1P

PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY KING'S COLLEGE MEETING

10-11 December 1976 DEMONSTRATIONS The oesophagus as an electric cable BY R. E. CIAIMLIS, M. FISHER and J. N. HUNT. Departments of Surgery and Physiology, Guy's Hospital Medical School, London SEl 9RT We have observed electric cable conduction phenomena in vivo in the human oesophagus. An axial array of nine annular electrodes spaced 6 mm apart was swallowed to a depth of 25 cm. Stepwise current transients (200,uA) were driven between right wrist and the proximal electrode. The voltage wave forms at the distal electrodes were recorded with respect to the left wrist. Recordings were made on a one-shot basis synchronized to the peak swallow of a 20 ml. bolus of saline thickened 30

VE2OmN

-

O-6

20L 5200N

E

10 lo 8 6

_T

20 mN

6

x 3 E

ZI* 4

1

~~~~~~~~TSo%200 mN

2 3 4 5 6 7 Electrode number (spacing 6 mm)

8

Fig. 1. Electrode steady-state voltage, VJ', and time to reach 50 % of that voltage, T50%, versus electrode position in the oesophagus, for saline concentrations 20 and 200 mN and stepwise current injection of 200 iPA. Each point is a mean of four observations, taken during four successive swallows.

with 1 % methyl cellulose by weight and maintained at body temperature. The steady-state amplitudes of the voltage wave forms and the time required to reach 50 % of those amplitudes (T50%) were determined for each electrode. The following relationships were observed: (1) For a given swallow the steady-state electrode voltages decayed nearly exponentially down the oesophagus (Fig. 1).

2P 2PROCEEDINGS OF THE (2) The time taken for the voltage transient to reach 50 % of its final value increased near linearly with distance down the oesophagus. (3) Increase of saline concentration increased the spatial decay constant, A, and reduced both the 50 % voltage rise time, Ti,,%, and the electrical impedance presented by the oesophagus, Z0. The spatial decay and the time delay are typical of cable conduction. The relationships between A, T5000, Z0, and saline concentration do not indicate a simple two element distributed model. We are in the process of developing distributed conduction models encompassing up to four shunt elements which we hope will describe the behaviour we have observed. Consideration is also being given to the problem of establishing the canonical form of such models given that the experimental data may be non-ideal and noisy.

The epidermo-dermal border in the type I slowly adapting mechanoreceptor in the skin of the rabbit BY L. H. BANNISTER and W. C. HAMANN. Department of Physiology, Guy's Hospital Mledical School, London SEl 9RT In the hairy skin of cats and rabbits there are two types of slowly adapting mechanoreceptors (Brown & Iggo, 1967; Iggo & Muir, 1969; Chambers, v. Duering, Andres & Iggo, 1972). The type I is associated with a thickening of the epidermis with Merkel cells at its dermal border. The Merkel cells themselves are attached basally to expansions of the

Fig. 1. Diagrammatic view of a type I touch receptor incorporating the two main features (A and B) diverting horizontal stretch forces away from the Merkel cell/end-plate complexes. A, folding of marginal epidermodermal junction; B, cup-like epidermal formation with interdigitating fibrous attachments.

terminal branches of afferent nerve fibres (nerve end-plates, Iggo & Muir, 1969). The type II receptor is associated with Ruffini endings in deeper layers of the skin (Chambers et al. 1972). One of the differences between the two types of receptors is the response characteristic to dermal stretch,

PHYSIOLOGICAL SOCIETY, DECEMBER 1976 3P which is a very effective stimulus for type II receptors, but not for the type I (Iggo & Muir, 1969). It was the aim of the demonstration to establish whether there are morphological features in the type I slowly adapting mechanoreceptor in the rabbit that could account for the absence of a response to dermal stretch. Small pieces of skin containing type I mechanoreceptors were obtained from anaesthetized rabbits. The specimens were either fixed in glutaraldehyde and prepared for the electron microscope in a conventional manner, or they were fixed in formaldehyde and stained for elastic fibres using the Verhoeff method. Observations on the structure of the tissues surrounding the Merkel cells support the view that the forces developed during dermal stretch are directed away from the Merkel cell/nerve end-plate complex and that their arrangement is suited for the action of perpendicular forces on the Merkel cell (Fig. 1). The authors would like to thank Mrs S. Lamb and Miss T. M. Anderson for excellent technical assistance. This study was supported by the Wellcome Trust. REFERENCES

BROWN, A. G. & IGGO, A. (1967). J. Phy8iol. 193, 707-733. CHAMBERS, MARGARET R., v. DUERING, MONIKA, ANDRES, K. H. & IoGo, A. (1972). Q. Ji exp. Physiol. 57, 417-445.

IGGO, A. & MUIR, A. R. (1969). J. Physiol. 200, 763-796.

TTX-resistant action potentials in crab nerve after treatment with Meerwein's Reagent BY P. F. BAKER and K. A. RUBINSON. Department of Physiology, King's College, Strand, London WC2R 2LS We have recently reported that chemical modification of crab nerve following reaction with carbodiimide and a suitable substrate renders the nerves insensitive to TTX without blocking nervous conduction (Baker & Rubinson, 1975). This method has a number of drawbacks and does not protect all the sodium channels in the treated nerve. We have now found that treatment of crab nerves with triethyloxonium tetrafluoroborate (Meerwein's Reagent) is far milder but gives greater protection against both TTX and STX. These TTX-resistant nerves were demonstrated. We also presented data on the binding of radioactive Meerwein's Reagent. Meerwein's Reagent was prepared by a standard method (Meerwein, 1966) and stored either at - 15° under dry ether or dry in liquid nitrogen. The radioactive reagent was prepared in a specially designed apparatus by reaction of AgBF4 + ['4C]ethyliodide + diethyl ether with 50 % (v/v)

PROCEEDINGS OF THE CH2Cl2 (Meerwein, Hedeiich & Wunderlich, 1958). All glassware was 'siliconized' with dimethyldichlorosilane to prevent the radiochemical reacting with the glass surface. The reagent was stored dry to prevent ethyl exchange. Meerwein's Reagent reacts with water having a pseudo first order half time of about 8 min and the products of the reaction, ethanol and ether, can be removed effectively by bubbling air through the reaction medium in a special vessel. The reaction is only slight pH dependent. It follows that nerves can be exposed to the reagent at physiological pH. The reaction does, however, generate acid and with reagent concentrations > 50 mm an automatic titrator was used to neutralize the acid generated. With reagent concentrations < 50 mm, hand addition of molar NaOH in the presence of 15 mm Tris buffer was adequate to control the pH. The nerve 4P

was first immersed in Tris-buffered artificial sea water and the reaction begun by addition of solid reagent. Normally the reaction time was 8-10 min. With concentrations of reagent 50 mm or more, blockage of toxin binding was complete; 15 mm blocked 30-50 % and 5 mm blocked 20 %. The reaction thus appears to be first order in reagent. The presence of TTX during exposure to Meerwein's reagent protects the TTX-binding sites from esterification. Although rendering nerves insensitive to TTX, Meerwein's Reagent seems also to react with a great many groups other than those directly associated with the sodium channels. The use of radioactive Meerwein's Reagent reveals appreciable labelling which if expressed in terms of membrane area amounts to 105 labelled sites/fum2. This labelling can be reduced to 2-5 x 104 by digestion of the labelled nerve with pronase under conditions which do not affect the TTX receptors of control nerves. The reaction's gentleness and operation over a wide pH range at relatively low concentrations recommends it for studying the effects of carboxylate, phosphate and phenolic anions in living systems. The work was supported by a grant to P. F. B. from the Medical Research Council. K. R. is a Postdoctoral Fellow of the National Institutes of Health (U.S.A.).

REFERENCES B A rR, P. F. & RUBINSON, K. A. (1975). Nature, Lond. 257, 412.

ME:ERwEIN, H., HEDERICH, V. & WUNDERLICH, K. (1958). Arch. Pharm., Berl. 291, 541. MEERWEIN, H. (1966). Org. Syn. 46, 113.

PHYSIOLOGICAL SOCIETY, DECEMBER 1976

5P Measurement of ionic diffusion and mobility in axoplasm isolated from giant axons of Myxicola BY P. F. BAKER and A. H. V. SCrAPiRA. Department of Phy8iology, King'8 College, Strand, London WC2R 2LS Axoplasm isolated from giant axons of the polychaete worm Myxicola provides a very convenient preparation of pure protoplasm for studies of the diffusion and mobility of substances of physiological and pharmacological interest. It is relatively simple to draw the isolated axoplasm into a polythene or cellulose-acetate tube of diameter roughly equal to that of the axon and to micro-inject axially into this a radioactive or fluorescent sample of the substance under investigation. In studies of longitudinal diffusion, the injected axoplasm is left undisturbed for varying lengths of time; and in studies of mobility is subjected to a longitudinal electric field. At the end of the experimental period, the polythene tube containing axoplasm is frozen on an aluminium block standing in a mixture of dry ice and acetone, and cut transversely into uniform segments of 1 mm or 2 mm in length, each of which is subsequently analysed. Values obtained for the longitudinal diffusion coefficient (cm2 sec-') of a number of substances of physiological and pharmacological interest are: Na, lx 10-5; Cl, 185x 10-5; Ca, lx 10-6; La3+, 0-93+0-05x10-6; Co2+, 32 + 0-8 x 10-6 and for mobility (cm/sec per V/cm) are Na, 3-63 + 0-12 x 104; Cl, 2-37 x 10-4; Ca, < 0-20 x 10-4; La3+, 0'13 + 0-08 x 104; Co2+ < 0-25 x 104. The figure for sodium is in good agreement with that obtained by Gilbert (1975) in Myxicola using a different method. Where comparison is possible the values obtained in axoplasm isolated from Myxicola axons are in close agreement with measurements on intact squid axons (Hodgkin & Keynes, 1957; Baker & Crawford, 1972) confirming that axoplasm suffers little damage during isolation. Both Na and Cl, and in a single experiment Rb, behave in axoplasm much as in free solution whereas Ca and La are strongly bound. Co is free to diffuse in axoplasm although the diffusing species seems not to carry a net charge. REFERENCES

BAKER, P. F. & CRAWFORD, A. C. (1972). J. Phygiol. 227, 855-874. GIBERT, D. S. (1975). J. Physiol. 253, 257-301. HODGKIN, A. L. & KzEYNs, R. D. (1957). J. Phy8iol. 138, 253-281.

6PBPROCEEDINGS OF THE Porous cellulose acetate tubing provides a suitable support for isolated protoplasm during studies under controlled conditions BY P. F. BAKER, D. E. KNIGHT and R. D. D. PATTNI. The Department of Physiology, King's College, Strand, London WC2R 2LS Pure samples of protoplasm, uncontaminated by extracellular fluid, can be obtained from giant axons of squid (Loligo) and polychaete worms (Myxicola). If prevented from drying out, for instance by being drawn into a tube of diameter roughly equal to that of the axon, isolated axoplasm can maintain an apparently normal consistency and physiological levels of a number of metabolic intermediates for many hours. For many experiments it is convenient to be able to alter the concentration of small molecular weight materials to which the protoplasm is exposed, without disturbing the macromolecules and organelles. This can be achieved by drawing the axoplasm into tubing which has been rendered porous and which can be exposed to media of defined composition. We have found that cellulose acetate tubing - similar in kind but of larger diameter than that used for internal dialysis (Brinley, Spangler & Mullins, 1975) - is very satisfactory. This cellulose acetate tubing (0.8 mm i.d., wall thickness 30 /tm, Fiber Spinning Services, F.R.L., Dedham, Mass., U.S.A.) can be rendered porous over a defined length by treatment with alkali (0.2 M-KOH for 16 hr at 200 C). After thorough washing, the porous tubing can be stored either dry or in distilled water until required. Tests show that after treatment with alkali, the tubing is freely permeable to substances of molecular weight less than about 1000, but only slowly permeable to substances of larger molecular weight. The half times for loss of a number of substances of relevance to physiological investigations are respectively 22Na 14 min; 45Ca, 1-7 min; Cr-EDTA, 4*0 min; Phenol Red, 3*5 min; ATP, 7 min; and FITC-labelled dextran (mol.wt. 3000), 4 hr. Isolated axoplasm can be introduced into the porous tubing for studies either of the bulk properties of protoplasm or of the uptake and loss of physiologically interesting molecules under controlled conditions. 6P

REFERENCE

BRINLEY, F. J., SPANGLER, S. G. & MuLLINS, L. J. (1975). J. gen. Phy8iol. 66,

223-250.

Laser measurement of particle size and movement in cells By P. F. BAKER, D. E. KNIGHT, R. W. PIDDINGTON and D. A. Ross. Department of Physiology, King's College, Strand, London WC2R 2LS Laser light is one of the simplest optical inputs to a system. It is monochromatic, coherent, parallel and plane-polarized; for a continuous-

PHYSIOLOGICAL SOCIETY, DECEMBER 1976 7P wave laser the photon emission times are Poisson distributed. The light emerging from a system illuminated by a laser may have some or all of these properties changed, and inferences can be made about the system by analyzing even a small fraction of this light such as that scattered at a particular angle in space. Brownian motion and active biological motion can be distinguished from each other and both can be quantified in ways which were previously impossible. The technique which we emphasized is called 'photon-correlation spectroscopy' (Fig. 1); the geometrical laws are the same as for light, sound or X-ray diffraction or holography. In each, the basic yardstick is the wave-length.

A~~~~~~~~~

B C

X

gJ

FJ~

IP DR

ig

Fr

C L

0

V=F/P R=Kr

Fig. 1. Photon correlation measurement of particle size and velocity. In A, light from a single incident laser beam is scattered by a sample. The intensity of the scattered light (viewed at a given angle) fluctuates due to movement of objects in the beam. Computer analysis of this fluctuating intensity gives an autocorrelation function (or a power spectrum). Further analysis gives information on the type of motion in the beam. In B, two beams set up interference fringes (F) at their intersection. In C, a particle moving at a velocity V across the fringes gives sinusoidal fluctuations to the scattered light at a period (P) equal to the period of the autocorrelation. V is obtained from F and P. In D, particles diffusing through the fringes give an exponential correlation of time constant r. The particle radius (R) is obtained from r and a constant (K) which incorporates F and the 'solvent' temperature and viscosity.

Experiments using laser spectroscopy have been done on a wide range of subjects including: diffusion of macromolecules and viruses, acoustic vibration, ultra-sonic air flow, blood flow, streaming plant cells, swimming

P PROCEEDINGS -OF THE 8P micro-organisms, muscle, adrenal gland and brain (see Piddington, 1976, for references). In our demonstration we showed data which support the following arguments: (1) Cytoplasm in characean plant cells is driven by the impulsive action of myosin-like cross-bridges at an average speed equal to or greater than the sliding-filament speed in fast muscle. (2) Catecholamine-containing vesicles in the adrenal gland are restricted in their amplitude of movement but vibrate at a speed governed by the viscosity of water. (3) Axoplasm from giant axons of the polychaete worm Myxicola is a solid gel which vibrates at low amplitude in low (physiological) levels of calcium but which liquefies and shows a tenfold or so increase in amplitude following exposure to millimolar concentrations of calcium. REFERENCE

PIDDINGTON, R. WV. (1976). In Calcium in Biological Sy8tems. ed. Du-NcAN, C. J. Symposium Soc. Exp. Biol. XXX, pp. 55-66. London: Cambridge University Press.

A system of multiple organ-baths BY R. N. PATEL and G. E. ROSE. Department of Pharmacology, King's College, Strand, London WC2R 2LS In a system of multiple glass organ-baths, it can be difficult to attain uniformity and convenience without expense. An economic and practical alternative is provided by the barrels of disposable plastic syringes, readily available in a large range of calibrated sizes.

Fig. 1. Detail of organ-bath mounting.

9P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 The 'baths' can be mounted, with ease, on a Perspex pipe, which also serves to supply the gas to a group. Fig. 1 details an efficient arrangement for the regulation of gas flow. The baths are filled by gravity-feed and emptied by a suction-line. The apparatus can readily be placed in a water-

bath of constant temperature. As demonstrated, the apparatus is in use to study transport processes in a large number of gut-sacs, prepared from both small and large intestines. A low resistance stimulator for brain slices BY M. S. BINGLEY. Department of Pharzmacology, King's College, London WC2R 2LS The electrical resistance of rat brain slices in vitro can be as low as 12 Q when measured from tissues sandwiched between two platinum grid electrodes. It was observed that a number of commercial stimulators have an internal resistance of more than 100 D. This will result in loss of voltage when these are connected to low resistance preparations. A stimulator of low resistance (0.1 Q) is required so that there is no change of output characteristics. One solution is to chop a low resistance source voltage with a mechanical chopper, at the appropriate frequency. This method is attractive in that it overcomes stability problems of an emitter follower. Since mechanical relays do not work well with asymmetric stimulation signals, a circuit has been designed incorporating two type 5A13(z) Carpenter relays. A selected square wave frequency (the master wave) is fed to one from an oscillator while the second relay is driven from a flip-flop circuit triggered by the master wave (Fig. l A). Current passes through the preparation only when both relays are closed at the same time. Relay 1 is set up so that it closes when the positive half cycle of the master wave is applied to it. The flip-flop is at the same time triggered by the rising edge of the master wave and produces a positive pulse which opens the second relay. By varying the time constant of the ffip-flop its pulse length is shortened relative to the master wave (Fig. 1B). At this point the second relay closes. Thus both relays are closed and the conduction path to the preparation is opened. When the master wave becomes negative again the first relay opens and the conduction path to the preparation closes. The frequency of stimulus to the preparation is that of the master wave which is a calibrated oscillator output, while its length is equal to the positive excursion of the master wave minus the pulse length

PROCEEDINGS OF THE

lop

I 1

1

1

39

Input stimulus to preparation

0 +

20 Closed 20

°

20 C

Input to relay 1

Open 21 21 21 0~--0~-+ L9BL 1 I Open U

Input to relay 2

Fig. 1. A, circuit diagram of stimulator showing flip-flop and relays. B, inputs to relays 1 and 2 and resultant stimulus to preparation.

of the flip-flop. By varying the latter (RV2) the pulse length can be changed since the master wave is symmetrical. A small degree of variable bias can be applied to the second relay (RV1) since its driving wave form is not symmetrical. A 10 Q resistor is inserted between the two relays to measure current. Pulse repetition rate for this stimulator extends from 15 to 60/s while pulse length can be varied from 500 ,ts to 5 ms.

The induction ad study of physical dependence on ethanol in mice BY C. ABU-MURAD, SUSAN J. BEGG, P. J. GRIFFITHS and J. M. LITTLETON Department of Pharmacology, King's College, London WC2R 2LS Groups of mice are housed in cages, enclosed by Perspex chambers of 1001. capacity, with free access to food and water. Into the chamber is pumped, at a rate of 2 1. min-, a mixture of room air and ethanol vapour. Environmental temperature is maintained at 27 + 1 C to prevent

lip PH YSIOLOGICAL SOCIETY, DECEMBER 1976 ethanol-induced hypothermia. The relative proportions of air and ethanol are chosen to achieve a concentration of ethanol in the chamber of 5-7 mg I.-' (measured by method of Griffiths, Littleton & Ortiz, 1974) on the first day of exposure. This initially causes locomotor excitation but locomotor depression and ataxia soon develop as blood ethanol concentrations rise. The concentration of ethanol vapour in the chamber is monitored at least twice daily and is increased in a stepwise fashion to about 15 mg 1.-i after 8 or 9 days of ethanol administration. This increase is necessary to maintain signs of intoxication, since both metabolic and cellular tolerance to ethanol occur during this time. This procedure maintains blood ethanol concentrations in the range 1-4 mg ml.-'. These high concentrations are associated with a mortality of 5-10 %. Ethanol is withdrawn after its administration for 10 days. Opening the Perspex chamber results in a rapid fall in the concentration of ethanol in inspired air. Blood ethanol concentrations fall to control levels in 2-3 hr. The withdrawal of ethanol in this way after 10 days of administration is associated with a characteristic syndrome of behaviour. A great increase in locomotor excitation, convulsions on handling, tremor, piloerection and erection of the tail may all be seen and an assessment made. of their severity. Increased voluntary consumption of ethanol (10 %, v/v) if it is provided as an alternative to drinking water also occurs. The withdrawal syndrome may last for 15 hr or more. Its severity in adult animals seems to be correlated with the blood ethanol concentration (estimated from that in expired air) immediately before withdrawal, but other factors are also important. We are currently using this model of ethanol dependence to study the neurochemical bases of tolerance, dependence and withdrawal, and also to assess the role of factors such as sex, age, strain, and environment in determining susceptibility to physical dependence on ethanol. This work was supported with the aid of grants from the Benescience Foundation, the Medical Council on Alcoholism, and the Mental Health Foundation. REFERENCE

GRIFFITHS, P. J., LITTETON, J. M. & ORTIZ, A. (1974). Br. J. Pharmac. 50, 489-498.

The chronic administration of L-nicotine by aerosol to rats BY J. M. LITTLETON and N. D. UMNEY. Department of Pharmacology, King's College, London WC2R 2LS Male Wistar rats (200-250 g) are housed in groups of 10 in wire mesh cages with free access to food and water. A rack of 2 cages is suspended

PROCEEDINGS OF THE 12P in an environmental chamber connected to a Dautreband micro-aerosol generator. Filtered room air is swept through the chamber, which is about 200 1. capacity, at a rate of approximately 1 air change per minute. A nicotine aerosol is generated from an aqueous solution of L-nicotine hydrogen tartrate (1 %, w/v) at pH 4-5. Aerosol droplet size is below 1 pm. Control groups are exposed to an aerosol generated from equimolar sodium hydrogen tartrate under otherwise identical environmental conditions. Exhaust air passes through a filter system into an exhaust duct. Using this apparatus rats are exposed to a concentration of L-nicotine in inspired air of approximately 1 jug 1.-i for a 5 min period every 30 min. Such a pulse of nicotine produces a concentration of nicotine in blood of about 70 ng ml.-' at the end of each period of administration. Nicotine concentrations in air are measured by trapping on Cambridge filter CGMI 13A and solubilization followed by estimation of nicotine by the gas liquid chromatographic method of Isaac & Rand (1972). Nicotine concentrations in mixed arterio-venous blood, obtained after decapitation, are measured by the method of Feyerabend, Levitt & Russell (1976). When rats are exposed to this regime of L-nicotine administration for several days behavioural changes are observed and may be assessed by simple tests. The cessation of pulse administration of nicotine in inspired air after its administration for periods of a week or more is associated with different behavioural changes. This method is designed to assess the neurochemical changes associated with chronic intermittent nicotine administration. Preliminary results suggest that changes in catecholamine metabolism in discrete areas of brain occur when L-nicotine is withdrawn from rats. It is hoped that these investigations may throw light on the neuronal and biochemical mechanisms associated with nicotine dependence. This work was supported with the aid of a grant from Gallaher Limited. REFERENCES FEYERABEND, C., LEVITT, T. & RUSSELL, M. A. H. (1976). J. Pharm. Pharmacy. 27, 434-436. IsAAc, P. F. & RAND, M. J. (1972). Eur. J. Pharmac. 8, 269-283.

Continuous monitoring of blood potassium demonstrated in an animal BY D. M. BAND and T. TREASURE. Sherrington School of Physiology, St Thomas's Hospital Medical School, Lambeth Palace Road, London SEl 7EH A potassium sensing electrode has been made on the tip of a catheter 1*5 mm in diameter and up to 40 cm long.

13P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 The sensor consists of a PVC membrane containing valinomycin and potassium tetraphenylborate as the ion selective components (Band & Kratochvil, 1974). This ion selective system has been demonstrated (Band et al. 1977) as an in-vitro electrode for rapid potassium measurement in whole blood samples. The aim of this further work was to produce a monitoring device for use in patients, in particular those in the intensive care unit following heart surgery. The catheter has been used for up to 10 hr in non-heparinized animals and responds rapidly to small changes in potassium. Although the aim was to produce a clinical measuring device, observations made during experiments to evaluate the catheter suggest it may be of value to physiologists in muscle blood flow studies and certain circulatory phenomena. We demonstrated the use of this catheter in an animal and the response of blood potassium to various physiological and pharmacological manipulations. REFERENCES

BAND, D. M. & KRAToCHV1i, J. (1974). J. Physiol. 239, 10P. BAND, D. M., KRATOCHVI, J. & TREASURE, T. (1977). J. Phy8iol. 265, 5-6P.

A ventral tract from the bed nucleus of the stria terminalis to the amygdaloid complex in the monkey (Mfacaca fascicularis): possible pathway in the regulation of ovulation By G. E. K. NOVOTNY. Institute fur Histologieund Neuroanatomie, Universitit GCttingen, Kreuzbergring 36, 3400 GCttingen Using a modification of the Glees silver impregnation (Novotny & Novotny, 1974; Novotny & Novotny, 1977), it is possible to visualize a system of approximately 200 fibre bundles lying between the bed nucleus of the stria terminalis (B.S.T.) and the baso-lateral part of the amygdaloid complex. In its course this fibre system passes under the anterior limb of the internal capsule, behind the anterior commissure and under the basal nucleus. From the general morphology of the bundles and the paucity of nerve cells in the traversed region, it may be postulated that the system is monosynaptic. Electron microscopical investigation of the bundles, just caudal to the anterior commissure, shows that they consist of unmyelinated fibres with a median diameter between 0'16 and 0-20,m. In 300 measurements no axons with a diameter greater than 0*55 ,um could be found, while 8% of the fibres had a diameter of less than 0'10 4m. Assuming the

14P 4PPROCEEDINGS OF THE sample to be representative of the total population, it is calculated that the total number of axons in this tract is 500,000. Fox (1943) described a connexion in the cat following a similar path, but from the amygdala to B.S.T. Other investigators, using degenerative techniques (Nauta, 1961; Valverde, 1965), specifically state that they were unable to verify this finding. It is therefore concluded, that the direction of conduction in the described pathway is from B.S.T. to the amygdala. .S t AL i22 . s s . v. . I. * ._ Ad ., .-.N.§ .-_. . ~.. 0.:%.

_i

Fig. 1. Electron micrograph of part of a fibre bundle caudal to the anterior commissure. Note compactness of bundle and lack of myelinated fibres.

Since the neurones of B.S.T. contain oestradiol (Pfaff & Keiner, 1973) and stimulation of B.S.T. or the amygdala leads to ovulation (Velasco & Taleisnik, 1969), it is conceivable that this tract is concerned in the regulation of ovulation. REFERENCES Fox, C. A. (1943). J. comp. Neurol. 79, 277-295. NAUTA, WV. J. H. (1961). J. Anat. 95, 515-531. NovoTNY, E. & NovoTNY, G. E. K. (1974). Stain Technol. 49, 273-280. NovoTNWY, G. E. K. & NovoTNY, E. (1977). Stain Technol. (in the Press). PFAFF, D. & KEINER, M. (1973). J. comp. Neurol. 151, 121-158. VALVERDE, F. (1965). Studied on the Piriform Lobe. Cambridge, Mass.: Harvard University Press. VELASCO, M. E. & TALEISNIx, S. (1969). Endocrinology 84, 132-139.

15P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 Ultrastructure of Cynomologus retina, with special emphasis on possible age related changes BY P. GLEES and P. E. SPOERRI. Institute of Histology and Experimental Neuroanatomy, University of Gdttingen, 3400 Cattingen, Federal Republic of Germany In continuation of our previous mitochondrial-lipofuscin age related studies (Hasan & Glees, 1973; Spoerri & Glees, 1973, 1974; Glees, Hasan & Spoerri, 1974; Glees, Spoerri & El-Ghazzawi; 1975), mitochondrial degeneration seems to be a precursor of lipofuscin which accumulates progressively with age. Comparing and contrasting these findings found in the central nervous system, we chose retina, being a special part of the C.N.S., with a view to study mitochondrial degeneration and lipofuscin accumulation. The form, the organization and the structure of the retinal elements of vertebrates is intensively described by Rodieck (1973). We examined the ultrastructure of the cellular components of the Cynomologus retina, using light microscopy for orientation by employing the Masson-Goldner trichrome stain. Particular emphasis has been given to the pigment granules. The common pigment granules found in the Cynomologus retinal epithelium are melanosomes. As lipofuscin and compound granules, consisting of both melanin and lipofuscin, were described in the human retinal epithelium (Feeney, Grieshaber & Alvarado, 1966), we further extended our investigations to senile animals. Even senile monkeys showed no obvious presence of lipofuscin in the pigment cells, solely composed of melanin. Nor did the mitochondrial bags of receptor cells show any signs of degeneration. It was expected that receptor cells, like neurones, would accumulate lipofuscin with age or reveal degenerative changes regarding their high mitochondrial content, but no changes comparable to neurones in the lateral geniculate body of the visual pathway could be detected. Supported by the 'Deutsche Forschungsgemeinschaft' Grant GL 28120. The technical assistance of W. Dresp and the photographic work of R. Dungan is gratefully acknowledged.

REFERENCES FEENEY, L., GRIESHABER, J. & ALVARADO, J. (1966). Invest. Ophthal. 5, 111. GI.sES, P., HAsAN, M. & SPOERRI, P. E. (1974). J. Phygiol. 239, 87P. GLEES, P., SPOERRI, P. E. & EL-GHAZZAWI, E. (1975). J. Hirnfor8oh. 16, 379-394. HAsAN, M. & GLEES, P. (1973). Acta anat. 84, 85-95. RODIECK, R. W. (1973). The Vertebrate Retina. San Francisco: W. H. Freeman and Company. SPOERRI, P. E. & GLEES, P. (1973). Exp. Geront. 8, 259-263. SPOERRI, P. E. & GLEES, P. (1974). Mech. Age Dev. 3, 131-155. 8 P H 2f66

16P 16PPROCEEDINGS OF THE Myelin changes induced by detraining BY R. SAMECK. Institute of Histology and Neuroanatomy, University of Gattingen, West Germany The adaptive behaviour of the peripheral nervous system paying particular attention to myelination was investigated in adult Wistar albino rats while performing a muscular training programme. Progressive swimming time induces supernumerary myelination of myelilnated, and myelination of unmyelinated, axons in dorsal and ventral lumbar roots and myelination of unmyelinated axons in dorsal and ventral lumbar roots and sciatic nerves, which in its extent appears to be correlated to duration and intensity of training (Sammeck, 1975). Conversely, if daily swimming over a period of two to three weeks (continuously for up to 4 hr) is stopped, and trained animals are transferred to a state of motor rest, adaptive myelin changes reverse, producing signs of fascicular degeneration involving wrinkling and folding of internodal sheaths firstly in nerve fibres of thicker calibre range. If training is not resumed within the first days following withdrawal of exercise, degenerative changes progress leading to the formation of ovoids and/or balls within the cytoplasm of axonassociated Schwann cells, similar to that described in the early stages of Wallerian degeneration but with most axons remaining intact. As the detraining period continues, axon-free Schwann cells laden with myelin debris enter the collagen-enriched endoneural space. Mononuclear cells, presumably of Schwann cell origin, accumulate in the immediate vicinity adjoining the vascular wall of blood vessels, presenting ruffling of basal laminae concomitant with microvesiculation of underlying plasmalemma and the occurrence of numerous vesicles within the cytoplasm. Veil-like basal laminae devoid of cells remain apparent in the perivascular space. Myelin-denuded single axons recongregate in later stages of detraining to form Remak bundles, abundantly present in normal laboratory rats. These adaptive changes following termination of swimming do not produce any major neurological impairment. This work was supported by 'Hermann und Lilly Schilling-Stiftung' (Stifterverband fur die Deutsche Wissenschaft, Essen).

REFERENCE SA Ecx, R. (1975). J. Phy8iol. 244, 7P.

Bite loads generated by the rat

BY M. W. RoBmNs. Department of Physiology, King's College, Strand, London WC2R 2LS

17P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 Doppler shift spectra in the assessment of peripheral arteries BY L. T. COTTON, V. C. ROBERTS, A. SUEz, A. L. STEVENS and M. H. THOMAS. Department of Biomedical Engineering, King's College Hospital Medical School, London, S.E.5 In an operation for lower limb ischaemia the surgeon must know where the major stenosis requiring correction lies in the peripheral arterial tree. Even the angiogram gives little information as to function and in some cases it can be difficult to decide whether to reconstruct the aorto-iliac or femoro-popliteal segment (Szilagyi, 1964). This demonstration showed a non-invasive method of assessing the peripheral arteries using ultrasound; in particular the arteries of the trunk (aorta to femoral) are compared to the arteries of the leg (femoral to posterior tibial). When a blood vessel is insonated with ultrasound the reflected sound has a frequency shift proportional to the velocity of the moving red cells. The pencil type probes used comprise a pair of piezo-electric crystals which transmit and receive the ultrasound. A 5 MHz probe is used to achieve the necessary tissue penetration for the deeper vessels and a 10 MHz probe is used to achieve optimum results from the surface vessels. The instrumentation analyses the complex frequency shift (caused by the moving blood) with due regard to the direction of flow. The output is presented either in the form of on line spectral plots (sonagrams), with time as the abscissa, frequency as the ordinate and spectral density as an intensity variation; or as single line, analogue processed mean and peak velocity traces (Woodcock, Gosling, King & Newman, 1972; Sainz, Roberts & Pinard, 1976). The arterial wave form may be quantified by digitizing its peak velocity envelope and calculating a Pulsatility Index (peak to peak/mean). In normal individuals the arterial velocity wave form becomes more pulsatile with distance from the heart. If the wave form becomes less pulsatile down a particular artery then an obstructing lesion such as atherosclerosis can be diagnosed in that artery. This is known as wave form damping. The pulse wave velocity (PWV) along an artery is calculated by measuring the foot to foot transit time of the arterial wave form between two points. The PWV increases with raised blood pressure and with age because of increased arterial wall stiffness and is slowed by severe arterial stenosis or occlusion (Gosling & King, 1974). In this demonstration two ultrasonic Doppler probes were used to record sonagrams from the arch of the aorta through the suprasternal notch, from the femoral, popliteal, posterior tibial and radial pulses in a volunteer subject. The sonagrams were displayed instantaneously. Pulse 8-2

18P PPROCEEDINGS OF THE wave velocity and pulse wave form damping are measured down the trunk (aorta to femoral), leg (femoral to posterior tibial) and arm (aorta to radial). Results in normal subjects show that the pulse wave velocities down the trunk and leg vary in individuals, but when related to the arm velocity as the subjects own control for age and blood pressure, the ratios are constant: WV down trunk 0.76; WV down leg - 1P44; WV down arm WV down arm the coefficient of variation is 10 %. Thus the PWV down the aorta is three quarters of that down the arm, while the PWV down the leg is one and a half times that down the arm. The wave form damping down the trunk is 0 5 and down the leg is 0 9. In studies on patients, wave velocity ratios and damping have been measured in diseased aorto-iliac and diseased femoro-popliteal segments and shown to be significantly different from controls, and to correlate well with angiograms. These non-invasive measurements, by localizing the major obstruction, can help in planning reconstructive surgery. Furthermore, assessment of the aorto-iliac segment using this method may avoid unnecessary

aortography. REFERENCES

GOSLING, R. G. & KING, D. H. (1974). In Cardiovascular Applications of Ultrasound, ed. RENEMAN, R. S. Amsterdam and London: North Holland Publishing Company. SAINz, A., ROBERTS, V. C. & PINARD, G. (1976). Ultrasonics 14, 128-131. SZILAGYI, D. E. (1964). J. Cardiovascular Surg. 5, 502. WOODCOCK, J. P., GOSLING, R. G., KING, D. H. & NEWMAN, D. L. (1972). In Blood Flow Measurement, ed. ROBERTS, V. C. London: Sector Publishing.

Altered responses to noradrenaline of vasa deferentia from genetically hypertensive rats By M. P. CAULFIELD, G. PATERSON and A.-R. WAYYES. Department of Pharmacology, King's College, London WC2R 2LS It has been suggested that the state of hypertension is maintained as a result of an alteration in the structure of vascular smooth muscle, which brings about an apparent increase in reactivity to various agonists (Folkow, Hallbick, Landgren, Sivertsson & Weiss, 1973). This pressureinduced hypertrophy of the arteriolar walls is itself thought to be triggered by an initial high blood pressure level. The cause of this initial rise in blood pressure remains to be elucidated.

19P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 Earlier studies carried out in this laboratory demonstrated that increased vascular reactivity in genetically hypertensive rats was not altered by short-term treatment with anti-hypertensive drugs (Wayyes & Paterson 1975). This would suggest that the increased reactivity is not entirely accounted for by pressure-induced arteriolar hypertrophy. It appears possible that any of the changes mentioned could be related to genetic abnormalities which affect other, non-vascular, tissues. Responses to transmural stimulation or to applied noradrenaline of the isolated vasa deferentia from genetically hypertensive rats (15-16 weeks old) were compared with those of age-matched normal Wistar rats. No apparent differences between the hypertensive and normal groups were observed in the responses of vasa deferentia to stimulation at various frequencies (0.5-32 Hz). However, responses to noradrenaline of vasa deferentia from the hypertensive rats differed from the responses of control preparations. The maximum responses (expressed as grams tension) of the hypertensive rat tissue (176 + 0 10 g) were significantly lower than those of controls, (2.14 + 0.10 g, P < 0.05). The sensitivities of the two groups to applied noradrenaline were not significantly different. In hypertensive rats treated from the age of seven weeks with antihypertensive drugs, the systolic blood pressure was found to be maintained at a normal level through eight weeks of treatment. Vasa deferentia from these animals were also found to have a differing maximum response to noradrenaline compared with control Wistar rats which had undergone identical drug treatment. Genetically hypertensive rats therefore seem to exhibit altered smooth muscle contractility which is not confined to cardiovascular smooth muscle. REFERENCES FOLKOW, B., HALLBXCK, M., LUNDGREN, Y., SIVERTSSON, R. & WEISS, L. (1973). Circulation Res. 32-33, supply. 1, 2-15. WAYYES, A. R. & PATERSON, G. (1975). Abstracts of The Sixth International Congress of Pharmacology, p. 569. Helsinki: IUPHAR.

A suction-operated transducer for monitoring tissue surface strains BY M. J. LAB and R. PRIcE. Charing Cross Hospital Medical School, Fulham Palace Road, London W6 8RF There are serious drawbacks in the methods used in the past for monitoring epicardial movements. Mercury-in-rubber strain gauges 'permanently' sutured on to the surface of the heart are orientated in one direction only (Forrester, Tyberg, Wyatt, Goldner, Parmeley &

20PPROCEEDINGS OF THE Swan, 1974) and cannot account for forces acting in different directions. A tripodal device, with delta rosette strain gauges (Dutertre, Cartier & Dieudonne, 1972), can provide tridirectional extensiometry but the feet have to be stabbed into the moving heart and may reach a variable depth. To overcome some of the shortcomings of the above techniques we have constructed a tripodal device which can "eadily, andreversibly, be attached to the heart by the vacuum (200-400 mmHg) generated by a conventional water pump. Epicardial movements, transmitted via the legs of the device to a flexible plate, result in extension or compression of threeelement rosette strain gauges on the upper and lower -surfaces of the plate. The signals are analysed by an amplifier specially designed for this device (Brown & Lab, 1977). 20P

Delta rosette strain gauge (micro measurements EA090 30YB-120) Conducting wires (from plate to ring)

8 BA screw and

nut

Flexible silicone rubber tube (from vacuum to

manifold)

Phosphobronze plate

(0.1

mm thick)

Perspex ring Flexible wires k (to amplifier) a

Hollow 'legs' Flexible tubing (to vacuum manifold)

Fig. 1. Diagram of tripodal transducer.

The device has three Perspex legs each of which is hollow and is screwed perpendicularly to one corner of a triangular plate of phospho-bronze shim. Each rosette is in the centre of one surface of the plate with each element of a gauge pointing toward a corner of the triangle. The plate is attached to a Perspex ring, surrounding the plate in the same horizontal plane, via the strain gauge wires embedded in silicone rubber cement.

21P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 These wires run from the ring to solder tags on the plate which are at the mid points of the sides of the triangle and so do not restrict movement of the area of the plate containing the gauges. Thin flexible tubing connects each leg to a manifold on the ring to which vacuum may be applied. The tubing also helps support the tripod. Near the manifold the gauge wires leave the ring for the amplifier. The physical and electrical characteristics of this transducer compare favourably with the earlier strain gauges and were demonstrated together with some of the details of the construction of the device, its calibration, and examples of its use. REFERENCES BROwN, A. W. S. & LAB, M. J. (1977). J. Physiol. 266, 21-22P. DUTERTRE, J., CARTIER, JEAN C. F. & DIEUDONNE, J. M. (1972). Med. & biol. Engng 10, 277-281. FORRESTER, J. S., TYBERG, J. V., WYATT, H. L., GOLDNER, S., PARMELEY, W. W. & SwAN, H. J. C. (1974). J. apple. Phy8iol. 37, 771.

A self-contained system for real time analysis of outputs from three element 600 Delta rosette strain gauges BY A. W. S. BROWN,* and M. J. LAB. Charging Cro03 Hospital Medical School, Fulham Palace Road, London W6 8RF A simple amplifier was constructed to analyse the signals obtained from two, three-element rosette strain gauges used in a tripodal device for measuring epicardial strains in the intact heart in sitU (Lab & Price, 1977). The rosettes are fixed to both sides of a plate and are identically orientated. The amplifier comprises three strain-gauge amplifiers (Fig. 1), one for each pair of co-directional arms. The outputs of the amplifiers provide length vectors V1, V2 and V3 for the movement of legs 1, 2 and 3 of the tripodal device. The amplifier gains are adjustable to give equal sensitivity for the three bridges, and each has an offset to allow suitable zero length setting. The vectors V1, V2 and V3 are at an angle of 1200 to each other, and may be resolved into components along arbitrary X and Y axes using operational amplifiers having gains matching the magnitudes of the projections of the vectors on to the X and Y axes chosen. For convenience, the X axis was chosen to be in the direction of V1 so it is only necessary to resolve the X and Y components of V2 and V3. The resultant vector along the X axis is therefore the sum of V1 and the projections on X of V2 and V3. These resultant vectors X and Y can be analysed in two different ways, depending * Present address: Experimental Workshop, Chailey Heritage (Craft School and Hospital), North Chailey, Lewes, Sussex.

22P A2PPROCEEDINGS OF THE upon experimental requirements. In mode I, the true resultant X and Y vectors are obtained. However, for symmetrical contraction of the epicardium, when V, V2 and V3 are equal, this gives a zero resultant vector. In mode II a resultant-shortening vector which is non-zero is obtained by always keeping the X and Y components of V3 and the X component of V2 positive. The resultant non-zero vector provides a useful dynamic monitor of magnitude and direction of epicardial movement, even if contractions are symmetrical. A simple switch allows the unit to be operated in either mode. The construction, the appropriate vector analyses, and some of the experimental applications of the unit were demonstrated. OUTPUTS

INPUTS

-X

V, V2

V3

-Y

1'r Offset

-15

+15

Fig. 1. Schematic diagram of rosette strain gauge amplifier. Bridge excitation balance is obtained separately, and is not wired into the system. An. dev., analogue devices; L, link in output socket allowing tape recordings of the vectors to be resolved into X and Y outputs; S, switch; R, convenient resistance of 10 K (20 K variable resistors used). REFERENCES

DUTETRE, J., JEAN, C. F. & DIEuDomNE, J. M. (1972). Med. & biol. Engng 10, 277-281.

LAB, M. J. & PRICE, R. (1977) J. Phy8iol. 266, 19-21P.

PHYSIOLOGICAL SOCIETY, DECEMBER 1976 23P Volume changes in brush-border vesicles detected by use of the Coulter counter By K. A. BuRTON, R. J. NAFTALI and M. W. SMrITH. A.R.C. Institute of Animal Physiology, Babraham, Cambridge, and Department of Physiology, King's College, London WC2R 2LS Vesicles prepared from intestinal brush-border membranes have been used to study the relationship between hexose transport and Na gradients. Sudden exposure of vesicles, previously equilibrated in 0-1 M D-manniutol to medium containing 0-1 M D-mannitol, 1 mM D-glucose and 01- M-NaCl 3-0

E

3-

A

EW

1-0

05 _ C

° 0-4 0.

T V.IV 03

E

-

02_ 0

1

2 Time (min)

3

4

Fig. 1. A, time course of mean-volume change of intestinal brush-borders isolated from new-born pigs. The error bars are S.D. of at least three separate determinations: *- , vesicles pre-loaded in 0-1 M D-mannitol; * *, vesicles preloaded in 0-1 M D-mannitol and 0-1 m NaCl. At zero time the vesicles were added to 0-1 M D-manxitol, 1 mM D-glucose and 0-1 M-NaCl; pH 7.5. In this series vesicles below 0-3 sum3 have been electronically removed from size-distribution. B, Murer & Hopfer's results (1974) showing 14C-labelled D-glucose uptake into brush-border vesicles preloaded with 0-1 M D-mannitol. of-- , following immersion at zero time in 1 mM D-glucose, 0-1 M D-mannitol and 0-1 M-NaCl; [ []shows D-glucose uptake into vesicles pre-equilibrated with Na.

24P 2PROCEEDINGS OF THE apparently leads to a transient accumulation of intravesicular glucose (Murer & Hopfer, 1974). The estimates of intravesicular sugar concentration depend on the implicit assumption that the vesicular volume remains constant during the course of uptake. It is now possible to test this assumption using a Coulter counter equipped with a particle size analyzer model P64 and a 50 #sm orifice probe. Vesicles prepared according to the method of Hopfer, Nelson, Perotto & Isselbacher (1973) were mixed in the Na-containing medium and the size distribution was determined at short intervals during the next 3 min. The mean vesicular volume is shown to increase then decrease, although the swelling phase is nearly outside the resolution time of the technique, and therefore can only be approximately determined. The vesicles have a skew distribution ranging from 0*2 to 4- 0 #m3: the modal volume varies from 0 3-0 9 #m3 following immersion in Na-mannitol solution. The time course of change in the mean vesicular volume is shown in Fig. 1A. These changes are sufficiently large to seriously affect the estimations of intravesicular sugar (Fig. 1B). Control vesicles reloaded in 0-1 M-NaCl and O AM D-mannitol then placed in identical medium showed no change in volume. Samples of the glutaraldehyde-fixed vehicles have been examined by electron microscopy and with the Coulter counter. The size of the vesicles determined by electron microscopy are similar to that found using the Coulter counter. REFERENCES HOPPER, U., NELSON, K., PEROTTO, J. & ISSELBACHER, K. J. (1973). J. biol. Chem. 248, 25-32. MURER, H. & HOPFER, U. (1974). Proc. natn. Acad. Sci. U.S.A. 71, 484-488.

Cytochemical localization of transport adenosine triphosphatase BY J. A. FIRTH. Department of Anatomy, King's College, London WC2R 2LS Enzyme cytochemical techniques for the subcellular localization of unspecified ATPases have been available for over a decade, but their usefulness has been impaired by the inability of such systems to demonstrate appropriate cation dependence and inhibition characteristics for identification with any particular ATPase of physiological interest. In particular, it seems certain that the use of glutaraldehyde fixation and of capture of inorganic phosphate by lead ions are incompatible with significant activity of Na+-K+-dependent transport ATPase. A novel approach exploits the fact that the K+-dependent phosphatase component of the transport ATPase reaction can be demonstrated alone.

25P PHYSIOLOGICAL SOCIETY, DECEMBER 1976 Using formaldehyde fixation, p-nitrophenyl phosphate substrate and phosphate capture by strontium ions, localization of a K+-dependent, ouabain-sensitive enzyme activity can be achieved. As the reaction is carried out at pH 85-.90, interference by alkaline phosphatases occurs; the effects of this can be minimized by suitable use of inhibitors. Biochemical and cytochemical data on the application of this approach to renal cortex and cerebral capillaries were presented. An in vitro investigation of physiological control mechanisms in the guinea-pig sphincter of Oddi BY S. BROOKS, J. HATLIDAY and H. KYHANGURA. Department of Pharmacology, King's College, London, W.C.2 This demonstration is intended to show the properties of an isolated perfused preparation of the guinea-pig sphincter of Oddi and its responses to various agents which have been implicated in normal physiological function. In the simple perfusion system (Brooks & Halliday, 1975) a constant inflow rate of 80 1l. min- results in a back pressure of between 1 and 7 cmH20. Activity in the tissue is inferred from changes in the inflow pressure. Since the perfused preparation includes a contractile pouch, inflow pressure may be influenced by changes both in pouch volume and sphincter resistance. This preparation has little inherent tone, therefore contractile effects such as motor responses, mediated via muscarinic receptors, are readily demonstrated. Field stimulation gives rise to a motor response which can be blocked by tetrodotoxin or muscarinic antagonists. Barium chloride or carbachol can be used to induce 'tone' and allow the study of relaxant effects. On such preparations cholecystokinin and prostaglandins E1 and E2 were without significant effect, at concentrations that produced pronounced contractile effects in gall-bladder preparations taken from the same animals. Noradrenaline and isoprenaline both induce relaxation, and the use of selective antagonists indicate that both a and ft adrenoceptors are present and that both, upon activation, cause relaxation. Morphological and histological studies demonstrate wide species differences in the terminal bile duct and sphincter of Oddi region. These observations, taken along with current knowledge of bile formation in this species (Shaw & Heath, 1974) and sphincter function in the dog (Tansy, Innes, Martin & Kendall, 1976) prompt speculation on the physiological control of bile flow in the guinea-pig. S. Brooks is in receipt of an M.R.C. Research Studentship.

26P

26PPROCEEDINGS OF THE REFERENCES

BROOKS, S. & HALLIDAY, J. (1975). Br. J. Pharmac. 55, 270P. SHAw, H. M. & HEATH, T. J. (1974). Q. Ji exp. Phy8iol. 59, 93-102. TANSY, M. F., INNEs, D. L., MARTIN, J. S. & KENDALL, F. M. (1976). Am. J. Dig. Di". 21, 233-241.

Role of the cervical lymphatic system in drainage of cerebrospinal fluid BY M. W. B. BRADBURY, C. C. H. COOK and RUTH HoPrINs. Department of Physiology, King's College, London WC2R 2LS Portocaval anastomosis in the rat - some effects on the brain and elsewhere BY M. W. B. BRADBURY and G. S. SARNA. Department of Physiology, King's College, London WC2R 2LS

COMMUNICATIONS Flux studies in perfused amphibian intestine By D. S. PARSONS. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU Frog small intestine artificially perfused through the vascular bed survives for many hours, thereby permitting a variety of measurements to be compared on the same animal. Measurement, in the steady state, of fluxes from blood to lumen and from lumen to blood, of sugars derived from oligosaccharides and of amino acids from peptides is simple (Parsons & Prichard, 1971; Boyd, Cheeseman & Parsons, 1975; Cheeseman & Parsons, 1976). Movements in the direction blood to lumen when the composition of the lumen is modified, e.g. by replacing the Na-, or adding phlorizin, suggest a recycling of sugars such as galactose (GAL) and 3-0-methyl glucose (3-MG). Changing the rate of perfusion through the vascular bed reveals that the net flux of 3-MG, lumen to blood, increases with the rate of perfusion, the increase being much more marked with Na absent from the lumen. A similar effect has been found with the amino acid, cycloleucine (I.R. Sanderson, unpublished observations). As an alternative to the steady-state experiments, a perturbation method may be employed: the tissue is loaded up either from the lumen or from the blood with a sugar (e.g. 3-MG) sometimes together with a marker (e.g. [14C]inulin or urea) added to the Ringer. The fluid on the loading side is then rapidly replaced with plain Ringer and the release of the substrate