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REFERENCES THOMAS, R. C. (1970). J. Phywiol. 210, 82-83P. ZEUTHEN, T. (1971). Acta phyaiol. acand. 81, 141.

Intracellular injection of horseradish peroxidase: a technique for demonstrating the soma, dendrites, axon and axon collaterals of a neurone By A. G. BROWN, P. K. ROSE* and P. J. SNOW.* Dcpartment of Physiology, Royal (Dick) School of Veterinary Studies,. University of Edinburgh, Edinburgh EH9 1QH This work was supported by a grant to A.G.B. from the M.R.C.

Measuring the efficiency of detecting patterns in random dots BY H. B. BARLOW AND A. VAN MEETEREN. Physiological Laboratory, Downing Street, Cambridge CB2 3EG COMMUNICATIONS Stimulus-secretion coupling in submandibular gland: role of cyclic AMP, cyclic GMP And calcium in regulating adrenoceptor mediated enzyme secwotPon BY JANET ALBANO, K. D. BHOOLA, M. CROKER, P. F. HEAP and M. J. C. LEMON. Departments of Pharmacology, Anatomy and Medicine, The Medical School, University of Bristol, Bristol BS8 1TD, and Metals Research, Royston, Hertfordshire S08 6EJ Experimental evidence suggests that calcium, adenosine, 3',5'-cyclic monophosphate (cyclic AMP) and more recently guanosine 3',5'-cyclic monophosphate (cyclic GMP) couple the stimulation by neurotransmitters of specific receptors on the plasma membrane to the secretion of enzymes into the acinar cell lumen in the submandibular gland. Correlations between enzyme (kallikrein) secretion, morphological changes and alterations in cyclic AMP and cyclic GMP levels evoked by adrenoceptor agonists were examined using isolated guinea-pig submandibular gland slices incubated at 370 C in a modified Krebs-Ringer solution gassed with 95 % 02-5 % C02. Cyclic AMP was measured by the binding protein method (Brown, Albano, Ekins, Sgherzi & Tampion, 1971), cyclic GMP by modification of the radioimmunoassay of Steiner, Parker & Kipnis (1972) and kallikrein by hydrolysis of benzoyl L-arginine ethyl ester (Bhoola & Dorey, 1971). *

Post-doctoral Fellows of the Canadian M.R.C.

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 lip The secretary response to noradrenaline was biphasic and dose-dependent. In concentrations of 40-400,uM, phenylephrine (a and partial ft agonist; see Jenkinson, 1973) was more active than isoprenaline (ft and partial a agonist), but neither was as potent as noradrenaline in stimulating kallikrein secretion. In contrast methoxamine (a agonist and ft antagonist) was completely ineffective in the same concentration. Both phentolamine (a antagonist) and propranolol (ft antagonist) were required at a concentration of 4 x 104 M for complete inhibition of the noradrenaline enzyme response. Morphological changes in vacuolation and a depletion of secretory granules in the acinar cells (measured with the Quantimet 720D television microdensitometer) were maximal in slices secreting kallikrein in response to noradrenaline and dibutyryl cyclic AMP. These changes were not observed in the intercalated and junctional ducts. The enzyme secretion evoked by adrenoceptor agonists and dibutyryl cyclic AMP showed a lesser dependence than acetylcholine to absence of (Calcium-free Krebs-Ringer containing 1 mm EGTA), and to increased concentrations (5 and 10 mM) of extracellular calcium. In gland slices secreting kallikrein the greatest rise in cyclic AMP at 1 and 30 min incubations was produced by noradrenaline (4 x 104 M); isoprenaline was almost as potent but methoxamine (4 x 104 M) was ineffective. Under these experimental conditions noradrenaline (4 x 104 M), only in the presence of 3-isobutyl 1-methylxanthine (0.5 mM), raised the intracellular levels of cyclic GMP at 2 min. Our results support the view that fl-adrenoceptor stimulated kallikreiln secretion and acinar cell vacuolation was mediated by cyclic AMP (see Lemon & Bhoola, 1975; Bhoola &; Lemon, 1975). The possible involvement of cyclic GMP in a-adrenoceptor evoked enzyme secretion is being further investigated. We thank the Medical Research Council and Wellcome Trust for financial support. REFERENCES

BHOOLA, K. D. & DoREY, G. (1971). Br. J. Pharmac. 43, 784-792. BHOOLA, K. D. & LEMON, M. J. C. (1975), J. Phyewl. 245, 121-122P. BIOwN, B. L., ALBANO, J., EKINS, R. P., SGHERZI, A. M. & TAMIION, W. (1971). Biochem. J. 121, 561-562. JENKINSON, D. H. (1973). Br. med. Bull. 29, 142-147. LEMON, M. J. C. & BHOOLA, K. D. (1975). Biochim. biophy8. Acta 385, 101-113. STEINER, A. L., PARKER, C. W. & KIPNIS, D. M. (1972). J. biol. Chem. 247, 11061112.

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Relation between blood levels of cortisol and duration of human labour By J. K. BURNS. Department of Physiology, University College, Galway A variety of factors contributes to human development and there are many causes of human birth defects (Burns, 1975). Prolongation of human labour, on a comparative basis, contributes to foetal hypoxia and brain damage in the new-born. A neuroendocrine cycle has been proposed to explain the longer duration of labour in the human species (Burns, 1971). An important question is, to what extent psychological or endocrine changes contribute to the development of human congenital distress (difficulty in labour). In a previous investigation we examined blood levels of ACTH during late pregnancy, using an immunoassay technique (Croughs, Tops & de Jong, 1973). We examined the relationship between these levels and durations of the first stage of labour in women. We found a direct correlation between plasma ACTH and the duration of human labour, and considered this to indicate a contribution by psychological stress to congenital distress. In the present investigation we measured the levels of cortisol in plasma during late pregnancy and examined the relationship between cortisol levels and the duration of human labour (first stage). Values for cortisol were markedly elevated in comparison with controls and this increase was highly significant (P < 0.01). We found a direct correlation (0.78) using linear regression analysis, higher levels of cortisol being associated with longer durations of labour. This finding is considered to further support the proposal that a relation exists between psychological stress, as indicated by plasma hormone levels, and human difficulty of labour. REFERENCES BURNS, J. K. (1971). Neuro-endocrine aspects of congenital distress. In P&ycho8omatic Medicine in Obstetric8 and Gynaecology, ed. MORRIS, N., pp. 222-225. Basel: Karger. BURNS, J. K. (1 975). Birth Defects and Their Causes. Galway: Hosty. CROUGHS, R. J. M., ToPs, C. F. & DE JONG, F. H. (1973). J. Endocr. 59, 439-449.

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975

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Endocrinology of pregnancy in the hyrax; plasma progesterone concentration and erythrocyte metabolism

BY S. GOMBE, R. B. HEAP and J. B. SALE. Departments of Animal Physiology and Zoology, University of Nairobi, Chiromo, P.O. Box 30179, Nairobi, Kenya and A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT The gestation period of the hyrax is long (7 5 months) for an animal with an adult body weight of only 2-3 kg. Studies of hystricomorph rodents, which also have long gestation periods relative to mature weight (Weir, 1974), have revealed the production in pregnancy of progesterone-binding globulin (Heap & Illingworth, 1974), a plasma protein with a progesteroneconserving effect and so far identified only in the Hystricomorpha. An aim of the present study was to examine whether such a mechanism occurred in another species with a relatively long gestation period. The study was also relevant to the question of the phylogenetic affinity of the hyrax and the elephant, and the role of progesterone in these species, since the progesterone concentration in the corpus luteum and plasma of the elephant has been found to be very low during gestation (Smith, Hanks & Short, 1969; Hanks & Short, 1972). Steroid hormones of pregnancy were measured in Heterohyrax brucei and Procavia habessinica. Progesterone and unconjugated oestrogens were determined by radioimmunoassay (Heap, Gwyn, Laing & Walters, 1973; Challis, Heap & Illingworth, 1971). The corpus luteum of pregnancy contained a significant concentration of progesterone (range, 5'8-24-2 ng/mg, 6 animals) and a high concentration of immunoreactive oestrogens (351840 pg/mg, 7 animals). The plasma concentration of progesterone in blood samples taken by cardiac puncture was low in all females studied. In pregnant females the values were less than 0 1-4'6 ng/ml. Plasma samples obtained from shot specimens contained a lower concentration of progesterone than those from anaesthetized females at similar times of the reproductive cycle, which implied a loss of activity after sampling since immediate separation of plasma was possible only for laboratory specimens and not for field samples. The loss of activity appeared to be related to steroid metabolism and uptake of progesterone by erythrocytes. During the incubation of whole blood or erythrocytes (0.5 ml.) with 2'5 juc [3H]progesterone for 3 hr at 370 C approx. 80 % of the steroid was metabolized. The enzyme activity was abolished by the addition of sodium fluoride. Thus the maintenance of gestation in the hyrax is characterized by a low plasma progesterone concentration, as in the elephant, and not by the

14P PPPROCEEDINGS OF THE presence of a progesterone-conserving mechanism, as in certain hystricomorph rodents. It is notable that from what is so far known about the endocrinology of pregnancy in the hyrax and elephant, neither of them appears to conform to any of the patterns described in other species. R.B.H. thanks the Inter-University Council for financial support and Dr G. M. 0. Maloiy for hospitality in the Department of Animal Physiology, University of Nairobi. The helpful co-operation of the Kenya Game Department is acknowledged.

REFERENCES CITALTaIS, J. R. G., HEAP, R. B. & ILLINGWORTH, D. V. (1971). J. Endocr. 51, 333345. HANKS, J. & SHORT, R. V. (1972). J. Reprod. Fert. 29, 79-89. HEAP, R. B., GWYN, M., LAING, J. & WALTERS, D. E. (1973). J. agric. Sci., Camb. 81, 151-157. HEAP, R. B. & ILLINGWORTH, D. V. (1974). Symp. Zool. Soc. 34, 385-413. SmrTH, J. G., HANKS, J. & SHORT, R. V. (1969). J. Reprod. Fert. 20, 111-117. WEm, F. (1974). Symp. Zool. Soc. 34, 265-299.

Transplacental total and ionic calcium gradients in conscious sheep BY S. S. CARLYLE.* Physiological Laboratory, Cambridge CB2 3EG The total plasma calcium concentration of the foetal sheep ([CaT]F) is higher than that of its mother ([CaT]M) in anaesthetized animals (Bawden, Wolkoff & Flowers, 1965). In the present study the [CaT] and plasma ionic calcium concentration ([Ca2+]) were measured in eight pairs of samples obtained from catheters in the foetal vena cava and maternal uterine vein of four conscious animals of 120-140 days gestation, at least 24 hr after operation. Blood (5 ml.) was drawn into a syringe containing 50 u. of heparin, and centrifuged, without exposure to air. The [CaT] was determined by either EDTA titration or atomic absorption spectrophotometry; the remainder was ultrafiltered through a semipermeable membrane. The filtration was carried out at 370 C, in a humidified atmosphere of CO2 in 02 (6 % for. maternal and 7 % for foetal samples) to minimize changes in protein-bound calcium (Robertson & Peacock, 1968). The [Ca2+] of the ultrafiltrate was determined with a calcium electrode (Orion, model 92-20), which was similarly warmed and gassed, in an apparatus modified from the description of Robertson & Peacock (1968); the electrode was calibrated with standard CaCl2 solutions before and after each pair of assays. *

Present address: Department of Veterinary Pharmacology, University of

Edinburgh, EH9 1QH.

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 15P The mean values for [CaT]M and [CaT]F (+ S.E.M.) were 7-7 + 030 mg. 100 ml.-' and 11-3 + 0-60 mg. 100 ml.-' respectively. The difference between the means was found to be significant (P < 0-001). The mean values of [Ca2+]M and [Ca2+]F were 4F4 + 0-24 mg. 100 ml.-1 and 3-4 + 0-24 mg. 100 ml-l. This difference was also significant (P < 0-01). Since the [Ca2+]F is lower than the [Ca2+]M, the gradient cannot be used to support the concept of active transport of calcium across the placenta (see Comar, 1956; Twardock & Austin, 1970). Nevertheless since most cells seem to have an internal ionized calcium level of around 106M (see Blaustein, 1974), transfer of calcium to the foetus probably involves its extrusion on the foetal side of the placental cells, irrespective of the plasma-to-plasma gradient. This work was carried out during the tenure of a Wellcome Trust Research Training Grant. The advice and encouragement of Dr R. S. Comline is gratefully acknowledged. REFERENCES BAWDEN, J. F., WOLKOFF, A. S. & FLOWERS, C. E. (1965). Obstet Gynec., N.Y. 25, 548-552. BLAUSTEIN, M. P. (1974). Rev. Phygiol. Biochem. Pharmac. 70, 33-82. CoMAR, C. L. (1956). Ann. N.Y. Acad. Sci. 64, 281-298. ROBERTSON, W. G. & PEACOCK, M. (1968). Clinica chim. Acta 20, 315-326. TWARDOCK, A. R. & AusnN, M. K. (1970). Am. J. Physiol. 219, 540-545.

The effects of adrenalectomy and hypophysectomy in the foetal lamb BY R. J. BARNES, R. S. COMLINE and MARIAN SILVER. Physiological Laboratory, Cambridge CB2 3EG We have compared the effects of foetal adrenalectomy and foetal hypophysectomy on the duration of gestation and foetal viability, on maternal and foetal plasma steroid concentrations, and on foetal tissue glycogen and plasma glucose levels. Ten hypophysectomies were performed at about 100 days and 13 adrenalectomies at 118-126 days gestation; all foetuses survived the operation. Hypophysectomy prolonged gestation in all single foetuses (8); they were delivered under anaesthesia at 162-178 days. Adrenalectomy was more capricious; 5 out of 8 foetuses survived beyond term (147 days) and were examined at 155-163 days; three died in utero at 143-147 days. The adrenalectomized foetuses were larger than the corresponding hypophysectomized animals, and did not have the prominent subcutaneous fat characteristic of the foetal lamb after hypophysectomy (Liggins & Kennedy, 1968). Pregnancy was not prolonged when either operation was performed on

16PPROCEEDINGS OF THE 16P one of a pair of twins. However, unlike hypophysectomized foetuses (Liggins & Kennedy, 1968), adrenalectomized twins were larger than their unoperated controls and in 3 of the 5 ewes in this group the operated twin died 12-48 hr before delivery, while its control was born alive. Total unconjugated oestrogen, progesterone and cortisol were estimated in peripheral maternal plasma taken during the course of gestation and in foetal and maternal samples at the termination of pregnancy. Maternal steroid concentrations in the operated animals were not significantly different from those found in a comparable group of controls and there was no obvious decline in maternal plasma progesterone in the post-mature groups. Foetal plasma cortisol values in both adrenalectomized and hypophysectomized animals were similar to those found in normal control foetuses before the pre-parturient rise in cortisol. Both foetal adrenalectomy and hypophysectomy resulted in changes in tissue glycogen content. Liver glycogen values, measured at term or later, were lower in the operated animals (20-40 mg/g) than in controls at term (60-90 mg/g). Skeletal and cardiac muscle glycogen concentrations were higher in the hypophysectomized foetuses than in the adrenalectomized animals: plasma glucose values were very similar in both operated and control foetuses. The comparatively poor viability of the adrenalectomized foetal lamb, first reported by Drost & Holm (1968), contrasts with that of the hypophysectomized foetus which lives and grows for 30-40 days after normal term. These observations together with the findings on tissue glycogen content, fat deposition and foetal body growth, suggest that alterations in the metabolism of the foetus rather than changes in the hormonal climate of the mother may help to explain the differences between hypophysectomized and adrenalectomized foetal lambs. REFERENCES DROST, M. & HOLM, L. W. (1968). J. Endocr. 40, 293-296. LIGGINS, G. C. & KENNEDY, P. C. (1968). J. Endocr. 40, 371-381.

Permeability of sheep placenta to unmetabolized polar nonelectrolytes BY R. D. H. BOYD, CAROL HAWORTH, T. E. STACEY and R. H. T. WARD.

Departments of Paediatrics and Obstetrics, University College Hospital Medical School, London, W.C.1 The permeability of the placenta to isotopic erythritol, mannitol, urea and 3-0-methyl glucose in the direction foetus to mother was studied in

17P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 23 sheep near term under a variety of conditions. Catheters were placed in the foetal circulation for isotope injection and blood sampling, and in a maternal femoral artery and uterine vein for measurement of the uterine arterio-venous difference. Animals were studied up to 1 week after recovery from anaesthesia. For each isotope studied in a given sheep, a permeability constant K (ml. min-') was calculated and the results were as follows: K + S.E.M. No. sheep Mol. wt Isotope 61-2+ 5-5 1-85+ 016 0-169 +0-036 62-1 + 7-0

Urea

Erythritol Mannitol

3-0-methyl glucose

K

11 12 6 11

60 122 182 194

]-[A]) -([(a)-(A)

[A] and [V] are maternal arterial and uterine venous whole blood and (a) and (A) foetal and maternal arterial plasma radioactivities respectively. 4 is uterine blood flow measured by the antipyrine technique. An individual K value is the mean of at least six measurements. Gel filtration of foetal plasma samples on Sephadex G 15 showed that the test molecules were free in solution at pH 7-4. Similar analysis of maternal arterial and uterine venous blood (except in the case of mannitol where maternal counts were too low) demonstrated that radioactivity remained in the relevant substance following transplacental passage. Comparison of bolus and constant infusion experiments with urea suggested there was no substantial 'third compartment' between foetal and maternal blood streams. The results for urea, erythritol and mannitol were highly consistent with pore theory (Solomon, 1968). In four individual sheep where these three isotopes were studied in quick succession, pore sizes derived from the equation for restricted diffusion and the experimental values ranged from 0-43 to 0-45 nM. K-3-0-methyl glucose was much higher under resting conditions than would be predicted from the proposed pore size. Unlike K-urea, K-3-0methyl glucose fell significantly when the foetal glucose concentration was raised by injection, and a regression of K-3-0-methyl glucose against foetal glucose concentration gave a significant inverse correlation in each study (n = 7 sheep). At 10-6 + 1-2 mm foetal arterial glucose concentration K-3-0-methyl glucose was half that found by extrapolation to zero glucose. 3-0-methyl glucose permeability was unaffected by quantitatively similar increases in foetal arterial mannitol or sodium chloride concentrations. The results given suggest the presence of a placental barrier having

PPROCEEDINGS OF THE 18P epithelial characteristics, between ewe and foetal lamb. Data are compatible with the existence of aqueous channels traversing the barrier of a size comparable to those proposed for some cell membranes, and some 10 times smaller than those suggested for capillary walls. In addition there is evidence for a specialized system enhancing the transfer of one glucose anologue from foetus to mother in a way that is subject to competitive inhibition. This work was supported by Birthright. REFERENCE

SOLOMON, A. K. (1968). J. gen. Physiol. 51, 335-364S.

Electrophysiology of endocrine sex BY R. G. DYER, F. ELLENDORFF and N. K. MACLEOD. A.R.C. Institute of Animal Phy8iology, Babraham, Cambridge CB2 4AT Injection of testosterone propionate into 4-day-old female rats produces an adult anovulatory syndrome. Conversely, cyclic ovulation occurs in male rats castrated on the first day of life and later provided with a female reproductive system. This aspect of endocrine sex results from hormonal action on cells in the rostral hypothalamus (for review see Gorski, 1971), possibly by influencing the connexions of neurones (Raisman & Field, 1971). In the present study we identified, by antidromic activation, neurones in the pre-optic and anterior hypothalamic areas which project directly to the region of the median eminence (Dyer, 1973) and have analysed the conrexions of these cells in both endocrine males and females. Extracellular action potentials were recorded from 582 neurones in 47 adult rats anaesthetized with urethane. Four groups of animals were used: normal males, females treated with 1 25 mg testosterone at 3 days of age (T.P. females), males castrated at day 1 (fales) and normal females. The cells were classified by their response to stimulation (biphasic pulses, < 2 mA; concentric bipolar electrodes) of the medial basal hypothalamus and then tested with similar pulses applied through an electrode in the cortico-medial region of the amygdala. The tests established whether the identified cells were influenced by orthodromic pathways from the site of stimulation. A marked sex difference was found among the 151 antidromically identified cells. In the females only 10 out of 41 (24 %) cells changed their firing rate after amygdala stimulation compared with 29 out of 52 (56 %) in the males (P < 0'01). Further, the fales were as unresponsive as normal females (only 31 % responding; P < 0.01 when considered with normal

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 19P males) and the T.P.-treated females were not significantly different (41 % responding) from normal males. Adjacent cells (n = 441), which were not antidromically activated, did not show these differences. Although there is substantial evidence that the electrical activity of the hypothalamus is modulated by systemic levels of a number of hormones (for references see Dyer, 1974) the present work suggests that a single exposure to a hormone can permanently modify neural transmission. F. E. is in receipt of a fellowship from the British Council and N. K. M. is supported by the Wellcome Trust.

REFERENCES DYER, R. G. (1973). J. Physiol. 234, 421-442. DYER, R. G. (1974). rn Progress in Brain Research 41, 33-147. Amsterdam: Elsevier. GoRsKI, R. A. (197 1). In Frontiers in Neuroendocrinology, ed. MARTINI, L. & GANONG, W. F., pp. 237-290. New York: Oxford University Press. RMsMAN, G. & FIELD, P. M. (1971). Science, N.Y. 173, 731-733.

Regional distribution of choline acetyltransferase and acetylcholinesterase in the rat amygdaloid complex BY Y. BEN-ARI,* P. R. LEwIs,t C. C. D. SHUTEt and R. E. ZIGMOND.4 M.R.C. Neurochemical Pharmacology Unit, Medical School, Cambridge CB2 2QD, and tPhysiological Laboratory, Cambridge CB2 3EG We have used a combination of biochemical and histochemical techniques to study the distribution of two marker enzymes for cholinergic neurones in the amygdaloid complex of the rat. The activity of choline acetyltransferase (CAT) was measured by a radiochemical assay (Fonnum, 1969) in ten selected regions dissected as previously described (Ben-Ari & Zigmond, 1975) from fresh frontal sections briefly stained with methylene blue. The distribution of acetylcholinesterase (AChE) was demonstrated by the thiocholine method as used by Shute & Lewis (1967). Large regional differences were found in the levels of both enzymes and the quantitative measurements of CAT closely matched the pattern of staining for AChE. Thus the nucleus of the lateral olfactory tract is intensely stained and has a high level of CAT (38.2 + 6-4 ,mol ACh/g . hr). The highest CAT activity (49.8 + 3.3) was found in the region called by Brodal (1947) the lateral posterior nucleus, which is an intensely stained structure that appears in the histochemical material to merge more caudally * Present address: Laboratoire de Physiologie Nerveuse du CNRS, Gif sur Yvette 91190, France. t Present address: Department of Pharmacology, Harvard Medical School, Boston, Mass., U.S.A.

A0PPROCEEDINGS OF THE 20P into another area, the basal nucleus, pars lateralis, also with high CAT (25.4 + 2.8). The lowest levels of CAT were found in the medial and central nuclei (4.7 + 0 5 and 9-3 + 0 7 ,cmol ACh/g . hr respectively), which stain only lightly for AChE. Besides the differences between nuclei, the histochemical staining showed large variations within certain individual regions such as the lateral posterior nucleus. Various subdivisions of this nucleus were therefore dissected out separately and analysed for CAT. Enzyme activity varied over a fivefold rage in good agreement with the pattern of staining. Our results illustrate the advantages of combining the anatomical resolution of a histochemical approach with the quantitative precision of a biochemical one. With the dual approach we have found a high degree of correlation between the distribution of CAT and AChE in the rat amygdala. Furthermore, the regional variation of enzyme activity seen in the lateral posterior nucleus emphasizes the possible existence of functional sub-divisions within a structure identified as a single entity by classical neuro-anatomical techniques. REFERENCES BEN-ARI, Y. & ZIGMOND, R. E. (1975). J. Phy8iol. 251, 4-5P. BRODAL, A. (1947). J. comp. Neurol. 87, 1-16. FoNNrm, F. (1969). Biochem. J. 115, 465-472. SHUTE, C. C. D. & LEwIs, P. R. (1967). Brain 90, 497-520.

Electrophysiological evidence for axon collaterals in the tuberoinfundibular system of the rat BY L. P. RENAUD. Division of Neurology, Montreal General Hospital, and McGill University, Montreal, Quebec, Canada The hypothalamic peptide hormones (factors) which mediate neural control of the adenohypophysis are elaborated from median eminence terminals of parvicellular 'tuberoinfundibular' neurones located in the mediobasal hypothalamus (Szentagothai, Flerko, Mess & Halasz, 1968). This communication reports that the patterns of activity evoked in presumed tuberoinfundibular neurones by median eminence, anterior hypothalamic area, preoptic area, and thalamic nucleus medialis dorsalis stimulation reflects the presence of local and distant central axon collaterals in the tuberoinfundibular system. Micropipettes filled with 3 % alcian blue dye in 0 5 M sodium acetate were utilized to record the activity and localize the positions of single hypothalamic units in pentobarbitone anaesthetized male rats. Tuberoinfundibular neurones (134 cells) identified by antidromic invasion from

2P 21P PHYSIOLOGICAL SOCIET Y, SEPTEMBER 1975 the median eminence were located within the ventromedial and arcuate nuclei and periventricular region. In twenty-four of twenty-nine spontaneously active cells studied, median eminence stimulation also evoked a decrease in excitability, with durations of 100-150 msec and at latencies coincident with but not necessarily dependent upon the antidromic spike. Similar responses were observed from six 'silent' tuberoinfundibular cells whose activity was maintained by iontophoretic application of L-glutamate (30-80 nA, 1DO m solution). These observations are interpreted to represent activation of a po-st-synaptic inhibitory mechanism via recurrent axon collaterals in the tuberoinfundibular system. Further indications of axon collaterals were provided by eight tuberoinfundibular neurones which displayed antidromic invasion from both the median eminence and from one other area: intrahypothalamic stimulation in the anterior hypothalamic area (2 cells); extrahypothalamic stimulation in the preoptic area (2 cells); and thalamic nucleus medialis dorsalis (4 cells). Hypothalamic peptides have now been localized within synaptosome fractions and nerve terminals in the central nervous system (Zimmerman, Hsu, Ferin & Kozlowski, 1974; Pelletier, Labrie, Puviani, Arimura & Schally, 1974; Pelletier, Labrie, Arimura & Schally, 1974; Hbkfelt, Effendic, Johansson, Luft & Arimura, 1974). The existence of hypothalamic and extrahypothalamic axon collaterals in the tuberoinfundibular system could provide one means for their widespread distribution in brain. Since these peptides appear to alter the excitability of central neurones (Dyer & Dyball, 1974; R~enaud, Martin & Brazeau, 1975), it is suggested that they may also be released at central synapses in association with activity in the tuberoinfundibular system. This work was supported by the Medical Research Council of Canada. REFERENCES

DYER. R. G. & DvBALL, R. E. J. (1974). Nature, Lond. 252, 486-488. HOKEELT, T., EFF~Nrnc, S., JoHLA.NsoN, 0., Lurr, R. & ARaIMURaA, A. (1974). Brain

Re8. 80, 165-169. PELETTiER~, G.; LABRIE, F., ARaimURA, A. & SCHALLY, A. V. (1974). Am. J. Anat. 144, 445-450. PELLETIER, G., LABRI.E, F., PuvIANI, R., ARiMEru.A, A. & SCHALLY, A. V. (1974). Endocrinology 95, 314-318. RENAUD, L. P., MARTIN, J. B. & BRAzEAu, P. (1975). Nature, Lend. 255, 233-235. SZENTIGOnHAI, J., FLERK06, B., MESS, B. & HALAsz, B. (1968). Hypothalamic Control of the Anterior Pituitary. Budapest: Akademiai Kiado. ZIMMERmAN, E. A., Hsu, K. C., FERIN, M. & Kozirowsi~c, G. P. (1974). Erndocrinology 95, 1 -1 1.

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Respiration and metabolic compartmentation in brain slices from a glia-deficient mutant, the Jimpy mouse BY P. KEEN, R. H. OSBORNE and U. M. M. PEHRSON. Departments of Pharmacology and Physiology, University of Bristol, Bristol BS8 1TD The Jimpy mouse described by Sidman, Dickie & Appel (1964) is a sex-linked recessive mutant which shows deficient myelination in the C.N.S. Mature oligodendrocytes are lacking in Jimpy mice (Farkas-Bargeton, Robain & Mandel, 1972), whereas other glial elements are only slightly affected and so this mutant provides an opportunity to study oligodendroglial functions. Brain slices (0.4 mm thick )were prepared by Mcllwain slicer from 20day-old Jimpy mice, which were showing intention tremor but no convulsions, and from normal litter-mates. Oxygen uptake of slices was measured in a Warburg manometer during 30 min incubation in Krebs-bicarbonate medium; K+ was then added to a final concentration of 56 mm and incubation continued for a further 30 min. The K+-stimulated increase in oxygen consumption by slices from normal animals was (%): cerebrum 46-8 + 7-9 (6), mid-brain 39'9 + 4-6 (6), medulla 39-6 + 5-6 (6) and cerebellum 98-6 + 25-9 (6) (mean +s.E.M.(n)). Slices of cerebrum of Jimpy mice showed a 12X1 + 3*4 (6) percentage increase and there was no significant increase in slices from mid-brain, medulla or cerebellum suggesting that K+-stimulated respiration may be specifically a function of oligodendroglia. Quantitative electron microscopic studies showed that, although mature oligodendrocytes were absent, there were more developing oligodendrocytes in the corpus callosum than in the spinal cord in Jimpy mice at this age, which may explain the small stimulation of respiration seen in the slices of cerebrum from these animals. In a second series of experiments slices were incubated with [1-14C]acetate for 10 min and the incorporation of 14C label into amino acids was determined by dansylation (Roberts & Keen, 1974). The relative specific activity of.glutamine (glutamate = 1.0) for slices from normal and Jimpy mice respectively were: cerebrum 4-97 + 0-19, 4*55 + 0'17; mid-brain 3*12+0*11, 2-61+0*09; medulla 1-89+005, 1-45+0-11 (mean+ s.E.M., n = 8). These results suggest that oligodendroglia do not play a major part in compartmentation. K+-stimulated respiration may represent uptake of K+ by glial cells (Hamberger & Henn, 1973) hence the demonstration here that this does not occur in Jimpy animals suggests that K+ removal from the periaxonal space may be predominantly a function of oligodendroglia. Compart-

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 23P mentation of amino acid metabolism, on the other hand, does not apparently reside solely in oligodendrocytes. R.H.O. is supported by the Mental Health Trust and U.M.M.P. by the Medical Research Council. REFERENCES

FARKAS-BARGETON, E., ROBAIN, 0. & MANDEL, P. (1972). Acta Neuropathol. (Berl). 21, 272-281. HAMBERGER, A. & HENN, F. A. (1973). In Metabolic Compartmentation in the Brain, ed. BALAzs, R. & CREMER, J. E., pp. 305-318. London: Macmillan. ROBERTS, P. J. & KEEN, P. (1974). J. Neurochem. 23, 201-209. -SIDMAN, R. L., DicKnE, M. M. & APPEL, S. M. (1964). Science, N.Y. 144, 309-311.

Time course of activity in intrafusal fibres in isolated cat muscle spindles BY I. A. BOYD. Institute of Physiology, University of Glasgow, Glasgow G12 8QQ The time course of contraction and relaxation of individual intrafusal fibres, in response to repetitive stimulation of the appropriate fusimotor axon in the muscle nerve, was determined by frame by frame analysis of moving film of isolated muscle spindles. The frequency of stimulation was that required to produce maximal contraction of nuclear bag fibres, i.e. between 50/sec and 100/sec. The mean contraction and relaxation times of fifteen nuclear chain fibre bundles were 0x48 and 0-56 sec, respectively. The time course of contraction of the two nuclear bag fibres in ten spindles was compared and in every spindle except one there was one 'slow nuclear bag fibre' and one 'fast nuclear bag fibre'. The exception contained two 'fast nuclear bag fibres'. The mean contraction and relaxation times for thirteen fast nuclear bag fibres were 0 50 and 0 59 sec, respectively, values which were not significantly different from those for nuclear chain fibres. However, the mean contraction and relaxation times of nine slow nuclear bag fibres were 0-81 and 1-15 sec, respectively, almost twice as long as for the other intrafusal fibres, this difference being highly significant statistically. Following a ramp and hold passive stretch some intrafusal fibres exhibit 'creep', the polar region of the fibre giving way so that the final extension of the primary sensory spiral round the fibre is less than it was at the completion of the stretch (Boyd & Ward, 1968). It has now been established that this creep is usually confined to the slow nuclear bag fibre in any spindle, while the fast nuclear bag fibre and the nuclear chain fibres behave in a purely elastic manner and follow the stretch without any creep-back.

PROCEEDINGS OF THE 24P The time from the end of the stretch to the completion of the creep in ten slow nuclear bag fibres varied from 05 to 2-6 sec with a mean of 1P69 sec. In all but two cases the creep time exceeded 1 sec. Presumably this creep contributes a mechanical component to the adaptation of the sensory discharge. More recent experiments on isolated spindles, activated via single fusimotor axons in the ventral spinal roots, have established that the slow nuclear bag fibres described above are 'dynamic nuclear bag fibres' and the fast nuclear bag fibres are 'static nuclear bag fibres' (Boyd, Gladden, McWilliam & Ward, 1975, and unpublished). REFERENCES

BOYD, I. A. & WARD, J. (1968). J. Physiol. 200, 104-105P. BoYD, I. A., GLADDEN, M. H., McWimLL>, P. N. & WARD, J. (1975). J. Physiol. 250, 11-12P.

Development of slow and fast muscles in chick embryos BY T. GORDON, R. PERRY,* N. C. SPURWAY and G. VRBOVA. Department of Physiology, University of Birmingham, and Institute of Physiology, University of Glasgow Differentiation of muscle fibres into slow and fast types proceeds only when innervation is intact (Askanas, Hee & Milhorat, 1972; Hanzlikova & Schiaffino, 1973). This indicates that the particular characteristics of the muscle fibres are imposed by their nerves. How the selection of muscle fibres by particular motoneurones is brought about is obscure. It is possible that during embryonic development, slow motoneurones innervate and activate muscle fibres a few days earlier than fast motoneurones, and that these will develop into slow muscle fibres. Consistent with this idea is the finding that -during embryonic development of the chick, myofilaments are formed earlier in slow muscle fibres than in fast ones (Gutmann, Hanzlikova & Holeckova', 1969). In the present investigation the development of the oxidative enzyme succinic dehydrogenase (SDH) was studied in the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscle of chick embryos. The muscles were excised from embryos of different ages and frozen transverse sections were used for visualizing SDH activity using a modification of the method described by Nachlas, Tsou, Souza, Cheng & Seligman (1957). At 15 days of incubation some enzyme activity could be detected in the * On study leave from the Department of Anatomy, University of Melbourne, Parkville, Victoria, Australia 3052.

25 PHYSIOLOGICAL SOCIET Y, SEPTEMBER 1975 25P slow ALD muscle, and none in the fast PLD. At 17 days of incubation SDH activity was well developed and uniformly distributed in ALD muscle fibres but almost absent in PLD. At 21 days, i.e. just before hatching, SDH activity in both muscles appeared to be similar. This is not surprising since at hatching the activity of this enzyme is the same in both muscles (Bass, Lusch & Pette, 1970). This earlier differentiation of ALD muscle fibres is also seen at the ultrastructural level. In particular, at 18 days, ALD has already a neatly ordered and quite closely packed filament array, and a well-formed sarcotubular system. The filament ordering is in fact neater than in the same muscle of the adult bird. Z lines for instance are straight in the 18-day-old embryo and will later become crooked. This point was previously noted by Shear & Goldspink (197 1). By contrast, the PLD from an 18-day-old embryo has more poorly formed fibrils, frequently prominent cytoplasmic spaces between them and only a rudimentary sarcotubular system. Histochemical and ultrastructural studies thus agree that the slow ALD develops before the fast PLD. This is consistent with the idea that the slow nerve fibres reach, innervate and activate muscle fibres some time before the fast nerve fibres; the fast nerve fibres will take over those muscle fibres that had not been innervated by the slow motor nerves. We are grateful to the Medical Research Council for supporting this work. REFERENCES

ASKANAS, V., HEE, D. & MILHORAT, A. T. (1972). J. Hi8tohem. Cytochem. 20, 849-850.

BASS, A., LusCH, G. & PETTE, D. (1970). Eur. J. Biochem. 13- 289-292. GuTmANw, E., HANZLtKOVk, V. & HoLECKOVk, E. (1969). Expl Cell Re8. 56, 33-38. HANZLIKOVA&, V. & SCHIAFFINO, S. (1973). Cell & Tiwie Re,8. 147, 75-85. NACHLAS, M., Tsou, M. K., SOUZA, E., CHENG, C., SELIGMAN, A. M. (1 957). J. Hi8tochem. Cytochem. 5, 420-436. SHEAR, C. R. & GJOLDSPINK, G. (1971). J. Morph. 135, 351-372.

Effects of cross-suturing the submandibular sympathetic trunk to the chorda in -cats By J. R. GARRETT and SHELA K. KEM-PLAY. Department of Oral Pathology, King's8College Hospital Dental School, London 5E5 8RX Cross-suture of nerves as a clinical and experimental tool- has a distinguished history. It is generally agreed that for the manoeuvre to be successful the two nerves involved must carry the same transmitter. In the present experiments the effects of suturing a post-ganglionic adrenergic nerve trunk to a preganglionic cholinergic trunk have been tested.

~~PAROCEEDINGS OF THE 26P26P The adrenergic sympathetic trunk to the submandibular gland was sectioned and the proximal stump was sutured to the distal stump of the glandular parasympathetic nerve, the chorda, after sectioning close to the lingual nerve. The animals were examined 3 weeks to 1 year afterwards. Secretory responses to i.v. methacholine (0.1-5 jug/kg) and adrenaline (0.1-20 jug/kg) and to nerve stimulation have been tested. Preganglionic, sympathetic responses were also assessed after eserine (2 jug per duct), and in some cases after atropine (25-75 jug/kg i.v.), or post-ganglionic sympathetic stimulation was tested before and after hexamethonium (2001800 jug/kg). The nerves were also examined histochemically. Preganglionic, sympathetic nerve stimulation evoked some secretion from all test glands. Formaldehyde-induced fluorescence indicated that adrenergic nerves had crossed the suture line at 3 weeks and entered the chorda, which is normally devoid of such nerves, but had not yet reached the gland. Subsequently, extensive adrenergic re-innervation of the glands resulted from nerves travelling via the chorda. Lingual nerve stimulation gave no responses in the early stages but after 6 months a slight secretion occurred and new cholinesterase-positive nerves were then found alongside the ducts. Despite adrenergic re-innervation, the test glands showed increased sensitivity to adrenaline compared with the control contralateral gland; this probably related to the parasympathetic decentralization. Methacholine sensitivities were divisible into two groups. Some animals showed increased sensitivity on the test side. The remainder surprisingly showed decreased sensitivity to methacholine, although the effective parasympathetic innervation remained deficient. Eserine, atropine or hexam~ethonium had no effects on the secretary responses to sympathetic stimulation in the group showing increased methacholine sensitivity, suggesting that their adrenergic re-innervation was mainly operating directly on glandular cells. In the group showing reduced methacholine sensitivity, eserine increased the sympathetic responses and atropine or hexamethonium reduced them. This suggests that part of the sympathetic response in these animals resulted from cholinergic effects which appeared to have been mediated by neurones in the chorda; correspondingly, fluorescent adrenergic axons were frequently observed close to ganglion cells in possible synaptic relationships. These results indicate that adrenergic axons can grow down the preganglionic cholinergic nerve trunk to re-innervate the gland and perhaps also create some functional 'heterogeneous' synapses with cholinergic ganglion cells. This work was supported by a Grant from King's College Hospital Joint Research Committee.

PHYSIOLOGICAL SOCIET Y, SEPTEMBER 1975

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The effect of lesions in the internal capsule and the sensorimotor cortex on servo action in the human thumb By JANE ADAM, C. D. MARSDEN, P. A. MERTON and H. B. MORTON. University Department of Neurology, Institute of Psychiatry and King's College Hospital, London SE5 8AF, The National Hospital, Queen Square, London, and The Physiological Laboratory, Downing Street, Cambridge CB2 3EG. It has been suggested that the servo-like responses in the long flexor of the thumb to unexpected perturbations during a voluntary movement, which have the relatively long latency of 40-50 msec, are mediated by a trans-cortical reflex pathway (Phillips, 1969; Marsden, Merton & Morton, 1973). We have investigated patients with unilateral lesions of this pathway. In an affected hand, the responses in the long flexor to stretch, halt and release were abolished or considerably attenuated. This was seen in three patients with vascular infarction of the internal capsule and in five patients with lesions of the sensorimotor cortex, due variously to middle cerebral artery occlusion, arterio-venous malformation, or a tumour. It has also been observed with isolated lesions of a posterior column (Marsden et al. 1973). In the most elegant case, the lesion was a small superficial tumour in the Rolandic fissure, and the responses largely recovered when this was excised. Immediately after the operation there was a severe cortical motor weakness, with hypotonia and brisk tendon jerks, and a profound cortical sensory deficit, with complete loss of appreciation of joint position, but preservation of crude touch, pain, vibration and temperature sensation. No response to stretch or release was detectable. As motor and sensory function improved post-operatively, the long-latency responses returned. At a stage when motor function had recovered completely but a sensory deficit persisted, the long-latency stretch reflex, although clearly present, was abnormal in that the early part of the response was relatively attenuated. This abnormality persisted even when joint-position sense and twopoint threshold had recovered and the only residual sensory abnormality was mild astereognosis and neglect with simultaneous bilateral stimulation. This loss or alteration in character of the long-latency stretch-reflexbased responses of the thumb after the lesions described is compatible with their dependence upon a long loop via the sensorimotor cortex. Additionally, the evidence suggests that the long-latency stretch reflex is a complex of at least two components, the earliest of which, at a latency of around 40-50 msec, is sensitive to dysfunction confined to the cortical sensory areas. 9

P HY 254

28P

A8PPROCEEDINGS OF THE

REFERENCES MARSDEN, C. D., MERTON, P. A. & MORTON, H. B. (1973). Lancet i, 759-761. PEnLIrs, C. G. (1969). Proc. R. Soc. B 173, 141-174.

Strychnine-sensitive Renshaw cell inhibition of group 1 A excited inhibitory interneurones BY G. BELCHER, J. DAVIES and R. W. RYALT. Department of Pharmacology, University of Cambridge, Hills Road, Cambridge CB2 2QD Renshaw cells have three known inhibitory functions. They recurrently inhibit motoneurones, mutually inhibit other Renshaw cells and also inhibit interneurones mediating reciprocal inhibition (see Ryall, Piercey & Polosa, 1972, for references). Recurrent inhibition is blocked by the iontophoretic administration of strychnine and is believed to be mediated by glycine but mutual inhibition is not blocked by strychnine. The effect of strychnine on the Renshaw cell inhibition of interneurones mediating reciprocal inhibition has not hitherto been investigated. Extracellular recordings were obtained from 50 interneurones excited at short-latency (average 1 13 msec) by group I volleys in muscle nerves in spinal cats anaesthetized with chloralose in which ventral roots L6, L7 and SI were transacted ipsilaterally. 40 % of such interneurones were inhibited by antidromic volleys in the ventral roots. The time course of this iiihibition corresponded with the duration of the Renshaw cell discharge to antidromic ventral root volleys. Maximal inhibition was evident 5-10 msec after the antidromic volley and the inhibition lasted for about 50 msec. On every neurone tested, the Renshaw cell inhibition was blocked by the iontophoretic administration of strychnine from a 0'01 M solution in 0*15 M-NaCl. The duration of the application required to block the inhibition varied with the iontophoretic current administering the strychnine (6-30 x 10-9 A) and from cell to cell. On the same cell it was observed that the reversal of glycine blockade by strychnine was more rapid than the block of synaptic inhibition for the same electrophoretic current ejecting strychnine from the micropipette. This indicates that the glycine receptors mediating the synaptic inhibition were located further from the micropipette than the receptors accessible to iontophoretically administered glycine. It is concluded that the transmitter, assumed to be glycine, liberated at each of the three known inhibitory synapses of Renshaw cells, interacts with strychnine-sensitive post-synaptic receptors at two of the three junctions and with a strychnine-resistant receptor at the third junction. This research was supported by a grant from the Medical Research Council.

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975

29P

REFERENCE

RYAIL, R. W., PIERCY, M. F. & POLOSA, C. (1972). Brain Re8. 41, 119-129.

Endocrine responses to hypoxia in the conscious calf BY S. R. BLOOM, A. V. EDWARDS, R. N. HARDY, KRYSTYNA W. MALINOWSKA and MARIAN SILVER. Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12, and the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG Young Jersey calves 3-5 weeks of age were habituated to wear a light, loosely fitting, 'helmet' constructed of aluminium and transparent polyethylene which was perfused with either air or nitrogen at 15 1./min. Perfusion of nitrogen through the 'helmet' produced severe hypoxia, associated with a reduction in Pco, and a rise in blood pH. Hypoxia was terminated after 8-10 min by which time spontaneous respiration had ceased in the majority of animals. The calves recovered rapidly after artificial ventilation had been applied for 1-2 min while air was perfused through the 'helmet'. The outputs of glucocorticoids and catecholamines from the right adrenal gland were measured using the 'adrenal clamp technique' (Edwards, Hardy & Malinowska, 1974) and the concentrations of glucose, insulin, glucagon and glucocorticoids in the arterial plasma were estimated at intervals. Severe hypoxia was found to produce a rapid but transient increase in plasma glucagon concentration, followed by a pronounced rise in plasma glucose concentration in animals with abundant liver glycogen. In contrast, no significant change in plasma insulin concentration occurred during hypoxia although plasma insulin rose subsequently in response to hyperglycaemia. The adrenal cortex was found to be extremely sensitive to hypoxia; the outputs of cortisol and corticosterone had risen by 522 + 64 ng kg-' min-' and 339 + 80 ng kg-' min-' (n = 6) respectively at the time the stimulus was discontinued (8-10 min) and achieved peak incremental values (cortisol: 791 + 174 ng kg-1 min-; corticosterone: 450 + 121 ng kg-' min') 10 min later. Thereafter the outputs of both steroids subsided steadily and had fallen to the resting range within 70 min. The release of adrenal steroids was reflected in the concentrations of cortisol and corticosterone in the arterial plasma and was accompanied by a pronounced increase in blood flow through the gland. Mean right adrenal blood flow rose from 215 + 23 ,ul. kg-' to a maximal value of 562 + 67 ul. kg-' min' 2 min after the stimulus was withdrawn. The extent of the increases in both steroid output and adrenal blood flow corresponded with those which occur in response to 9-2

30P PROCEEDINGS OF THE supramaximal doses of ACTH in calves of the same age (Edwards, Hardy & Malinowska, 1975). Secretion of catecholamines from the adrenal medulla was not observed until the PO2 of the arterial blood had fallen below 15 mmHg. However, hypoxia of this severity stimulated release of both adrenaline and noradrenaline (adrenaline: 974 + 410 ng kg-' min-, range 268-1827; noradrenaline: 532 + 252 ng kg-1 min-, range 60-1149; n = 4) at rates comparable with those recorded during maximal stimulation of the sympathetic innervation or asphyxia in anaesthetized calves of the same age (Silver, 1960; Comline & Silver, 1966). It is concluded that, although the adrenal medulla remains insensitive to relatively large reductions in the PO, of the arterial blood, intense hypoxia represents a maximal stimulus for the release of both glucocorticoids and catecholamines from the adrenal gland. This work is supported by grants from the Agricultural Research Council, the Medical Research Council and the British Diabetic Association. We are also indebted to Dr D. M. Burley (CIBA) for his continued support. REFERENCES ComLIBE, R. S. & SILVER, M. (1966). J. Phy8iol. 183, 305-340. EDWARDS, A. V., HARDY, R. N. & MALINowsKA, K. (1974). J. Physiol. 239, 477-498. EDWARDS, A. V., HARDY, R. N. & MALiNowsKA, K. (1975). J. Phy8iol. 245, 639-653. SILVER, M. (1960). J. Physiol. 152, 14-29.

The release of catecholamines from perfused canine adrenal glands by corticosteroids BY J. A. J. H. CRIrTcLEY, C. G. HENDERSON, L. E. F. MOFFAT, A. UNGAR, J. WAITE and CIRISTINE P. WEST. Department of Pharmacology, Univeraity of Edinburgh, 1 George Square, Edinburgh EH8 9JZ We have shown (Critchley & Ungar, 1974) that intravenous administration of corticotrophin in dogs causes the release of adrenaline and noradrenaline from the adrenal medulla. This release is inhibited by cycloheximide, a drug that blocks the secretion of corticosteroids from the adrenal cortex. This observation raises the possibility that the output of corticosteroids from the cortex may regulate that of catecholamines from the medulla in parallel with neural influences. We have now studied the influence of hydrocortisone on the output of catecholamines from isolated perfused adrenal glands. Adrenal glands were excised from dogs under pentobarbitone anaesthesia, and perfused retrogradely through the adrenolumbar vein with Locke's solution at a constant flow of 2 ml./min. The effluent from the cut ends of the arteries was collected in 30 sec fractions by an Unicam AC60

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 31P analyser, which made estimations of catecholomine concentrations by the trihydroxyindole fluorimetric method. After collection of samples from the resting gland hydrocortisone was infused for 8 min at one of three rates. The results are shown in Table 1. In three glands similar responses were obtained with deoxycorticosterone. TABLE 1. Results from 8 adrenal glands having a mean resting output of 202 + 24 ng/min Hydrocortisone sodium succinate infused at Ir

Incremental output of catecholamines

A

30 jug/ml. 48 + 7

50 jug/ml. 253 + 54

100 ,sg/ml. 610+117

(ng/min) These results support the theory that corticosteroids may play a part in regulating the release of adrenal catecholamines as well as the synthesis of adrenaline. REFERENCE

CmRTciuRY, J. A. J. H. & UNGAR, A. (1974). J. Phy8iol. 239, 16-17P.

Suppression of histamine leucocytosis by metiamide BY C. F. CODE Mayo Clinic and Foundation, Rochester, Minnesota, U.S.A. Tasaka & Code (1964) have demonstrated that histamine administered to dogs or rabbits in beeswax mixture to prolong the action ofthe histamine, induces a leucocytosis due entirely to increased numbers of circulating neutrophils. In the present study, 6 dogs received 30 mg histamine in beeswax at 8 a.m. on 3 consecutive days and all developed, each afternoon, a neutrophil leucocytosis. In a second test, identical to the first except that the 6 dogs were each given 100 or 200 mg metiamide by mouth at 8 a.m., noon and 4.30 p.m., the afternoon neutrophil counts were uniformly suppressed. In another group of 8 dogs, continuous i.v. histamine for 12 hr produced a neutrophil leucocytosis of variable magnitude; continuous intramuscular histamine in the same dose (3 ,umole/hr for the first 6 hr and 6 ,umole/hr the second) produced a more consistent and greater neutrophil leucocytosis. The I.M. injections of histamine were started at 6 a.m. and by noon the leucocytosis was usually apparent; by 4 p.m. it was always present. On another occasion, the same continuous I.M. injection of histamine was given to each dog, but each also received metiamide by mouth (100 or 200 mg at 5 a.m., 9 a.m., noon and 3 p.m.). In addition, four of them were given two or three doses of 100-200 mg on the day prior to the test. The neutrophil leucocytosis was reduced in every animal given metiamide

32P PROCEEDINGS OF THE and in half of them it was eliminated completely. The experiments indicate that histamine produces a neutrophil leucocytosis. The irregularity of the leucocytosis to i.v. histamine, as compared to its uniformity when given I.M. suggests that histamine releases a neutrophil leucocytic factor in muscle which in turn releases increased numbers of neutrophils into the circulation. The effect is specific for the neutrophils because the other formed elements of the blood are not increased. Metiamide may block the release of the leucocytic factor by histamine (an H2 receptor effect) or it may block the action of histamine or of the leucocytic factor in the bone marrow, or it may simply have a non-specific suppressive action on the release of neutrophils from bone marrow in dogs. I wish to thank Delbert Minsky for technical assistance. REFERENCE TASAKA, K. & CODE, C. F. (1964). Fedn Proc. 23, 471.

Measurement of blood flow in rabbit skin homografts and autografts using a 133Xe clearance technique BY G. P. LEWIS, M. J. PECK, T. J. WILLIAMS and BEVERLEY A. YOUNG. Department of Pharmacology, Institute of Basic Medical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN An understanding of the mechanisms underlying vascularization and rejection of grafts requires precise measurements of blood flow changes in grafted tissue. This paper describes the application of a 133Xe clearance technique (Sjersen, 1969) to the study of blood flow changes in skin grafts. Autografts and homografts of skin were made on to rabbit hind limbs (Jasani & Lewis, 1971). Each day subsequently, rabbits were anaesthetized, grafts injected in turn with 0-05 ml. 133Xe in saline, and the washout of radioactivity from each graft monitored for 30 min using a collimated yscintillation detector. Fig. 1 shows that the clearance plot of a homograft 1 day after grafting is monoexponential. On days 5, 6 and 7, the curves contained in addition a faster component which could be derived by curvepeeling. Fig. 1 shows the curve on day 6 with its two derived exponential. Biexponential curves were also produced in normal skin. On day 8, homograft curves returned to monoexponentials (Fig. 1) corresponding to visible signs of rejection, whereas autograft curves remained biexponential. Changes in slope of the slow phase (phase II) may reflect changes in graft thickness and vascularity of the subcutaneous graft bed. We conclude that the slope of the fast component (phase I) of the 133Xe clearance curve provides a measure of blood flow in grafted skin. This work is supported by the M.R.C.

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975

33P

100 50

Z_C 10 0

x*

I Homograft, day "i*1 k=719x102min'

X1

phase I I,

cere l c xk=63X102 min-'

0010.5

0

0

1

0

dsgmdhay8ay 6

Homo.v day 6

I k3.3x1pnsphase

phas

kc-6x 10-1 min0

5

10

0

2

3

e dae6 1m75,

p10-1 min-'

minkt1 15

20

25

30

35

Time (min)

Fig. 1rlXe clearance curves in one homograft 1, 6 and 8 days after grafting. Inset shows the changes in rate constants for the homograft, phases I and IL REFERENCES

JASANI, M. K. & LEwiis, G. P. (1971). J. Phy~iol. 219, 525-554. SJERSEN, P. (1969). Circulation Re8. 25, 215-229.

The effect of alarm on the spontaneous activity of neurones in the cerebral cortex of the unrestrained cat By A. C. WEBB. National Institute for Medical Research, Mill Hill, London NWZ 1AA Interval histograms derived from the activity of single cortical neurones can be described by log-normal distributions (Burns & Webb, 1975). They can therefore be defined in terms of only two parameters, a modal interval, and a geometric standard deviation (GSD). When an animal falls asleep, the values of these parameters change predictably. The mode becomes shorter, and the' GSD becomes bigger (Burns & Webb, 1975. The experiments reported here are intended' to find out whether the two parameters are also related to changes in arousal which occur while the animal is awake. The specific question to be answered was as follows: does the mode lengthen and the GSD become smaller when a relaxed animal is suddenly alarmed? Single unit recordings were made from cells in the post-lateral and suprasylvian gyri of the cerebral cortex of the unrestrained cat (Burns, Stean &

34P 34PPROCEEDINGS OF THE Webb, 1974). During the experiment the animal was placed in a large Perspex box. The nozzle of a compressed air line protruded into one corner of the box. The work was carried out in complete darkness, and the cat was observed through an infra-red telescope. This was done to avoid any direct effects on neuronal activity which changes in eye-movements might produce. The animal was first allowed to sit quietly in the dark, while a control record was made. Then the compressed air line was turned on, making a hissing noise. The animal was considered to be alarmed when he stood up, and 'pointed' towards the nozzle of the air line. Interval distributions were constructed from two stretches of record, one made while the animal was sitting quietly in the dark, and the other taken just after the cat had reacted to the sound of compressed air. Best-fit lognormal curves were calculated, and the values of the defining parameters of the pair of records were compared. Those neurones in visual cortex which initially displayed a low rate of spontaneous activity, stopped firing altogether during alarm. The remainder of the cells showed systematic changes in the values assigned to the mode, and to the GSD. The absolute magnitude of these changes was variable (as, indeed, was the behavioural reaction of the cats themselves), but, in every case the mode lengthened, and the GSD became smaller. These changes are opposite in direction from the changes which accompany the transition from wakefulness to sleep. It therefore appears that 'level of arousal' is represented in the cortex as a continuum of possible modal intervals and possible GSDs. If the activity of all cortical cells is 'biased' in this way, then the special functions of each neurone must be superimposed on this bias. REFERENCES

BURNS, B. DELISLE, STEAN, J. P. B. & WEBB, A. C. (1974). Electroenceph. din.

Neurophy8iol. 36, 314-318. BURNS, B. DELISLE & WEBB, A. C. (1975). J. Physiol. 248, 44-45P. BURNS, B. DELISLE & WEBB, A. C. (1975). J. Physiol. 252, 18-19P.

Slow waves in neural records BY A. G. H. BLAXELEY and R. 0. STEPHEN. Department of Pharmacology and Institute of Physiology, University of Glasgow We have observed low frequency waves in the activity recorded from populations of active neurones. The usual explanations for such activity, involving synchronization of units by anatomical interconnexions, were impossible. We offer an alternative explanation applicable to any large neural population. Statistical addition of independent random events can produce slow-

PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 35P wave summated responses, e.g. 1ff noise in semi-conductors (Thornton, de Witt, Gray & Chennette, 1966) and fluctuation in nuclear reactions (Stephen, 1963). The mathematical analysis used in these examples, applied to populations of neurones firing independently at nearly the same frequency with interspike intervals subject to moderate variation, predicts that summing enough units at the electrodes produces a signal having two components corresponding in frequency to the frequency components in the spikes and to the mean interspike frequency. s.M(msc.

N 1K

50

10K KIA

0-1 K

11111

1K

100

10 K

200

10K S

1 sec

Fig. 1. Model of neural recording S.D. = S.D. of unit interspike interval; = population size; St = 'stimulus' on; amplitude scale, arbitrary.

f = 2 Hz; N

To demonstrate this we modelled, on a computer, populations of neurones. Each 'neurone' equally contributes a 1 msec square pulse to the recorded response. Fig. 1 shows the computer-simulated recordings of constant discharges initiated by an 'afferent stimulus', and the effect of changing the variance of the spike distribution and population size. In other situations where slow waves are observed and the activity in single units is found to be uncorrelated this explanation will apply. REFERENCES D.Phil. R. 0. (1963). Thesis, Oxford University. STEPHEN, THORNTON, R. D., DE Wnr, D., GRAY, P. E. & CHENNETTE, E. R. (1966). Characteri8tic8 and Limitations of Tranviator&. New York: John Wiley.

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6PROCEEDINGS OF THE

Effects of cholinergic drugs on catechol evoked convulsions BY A. ANGEL and D. G. DEWHUMsT.* Department of Physiology, University of Sheffield After catechol, electrical stimulation at the wrist evokes, in the rat deeply anaesthetized with urethane, three brief action potentials in forelimb flexor muscles: the first representing a spinal reflex, the second a cortical reflex (Angel, Lemon & Orpwood, 1968) and the third possibly a spino-bulbo-spinal reflex. Previous work (Angel & Dewhurst, 1975) indicated that spontaneous convulsions induced by injection of catechol may be dependent upon a central cholinergic mechanism. This has been investigated by studying the effects of drugs which modify cholinergic transmission on the early components of the evoked muscle response. Atropine (40 mg/kg) and hyoscine (40 mg/kg) increased the amplitude and slightly decreased the latency, while eserine (40 fig/kg) and neostigmine (25 fg/kg) caused a large increase in amplitude of the spinal reflex. The probability of occurrence of this first response was unaffected by any of the drugs used. The probability of occurrence, amplitude and latency of the second component of the evoked muscle response were unaffected by neostigmine, atropine methyl nitrate (20 mg/kg) and hexamethonium (10 mg/kg). Eserine significantly increased both amplitude and probability of occurrence, while atropine and hyoscine significantly decreased the frequency of occurrence, with mecamylamine giving only a slight attenuation. These results indicate that the pathway involved in the cortical component of the evoked response contains, or is modulated by, at least one cholinergic synapse, and further that the action of acetyl choline at this site is predominantly muscarinic. Angel (1969), in a study of the dorsal column sensory pathway, showed that the increased corticofugal discharge after catechol was a consequence of a large increase in the thalomo-cortical volley and transmission through the ventrobasal thalamus is modulated by cholinergic pathways (Anderson & Curtis 1964a, b). Further Angel & Lemon (1973) concluded that catechol also activates the brain stem reticulo-spinal system, transmission through which is also thought to be cholinergic (Shute & Lewis, 1963; McCance, Phillis & Westerman, 1968; Bradley & Dray, 1972). The possibility that catechol is acting at either or both of these sites is being considered. * M.R.C. scholar.

PH YSIOLOGICAL SOCIETY, SEPTEMBER 1975

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REFERENCES ANDERSON, P. & CuIRrs, D. R. (1964a). Acta physiol. 8cand. 61, 85-99. ANDERSON, P. & CUIRTIS D. R. (1964b). Acta phygiol. 8cand. 61, 100-120. ANGEL, A. (1969). Electroenceph. clin. Neurophy&iol. 27, 392-403. ANGEL, A. & DEWHURST, D. G. (1975). J. Physiol. 252. 19-20P. ANGEL, A. & LEMON, R. N. (1973). Electroenceph. olin. Neurophysiol. 34, 369-378. ANGEL, A., LEMON, R. N. & ORPWOOD, R. (1968). J. Physiol. 198, 10-11P. BRADLEY, P. B. & DRAY, A. (1972). Br. J. Pharmacy. 45, 372-374. MCCANCE, I., PHIInES, J. W. & WESTERMAN, R. A. (1968). Br. J. Pharmacy. Chemother. 32, 635--651. SHUTE, C. C. D. & LEWIS, P. R. (1963). Nature, Lond. 199, 1160-1164.

Coupling between cutaneous nerves BY B. MATTHEWS. Department of Physiology, University of Bristol, BS8 1TD Evidence is presented which suggests that in skin, as in teeth (Matthews, 1975), an impulse propagated towards the terminals of one afferent nerve may generate an orthodromic impulse in another. The preparation is shown in Fig. 1 A. Ten fibres in seven cats responded in an unusual way to electrical stimulation. An action potential with a

B S1, 2 0 V

S1, 3-0 V

S2, 2 0 V

0.3

S2, 3 0 V 10 msec

J mV S

Fig. 1. A, experimental set-up. B, responses of a saphenous nerve filament containing two fast units and one of variable latency. Five traces were superimposed in each record. Stimuli (50 Usec and intensities shown) were applied at the start of each sweep to S1 or S2. S1-S2, 28 mm; S1-R, 35 mm.

PROCEEDINGS OF THE 38P long latency was evoked at threshold but, as the stimulus was increased, a critical intensity was reached at which the latency decreased markedly. Six units responded in this way to stimulation at either SI or S2 and four did so at both sites. In the latter group, the long latency response from SI was more delayed than that from S2 (Fig. 1B), despite the apparently shorter conduction distance. Each unit responded to innocuous mechanical stimulation of skin. Cooling the receptor field increased the delay of the long, but not the short, latency response.. The long, but not the short latency response was blocked by section of the nerve trunk peripheral to S2. Conduction velocities calculated from the shorter latencies were 09-22 msec-1. Supported by the M.R.C.

REFERENCE MATTHEws, B. (1975). J. Phy~iol. 252, 25P.

Action of neurotransmitter substances on parotid amylase secretion BY 0. H. PETERSEN* and N. UEDA.* Institute of Medical Phy0iology C, University of Copenhagen, Denmark The existence of three receptor sites on the parotid acinar cell membrane has been described (Petersen & Pedersen, 1974). The effects of a-adrenoceptor, f8-adrenoceptor and cholinergic receptor excitation on amylase secretion from mouse parotid segments have also been studied. Isoprenaline evoked a large response, but the amylase output only rose slowly following start of stimulation. In contrast, phenylephrine and acetylcholine (ACh) evoked small but steep increments in amylase output (Petersen & Ueda, 1975). Fig. 1 shows two typical tracings from experiments on superfused mouse parotid segments. The combination of ACh and isoprenaline stimulation resulted in both a steep and large response. The increase in amylase output following ACh stimulation was similar during control conditions and during maximal and sustained isoprenaline stimulation. Very similar findings were obtained with a-adrenoceptor activation. The amylase secretion in response to ACh and a-adrenoceptor activation was totally dependent on the presence of external Ca, whereas the isoprenaline response was hardly affected by severe reductions in external Ca concentration. *

Present address: Department of Physiology, The University, Dundee DD1 4HN,

Scotland.

39P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 There are presumably 2 classes of receptors on the parotid acinar cells: the ac-adrenoceptors and the cholinergic receptors which are Ca-sensitive and the f8-adrenoceptors which are relatively Ca-insensitive. There does not seem to be any important interaction between the two receptor types in the kind of experiments reported here. I

ACh 10-6M L Isoprenaline 10-1 M

L

ACh 10-6M-

10 u/ml.---

L10 minI |

Adrenaline 10-5M

Isoprenaline 10-SM

-Isoprenaline 10-6 M

10 -uml.

--

Basal secretion

.-_ _ _ _

_

-

_

-

-._------0 _

---

'-10 minm-

Fig. 1. The tracings represent the concentration of amylase in the effluent from a flow cell, in which several segments of mouse parotid were placed, measured with an automated continuous fluorescence method (Matthews, Petersen & Williams, 1974). Calibration is shown and the horizontal bars indicate the periods of stimulation. REFERENCES

MAirHEws, E. K., PETERSEN, 0. H. & WILLIA s, J. A. (1974). Analyt. Biochem. 58, 155-160.

PETERSEN, 0. H. & PEDERSEN, G. L. (1974). J. membrane Biol. 16, 353-362. PETERSEN, 0. H. & UEDA, N. (1975). J. Physiol. 250, 1-2 P.

PROCEEDINGS OF THE

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The effect of low-sodium solution and lanthanum on the sodium activity of crab muscle fibres BY R. D. VAUGHAN JONES.* Department of Physiology, University of Bristol Until now, only brief measurements have been made of the intracellular sodium activity, aNa,, of muscle fibres (Caldwell, 1968). Fig. 1 shows an experiment where aNa, was continuously recorded in a crab (Carcinus maenas) muscle fibre using a recessed-tip Na+-sensitive micro-electrode (Thomas, 1972). |20 minX > 50 E

3 mM- LaCI, E

3 mm-LaCI,

8

4

I'd Na

,-0 Na _010' 10Na

~~~~~~Na Fig. 1. Effects on membrane potential (M.P.) (upper trace) and on aNa1 Na

(lower trace) of reducing [Na]0 to one-tenth normal (50 mM; Li-substituted). The osmotic strength of all solutions was constant. pH of all solutions was 7.4.

Lowering [Na]0 from 500 mM to 50 mm, by substituting Li for Na, rapidly and reversibly decreased aNa, by about 4 mm within 5 min. Addition of LaCl3 (3 mM) stimulated a similar fall in aNa1 and nearly abolished subsequent responses to low [Na]0. The response of aNa1 to low [Na]0 is very similar in Tris-substituted solutions, is virtually unaffected by ouabain (2 x 10-4 M, 60 min) or zero [Ca] + 1 mM EGTA (90 min) but is greatly reduced by external CoCl2 or MnC12 (50 mM). If the rapid decrease of aNa1 represents an efflux of Na+ from the fibre, it must be both active, because Na+ would be moving against an electrochemical gradient, and coupled to the movement of another ion, since there is only a small change in membrane potential. Alternatively, the effect may be caused by Naj+ moving into another cellular compartment inaccessible to the Na+-sensitive electrode but reasonably accessible to the external environment to account for the rapid effects of both low [Na]0 and LaCl3. *

Present address: Department of Pharmacology, University of Oxford.

PH YSIOLOGICAL SOCIETY, SEPTEMBER 1975

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REFERENCES CAL wEIJ, P. C. (1968). Phyriol. Rev. 48, 1-64. THOMAS, R. C. (1972). J. Physiol. 220, 55-71.

Heat production and chemical change in frog sartorius; a comparison of R. pipiens with R. temporaria BY JOAN DAWSON, D. GOWER, M. K. KRETZSCHmAR and D. R. WiuUKE. Department of Physiology, University College London, Gower Street, London WC1E 6BT This laboratory has reported that in R. temporaria the heat production associated with isometric tetanus at 00 C cannot be accounted for by known chemical reactions (Gilbert, Kretzchmar, Wilkie & Woledge, 1971; Curtin, Gilbert, Kretzschmar & Wilkie, 1974). Homsher, Rall, Wallner & Ricchiuti (1975) have recently reported that a similar quantitative discrepancy does not appear to exist in R. pipiens although they confirmed our results in R. temporaria. We have now ourselves measured heat production and chemical change using R. pipiens imported from Massachusetts. TABLE 1. Production of heat and work compared with phosphocreatine break-down in sartorii from R. pipiens and R. temporary. All muscles unpoisoned at 00 C and in 02, 5 % CO2 bicarbonate Ringer. 5 sec isometric tetanus; muscles for chemical analysis frozen immediately after last stimulus

R. temporary

R. pipien8 -

Mean + S.E. n = number of expts.

(h+w) Crt

mJI4jtmol Crt A (PCr/Crt) x 100

Homsher et al. (1975, Table II)

2*21 + 0X08 (13)

5X4 ± 0-3 (22) Crtl(muscle weight) 28X70 ± 0X55 (30) (mmol/kg) Excess energy

(kJ/mol Crt) 'AHm'

=

- 0-20 + 0-15

A

Curtin &

This paper

Homsher et al. (1975, Table III)

2*9 + 0.1* (5)

3-50+ 0-10 (8)

3.3 + 0.2 (6)

4-2+0-6

4-7+0±5

4-7+0±8

(10)

29-3±0-6

(13) 33-91 ± 0 85

(12) 29X9 ± 0-7

(10)

(8)

(6)

1-06 + 0-30

1-40 + 0-24

1*20 + 0-42

Woledge (1975)

-44.7 kJ mol-'

1-70 + 0-35 1-90 ± 0-20 1-50 + 0-24 0.37 ± 0-13 = -34 kJ mol-1 * Average of results using direct and Peltier methods of calibration. h = heat produced, w = work produced, Crt = total creatine (free creatine plus

AHm

phosphocreatine), PCr = phosphocreatine.

42P PROCEEDINGS OF THE 42P Our results do not support the conclusion that there is a species difference. As shown in Table 1, our measurement of energy production in R. pipiens is significantly (P 0-001) greater while that of PCr break,down is somewhat (P 0.1) less than reported by Homsher et al. (1975). If the comparison between energy production (h + w) and phosphocreatine breakdown (APCr) is based on the ratio ((h + w)/APCr = 'Aflm' = - 44.7 kJ mol-1) adopted by Homsher et al. then the excess energy production indeed disappears in their, but not in our, experiments on R. pipien8. However, the calorimetric determinations by Woledge (1972), now confirmed by later experiments, show that the in vivo heat of hydrolysis of phosphocreatine (AHm) should be -34 kJ mol-1. We conclude therefore that in R. pipiens, as in R. temporaria, the output of energy is always in excess of that which can be accounted for by contemporarious hydrolysis of phosphocreatine. -

We wish to acknowledge the support of the Muscular Dystrophy Association of America and the Medical Research Council.

REFERENCES

CuwRmIN, N. A., GILBERT, C., KRETZSCHMAR, K. M. & WLxiE, D. R. (1974). J. Physiol. 238, 455-472. & WOLEDGE, R. C. (1975). J. Physiol. 246, 737-752. GILBERT, C., KRETZSCHMAR, K. M., WuIKCE, D. R. & WOLEDGE, R. C. (1971). J. Physiol. 207, 163-193. HoMsHER, E., RALL, J. A., WALLNER, A. & Ricchi, N. A. (1975). J. gen. Phy9iol. 65, 1-21. WOLEDGE, R. C. (1972). Cold Spring Harb. Symp. quant. Biol. 37, 629.

CURTJN, N. A.

Heat production during small length changes in active muscle BY S. H. GILBERT and Y. MATSUMOTO. Emory University, Atlanta, Georgia 30322, U.S.A. Recent observations concerning the tension responses to small length changes in single muscle fibres (Huxley & Simmons, 1972) suggest that the examination of thermal responses in whole muscles under similar conditions could be productive. Also, the failure of previous workers to observe directly a thermoelastic absorption of heat during a tension rise in active muscle (Woledge, 1961, 1971) suggests that previous interpretations of such experiments are somewhat questionable. Small releases and stretches (0.5 % lt < Al < 3 0 % lo) were applied to R. pipiens sartorii stimulated tetanically via the nerves at 00 C during the isometric tension plateau. Releases of 0 5 % lo (or less) resulted in a thermal response (AQ) superimposed on the isometric maintenance heat whose time course followed closely the time course of the tension change. No

43P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 shortening heat (h8) was observed. The thermal response to larger releases (Al > 1.0 % lo) consisted of two separate phases, an initial burst of extra

heat accompanying the drop in tension (AQ), followed by a second burst of h8 during tension recovery. These two phases were separated by a momentary decline in heat rate during the earliest part of tension recovery. AQ was always proportional to the amount of tension change (AP), and the thermoelastic heat: tension ratio/I was - (5.2 + 0 1) x 10-3 in 32 determinations from four sets of muscles. Comparisons between the time course of the recorded h8 and that calculated from the time course of tension recovery, the length-tension curve of the series elasticity for that muscle pair and Hill's h8 coefficient a indicate that the earliest and latest stages of tension recovery are not accompanied by h.. The muscles seem able to shorten by about 1 % lo without producing h8. In later experiments stretches and releases of similar amounts were applied in successive contractions in the same muscles. Thermal responses to the stretches consisted also of two phases. The first phase was an absorption of heat (- AQ) during the increase in tension as the muscle was stretched, which was always proportional to AP. The pooled R was - (4.6 + 0 2) x 10-3 for 13 stretches and 12 releases on two sets of muscles. The second phase of the thermal response was a burst of heat as tension fell just after the end of the stretch. There seemed to be more heat produced during the latter phase than could be accounted for as degradation of store elastic energy. REFERENCES HuxLEY, A. F. & SiMMONS, R. M. (1972). Cold Spring Harb. Symp. quant. Biol. 37, 669-680. WOLEDGE, R. C. (1961). J. Physiol. 155, 187-208. WOLEDGE, R. C. (1971). Prog. Biophy8. & molec. Biol. 22, 39-74.

Dose-response curves with labelled sodium and labelled decamethonium in rat muscle BY R. CREESE, G. I. FRANKLIN, P. P. A. HUMPHREY* and L. D. MITCHELL. Department of Physiology, St Mary's Hospital Medical School, London W21PG The effect of depolarizing drugs on the influx of sodium can be demonstrated in rat diaphragm by short exposure (15-30 sec) to a solution containing 24Na plus depolarizing drug in the presence of tetrodotoxin (0.1gM), followed by a wash in inactive solution so that the excess radioactivity at the junctional region can be detected by slicing and counting the * Present address: Pharmacology Department, Allen and Hanburys Ltd, Ware, Herts.

PROCEEDINGS OF THE 44P tissue (Creese & Mitchell, 1975). In the case of decamethonium and similar compounds the entry of labelled drug can also be measured (Taylor & Nedergaard, 1965; Creese & England, 1970), and this provides another method for assessing the change in permeability at the end-plate. With 24Na the increased influx was dependent on the concentration of depolarizing drug, and rose to a plateau at 100 AiM for decamethonium and also for carbachol, with Hill coefficients (Rang, 1975) between 1-5 and 1 8 in different assays. The depolarizing action of decamethonium, recorded by a combination of external electrodes (Humphrey, 1973) and internal electrodes, gave a half maximal effect at 4-7 AM (95 %, limits 3-8-12-2 AM), which is compatible with results obtained with 24Na and also with [3H]methyl decamethonium. Measurement of influx by isotope methods enables the effects of the drug to be determined over a wide range of concentrations, and may be compared with dose-response curves derived from conductance methods (Rang, 1975; Dreyer & Peper, 1975). This Research was supported by a grant from the Medical Research Council.

REFERENCES CREESE, R. & ENGLAND, J. M. (1970). J. Physiol. 210, 345-361. CREESE, R. & MITCHELL, L. (1975). J. Physiol. 246, 44-45P. DREYER, F. & PEPER, K. (1975). Nature, Lond. 253, 641-643. HUMPHREY, P. P. A. (1973). Br. J. Pharmac. 47, 636-637P. RANG, H. P. (1975). Q. Rev. Biophys. 7, 283-399. TAYLOR, D. B. & NEDERGAARD, 0. A. (1965). Physiol. Rev. 45, 523-554.

Effects of atropine on membrane currents and twitch tension in bullfrog atrium By B. E. BLOOD, W. GILES and S. J. NOBLE. University Laboratory of Physiology, South Parks Road, Oxford OX1 3PT Atropine is considered to be a competitive antagonist to acetylcholine in cardiac muscle. In frog atrial muscle, acetylcholine markedly reduces the slow inward current (Giles & Tsien, 1975). It was therefore expected that atropine would simply block this effect. Voltage-clamp experiments have been performed using a double sucrose gap technique (Brown & Noble, 1969) in order to study the effects of atropine on the membrane currents in atrial trabeculae from Rana catesbeiana. In parallel experiments, changes in twitch tension were recorded using a piezo-electric tension recorder (Blood & Spindler, 1975). Following either the application of atropine (10-7-10-5 M) alone, or its use to block relatively low doses of acetylcholine (< 10-7 M), unexpected results were observed:

45P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 (i) The slow inward current was significantly increased over control values (see Fig. 1). I (x 10-' A)

Control

*

o x

20 (TTX2-0x106g/mI) X O-*M I ) 15 ACh (5-O Atropine (10-TMm)

-90 -70 -50 3

10

-

10

30(mV)

-30J Fig. 1. Effects of acetylcholine and atropine obtained in the presence of tetrodotoxin (2.0 x 10-6 g/ml.) on the slow inward current. The peak inward or minimum outward current was measured and was plotted as a function of applied voltage, without correction for leakage currents.

(ii) A positive inotropic effect was observed. It therefore appears either that atropine is blocking endogenous as well as applied acetylcholine or that atropine has an additional intrinsic pharmacological action on frog atrial muscle. We acknowledge support from the M.R.C. and the Canadian Heart Foundation and M.R.C. REFERENCES

BLOOD, B. E. & SPINDLER, A. J. (1975). J. Physiol. 252, 2-3P. BROWN, H. F. & NOBLE, S. J. (1969). J. Phyaiol. 204, 717-736. GmEs, W. & TsIEN, R. W. (1975). J. Phyaiol. 246, 64-66P.

The effects of ouabain on interval duration relationships in sheep Purkinje fibres By I. COHEN, J. DAUT and D. NOBLE. University Laboratory of Physiology, Oxford OX1 3PT The duration of the Purkinje fibre action potential usually shortens when the interval between the test stimulus and the conditioning stimulus (C-T interval) is decreased. This shortening is largely due to a maintained activation of the repolarizing current iI (Hauswirth, Noble & Tsien, 1972). Occasionally, however, the test action potential lengthens as the C-T interval is reduced. We report here the investigation of such a preparation.

46PPROCEEDINGS OF THE 46P In normal Tyrode the action potential lengthened by 9-5 % following a 3-8 sec C-T interval, and was maximally lengthened by 60 % at a C-T interval of 800 msec. The peak of the action potential is also increased as the C-T interval is shortened. This effect is maximal at an interval of 450 msec, where the overshoot is increased by 27 mV. At a-recent meeting of the Society (Cohen, Daut & Noble, 1975) we presented evidence for the existence of a restricted extracellular space in Purkinje fibres. This space would normally exchange a high percentage of its total [K] every second. If the action potential stimulated the Na+-K+ exchange pump (either through elevated [K+] outside, elevated [Na+] inside or reduced [Na]0) [K]0 could decline below resting values transiently and the action potential would be lengthened through the well known decrease of gE with lowered [K]O. The increase in overshoot would be due to an increase in the Na+ gradient due to pumping of Na+ out of the cell. It is clear that any such model would require a lag in pump response to its own alteration of ion gradients. To test this hypothesis 106 M ouabain was applied in order to partially block the Na-K exchange pump. The action potential following drug application is shortened at C-T intervals of 3-8 and 4-8 sec, and maximally lengthened by only 16 % at an interval of 800 msec. The maximum increase in overshoot is also reduced. It is only 15'6 mV at a C-T interval of 600 msec. It is possible that some of the arrhythmic effects of the cardiac glycosides are due to alterations of ion balance in restricted extracellular spaces. Further study is needed to assess the clinical significance of these effects. We should like to acknowledge the support of the Muscular Dystrophy Association of America, the Rhodes Foundation and the Medical Research Council. REFERENCES COHEN, I., DAUT, J. & NOBLE, D. (1975). J. Phyaiol. 249, 42-43 P. HAusw=RHr, O., NOBLE, C. & TSIEN, R. W. (1972). J. Phy8iol. 222, 27-49.

Factors affecting the time course of decay of excitatory postsynaptic currents at a glutamate synapse By R. ANWYL and P. N. R. USHERWOOD. Department of Zoology, University of Nottingham, Nottingham NG7 2RD We have examined the effect of membrane potential, temperature and glutamate uptake blockers on the time course of decay of the e.p.s.c.s at the insect neuromuscular junction. Small muscle fibres of the multiterminally innervated extensor tibiae muscle of the locust were voltage clamped using a conventional 2-micro-electrode method. The fibre membrane can be

47P PHYSIOLOGICAL SOCIETY, SEPTEMBER 1975 successfully space clamped using this method because of the high length constant/length ratio of the fibres. However, the clamping of the excitatory post-synaptic potentials was often improved by perfusing the preparation with Ca-free saline and restoring neuromuscular transmission by the iontophoresis of Ca2+ on to a single nerve terminal close to the two clamping electrodes. The decay phase of the e.p.s.c. was exponential after a short initial nonexponential phase. The time constant of the decay phase averaged 2*8 msec. Varying the membrane potential between + 60 and - 160 mV did not alter the decay time of the e.p.s.c. This finding is similar to that at the squid giant synapse (Llinas, Joyner & Nicholson, 1974) where the post-synaptic membrane is also sensitive to L-glutamate (Miledi, 1969). However, at the frog neuromuscular junction, the decay of the end-plate current is approximately three times faster at +50 mV than - 100 mV (Magleby & Stevens, 1972). Reducing the temperature caused an increase in the decay time of the e.p.s.c. The Q10 for the decay time constant averaged 16 between 100 C and 20° C. This compares with a Q10 of approximately 3 for the decay time constant of the e.p.s.c. at the frog neuromuscular junction (Magleby & Stevens, 1972). Low Na, p-chloromercuriphenylsulphonate, 2-4-dinitrophenol, ouabain, L-aspartate, L-cysteate and L-glutamate have been shown to inhibit the high affinity uptake system for L-glutamate in the vertebrate C.N.S. (Balcar & Johnston, 1972). These agents caused only a very small or no increase in the time course of the e.p.s.c. at the insect neuromuscular

junction. The low Q10 and the lack of effect of uptake inhibitors on the decay time constant of the e.p.s.c. suggest that the termination of the transmitter action may be mainly by diffusion. REFERENCES

BALCAR, V. J. & JOHNSTON, G. A. R. (1972). J. Neurochem. 19, 2657-2666. LLINAS, R., JOYNER, R. W. & NICHOLSON, C. (1974). J. gen. Phy8iol. 64, 519-535. MILEDI, R. (1969). Nature, Lond. 223, 1284-1286. MAGLEBY, K. L. & STEVENS, C. F. (1972). J. Physiol. 223, 173-197.

The time course of action of L-glutamate at the excitatory neuromuscular junction in Maia squinado By A. C. CRAWFORD and R. N. McBURNEY. Physiological Laboratory, Cambridge CB2 3EG In a previous communication to the Society (Crawford & McBurney, 1975) we reported that L-glutamate caused a post-synaptic depolarization

48P 4PROCEEDINGS OF THE and an increase in membrane voltage and current 'noise' when applied to neuromuscular junctional regions of muscle fibres from the walking legs of the spider crab, Maia squinxdo. This paper reports experiments which examine the relationship between the duration of the 'elementary' current event caused by L-glutamate and the time course of the response to quantal packets of the natural transmitter. The time courses of the miniature excitatory junctional currents (m.e.j.c.s) were determined by recording their associated extracellular voltage changes with a micropipette (10-15 ,sm in diameter) filled with crab saline. In each fibre we also recorded the extracellular voltage 'noise ' caused by the application of L-glutamate to the junctional region. The decays of the m.e.j.c.s were then compared with the average duration of the 'elementary' event as determined from the half-power frequency of the spectral density function which was calculated from the 'noise' record. A considerable variation exists in the form and duration of m.e.j.c.s. While many m.e.j.c.s had a fast (< 0 5 msec) growth phase and a slower exponential decay (time constant approximately 1 msec) similar to those recorded at the frog neuromuscular junction (Katz & Miledi, 1973), other m.e.j.c.s were much slower in time course and had differing forms (cf. Gage & McBurney, 1975). Good agreement was found between the duration of the 'elementary' event and the rate of decay of the fastest decaying m.e.j.c.s. These results indicate that the decay phase of these 'fast' m.e.j.c.s is controlled by the rate of closure ofthe post-synaptic conductance channels following receptor activation by the transmitter. The results strongly support the existing evidence (Kravitz, Slater, Takahashi, Bounds & Grossfeld, 1970) that L-glutamate is the transmitter substance at crustacean neuromuscular junctions. We conclude that during most m.e.j.c.s the time course of the transmitter concentration changes in the synaptic cleft must be complete within 0-5 msec. This is perhaps surprising since no enzyme is present to destroy L-glutamate at the synapse (Kravitz et al. 1970). Indeed the slower and more variable m.e.j.c.s may arise from prolonged transmitter action. R.N.McB. is in receipt of a Florey Fellowship from the Royal Society. REFERENCES CRAWFORD, A. C. & MCBURNEY, R. N. (1975). J. PAPyiol. 251, 73-74P. GAGE, P. W. & MCBURNEY, R. N. (1975). J. Physiol. 244, 385-407. KATZ, B. & MIETTDI, R. (1973). J. Physiol. 231, 549-574. KRAVITZ, E. A., SLATi;R, C. R., TAKA1AsmI, K., BOUNDS, N. D. & GaROSSFELD, R. M. (1970). In Excitatory Synaptic Mechaniem8, ed. ANDERSEN, P. & JANSEN, J. pp. 8594. Oslo: Universitetsforlaget.

Proceedings: Stimulus-secretion coupling in submandibular gland: role of cyclic AMP, cyclic GMP and calcium in regulating adrenoceptor mediated enzyme secretion.

PROCEEDINGS OF THE loP REFERENCES THOMAS, R. C. (1970). J. Phywiol. 210, 82-83P. ZEUTHEN, T. (1971). Acta phyaiol. acand. 81, 141. Intracellular in...
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