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COMMUNICATIONS Metabolic clearance rate of cortisol and aldosterone during controlled hyperthermia in man BY K. J. COLLINS, J. D. FEW and J. P. M. FINBERG.* M.R.C. Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT Adrenocortical secretion is stimulated in man by high environmental temperature conditions as indicated by raised plasma concentrations of cortisol (Collins, Few, Forward & Giec, 1969) and aldosterone (Kosunen, Pakarinen, Kuoppasalmi & Aldercreutz, 1976). Plasma concentrations of these hormones are also determined by the respective metabolic clearance rates (MCRs), and in the heat, hepatic clearance is influenced by a reduced splanchnic blood flow. In order to study the effects of acute heat exposure on changes in MCR of cortisol and aldosterone, seven unacclimatized young adults were investigated during controlled hyperthermia using a temperature-controlled bed (Fox, Crockford, Hampton & MacGibbon, 1967). Four subjects were studied at rest for 2 hr in neutral temperature conditions (aural temperature 37-18 + 0.170 C S.D.) followed by rapid elevation of deep body temperature to 38-14 + 0.140 C, which was then maintained for 1-5 hr. Three control subjects were investigated in the bed for the same period without heating. MCRs were measured by an initial intravenous injection followed by continuous infusion of [1,2,6,7-3H] aldosterone and [4-14C]cortisol, and hepatic clearance by intravenous injection of a single bolus of indocyanine green (JCG) once in the neutral phase and once during controlled hyperthermia. In three subjects with deep body temperature elevated by 1-08 + 0 03 0C, cortisol MCR remained constant in the neutral and hyperthermic stages at 11 9 + 3'9 and 11-9 + 4*6 l. hr-' respectively. At the same time, aldosterone MCR was significantly reduced from 77 + 181. hr-1 in neutral to 56*9 + 3-8 1. hr-1 in hyperthermia. In the fourth heated subject, in whom a rise of deep body temperature of only 0 6° C was produced, and in the three control subjects, there was no change in either cortisol or aldosterone MCR. Hepatic clearance was reduced during heating as shown by a significant increase in ICG half-life from 3 0 + 1'06 min in the neutral stage to 4-4 + 0 17 min in hyperthermia. Plasma aldosterone concentration may therefore be raised during heat exposure partly as the result of a reduction in MCR. Cortisol MCRs were not similarly reduced in the heat when plasma cortisol concentrations * Present address: Department of Physiological Chemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110, U.S.A.

8P PROCEEDINGS OF THE increased. While these differences in MCR may reflect the higher binding affinity of plasma proteins for cortisol, we have evidence from only one experiment in the heat, when plasma cortisol concentration increased to 33 jug 100 ml.-', which suggests that aldosterone MCR can be increased by displacement of aldosterone from plasma proteins by cortisol. REFERENCES

COLLINS, K. J., FEW, J. D., FORWARD, T. J. & GIEC, L. A. (1969). J. Physiol. 202,

645-660. Fox, R. H., CROCKFORD, G. W., HAMPTON, I. F. G. & MACGIBBON, R. (1967). J. apple. Phy8iol. 23, 267-275. KOSUNEN, K. J., PAXARINEN, A. J., KUOPPASALMI, K. & ADLERCREUTZ, H. (1976). J. apple. Phy8iol. 41, 323-327.

Temperature regulation in anorexia nervosa BY C. T. M. DAvIEs, L. FoimIN and C. THOREN. Medical Research Council Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, and Cardiac Laboratory, St Goran's Children's Hospital, Stockholm, Sweden The thermal responses to prolonged exercise (1 hr) on a stationary bicycle ergometer have been studied in ten anorexic patients and 5 normal children. The patients were female (except for 1 case) with a mean age of 15 2 yr and had < 10% of their body weight as fat. At the time of measurement they were not receiving any form of drug therapy. The basic experiments were conducted in a moderate environment (Tdb = 23 6 + 0 840 C, TWb = 14-1 + 2.20 C) at approximately 65 % maximal aerobic power output (V0omax) on the patients and control subjects. In addition, two patients were studied in a warm (Tdb = 32*2 + 0.40 C, TWb = 17b6 + 1.400 C) and one patient in cool (Ttb = 1 1*7 + 0 4° C, Tb = 6 4 + 0.50 C) conditions and two patients after being heated for 30 min in a sauna at 500 C for 20 min. Measurements included metabolic (M) and total heat (Ii) production, evaporative sweat rate (E), rectal (Tre) and mean skin (Tsk) temperatures and an estimate of peripheral tissue heat conductance (K). The results showed that for a given M, Tre and Tsk were higher and E and K lower in the patients than the control subjects. The inter-subject variation of Tre could be reduced by considering the relative (% rmax) rather than the absolute work load. The patients dissipated a greater proportion of H by convection and radiation than did controls, but there was some evidence of selective vasoconstriction in the extremities at rest (Wakeling & Russell, 1970) and during exercise in anorexia. Passively heating the anorexia patients before exercise increased E resting Tre but did not affect the 'plateau' value of Tre obtained at the end of work

9P PH YSIOLOGICAL SOCIETY, FEBRUARY 1977 (cf. Bittel & Henane, 1975). In the warm environment Tre rose to the same level as observed at 220 C, but in cool conditions Tre failed to rise above the resting value. It was concluded that anorexia patients can regulate their body temperature in a thermally neutral environment, and therefore our findings lend no support to the theory of Mecklenburg, Loriaux, Thompson, Anderson & Lipsett (1974), which postulates a loss of central nervous control of temperature regulation. However, the loss of body fat which reduces thermal insulation in patients with the disease may decrease the range (prescriptive limit) of ambient temperatures within which Tr, can be maintained during exercise. REFERENCES

BITTEL, J. & HENANE, R. (1975). J. Physiol. 250, 475-490. MECKLENBURG, R. S., LoRiAux, D. L., THOMPSON, R. H., ANDERSEN, A. E. &

LIPSETT, M. B. (1974). Medicine, Baltimore 53, 147-159. WAXELING, A. & RUSSELL, G. F. M. (1970). Psychological Medicine 1, 30-39.

The assessment of levels of heat tolerance BY 0. G. EDHOLM, A. J. HACKETT, M. Y. SUKKAR* and J. S. WEINER. M.R.C. Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, and *Department of Physiology, Faculty of Medicine, University of Khartoum, Sudan It is well established that continuous or repeated exposure to heat brings about an improved tolerance to heat as judged by the increased work output and the progressive reduction of the initially high levels of core and skin temperatures and of heart rate. These improvements are all associated with a more rapid onset of sweating and an increased sweat rate. The course of this acclimatization process has been followed in climatic chambers on subjects performing standardized work at high external temperatures for periods up to 4 hr. To elicit a maximal sweat response from a group of 20 fit young soldiers required 16 days of repeated exposure for 4 hr at 40° C dry bulb, 320 C wet bulb, air movement 25 cm/sec and a work/rest routine of 464 kJ/m2 . hr (0. G. Edholm, R. H. Fox, R. Goldsmith and H. S. Wolff, unpublished). Similar chamber tests have shown that individuals leading an active life in the tropics acquire a high degree of 'natural' acclimatization (Hellon, Jones, Macpherson & Weiner, 1956). As an alternative to the climatic chamber, Fox (1969) introduced his mobile controlled-hyperthermia test-bed by means of which the core temperature is rapidly raised and maintained at 38° C for 1 hr. The sweat responses of different groups of subjects can thus be obtained on a strictly comparable basis; data for some 20 different groups living in a variety of

ADp lop PROCEEDINGS OF THE climates are now available. For unacclimatized subjects the Fox test gives a mean value for the 'initial' sweat rate of 4-5 ml./min; for highly acclimatized subjects a value of 18*2 ml./min has been recorded (Fox, Lofstedt, Woodward, Eriksson & Werkstrom, 1969). Acclimatization by the daily application of the controlled hyperthermia technique (but with a procedure differing from the Fox bed) has been demonstrated by Fox, Goldsmith, Kidd & Lewis (1963). We have now established that the progressive increase of sweat output during heat treatment by controlled hyperthermia (from the experiments of Fox et al. 1963) and by the chamber technique of Edholm and his colleagues are very closely related (r = 0.9). We have used this relationship to express the result of a single Fox bed test in terms of an equivalent number of days of acquired heat acclimatization. To test this relationship we have used data obtained on two groups of young Sudanese soldiers. The control group (n = 8) gave a mean 'initial' sweat rate, in the Fox test, of 10 63 ml./min. This is equivalent to an acclimatization value of 8 days. The second group (n = 14) was subjected to five successive days of heat treatment by raising core temperature to 38.50 C by exercise in vapour barrier suits in indoor unventilated conditions. The Fox test for this group gave a mean 'initial' sweat rate of 14*97 ml./min. This value represents a heat acclimatization equivalent of 13 days. The two Fox tests thus satisfactorily confirm the 5 days' enhancement of the intensity of heat acclimatization. The data from the Fox bed tests on the different geographical and ethnic groups can be used in a similar way to assess their heat tolerance levels. REFERENCES

Fox, R. H., GOLDSMITH, R., KIDD, D. J. & LEWIS, H. E. (1963). J. Phy8iol. 166,

530-547.

Fox, R. H. (1969). In Human Biology: A Guide to Field Methods, ed. WEINER, J. S. & LoURIE, J. A., pp. 359-374. Oxford: Blackwells. Fox, R. H., L6FSTEDT, B. E., WOODWARD, P. M., ERIKSsoN, E. & WERKSTROM, B. (1969). J. appl. Phy8iol. 26, 444-453. HELLON, R. F., JONES, R. M., MACPHERSON, R. K. & WEINER, J S. (1956). J. Physiol. 132, 559-576.

Hyperthermic effects of morphine in the rat spinal cord BY T. A. RUDY and T. L. YAKSH. Department of Pharmacology, University of Wiscon8in, Madi8on, W.I., U.S.A. To study the effect of morphine in the spinal cord on the thermoregulatory response of the unanaesthetized rat, animals were prepared with catheters inserted through a slit in the cisternal membrane to the level of the lumbar subarachnoid space (Yaksh & Rudy, 1977a). Morphine was

PH YSIOLOGICAL SOCIETY, FEBRUARY 1977 lip administered in a volume of 10 l1. Colonic and tail temperatures (a measure of vasodilation) were monitored by thermistor probes. Ra's were restrained in hemicylindrical wire mesh containers. Morphine sulphate, administered intrathecally, yielded a dose-dependent (2-45 #ug) hyperthermia with the mean maximum response (± s.E.) for the extreme doses being 0 4 + 0.20 C and 2 1 + 0.5° C respectively (n = 4-6). The time of onset was rapid (3-10 min) and the duration was dosedependent (approximately 6 hr for the 45 ,sg doses). Naloxone (1 mg/kg, i.P.) completely antagonized the hyperthermia while indomethacin (5 mg/kg, i.P.) had no effect. Experiments carried out with tritiated morphine indicated that following an intrathecal injection'of the narcotic, background levels of radioactivity failed to rise, indicating that the drug remained localized within the spinal subarachnoid space (Yaksh & Rudy, 1977b). To ascertain whether the hyperthermic response was regulated, animals were injected with morphine sulphate (45 jug) during exposure to one of three ambient temperatures (320, 220 or 40 C). The mean rise in core temperature was 2-1 + 0.50 C, 2-0 + 0.60 C and 1*9 + 0.60 C (n = 3) at the three ambient temperatures, respectively. In the warmer environments, the animal's tail temperature consistently fell to within 1-2° C of the ambient temperature. At the 40 C temperature, the animals were maximally vasoconstricted from the start. Shivering and piloerection were likewise observed in all animals at the lower temperatures. After 60 min, naloxone was administered and a transient vasodilatation, coincidental with the return of the animal's core temperature to its premorphine level, was consequently observed. These observations suggest that the hyperthermic response was the result of a co-ordinated thermoregulatory effort on the part of the rat, elicited by a local action of morphine in the spinal cord. The possibility that within the spinal cord there exists an endogenous opiate sensitive system capable of modulating the transmission of nociceptive stimuli (Yaksh & Rudy, 1976, 1977b) leads us to the hypothesis that the observed hyperthermia may have occurred as the result of an alteration in the content of the sensory message arriving at the classical thermoregulatory centres in the hypothalamus. The possibility that other drugs, when administered systemically, alter the set point in this manner may be of considerable importance in understanding the pharmacology of the thermoregulatory system. REFERENCES YAKSH, T. L. & RUDY, T. A. (1976). Science, N.Y. 192, 1357-1358. YAKSH, T. L. & RUDY, T. A. (1977a). Phy8iol. Behav. (in the Press). YAKSH, T. L. & RUDY, T. A. (1977 b). J. Pharmac. exp. Ther. (in the Press).

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The excretion of magnesium and calcium by the dog kidney during an acute increase in the plasma sodium chloride concentration BY R. L. WARREN and C. S. WILCOX. The Courtauld Institute of Biochemistry and the Department of Pharmacology and Therapeutics, The Middlesex Hospital Medical School, London, and the Medical Unit, St Mary's Hospital Medical School, London W2 iN Y In many circumstances the rates of renal excretion of magnesium (UMgV) and calcium (UcaV) are positively correlated with that of sodium (Walser, 1973). However, the effects of an acute increase in plasma sodium concentration (PNa) on these relationships has not been defined. Experiments were performed on greyhounds anaesthetized with pentobarbitone sodium. The methods used have been described previously (Wilcox, 1976, 1977). One kidney (referred to as 'transplanted') was acutely denervated by resection and reanastomosis of its vessels to one of the animal's carotid arteries and jugular veins. A needle in the arterial anastomosis transmitted saline at a fixed rate of 0x1 ml. min-' kg body weight-' throughout the experiments. The other kidney (referred to as 'untouched') remained in situ. In 12 experiments, when 0 154 M sodium chloride solutions were infused, the mean value for PNa was 145 + 3 (± S.D.) m-mole 1.-i and the two kidneys excreted both calcium and magnesium at similar rates (P > 0.2). When the sodium chloride concentration of the infusion was increased to 1-232 M for 1 hr the mean PNa of blood perfusing the 'untouched' kidney increased to 153+4 and that perfusing the 'transplanted' increased to 165+4 im-mole 1.-i. In all experiments, the infusion of hypertonic saline led to an increase in UMgV and UCaV by both kidneys. However, UmgV and UcaV by the two kidneys were now different, being greater (t > 4*5; n = 24; P < 0.005) by the 'transplanted' kidneys despite a significant (t = 2-64; P < 0.01) reduction in their GFR relative to their 'untouched' pair. Combining all results obtained during infusion of hypertonic saline solution, there was a close dependence of both UMgV (r = 0-84; n = 112, P < 0.001) and UcaV (r = 0*85; n = 116; P < 0.001) on PNa* In 11 experiments plasma magnesium concentration was varied over a sevenfold range by adding magnesium chloride solutions to the saline infusions. Magnesium excretion was greater when PNa was increased at each level of plasma magnesium concentration. These findings suggest that an acute rise in plasma sodium chloride concentration increases UMgV and UcaV by an action on the renal tubules.

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This work was supported by grants from the National Kidney Research Fund and the Wellcome Trust.

REFERENCES WALSER, M. (1973). In Handbook of Physiology, section 8: ed. ORLOFF, J. & BERLINER, R. W., pp. 555-586. Washington, D.C.: American Physiological Society. WILCOX, C. S. (1976). J. Physiol. 258, 91-92P. WILCOX, C. S. (1977). J. Physiol. 265, 6P.

Glutamine and glucose as fuels for the fed, fasted and acidotic rat small intestine BY P. J. HANSON and D. S. PARSONS. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU In an attempt to identify physiological substrates of metabolism of the rat small intestine in conditions such as fasting, the metabolism of glucose and glutamine was studied using an in vitro preparation of intestine perfused in situ through the vascular bed with a medium containing erythrocytes (Hanson & Parsons, 1976). Initial concentrations of glucose and glutamine in the perfusate plasma were 7-5 and 1-5 mm respectively. No substrates were added to the fluid passing through the lumen. Fasting for 48 hr nearly halved the rate (Ctmole g dry wt-'. hr-1) at which glucose was utilized by proximal jejunum, but the utilization of glutamine and the production of alanine were moderately increased. Fasting, however, reduces the weight of mucosa per cm of jejunum, and if the results were expressed with respect to intestinal length, which gives a better indication of the metabolic activity of the whole jejunum in vivo, glutamine utilization and alanine production were unchanged by fasting, whilst glucose utilization was reduced by 66 %. In fed rats glutamine utilization was 44 % of the rate of glucose utilization, but in fasted rats it was 112 % of glucose utilization. Glutamine may therefore make an important contribution towards the energy requirements of the fasting jejunum, for 57 % of glutamine may be oxidized to CO2 (Windmueller & Spaeth, 1974). There was some increase in the utilization of glutamine (#mole g dry wt.-1 hr-1) by jejunum from unfasted rats made acidotic by including NH4Cl (15 g/l.) in their drinking water for 7-11 days. However, such treatment produced no change in the utilization of glutamine by a given length of intestine, and in vivo there may be a fall in glutamine utilization, for the concentration of glutamine in the plasma of acidotic rats is reduced by 39 %, and this fall may influence the rate of glutamine metabolism. Fasting for 48 hr does not alter the concentration of glutamine in

14P PROCEEDINGS OF THE the plasma. Glucose utilization and alanine production were unaffected by the induction of acidosis. In unfasted animals glutamine utilization by mid-ileum was 72% of the rate found with proximal jejunum. However, with a plasma glucose concentration close to physiological levels, there was no difference between jejunum and ileum in the rate of glucose utilization, irrespective of whether glutamine was present in the perfusate. This apparent absence of a gradient of glucose utilization is in contrast to results obtained in vitro with non-vascularly perfused intestine (Hanson & Parsons, 1976) in which glucose is initially taken up by the luminal pole of the intestinal epithelia] cells. This work was supported by a grant from the Medical Research Council.

REFERENCES HANSON, P. J. & PARsoNs, D. S. (1976). J. Physiol. 255, 775-795. WINDMUELLER, H. G. & SPAETH, A. E. (1974). J. biol. Chem. 249, 5050-5079.

The case against an invariable direct innervation of sweat glands and for a general sudomotor mechanism By H. Y. ELDER, D. M. JENKINSON and I. MONTGOMERY. Departments of Physiology, The Hannah Research Institute, Ayr, and the University of Glasgow, G12 8QQ

The logical analysis of the canine electroenterogram By T. G. BARNETT and D. L. WINGATE. Department of Physiology and Academic Unit of Gastroenterology, London Hospital Medical College, Turner Street, London El 2AD In the conscious dog with implanted serosal electrodes, the spatial and temporal distribution of intestinal spike activity is a sensitive index of fasting and post-prandial states (Szurszewski, 1969; Code & Marlett, 1975) and of the effects of gastro-intestinal hormones (Marik & Code, 1975; Wingate, Ruppin, Green, Thompson, Domschke, Wunsch, Demling & Ritchie, 1976). The formidable problem of analysing prolonged recordings from multiple sites can be solved by a technique comprising slow tape recording with rapid replay, active bandpass filtering, and digitization of recorded spikes (Barnett & Wingate, 1976); by counting of the digitized spikes, quantitative analysis is possible and, in theory, useful. In practice, an obstacle was encountered. An infused peptide which abolished cyclical activity in the fasted dog did not alter the total incidence of spikes during the infusion in comparison with a similar period of saline infusion, even

15P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 though a marked change in the pattern of spike activity was apparent. Clearly, the signal processing system should be capable of pattern recognition; the availability of digital pulses corresponding to recorded spikes suggested the use of digital logic for this purpose. Three types of pattern were defined, broadly corresponding to the three phases described by Code & Marlett (1975). Type 1 is absence of spike activity for more than four cycles of the basic electrical rhythm (BER). Type 3 activity is repetitive spike bursts phase-locked to the BER for more than three BER cycles. All other activity is, by our definition, Type 2 activity which is equivalent to Code and Marlett's Phase 2: 'persistent but random action potential activity'. The logic analyzer consists of three monostable multivibrators, whose input is the digital pulses corresponding to recorded spikes, and whose output is passed to a series of logic (NOR and AND) gates. Specific permutations of the logic levels of the three monostables, produced by different patterns of spike activity, operate the logic gates so as to assign clock pulses, and digitized spikes into activity counters allocated for Types 1, 2 and 3. The application of digital logic to the problem of pattern recognition provides an objective quantitative method for characterizing the response of the intestine of the conscious animal to a variety of physiological and pharmacological stimuli. The support of the Wellcome Trust is gratefully acknowledged. REFERENCES

BURNETT, T. G. & WINGATE, D. L. (1976). J. Phy8iol. 256, 6-7P. CODE, C. F. & MARLETT, J. A. (1975). J. Phy8iol. 246, 289-309. MAR=K, F. & CODE, C. F. (1975). Gadtroenterology 69, 387-395. SzaszwsKi, J. H. (1969). Am. J. Phy8iol. 217, 1757-1763. WINGATE, D. L., RurPIN, H., GREEN, W. E. R., THOMPSON, H. H., DoMscbm, W., WuNScH, E., DEMLING, L. & RITCHIE, H. D. (1976). SCand. J. Ga8troenterol. 11, supply. 39, 111-118.

The influence of age on the influx of ketone bodies into the brain of the rat BY P. M. DANIEL,* E. R. LOVE, S. R. MooRHousE and 0. E. PRATT. Department of Neuropathology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF Although the major fuel for the brain is glucose, the ketone bodies, acetoacetate and 3-hydroxybutyrate, can be used to a limited extent as an alternative source of energy. * Present address: Department of Applied Physiology and Surgical Science, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2 3PN.

16P PROCEEDINGS OF THE 16P In rats aged from 1 to 16 weeks, influx into the brain was measured by maintaining a constant level of radioactively labelled ketone body in the circulation for 3 min, or less, by a controlled injection (Daniel, Donaldson & Pratt, 1975), and determining the radioactivity entering the brain (Daniel, Love & Pratt, 1977). The influx of both ketone bodies was high during the first three weeks of life and fell with increasing age, e.g. 3-hydroxybutyrate fell from 86 + 16 nmol min-' g-1 of brain at 1-3 weeks to 5 + 1 nmol min' g-1 at 8-16 weeks. Since the concentrations of ketone bodies in the blood fall over this age range and since their influx is almost directly proportional to their concentration in the blood (Daniel et al. 1971), the reduction in influx must be due, in part, to the concomitant diminution in their concentration in the blood (two- to threefold). In an attempt to increase the influx in adults up to that found in rats 1-3 weeks old, the blood levels of ketone bodies in the adults were raised (either by fasting or by injecting 3-hydroxybutyrate) to levels comparable to those normally found in the young. The influx in these adults rose but only to about 20 % of the value found in the young. Our results are best explained if ketone bodies enter the brain by means of a carrier-mediated transport system, the activity of which is increased when high levels of circulating ketone bodies are present for a considerable period, as during suckling. REFERENCES DANIEL, P. M., DONALDSON, J. & PRATT, 0. E. (1975). Med. & biol. Engng 13,

214-227. DANIEL, P. M., LoVE, E. R., MOORHOUSE, S. R., PRATT, O. E. & WILSON, P. (1971). Lancet ii, 637-638. DANIEL, P. M., LovE, E. R. & PRATT, 0. E. (1977). Proc. R. Soc. B 196, 85-104.

The effects of hyperbaric oxygen (2 ATA) on the haemodynamic, metabolic and electrocardiographic consequences of acute myocardial ischaemia in anaesthetized greyhounds BY I. MCA. LEDINGHAM, R. J. MARSHALL and J. R. PARRATT. Department of Surgery, University of Glasgow, and Department of Physiology and Pharmacology, University of Strathelyde, Glasgow G1 1XW We have previously shown that, in anaesthetized greyhounds breathing oxygen at 2 ATA (OHP), acute ligation of the left anterior descending coronary artery results in a lower infarct blood flow and more marked lactate production than in dogs breathing an air equivalent (Ledingham, Marshall & Parratt, 1973). In the present study we have investigated whether OHP modifies the haemodynamic, metabolic and electrocardiographic indices of myocardial ischaemia when administered 1-5-2 h after

17P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 ligation, a situation which is more relevant to any possible clinical application of OHP in patients with acute myocardial infarction. All experiments were carried out at 2 ATA in a large pressurized chamber using greyhounds (20-28 kg) anaesthetized with trichlorethylene. The main effects of acute coronary artery ligation were significant (P < 0.05) decreases in cardiac output, myocardial contractility, and in the oxygen content and pH of blood sampled from a local coronary vein draining the ischaemic region. There was also lactate production by the ischaemic zone. The most pronounced haemodynamic effects produced by changing the inspired gas mixture from air equivalent (Pa, 0,95 + 7 mmHg) to oxygen (Pao2 917 + 66 mmHg) 15-2 h after coronary artery ligation, were decreases in cardiac output (1-5 + 0*2 to 1 1 + 0. 1I./min) and in blood flow to both normal (51 + 6 to 43+ 5 ml./min) and ischaemic (24 + 6 to 14 + 3 ml./100 g. min) myocardial regions. Oxygen consumption by, and availability to, the ischaemic zone, however, were not changed significantly, the increase in arterial oxygen content that results from oxygen breathing being almost exactly balanced by the reduction in coronary blood flow. This was reflected in the absence of changes in lactate handling and in ST-segment depression. Evidence was also obtained, using epicardial ST-segment mapping following brief (3-5 min) coronary occlusions, that OHP does not modify the magnitude or extent of myocardial ischaemia. These results suggest that OHP does not beneficially modify myocardial ischaemia and this may be due to the fact that, as in normal myocardium, oxygen strongly constricts the coronary vessels in the acutely ischaemic region, thus limiting oxygen availability. We wish to thank the Scottish Hospitals Research Trust for generous financial support. REFERENCE

LEDINGHAM, I. MCA., MARSHALL, R. J. & PARRATT, J. R. (1973). J. Physiol. 231, 100-101 P.

Blood flow in the feet of patients recovering from fractures of the tibia and fibula BY F. J. IMMS, A. A. MCGREGOR, S. P. PRESTIDGE and KARRAN RUDGE. M.R.C. Environmental Phyaiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT Patients recovering from fractures of the lower limb have increased flow of blood through the tissues of the calf (Imms, Lorde, Prestidge & Thornton, 1976). Blood flow has now been measured in the foot. o0

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18P PROCEEDINGS OF THE 18P The subjects were nineteen servicemen aged 16-35 who were undergoing a full-time course of treatment at the Joint Services Medical Rehabilitation Unit. They had suffered unilateral fractures of the tibia and fibula 178 (range 67-662) days prior to admission to the Unit and had been immobilized in plaster of Paris (POP) until 22 (range 3-85) days prior to admission. Blood flow was measured simultaneously in the two feet using venous occlusion plethysmographs filled with water at 32 + 1 C. In five control subjects the blood flows (means+ S.E.M.) in the two feet were similar (right 3-58 + 0*60 ml. 100 ml.-1 min'; left 3*68 + 0.57). In 30 experiments on nineteen subjects the blood flow in the foot of the injured leg (4.86 + 0.31) was significantly higher (P < 0.001) than that of the noninjured limb (3.42 + 0-31). This increase in blood flow was still present in seven subjects who were re-examined approximately 4 months after the end of rehabilitation. The elevations of blood flow were not correlated with the time which had elapsed from the removal of POP or from commencing rehabilitation (r = 0-036; P = 0 005; n = 37). Since bone constitutes about 40 % of the volume of the foot (Allwood & Burry, 1954), then any marked changes in bone blood flow might cause measurable changes in blood flow of the foot as a whole. The possibility that changes in blood flow in the foot were related to changes in calcification of bones was investigated. Calcification of bone was assessed from radiographs, which were calibrated by incorporating a stepped aluminium wedge in each exposure. Radio-opacity of bone was reduced by 13*3 + 1-4 % in the lower tibia and by 14-3 + 2-9 0/ in the calcaneum of the injured leg. In seven subjects reviewed 4 months after the end of rehabilitation the radio-opacity was reduced by only 8'9 %, and there were significant correlations between reduction of opacity and the time elapsed since removal of POP or beginning rehabilitation. There were no correlations between the elevations of blood flow in the injured limb and the reductions in radioopacity of either the tibia or the calcaneum. It is concluded that changes in blood flow in the foot are not related to the degree of osteoporosis in the ankle. REFERENCES ALLWOOD, M. J. & BuRRY, H. (1954). J. Py8yiol. 124, 345-357. IMMs, F. J., LORDE, D. A., PRESTIDGE, S. P. & THORNTON, C. (1976). Clin. Sci.

51, 297-302.

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PHYSIOLOGICAL SOCIETY, FEBRUARY 1977

Validation of a submaximal exercise test BY C. T. KAPPAGODA, R. J. LINDEN and J. P. NEWELL. Department of Cardiovascular Studies, University of Lee&s, Leeds LS2 9JT During the course of an investigation into the 'cardio-respiratory fitness' of patients recovering from cardiac surgery a test of 'fitness' was required. The submaximal heart rate-oxygen consumption relationship has formed the basis of several such indices of 'cardio respiratory fitness' (e.g. Spiro, et al. 1974) but we were unable to find a complete validation of any of these indices. The validity of any index of physical fitness can only be established by demonstrating, in individual subjects, a systematic change in the index after a programme of physical training; with an opposite change on cessation of training. The present investigation was undertaken to obtain this evidence for the submaximal heart rate-oxygen consumption relationship by performing a longitudinal study in a group of normal human volunteers. Ten subjects completed 16 weeks of daily training according to the '5BX, XBX Canadian Airforce Exercise Programme' (Penguin Books, 1964). A submaximal exercise test was performed before training, 8 weeks after training and on completion of training. Six subjects were retested 4 weeks after cessation of training and four subjects were retested 4 weeks after continuing training three times per week as recommended for maintenance of fitness by the 5BX, XBX programme. The submaximal exercise test consisted of three pairs of increasing discontinuous loads on an upright bicycle ergometer. Heart rate was measured from a three lead electrocardiogram and the oxygen consumption by an open-circuit flow through technique (Kappagoda, Stoker & Linden, 1974). Both measurements were made under steady-state conditions. The data from each exercise test was analysed so that the results from each individual could be considered as a series of regression lines relating heart rate to oxygen consumption. These regression lines were compared using standard statistical methods (Snedecor & Cochran, 1967). In all ten subjects the period of training was associated with a statistically significant (P < 0.01) diminution in the elevations, as expressed by the adjusted means, of the regression lines. The slopes of the corresponding regression lines were diminished in six subjects (P < 0.05), increased in one (P < 0.05) and unchanged in the remaining three. The six subjects who ceased training showed a significant increase in elevation as compared to the trained state (P < 0-01). On the maintenance schedule none of the four subjects showed a 10-2

20P 2PPROCEEDINGS OF THE statistically significant increase in elevation suggesting no deterioration in fitness over the four weeks. It is concluded that the elevation of the submaximal heart rate-oxygen consumption relationship can be used as an index of 'cardio-respiratory fitness' when sequential tests are performed in an individual subject. In addition, the 5BX, XBX programme can be recommended as a training schedule. REFERENCES

KAPPAGODA, C. T., STOKER, J. B. & LINDEN, R. J. (1974). J. appl. Physiol. 37, 604-607. PENGUIN BOOKS (1964). Physical Fitness. SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical Methods, 6th ed. Iowa State University Press. SPIRO, S. G., JUNIPER, E., BOWMAN, P. & EDWARDS, R. H. T. (1974). Clin. Sci. & Mol. Med. 46, 191-206.

Subdivision of 8-adrenoceptors in the cardiovascular system of the rat By F. J. IMMs,* R. L. B. NEAME and D. A. Powis. Department of Physiology, St Bartholomew's Hospital Medical College, Charterhouse Square, London ECiM 6BQ and * M.R.C. Environmental Physiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 7HT The cardiovascular response to infused noradrenaline in the anaesthetized rat is unusual in so far as the induced rise in arterial blood pressure is brought about entirely by an increase in cardiac output with no simultaneous change in total peripheral vascular resistance (TPR). It has been suggested that fl-adrenoceptors are important in mediating this response (Imms, Neame & Powis, 1974). In some mammals /-adrenoceptors have been subdivided into /,% (cardiac) and /82 (peripheral vascular) types according to their susceptibility to blockade. Because of the particular significance of fl-adrenoceptors in the rat we have investigated whether a similar subdivision can be made in this species. Male rats of an SPF Wistar-derived strain were anaesthetized with pentobarbitone sodium (60 mg kg-1 i.P.) and prepared for measurement of arterial B.P., cardiac output and heart rate (Imms et al. 1974). TPR was calculated as the dividend of arterial B.P. and cardiac output. Isoprenaline (Isoprenaline Sulphate B.P., Macarthy) was infused intravenously at 50 ng min- both before and after f-adrenoceptor blockade with either propranolol ('Inderal', ICI; 0 01, 0'03 or 0 1 mg kg- i.v.) or with practolol (Eraldin, ICI; 0-1, 1*0 or 3'0 mg kg-1 i.v.). Before blockade, isoprenaline caused a fall in arterial B.P. of 31 + 4 mmHg

PH YSIOLOGICAL SOCIET Y, FEBRUAR Y 1977 21P (mean + S.E.M.) from a control level of 133 + 4 mmHg. This was due to a reduction in TPR of 608 + 64 from 1600 + 78 mmHg ml.-. min 103 which was partially offset by an increase in cardiac output of 23*9 + 5-1 from 85-0 + 6-0 ml. min-. Heart rate rose by 72 + 9 from 423 + 6 beats min-'. Since baroreceptor reflexes in the anaesthetized rat are weak (De Jong & McLeod, 1967; Imms, Neame & Powis, 1977), changes in heart rate during isoprenaline infusion have been taken as an index of fl1-adrenoceptor stimulation and changes in TPR as evidence of fl2-adrenoceptor stimulation. Increasing doses of propranolol progressively reduced the positive chronotropic response to isoprenaline and at the same time reduced the decrement in TPR. Expressed in terms of percentage reduction in response to isoprenaline, increasing doses of propranolol caused equivalent decreases in each variable, e.g. 0 01 mg kg-' propranolol reduced the chronotropic response by 30 % and the vasomotor response by 37 %. Practolol, a selective ,l1-adrenoceptor blocking agent in the dog (Hainsworth, Karim & Stoker, 1974), also decreased the chronotropic and vascular response to isoprenaline, but at all doses the blockade of the cardiac response exceeded that of the vasomotor response. Thus 0 I mg kg-1 practolol reduced the chronotropic response by 18 % and the vascular response by 1 %; 3*0 mg kg-1 practolol reduced the responses by 84 % and 47 % respectively. Although some discrimination can be obtained between those fl-adrenoceptors mediating the changes in heart rate and TPR in the rat, the distinction obtained with practolol is not as marked as that demonstrated in the dog (Hainsworth et al. 1974). REFERENCES DE JONG, W. & McLEOD, S. M. (1967). Eur. J. Pharmac. 1, 93-98. HAINSWORTH, R., KARIm, F. & STOKER, J. B. (1974). Br. J. Pharmac. 51, 161-168. IMMs, F. J., NEAME, R. L. B. & Powis, D. A. (1974). J. Phyaiol. 241, 47-49P. IMMs, F. J., NEAME, R. L. B. & Powis, D. A. (1977). Br. J. Pharmac. (in the Press).

Some observations on sinus arrhythmia BY JANET CORT, 0. G. EDHOLM, T. LOBSTEIN and R. WEBB. School of Environmental Studies, University College London, WC1E 6BT In the course of investigations into possible relationships between personality and physiological characteristics, observations have been made on sinus arrhythmia in a group of eighteen subjects, aged 18-25. The differences between individuals were large, with the mean amplitude of heart rate change during a respiratory cycle ranging from 2 to 40 beats. Many subjects also had a wide range of sinus arrhythmia from one cycle to the next. The standard deviation of the amplitude over a period of ten

22P PROCEEDINGS OF THE respiratory cycles for the eighteen subjects ranged from 1 0 to 11.8. Davies & Nielsen (1967) have shown that amplitude and respiratory cycle length are related; in the present studies the mean amplitude of the sinus arrhythmia and the mean respiratory cycle length are related, but not the individual respiratory cycles and the amplitude of the associated heart rate change. It has been shown by a number of workers, including Katsona & Felix (1975), that sinus arrhythmia is due to inhibition of the vagus during inspiration. It was therefore surprising that there was no relationship between mean heart rate and the amplitude of sinus arrhythmia. Furthermore, in a group of eighteen elderly subjects (69-87 yr old), a number had virtual absence of sinus arrhythmia but low heart rates, whereas some elderly subjects who had heart rates of 70-90 also had well-marked sinus arrhythmia. There were no significant correlations between personality characteristics and the amplitude of sinus arryhthmia. This research was supported by a grant from the Medical Research Council. REFERENCES

DAVIES, C. T. M. & NIELSEN, J. M. M. (1967). J. apple. Phyaiol. 22, 947-955. KATsONA, P. G. & FELIX, J. (1975). J. apple. Phyaiol. 39, 801-805.

Modification of the Falck-Hillarp Technique with Intravital trypan blue to differentiate elastic fibres from noradrenergic endings By A. MCINNES.* Institute of Physiology, University of Glasgow, Glasgow G12 8QQ The Falck-Hillarp technique (Falck, Hillarp, Thieme & Torp, 1962) is a sensitive histochemical technique for localizing noradrenaline and hence sympathetic nerve endings. Unfortunately elastic tissue has an autofluorescence which is very similar to the formaldehyde-induced fluorescence (FIF) of noradrenaline and this may in certain situations be mistaken for it. The internal elastic lamina of an arteriole is unlikely to be mistaken for sympathetic nerve endings but there are situations where the anatomy is not so distinctive. One such situation is the muscle spindle, where elastic fibres surround the intrafusal fibres and sympathetic nerve fibres may lie close to them. Quenching the FIF with water or sodium borohydride is said to remove the FIF but not the autofluorescence, but this method was not found to be sufficiently discriminating as the elastic tissue autofluorescence, while more resistant, was quenched also by these agents. * Present address: Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow G51 4TF.

23P PHIYSIOLOGICAL SOCIETY, FEBRUARY 1977 Elastic tissue can be distinguished from FIF by microspectrofluorimetry but the method is laborious for distinguishing numerous small points which may be close together. Lande & Waterson (1968) showed that the internal elastic lamina of isolated arteries could be made to fluoresce red by staining supravitally with Evans blue. This technique has now been developed. Cat tenuissimus muscles were stained intravitally with trypan blue (C. Lamb Ltd). Sufficient of 1 % solution (w/v) to give a total dose of 15 mg/kg was injected intravenously and allowed to circulate for 1 hr. The muscle was then removed and prepared by the Falck-Hillarp technique. All connective and elastic tissue including that at the poles of the muscle spindles then fluoresced red. The FIF of noradrenaline was yellow green as before. Thus elastic tissue could be easily differentiated from sympathetic nerve endings. Further, the background structures were more easily identifiable since trypan blue stains intracellular material green and extracellular material red. The method was also found to preserve tissue structure better. Supravital rather than intravital staining with trypan blue may also be employed but is not advised as penetration of the dye may be inadequate in the region of interest. This work was conducted during the tenure of the Henry Faulds Research Fellowship of the University of Glasgow. REFERENCES FALcK, B., HiLA.Rp, M. A., THIEME, G. & TORP, A. (1962). J. Hietochem. Cytochem. 10, 348-354. LANDE, J. S. DE LA & WATERSON, J. G. (1968). J. Hfi8tochem. Cytochem. 16, 281-282.

The discharge of mechanoreceptors in the cat knee joint at intermediate angles BY W. R. FERRELL. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ Boyd & Roberts (1953) found that the discharge of slowly adapting mechanoreceptors in the cat knee joint could be correlated with joint angle. However, Burgess & Clark (1969, 1975), recording in the dorsal spinal roots, found that 95 % of such mechanoreceptors discharged at one or both extremes of movement but not at intermediate angles, while only about 5 % discharged through intermediate positions. The status of these 'mid-range' units as joint receptors was suspect, as some could be localized to the adjacent popliteus muscle, and their resting discharge was increased by an intravenous injection of succinylcholine (SCh) in a dose sufficient to excite muscle spindle afferents (0.1-0.2 mg). Thus Burgess & Clark concluded that most mid-range units are muscle afferents from popliteus,

24P PROCEEDINGS OF THE 24P and that articular mechanoreceptors are incapable of continuously signalling joint angle. The present experiments were performed to determine whether purely articular mechanoreceptors can provide adequate information about joint angle at intermediate positions. In a sample of fifty-two single units isolated in cat dorsal roots, 13 % of the slowly adapting knee joint mechanoreceptors were mid-range units. Direct recording from the whole posterior articular nerve of the cat knee joint revealed that there were always units active at intermediate angles, with a mean of 4-2 + 04 units (S.E. of mean) in nine cats. Tetanic contraction of surrounding muscles produced little alteration of the range of activity of most knee joint mechanoreceptors, and section of quadriceps femoris did not significantly alter the mean number of active mid-range units in eight cats. Muscle afferents from popliteus did not make a significant contribution to the articular nerve. The mean number of spontaneously discharging mid-range units before and after removal of popliteus was 5-5 + 0O8 and 5.3 + 0.8 respectively (seven cats). Intravenous injection of 0- 1-0 2 mg SCh was sufficient to affect the discharge of purely articular mechanoreceptors, as this dose usually increased articular nerve activity at intermediate angles after removal of popliteus, and also increased the resting discharge in dorsal root filaments of mid-range units specifically located to the joint capsule. These results suggest that there are adequate articular mid-range units in the cat knee joint to provide useful information about joint angle. REFERENCES BOYD, I. A. & ROBERTS, T. D. M. (1953). J. Phyaiol. 122, 38-58. BURGESS, P. R. & CLARK, F. J. (1969). J. Phy8iol. 203, 317-335. BURGESS, P. R. & CLARK, F. J. (t975). J. Neurophysiol. 38, 1473-1484.

The response of cat muscle spindle primary sensory endings to stretch during infusion of suxamethonium BY M. B. DUTIA.* Institute of Physiology, University of Glasgow, Glasgow G12 8QQ The dynamic nuclear-bag fibre (DNB) of the cat muscle spindle is more sensitive to ACh than the static nuclear-bag fibre (SNB) (Gladden, 1976). Changes in the dynamic and position sensitivities of six soleus muscle spindle primary sensory endings were studied during intravenous infusion of suxamethonium (1o00 #g/kg. min), to examine the effect of the contraction of the nuclear-bag fibres on the Ia discharge. During infusion, the de-efferented muscle was subjected to sequential *

Wellcome Research Scholar.

PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 25P short-duration ramp-and-hold stretches. The dynamic and position responses to each stretch were recorded from a 'single-fibre' I a afferent in the dorsal roots (Fig. 1). The dynamic sensitivity was always seen to increase first (A), with a smaller increase in position sensitivity, usually 40-50 see from start of infusion. Later, usually 100-120 see from start of infusion, an independent sharp rise in position sensitivity occurred (B), and the dynamic index decreased. Dynamic response

(D)

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20

30

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20

70

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' Time' (sec) 80 90 100

110

120

I 30

40 Stretch number

50

60

Fig. 1. Dynamic and position responses of a soleus Ia afferent (inset) during SCh infusion.

The increase in dynamic index at A probably results from the contraction of the lower-threshold DNB. The later contraction of the SNB no doubt causes the large increase in position sensitivity at B. Since position sensitivity rises independently of dynamic sensitivity at B, and during recovery falls independently, 'pace-maker switching' (Matthews, 1972) probably occurs between the Ia spirals on the two nuclear bag fibres of the cat spindle. REFERENCES

GLADDEN, M. H. (1976). Prog. Brain Res. 44, 51-59. MATTHEWS, P. B. C. (1972). Mammalian Muscle Receptors, ch. 5. London: Arnold.

Real and apparent y axon contraction sites in intrafusal fibres BY ELINOR R. ARBUTHNOTT,* I. A. BOYD, MARGARET H. GLADDEN and P. N. MCWILLIAM. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ and Department of Physiology, Trinity College, Dublin In a previous correlation of the ultrastructure of an isolated spindle with its behaviour (Arbuthnott, Boyd & Gladden, 1976) a P2 plate with irregular *

Muscular Dystrophy Group Research Fellow.

26P PPROCEEDINGS OF THE junctional folds was found as expected on one nuclear bag fibre 2*4 mm from the spindle equator where focal contraction had been observed during stimulation of a dynamic y axon. A static y axon produced contraction in all the other intrafusal fibres, and trail terminations without junctional folding were found on most of these fibres. In similar experiments, however, Banks, Barker, Bessou, Pages & Stacey (1976) failed to find motor nerve endings at some sites of focal contraction occurring far out in the spindle pole during dynamic y axon stimulation. We have now sectioned one pole of a second spindle in which two focal contraction sites were seen in a dynamic nuclear bag fibre (D.N.B.), 1*4 and 2-6 mm from the equator, during stimulation of a dynamic y axon (Boyd, Gladden, McWilliam & Ward, 1977). The D.N.B. was marked intracellularly with Alcian Blue dye. A motor ending was found on the marked D.N.B. at 1*4 mm. No ending was present at the more distal focus, nor was any other ending found anywhere at this pole of the D.N.B. During stimulation of the dynamic y axon, movement of the D.N.B. observed 04 mm from the equator occurred in two overlapping phases. We suggest that if a nonpropagated potential produces focal contraction at a P2 plate in the intracapsular region of a D.N.B. fibre, stretch activation of the more polar region might result in maximal sarcomere shortening at a site where there is no motor ending, slightly later than the main contraction. In the same spindle pole a static y axon caused contraction of the static nuclear bag fibre (s.N.B.) and chain fibres at 09 and 1-2 mm from the equator, respectively; motor endings were found precisely at both these points. However, both these supposedly 'trail' endings had marked junctional folding so we agree with the recent view of Barker (1976) that the presence or absence of folding is not diagnostic of motor endings of dynamic y axons and static y axons, respectively. The D.N.B. had no or only faint M-lines in the region 0S3-1*5 mm from the equator which was sectioned longitudinally, while the S.N.B. and chain fibres had prominent M-lines. Unlike the D.N.B., the S.N.B. had prominent elastic fibres round its polar region (Gladden, 1976). REFERENCES ARBUTHNOTT, E. R., BOYD, I. A. & GLADDEN, M. H. (1976). Prog. Brain Re8. 44, 61-65. BAtxs, R. W., BARKER, D., BESsoU, P., PAGES, B. & STACEY, M. J. (1976). J. Physiol. 263, 180P.

BAKER, D. (1976). Prog. Brain Res. 44, 65. BOYD, I. A., GLADDEN, M. H., MCWILiAm, P. N. & WARD, J. (1977). J. Physiol. 265, 133-162. GLADDEN, M. H. (1976). Prog. Brain Res. 44, 51-59.

27P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 Alternation between occlusion and summation during combined fusimotor excitation of Ia afferents BY M. HULLIGER, P. B. C. MATTHEWS and J. NOTH. University Laboratory of Physiology, Parks Road, Oxford OX1 3PT During large-amplitude sinusoidal stretching primary spindle afferents show an asymmetrical response pattern during combined static and dynamic fusimotor stimulation. Static action dominates the trough, dynamic action the peak of the response- the former with complete occlusion, the latter with some degree of summation. Trough occlusion was found irrespective of the level of the trough response during dynamic activation alone, provided the static were above the dynamic firing levels; such variation was achieved by adjusting the amplitude and frequency of stretching. 100-

,

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Fig. 1. Cycle histograms with the averaged responses to sinusoidal stretching (1 mm at 0-32 Hz) during dynamic (A, B) and combined static and dynamic stimulation (C, D). The pattern fD Of stimulation (see bottom) was constant in A, C, and rectangular (phase locked to the stretch cycle) in B, D. In C, D histograms during combined and static stimulation alone (Vs., rate 57/sec) are superimposed to show the occurrence of trough occlusion irrespective of the dynamic-induced trough response pattern.

28P PROCEEDINGS OF THE 28P The trough response during dynamic stimulation could be raised close to the static firing levels with rectangular patterns of dynamic activation, locked to the release phase of the stretch cycle (Fig. 1 B). During combined stimulation trough occlusion occurred again (D, C), irrespective of the presence (B) or absence (A) of trough firing due to dynamic action alone. This agrees with the concept of two or more pace-makers determining the afferent response by competitive interaction.

Histochemical profiles of rat muscles chronically tenotomized during development BY N. J. W. RUsSELL. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ The histochemical profiles of seven intertransverse caudal (ITC) muscles from 8-week-old normal male albino rats were compared with the same number of muscles taken from 8-week-old animals in which the ITC muscle had been tenotomized at birth. As would be expected of a growing muscle composed of predominantly fast-twitch fibres, the tenotomized ITC muscles were not markedly atrophic. However, comparison of cross-sectional areas of unfixed sections showed the mean cross-sectional area of the fibres in tenotomized muscle to be smaller (1409.9 /Zm2 + 27.7 s.E.) than in normal muscle (2410.5 ,um2 + 49*8 s.E.).

Freshly excised ITC muscles were treated to demonstrate alkalipreincubated myosin adenosine triphosphatase (mATPase) and its acidpreincubated reversal (Guth & Samaha, 1970) and succinic dehydrogenase (SDH). Descriptions are based upon profiles obtained in serial sections. In the normal ITC muscle three fibre types can be distinguished: (a) SDHdense, alkali-pale (low mATPase activity), acid-dark fibres, comprising about 15 % of the muscle, (b) SDH-intermediate, alkali-dark (high mATPase activity), acid-pale fibres, comprising about 10 % and (c) SDHpale, alkali-dark (high mATPase activity), acid-pale fibres comprising about 75 % of the muscle. This overall histochemical profile is appropriate for a phasic fast-twitch muscle in the rat (Close, 1972). In contrast, the muscles subjected to tenotomy during post-natal development show abnormal mATPase histochemical profiles while retaining normal SDH staining reactions. The proportions of fibres in the three SDH categories are unaltered in tenotomized muscle. However, the 15 % which are SDH-dense show an acid-pale and alkali-dark mATPase reaction, which is the reverse of normal. The majority of fibres in tenotomized muscle are SDH-pale and SDH-intermediate, and show high

29P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 mATPase activity in alkali-preincubated sections, but, in contrast to normal, they also show a dark reaction in acid-preincubated sections. Salmons & Vrbova (1969) have recently demonstrated the significance of impulse activity in the regulation of adult skeletal muscle types and it may be suggested therefore that, during development, the alteration in the extent or pattern of impulse activity imposed by tenotomy has prevented the muscle fibres from attaining the mATPase characteristics normally associated with the specific fibre types of adult muscle. REFERENCES

CLOSE, R. I. (1972). Phy8iol. Rev. 52, 129-197. GUTH, L. & SAMAHA, F. J. (1970). Expl Neurol. 28, 365-367. SALMONS, S. & VRBOVA, G. (1969). J. Phy8iol. 201, 535-549.

The A action of catecholamines on the smooth muscle of guinea-pig taenia coli BY EDITH BULBRING and A. DEN HERTOG. University Laboratory of Physiology, Parks Road, Oxford OXi 3PT, and Department of Pharmacology, State University, Groningen, The Netherlands In the smooth muscle of guinea-pig taenia coli the a and ft actions of catecholamines are synergistic. Both abolish spontaneous spike activity and cause relaxation. The a action is associated with a decrease in membrane resistance and hyperpolarization, mainly due to increased K-conductance. The ft action abolishes the pace-maker potential and usually causes some hyperpolarization but no change in membrane resistance (Bulbring & Tomita, 1969a, b), and reduces the size of the phasic contraction following an evoked action potential. The mechanism of the ft action has been further examined by observing the effects of isoprenaline (2.2 x 10-7 to 3-6 x 10-6 M) in the presence of an a blocker, phentolamine (6.3 x 10-6 M), using the double sucrose gap method. The reduction of phasic contractions by isoprenaline did not appear to be the consequence of membrane hyperpolarization since repolarization by electric current application did not antagonize the ft action. The possibility that an electrogenic component of Na/K pumping might be involved in the hyperpolarization was tested by applying isoprenaline after removal, and during readmission, of external potassium; it was found that the ft effect remained unchanged. In low [CI-]o (13.3 mM), when Rm was higher than in normal Krebs solution, isoprenaline caused a larger hyperpolarization. Similarly, hyperpolarization diminished linearly with diminishing Rm in increasing

30PPROCEEDINGS OF THE [K+]o (0.6-29-5 mM). However, in low [Ca2+]O (0.5 mM), when Rm was higher than normal, the isoprenaline effect was smaller, and in high [Ca2+]O (7.5 mM), when Rm was lower, isoprenaline caused a larger hyperpolarization than in 2*5 mm [Ca2+]0. Furthermore, in the presence of isoprenaline, '7Ca efflux was increased by about 35 % above the control value. The results suggest that the response of the taenia coli to the , action of isoprenaline may be due to activation of a Ca pump operating electrogenically, since the hyperpolarization, occurring without change in membrane conductance, is associated with an acceleration of the outward movement of calcium. The reduction in the size of phasic contractions may be caused by a decrease of available intracellular calcium. 30P

REFERENCES

BULBRING, E. & TOMITA, T. (1969a). Proc. R. Soc. B 172, 89-102. BULBRING, E. & TOMITA, T. (1969b). Proc. R. Soc. B 172, 103-119.

Action of ouabain on Na-free contractures in mammalian ventricular muscle By F. BREMNER, C. H. FRY and J. A. S. McGUIGAN. Institute of Physiology, University of Bern, Switzerland, and Cardiothoracic Institute, 2 Beaumont Street, London, W. 1 Sheep and calf ventricular trabeculae do not develop marked Na-free contractures (Bassingthwaighte, Fry & McGuigan, 1976). However, if these trabeculae were perfused with Tyrode solution containing ouabain (10-6 to 10-7 M), Na-free contractures could be observed, the amplitude increasing with time of perfusion to more than twice the control twitch tension. Upon removal of ouabain, Na-free contractures of constant height could be elicited for several hours. The contractures relaxed spontaneously and were accompanied by either a small hyperpolarization or no change in transmembrane potential. The amplitude depended on the external concentrations of Na and Ca. Similar contractile events were observed if 150 mM-KCl/l. were added to Tyrode or the Na-free solution. The spontaneous relaxation was slowed by lowering the perfusate temperature or by adding chlorpromazine (10-6 M). Pretreatment with tetracaine (2-10 mM) reduced the contracture height, whilst addition of tetracaine during the contracture depressed it. Caffeine (5 mM) could further increase tension, either at the peak of the contracture or after it had spontaneously relaxed, even in the presence of zero external Ca. Repriming of the Na-free contractures occurred with a time constant of about 2 min, suggesting that some mechanism other than extracellular

31P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 diffusion was involved in tension generation. These findings may be interpreted to mean that the Na-free contractures are due to the release of Ca from the sarcoplasmic reticulum and not due to a direct influx of Ca through the surface membrane. If the intracellular Na was increased by perfusion with a cooled Tyrode solution much smaller Na-free contractures were elicited. Moreover, if the trabeculae were perfused with ouabain-containing, Na-free Tyrode, contractures could be elicited by raising the external Ca. The amplitude of the latter contractures was again dependent on the perfusion time in ouabain. To explain these results we suggest that ouabain has a dual action. Ouabain will increase the internal Na by blocking the Na pump and this, via the Na/Ca exchange mechanism, will increase the internal Ca (see Reuter, 1974). However, since Na loading does not mimic the ouabain effects and the ouabain action can also occur in Na-free solutions, we suggest that ouabain also alters the kinetics of Ca release or storage properties of the sarcoplasmic reticulum. This work was supported by grant no. 3.758. 72 from the Swiss National Scientific Foundation. C.H.F. was also supported by the Royal Society. REFERENCES

BASSINGTHWAIGHTE, J. B., FRY, C. H. & McGuIGAN, J. A. S. (1976). J. Phy8iol. 262, 15-37. REiUTER, H. (1974). Circulation Re8. 34, 599-605.

The effects of hypertonic fluids on the contractility of isolated frog atrial trabeculae BY R. A. CHAPMAN. Department of Physiology, University of Leicester, LE1 7RH Increasing the tonicity of the bathing fluid (up to a fourfold rise in the relative tonicity due to added sucrose or Tris-HCl) has complicated effects on the contractile responses of frog atrial trabeculae. These effects can be resolved into: an initial rapid sustained but reversible fall in resting tension, a delayed rise in contractility, and a further increase in contractility on return to isotonic fluid (off response). The initial relaxation has a t, of 3-5 sec and is unaffected by prolonged perfusion with Ca-free solution or by local anaesthetics. Its size increases linearly with the stretch applied to the muscle, so that it resembles the response of the 'sarcolemmal preparation' of Lannergren & Noth (1973) and may therefore be due to an effect of the hypertonic solution on the tensile properties of some element in parallel with the contractile apparatus.

32PPROCEEDINGS OF THE 32P The delayed effect shows itself as a progressive rise in the tension developed by the regularly evoked heart beat or as a delayed contracture in Na-free or K-rich media. The contracture and the potentiation of the heart beat reach a maximum at a relative tonicity of 3, at higher tonicities contraction is rapidly suppressed. The hypertonic contracture is inhibited by local anaesthetics but still develops in Ca-free, Na-free solution and is therefore like the hypertonic contractures found in skeletal muscle fibres (Lannergren & Noth, 1973). The off responses take the form of a transient rise in the strength of the heart beat or as a transient contracture in Na-free or K-rich fluids which occur on return to the isotonic solution. The off contracture is insensitive to local anaesthetics and to the removal of the bathing Ca2+ in Na-free fluid. The strength of this contracture shows a reciprocal relationship to the strength of the preceding hypertonic contracture. The results suggest that both the hypertonic and the off contracture originate from the release of Ca from an intracellular store, while the effects of local anaesthetics and the reciprocal relationship between the contractures indicate that this store is more complicated than previously supposed. Perhaps a functionally divided sarcoplasmic reticulum of the sort proposed for skeletal and mammalian cardiac muscles is present in frog heart (Winegrad, 1970; Chapman & Leoty, 1976). Indeed, such a division would seem to be a necessary requirement of any model of excitation-contraction coupling that incorporates a Ca-induced Carelease as part of its mechanism. REFERENCES

CHAPMAN, R. A. & LEOTY, C. (1976). J. Phy8iol. 256, 287-314. LANNERGREN, J. & NOTH, J. (1973). J. gen. Physiot. 61, 158-175. WINEGRAD, S. (1970). J. yen. Physiol. 55, 77-88.

Calcium entry across the post-junctional membrane during transmitter action BY R. MILEDI, I. PARKER and G. SCIALOW. Department of Biophysics, University College London, Gower Street, London WC1E 6BT During transmitter action, an influx of calcium ions across the postsynaptic membrane of the squid giant axon has been demonstrated using aequorin (Kusano, Miledi & Stinnakre, 1975), and there is evidence for a similar influx at the neuromuscular junction (see, for instance, Takeuchi, 1963; Katz & Miledi, 1969; Evans, 1974). We have used the calcium indicator dye arsenazo III (Dipolo et al. 1976; Brown et al. 1975) for recording changes in intracellular calcium levels in the end-plate region of muscle fibres from the cutaneous pectoris of Rana temporaria.

33P PHYSIOLOGICAL SOCIETY, FEBRUARY 1977 Arsenazo III was injected into a fibre close to the end-plate by ionophoresis from a micro-pipette, and the calcium-induced changes in light absorption of the dye were recorded by focusing a spot of light on to the end-plate. Simultaneous measurements of light transmission through the fibre was made at two wave-lengths, using interference filters and photomultipliers. At one wave-length (602 nm) the dye shows a large change in absorbance in the presence of calcium, whilst at the other (532 nm) the change is small. By subtracting the two signals a record is obtained which reflects changes in calcium concentration, but is relatively unaffected by movement of the fibre. The end-plate was voltage-clamped, and the clamp current recorded in response to either local nerve stimulation or ionophoretically applied acetylcholine. Following transmiter action, a change in light absorption of the dye lasting several hundred milliseconds was seen. The size of the calcium response was increased by raising the calcium concentration in the bathing solution, and by hyperpolarizing the fibre. A non-linear relationship exists between calcium influx and membrane potential, with the calcium response produced by ionophoretic application of acetylcholine increasing steeply at potentials more negative than about -90 mV. Addition of cobalt to the bathing solution reduces the non-linearity, and a small calcium response remains which increases linearly with increasing hyperpolarization. REFERENCES

BROWN, J. E., COHEN, L. B., DE WEER, P., PINTO, L. H., Ross, W. N. & SALZBERG, B. M. (1975). Biophys. J. 15, 1155-1160. DIPOLO, R., REQUENA, J., BRINLEY, F. J., MULLINS, L. J., SCARPA, A. & TIFFERT, T.

(1976). J. gen. Phy~iol. 67, 433-467. EVANS, R. H. (1974). J. Physiol. 240, 517-533. KATZ, B. & MILEDI, R. (1969). J. Physiol. 203, 689-706.

KuSANO, K., MILEDI, R. & STINNAKRE, J. (t975). Proc. R. Soc. B 189, 49-56. TAKEUCHI, N. (I963). J. Phypiol. 167, 141-155.

Individual e.p.s.p.s in chronic spinal cats BY L. MENDELL and S. G. NELSON. Duke University, Durham, North Carolina, U.S.A. Individual excitatory post-synaptic potentials (e.p.s.p.s) evoked by the action of single medial gastrocnemius (MG) I a afferent fibres in MG motoneurones have been recorded in the L7 segment of anaesthetized cats made spinal at T1 3 or L5. Comparisons were made with data obtained previously under identical conditions in cats with intact spinal cords (Scott & Mendell, 1976) using spike-triggered averaging (Mendell & Henneman, 1971). In intact animals, individual e.p.s.p.s average 94 + 8 ,uV (S.E. of mean, n = 70). Similar results were obtained in four animals transacted at T13,

34P PPROCEEDINGS OF THE 12-30 days previously under Nembutal anaesthesia. However, in five L5transected animals (12-91 days previously), the mean e.p.s.p. amplitude increased significantly (P < 0*01; 2-tailed t test) to 320 + 33 ,uV (n = 57). In addition, several very large (400-1200,V) individual e.p.s.p.s were observed contrasted with virtually none in normal or T13 transacted preparations. These large e.p.s.p.s almost always had very brief rise times and half widths, unlike the largest individual e.p.s.p.s in intact anaesthetized cats (Mendell & Weiner, 1976). Other individual e.p.s.p.sin these same motoneurones were not necessarily brief or large. Acute transaction at L5 resulted in significantly increased mean e.p.s.p. amplitude (234 + 30 ,V; n = 68; P < 0.001). A similar increase was observed in animals acutely transacted at T13 (313 + 29 ,uV; n = 62; P < 0.001). Some variability between animals was observed. Large, brief e.p.s.p.s were observed acutely following transaction at either level. E.p.s.p. enhancement following acute spinal cord transaction does not depend on the level of transaction. The chronic changes may result from a different process since they occur after L5 but not T13 transaction. Since most degeneration on motoneurones occurs within two segments of a more rostral transaction (McLaughlin, 1972), the loss of synaptic input on MG motoneurones may cause the chronic changes perhaps via denervation supersensitivity and/or collateral sprouting. It is also possible that tonic presynaptic disinhibition of I a fibres occurs causing enhanced e.p.s.p.s. Ischaemia can increase e.p.s.p. amplitude (Eccles, L0yning & Oshima, 1966) but by much smaller amounts than observed here. Input resistance of motoneurones developing the largest e.p.s.p.s was within normal limits suggesting that post-synaptic changes are not responsible, although highly localized ones cannot be ruled out. Supported by U.S. Public Health Service. REFERENCES

Eccims, R. M., L0YNING, Y. & OSHIMA, T. (1966). J. Neurophy8iol. 29, 315-332. MCLAUGHLIN, B. (1972). J. comp. Neurol. 144, 475-500. MENDELL, L. & HENNEMAN, E. (I971). J. Neurophy8iol. 34, 171-187. MENDELL, L. & WEINER, R. (1976). J. Physiol. 255, 81-104. SCOTr, J. & MENDELL, L. (1976). J. Neurophy8iol. 39, 679-692.

Strength and decay of McCollough effects BY C. C. D. SHUTE. Physiological Laboratory, University of Cambridge When McCollough effect (ME) (= percentage extinction of green light) decay is plotted as the decrement d below initial strength MEo after t minutes by the match interference method (Shute, 1977), the curve forms

PH1YSIOLOGICAL SOCIETY, FEBRUARY 1977 35P the cubic hemi-parabola, d = at+. For this subject, a normally equals -LMEO (Fig. 1A, ME; 10, 2). Another colour intensity method using different units (Riggs, White & Eimas, 1974) yields a similar curve with a i j, but that obtained with the method of MacKay & MacKay (1975) is of quite different shape (Fig. IA, Ml). The discrepancy is resolved if the match interference and MacKay methods have the same threshold for weak MEs and in each case the smallest detectable value (S) is obtained after about ME M 60 1-5

40

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M2 15

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ME S 05 C

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Fig. 1. A, ME and M2 (modifications of MacKay's MI) decay curves reaching smallest detectable value S after time t = 400 min. B, M2 plotted against ME passes through origin and S. C, Linear plots of log d (ME decrement) against log t (2) and d' (MacKay decrement) against log d (1) and log t (3). D. ME stimulus intensity curve (I = percentage intensity when MEo = 50).

the same time lapse. The MacKay curve then has the form M2 in Fig. 1 A. M2 plotted against ME gives a smooth curve (Fig. IB). The MacKay decrement (d') for M2 is related linearly to log d (gradient 1) and log t (gradientJ Fig. IC; 12 3). Polaroid strips with transmission axes at measured angles were used to relate ME strength to stimulus intensity. Values satisfied the equation ME = bIt, where I = percentage of stimulus intensity giving a strength = of MEo with no polaroid, and b -7MEo. ME decay rate is nearly halted by withholding red and increased after red excess to a value given by a = 2ME0.

36P

36P

PROCEEDINGS OF THE REFERENCES

MAcKAY, D. M. & MAcKAY, V. (1975). Q. J1 exp. P8ychol. 27, 225-233. RIGGS, L. A., WmTE, K. D. & ETMAS, P. D. (1974). Percept. & P8ychophy8. 16, 535-542. SHUTE, C. C. D. (1977). J. Phyeiol. 269 (in the Press).

Innervation of stretch receptors in the extra-thoracic trachea By D. BARTLETT JR., J. P. MORTOLA and G. SANT'AMBROGIO. Departments of Anesthesiology and Physiology and the Division of Neurosurgery, University of Texas Medical Branch, Galveston, Texas, and the Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. We have studied the innervation of the stretch receptors in the extrathoracic trachea of the dog. Ten dogs were anaesthetized with sodium pentobarbitone (30 mg/kg i.v. initial dose). An L-shaped cannula was inserted as low as possible in the neck and its cuff inflated just below the thoracic inlet. A Foley's catheter was inserted through the mouth and the larynx and fixed by inflating its cuff at the level of the cricoid cartilage. The extra-thoracic segment of the trachea was thus isolated and could be either inflated or deflated. Unitary action potentials were recorded from the vagus nerves and identified as originating in extra-thoracic trachea by inflating its isolated segment. Once an extra-thoracic tracheal receptor was found, its precise location was determined by inflating the cuff of the Foley's catheter at various levels in the tracheal segment. All ten dogs were studied with the chest widely opened and were artificially ventilated. The nervous pathway followed by the action potentials was determined by cold block of the ipsilateral recurrent laryngeal nerve (RLN). On the left side all the 18 receptors studied had their activity going through the ipsilateral RLN: 78 % of them joined this nerve in the neck and the rest within the thorax. These latter receptors were located mainly in the lower extra-thoracic trachea. On the right side only 45 % of the 18 receptors studied were found to follow the RLN whilst the others reach the vagus nerve through a different pathway. These latter receptors were located in the lower part of the extra-thoracic trachea. Therefore sectioning both the RLNs would interrupt about three quarters of the fibres coming from the extra-thoracic trachea if done just before their entrance into the main vagus nerves. With section of the nerves exclusively outside the thorax, at the level of the thoracic inlet, the de-afferentation would be about 60 % of the total supply.