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PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY LEEDS MEETING

12-13 January 1979 DEMONSTRATIONS The measurement of ionized calcium concentration in chicken plasma during eggshell formation and in paired samples of fetal and maternal ovine plasma By M. R. LUCK. Department of Animal Physiology and Nutrition, University of Leeds, Leeds LS2 9JT Calcium activity in the blood is represented by the ionized calcium fraction, and in mammals this can be as much as 50 % of the total blood calcium (Radde, Parkinson, Skeepers & Luckham, 1971). In laying hens, because of the presence of calcium binding yolk proteins, only about 20 % of the plasma calcium is ionized (Luck & Scanes, 1979) but it is from this fraction that the egg-shell calcium is derived (Urist, Schjeide & McLean, 1958). A method has been devised for measuring ionized calcium in avian plasma over the laying cycle using an anaerobic technique with a calcium specific electrode. The method has also been used to study the fetal-maternal calcium activity gradient in the sheep. Anaerobic sampling was achieved using a 2 ml. syringe by filling the dead space between the plunger end and the needle tip with Tris buffer (pH 7-4, containing 100 units of heparin when sampling from laying hens). The full syringe was capped and centrifuged with the capped end towards the centre of spin. To maintain anaerobic conditions the plasma was not decanted but was injected directly from the syringe into the electrode system. A Calcium Selectrode (F2112, Radiometer, Copenhagen) was adapted for flowthrough operation by means of a specially constructed Teflon cap. The chamber thus formed around the end of the electrode was connected in series with a reference calomel flow-through electrode (Orion Research, Cambridge, Mass.), the output being displayed as a relative millivolt (mV) reading on a digital pH meter (Orion Research). Each sample was assayed as duplicate 0*5 ml. aliquots of plasma, the ionized calcium concentration being obtained from the mean relative mV value using a curve constructed from CaCl2 standards (0.25-2-00 mm in buffer). Between each measurement the buffer was pumped through the electrodes as a wash and as a zero reference. Standard CaCl2 solutions had a coeXcient of variation of 4-5 % (n = 5). Repeated samples of plasma from a single laying hen showed a coefficient of variation of 7-4 % (n = 5). Samples could be stored for 10 hr at 4 'C before assay without significantly affecting the measured ionized calcium concentration. In the laying hen the plasma ionized calcium concentration fluctuated sinusoidally over the laying cycle with a significant fall associated with the period of shell calcifica0022-3751/79/3300-0000 $01.00 © 1979 The Physiological Society I9

PHY 290

2P 2PROCEEDINGS OF THE tion. Maximum levels (mean 1P60 mM) occurred 3-6 hr after oviposition with mintimum levels (mean 1-14 mM) at 3-6 hr prior to oviposition of the following egg. The ionized calcium concentration in ovine fetal plasma was found to exceed that in maternal plasma (fetal: maternal ratio of 1 6:1). This fetal-maternal difference reflects a similar trend in plasma total calcium concentration and ultrafilterable calcium concentration. The percentage ionization of the total calcium in fetal plasma was very similar to that in maternal plasma despite fetal plasma protein concentrations which are only 50% of maternal values. This strongly suggests that the calcium gradient which exists in favour of the fetus is not the result of increased binding of calcium by fetal plasma proteins. Supported by the British Egg Marketing Board. REFERENCES LUCK, M. R. & SCANES, C. G. (1979). Comp. Biochem. Physiol. (in the Press). RADDE, I. C., PARKINSON, D. K., SKEEPERS, J. & LUCKHAM, A. (1971). Clin. Chem. 17, 1002-1006. URIST, M. R., SCHJEIDE, 0. A. & MCCLEAN, F. C. (1958), Endocrinology 63, 570-585.

The pond-snail brain - an answer to a neurophysiologist's prayer? BY J. MiLLs and W. WINLOW, Department of Physiology, University of Leeds, Leeds LS2 9JT The central nervous system of the pulmonate mollusc Lymnaea stagnalis (L.) has several attractive features for students of neurobiology. Unlike the brains of many of the terrestrial snails the brain of Lymnaea is surrounded by relatively little connective tissue and its excision and preparation for experimental purposes is therefore relatively simple. The somata of many large identifiable neurones and neuronal clusters lie on the external surfaces of the ganglia and are specific in their position and coloration (anything from bright orange to white). Furthermore, individual neurones are easily impaled with micro-electrodes and have quite specific electrophysiological properties. The external location of the cell bodies on the ganglia also makes them amenable to micro-iontophoretic application of neurotransmitters, drugs, etc. Either cobalt chloride or the dye Procion yellow may be injected ionophoretically into identified cells in order to elucidate axonal branch patterns or dendritic structures (Fig. 1 A). In addition, the short peripheral nerves facilitate retrograde filling of cell bodies in the central ganglia and this is often a useful preliminary step in mapping studies (Fig. 1 B). However, because of the awkward position of nerves on the pedal ganglia the conventional 'wick' method cannot be employed and 2 M cobalt chloride is introduced on to the cut nerve endings through a glass suction pipette. Another useful facet of the brain of Lymnaea is the relative lack of condensation of the ganglia of the C.N.S. Interganglionic interactions among neurones may therefore be studied with ease. We have not yet carried out whole animal experiments, but Hekstra & Lever (1960) indicate that Lymnaea is well able to withstand and survive drastic surgical manipulation. It is also cheap and easily maintained in the laboratory. Finally it has

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PHYSIOLOGICAL SOCIETY, JANUARY 1979

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Fig. 1. Camera lucida drawings of (A) cobalt-impregnated giant neurone of right pedal

ganglia (S, statocyst) and (B) cell bodies impregnated with cobalt via the right medial pedal nerve (shaded neurones, dorsal; unshaded neurones, ventral).

an interesting - though admittedly slow - behavioural repertoire and ought to prove useful for neurophysiological studies on the control of specific behaviours, as has a related species (Kater, 1974). Supported by an S.R.C. project grant to W.W. REFERENCES

HEKSTRA, G. P. & LEVER, J. (1960). Proc. K. ned. Akad. Wet. C 63, 271-282. KATER, S. B. (1974). Am. Zool. 14, 1017-1036. 19-2

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PROCEEDINGS OF THE

Microperfusion of isolated renal tubule segments in vitro in the rabbit BY D. J. POTTS. Department of Physiology, Leeds University, Leeds LS2 9JT Segments of renal tubules are dissected from the kidneys of freshly killed New Zealand white rabbits and perfused by a modified version of the technique originally described by Burg, Grantham, Abramow & Orloff (1966). The tubule segment is mounted upon two pipette assemblies which allow fluid to be perfused into one end of the tubule and collected from the other end; the tubule is maintained in a separate bathing fluid which can be rapidly changed. The perfusion pipette assembly consists of four concentric glass pipettes with tips constructed to hold the tubule, to perfuse fluid into its lumen, to exchange the perfusion fluid, and to support it in a way that provides a good seal between tubule and pipette. The collecting pipette assembly consists of three concentric pipettes to hold the tubule, to collect and measure the volume of the perfusate, and to support and seal the tubule. Both pipette assemblies are mounted upon micromanipulator stands situated on either side of an inverted microscope which carries the bath upon its stage. The bathing fluid is maintained at 37 'C and stirred continuously by bubbling with a suitable gas mixture. It has been found necessary to close the bath in order to maintain the gas tensions of the bathing fluid. Tubule segments dissected from any part of the tubule, from any depth of the kidney, may be perfused and bathed with any chosen solutions whilst the 'reabsorption' rate and the transport of ions and solutes in either direction across the tubule wall are measured. Transtubular potential differences are measured from agar bridges in contact with the perfusion and bathing solutions. Perfusion of a segment of proximal convoluted tubule and the associated techniques of micropipette construction, dissection, and microvolumetric measurement were demonstrated. Supported by the Medical Research Council.

REFERENCE BUnG, M., GRANThAM, J., ABimAmow, M. & ORLOFF, J. (1966). Am. J. Physiol. 210, 1293-1298.

The collection of blood and urine from the conscious dog BY KAREN LEE and R. A. SUMMER11JL. Department of Physiology, University of Leeds, Leeds LS2 9JT A method of stimulation of aortic cbemoreceptors in the dog BY R. HAINSWORTE, F. KARIM, 0. A. SoroLA and L. M. WOOD. Departments of Cardiovascular Studies and Physiology, University of Leeds, Leeds LS2 9JT There have been few studies on aortic chemoreceptor reflexes presumably because of the relative inaccessibility of the receptors. The method described here enables us to stimulate most aortic chemoreceptors without obstructing the circulation.

5P PHYSIOLOGICAL SOCIETY, JANUARY 1979 A dog is anaesthetized, the left chest widely opened and the descending aorta mobilized by dividing the upper four pairs of intercostal arteries. Threads are placed round the brachiocephalic artery and the ascending aorta. About 3 cm of the left subclavian artery is mobilized. The animal is given heparin and the circuit (Fig. 1), filled with dextran or blood, is connected as follows. A temporary bypass, to perfuse Constant pressure

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The head is perfused at constant flow through cannulae inserted into the left subclavian artery and the central end of a common carotid artery. The carotid sinus regions may also be isolated. Finally, a pouch is made that contains most of the

PROCEEDINGS OF THE 6P arteries to the aortic chemoreceptors (Coleridge, Coleridge & Howe, 1970), by tying the ascending aorta on to the cannula and tying the brachiocephalic artery on to a tube inserted through a carotid artery. The pouch can be perfused with arterial blood, venous blood from the inferior vena cava or blood from an oxygenator. This preparation, which effectively isolates the stimulus to the aortic chemoreceptors, as confirmed by injections of dye or indian ink, allows the study of cardiovascular reflex responses. This work was supported by the British Heart Foundation, M.R.C., and the Wellcome Trust. REFERENCE

COLETRDGE, H. M., COLERIDGE, J. C. G. & HowE, A. (1970). Circulation Re8. 26, 235-247.

A method for determination of vascular capacitance changes in a vascularly isolated region of the dog BY R. HAInswORTH, F. KARIM, K. McGREGOR and L. M. WOOD. Departments of Cardiovascular Studies and Physiology, University of Leeds, Leeds LS2 9JT If a region of the body is vascularly isolated, perfused at constant flow and drained from a vein at constant pressure, a transient change in venous outflow signifies a change in vascular capacitance. The capacitance response can be quantified by integration of the change in outflow with respect to time. This can be done by measuring the area from the outflow trace. The main difficulty is in estimating this area since techniques such as planimetry are laborious and cannot readily provide information during the course of an experiment. The method we describe involves immediate on-line computation of volume changes. A dog is anaesthetized and a hind limb is isolated from the rest of the body by tying the three main muscle groups at the upper end of the leg but avoiding damage to the femoral vessels and the femoral and sciatic nerves. The limb is perfused through the femoral artery at a constant flow which is monitored by an electromagnetic flowmeter. The outflow drains from the femoral vein into a reservoir passing either through a flowmeter transducer or a bypass. To determine a capacitance response the following sequence is followed and the flowmeter output from each stage is stored in separate data files of an on-line computer. A manual switch changes the file. (1) Zero flow is recorded by clamping the flowmeter tube and diverting the blood along the parallel channel. (2) A known calibrating volume of blood is injected through the flowmeter. (3) Outflow is diverted through the flowmeter and a baseline flow value is stored. (4) The output of the flowmeter during an intervention or series of interventions is stored in up to eight files. The computer is programmed to integrate the signals stored in each file and to subtract from each an integrated signal equivalent to a control value. These signals are expressed as changes in volume by reference to the initial calibrating volume. Thus up to eight sequential changes in volume are obtained. The advantages of this method are that repeated flow calibrations are performed thus allowing for effects of changes in haematocrit or flowmeter zero and sensitivity,

7P PHYSIOLOGICAL SOCIETY, JANUARY 1979 results are immediately available, and observer error as may occur with planimetry is eliminated. This work was supported by the British Heart Foundation, M.R.C., and the Wellcome Trust.

Estimation of cardiac output in man by a single breath method BY T. G. DOBIE, R. HAINSWORTH, A. F. HOLTON and M. MOHAMMED. Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT During a prolonged expiration the tension of CO1 in the alveoli rises towards that in mixed venous blood. This results in a progressive reduction in CO2 exchange as shown by a reduction in the instantaneous respiratory exchange ratio (Rin8). Cardiac output can be determined from the following equation which is a modification of the Fick equation: = o/4/7 (slope of PCO2--Rinst plot), (1) where 4 is cardiac output (1. min-'), To, is oxygen uptake (s.t.p.d. ml. min-) and 4-7 is slope of CO2 dissociation curve (ml. l.-' mmHg-). Details of the derivation of this equation are given by Kim, Rahn & Fahri (1966) and Buderer, Rummel, Sawin & Mauldin (1973). The method we describe involves the use of a continuous flow system for determining oxygen uptake and a mass spectometer and a computer for determining the slope of the Pco. - R1lt line. Apparatus required. Alveolar air tube: this is a steel tube, 2-5 cm diameter, 90 cm length, with a 2-way valve and mouthpiece at one end which allows inspiration from room air and expiration down the tube. Beyond the valve there is a sliding shutter which when lowered restricts expiration through a hole 3 mm diameter. A sampling tube is attached proximal to the restriction. Mass spectometer: a V. G. Micromass 501 draws air continuously from the sampling tube to determine Po2 and P(o,. Computer: the slope of the PCoS/RIng curve is determined on-line by a PDP-12 computer (D.E.C.). Oxygen uptake: this is determined by use of a continuous flow method with a Servomex type OA 137 oxygen analyser (Kappagoda & Linden, 1972). Procedure. The subject must be in a steady state of rest or exercise. He then performs the single-breath manoeuvre, inspiring room air and expiring down the tube. After the initial expiration of dead space air the shutter is lowered to prolong expiration over about 10 sec. The expired air is sampled by the mass spectometer the output of which is fed into the computer. Next, the subject is connected to the oxygen uptake apparatus and the steady-state value of Io, is noted. Finally, the single breath procedure is repeated. The value of Vo2 is fed into the computer via a teletype. Cardiac output is computed, according to equation (1), for each of the single breath values, and the two values are averaged. This work was supported by the British Heart Foundation. M.M. is an Iraqi Government Scholar. REFERENCES BuDERER, M. C., RUMMEL, J. A., SAWIN, C. F. & MAULDIN, D. G. (1973). Aerospace Med. 44, 756-760.

KAPPAGODA, C. T. & LINmEN, R. J. (1972). Cardiovascular Re8. 6, 589-597. KIM, T. S., RAHN, H. S. & FAnnI, L. E. (1966). J. appi. Phy8i. 21, 1338-1344.

PROCEEDINGS OF THE

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A twin-barrelled tungsten-glass micro-electrode for extracellular recording from neurones BY S. DoNOGHUE* and C. KIDD. Departments of Physiology and Cardiovascular Studies, University of Leeds, Leeds LS2 9JT Several techniques for fabricating tungsten-glass micro-electrodes have been described previously (e.g. Freis & ZieglAnsberger, 1974; Ainsworth, Dostrovsky, Merrill & Millar, 1977). However, the micro-electrode puller requires modification and tip marking requires an additional coating to the tip. We have devised a simple technique which uses a conventional micro-electrode puller to provide a tungstenglass micro-electrode and which provides an additional adjacent glass micropipette which is available for ionophoretic application of drugs or a marker dye, e.g. pontamine sky blue (Hellon, 1971). A 10 mm section of a straight length (38 mm) of tungsten wire (diam. 250 jsm) is immersed in saturated KNO3 solution (> 200 g/I00 ml.) and etched by passing 12 V a.c. between the electrode and a carbon rod. The initial current is adjusted to 0-6-0*7 A using series resistors and monitored until it falls to 58 % of the initial value. A smooth taper is achieved by bubbling this solution with air. The wire is cleaned and dried. The unetched end is pushed into a 75 mm length of 'theta' borosilicate glass tubing (Clarke Electromedical) which has the opposite end plugged with Plasticine. The tube and wire are mounted in a vertical micro-electrode puller and adjusted so that the shoulder of the wire is level with the top of the furnace. The glass then seals to the tungsten wire as the tubing is heated and pulled. Appropriate valves for heater and solenoid currents are found by experiment. Excess tungsten wire protruding from the tip is etched off in a dilute KNO3 solution (3 g/100 ml.) with 12 V a.c. The adjacent barrel is filled with pontamine sky blue. The over-all tip diameter of the composite electrode is 10-20 /sm. The impedance of the tungsten barrel ranges from 100 to 700 kQ at 100 Hz. The resistance of the second barrel when filled with 2 % pontamine sky blue in 0-5 M sodium acetate solution is 1-10 M.Q. The manufacture and use of the electrodes was demonstrated. We are grateful for the support of the M.R.C., the Wellcome Trust and the British Heart Foundation.

REFERENCES

AINswoRTH, A., DosTRovsKY, J. O., Mki&Riuu, E. G. & MILLAR, J. (1977). J. Physiol. 269, 4P. Fnxis, W. & ZIEGLANSBERGER, W. (1974). Expl Brain Re8. 21, 441-445. HELLON, R. F. (1971). J. Phypiol. 214, 12P. *

Present address: Department of Physiology, University of Birmingham.

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Brain stem neuroses responding to stimulation of cardiac and pulmonary branches of the vagal nerves in the cat BY S. DoNOGIEuE*, C. KIDD and P. N. McWnTLmM. Department of Cardiovascular Studies and Physiology, University of Leeds, Leeds LS2 9JT Recently we (Donoghue, Fox, Kidd & Koley, 1977) have described the location of neurones within the brain stem which are excited by unmyelinated fibres in the cardiac branches of the vagal nerves. The purpose of this demonstration was to illustrate the techniques for stimulating and recording from these neurones and the criteria used to differentiate recordings from presynaptic and post-synaptic elements excited by C-fibres in the central nervous system. Experiments are carried out on cats anaesthetized with sodium thiopentone (30 mg/kg) or chloralose (80 mg/kg). The thorax is opened by removing ribs 3-7 and the lungs ventilated with a Starling Ideal pump. The upper right lobe of lung is removed and the azygos vein cut between ligatures. The cardiac and pulmonary branches of the thoracic vagal nerves are identified and the peripheral ends tied with fine thread. A small sheet of polythene is placed beneath the nerves which are then placed over pairs of fine chlorided silver wires for stimulation. The electrode-nerve assembly is then stabilized by filling a trough, created by lifting the edges of the pleura, with Silgel 604 (Donoghue, Fox & Kidd, 1977). The dorsal surface of the brain stem is exposed and the cerebellum gently retracted. Extracellular recordings of single neurone activity are made using the double-barrel tungsten-glass microelectrodes (Donoghue & Kidd, 1979) with conventional amplification and display systems. We are grateful for the support of the M.R.C., the Wellcome Trust and the British Heart Foundation. REFERENCES

DONOGHUE, S., Fox, R. E. & KIDD, C. (1977). J. Phy8iol. 269, 22P. DoNoGHUx , S., Fox, R. E., KIDD, C. & KoLEY, B. N. (1977). J. Phy8iol. 270, 44-45P. DONOGHruE, S. & KIDD, C. (1979). J. Phy8iot. 290, 8-9 P. * Present address: Department of Physiology, University of Birmingham.

The identification of the cell bodies of vagal efferent fibres of pulmonary and cardiac origin in the cat using horseradish peroxidase By C. KIDD and P. N. MCWITTLAM. Departments of Cardiovascular Studies and Physiology, University of Leeds, Leeds LS2 9JT Previous studies in which horseradish peroxidase (HRP) has been used to identify the location of the cell bodies of thoracic vagal efferent fibres have relied on the injection of HRP into the regions of the sino-atrial and atrio-ventricular nodal tissue (Todo, Yamamoto, Satomi, Ise, Takamata & Takahashi, 1977). However, HRP solution escaping from the injection site will be taken up by any nerve terminals or damaged axons and transported retrogradely to the cell bodies. This is a serious disadvantage of this technique. We restrict the access of HRP to the cut ends of fine vagal branches and great care is taken to ensure that there is no spillage of the material on to adjacent tissues.

lop PROCEEDINGS OF THE Experiments are carried out on cats anaesthetized with sodium pentobarbitone (40 mg/kg). The thorax is opened by removing ribs 3-7 and the lungs are ventilated with a Starling Ideal pump. The fine thoracic vagal branches passing to the heart and lungs are identified and freed (0-5-1 cm). The cut central end is placed on a small (3 x 7 mm) black plastic plate and the edges are sealed with a wall of silicone grease through which the nerve passes. The edges of the pleura surrounding the plate and nerve are lifted to create a pool which is filled with warm (37 0C) paraffin oil to immerse the plate and nerve. A small (2-3 1d.) drop of 30 % HRP (Sigma, type VI) in 0.9% NaCl solution is placed on the end of the nerve where it remains for 30-40 hr. Cardiovascular and respiratory parameters are continuously monitored and anaesthesia maintained during this time. At the end of this period heparin (1000 i.u., i.v.) is given and the brain perfused with 200-300 ml. 0 9 % NaCl solution followed by a similar volume of 1.25% gluteraldehyde in phosphate buffer (pH 7.4). The brain stem is removed, stored overnight in fixative and serially sectioned with a freezing microtome at 10-50 #um. The sections are then processed for the detection of HRP using the p-phenylenediamine dihydrochloride/pyrocatechol technique (Hanker, Yates, Metz & Rustioni, 1977). The demonstration showed details of the technique. We gratefully acknowledge the support of the M.R.C., the Wellcome Trust and the British Heart Foundation. REFERENCES

HANKE, J. S., YATES, P. E., MmTz, C. B. & RusTIoNI, A. (1977). Hitochem. J. 9, 789-792. TODO, K., YAMA oro, T., SATOMI, H., IsE, H., TAKATAMA, H. & TAKAHASH, K. (1977). Brain Re8. 130, 545-550.

Chloralose anaesthesia using a continuous infusion system in dogs BY C. T. KAPPAGODA, R. J. LINDEN and D. A. S. G. MARY. Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT It is now established that stimulation of atrial receptors by the distension of small balloons at the pulmonary vein-atrial junctions in chloralose anaesthetized dogs results in a reflex increase in heart rate and a reflex increase in urine flow (Linden, 1976). In experiments investigating these reflexes, care is taken to maintain a steady state of light anaesthesia. Thus in earlier experiments, and following the initial intravenous infusion of a solution of z-chloralose (dose 0*12 g/kg, Etablissements Kuhlmann, Paris), anaesthesia was maintained by further intermittent intravenous infusions of chloralose (approximately 10 mg/kg every 10 min). Eventually a method of induction and maintenance of anaesthesia which involved a continuous intravenous infusion of chloralose solution has been developed and used, and was the

subject of this demonstration. This work was supported by the British Heart Foundation and the Wellcome Trust. REFERENCE

LINDEN, R. J. (1976). Cardiology 61, 7-30.

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Atrial receptors which effect reflex inhibition in renal nerves in dogs BY R. J. LINDEN, D. A. S. G. MARY and D. WEATHERILL.* Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT Distension of small balloons at the pulmonary vein-atrial junctions in anaesthetized dogs has been shown to stimulate atrial receptors which discharge into myelinated and non-myelinated vagal fibres (Kappagoda, Linden & Sivananthan, 1977). Using graded cooling of the cervical vagi, the response in myelinated fibres to distension of the balloons was blocked at temperatures which were different from those at which the response in non-myelinated fibres was blocked, findings which allowed the conclusion that only myelinated fibres were involved in the reflex increase in heart rate and a reflex reduction in activity in efferent renal nerves observed in response to distension of the balloons (Kappagoda et al. 1977; Kappagoda, Linden, Mary & Weatherill, 1978). The demonstration illustrated the use of a graded cooling of the vagi in an attempt to identify the afferent limb of the reflex inhibition of activity in renal nerve during stimulation of atrial receptors. Dogs are anaesthetized with chloralose, artificially respired and immobilized with gallamine. Small balloons are positioned in the left upper and middle pulmonary vein-atrial junctions and within the left atrial appendage. Cooling thermodes (2.5 cm wide) are applied to both cervical vagi in pools of paraffin. The activity in efferent sympathetic nerves on the surface of the left renal artery is recorded, along with e.c.g., and pressures in the femoral artery, left atrium and trachea. The reflex increase in heart rate is prevented by the administration of bretylium tosylate

(10 mg/kg).

The magnitude of the renal nerve inhibition to balloon distension is studied with the vagi warm (32-37 'C), with the vagi cool (18 or 12 'C) and finally with the vagi rewarmed to demonstrate that the effect of cooling the vagi is similar to that on the response of increases in activity in myelinated vagal nerves during balloon distension. This work was supported by the British Heart Foundation and the Wellcome Trust.

REFERENCES KAPPAGODA, C. T., LINDEN, R. J., MARYv, D. A. S. G. & WEATHERILL, D. (1978). J. Physiot. 280, 61-62P. KAPPAGODA, C. T., LINDEN, R. J. & SIvANANTHr , N. (1977). J. Phy8iol. 266, 89-90P. *

M.R.C. Research

Fellow.

The temperature in the vagal core during graded cooling in the dog BY C. T. KAPPAGODA, R. J. LINDEN, N. SIVANANTHAN and N. SREEHARAN. Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT In 1977, Kappagoda, Linden & Sivananthan introduced graded cooling of the cervical vagi in anaesthetized dogs, showing that the increase in activity from atrial receptors with myelinated fibres was blocked at temperatures which were different from those at which the response in non-myelinated fibres was blocked. This tech-

1 2PAPROCEEDINGS OF THE 12P nique allowed the conclusion that only myelinated fibres were involved in the reflexes of increase in heart rate, increase in urine flow and reduction in activity in the renal nerves (Kappagoda, Linden & Sivananthan, 1978; Kappagoda, Linden, Mary & Weatherill, 1978). The demonstration illustrated a method of cooling the nerve so that difference between the temperature at the surface and the centre of the vagus nerve in the dog is not significant. This work has been supported by the British Heart Foundation and Wellcome Trust. REFERENCES KAPPAGODA, C. T., LINDEN, R. J., MARY D. A. S. G. &WEATHRILL, D. (1978). J. PIy8iol. 280, 61-62P. KAPPAGODA, C. T., LINDEN, R. J. & SivAxANTAN, N. (1977). J. Physiol. 266, 89-90P. KAPPAGODA, C. T., LINDEN, R. J. & SIvAwAwTHAN, N. (1978). J. Physiol. 282, 49P.

Effect of graded vagal cooling on the myelinated nerve response to atrial receptor stimulation in the dog BY C. T. KAPPAGODA, R. J. LINDEN, N. SIVANANTHAN and N. SREEHARAN. Department of Cardiovascular Studies, University of Leeds, Leeds LS2 9JT Distension of small balloons at the pulmonary vein-atrial junctions in anaesthetized dogs, stimulates atrial receptors which discharge into myelinated and nonmyelinated vagal nerve fibres. Kappagoda, Linden & Sivananthan (1977) used graded cooling of the vagus nerve to identify the type of vagal fibres involved in the reflex increase in heart rate; the blocking effect of cooling on the increase in heart rate was the same as that on the response of increase in activity in myelinated vagal fibres during balloon distension. The demonstration illustrated the effect of vagal cooling of myelinated nerve fibres on the response to balloon distension. Dogs are anaesthetized with chloralose, artificially respired and immobilized with gallamine. Small balloons are positioned in the left upper and middle pulmonary vein-atrial junctions and the left atrial appendage. A cooling thermode (2.5 cm wide) is applied to the left cervical vagus in a pool of paraffin. The activity in an afferent vagal nerve fibre is obtained at a site 2-3 cm cephalad to the cooling thermodes and recorded along with e.c.g and pressures in the femoral artery, left atrium and trachea. The conduction velocity of the fibre studied is determined by electrical stimulation of the cervical vagus 5-7 cm caudal to the recording site, using a pair of silver electrodes. The atrial receptors are then stimulated, and the increases in activity in the myelinated vagal fibre is studied with the vagus warm (32-37 TC), cooled in steps of 2 'C and finally with the vagi warm again. The experiment demonstrated that the responses in myelinated vagal fibres to balloon distension are slightly reduced at 18 0C and considerably reduced or abolished at 12-9 0C. This work was supported by the British Heart Foundation and the Wellcome Trust. REFERENCE

KAPPAGODA, C. T., LINDEN, R. J. & SIVwANTH.N, N. (1977). J. Physiol. 266, 89-90P.

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Apparatus for studying the mechanical properties of skinned muscle fibres BY R. GOODSHIP-PATIENCE, M. G. HIBBERD*t and B. R. JEWELIt. Department of Physiology, University College London, Cower Street, London WC1E 6BT This system was developed for studying steady-state and transient mechanical properties of skinned muscle fibres. The muscle bath and tension transducer designs are modifications of those originally used by Drs M. Ferenczi, Y. Goldman and R. M. Simmons (see Ferenezi, 1978, for further details). Fibres are immersed in one of ten baths machined into an anodized and chromate-finished aluminium block (Fig. 1 A). The block is recessed in a mount that allows it to be lowered, moved sideways and

1 cm~~~~~1c

Fig. 1. A, perspective drawing of the muscle baths (MB). Two parallel channels (C) in the block allow water to be circulated for temperature regulation. B, Arrangement of the servo-controlled lever (L) and force transducer (FT) around a single muscle bath. Slots in the side walls allow access for two hardened stainless-steel pins (S) to which the preparation is attached. A tripod made of carbon fibre (A.E.R.E., Harwell) is glued to a magnesium alloy bracket (B) which grips the spindle of the servo motor (SM). The degree of damping applied to FT can be adjusted with the screws e.

raised, once a new bath is correctly positioned. Each bath has two glass floors separated by dry air to prevent condensation when the block is cooled. Laser light directed through these floors allows sarcomere lengths to be determined from the resulting diffraction pattern. The preparation is suspended between a servo-controlled motor and a force transducer (Fig. 1 B). A moving-coil galvanometer (G1OOPD; General Scanning Inc. Watertown, Mass.) has been adapted to control muscle length by adding the lever shown in Fig. 1 B. In the servo-controlled mode the linear compliance at the lever tip is 2 7,um/mN. Critically damped length steps of 45 #um can be achieved in 500 ,usec. *

M.R.C. Scholar.

t Present address: Department of Physiology, University of Leeds, Leeds LS2 9JT.

14P 14BPROCEEDINGS OF THE Force changes are recorded with an AE801 strain gauge (Akers Electronics, Horten, Norway) coupled to a bridge amplifier based on a Burr Brown 3500E operational amplifier. The latter has low input noise, offset voltage and temperature drift characteristics. Rapidly unloading the force transducer causes a damped oscillation at 5 kHz which is eliminated by adjusting the oil-filled gap between the transducer element and a glass plate (G). Resolution is better than 10 1sN over a few seconds and baseline drift is unimportant when steady forces of 100-1000 suN are measured over a few minutes. The overall compliance of the force transducer is 0'4 ,um/mN. This work was supported by the British Heart Foundation. REFERENCE

FERENOZI, M. (1978). Ph.D. Thesis, University of London.

Automatic system for continuous measurement of oxygen consumption of animals weighing down to 1 gram BY W. L. DAVIES, M. C. ELPHICK, F. JOHNSON and M. TAYLOR. Medical School, Queen'8 Medical Centre, Nottingham The apparatus operates at constant volume on the principle of continuously replacing consumed 02 using a needle valve. Respired CO2 is removed by soda lime. The 02 flow that maintains constant pressure is a measure of the animal's rate of 02 consumption. The apparatus is shown in Fig. 1. It was designed to measure thermo-

Keyboard

F |Water

T

~~~Fan

_ |

1

Soda lime

Animal

chamber

Reference chamber

Ftm0anM Soda lime

a

Microprocessor

circulator

Recorder

Valve

Fig. 1. Diagram of the apparatus. The aluminium block is 20 cm high and is shown containing a new-born rat.

15P PHYSIOLOGICAL SOCIETY, JANUARY 1979 genic responses of new-born rabbits, hamsters and mice to cold exposure. Two identical chambers are cut into a solid block of aluminium; one contains the animal and the other serves as a reference chamber. A differential pressure transducer (Furness Controls Ltd, +25 mm H20) is connected between the two. Water is pumped from a thermostated bath through channels cut in the block. The ends of the two chambers are closed with thick plates of Perspex. The air in each chamber is circulated by miniature fans mounted at the top of a cylinder containing soda lime. The perforated base serves to maintain the temperature of the air. Oxygen is fed to the valve (Negretti and Zambra Ltd.) via a low-pressure regulator (Brooks Instruments). Tht, valve has a potentiometer ganged with it and is operated by a small d.c. motor. The valve is controlled by a microprocessor (Intel 8080 A). The microprocessor makes predictive control adjustments to the valve when the pressure between the two chambers differs. It also computes the 02 consumption, which it plots linearly on a chart recorder. The working range is from 0 01 to 5 ml./min and the apparatus can operate continuously for 24 hr periods. Since the volumes of the two chambers are matched it is possible to measure 02 consumption while the temperature is being changed.

An automated long-term calorimeter for rats BY G. ARMITAGE, G. R. HERVEY and G. TOBrN. Department of Physiology, University of Leeds, Leeds LS2 9JT Regulation of energy balance and therefore body weight is only evident over periods considerably longer than a day, and it is difficult, especially in animal experiments, to estimate total energy expenditure from short sample periods. The calorimeter demonstrated was designed to measure the energy output of up to four treatment groups of one to four rats effectively continuously, apart from a short daily 'down period'. It uses the 'flow-over' principle: air is drawn through animal chambers and 02 uptake and CO2 output derived from the flow rate and concentration differences. Flow is measured by a mass flowmeter (Hastings EA22) which is independent of temperature and pressure; it is checked by a wet gasmeter, itself calibrated from volumetric glassware. Oxygen (Servomex OA184) and CO2 (Grubb Parsons IRGA 20) analysers are differential, with full scale 1 % concentration difference. Reference air and 'span gas' checks are made at intervals. The animal chambers are Perspex boxes 37 x 37 x 15 cm internally. They are connected in turn for ca. 10 min to the flowmeter and analysers and are ventilated at the same rate for the rest of the cycle by individual pumps. They have internal stirring fans and moisture absorbers. A flow rate of ca. 3 I./min is appropriate to four rats. A fifth, empty, chamber provides a 'blank' in each cycle; admission of an N2-CO2 mixture to this provides further calibration. Temperature and humidity are monitored at various points and the samples to the analysers barostatted. The chambers are provided with standard 'metabolism cage' arrangements for feeding, and collecting excreta. They are in a room with controlled temperature, humidity and lighting; the rest of the equipment is in an adjacent room.

16P

6PPROCEEDINGS OF THE Wet/dry thermistor

wall

_

Fig. 1. Flow of system. d.d., digital display; m.f.m., mass flowmeter; v.f.m., visual flowmeter; f.f., sintered filter; r.h., relative humidity meter; s.v., solenoid valve; wgm, wet gas meter.

Initially operation has been by an electromechanical switching system and data collection by a potentiometric recorder. Control by an on-line mini-computer (DEC PDP-8/A) is being implemented. The M.R.C. supported the project.

A preparation for the investigation of reflex interactions between pelvic viscera in the cat BY K. FLOYD, S. B. McMAHoN and J. F. B. MopRIsoN. Department of Physiology, University of Leeds, Leeds LS2 9JT The demonstration consisted of a simple preparation that allows easy access to the nerves which innervate the pelvic viscera of the cat. Division and separation of the symphysis pubis and limited surgery in the lower abdomen give good exposure of the hypogastric, pelvic and pudendal nerves, and their branches. The preparation allows electrical stimulation of nerves and recording of neuronal activity with simultaneous monitoring of the motility of the bladder, bowel and sphincters.

PHYSIOLOGICAL SOCIETY, JANUARY 1979

17P

Vascular responses in the hind limb BY D. COTTERELL, F. KMRI AND M. WOOD. Department of Physiology, University of Leed8, Leeds LS2 9JT COMMUNICATIONS Energy expenditure of rats tube-fed at different energy levels BY G. ARMITAGE, G. R. HERVEY and G. TOBIN. Department of Physiology, The University of Leeds, Leeds LS2 9JT Although changes in voluntary food intake can certainly regulate energy balance, the ability to regulate by variation of energy ouptut is uncertain. Energy output of 6-month-old female PVG/C x WAG/C rats has been measured continuously (excepting ca. 1 hr daily) for 3 and 2 months in two experiments, with an indirect 'flow-over' 4-channel calorimeter. Metabolizable energy intake was

150

-

,D

0

C

w

100

150

200

Energy intake (kJ/rat per day)

Fig. 1. Energy output v8. intake. M, 0, First experiment, ad lib. (Oxoid) and tube-fed (synthetic diet); U, @, second experiment, ad lib. (synthetic) and tube-fed (synthetic). Four rats per cage.

determined by bomb calorimetry. In one experiment carcasses were analysed. Initial voluntary energy intake was ca. 145 kJ/rat per day; thereafter the rats were tube-fed 100-200 kJ/rat per day. During Expt. 1 the two treatment groups of eight rats apparently retained 542 and 927 kJ/rat while mean weights changed by - 8 and + 18 g; the mean difference n energy content of the carcasses was 580 kJ. In Fig. 1 energy output is plotted against intake over periods of 4-5 days commencing ca. 10 days after changes in intake. Over these periods weight change was insignificant and not related to energy balance, but there was on average an apparent