerva-tion and neurotransmission led to a collabo-rative study by Caryl Hill, Hirst and vanHelden of the development of functionalsynapses in arteries and arterioles. The ear-liest postnatal vascular responses to sympa-thetic nerve stimulation were found to differphysiologically and pharmacologicallyfrom those recorded later during develop-ment. This suggested that these blood ves-sels could be controlled by the sympatheticnervous system early during development,prior to the formation of mature synapses,and that sympathetic nerves might have atrophic influence in shaping the nature ofthe mature postsynaptic response. Subse-quent investigations included an electro-physiological and anatomical study of therestoration of sympathetic responses duringneural regeneration, and a pharmacologicaland anatomical analysis of the developmentof the sympathetic innervation of entericneurones of the gut, the latter in collabora-tion with Meng-Chong Ngu of the Depart-ment of Clinical Science. Together, thesestudies confirmed the selectivity of the

Synaptic Transmission in the Mammalian Peripheral Nervous System

by Caryl Hill

processes underlying innervation by sympa-thetic nerve fibres during development andregeneration.

In a series of experiments in collabora-tion with Gerald Silverberg, visiting fromStanford University, in 1984 Hirst, vanHelden and Hill provided evidence thatsympathetic innervation could indeed havea trophic influence on the function of vascu-lar smooth muscle. Using cerebral arteri-oles, experiments were aimed at measuringthe kinetics of smooth muscle voltage-dependent calcium currents which areresponsible for muscle action potentials andcontraction. A direct correlation was foundbetween the density of innervation and thevoltage dependent excitability of the smoothmuscle cells, suggesting that the density ofcalcium channels was in some fashion influ-enced by the pattern of sympathetic inner-vation.

Hill’s interest in autonomic neurotrans-mission during development, together withher continuing studies on neuronal growthfactors in the laboratory of Ian Hendry (seep. 441) led to a comparison of the sympa-thetic innervation of different target tissuesin the gastrointestinal tract. Different sub-populations of sympathetic neurones werefound to exist from the earliest stages ofneurite outgrowth, which innervated differ-ent target tissues and depended on differenttrophic factors for their survival duringdevelopment. This finding, in 1985, wasone of the earliest demonstrations that sym-pathetic ganglia comprised a heterogeneouspopulation of neurones projecting axons toappropriate target cells with a great deal ofprecision. Subsequently, the axonalprocesses of sympathetic neurones wereshown to branch in the vicinity of targetcells, the excess collateral branches beinggradually pruned over time. Neuronal com-petition was identified as an important fac-tor in this reduction of collaterals duringdevelopment.

Following the 1988 JCSMR Review,Hill established an independent laboratoryin order to extend her studies of autonomicneurotransmission in a variety of target tis-sues. Molecular biological studies concern-

ing the expression of transmitter receptorsduring development were commenced in1991 with Maria Vidovic. The aim was todetermine whether the physiological andpharmacological changes in synapticresponses observed during developmentwere due to neural induction of receptorexpression. Comparative data from differ-ent tissues showed, however, that receptorexpression occurred before there was a sig-nificant entry of nerve fibres into target tis-sues, and was maintained in tissues whichhad been denervated at birth. Thus, modifi-catons in receptor types mediating func-tional responses of target cells werepostulated to result from selective interac-tion of the nerves with appropriate receptorson smooth muscle cells. This proposal isconsistent with a later demonstration byShaun Sandow that very close associationsbetween the transmitter release sites and thepostsynaptic muscle membrane are presentat the times when responses of an immaturetype can be recorded. Thus, modification ofphysiological responses during develop-ment may result from close range interac-tions and the possible interchange of growthfactors.

Another major investigation during the1990s concerned the receptor-activated sig-nal transduction mechanisms initiated byperivascular sympathetic nerve stimulation,undertaken by Hill and Dianna Gould. Thefindings indicated that the contraction ofsmooth muscle in some small arterioles wasthe consequence of the release of intracellu-lar calcium rather than, as generallyaccepted, the influx of calcium throughvoltage activated channels in the musclemembrane. This was a particularly impor-tant observation which suggested that drugsdesigned to reduce the constriction of smallarterioles by blocking these calcium chan-nels may be not be very effective, andpointed to the need to develop drugs aimedat the intracellular calcium store/releaseprocess.

These results, and those from other lab-oratories, have provided evidence for theheterogeneous nature of the physiologicalmechanisms and receptors involved in auto-

437

16. Neuroscience

438


nomic nerve-mediated responses in differ-ent vascular beds. Hill and JacquelinePhillips made a comparative study ofmRNA expression of receptors mediatingvasoconstriction and vasodilation in fourdifferent types of blood vessel. Differentialgene expression appears to be important inthe heterogeneity of vascular responsessince a unique array of receptors is presentin each type of vessel, some expressed incommon, others specific for a particulartype of vessel.

Another factor determining the hetero-geneity of vascular responses is structuraldiversity within the vascular wall. Differentsized vessels have different numbers of lay-

ers of smooth muscle cells, and the coordi-nation of vasoconstriction and vasodilationrelies on cellular coupling through struc-tures called gap junctions. These are ionchannels comprised of constituent mole-cules called connexins. Recent evidenceindicates differences between the incidenceof gap junctions and the subtypes of theconstituent connexins in different vascularbeds and different sized vessels. Geneti-cally modified mice are to be used to furtherexamine the role of gap junctions and con-nexins in determining vascular responsescontrolled by the autonomic nervous systemin health and disease.

Further Reading

Eccles, J.C. (1964). The Physiology of Synapses. Springer-Verlag, Berlin.Hill, C.E. (1985). Selectivity in sympathetic innervation during development and regeneration in the

rat. Experientia, 41, 857-862.Hill, C.E., Phillips, J.K. and Sandow, S.L. (1999). Development of peripheral autonomic synapses:

neurotransmitter receptors, neuroeffector associations and neural influences. Clinical andExperimental Pharmacology and Physiology (Brief Reviews), 26, 581-590.

Hirst, G.D.S. and Edwards, F.R. (1989). Sympathetic neuroeffector transmission in arteries andarterioles. Physiological Reviews, 69, 546-604.

George David Scarcliffe Hirst (1942–) graduatedBSc Pharmacy in 1965, BSc Pharmacology in1966 and PhD in 1969 at the University of Leeds.After a two year postdoctoral fellowship at theUniversity of Edinburgh he moved to MonashUniversity in 1971 and held a series ofappointments, including a Queen Elizabeth IIResearch Fellowship (1973–75) and a SeniorLectureship (1978–81). From 1981–1984 he wasa Fellow in the Department of PharmacologyJCSMR, and in 1985 returned to Melbourne,initially as a Visiting Scientist at the Baker MedicalResearch Institute. Subsequently, he wasappointed a NHMRC Senior Research Fellow(1985–1988), Principal Research Fellow(1989–1992) and Senior Principal ResearchFellow (1993–) in the Department of Zoology,University of Melbourne. His seminalcontributions to autonomic neurotransmission ledto his election to Fellowship of the AustralianAcademy of Science in 1993.

439

16. Neuroscience

In 1974 Ian Hendry introduced a new line ofresearch in the Department of Pharmacol-ogy, the study of the factors which deter-mine that developing neurones makecontact with the correct target cells. Nervegrowth factor (NGF) was the first proteinshown to promote the growth of developingneurones, and Hendry had demonstrated inCambridge that the retrograde axonal trans-port of NGF was critical in the developmentin vivo of mature adrenergic neurones ofsympathetic ganglia. At the time, NGF wasgenerally considered to act as a mitogen, aproposition which was challenged by thefindings that this protein enhanced the sur-vival and hypertrophy of neurones, but nottheir hyperplasia, and did not increase thecellular mitotic index. Subsequently, in

Canberra, Hendry and Sue Ebbott showedthat NGF was also retrogradely transportedin axons of the central nervous system. Asfurther information became available, thegeneral term ‘retrophins’ was proposed in1980 to describe neurotrophic factorsreleased by target tissues which, after retro-grade transport, were responsible for thesurvival of the neurones transporting them.

In collaboration from 1975 with CarylHill, then a Postdoctoral Fellow in theResearch School of Biological Sciences andwith previous experience of growing dissoci-ated sympathetic ganglion neurones with theirtarget tissues in culture, Hendry studied thefactors controlling the phenotype of develop-ing neurones. Under tissue culture conditions,these normally adrenergic neurones were

Caryl Elizabeth Hill (1949–) graduated BSc(Hons)from the University of Melbourne and gained herPhD in Zoology in 1974. After a postdoctoralappointment in the Research School of BiologicalSciences, ANU, and an Overseas ResearchFellowship of the National Heart Foundation ofAustralia at University College London, she joinedIan Hendry’s group in the Department ofPharmacology, JCSMR, in 1978 as a NHFResearch Fellow to study neuronal growthfactors. Appointed a Research Fellow in 1982,Senior Research Fellow in 1985 and SeniorFellow in 1994, she has been Leader of theAutonomic Synapse Group in the Division ofNeuroscience since 1995.

Nerve Growth Factors

by Ian Hendry

found by enzyme assays to be cholinergic.Following the appointment of Hill to theDepartment in 1978, a factor released by thenon-neuronal cells in the culture was shownto be responsible for this change in pheno-type. In 1980, glucocorticoids were foundto modulate the nature of the neurones indeveloping sympathetic ganglia by sup-pressing the formation of this factor.

Nerve growth factors for parasympa-thetic (cholinergic) neurones had beendemonstrated to be present in muscle in1977 by Ian McLennan. In the followingyear he showed that removal of limb buds infoetal animals resulted in the death ofmotoneurones which would have innervatedthe missing limb. This observation,together with the finding that there was afactor in cardiac tissue that supported thesurvival of parasympathetic neurones, led toan attempt to purify the nerve growth factorfor motoneurones which are also choliner-gic neurones. A high molecular weightcomplex protein was isolated from bovineheart muscle that promoted the survival ofparasympathetic neurones in tissue culture,and a monoclonal antibody was generatedthat blocked this in vitro effect and thedevelopment of the parasympathetic systemin mice. In 1989, this factor was shown tobe identical with ‘acidic fibroblast growthfactor’, a protein with diverse effects actingas a mitogen for fibroblasts and vascularendothelium and as a survival factor forneurones.

By this time a number of neurotrophicfactors had been described, and Hendry andhis colleagues returned to their earlier inter-est in the role of retrograde transport in neu-ronal development. In collaboration withPerry Bartlett and Mark Murphy, of theWalter and Eliza Hall Institute in Mel-bourne, the retrograde transport of‘leukaemia inhibitory factor’ by the sensorynervous system was demonstrated in 1992.

Further progress followed the appoint-ment in 1988 of Michael Crouch (see p.267), who brought expertise in second mes-senger systems involved in the actions ofgrowth factors. GTP-binding proteins wereshown to be involved in the survival of neu-

rones induced by neurotrophic factors.This, coupled with the finding that some ofthese factors were not transported retro-gradely, led to the concept that retrogradelytransported long-acting second messengersconvey information from target tissues toinnervating neurones. From 1992 Crouchconcentrated on growth factors which regu-lated cell proliferation. These studies sug-gested that G-proteins might function asintracellular signalling molecules. Subse-quently retrograde axonal transport of thealpha sub-unit of GTP-binding proteins wasdemonstrated, and in 1994 GTP-bindingproteins were shown to be translocated tothe nuclei of sensory neurones after retro-grade axonal transport.

In order to explore the role of the GTP-binding protein Gz, Hendry and his group,in collaboration with Klaus Matthaei (see p.464), used molecular biological techniquesto generate a line of mice deficient in theprotein Gz. Although no obvious abnor-mality of the nervous system could bedetected, this animal was hypertolerant toopiates and the project is continuing.

From 1994 a detailed analysis of the sec-ond messengers which were transported ret-rogradely was undertaken, a project whichincluded Sven Johanson, Selina Bartlett andAnna Reynolds. A number of second mes-sengers were found to be involved, not onlyby being transported themselves like GTP-binding proteins but also in the regulation ofthe actual transport process. Use was madeof the retrograde transport of iodinated NGFfrom the anterior chamber of the eye to thesympathetic and sensory ganglia to assessthe effect of many compounds which mod-ify the second messenger cascades. Theimportance was shown of the enzymephophatidylinositol 3-kinase for neuronalsurvival and retrograde axonal transport. Inlater experiments by Shaun Sandow usinggold-labelled NGF to examine the ultra-structure of the organelles containing NGF,and by Michael Weible and Kathrina Hey-don using rhodamine-labelled NGF toexamine the in vivo movement of trans-ported particles, a description of a retro-grade signalling organelle at the electron

440


441

16. Neuroscience

microscopic level became possible. Theelucidation of the proteins contained in thisstructure and its formation at the nerve ter-

minal will be essential to the understandingof the role of retrograde axonal transport inneuronal survival and differentiation.

Further Reading

Crouch, M.F. and Hendry, I.A. (1993). Growth factor second messenger systems: Oncogenes and theheterotrimeric GTP-binding protein connection. Medical Research Review, 13,105-123.

Hendry, I.A. and Hill, C.E. (1980). Retrograde intra-axonal transport of target tissue derivedmacromolecules. Nature, 287, 647-649.

McLennan, I.S., Hill, C.E. and Hendry, I.A. (1980). Glucocorticoids modulate the choice oftransmitter in the developing superior cervical ganglion. Nature, 283, 206.

Ian Alexander Hendry (1944–) graduatedBSc(Med) (1965), MBBS (1969) and DSc (1992)from the University of Sydney. After a year’sjunior residency he sought a career in research,obtaining a PhD from the University of Cambridgein 1972. After postdoctoral fellowships at theBiozentrum of the University of Basel andStanford Medical School, California, he returnedto Australia in 1974 as a Queen Elizabeth IIFellow in the JCSMR Department ofPharmacology. He was appointed a SeniorResearch Fellow in 1976, Senior Fellow (1981),and Leader of the Developmental NeurobiologyResearch Group in the Division of Neuroscience(1989). In 1995 he was appointed Professor inthis Division.

The Neurophysiology of Vision

by Peter Bishop

From the time Professor Peter Bishop (seep. 82) arrived at the John Curtin School in1967 the neurophysiology of vision becamea major component of the research of theDepartment of Physiology, with concernsabout visual optics, the formation of images

in the eye and the organization of the visualsystem as a whole.

The concept of the receptive field and itsproperties formed the basis of much of thiswork. The receptive field of a single neuronis defined as the region in space where

442


objects cause an increase or decrease in therate of a cell’s discharge. Through theirreceptive fields, striate cells in the cerebralcortex respond to particular features ofobjects, such as lines, edges, corners and soon. It is only much later along the visualpathway in the brain that cells respond tocomplete objects such as the human face.

Based on the concept of receptive fielddisparity, he, with his colleagues at SydneyUniversity, had already established the basicneural processes underlying binocular sin-gle vision and depth discrimination (stere-opsis), and at the John Curtin School in1967 he made binocular vision a majorcomponent of the Department’s researchendeavour. This short essay gives a briefoutline of this aspect of the research. Until1975 most of his research on this and otheraspects of vision was done in collaborationwith Geoff Henry (see p. 448) and other col-leagues, but after that Henry worked inde-pendently.

Because the two eyes are horizontallyseparated in the head, each eye sees a givenobject from a slightly different vantagepoint, leading to small horizontal differ-ences in the relative positions of theirimages on the retinas of the two eyes.Hence the concept of receptive field dispar-ity. The impulses from retinal ganglioncells with receptive fields in the two eyestravel along the optic nerves and pathwaysin the brain before finally coming togetherin single cells in the primary visual cortex -the striate cortex. In this way each binocu-larly activated striate cell responds selec-tively to one and the same object feature ineach eye (binocular single vision) eventhough the location of the images on the tworetinas may not correspond exactly. Eachstriate cell necessarily has the sameresponse properties from the two receptivefields, although there are cell-to-cell differ-

ences that depend upon the object beingviewed.

When the two eyes are turned towards apoint in space the images of that pointoccupy the same relative position on the tworetinas, i.e., they are corresponding retinalpoints. A frontal plane positioned to passthrough this fixation point then defines theprojection of other corresponding pairs intospace and may be used as a reference plane.Points lying closer or further than this planefall on non-corresponding or disparate partsof each retina.

In the population of striate neurons somefire with a maximum response to objects onthe reference plane while others show apreference for closer or more distant points.Thus each striate neuron responds selec-tively to an object feature located at a par-ticular depth. Work in the JCSMRlaboratories showed that for each cell themaximum response occurs when the recep-tive fields for each eye was in spatial regis-ter. Also that the cell’s response dippeddramatically with small changes from thisin-register position. This narrow range ofeffective responses brings precision to cell’scapacity to estimate depth, and our work didmuch to explain how cortical inhibitorymechanisms act to restrict the out-of-regis-ter responses. With the in-register locationvarying from cell to cell, the population ofstriate neurons provides for a precise esti-mate of a wide range of different depths.

All the above observations were madeon the anaesthetised cat preparation and it isclearly beyond the scope of this brief essayto give the experimental details on whichthe observations were based. One detailthat reveals the precision of the system,however, is that there is a significant changein the discharge rate from striate cells whenits two receptive fields are out of register byas little as 5 min. arc.

Further Reading

Bishop, P.O. (1973). Neurophysiology of binocular single vision and stereopsis. Handbook ofSensory Physiology, Vol. VII/3A (R. Jung, ed.), Springer-Verlag, Berlin. pp. 255-305.

Maske, R., Yamane, S. and Bishop, P.O. (1986). Stereoscopic mechanisms: binocular response ofstriate cells of cats to moving light and dark bars. Proceedings of the Royal Society of London,B229, 227-256.

443

16. Neuroscience

The initial processing of visual informationoccurs in the retina, a complex laminatedstructure of rod and cone photoreceptors,interneurones and retinal ganglion cells(RGCs). The axons of ganglion cells, viathe optic nerve, the optic decussation andthe optic tract, synapse with neurones in thelateral geniculate nucleus (LGN), the axonsof which project via the optic radiation toregions of the cerebral cortex concernedwith vision. Within the Department ofPhysiology from 1967–1996 William Lev-ick (see p. 133), and from 1970–1976Jonathon Stone, investigated RGCs andtheir central linkages

Specificity of Retino-GeniculateConnections

The very essence of organization in the cen-tral nervous system resides in the patterns ofinterconnections made by individual neu-rones at different functional levels. Since

the late 19th century such patterns havebeen inferred indirectly, and often incor-rectly, from morphological studies. Forexample, in the LGN, the main link betweenretina and visual cortex, it was known thatindividual LGN neurones were studded withthousands of synapses. This had led to thebelief that the LGN was a major centre ofintegrative activity. An entirely differentpicture emerged from a neurophysiologicalattack on the issue carried out by WilliamLevick, Brian Cleland and Mark Dubin in1971. By making simultaneous single-cellrecordings from an LGN neurone and a suc-cession of RGCs, it was established thatessentially every output impulse from theformer was attributable to an incomingimpulse from one (8% of dual recordings)or just a very small number (up to 5) ofRGCs. In one stroke, the notion of massiveconvergence was swept aside. A large pro-portion of the thousands of synapses on an

Highlights of Retinal Research

By William Levick

Brian Geoffrey Cleland (1937–) received his BEdegree from the University of New South Wales in1959, and MS (1964) and PhD (1967) fromNorthwestern University, Illinois, where he carriedout research on fundamental visual properties ofretinal ganglion cells with R.W. Jones andChristina Enroth-Cugell. After a postdoctoralfellowship at Northwestern University for a furtheryear, he was appointed to the Department ofPhysiology, JCSMR, in 1968 as a ResearchFellow, and was promoted Fellow in 1972 andSenior Fellow in 1981. His major researchinterests included retino-geniculate connectivity,interneurones of the lateral geniculate nucleusand the gradients of visual acuity of retinalganglion cells. In 1983 he moved to theDepartment of Physiology at the University ofSydney as a Senior Lecturer. In 1990, after asecondment into the electronics industry, he tookup a permanent position with Telectronics Pty Ltd(Sydney).

444


LGN neurone must be coming from only asingle RGC or a very small number of them.

This result attracted wide and persistentattention because it provided much-neededlinkages between methodologically differ-ent fields. The linkage with morphologycame through the identification of sustainedand transient receptive field classes with theclassical axonal conduction velocity groups(T1, T2) in the primary optic pathway andthus with axonal diameter and cell body

size. A linkage with visual psychophysicscame from the identification with the lin-ear/nonlinear (X/Y) classification of gan-glion cells and thus with the popular notionthat the visual system operated to someextent as a Fourier analyzer (necessarily lin-ear) of the visual scene. A linkage with cor-tical neurophysiology came from thedemonstration that the sustained and tran-sient classes were separately projected toprimary visual cortex.

Further Reading

Cleland, B.G., Dubin, M.W. and Levick, W.R. (1971). Sustained and transient neurones in the cat’sretina and lateral geniculate nucleus. Journal of Physiology, 217, 473-496.

Functional Classification of RetinalGanglion Cells

The notion of ‘receptive field’, originatingin the 1930s, is a fundamental concept in theneurophysiological account of the mecha-nism of so-called ‘early’ vision. In its sim-plest form, the concept is expressed as ‘thepatch of visual field within which theimpulse discharge of a visual neurone maybe influenced by presentation of a visualstimulus’. At the retinal level, much more isnowadays implied by the concept, largely asa result of the unfolding, in the laboratoriesof William Levick and Jonathon Stone, ofthe wide diversity of RGC classes. Thus thecurrent textbook bundling of ganglion cellsinto just two classes (On-centre and Off-centre) dating from the 1950s is long over-due for revision. Depending on how theyare counted, there are upwards of thirteendifferent classes of cat RGCs.

There are several key factors that under-

pinned the new view. Perhaps the mostimportant was the evaluation of a suffi-ciently large reference sample of recordings(thousands of contact hours) so that rarelyencountered classes could be convincinglydistinguished from artefactually damagedversions of commonly encountered classes.Another key realization was that recording‘encounters’ cannot be equated withanatomical prevalence. The anatomicallynumerous (~45%) small-bodied cells pres-ent much smaller cross-sectional areas tofine recording electrodes of all types. Yetthe dendritic trees of the individual mem-bers of each class may be sufficiently largeas to completely tile the retina and so supplya complete veridical image-analysis mes-sage to higher centres. This is an importantfeature of a genuine class. What the multi-ple classes are doing is supplying a multidi-mensional analysis of the visual scene tohigher centres via an inherently parallelvisual pathway.

Further Reading

Levick, W.R. and Thibos, L.N. (1983). Receptive fields of cat ganglion cells: classification andconstruction. Progress in Retinal Research, 2, 267-319.

445

16. Neuroscience

Optic Decussation

Decussations in the central nervous systemare enigmatic morphological arrangementsin which collections of similarly sourcednerve fibres from one side meet their mir-rored counterparts from the other side at themidline as they cross to the opposite side ofthe brain or spinal cord. The decussation atthe optic chiasm of mammals has a remark-able specialization in that it is incomplete inspecies with more frontally directed eyes.This feature was recognized more than 270years ago by Isaac Newton as being associ-ated with the possibility of binocular singlevision. He noted that points in the temporal‘half-retina‘ of the right eye shared similarlydirected lines of sight in the external visualworld as corresponding points in the nasal‘half-retina‘ of the left eye. It would there-fore make sense for nerve fibres of temporalorigin to remain uncrossed at the decussa-tion so as to keep company with the cross-ing nasally originated fibres of the oppositeeye having similar visual directions.

The neurophysiological demonstrationof the coming-together of signals from

crossed and uncrossed pathways on singleneurones of the visual cortex came 230years later. It constituted the basis for themost evident aspect of binocular vision,namely, although two eyes are doing theviewing, the percept we experience is of asingle visual world. A second, more subtleaspect of binocular vision is that the percepthas a three-dimensional character (stereop-sis). The neurophysiological basis for thiswas developed in Peter Bishop’s laboratory(see p 442) and elsewhere. The recognitionof the many classes of retinal ganglion cellsled to the exploration of their individualdecussation patterns in the laboratories ofWilliam Levick and Jonathon Stone. Sur-prisingly, it emerged that at least three dif-ferent patterns existed. This result led to theproposal that the role of one of the classes,‘Brisk-Transient(Y)’, in binocular vision isto provide the basis for single vision andstereopsis in a region closer to the subjectthan that provided by the more numerous’Brisk-Sustained (X)’ class. Ten years later,this proposal received resounding independ-ent support from a study at the LGN andcortical levels.

Jonathan Stone (1942–) graduated BSc Med(1962), PhD (1966) and DSc (1977) from theUniversity of Sydney and began his research careerin visual neurophysiology with Peter Bishop inSydney University’s Department of Physiology. ALectureship in the Department was followed bythree postdoctoral years abroad, and in 1970 hereturned to Australia as a Research Fellow in theDepartment of Physiology at JCSMR (SeniorResearch Fellow, 1972–76). Here he continued hisinterests in the functional properties of subclassesof retinal ganglion cells, and of neurones in thelateral geniculate nucleus and visual cortex, whichled to the development of the concept of parallelprocessing in the visual system. From 1976 he wassuccessively Senior Lecturer, Associate Professorand Professor in the School of Anatomy, Universityof New South Wales and in 1987 was appointedChallis Professor in the School of Anatomy (Headof Department, 1987–92) in the University ofSydney. Since 2000 he has been Head of theDepartment of Anatomy and Histology, and from2001 Director of the Institute for BiomedicalResearch in the University of Sydney. His manycontributions to vision research were recognized in1984 by his election as a Fellow of the AustralianAcademy of Science, of which he was BiologicalSecretary from 1986–1990.

446


Neurophysiology of a Scotoma

Clinically, a scotoma is defined as a blindpatch in the visual field. Pathological sco-tomata may be associated with a diseaseprocess at any level of the visual systemfrom retina to visual cortex. The surprisingfeature of a scotoma is that it is often hardlynoticeable to the subject, even when asmuch as half the visual field is blanked out(‘hemianopia’). Accounting for the perceptof a scotoma in terms of neurophysiologicalmechanisms at the level of the visual cortexor lateral geniculate nucleus is subject toconsiderable uncertainties because of lackof knowledge of what is happening at theretinal level. The condition of feline centraldegeneration presented William Levick andLarry Thibos with an exceptional opportu-nity for the required analysis. In its maturestabilized state the lesion is characterized by

an essentially complete loss of photorecep-tors over a sharp-edged patch of centralretina. The brightly reflecting tapetumenabled clear visualization of the lesion out-line by ophthalmoscopy and back-projec-tion into the external visual field.Moreover, it has long been known that thereceptive fields of ganglion cells areapproximately centred on the cell-bodyrecording. Thus, in contrast to studies athigher levels, directly visualized placementof the recording electrode enabled preciseselection of cells for study with dendritictrees (and expected receptive field centres)in known relation to the edge of the lesion.Measurement of antidromic latency enabledinference of receptive field class, even whenvisual performance was corrupted.

The main result of the work was thatganglion cells with cell bodies more than 1°inside the border of large lesions were blind,

Further Reading

Levick, W.R. (1977). Participation of brisk-transient retinal ganglion cells in binocular vision – anhypothesis. Proceedings of the Australian Physiological & Pharmacological Society, 8, 9-16.

Pettigrew, J.D. and Dreher, B. (1987). Parallel processing of binocular disparity in the cat’sretinogeniculate pathways. Proceedings of the Royal Society of London, B232, 297-321.

Larry N. Thibos (1947–) received his BS (1970)and MS (1972) degrees in Electrical Engineeringfrom the University of Michigan and PhD (1975) inPhysiological Optics from the University ofCalifornia, Berkeley, where he carried outresearch on the retinal processing of spatio-temporal intensity variations. After working as apostdoctoral fellow at the National Institutes ofHealth, Bethesda, in 1975 he joined theDepartment of Physiology, JCSMR, as a VisitingFellow. He was appointed as a Research Fellow(1977–1982) and to a Sulman Fellowship in 1983.His major research interests included informationprocessing by the retina and the role of retinalorganization and visual optics in setting the limitsto visual performance. In 1983 he moved to theSchool of Optometry, University of Indiana asAssistant Professor of Visual Science, withpromotions to Associate Professor in 1989 andProfessor of Optometry and Visual Science in1994.

447

16. Neuroscience

although not always silent. Over an approx-imately 2° wide transition zone centred onthe lesion edge the majority of cells hadvariably reduced visual sensitivity. Thus,although the edge of the photoreceptorlesion is sharp, the representation of it in theganglion cell array was fuzzy. That thefuzziness was associated with the size of thereceptive field centres was clearly broughtout by a simple modelling of the effects ofvariable truncation of receptive fields by thelesion. A further important result was thatganglion cells with the normal distributionof axonal conduction properties were foundin plentiful number and ostensibly normalproportions (judged by conduction groups)within long-standing (years) lesions. Thisindicates that ganglion cell survival in theabsence of photoreceptors is not criticallydependent on hypothetical anterogradely

transported trophic factors that mayemanate from the photoreceptor layer.

The effects at the cortical level of thisparticular type of lesion have not been stud-ied. Instead, others have created localizedlesions of the photoreceptor array by meansof a high-powered laser. A strikingly differ-ent picture has emerged in that a sizeableproportion of cortical neurones that wouldnormally receive visual input from theaffected region instead become sensitive toa patch of visual field (ectopic receptivefield) positioned outside the boundary of thelesion. Whether this cortical adult topo-graphic plasticity is related to the characterof the photoreceptor lesion (laser ablation ishyperacute; central retinal degenerationtakes months to years) is the subject of fur-ther ongoing investigations.

Further Reading

Levick, W.R. and Thibos, L.N. (1993). Neurophysiology of central retinal degeneration in cat.Visual Neuroscience, 10, 499-509.

Working with a succession of talented Visit-ing Fellows, Geoffrey Henry made a num-ber of fundamental discoveries and oneimportant practical discovery in visual neu-roscience. Two of the fundamental discov-ery and the practical discovery are describedin the following three essays

The Discovery of New Visual Cells in theBrain

In January 1968 Geoff Henry began to workin association with Peter Bishop to classifythe response characteristics of individual

cells in the primary visual area of the cere-bral cortex. A year later they were joined byBogdan Dreher from Poland.

After leaving the eyes, signals cominginto the brain undergo their first major pro-cessing step in the primary visual area of thecerebral cortex, which covers the surface ofthe occipital lobe at the back of the brain.To understand how information is processedat this early stage, it is essential to classindividual cells from their visual responses.In attempting this task the Canberra groupcame up against the prevailing classification

Discoveries in Visual Neuroscience

by Geoffrey Henry

448


that had been proposed by Hubel andWiesel, who were to receive the Nobel Prizein 1981. In this classification, each cell’sresponse was interpreted from its perceivedposition in a functional hierarchy. Theacceptance of this scheme virtually closedthe door on the discovery of new cell types,since it only allowed for simple cells to pro-vide the input to complex cells which in turnfed onto hypercomplex cells.

The JCSMR group, however, refused to

be hamstrung by this restriction and, duringthe time of Dreher’s stay, they proposedseveral new cell types and demonstratedthat the hierarchical theory did not tell thewhole story. The first of the new cell typesdid not run counter to the hierarchicalscheme since three varieties of the simplecell were detected, each displaying distinc-tive types of binocular interaction. Thenext new cell, however, was damaging tothe scheme since its properties met the

Left Geoffrey Herbert Henry (1929–) graduated in science and optometry from Melbourne University in1952, MAppSc in 1965 and DSc in 1975. During the 15 years from 1952, he practiced optometry,undertook postgraduate studies and was Chairman of the College of Optometry in Melbourne University.Awarded a Churchill Fellowship (1965) to study at Indiana and Cambridge Universities, he began hisacademic career in 1968 with his appointment to work with Peter Bishop at JCSMR. This associationcontinued until 1976 and in 1979 he was appointed to the position of Senior Fellow. In 1991 he wasappointed Director of the Centre for Visual Science at ANU. He retired from JCSMR in 1994. Henrywas awarded the Glen Fry Award of the American Optometric Association, the Rimpac Award and madean Honorary Fellow of the National Vision Research Institute of Australia. He is now a Visiting Fellow atANU.

Right Bogdan Dreher, (1941–) graduated MSc from the University of Warsaw and received his post-graduate training at the Nencki Institute of Experimental Biology in Warsaw (PhD 1968) where he was aResearch Fellow for two years before filling a similar position at the JCSMR from 1968 to 1972. He thenwent to Sydney University in 1972 where he progressed from Research Fellow in the PhysiologyDepartment to be made Professor in Visual Neuroscience in 1995. He was awarded a DSc from SydneyUniversity in 1989. His interests have remained in the visual system but have ranged from theembryological development of retinal receptors to the response characteristics of cells in the highestcentres of the visual cortex. He is now regarded as a world authority in these fields and is great demandas an international visitor, lecturer and author.

449

16. Neuroscience

requirements of an implausible cell, one thatbelonged to a simple hypercomplex class.This provoked hostility from the advocatesof the hierarchy but they were placatedwhen, some years later, the authors of thescheme also found these cells.

Another cell type also posed problemssince its properties appeared to be thoseexpected from a mix of simple and complexcells. Later experiments in Henry’s labora-tory, with Alan Harvey and Jenny Lund,showed that these cells projected to a highercortical area where they terminated on a cellother than one with hypercomplex proper-

ties. It was also demonstrated later by JeanBullier and Henry that complex cells werefrequently the first cells in the processingsequence in the primary visual area.

These results provided evidence that ifthe hierarchical scheme was to prevail, thenother flow paths must exist to bypass differ-ent stages of the sequence. The role ofthese abbreviated paths has remained a mys-tery, but one that will need to be resolved inorder to understand the entire contributionof the primary visual cortex in the act ofseeing.

Further Reading

Henry, G.H. (1977). Receptive field classes of cells in the striate cortex of the cat. Brain Research,133, 1-28.

The Paths Taken by Visual StreamsEntering the Brain

From the early days of the visual scienceprogram at the JCSMR it had been demon-strated that information emerging from theeyes passed into the brain along a number ofparallel streams. One question remainedunanswered, what happened to thesestreams once they entered the visual cortex?

Jean Bullier and Henry set out to resolvethis question. Their approach was to initiatea signal (a nerve spike) at one point alongthe incoming stream and then attempt torecord the new signal at another point,either further up or down the stream. Thetime of arrival and the shape of the signalgave clues of the stream-carrying signal andalso the number of cells it had traversed inreaching the recording site. Usually therecording electrode was close to the body ofa cortical cell and it was also possible to

classify the cell according to its visualresponses.

These physiological experiments werecomplemented by histological experimentsapplying a similar principle. A tracer sub-stance such as the enzyme, horseradish per-oxidase, was injected into a pathway orstream and followed in microscopic sectionsas it moved away from the injection site.The size of the revealed fibres allowed thestream and its destination to be identified.

These studies opened the way to almostall the modern concepts of flow pathsthrough the visual cortex. Tables were pre-pared setting out the response characteris-tics for cortical cells in different streams andat different stages in the processingsequence in the primary visual area. Theaccuracy and comprehensive nature of thesetables has ensured that they have beenwidely adopted and that they have retainedtheir currency to the present day.

Further Reading

Henry, G.H. (1986). Streaming in the striate cortex. In Visual Neuroscience, (J.D. Pettigrew, W.R.Levick, and K.J. Sanderson, eds.). Cambridge University Press, Cambridge, pp. 260 –279.

450


A New Instrument for the EarlyDiagnosis of Glaucoma

By the early 1980s the laboratories at theJCSMR had unearthed the cell types con-tributing to the different streams passingfrom the retina up into the primary visualarea of the cerebral cortex of the cat and themonkey. At the same time Professor LiamBurke, of Sydney University, had shownthat it was possible to selectively knock outone of these streams, that composed of largefibres, by applying pressure to the opticnerve.

Believing that elevated eye pressure inglaucoma would similarly degrade the per-formance of the large fibre stream Ted Mad-dess and Henry decided to compare theperformance of this stream in human sub-jects with and without glaucoma, a majorcause of blindness around the world. Mad-dess, who had previously worked mainly oninsect vision, soon came up with a methodto test the integrity of the large fibre path-way in humans. Based on the observationthat the measured response in this pathwaywas not directly proportional to the strength

of its visual input, he was able to isolate thecontribution from the large fibres and thento test their threshold, i.e., he measured thebrightness of a test pattern at which thepathway just started functioning. As antici-pated, this threshold value was significantlyhigher in subjects with glaucoma, appar-ently because of damage caused to thelarger fibres.

After many clinical tests, an agreementwas drawn up with an American instrumentcompany to manufacture a new clinicalinstrument called the Frequency DoublePerimeter. The term ‘frequency double’comes from an illusion, attributed to thelarge fibre stream, that results in seeingtwice the number of bars in a coarse gratingwhen it is flashed on and off rapidly. Theinstrument has proved particularly valuablein the early detection of glaucoma, since notonly are the large fibres the first to sufferdamage, but they also make such a smallcontribution to acute vision that theirabsence results in a barely noticeable visualloss.

A number of clinical evaluations from

Jean Bullier, (1949–), a graduate the ÉcoleSupérieure d’Electricité in Paris, graduated PhDfrom Duke University in North Carolina, and thenspent three years from 1978 in Geoff Henry’slaboratories in the JCSMR. He examined the flowpathways of the different visual steams as theyentered and distributed throughout the primaryvisual area of the cerebral cortex. Eventually thisbecame a lifelong study for Bullier, although hehas not stopped in the primary visual area but hascarried his interest on into higher visual centres inthe brain. Between 1981 and 1998 he worked inthe INSERM laboratories in Lyon. In 1993 herose to be Directeur de Researche and head ofthe team working on ‘Structure et fonction ducortex visuel’. He was awarded the Silver Medalof the French National Centre of ScientificResearch (CNRS) in 1997 for his contributions tothe unraveling the brain’s visual pathways. In1999 he was appointed Director of the CNRSCentre for Brain Research and Cognition at theUniversité Paul Sabatier in Toulouse.

451

16. Neuroscience

around the world have now supported theexperimental findings of the new instru-ment, and it has become an essential part of

the clinical armory for assessing the healthof the large fibre stream.

Ted Maddess (1956–) completed hisundergraduate training at the University of BritishColumbia and his PhD (1985) and a year ofpostdoctoral study in the Research School ofBiological Sciences (RSBS). During this time heinvestigated temporal adaptation in the visualresponses recorded from the insect eye. Heworked as a Postdoctoral Fellow in the JCSMRfrom 1986 to 1989, funded by a Canadian MedicalResearch Council Fellowship. With Geoff Henry,he followed a new line of research that led todevelop a clinical method for the early detectionof glaucoma. For this work, which resulted in thecommercial production of a highly successfulinstrument, he received the Rimpac Award forinnovative research in Australian science and theAustralian Technology Prize. On leaving theJCSMR, he reactivated his PostdoctoralFellowship with RSBS and was appointed aResearch Fellow in 1995 and a Fellow in 1999.He is currently the head of the BiotechnologyTransfer Unit in RSBS.

Further Reading

Maddess,T. and Severt, W.L. (1999). Testing for glaucoma with frequency doubling illusion in thewhole, macular and eccentric visual fields. Australian and New Zealand Journal ofOphthalmology, 27, 198-200.

Functions of the Supplementary Motor Area of the Primate Cerebral Cortex

by Robert Porter

In 1951, the Canadian neurosurgeon WilderPenfield and his associates in Montreal, bystimulation of the human brain in patientsundergoing neurosurgery, identified aregion in front of the classical ‘motor’ cor-tex from which complex movements and theassumption of postures could be produced.In the assumption of postures, ‘the muscula-ture of both sides of the body is employed

appropriately to bring about the change inposition’. To understand more about theneurophysiology of this ‘supplementarymotor area’ of the cerebral cortex, in 1979Cobie Brinkman and Robert Porter (see p.118) at Monash University had recorded thedischarges of individual neurones in thisregion of the brain in conscious cooperatingmonkeys while the animals performed a

452


number of movement tasks for which theyhad been trained. In contrast to neurones inthe motor cortex, a large proportion of thosein the supplementary cortex dischargedimpulses in association with movementwhether the task was performed by the leftor the right hand. Furthermore, these dis-charges occurred before the motor perform-ance and appeared to be related to the samemuscular actions on each side.

Later, the potential significance of theseobservations was highlighted when P.E.Roland and his associates in Copenhagenstudied regional cerebral blood flow inhuman subjects performing a learnedsequence of finger-thumb touching move-ments. Repetition of the learned move-ments using one hand was associated withan increase in blood flow in the ‘hand’region of the contralateral sensorimotorcerebral cortex. In addition there was anincrease in blood flow in the vicinity of thesupplementary motor area bilaterally. If,without making a movement, the subjectsthought through the sequence of movementsby rehearsing them in their heads, the bloodflow again increased in both supplementarymotor areas but not in the sensorimotor cor-tex. This bilateral association was alsodemonstrated to occur when the skilledmovements were those accompanyingspeech or movements of the feet. Thus, thedischarges of supplementary cortical neu-rones recorded in monkeys seemed likely tobe a correlate of the preparation andsequencing of instructions which this areathen delivered to sensorimotor cortical neu-rones if skilled movements were to be exe-cuted.

Moving to the JCSMR ExperimentalNeurology Unit in 1980, Brinkman thenconducted a detailed re-evaluation of theinfluence of the supplementary motor cortex

on movement performance by unilaterallyremoving it, and subsequently assessing theuse of the hand in the production of delicateand precisely controlled movement of thefingers. For a few days post-operatively,monkeys exhibited a bilateral clumsiness offinger movements used in a food collectiontask. There was a disintegration of the pre-cise ordering of a whole range of posturaladjustments and finger movements, whichresolved after a few days and could nolonger be detected.

The performance of a task that requiredcoordination of both hands, however, waspermanently impaired by unilateral removalof the supplementary motor cortex. Mon-keys had learned to retrieve food pelletsfrom holes in a perspex board by pushing afinger of one hand from above and catchingthe pellet in the other hand cupped beneaththe board. This performance required theuse of the two hands to engage in differentpostures simultaneously and to cooperate insharing in components of the food retrievaltask. Even with full visual control, mon-keys with only one intact supplementarymotor cortex made frequent mistakes. Acharacteristic mistake was to use the handnormally cupped beneath the hole to pushfrom above and to simultaneously pushfrom below with a similar action of theother hand. Instead of cooperating andsharing the task by performing differentactions, both hands appeared to be in receiptof the same instructions, presumably fromthe single intact supplementary motor area.

This investigation, and later clinicalobservations in humans, established that theprimate supplementary motor cortex has animportant role in organising the preparationof motor programs for bilaterally coordi-nated skilled motor tasks.

Further Reading

Brinkman, J. (1984). Supplementary motor area of the monkey’s cerebral cortex: short- and long-term deficits after unilateral ablation and the effects of subsequent callosal section. Journal ofNeuroscience, 4, 918-929.

Porter, R. and Lemon, R. (1993). Corticospinal Function and Voluntary Movement. Clarendon Press,Oxford.

453

16. Neuroscience

Biophysical analyses of the ionic fluxesunderlying action and synaptic potentials ofexcitable cells have been based on the volt-age dependence of these events over a rangeof membrane potentials. For large cells itmay be possible to use one intracellularmicroelectrode to record action or synapticpotentials and another to pass electrical cur-rents through the membrane, so ‘clamping’its potential at preselected voltage levels.Such a voltage-clamp technique enablesdirect measurement of the ionic currentsresponsible for action or synaptic potentials,and the dependence of these currents onmembrane potential provides clues as to thenature of the ions involved. Single micro-electrodes can also be used, particularly forsmall cells, provided that a very rapidswitching rate can be achieved between therecording and current passing mode.

This research had its origins at Monash

University in 1980, when Stephen Redman(see p. 135) and David Hirst (see p. 438)tried to voltage clamp smooth muscle cellsusing a conventional two electrodeapproach. To maintain two intracellularelectrodes in close proximity in a smoothmuscle cell proved very difficult to achieve.As a consequence Alan Finkel, then a PhDstudent, and Redman began discussing ideason how to make a single electrode voltageclamp with sufficient bandwidth to be use-ful for clamping fast synaptic potentials inneurones. A switched single electrodeclamp design had previously been publishedby others, but its switching rate was tooslow to be useful for clamping fast synapticevents. When a current pulse is applied tothe electrode, the resulting voltage acrossthe cell membrane cannot be reliably meas-ured until the electrode capacitance,charged by the current pulse, has dis-

Development of the Single Electrode Voltage Clamp

by Stephen Redman

Alan Simon Finkel (1954–) received the degreesof BE and PhD from Monash University, the latterin 1981 for research covering electricaltransmission and synaptic activity in brain tissue.For two years he conducted postdoctoral researchin the JCSMR Experimental Neurology Unit, andin 1983 moved to San Francisco to establish AxonInstruments Inc. He was instrumental in thedevelopment of all aspects of the products of thecompany, which grew rapidly to become a majormanufacturer of instruments and software forcellular neurophysiological research. Axon is alsonow becoming established in genomics and high-throughput drug screening. In 1987 Finkelreturned to Australia where in Melbourne hecontinues his roles of full-time Chief ExecutiveOfficer and Vice-President of Research andDevelopment of Axon. In 1994, under hisguidance, Axon founded Optiscan Pty. Ltd., anAustralian company dedicated to the developmentof ultrahigh-magnification endoscopes for imagingindividual cells in the human body. Finkel iscurrently a member of the Academic AdvisoryCommittee of the Electrical and ComputerSystems Engineering Department of MonashUniversity and the Medical Research Board of theJCSMR.

454


charged. The first ideas tried at Monash tospeed up the discharge process turned out tobe impractical.

Finkel and Redman moved to theJCSMR Experimental Neurology Unit inSeptember 1981 and this project became thetop priority. The final successful designrelied on a theoretical analysis of the circuitconditions throughout the switching cycle,including the cellular contributions, carefuldesign of the electronic circuits, and fabri-cation of the intracellular electrodes to min-imize their capacitance. The theoreticalanalysis provided a clear understanding ofthe correct way to use the instrument and toavoid measurement artefacts.

The prototype clamp was used to meas-ure synaptic currents evoked in spinalmotoneurones in vivo by stimulating singleGroup 1a afferent axons. Only synapticconnections made on the soma were suitablefor voltage clamping, as these provided anundistorted record of the synaptic current.The synaptic potentials evoked at thesesynapses were recognized by their timecourse, having the fastest rise and decay

times of all synaptic potentials evoked inmotoneurones. As somatic connectionswere found very infrequently, it took manyexperiments to accumulate a few record-ings. These measurements were the firstever made of a synaptic current in a mam-malian central neurone, and were used toderive the open time of the excitatorysynaptic channel, its voltage dependenceand to unequivocally demonstrate a reversalpotential of 0mV.

Finkel then collaborated with Hirst andvan Helden of the Department of Pharma-cology to measure the synaptic currentevoked in arterial smooth muscle by stimu-lation of sympathetic nerves (see p. 436).Again, this was the first time a synaptic cur-rent had been measured in smooth muscle.The international neuroscience communityrapidly learned about this fast single-elec-trode clamp suitable for measurements oncentral nervous system neurones, andappropriate equipment soon became avail-able commercially when Finkel moved toCalifornia in 1983 and established AxonInstruments Inc.

Further Reading

Finkel, A.S. and Redman, S.J. (1983). The synaptic current evoked in cat spinal motoneurones byimpulses in single group 1a axons. Journal of Physiology, 342, 615-632.

Finkel, A.S., Hirst, G.D.S. and van Helden, D.F. (1984). Some properties of excitatory junctioncurrents recorded from submucosal arterioles of guinea-pig ileum. Journal of Physiology, 351,87-98.

Quantal Analysis of Synaptic Transmission in Central Neurones

by Stephen Redman

A characteristic morphological feature ofchemical synapses is the presence of vesic-ular organelles within the presynaptic termi-nal. These vesicles contain the chemicaltransmitter of the particular synapse, and theprocess of neurotransmission involves therelease of the contents of a vesicle into the

synaptic cleft, rapid diffusion of transmittermolecules to activate specific postsynapticreceptors and the opening of ion channelscharacteristic of the type of synapse. Theresultant ionic current modifies the restingpotential and the excitability of the postsy-naptic membrane.

455

16. Neuroscience

Quantal analysis is a statistical proce-dure used to identify the ‘quantum’ ofsynaptic transmission, i.e., the current (orvoltage) generated by the release of trans-mitter from a single synaptic vesicle. Quan-tal analysis can also provide information onthe number of sites at which transmitter isbeing released, and the probability of trans-mitter release at these sites. The methodrelies heavily on quantisation of the synap-tic currents recorded in response to repeatedactivation of a synaptic connection, i.e., theamplitudes must be distributed as a series ofpeaks and troughs, with the separation ofthe peaks corresponding to the quantalresponse.

While at Monash University, StephenRedman, in collaboration with Bruce Walm-sley, Frank Edwards and Julian Jack, devel-oped a method to extract the quantalparameters at the excitatory synapsesbetween a single Group Ia afferent fibre andspinal motoneurones in vivo. The methodwas based on deconvolving the measuredamplitude distribution of an intracellularlyrecorded excitatory postsynaptic potential(EPSP) with the baseline noise for thatEPSP. This procedure worked well forthese particular synapses, where the quantalresponse was usually several times largerthan the recording noise. When thismethod, however, was applied later in Can-berra by Redman and Christian Stricker toevoked excitatory responses in the hip-pocampal cortex in vitro, the quantum tonoise ratio was smaller than that found in

the spinal cord in vivo and the method wasopen to criticism.

Stronger statistical tests for quantisationwere required, and the collaboration ofDaryl Daley from the Centre for Mathemat-ics and its Applications in the ANU Schoolof Mathematical Sciences was sought.Daley showed how the Wilks criterioncould be used to establish an objective sta-tistical test for quantisation within specifiedconfidence limits. If quantisation was pres-ent, it was then possible to use the Wilks cri-teria to extract the quantal parameters fromthe amplitude distribution of the synapticresponses. Finally, bootstrap techniqueswere used to obtain confidence limits forthese quantal parameters. These proceduresestablished a degree of statistical rigor toquantal analysis which had previously beenabsent.

These new methods were applied tosynaptic transmission in the hippocampus,between Schaffer collaterals and CA1pyramidal cells. Quantal parameters wereobtained from control recordings, and fromrecordings taken after conditioning stimula-tion which enhanced the synaptic strength(long-term potentiation). Stricker, Redmanand Anne Field showed that long-termpotentiation is caused partly by an increasein the size of the quantal current evoked atexcitatory postsynaptic receptors, togetherwith an increase in the average number ofquanta in each response. The mechanismscausing the increased number of quanta arestill unclear.

Further Reading

Redman, S.J. (1990). Quantal analysis of synaptic potentials in neurones of the central nervoussystem. Physiological Reviews, 70,165-198.

Stricker, C., Redman, S.J. and Daley, D. (1994). Statistical analysis of synaptic transmission:Model discrimination and confidence limits. Biophysical Journal, 67, 532-547.

Stricker. C., Field, A.C. and Redman, S.J. (1996). Changes in quantal parameters of EPSCs in ratCA1 neurones in vitro after the induction of long-term potentiation. Journal of Physiology, 490,443-454.

456


Research which was initiated at the Univer-sity of New South Wales by Peter Gage (seep. 133) and his colleagues, prior to his tak-ing up appointment as Professor and Headof the Department of Physiology JCSMR in1984, is providing insights into the causeand possible reduction or prevention ofischaemic cell damage that occurs during aheart attack or a stroke. Using hippocampalslice techniques developed in his laboratoryat the University of New South Wales,together with single electrode voltage clamptechniques developed at Monash Universityin Stephen Redman’s laboratory (see p.453), Gage and Christopher French, a PhDstudent, studied voltage-activated currentsin pyramidal neurones in hippocampalslices. An effect of the sodium channelblocker tetrodotoxin (TTX) suggested thathippocampal neurones had an unusual per-sistent sodium current which was not inacti-vating during prolonged depolarizations.

This project was taken up in Canberra in1984 by Pankaj Sah, a PhD student, andPostdoctoral Fellows Alastair Gibb andKevin Buckett, who for the first time com-pletely characterized these persistentsodium currents using improved patchclamp techniques. Another PostdoctoralFellow, Graham Lamb, used the vaselinegap technique to demonstrate a persistentsodium current in voltage-clamped skeletalmuscle. Results using denervated striatedmuscle suggested that denervation fibrilla-tion was due to the persistent sodium cur-rent. Subsequently, similar currents wererecorded from cardiac muscle by Yue-kunJu, a PhD student, and David Saint, a Post-doctoral Fellow.

These exciting basic scientific findingsrelated to a small sodium current very resist-ant to inactivation had not been described inthe earlier classical papers of Hodgkin andHuxley, probably because of its small size.This current certainly has a role in the repet-

itive action potentials of neurones generatedduring small depolarizations such as excita-tory synaptic potentials, and may also boosttransmission along neuronal dendrites.Gage and his collaborators then becameaware of papers claiming that drugs block-ing membrane sodium channels couldreduce cell damage produced by hypoxia.These observations, however, had receivedlittle attention since classical, TTX-sensi-tive, sodium channels were considered tobecome inactivated during hypoxia-induceddepolarization, and were thus unlikely to beinvolved in cell damage. Further experi-ments using cardiac muscle and neurones,however, showed that the persistent sodiumcurrent was actually boosted by hypoxia.

Thus one of the first events leading tocell damage during ischaemia was uncov-ered serendipitously. When arteries in theheart are blocked, heart cells deprived ofoxygen are damaged and patients often dieas a consequence of irregular heart contrac-tions (arrhythmias). Eventually, hypoxiakills heart cells, so reducing the heart’spumping efficiency and leading to conges-tive heart failure. Similarly in the brain,strokes are the result of blocked arteries orbleeding which deprives neurones of oxy-gen and kills them. For some time it hasbeen known the cells deprived of oxygenare killed by an intracellular accumulationof calcium. During hypoxia calcium mayenter cells because of increased calciumchannel activity or through channels acti-vated by abnormally high extracellular lev-els of glutamate. Clinical trials, however,have shown that drugs which block calciumchannels or glutamate-activated channelsare not very effective in treating strokepatients.

There is evidence that cellular accumu-lation of calcium during hypoxia can beblocked by preventing the entry of sodiumions, and Gage and his colleagues believe

A Persistent Membrane Sodium Channel

by Peter Gage

457

16. Neuroscience

that calcium accumulation depends on theincreased activity of persistent sodiumchannels. Current research is focussing onthe link between hypoxia and increased per-sistent sodium channel activity, in order to

find a selective method for blocking thesechannels. Suppression of this sodium entrymay provide a new method for reducing celldamage during heart attacks and strokes, soimproving the prognosis for patients.

Further reading

Ju, H.-K., Saint, D.A. and Gage, P.W. (1996). Hypoxia increases persistent sodium current in ratventricular myocytes. Journal of Physiology, 197, 337-397.

Muscle Research at the JCSMR

by Angela Dulhunty

Research into skeletal muscle physiologybegan at the JCSMR in 1958 with innova-tive work by Arthur Buller, John Eccles andRosamond Eccles on cross-innervationwhich showed that the central nervous sys-tem was responsible for determiningwhether a skeletal muscle was a fast-twitchdynamic muscle or a slow-twitch tonic mus-cle. Russell Close, appointed a ResearchFellow in 1960, followed Eccles’ lead instudying fast and slow twitch muscle. Theemphasis of his research was more thedynamic properties of the muscles, ratherthan their regulation by innervation, and hedeveloped methods for studying intracellu-lar calcium transients in single musclefibres.

Angela Dulhunty joined the Departmentof Physiology in 1984 and, although she didnot collaborate with Close, they had com-mon interests in the properties of fast- andslow-twitch muscle. Her interests then andnow have been focussed on the process ofexcitation-contraction coupling, which isthe mechanism responsible for turning themuscle surface membrane action-potentialinto calcium release from internal stores inthe muscle’s sarcoplasmic reticulum. Mus-

cle contraction depends entirely on thatrelease of calcium ions.

The activities of the Muscle ResearchGroup from 1984 to 1998 evolved with theintroduction of new techniques and the dis-covery of key proteins in the moleculartransduction of the action potential into cal-cium release from the sarcoplasmic reticu-lum. In the early 1980s little was knownabout the mechanism of excitation-contrac-tion coupling or the nature of the moleculesinvolved in the process. In 1984 the groupcompleted the first, technically challenging,studies of asymmetric charge movement inmammalian muscle. These studies requiredthe insertion of three microelectrodes inclose proximity within 50µm of the tendonend of a muscle fibre, and then sophisticatedanalyses of recorded electrical signals. Theexperiments revealed minute electrical cur-rents generated by the movement of parts ofvoltage sensitive molecules through themembrane in response to a change in mem-brane potential. These minute electrical sig-nals were the first step in the process ofexcitation-contraction coupling.

In 1987 experiments were completedwhich led to the confident prediction that

the dihydropyridine receptor calcium releasechannel was the voltage sensor for excita-tion-contraction coupling, and that themovement of parts of the protein through themembrane generated the charge movementrecorded earlier. Our predictions were con-firmed in 1989 when Beam and colleaguesshowed that deletion of the dihydropyridinereceptor abolished excitation-contractioncoupling. During the same period(1984–1987) members of the Group, using acombination of freeze-fracture and thin sec-tion electron microscopy, identified novelstructures in the triad junction that wereassociated with excitation-contraction cou-pling. The triad junction in muscle sepa-rates the surface/T-tubule membrane andthe sarcoplasmic reticulum. The T-tubulemembrane of the triad contains the dihy-dropyridine receptor voltage sensor, whilethe adjacent sarcoplasmic reticulum mem-brane contains the calcium release channelwhich allows calcium to flow from thelumen of the reticulum following the sur-face action potential. Structures were iden-tified that were continuous betweenT-tubule and sarcoplasmic reticulum mem-branes, and presumably represented amacromolecular complex containing thedihydropyridine receptor and the calciumrelease channel.

In the late 1980s the calcium releasechannel in the sarcoplasmic reticulum wasidentified and purified, largely due to itshigh affinity for the plant alkaloid ryan-odine, hence the protein became know asthe ‘ryanodine receptor’. The Groupquickly developed the lipid bilayer tech-nique for recording single channel activityfrom ion channels in the sarcoplasmic retic-ulum including ryanodine receptors. Dis-coveries during the 1990s have been relatedfirstly to the regulation of ryanodine recep-tor by cytoplasmic factors and by parts ofthe dihydropyridine receptor and secondlyto anion channels in the sarcoplasmic retic-ulum membrane.

The first studies were devoted to the reg-ulation of the ryanodine receptor channelactivity by calcium and magnesium ions.Activation of the ryanodine receptor by

Ca2+ is the basis of excitation contractioncoupling in cardiac muscle, while inhibitionby magnesium is important in suppressingskeletal ryanodine receptor activity whenthe muscle is not contracting. The findingsshowed clearly for the first time how theCa2+ activation and Mg2+ inhibition sitesdiffered in skeletal and cardiac muscle, andthat two different sites on the protein wererequired for Ca2+ activation and Mg2+inhibition sites. At the same time othermembers of the Group were conductingground-breaking work into the effects ofsulfhydryl modification on the activity ofthe ion channel, and others were studyingthe all important regulation of the channelby the FK506 binding protein FKBP-12.FKBP-12 regulates the co-ordinated open-ing of the ryanodine receptor channel by thefour subunits of the protein and is importantin excitation-contraction coupling.

Three types of anion channels werefound in the sarcoplasmic reticulum mem-brane. The first, a channel identical to themitchondrial voltage dependent anion chan-nel, was rarely seen, but was resident in thesarcoplasmic reticulum and was localised tothis organelle using ultrastuctural tech-niques. A second channel, the large chlo-ride channel, is constitutively open inbilayers and contained a myriad of conduc-tance states. The third type of channel, thesmall chloride channel, is highly regulatedby Ca2+, by inositol phosphates, by ATPand by redox state. The small chloridechannel has a high phosphate permeability,and is considered to allow phosphate move-ment into the sarcoplasmic reticulum andthus to be important in muscle fatigue.

In the late 1990s work continued withthe ryanodine receptor ion channel, but themain focus has been on the II-III loop of thedihydropyridine receptor which is the partof the voltage sensor essential for excita-tion-contraction coupling. It is currentlybelieved that a protein/protein interactionbetween the II-III loop of the dihydropyri-dine receptor and the ryanodine receptor isan essential step in excitation-contractioncoupling. The conformational change in themembrane-spanning segment of dihydropy-

458


459

16. Neuroscience

ride receptor (measured as asymmetriccharge movement) is transmitted to the II-III loop of the protein and thus communi-cated to the calcium release channel. Theeffects of peptides corresponding to differ-ent parts of the II-III loop have been exam-ined on ryanodine receptor channel activity,and strong activation of the channel by twodistinct parts of the loop have been found.Using Nuclear Magnetic Resonance thestructural requirements for activation of theryanodine receptor by these fragments ofthe II-III loop have been determined.

In summary, muscle research at theJCSMR has evolved over the past 40 yearsfrom the definition of the macroscopicproperties of skeletal muscle in the wholeanimal and its regulation by the central

nervous system, to the elucidation of excita-tion-contraction coupling in intact fibres, tothe molecular events that occur in the mus-cle’s membranes during excitation-contrac-tion coupling. The molecular mechanismsinvolved in excitation-contraction couplingwill continue to be studied into the 21st cen-tury and our insight into the processes inskeletal and cardiac muscle contraction willcontinue to expand. With increased knowl-edge of molecular structure and functionnew and more effective drugs are likely tobe developed to combat heart failure, mus-cular dystrophy and other diseases leadingto muscle weakness, and also drugs for ter-minating episodes of malignant hyperther-mia.

Further Reading

Dulhunty, A.F. (1992). The voltage-activation of contraction in skeletal muscle. Progress inBiophysics and Molecular Biology, 57, 181-223, 1992.

Dulhunty, A.F., Junankar, P.R., Eager, K.R., Ahern, G.P. and Laver, D.R. (1996). Ion channels inthe sarcoplasmic reticulum of striated muscle. Acta Physiologica Scandinavica, 156, 375-385.

Angela Fay Dulhunty (1946–) graduated BScfrom the University of Sydney in 1969 andreceived her PhD from the University of NewSouth Wales in 1973 (DSc 1988). Following a twoyear Muscular Dystrophy Postdoctoral Fellowshipat The University of Rochester Medical Centre,she was a Lecturer in the Department of Anatomy,University of Sydney (1976–1980), SeniorLecturer (1980–1984). From 1983–1984 she wasa Fellow in the Nerve Muscle Research Centre atthe University of New South Wales. Appointed aFellow in the Department of Physiology, JCSMR,in 1984, Senior Fellow in the Division ofNeuroscience in 1988, and Professor in 1997, shehas continued her research into muscleexcitation-contraction coupling as Leader of theMuscle Research Group since 1989, from August1998 in the School’s Division of Biochemistry andMolecular Biology.

Accueil BIU Santé — BIU Santé, Paris - 16 Neuroscience · 2007-03-29 · Foster, S.G. and...

Documents

Transcript of Accueil BIU Santé — BIU Santé, Paris - 16 Neuroscience · 2007-03-29 · Foster, S.G. and...