[From the Proceedings of the Physiological Society, 20-21 April 1979]

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DEMONSTRATIONS Dynamics of the light reflex and the influence of age on the human pupil measured by television pupillometry BY P. R. BOURNE, SHIRLEY A. SMITH and S. E. SMITH. Departments of Pharmacology and Bioengineering, St Thonus's Hospital and Medical School, London SEl 7EH Television pupillometry provides a measurement of pupillary diameter every 20 msec so that reflex responses to light can be studied accurately. The cameras are infrared sensitive enabling measurement of resting diameters in darkness. Sixty healthy subjects, thirty men and thirty women, mean age 40 yr (range 1867 yr) were dark-adapted by wearing red goggles for 30 min. Vertical pupil diameters were measured with a Whittaker Corporation Series 1800 binocular television pupillometer with the subject focusing on infinity. Light flashes of duration 500 msec were directed at one eye with the light beam focused to a diameter of 1.8 mm in the plane of the pupil. Light stimulation was thus 'open loop' in that the beam was too small to be reduced by a constricting pupil. The intensity of the light was adjusted by insertion of Wratten neutral filters of different density between the source (Sylvania 2 W tungsten arc lamp) and the focusing lens system. Each individual's threshold of visual perception was measured by a forced choice method (Fison, Garlick & Smith, 1979). Ten light flashes, of intensity 106 times greater than the perception threshold, were given at 8 sec intervals and the resulting reflex responses recorded on magnetic tape for subsequent computer analysis. The latency, magnitude and time to the peak of the reflex and the resting diameter were obtained from the averaged response by computer. The first derivative yielded the maximum velocities of constriction and dilatation and the time to the maximum constriction velocity. Each of these variables was assessed for dependence on age, sex and other selected independent variables by sequential multiple linear regression analysis. Increasing age was found to reduce resting diameter, to increase times to maximum constriction velocity and to the reflex peak, and to decrease maximum constriction velocity. Except for time to peak response, the dynamic variables were strongly dependent on light reflex amplitude. REFERENCE

FIsoN, P. N., GARLICK, D. J. & SMITH, S. E. (1979). Br. J. Ophthal. 63, 195-199.

0022-3751/79/4580-0000

$01.00 © 1979 The Physiological Society

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A technique for quantitation of cardiac arrhythmias BY S. C. DENNIS, M. W. STOATE and C. B. WALDRON. Myocardial Metabolism and Bioengineering Departments, The Rayne Institute, St Thomas's Hospital, London SEl 7EH The investigation of the pathenogenesis of irregular and unpredictable disturbances of cardiac rhythm necessitates collection and analysis of large quantities of data. To process and quantify this information a computer linked arrhythmia analysis system (Fig. 1) for use with isolated perfused heart preparations has been developed. This system is based on statistical analysis of R-R intervals. An e.c.g. signal relayed into a narrow band filter (R-wave detector in Fig. 1), tuned to respond to the Coronary perfusion

Bipolar epicardial e.c.g.

Catheter (left ventricular pressure) Isolated male

E.c.g. amplifier R wave

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detector]

Pressure transducer 1 mm event marker

Amplifier

Two-channel oscilloscope

(monitor)

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Computer Fig. 1. System for monitoring electromechanical heart function.

frequency of the QRS complexes, triggers the generation of square pulses corresponding to R waves which are stored on magnetic tape for subsequent computer analysis. Preliminary processing of data, conducted at twice real time, evaluates for each one minute of real time, the number of beats counted, the mean R-R interval in seconds, the standard deviation and finally, as a secondary check of input data, prints a 100 msec bin width histogram of R-R intervals. The independent analysis of a series of one minute periods offers two advantages. It permits temporal analysis of arrhythmia development and minimizes R-R interval standard deviations arising simply out of slow alterations in heart rate. Data sets relevant to the time period of a particular perfusion condition are then grouped. For each group, the group mean R-R interval and group 'residual' standard deviation are derived from a one-tailed analysis of variance. 'Residual' standard deviation is

3P PH YSIOLOGICAL SOCIETY, APRIL 1979Y the root mean square of the variance remaining after subtraction of between set variation from total variation. As such, use of this term again overcomes variations between data sets caused by fluctuations in heart rate. Finally, to normalize group 'residual' standard deviations against gross alterations in group means, as is seen for instance between rates of normally and ischaemically perfused hearts, a coefficient of residual variation is computed. It is this expression of the group 'residual' standard deviation as a percentage of the group mean value that is employed as the unit of arrhythmia. For comparison between different experimental series average values of arrhythmia units derived from a number of hearts are employed.

An isolated, globally perfused, rat heart model of myocardial ischaemia BY A. S. MANNING. The Rayne Intitute, St Thoma's Hospital, London SEl 7E Currently, there is great interest in the proposition that certain pharmacological agents may be used to reduce tissue damage during evolving myocardial infarction. Investigation of this possibility requires the availability of appropriate models of myocardial ischaemia. A number of in vivo and ex vivo models have already been reported and these include, selective ligation of a coronary artery (Bester, Basjusz, & Lochner, 1972), embolization of coronary vessels with microspheres (Stam & De Jong, 1977) and preventing coronary perfusion during diastole by placing a one-way valve in the aorta of an isolated rat heart (Rovetto, Whitmer & Neely, 1973). In determining the suitability of any model of myocardial ischaemia certain criteria should be considered: (1) The model should be ischaemic as opposed to hypoxic only. Essentially ischaemia represents an imbalance between the myocardial demand for, and the vascular supply of, coronary blood. Not only does this create a deficit of oxygen, substrates and energy in the tissue, but also may result in an insufficient capacity for the removal of potentially toxic metabolites. The total cessation of coronary flow is not a prerequisite of myocardial ischaemia, and rarely occurs clinically. Anoxia and hypoxia alone are totally different from ischaemia and are conditions less frequently observed clinically. In anoxia or hypoxia the oxygen delivery to the myocardium is reduced by removing all or some of the oxygen in the coronary supply. Thus while the Po, is reduced, coronary flow may be normal or even elevated and substrate delivery and metabolite removal may also be normal. (2) The reduced flow reaching the ischaemic tissue should be fully oxygenated and contain a mixture of substrates normally found in blood. (3) The model should allow the measurement of a number of independent indices of tissue function and survival. Few isolated heart models fulfil these criteria. We have therefore developed a model of severe myocardial ischaemia, in which coronary flow is reduced (0.1 ml./ min/g) and the perfusate which is fully oxygenated, contains glucose, fatty acid and albumin at physiological concentrations (4, 0*5 and 0 3 m-mole/l. respectively) with a relevant binding ratio of 1-2: 1. Thermostated cannulae (to maintain accurate and constant perfusion temperature) and hollow-fibre oxygenators (to oxygenate fully,

4P PPROCEEDINGS OF THE highly proteinaceous perfusates without foaming or denaturation) have been developed for use with the model. We have measured during a 6 hr period of ischaemia, changes in heart rate, left and right ventricular pressure and electrocardiographic activity. Metabolic changes have been assessed by measurement of tissue levels of ATP, ADP, AMP, cAMP, creatine phosphate and glycogen. Additional assessments of the progression and nature of the ischaemic injury were obtained from measurements of myocardial enzyme (creatine kinase) leakage and ultrastructural changes. Significant enzyme leakage could be detected after 90 min. This leakage increased throughout the 6 hr experimental period. When corrected for differences in clearance and distribution (Hearse, 1977), the extent and profile of enzyme leakage was comparable to that shown in experimental hearts in vivo and in man. Similarly, progressive ultrastructural damage occurred during the ischaemic process and this was consistent with the observed pattern of enzyme leakage and biochemical disruption. In conclusion, the model which we have developed and characterized appears to mimic the biochemical, functional, structural and enzymic changes which are known to occur in the human heart during acute regional ischaemia. We would therefore propose that the model is of value in the investigation of interventions designed to protect the ischaemic myocardium. REFERENCES

BESTER, A. J., BAsJusz, E. & LocmEER, A. (1972). Cardiova8c. Re8. 6, 284-294. HEARE, D. J. (1977). J. Mol. Med. 2, 185-200. RovETro, M. J., WHTMER, J. T. & NEELY, J. R. (1973). Circulation Re8. 32, 699-710. STYm, H. & DE JONG, J. W. (1977). J. mol. cell. Cardiol. 9, 633-650.

Myocardial enzyme leakage: clearance via lymphatic and coronary routes in the isolated rat heart By D. J. HEARSE and S. M. HUMPHREY. The Rayne Institute, St Thomas'8 Hospital, London SEl 7EH Myocardial ischaemia or anoxia results in the leakage of cytoplasmic enzymes to the extracellular space and their appearance in the circulation is used in the detection of tissue injury (see Hearse & de Leiris, 1979). Enzyme leakage can also be used for the quantitation of infarct size during evolving myocardial infarction andthe equations upon which these calculations are based are dependent upon the characteristics of enzyme clearance from the heart. Since the rate of clearance may be determined by the route of transportation, it is important to ascertain whether enzymes leak via rapid venous drainage, slow lymphatic channels or both. Since the isolated rat heart is used extensively for the study of enzyme leakage it is important to resolve this question in this preparation. An isolated rat heart model for the separate collection of coronary and lymphatic effluent and the assay of enzyme leakage was developed based upon the preparation of de Deckere & ten Hoor (1977). The aorta is cannulated and coronary perfusion (10 ml./min . g wet weight) with oxygenated (95 % 02 + 5 % CO2) bicarbonate buffer (pH 7-4 at 37 'C) initiated. The pulmonary artery is then cannulated and the caval and pulmonary veins tied off. Coronary flow exits to the right heart and is ejected

5P PHYSIOLOGICAL SOCIETY, APRIL 1979 via the pulmonary artery. Perfusion fluid that has left the vascular bed and passed via the interstitial space to the cardiac lymphatics accumulates on the surface of the heart and drips from the apex. The flow rate ofthis fluid, which is highly proteinaceous, is less than 1 % of coronary flow. If hearts are subjected to 300 min of substrate-free anoxic (95 % N2+5 % CO2) perfusion and the coronary and lymphatic fluid assayed for creatine kinase (i.u./min. g dry wt) the leakage profiles in Fig. 1 are observed. During the first 50 min there is a constant but small leakage of enzyme to the coronary fluid and a greater but 22 5

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Fig. 1. Creatine kinase leakage from the isolated perfused anoxic rat heart, (U) coronary route (@) lymphatic route. Each point is the mean of 6, S.E. of the mean indicated by bars.

transient leakage to the lymph. Between 100 and 300 min substantial enzyme is cleared via both routes. While the enzyme activity in the lymph can be 100-fold greater (by volume) than in the coronary fluid the disparity of flow rates results in comparable amounts being cleared by each route (approx. 40 % and 60 % respectively). REFERENCES DE DECxERE, E. A. M. & TEN HOoR, F. (1977). J. mol. cell. Cardiol. 9, 13P. HEARSE, D. J. & DE LEmIs, J. (1979). Enzymes in Cardiology: Diagnosis and Research. Chichester: John Wiley.

A multiple biopsy gun for the study of three-dimensional metabolic geometry By D. M. YELLON. The Rayne Institute, St Thomas's Hospital, London SEl 7EH The zone of regional ischaemia occurring during acute myocardial infarction is highly heterogeneous. Surrounding the severely ischaemic core and separating it from normal tissue is a zone of moderately ischaemic tissue. This area, which has been designated the 'borderzone', is the target for various interventions designed to reduce infarct size. Characterizing this zone is critical to our understanding of regional ischaemia and to the rational development of therapeutic agents. Consequently single transmural biopsies have been taken and analysed in an attempt to define the 'borderzone' (Braimbridge & Niles, 1964) but this has been limited to the relatively low number of ventricular biopsies that can be successfully retrieved over a short

6P 6PROCEEDINGS OF THE period of time. The advent of the multiple biopsy drill (Hearse & Chappell, 1976) increased the resolving power by permitting simultaneous sampling and rapid freezing of 29 transmural biopsies spaced over an area of 36 x 52 mm. This technique was hindered somewhat in that sampling took several seconds, the rotating cutters twisted the biopsies during drilling and mechanical considerations prevented cutting tubes being any closer than 4 mm apart, leaving 'interspaces' that could not be sampled or assayed (Hearse, Opie, Katzeff, Lubbe, Van der Werff, Peisach & Boulle, 1977). To overcome these problems a non-rotating high-velocity projectile has been developed that is capable of retrieving forty adjacent biopsies (3 x 3 mm) over an area of 32 x 20 mm. The biopsy cutter is composed of a lattice of intersecting cutting blades 30 mm deep fused to form a cutting grid. The cutter is fired (105 mm/sec) from a modified masonry impact gun using 'low'-velocity cartridges of 6.8/11 M calibre. The velocity and momentum of the cutter is sufficient to cut forty transmural biopsies of the left ventricular wall. The resistance of the heart tissue is sufficient to force biopsies into the respective chambers of the pre-cooled matrix. Due to the sharpened edge of the cutting surface and design of the projectile head, the biopsies retrieved are directly adjacent to one another, thereby eliminating sampling 'interspaces'. After firing, the cutter, together with the biopsies, are immersed in Arcton '21' surrounded by liquid nitrogen to give a freezing temperature of - 135 0C. This process, from the time of firing to that of freezing, takes 3 sec. The biopsies can then be pressed out of the grids, epicardial and endocardial surfaces identified (with appropriate sectioning if required) and the tissue taken for analysis. In this way a high-resolution three-dimensional model of the metabolic geometry of the 'borderzone' can be constructed. REFERENCES

BRAIMBRIDGE, M. V. & NILES, R. N. (1964). J. thor. card. Surg. 47, 685-686. HEARSE, D. J. & CHAPPELL, D. A. (1976). J. mol. cell. Cardiol. 8, 803-809. HEARSE, D. J., OPIE, L. H., KATZEFF, I. E., LU-BBE, W. F., VAN DER WERFF, T. J., PEISACH, M. & BouwLI, G. (1977). Am. J. Cardiol. 40, 716-726.

Investigation of circulation model design for clinical application BY P. R. BOURNE and R. I. KITNEY. Bioengineering Department, St Thomas's Hospital, London SEl 7EH, and Department of Physiology and Biophysics, Chelsea College, London SW3 6LX Traditional methods of analysing pressure and flow measurements in clinical cardiology are mainly empirical and do not lead directly to a physiological description of circulatory state. Mathematical models of the circulation can be used to give a more direct estimate of the parameters of interest (Aaslid, 1974). This is done by comparing model outputs with the corresponding patient measurements and adjusting the model parameters to minimize the differences between them. The adjusted model parameters then represent direct estimates of the corresponding physiological ones. We are studying this method as an alternative way of interpreting simultaneous arterial pressure-flow measurements to that of calculating hydraulic input impedance. There is evidence that peripheral vasodilators may have a beneficial effect in heart failure (Chatterjee, Massie, Rubins, Gelberg, Brundage & Ports, 1978). One

7P PHYSIOLOGICAL SOCIETY, APRIL 1979 application of this model technique will be to evaluate the effect of these drugs on both arterial compliance and peripheral resistance. We have developed methods for matching digital computer models to clinical data (Bourne & Kitney, 1978) and have used these to investigate the detail needed in a model to achieve a good match. Four models have been studied including one of our own design. The measured data consisted of the femoral artery pressure and the ascending aortic flow recorded for one heartbeat. The aortic flow measurements were used as the driving function for the model. The femoral artery pressure was compared with that produced by each model and the root mean square (r.m.s.) difference between measured and simulated values calculated. This matching error was used as the criterion by which the models were assessed. We have confirmed that a single compartment arterial model with the haemodynamic parameters lumped together will not reproduce the peripheral pressure pulse amplification which is characteristic of the major arteries. Our own model divides the systemic arterial system into eleven segments. Each segment is represented by arterial-wall compliance, resistance to steady flow and blood inertia. The arterial branches terminate in peripheral resistances. We have shown, with studies on seven patients, that this model will give a good match to the femoral artery pressure with an error usually in the range of 2-4 mmHg r.m.s. To do this it was necessary to adjust overall arterial compliance, peripheral resistance and blood inertia. Studies on two other models show that increasing the number of segments does not significantly improve this match. Our model has no arterial wall damping term. We have found that incorporation of damping makes less difference to the matching error than the assumptions about the geometry of the major arteries. However, there are insufficient measuring sites that could be used simultaneously on a patient to characterize the distribution of the arterial parameters along the arteries. So we conclude that for this set of clinical data there is no justification for including wall damping terms. REFERENCES

AASLID, R. (1974). Ph.D. thesis, Division of Engineering and Cybernetics, University of Trondheim.

BoURNE, P. R. & KITNEY, R. I. (1978). Med. biol. Engng & Comput. 16, 689-696. CHATTERJEE, K., MASSIE, B., RUBINS, S., GELBERG, H., BRUNDAGE, B. H. & PORTS, T. A. (1978). Am. J. Med. 65, 134-145.

A sensitive alarm system detecting a change in the heart rate of anaesthetized and paralysed preparations in neurophysiological experiments By HisAKo IKEDA and J. S. MUCKELROY. Vision Research Unit (of Sherrington School), The Rayne Institute, St Thknoms's Hospital, London SEl 7EH In order to regulate the level of anaesthesia in a paralysed animal it is necessary to assess its autonomic responses. Could the detection of a slight increase in the heart rate be used to indicate a lightening of anaesthesia or distress? We felt a sensitive e.c.g. alarm system was necessary, since even though e.c.g. is monitored continuously during an experiment, we may forget to check the e.c.g. during the tense moments of cell recordings. Since the heart rate of cats varies from animal to animal, ranging between 3 and 5-5 Hz (240-330/min), the alarm system should be flexible enough to set the levels at which the alarm is activated.

PROCEEDINGS OF THE 8P The e.c.g. output from an a.c. amplifier is regulated to about 1 V peak to peak, using a 741 operational amplifier. This enables the input voltage to the alarm system from different animals to be relatively constant. The output of the regulating amplifier is connected to a buzzer via a Schmitt trigger operational amplifier comparator for an auditory monitor of the heart rate. e.c.g. pulses from Schmitt trigger output

Differentiating circuit (741 operational amplifier type) Low alarm

Retriggerable monostable (T. T. L. 74123) T.C. 200-700 mrsec

High alarm Monostable A (T.T. L. 74121) T.C. 1-4 msec

Monostable B (T.T.L. 74121) IT. C. 84 -300 msec And gate

Retriggerable monostable (T.T.L. 74123) T.C. 140 msec Or gate Buzzer Fig. 1. Block diagram of an alarm system detecting a change in the heart rate.

The 'quickening'- and 'slowing'-rate window are adjustable very accurately, depending on the initial heart rate of the animal. Fig. 1 shows the block diagram of the circuit. The principle of the system is to differentiate the signal at the output of the Schmitt trigger and measure the interval between successive heart beats. The level for the alarm associated with an increase in the heart rate is determined by a monostable (B) with a time constant, adjustable between 4 and 11 Hz. The output of the monostable (B) is fed via an 'AND' circuit to a retriggerable monostable to activate the alarm. To avoid any false alarm, a fixed time constant monostable (A) is necessary to delay the triggering of the monostable (B) until the end of the pulse from the differentiating circuit. The alarm associated with a decrease in the heart rate is a single retriggerable monostable with an adjustable time constant ranging between 1.5 and 5 Hz. This work is supported by the Endowmont Funds of St Thomas's Hospital.

Route for cerebrospinal fluid flow out of the cerebral ventricles in amphibians BY H. C. JONES. Biomedical Research Unit and Department of Zoology, University of Hull HU6 7RX The cerebral ventricles of amphibians have long been thought to be totally enclosed by the brain and its membranous roof. This has posed the problem: by what route does the cerebrospinal fluid (c.s.f.) reach the subarachnoid space in these animals? A recent light microscopical study (Jones, Dolman & Brocklehurst, 1978)

PHYSIOLOGICAL SOCIETY, APRIL 1979 9P has shown the caudal end of the roof of the fourth ventricle in anuran amphibians, the posterior tela (Herrick, 1935), to consist of an incomplete ependymal membrane. Furthermore, after infusion of fluorescein-labelled dextran (mol. wt. 150,000) into the lateral ventricle of Rana pipiens, the marker is seen to pass out of the ventricular system into the subarachnoid space through the posterior tela (Jones, 1978). The roof of the fourth ventricle has been prepared for scanning electron microscopy in five amphibian species: Rana pipiens, Rana catesbeiana, Xenopus laevis,

Fig. 1. Sce nning electron micrograph of the posterior tela in Rana pipiens. Ependymal cells (E), either squamous ox round in shape, are interrupted by numerous fenestrations some of which have accompanying pial fenestrations (arrows). Bar = 20 jim.

Salamandra salmandra and Ambystoma mexicanum. Intravascular fixation with a glutaraldehyde/paraformaldehyde mixture was followed by post-fixation of the trimmed hind brain in osmium tetroxide. Dehydration was in 2,2-dimethoxypropane followed by acetone. Specimens were critical point dried in C02, coated with gold and viewed in a Cambridge Stereoscan 600. The ventricular surface of the posterior tela from all species examined contains clearly defined pores or fenestrations. These are numerous interruptions in the ependymal epithelium (50-100 per animal) and vary in size from 5 to 100 sum, with some exceptionally large caudal pores reaching 200 or 300 Atm. Each fenestration is bordered by a variable number of ependymal cells with clearly defined intercellular margins. In most species the ependymal cells are squamous and low but in Rana they can also be extremely rounded. The fenestrated ependyma is overlain on the dorsal, or subarachnoid, side by a layer of pial cells loosely arranged in flat sheets which probably does not form a significant barrier to c.s.f. flow. In some species the pial layer is also fenestrated in places, giving complete continuity between the ventricles and subarachnoid space. It is suggested that the ependymal fenestrations allow the bulk flow of c.s.f. out of the ventricular system into the subarachnoid space in amphibians. The financial support of the Wellcome Trust is gratefully acknowledged.

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pPROCEEDINGS OF THE REFERENCES

HERRICE, C. J. (1935). J. comp. Neurol. 61, 297-346. JosES, H. C. (1978). Cell & Ti88ue Re8. 195, 153-167.

JoiNs, H. C., DoTxMA., G. S. & BROCKLEHURST, G. (1978). J. Zool., Lond. 185, 341-354.

Afferent discharge during voluntary movement BY A. PROCHAZKA and P. WAND. St Thomas's Hospital Medical School, London SEl 7EH, and Max-Planck-Inetitut for Experimental Medicine, Gottingen, West Germany The discharges of single afferents are recorded from implanted dorsal root electrodes during voluntary movements in the cat. Recordings from muscle spindle afferents have indicated that for muscle velocities exceeding 02 resting lengths per second (lr/sec), the modulations of spindle firing rate are largely determined by the variations in muscle length. Below 0-2 Ir/sec variations in fusimotor action can significantly contribute to the modulation (Prochazka, Stephens & Wand, 1979). When active muscle shortening was unexpectedly impeded, spindles in the shortening muscles increased their firing rates, but the increases were not as large or abrupt as might have been expected had there been strong co-activation of fusimotor and skeletomotor neurones. We are currently using imposed sinusoidal displacements of the limb in order to study the variations in dynamic sensitivity of spindles accompanying variations in extrafusal muscle activity. REFERENCE

PROCHAZKA, A., STEPHENS, J. A. & WAND, P. (1979). J. Phy~iol. 287, 57-66.

A gravimetric venous occlusion plethysmograph BY T. K. COWELL,* P. A. HURST, D. L. RUTT and D. SIZELAND. Departments of Bioengineering* and Surgery, St Thomas's Hospital, London SEl 7EH Hill, Jackson and Matthews have described a gravimetric plethysmograph and have evaluated its clinical use (Hill, Jackson & Matthews, 1974; Jackson & Matthews, 1977). We were impressed by the principle of gravimetric plethysmography, i.e. the measurement of limb blood flow by estimating its weight gain in response to venous occlusion, and have designed a new apparatus for achieving this. Our device utilizes a horizontal rigid beam hung from its centre point by a vertical aluminium-alloy spring plate, to either side of which are bonded semi-conductor strain gauges. Two further spring plates constrain the beam horizontally which together with the vertical plate form a frictionless bearing axis about which the beam can rotate. Differential loading on the beam produces deformation of the strain gauges which form two arms of a Wheatstone bridge, connected to a Bryans Southern pen recorder. The beam is supported by vertical aluminium arms bolted to a modified operating table upon which the patient lies.

PHYSIOLOGICAL SOCIETY, APRIL 1979 lip Each limb is suspended from either end of the beam in a sling mounted on a movable clamp. The position of the limbs can be adjusted to balance the beam with limbs of different sizes or to cancel out respiratory fluctuations in blood flow. Mechanical damping devices are incorporated to minimize deflexions due to inherent rhythmicity in the beam or to patient movement. Leg supports are used and these obviate the need for sedation.

JDg. 1.

Occlusion cuffs are placed around the leg proximal to the area in which blood flow is to be measured. These are inflated automatically to above venous pressure and deflexions in the trace due to weight gain in the test leg reflect arterial inflow. Calibration consists of measuring the deflexion(s) produced by a known weight (w, g) placed at the centre of maw of the tissue in which flow is measured. The deviation (d) of the trace over time (t, sec) is measured using only the initial slope of the trace as the subsequent slope reflects venous distension and capillary filtration. A measurement of the volume of the test limb (v, ml.) is made by water displacement. A calculation of arterial inflow can then be made according to the formula flow = (100 w/vt8) x d (g/100 ml. min). The illustration shows the device in use. REFERENCES

HL, C. D., JACKSON, J. R. & MIATTHEws, J. A. (1974). J. Phyjiol. 241, 71-72P. JACKsoN, J. R. & MArruEws, J. A. (1977). Br. J. Surg. 64, 876-882.

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Forward and backward correlation of motoneurone synaptic noise and muscle afferent activity By P. J. HARRIsON and A. TAYLOR. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH The technique of spike triggered averaging introduced for the study of I a afferent monosynaptic connexions by Mendell & Henneman (1971) has yielded a wealth of detail about the spindle primary projection (Watt, Stauffer, Taylor, Reinking & Stuart, 1976; Scott & Mendell, 1976) and about the secondary and other connexions (Kirkwood & Sears, 1974; Stauffer, Watt, Taylor, Reinking & Stuart, 1976). However, technical difficulties make data gathering by this method slow and costly. Certain advantages are obtained by relaxing some of the constraints of the method.

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Fig. 1. Averaged rectified neurogram from medial gastrocnemius nerve (6025 sweeps) triggering from individual e.p.s.p.s in medial gastrocnemius motoneurone of fast, fatiguable (FR) type. The time scale has been converted to one of conduction velocity assuming 0 7 msec synaptic delay and intra-cord conduction time.

Thus extracellular spike triggered averaging (Taylor, Stephens, Somjen, Appenteng & O'Donovan, 1978) shows the monosynaptic projection of single afferents to a pool of target cells, without intracellular recording. Another approach, which was demonstrated, is to average the asynchronous activity recorded from a muscle nerve with the averager triggered by the rising edges of intracellularly recorded individual e.p.s.p.s. The neurogram is filtered (200 Hz to 5 kHz), delayed by 6 msec, and rectified before averaging and the trigger is derived from positive deflexions in the intracellular synaptic noise (filtered 32 Hz to 3-2 kHz). An example of the resultant record is shown in Fig. 1. Similar results have been obtained when the triggering level was set to be crossed by only the largest on-going e.p.s.p.s. It is evident therefore that these can be attributed to monosynaptic effects of impulses in large muscles afferents. P.J.H. was aided by the Carlo Campolia fund. REFERENCES

KIRKWOOD, P. A. & SEARS, T. A. (1974). Nature, Lond. 252, 243-244. MENDELL, L. M. & HENNXMAN, E. (1971). J. Neurophy8iol. 34, 171-187. ScoTT, J. G. & MENDELL, L. (1976). J. Neurophyeiol. 39, 679-692. STAUFFER, E. K., WATT, D. G. D., TAYLOR, A., REiNKING, R. M. & STUART, D. G. (1976). J. Neurophysiol. 39, 1393-1402. TAYLOR, A., STEPHENS, J. A., SOMJEN, G., APPENTENG, K. & O'DoNovAN, M. J. (1978). Brain Re8. 140, 344-348. WATT, D. G. D., STAUFFER, E. K., TAYLOR, A., REnEXING, R. M. & STUART, D. G. (1976). J. Neurophysiol. 39, 1375-1392.

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Methods used to compare monosynaptic and multisynaptic projection of spindle afferents to jaw elevator motoneurones of different types BY K. APPENTENG, T. MORIMOTO and A. TAYLOR. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH The monosynaptic projection of jaw elevator muscle spindle afferents in the cat has been shown to be weak and sparse (Appenteng, O'Donovan, Somjen, Stephens & Taylor, 1978). Despite this, powerful tonic stretch reflexes can be obtained from these muscles and it is therefore of interest to see if supplementary multisynaptic pathways exist and if so whether they are at all specific in the motoneurone types involved. Cats anaesthetized with sodium pentobarbitone are placed in a stereotaxic frame. Electrodes are implanted for stimulating masseter and mandibular nerves in continuity and an electromagnetic vibrator connected to a screw in the mandible. After cerebellectomy the motor nucleus of the fifth nerve is explored with a micropipette and motoneurones identified antidromically. In each motoneurone the synaptic effects are recorded of graded masseter nerve stimulation and of spindle volleys set up by minute transient muscle stretches. The motoneurone type is then examined by intracellular stimulation and recording of force on the mandible. So far we have some evidence that there is a reciprocal relationship between the strength of monosynaptic and of multisynaptic e.p.s.p.s in different motoneurones. Supported by the Research Endowments of St Thomas's Hospital. REFERENCE

APPENTENG, K., O'DONOVAN, M. J., SOMJEN, G., STEPHENS, J. A. & TAYLOR, A. (1978). J. Phyaiol. 279, 409-423.

An electrode system for plotting ionized plasma calcium and pH BY D. M. BAND, L. BEYNON and P. SKINNER. Sherrington School of Phy8iology, St Thoma-s's Hospital Medical School, London SEl 7EH Several studies have shown that the plasma ionized calcium, in vitro, varies as a function of pH and that this variation is largely due to an effect on the plasma proteins (Fogh-Andersen, Christiansen, Komarmy and Siggaard-Andersen, 1978; Band & Broadway, 1979). Hass (1956) suggested that pathological serum proteins may react with calcium in a fashion different from normal serum proteins and that this may be a factor in pathological calcification. If this were true then the form of the pH dependence of the ionization could be of great importance in determining the equilibrium of the plasma ionized calcium with the bone mineral both in health and disease. Investigation of this possibility has been hampered by the lack of a suitable electrode. We have threfore modified the calcium cell demonstrated by Band, Fry & Treasure (1978). A glass pH electrode, calcium electrode and a common reference are connected in a recirculation system incorporating a silicone rubber coil. Carbon dioxide passes over the coil and diffuses into the blood producing a progressive ' respiratory' acidosis. The pH and calcium activity are displayed as an X Y plot. The cell is temperature regulated and requires approximately 1*5 ml. of plasma.

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BAND, D. M. & BROADWAY, J. (1979). J. Phyeiol., 289, 38P. BAND, D. M., FRY, C. H. & TREASURE, T. (1978). J. PhyJ8iol. 276, 1-2P. FOGH-A.NDERSEN, N., CHRISTIANSEN, T. F., KOMARMY, L. & SIGGAARD-ANDERSEN (1978). Clin. Chern. no. 2419, 1545-1552. HASS, G. M. (1956). In The Biochemietry and Phyeiology of Bone, ed. BouRNE, G. H., p. 781. New York: Academic Press.

A telemeter for ion selective catheter tipped electrodes BY D. M. BAND, T. K. COWELL and T. TREASURE. Sherrington School of Physiology and Department of Bioengineering, St Thomas's Hospital, London SEl 7EH A telemeter has been developed specifically for use with the potassium sensing catheter described by Treasure & Band (1977). The system comprises a magnetically coupled short-range transmitter-receiver link. Signal modulation is achieved by a blocking oscillator the pulse repetition frequency of which is proportional to the applied signal voltage. The transmitter consists of a dual F.E.T. voltage to current amplifier the output of which modulates the blocking frequency of a P.N.P. transistor L.C. oscillator. The blocking frequency is typically 200 Hz for a zero input signal varying linearly over about + 40 %. At the receiver (a standard car radio) the signal generates a train of constant width pulses from which a recorder output is obtained by low pass filtering. The system has some advantages over the more conventional F.M. telemeter circuits. The low-power drain gives a battery life of about 6 weeks continuous operation. In a constant-temperature environment the d.c. stability is adequate for ion-selective electrode work. Although limited to the confines of the loop receiving aerial, the signal is not easily masked by tissues. For normal recording from a static preparation the absence of any ground or common connexion to the recorder reduces the problem of interference met when using high-resistance electrodes. The complete probe can be encapsulated, sterilized, and of course ensures complete patient isolation for electrical safety. REFERENCE TREASURE, T. & BAND, D. M. (1977). J. med. Eng. Technol. 1, 271-273.

Electron microscope observations on the cytoplasmic organelles in the superior rectus oculi muscle of human fetuses BY J. FENTON, H. J. GAMBLE and J. LOUISE SCHEUER. Departments of Anatomy and

Morbid Anatomy, St Thomas's Hospital Medical School, London SEl 7EH Superior rectus oculi muscles from human fetuses of 5, 9-2, 12 and 24 cm crownrump length have been examined by electron microscopy (the preparative technique is given in Gamble, Fenton & Allsopp, 1978). Myofilaments were well differentiated; occasionally, when muscle cells were cut longitudinally, M bands were also well preserved. Droplets of amorphous material, tentatively identified as yolk were

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present in the smallest specimen and glycogen was present in this and in the 9-2 and 12 cm specimens. Smooth endoplasmic reticulum was neither conspicuous nor extensive but did sometimes show local concentrations alongside the Z bands. Transverse tubules opening to the surface were very rarely seen but similar tubules, longitudinally disposed, were to be found in association with the smooth endoplasmic reticulum so that diads, triads, quadrads and even pentads were present in all four specimens.

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Fig. 1. Cytoplasmic organelles in muscle fibres from 12 cm crown-rump length fetus. A, where filaments were transected; B. where filaments were sectioned longitudinally.

Other cytoplasmic organelles included 'dense bodies' similar to those seen in fetal

Schwann, endothelial and other cells, and some of these, on closer examination, exhibited an extremely regular internal structure (Fig. 1A). A membrane, usually complete and sometimes double, surrounded an aggregation of electron dense structures set in an orderly hexagonal array. The whole organelle had diameters of 0.40-2 x 0-3-0-15 ,um and the dense filaments (or particles) within were of approximately 6 nm diameter and set at the corners of equilateral triangles whose sides measured approximately 10 nm. Nearly all of the organelles of this kind so far seen have been found in the 12 cm specimen and may be interpreted as containing dense filaments (or stacks of dense particles) whose axes nearly correspond with that of the muscle cell. Others have been found where what appear to be filaments have been sectioned longitudinally (Fig. 1 B); as in some of the transversely sectioned examples, the overall diameter approximates to 0 4 x 0 3 rum so that it seems likely that the organelles are of rather ovoid form. Their nature is wholly obscure.

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REFERENCE GAMBLE, H. J., FENTON, J. & ALLSOPP, G. J. (1978). J. Anat., Lond. 126, 567-589.

Measurement of micromolar levels of calcium with the aid of an ionsensitive electrode BY C. H. FRY. Sherrington School of Physiology, St Thomas' Hospital Medical School, London SEl 7EH

The stimulus locked interval histogram: a method that may allow investigation of Renshaw inhibition in man. BY A. K. DATTA* and J. A. STEPHENS. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH In previous demonstrations to the Society we have shown how post-stimulus time histograms may be used in the study of motor unit reflex responses in man. Using this technique, the size and timing of the different components of a reflex are revealed as fluctuations in the probability of unit firing following each stimulus (Stephens & Usherwood, 1976; Buller & Stephens, 1977). In this demonstration we showed the use of another related statistical technique but one which can be used to examine the total reflex effect of a given stimulus rather than its individual components. Developed originally for the study of cutaneous reflex effects (Kranz, Adorjani & Baumgartner, 1973; Datta & Stephens, 1979), the technique on this occasion was used to show the effects of a synchronous muscle afferent input on motor unit firing. The subject's hand is immobilized and a motor unit action potential isolated for recording using an e.m.g. needle inserted into first dorsal interosseous muscle. As a preliminary to the main part of the demonstration it is necessary to establish the latency of reflex firing of the motor unit under study following synchronous muscle receptor stimulation. For this purpose the subject is required to maintain the unit firing steadily and a post-stimulus histogram accumulated of the time of occurrence of motor unit spikes following brief mechanical pulses applied to the muscle belly (Buller & Stephens, 1977). The required reflex latency is given by the time to the large increase in probability of firing which appears in the post-stimulus time histogram. Once this measurement has been made the main part of the demonstration can begin. Aided by a frequency meter, the subject is required to maintain the unit under study firing steadily at 10 pulses/sec. A brief mechanical pulse is applied to the muscle belly 50 msec after every third or fourth unit action potential. If the next unit discharge occurs at a time corresponding to the reflex latency determined previously then this reflex elicited spike is allowed to start an interval timer that is stopped by the next naturally occurring spike. This procedure is repeated until a histogram of 300 intervals has been accumulated. In all units studied to date, intervals collected in this way have been on average 20-30 msec longer than intervals *

Medelec Researoh Assistant.

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collected in the absence of muscle receptor stimulation. Once having caused the motoneurone to fire, the remaining effect of the muscle afferent volley is as if to prolong the succeeding interspike interval. Several mechanisms can be put forward to account for this observation, including the possible reflex effects of tendon organ stimulation by the muscle tap or -of the pause in spindle afferent discharge that must follow the synchronous activation of these receptors. While we cannot rule out these effects, the same result can be obtained for units in second dorsal interosseous following their reflex excitation by taps applied to relaxed first dorsal interosseous. This suggests to us that another type of mechanism might be involved. One attractive possibility is that the delayed firing of the motoneurone following a reflexly elicited discharge is due to Renshaw cell activity. The muscle afferent volley set up by each tap must elicit the near-synchronous reflex firing of many motoneurones including the one under study. If firing of these cells combines to excite Renshaw cells then inhibitory convergence from these cells would presumably produce some temporary reduction in the net excitatory drive on to the motoneurone under study and thus prolong its next interspike interval. REFERENCES BULLER, N. P. & STEPHENS, J. A. (1977). J. Physiol. 271, 1-2P. DATTA, A. K. & STEPHENS, J. A. (1979). J. Physiol. 292, 16P. KRANz, H., ADORJANI, C. & BAUMGARTNER, G. (1973). Brain 96, 571-590. STEPHENS, J. A. & USHERWOOD, T. P. (1976). J. Physiol. 258, 49-51P.

The formation of a pericardial like fluid from the epicardial surface of the isolated perfused rat heart BY G. J. TYSALL* and M. B. SEGAL. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH The mechanism of production of pericardial fluid is still a matter of some debate. The composition of pericardial fluid closely resembles that of an ultrafiltrate of plasma (Gibson & Segal, 1978). These findings would indicate that pericardial fluid is formed by a passive hydrostatic process. Miller, Pick & Johnson (1971) observed that occlusion of the cardiac venous drainage produced copious quantities of a lymph like fluid on the epicardial surface of the heart. To investigate the factors which affect the production of pericardial fluid the isolated perfused rat heart was used. The perfusion method is based on that of Langendorff for the non-working heart and on that of Hearse & Chan (1972) for the working heart. The coronary outflow is collected by means of a cannula in the pulmonary artery and loss from the right atria is prevented by ligation of both vena cavae. Fluid is collected from the epicardial surface of the heart over timed intervals, for analysis and comparison with that of the perfusing medium. The effect of variation in hydrostatic and colloid osmotic pressure on the rate of formation and composition of this pericardial like fluid was demonstrated. Preliminary studies have shown that the rate of formation of fluid on the epicardial surface of the heart does indeed vary with the hydrostatic pressure. As yet no *

S.R.C. Scholar.

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PROCEEDINGS OF THE differences have been observed between the ionic composition of the perfusion fluid and that of the pericardial like fluid formed on the surface of the heart. This project is supported by the British Heart Foundation. REFERENCES

GIBSON, A. T. & SEGAL, M. B. (1978). J. Physiol. 277, 367-377. HEARSE, D. J. & CmAN, E. B. (1972). Biochem. J. 128, 1125-1133. MILLER, A. J., PICK, R. & JOHNSON, P. J. (1971). Am. J. Cardiol. 28, 463-466.

Histochemistry of developing muscle in rabbit and cat BY ANTHEA ROWLERSON. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH The influence of insulin on mixed protein synthetic rate in the sheep placenta in utero By SYLVIA CHRYSTIE, JANE HORN, D. NOAKES, M. STERN and MAUREEN YOUNG. Department of Gynaecology, St Thomas's Hospital, London SEl 7EH

COMM7UNICATIONS The effect of vascular perfusion of the choroid plexus on the secretion of cerebrospinal fluid BY R. DEANE and M. B. SEGAL. Sherrington School of Physiology, St Thomas's Hospital Medical School, London SEl 7EH Many epithelia have a complex microcirculation yet the role of this structure and the effect of vascular perfusion has largely been ignored in the study of secretion by these tissues. In a previous communication we have shown that the transport of sugars across the isolated perfused choroid plexus of the sheep was sensitive to the rate of vascular perfusion (Deane & Segal, 1978). A similar finding has been observed in the perfused frog small intestine (Boyd & Parsons, 1978). The effect of perfusion on the rate of c.s.f. secretion and on ion transport has been investigated. A reduction in the rate of vascular perfusion from 0*63 + 0-08 to 0-25 + 0 04 ml./min (n = 5) caused a decrease in the secretion of c.s.f. from 21-37 + 2*69 to 9.15 + 1-51 1l./min. The rate of sodium and chloride transport from blood to c.s.f. was also reduced; 5-88 + 1-71 /equiv Na+/min to 3-74 + 0-061 ,equiv Na+/min and 7-02 + 1.03 /equiv Cl-/min to 4.65 + 0.52 ,equiv Cl/min. All values expressed as mean + S.E.M.

The degree of inhibition of c.s.f. secretion by Diamox (acetazoleamide, 10-7 M) was shown to be sensitive to the rate of vascular perfusion, the inhibition being increased at the lower rate of perfusion. Vascular perfusion would thus appear to increase the rate of secretion of c.s.f. by the choroid plexus and to increase both the transport of ions and sugars. A possible explanation of these observations could be that vascular perfusion causes hydrostatic