ROYAL FREE MIEETING 15 - 16 February 1991 These abstracts were acceptedl by memibers of the Society present at the Meeting

P>hy.xio1. (199!) I' /. 4-'),S. BI?oydi Fr(1' I.lq(yJ 1.)1 PRO(W1EDING/8 OF THE PH YSIOLOGIC( -4IL SOIETY .1.


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Extracellular recordings from lower thoracic and lumbar sympathetic preganglionic neurones in the anaesthetized rat 8-Y. Zhou and A.P. (Gilbev I)epartment of Physioloyy. Royal F,ee Ho.spital S'chool of Medicine. Rowlanl Hill ASieet. Londloni NIV3 2PF A preparation is being demonstrated which is used to study the physiology and pharmacology of symmpathetic preganglionic neurones (SPNs) in T13-L2 spinal segments which project into the lower lumbar svynpathetic chain. Sprague-Dawley rats (250-350 g) are anaesthetized with an I.P. inject,ion of sodium pentobarbitone (60 mg/kg) and supplementary doses of anaesthetic given as indicated (see Gilbey & Stein, 1991) in the f'orm of I.V. a-chloralose (510 mg). The trachea is cannulated and catheters placed in a femoral artery and vein for monitoring blood pressure and administration of drugs, respectively. Animals are artificially respired with 0.)-enriched room air following paralysis with gallamine (16 mg/kg) (Marks et al. 1990). End-tidal CO.) (ADC, fast response CO.) analyser), and blood gases and pH (Corning 158 pH/blood gas analyser) are monitored. All animals are vagotomized and given a pneutmothorax. An endexpiratory pressure of 2-3 mmH2.O is applied to the expiratory line to prevent

atelectasis. The left lumbar sympathetic clhain is exposed via a ventral laparotomy and stainless steel wire electrodes wrapped around it between L4 and L5 ganglia. The chain and electrodes are then embedded in silicone-based dental impression material (Provil L, Bayer Dental) and the laparotomny repaired. A dorsal laminectomy of T 11 and T12 vertebrae is performed to expose spinal segments T12-L2. The spinal cord is secured by attaching muscle clamps to mid-thoracic and lower lumbar vertebrae and lateral clamips at Ti 1-12 vertebrae. The head of the animal is fixed in a stereotaxic fiame. Electrical stimulation of the svmpathetic clhain enables SPNs to be identified antidromically. The physiological characteristics of the SPNs are studied; e.g., their respiratory modulation and baroreceptor-related inputs. Action potentials are recorded through a 4M1 NaCl-filled barrel of a multi-barrelled microelectrode assembly. Other barrels are filled with drug solutions for pharmacological experiments. I of thIe I R(C & I3H F is g' iateftilII ac kio led(ged. 'I'll e Il)u)oit REFERENCES

GiIbey. M.P. & Stein R.). (1991). .J. Marks. S.A.. Stein. R.D.. Dashwood.

IPhysiol. 432. 427-444

.R. & Gilbevy. MI.P. (1990). IB'(iwi R1,. 530. 320-:324.


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I}/!P/hyiolo(yic(dW Soci(y ROYAL FREE MEETING 1^5-16 FEBRI`ARY 1991 I'o/.

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Anterograde labelling of some short CNS pathways under acute experimental conditions in the rat and cat J. Deuchars. R.Al. Svkes. HI. (Choudlhurv anid P.N. Izzo D)epartment of Physioloyy. Royal Free Hospital School of Medicine. Rowland Hill Street. ' London NWV32SPF This technique utilizes the neuronal uptake and transport of biocytin previously described by King et al. (1989). lonophoretic injection of biocytin (5% solution in 2MI sodium methylsulphate) can be placed into electrophysiologically identified regions. Due to the fast transport of the biocytin and the sensitivity of the detection system involved (Izzo, 1991) projections firom the injection site can be traced 2-3cm folloNwing post-injection survival times of 6-12 h while the animal is maintained under anaesthesia. This technique allows for more elaborate physiological identification of the injection site as post-operative recovery is

avoided. r FERP E NCE(ES IzZ,. P.N. (1991). .J. Nellro.sci. .1leth/. (ill the Piess). King. M.A.. LIouis. P.Al.. Hounter. B. E. & Walker. DAW. (1989). IBrjti I?e,s. 497. 3(61-367.

Plhyjiol. (1991)1 Io/. 4.,X. B?oyda1 *'l Jhlety( 11;-16 Fb mury OF THE PHYSIOLOGICAL SOCIETY' .J.




Integrating cellular and hemodynamics events in didactic simulations of cardiovascular dynamics J. Lefevre and P. V'an Roev Sherrington School of Physiology and )Department of Enidocrini0oloy.y UJIDS, St Thomaas' s Hospital. Lamitbeth Palace Road. Lonldon SE] 7EH

AMany models have been built to teach cardiovascular pulsatile dynamics in system components or in the whole loop. However, these models represent only the hemodynamics aspects and neglect the underlying cellular processes and their integration in the wNhole system. WA"e have therefore developed a hierarchy of models starting writh cardiac muscle, then building models of cardiac cavities and integrating all the components in models of the whole loop. At each level, models of various complexity are used. For instance, muscle models may be dynamic springs, threeelement models or sliding filament models; excitation-contraction may be due to some periodic function or to compartmental models of the calcium mechanisms; cardiac chambers may be represented by one or more cylindrical segments, and arterial beds may be lumped or separated in various components. A modelling, simulation and authoring software has been written to develop or modify these models and to write coursewares about their behaviours. When using this software, the students run simulations and receive explanations, help, hints and questions. Attention is paid to interlevel correspondences (e.g. difference between afterload in muscle experiments and in a cardiac chamber, differences between the behaviour of the ventricle in isolated or integrated conditions, effects on septum and pericardium). When using any of the model, the students may run any conceivable experiment, change all the parameters and visualize up to 20 curves simultaneously. This experimental freedom may be constrained by the lessons designer to avoid conceptual overload in beginning students. Our goal is to teach how knowledge about the parts can be integrated into predictive knowAledge about the wAhole. W1'e want thus to foster in students mental models that allow synthesis of knowledge for problem solving. Students are not bothered by computer trivia, by mathematical abstractions or by quantitative details. They concentrate on animations and plots and they try to explain intuitively their results by building qualitative causal explanations on why the model behaves as it does. Our approach stresses the active development of qualitative mental models based on quantitative simulations. AMore than 1000 students have used these models during their premedical years at Louvain University (Brussels. Belgium).

1'roceedinqps of 7'The 1'hky.iologlical sSociety ROYAL FREE MEETING 15-16 FEBRUARY 1991

.J. hqysiol. (1991) 1l/. 438.


Measurement of the ionic composition of urine with ion-selective electrodes S.E.M. Langley, J. Graves and C.H. Fry Department of Physiology, UMDDS (St Thomas's Camtpus), Lambeth Palace Road, London SE1 7EH The ionic composition of urine is likely to determine the tendency for crystalline precipitates to be generated in the urinary tract. This process is believed to be one of the underlying factors in the pathogenesis of urinary tract stone formation. However, until now most measurements have been of the total

metal concentrations and the correlation between these values and stone formation is equivocal (Ryall & Marshall, 1990) We wish to demonstrate a system to measure simultaneously the free ionic concentrations of several ions in undiluted urine using dip-cast ion-selective electrodes (Band, Fry & Treasure, 1978). Measurement of urinary [Ca2+], [Mg2+] and pH have been possible, with a [Na+] in the range 50-250 mM. The electrodes are placed in a water-jacketed Perspex chamber, maintained at 37°C. The urine sample is placed in the chamber and additions can be made through a small hole in the roof. Using this system we have measured the change of urinary free [Ca2+], at constant pH, upon incremental additions of CaC12. A non-linear relationship was observed in which the free [Ca2+] remained fairly constant during initial CaCl2 additions. The shape of the relationship was dependent on the pH, becoming progressively non-linear as pH increased. Under these conditions the Ca2+ and H+ selective electrodes exhibited near Nernstian responses, >28 and >55mV/decade respectively. At both high pH and total [Ca] crystalline precipitates were frequently formed. This phenomenon could be more accurately measured by recording the degree of light scattering by the sample. Light from a 12 W bulb was shone through the sample and the intensity measured at the opposite side of the chamber with a general purpose photodiode (RadioSpares no. 305-462). Precipitation was recognized as a fall in photodiode output. It has also proved possible to measure free ionic [Mg2+] in these samples using a novel Mg2+ sensor (ETH 7025). In 150 mM NaCl the electrode responded equivalently to Mg 2+ and Ca2+. In the abscence of added Ca2+ the Mg2+-electrode had a slope of approximately 25 mV/decade, between 1 and 20 mM MgCl2. WNTe are grateful to Prof. W. Simon for the gift of E'I'H 7025. REFER ENCES

Baned. D.M.. Fry. '.H. & rureasure. r. (1978) J. I'hysiol. 276, 1-21'. Ryall. R.L. & Marshall. V.R. (1990). In Renal Tract Stone. ed. J.E.A.Wickham & A.C.Buck. Churchill Livingstone. London. pl) 307-331.

' 1 l3-IG FIrim)uy!/ 1/991 Phyxio1. (1991) 1 '/. 4.A.S. IoyW/dF(1i .lIlU PROC'EEDINA.(tS OF THE PH YS.IOLOG(IC.AL SOCIETY



The use of Mag-Fura-2 to measure the intracellular Mg2+ concentration [Mg2+Jj in isolated guinea-pig ventricular myocytes A. Buri C.H F'v* . J.A.S. M1c(Guiaan. 1). Noble. T. Powelland(1 VA1V'. Twist


I DS1)5 (St Thomas.s Hos/pital). Lambeth Palace RPoal. *D)epartmjent of London SE] 7EH ani(l The Laborator y of Physiololy. I Unirersity of Oxford. South Parks Ioa(l. Oxford 0X1 .31'T

Experiments used isolated guinea-pig ventricular mnyocytes. incubated wsitlh the A.M ester of Mag-Ftuia-2 (5 pMl) and performed On ani inverted stage microscope (Nikon, t)iaphot) with qIuartaz ol)jectives. Cells wer-e alternately excited at 340 and 380( nm and emitted light was collected betweeni 510 and 580 nmn (Cairn Instruments, Sittingbourne, Kent). The ratio of emitted light when excited at 340 or 380 nm was used as an index ofthe [Mg2+]1. Loaded myocytes were superfused at 37°C with a TN-rode solution containing 24 mn\M NaHCO:j and gassed with 95%02./5%CO2 (pH 7.35 ± 0.03). An increase of MAlg2+]i, as assessed by an increase of the ratio signal. occurred upon raising extracellular [Mg] or decreasing extracellular [Nal (e.g. Buni & McGuigan. 1990). Superfusion with Tv rode containing 10 mMAI NH4(l increased pHi anid acidified the sarcoplasm upon removal. NH4Cl also decreased the Mag-Fura-2 ratio upon addition and increased it upon removal (Freudenrich et al. 1990). Separate experiments with Fura-2 indicated that this intervenitioni also decreased [Ca2+ upon addition and increased it upon removal. Calibration of AMag-Fura-2 used the free acid form and Nas carried out in a solution containing, in mAM; KCl 140, HEPES 20 (pH 7.0), NaCl 10, EGTA 5, .MgCl2. 0-80. An apparent K(1 for AMag-Fura-2 of 22 ± 2.4 mMAl (S.D. n = 7) was obtained. Variation of the p(a of the calibrating solutions between 8.0 and 5.7 revealed a Ca-sensitivitvy of the probe at [Mg] betw!een 0.5 and 2.0 mM. Myocyte autofluorescence, in the absence of Mag-Fura-2, was measured under identical conditions and introduced considerable uncertaintv in the measurement of the fiuorescence ratio, varying widely from cell to cell but representing about 21% of the total emitted signal when excited at 340 nm. The [Alg2+li measured with ion-selective electrodes is about 1 mAl (Buri & AMeGuigan, 1990) so that AMag-Fura-2 measurements were carried out over an insensitive range with this experimental system. The Ca2+ sensitivity makes interpretation of the fiuorescent signal difficult whe n pHi or [Na+]i alters because of associated changes of [Ca2+]1i. eaegi,atefu,l to the AI (C and the BH F

fOr financial assistance.

RP EFERP EN(ES BI3Ii. A. & MceGuigan. J.A.S. (1994)). Kxperiieatotf Phyxsiolof/y 75. 751 7(71. ( .(.1t11ni l. E.. L\Y L.A.. Lndnn. L. E. & Liebeieian. .1. (1 99()). AmJ.-1m.//J.hsio/.. FA S Fliedentlclih. CC 4. A2 93.



Phy/siol. (1991)

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1Physiolorlicld Societl



Anatomical identification of the origin and chemistry of synaptic inputs onto vagal motoneurones in the rat and cat J. Deuchars and P.N. Izzo J)epartment of Phy.siology. Royal Free Hospital School of Medicine. Rowland Hill Street. London N,V3 2PF

Proceedings of the Physiological Society. Royal Free meeting, 15-16 February 1991. Abstracts.

ROYAL FREE MIEETING 15 - 16 February 1991 These abstracts were acceptedl by memibers of the Society present at the Meeting P>hy.xio1. (199!) I' /. 4...
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