Haematology, The London Hospital Medical College, London, E. 1

P4PPROCEEDINGS OF THENeglected litters squeal far less than those which are attended to by the

mother. During the pilot study, one primigravid female appeared unawareof her duties, leaving babies scattered around the cage, and one multi-gravid female gave birth to a small, partially dead litter with somedystochia and delays during which she left the nest. In both cases thebabies quickly cooled, slowed in their movements, and squealed only infre-quently. Such litters can be recognized when found, but to reduce thechance of poor mothering influencing the method, it is worthwhile to usemultigravidae with a good mothering record.The adult rats remained silent throughout the study.

REFERENCE

TAYLOR, P. M. (1960). J. Phygiol. 154, 153-16S.

Thermography in the new-bornBY A. M. Fox, D. L. KENNAIRD and A. J. WADE. Department of Physio-logy, The London Hospital Medical College, London, E. 1

The use of the superfused rat cremaster preparation and gelfiltration of radioiodinated polyvinyl pyrrolidone for the measure-ment of vascular permeability to macromoleculesBY L. J. F. YOULTEN. Department of Physiology, The London HospitalMedical College, London, E. 1

The use of the Coulter Model 'F' particle counter for haemato-logical measurementBy R. G. FEWELL, G. C. JENKINS and A. J. WILKINSON. Department ofHaematology, The London Hospital Medical College, London, E. 1

COMMUNICATIONS

A possible diurnal rhythm in rat pancreatic secretionBy J. BARROWMAN, D. BROGAN, J. FORDEAM, M. HATHORN, A. MOTT,P. RAmILY and R. TIPTAFT. Department of Physiology, The LondonHospital Medical College, London, E. 1A diurnal rhythm in the flow of human pancreatic juice was first de-

scribed by Pfaff (1897). It has been found in man that secretion is at amaximum in the early afternoon and a minimum at night (Mahaffey &Haynes, 1953). Such secretion may be greatly influenced by food intake.

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PHYSIOLOGICAL SOCIETY, FEBRUARY 1970

However, this rhythm can be detected in patients with pancreatic fistulaewho receive only water by mouth (Lines & Ranger, 1969). In rats withchronic pancreatic fistulae Grossman (1958) noted a greater output ofpancreatic juice by day than by night.

'60-

0

a..

0L 40

E .--tMean

0

._

@! 2-0-I I I

E .mean.

0 ' I

9 p.m. 12 3 a.m. 6a.m. 9 a.m. 12 3 p.m. 6 p.m. 9 p.m. 12Time

Fig. 1. Weight of pancreatic juice, sodium and protein concentrations, andtotal protein output over 24 hr (means for 3 hourly collections in five rats).

Diurnal rhythms of pancreatic secretion have been investigated in ratsnourished by intravenous infusion in an attempt to minimize gastro-intestinal stimuli to the pancreas. Biliary and pancreatic fistulae wereestablished in male rats of approximately 250 g. They were given dailyabout 30 ml. of glucose-saline by steady intravenous infusion. Obser-vations were started 36 hr after operation. Pancreatic juice was collectedover dry ice in 3 hr periods. Throughout the experiment the condition ofrats was carefully studied. The juice was subsequently thawed, weighedand analysed for protein and sodium content. Results were accepted from

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6PROCEEDINGS OF THE

animals in which no progressive changes in flow rate were noted over aperiod of days.

Analysis of variance carried out on a complete 24 hr period in each offive different rats showed a significant difference in weight of secretion,protein concentration and protein output within the 24 hr, the maximumin each case occurring during the hours of daylight and the minimum atnight. Sodium concentrations in the samples showed a tendency to followthose of protein. These results point to a daily rhythm in secretion of therat pancreas which is independent of gastro-intestinal stimulation.

REFERENCES

GROSSMAN, M. I. (1958). Am. J. Phy8iol. 194, 535-539.LINEs, J. G. & RANGER, J. (1969). J. Phy8iol. 200, 57-58P.MARAFEY, J. H. & HAYNEs, B. W. (1953). Am. Surg. 19, 174-181.PFAFF, F. (1897). J. Boston Soc. med. Sci. 2, 10-18.

The gastric lipase of the ratBY J. BARROWMAN and S. JANE DARNTON. Department of Physiology, TheLondon Hospital Medical College, London, E. 1

Haematological effects of moderate blood loss in healthy medicalstudentsBY S. JANE DARNTON, M. K. S. HATHORN, A. WIRINSON and L. J. F.YOULTEN. Department of Physiology, The London Hospital Medical Collegeand the Haematological Laboratory, The London Hospital, London, E. 1

In November 1968, medical students in their first term at this collegeinvestigated the haematological and other changes in themselves followingthe loss of 300-400 ml. of blood, taken for the blood bank. The students'investigations suggested inter alia that a reticulocyte response might bepresent as early as 24 hr after blood loss. The exercise was thereforerepeated in November 1969 by the new student entry and the investiga-tions of the blood changes in twenty male and ten female volunteerscarried out in detail by the authors, in parallel with the students' owninvestigations. Haemoglobin concentration, red and white cell counts, andmean red cell volume were measured by a 'Coulter' counter, model 'S',in the clinical haematology laboratory of The London Hospital, on venousblood samples taken at the time of blood donation and at 3 hr, 1, 3, 7 and14 days thereafter. In addition, reticulocyte counts and differential countswere performed by conventional methods. None of the subjects took oraliron during the experiment.

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PHYSIOLOGICAL SOCIETY, FEBRUARY 1970 17PThe results for some of the measurements are shown in Fig. 1. At 3 hr

after bleeding there was no significant decrease in the red cell count, thehaemoglobin concentration, or the haematocrit, which had all fallen after24 hr, reaching even lower values at 3 days. They then rose but had not

.52____- r 2

5.0FLIE 4-8F

444 F 24E0

0380,A~ ~ ~ ~ ~~~~2

E36 I- UbN.I1 I I I~: Y03hr1ld 3d 7d 14d

Time after blood donation

Fig. 1. The mean changes after a loss of 300-400 ml. blood in twentymale subjects (-) and in ten female subjects (---). Open circles showthe control values at the time of blood loss and those values at other timesnot significantly different from the control mean. Closed circles representmeans at collection periods significantly different from the control meanfor that parameter (P < 0 01).

returned to control levels by 14 days. The earliest reticulocyte responseoccurred at 24 hr, and the count remained elevated for the remainderof the 14-day period.The co-operation of the medical, nursing and lay staff of the North-East Metro-

politan Regional Blood Transfusion Centre is gratefully acknowledged.



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Active potassium transport in the immature red cells of anaemicsheep

By J. C. ELLORY and ELIZABETH M. TUCKER. A.R.C. Institute of AnimalPhysiology, Babraham, CambridgeWhen sheep are subjected to a sudden anaemia, induced by phlebotomy,

there is a massive influx of immature red cells and reticulocytes into thecirculation, reaching a maximum 3-5 days after bleeding. Using thecentrifugation technique developed by Drury & Tucker (1963), modified toreduce white cell contamination, it is possible to collect a young cellfraction (containing 20-40% reticulocvtes) from the blood of anaemicsheep. It is known that even in sheep with low potassium type (LK) redcells, these young red cells have a high internal K+ concentration, and it

TABLE 1. Active potassium transport in mature and immature redcells from anaemic LK sheep

Anti-LYoung cell Mature treatedfraction erythrocytes erythrocytes

Reticulocytes (%) 261± 4-0 0 0K+ concentration (mm/l. 711 + 7-8 15*1 + 14cells)

Ouabain-sensitive K+ 3.20+ 039 0.113 ± 0009 0.614+ 0-051uptake (m-moles/l. cells. hr)Na+-K+ activated ATPase 0 49 + 007 0-025 + 0002 0-137 + 0.014(4Lt-moles Pi/mg protein.hr)Bound ouabain 186± 28 8-4+ 1.7 25.4+ 3*1(molecules/cell)Values are the mean ± S.E. of three determinations on each of fotur sheep except

for ATPase and ouabain-binding which are the mean of four determinations on onesheep.

has been suggested (Ellory & Tucker, 1969) that LK sheep erythrocytesdevelop from reticulocytes by the suppression of part of the potential foractive transport by the appearance of the L antigen (previously called m)on these cells. In the present experiments all three parameters of thesodium pump measured in the young cell fraction (Table 1) gave very highvalues compared with either mature LK cells or LK cells sensitized withanti-L. When the reticulocyte-enriched fraction was tested serologicallyfor the presence of the L antigen, there was a normal positive reaction,changing to a weaker L reaction 10-15 days after bleeding, consistent withthe appearance of a different population of cells.Thus the situation exists that young red cells have a very high rate of

active potassium transport and yet carry the L antigen. It therefore seems

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that if the L antigen is directly concerned with suppressing part of thepump in mature LK erythrocytes, its effect is mediated by changes in theimmature red cell during the final maturation process.

REFERENCES

DRURY, A. N. & TuCKER, E. M. (1963). Res. vet. Sci. 4, 568-579.ELLORY, J. C. & TuCKER, E. M. (1969). Nature, Lond. 222, 477-478.

Exchange diffusionBY H. G. BRrrTON. Department of Physiology, St Mary's Hospital MedicalSchool, London, W. 2

Excitatory input from the colon to the inferior mesenteric ganglionBY P. J. CROWCROFT, MOuLIE E. HOLMAN and J. H. SZuRSZEwWS1I.* Depart-ment of Physiology, Monash University, Victoria, Australia

Intracellular recording from neurones of the inferior mesenteric ganglionof guinea-pigs has shown that they receive a continual excitatory synapticinput from the colon. This confirms, at the single unit level, earlier morpho-logical (Kuntz, 1940) and physiological (Kuntz & Saccomanno, 1944)evidence for the existence ofenteric neurones that send fibres to prevertebralautonomic ganglia.Young guinea-pigs of either sex were stunned and bled. The inferior

mesenteric ganglion, its presynaptic inputs and the colonic nerves with6-8 cm of distal colon were dissected and placed in a two-compartmentisolated organ bath. The colonic segment was pinned out in one compart-ment and the ganglion with its inputs in the other. Both compartmentswere separately perfused with modified Krebs solution (Blackman et al.1969) warmed to 33350 C.In fifty-five of sixty-four cells, continual synaptic activity was recorded

when the colon was attached to the ganglion. This activity consisted ofexcitatory synaptic potentials of variable amplitude. Frequently, theseexceeded threshold for initiation of action potentials. The characteristicsof the excitatory synaptic potentials were similar to those that resultedfrom subthreshold presynaptic nerve stimulation. We were unable toobserve rhythmic patterns of the input or to correlate it with spontaneouscontractions of the colon. However, when the entire segment was distendedfor 5-8 sec, the frequency of the synaptic input and action potentialactivity increased. Following release of the stimulus, there was a transientperiod when both synaptic input and motor activity were reduced.

* Fulbright-Hays Scholar.

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PPROCEEDINGS OF THE

When the nerve fibres running from the colon to the ganglion were cutduring intracellular recording, the synaptic activity was immediatelyabolished. Tetrodotoxin (5 x 10-7 g/ml.) added to the colon side of thebath also abolished the synaptic activity, proving that the action potentialswhich eventually lead to the release of the transmitter in the ganglionare initiated in the colon. Addition of dihydro-f,-erythroidine (5 x 10-g/ml.) to the ganglion side of the bath blocked both the response to pre-ganglionic nerve stimulation and the synaptic activity from the colon.This indicates that both inputs are mediated through similar cholinergicsynapses.The data suggest that the synaptic input may be coming from centrally

directed processes of cholinergic neurones of the enteric plexuses of thecolon. Since the enteric neurones of the colon receive an abundant nor-adrenergic innervation (Norberg, 1964), then their activity might be limitedby the inhibitory action of noradrenaline released at the peripheral ter-minals of inferior mesenteric ganglion cells. This hypothesis is supportedby our findings that preganglionic nerve stimulation causes a transientinhibition of synaptic activity and further, that application of noradrena-line (10 g/ml.) to the colon side of the bath abolishes the incomingactivity.These experiments provide evidence for a mechanism by which the

sympathetic system exerts an inhibitory control on the cholinergic ganglioncells of the colon.

REFERENCES

BLACKMAN, J. G., CROWCROFT, P. J., DEVINE, C. E., HOLMAN, M. E. & YONEMUTRA,K. (1969). J. Phy8iol. 201, 723-743.

KuNTz, A. (1940). J. comp. Neurol. 72, 371-382.KuNTz, A. & SACCOMANNO, 0. J. (1944). J. Anat. 7, 163-170.NORBERG, K.-A. (1964). Int. J. Neuropharmac. 3, 379-382.

The fine structure of avian vascular muscle and its electrical con-stants obtained with intracellular micro-electrodesBy T. B. BOLTON and H. NISHIHARA. Department of Pharmacology,University of OxfordAlthough it is possible to record intracellularly from vascular smooth

muscle cells (Funaki & Bohr, 1964; Cuthbert, Matthews & Sutter, 1965;Nakajima & Horn, 1967), it is difficult to maintain an impalement with amicro-electrode for any length of time as the cells are generally small andcontract when stimulated. Thus, until now, no study of the electricalconstants of vascular smooth muscle has been reported.Experiments were performed on the longitudinal muscle of the fowl

anterior mesenteric artery. This tissue was chosen because it showed

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electrical activity which suggested that there were low resistance pathwaysbetween cells (Bolton, 1968) and because this muscle can be studied fromthe adventitial side of the artery without cutting strips or otherwisedamaging the tissue in the vicinity of the region from which records areto be made. Furthermore, the longitudinal disposition of the muscle fibresallowed it to be subjected to a longitudinal potential field in an apparatusdescribed by Abe & Tomita (1968).

Electrotonic potentials were recorded in response to applied rectangularstimuli from cells in an extrapolar position both in isotonic salt solutionat 310 and also at higher temperatures in hypertonic salt solution in whichthe contractile power of the tissue was diminished. The relationshipbetween the size of the electrotonic potential and the applied voltagegradient was linear for hyperpolarizing pulses except when the electro-tonic potentials were very large. Thus, this arterial muscle, like othersmooth muscle (Tomita, 1970), showed cable properties.By obtaining electrotonic potentials from several cells at increasing

distances from the anode in the same preparation, it was found that thespace constant, A, measured as the distance at which the electrotonicpotentials declined to a relative size of 1/e, was about 2-8 mm. Similarly,by measuring the time taken for electrotonic potentials to reach half theirmaximum size it was possible to calculate the time constant, r, of thetissue since the slope of the line relating this parameter to distance fromthe anode is r/2A (Hodgkin & Rushton, 1946). The time constant wasfound to be 470 msec at 31° C.An electron microscope study of the artery was made to try to identify

the site of low-resistance pathways between cells. The cells from whichelectrical recording was made were about the same size as those of taeniacoli (Nishihara, 1970) but were unusual in being enmeshed in a well-developed collagen framework. Cells were generally keyed together bylarge projections of one cell into another and nexuses were common.As in other smooth muscle cells, it is probable that either or both of theseregions are the site of low-resistance pathways between cells (Dewey &Barr, 1962).

This work was supported by a grant from the Medical Research Council.

REFERENCES

ABE, Y. & TOmrTA, T. (1968). J. Phy8iol. 196, 87-100.BOLTON, T. B. (1968). J. Phy8iol. 196, 283-292.CUTHBERT, A. W., MA1-1ws, E. K. & SUTTER, M. C. (1965). J. Phy8iol. 176,

22-23P.DEWEY, M. M. & BARR, L. (1962). Science, N.Y. 137, 670-672.FuJNAx, S. & BoHR, D. F. (1964). Nature, Lond. 203, 192-194.

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PROCEEDINGS OF THEHODGKIN, A. L. & RUSHTON, W. A. H. (1946). Proc. R. Soc. B 133, 444-479.NAxAJIMA, A. & HORN, L. (1967). Am. J. Phy8iol. 213, 25-30.NISHARA, H. (1970). J. Anat. (In the Press.)TOMITA, T. (1970). In Smooth Muscle, eds. BULBRING, E., BRADING, A. F., JoNEs,

A. W. & TOMITA, T., pp. 197-243. London: Arnold.

Human temperature regulation after severe exercise and ethanolBY J. S. J. HAIGHT* and W. R. KEATINGE. Department of Physiology,The London Hospital Medical College, London, E. 1

Hiliwalkers who die from exposure usually do so after collapsing fromexhaustion in cold surroundings (Pugh, 1966). Prolonged physical exercisewith food and without ethanol does not impair man's ability to maintainhis deep body temperature in the cold (Haight & Keatinge, 1969). Indeed,volunteers maintained slightly higher temperatures than normal underthese circumstances.The purpose of the present experiments was to see whether ethanol

taken after exercise might produce sufficient hypoglycaemia to impair themechanisms by which body temperature is regulated in the cold. Thisseemed possible in view of the fact that ethanol reduces the rate of gluco-neogenesis from some precursors (see Krebs, 1968 for review), and thatexercise depletes carbohydrate reserves.

Six healthy young volunteers aged 19-26 years, on a normal diet,exercised for as long as they were able, with short rest pauses, at an averageof 71 % of their maximum oxygen capacities. They received no foodduring the work which lasted an average of 3 hr 20 min. After the exertionthey drank a small quantity of ethanol (0.34 g/kg body weight), rested witha blanket round them in air at 19.70 C for 23-37 min, and then sat wearingonly shorts in a cold room (14.40 C) for 30 min. Deep body temperaturesand metabolic rates were measured before and during the cold exposure.The subjects had no significant metabolic responses to the cold and their

mean rectal temperature (six subjects) and oesophageal temperature (threesubjects) fell to 34. 8 C and 34.10 C respectively by the end of the exposure.Their blood glucose concentration measured by the glucose oxidase methodbefore and after the cold was on average 2-2 /,M/ml. (39.6 mg/100 ml.).

In one additional set of experiments the volunteers received the ethanolbut did not exercise, and in another set were given glucose (0.94 g/kgbody weight) as well as ethanol after exertion. In both cases metabolicrates subsequently rose in the cold room. At the end of these exposuresmean rectal temperature was not below 36.70 C, mean oesophagealtemperature not below 36.00 C and mean blood glucose not below 3-3/Sm/ml. (59.4 mg/100 ml.).

* M.R.C. Research Scholar.

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The results indicate that severe exercise followed by the consumptionof a small amount of ethanol can produce hypoglycaemia of sufficientseverity to prevent the maintenance of body temperature in a moderatelycold environment.

REFERENCES

IIAIGHT, J. S. J. & KEATINGE, W. R. (1969). J. Phy8iol. 206, 20P.KREBS, H. A. (1968). Advances in Enzyme Regulation 6, 467-480.PUGH, L. G. C. E. (1966). Br. med. J. 1, 123-129.

Preliminary observations on the application of thermography tothe study of brown adipose tissue in the human new-bornBY D. P. G. BOLTON, A. M. Fox and D. L. KENNAIRD. The PhysiologyDepartment, The London Hospital Medical College, London, E. 1

Thermographic techniques employed in clinical medicine achievepictorial representation of small temperature gradients over the skinsurface. Brown adipose tissue is a site of neonatal metabolic heat pro-duction and the results of Dawkins & Hull (1964) indicate subcutaneouswarming over this tissue. Contact thermometry is fallacious and thermo-graphic techniques, using an AGA 'Thermovision' (Bjork, 1967), wereexplored.

Ideal conditions for clinical thermography of adults involve equilibra-tion of the naked subject for at least 15 min with a stable ambient tem-perature of 210 C, the subject being immobile and untouched during thisperiod. Similar conditions are difficult to achieve in the new-born.Attempts to study surface thermographic patterns in a thermoneutral

environment were non-productive due to the limitations of the system.Comparisons between thermographs made at different times are hazardous;the only satisfactory controls utilize either other parts of the same bodyor a modified Leslie cube as reference. Caution is needed in the interpreta-tion of thermographic appearances due to thermal asymmetry, bonyprominences, metabolically inactive fat and instabilities either of theenvironment or within the electronic circuitry employed. Anomalies areproduced by opposed skin folds as in the neck and axilla, so that althoughthese are known to be important areas of brown adipose tissue it is notpracticable to study them.The interscapular deposit of brown fat is suitable for thermographic

observation, being little influenced by environmental changes and sur-rounded by a mass of less active tissue.A preliminary study of twenty neonates was made. With increasing

experience a standard technique was evolved and repeatable appearancesrecognized.

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PROCEEDINGS OF THE

Thermographs were made of the naked infant, sitting supported, after20 min exposure to a suberitical temperature, usually 210 C. Care wastaken not to handle skin near the region to be studied. The environmentwas as draught-free and thermostable as possible.Under these conditions an interscapular warm zone could be detected;

this could not be seen in adults and it is suggested that this may representmetabolically active brown fat. Radiant areas are also seen on the nape ofthe neck and the axillae, but their interpretation is uncertain.

Should this technique be established as a means of detecting meta-bolically active brown fat, it could also be used to detect fat depletion; ashypoxia probably inhibits brown fat thermogenesis it might also beutilized in the assessment of hypoxic states. No definitive method for theuse of thermography in this context can yet be recommended.

REFERENCES

BJORK, NILs A. (1967). J. Radiol. Elect. 48, 30-33.DAWKINS, M. J. R. & HULL, D. (1964). J. Physiol. 172, 216-238.

A thermographic study of the dorsal surface temperature of thenew-born rabbitBY K. W. CROSS and A. J. WADE. The Physiology Department, The LondonHospital Medical College, London, E. 1A closed circuit television system with an infrared sensitive camera has

recently been made available to us by AGA Thermovision. This enabled usto make a short study principally aimed at evaluating the potential of theinstrument for use in paediatrics. The camera gives a continuous pictorialdisplay of radiant heat emitted from surfaces (Bjork, 1967). Comparisonsof surface temperature were made by using two, independently variable,reference circuits, described as isotherms. It is essential that one of theseshould be trained on a reference 'black body surface' in every field ofview, if surface temperature is to be estimated, or if comparisons are to bemade of the isothermal patterns in separate photographic records.

Results published previously (Dawkins & Hull, 1964) on subcutaneoustemperatures over the dorsal surface of the new-born rabbit led us to hopethat studying the same surface of the new-born infant (Bolton, Cross, Fox& Kennaird, 1970) and rabbits with this remote and harmless instrumentmight reveal a rise in the relative, or absolute, temperature of the skinsurface above deposits of brown fat when these were stimulated bycooling. We also considered the possibility that the topography of tem-perature gradients might change under warm and cool conditions.The apparatus used to hold the rabbit was intended to minimize

fluctuations in heat loss from surfaces. A restraining chamber, with an

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infrared transparent sheet (Platee Plastic 'Cryovac XL') closing theviewing end, was constructed so that a rabbit could be held on the baseplate while the chamber was sloping into a constant temperature water-bath ( ± 0 050 C). Air passed around the rabbit and through the chamber,after being heated to the temperature of the surrounding water, at 8 1./min.The surface temperatures assumed a definite pattern provided that theanimal remained still for about 10 sec. During bouts of movement, re-cording was impossible, as heat losses altered the surface temperaturesdramatically. Rectal, environmental, and water-bath temperatures werecontinuously monitored by thermocouples.In an environment at 30-31o C, the temperature over the nape falls

more slowly than that of the rectum if the animal is responding to cooling.This differential is lost on returning to a thermoneutral environment, orfollowing the prevention of the metabolic response to cooling by hypoxiaor propranolol. There was no evidence of a local increase in metabolicactivity above the nape of a rabbit with depleted brown fat which wasexposed to a cool environment.We have been unable to demonstrate a topographical difference in the

warmest areas of the dorsal surface under various experimental conditions.The nape was invariably the warmest area. The distribution of warmtharound the nape was not delineated by the areas of subcutaneous brownfat.

REFERENCES

BOLTON, D. P. G., Fox, A. M. & KENNAIRD, D. L. (1970). J. Physiol. 208, 23P.BJORK, N. A. (1967). J. Radiol. Elect. 48, 30-33.DAWxIS, M. J. R. & HULL, D. (1964). J. Physiol. 172, 216-238.

Lung volume and mixing efficiency in the new-born infantBY D. P. G. BOLTON and K. W. CROSS. The Physiology Department, TheLondon Hospital Medical College, London, E. 1

It is far from simple to measure lung volumes in new-born infants as allthe established techniques for use on adults demand a modicum of co-operation on the part of the subject. The nitrogen washout technique hasbeen chosen for modification as additional information on mixing efficiencyin the lungs is forthcoming.The method is outlined in the Demonstration at this meeting (Bolton,

Cross & Kennaird, 1970).If the nitrogen remaining in the lungs after each breath is plotted on a

logarithmic scale against the accumulated tidal volume on a linear scalethe curve approximates to a straight line. A constructed straight line fromthe starting point to cut the 1 % line at a running tidal volume of 5-05

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2PROCEEDINGS OF THEtimes the F.R.C. (or turnovers) would be the decay curve if 100% effi-ciency obtained, with the particular ratio (1: 5) of tidal volume to F.R.C.seen in infants. A straight line down to 10 1 turnovers would be a 50%efficiency line, constant throughout the washout, and would represent'stratified inhomogeneity' (Cumming, 1967) in the lungs, where gaseousdiffusion of nitrogen molecules has to take place over a finite distance from

111*4!

X_

E

bO

Rx~~~~

bo

* 1114E \a'~~~~~~~\

z ~~~~~~~~~~~xz ~~~~~~~~~~~X

A ~~~~~x1 2 3 4 5 6 7 8 9 \0 x 11 12 Turnovers

1*114 I200 400 600 800 1000

Accumulated tidal volume (cm3)

Fig. 1. Nitrogen washout: nitrogen remaining in lungs (log. scale) againstaccumulated tidal volume. A, 100% mixing efficiency line; B, 50%mixing efficiency line. Points (x) from curve of baby L.J.P. After thefirst two tidal volumes, only alternate points have been plotted.

the depths of the alveoli to gas which is physically exchanged by venti-latory movement. A marked curve away from this line would indicatethat the mixing efficiency was falling off late in the washout and nitrogenwas being retained in particular zones of the lungs; 'zonal inhomogeneity'.We have investigated some thirty normal babies in this way and found

an average F.R.C. of 90 cm3 and mixing efficiencies characteristically ofthe stratified type of about 50% (see Fig. 1).Normal adult curves are very similar but with efficiencies of about 70 %.

It is suggested that this discrepancy is due to the higher respiratory rateof the infant allowing less time for diffusion in the lungs.

REFERENCES

BOLTON, D. P. G., CRoss, K. W. & KENNAIRD, D. L. (1970). J. Physiol. 208, 6P.GUMMING, G. (1967). Resp. Physiology, 2, 213-234.

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PH YSIOLOGICAL SOCIETY, FEBRUARY 1970

Oxytocinase levels in the female rabbit hypothalamus after matingand after the injection of antiovulatory compounds

BY DONAA. FRITH and K. C. HOOPER. Department of Physiology, Uni-versity of Sheffield, Sheffield, S10 2TN

Recent work on certain peptidases in the hypothalamus showed thatsome of the stimuli which are known to alter gonadotrophin release fromthe anterior pituitary also change enzyme activity in the hypothalamus.Consequently it was suggested that there may be some link betweenenzyme activity and gonadotrophin release (Hooper, 1968). In the rabbit,mating is known to initiate the release of gonadotrophin which results inovulation 10 hr later; it was thought probable therefore, that if there wasa relation between enzyme activity in the hypothalamus and the release ofgonadotrophin, changes in enzyme activity might be detectable in thefemale.

Rabbits were mated and killed from i hr to 24 hr later and the peptidaseactivity in a particulate fraction and the supernatant fraction of the hypo-thalamus was estimated. The activity of the supernatant peptidase haddecreased 2 hr after mating, returning to approximately normal levelsby 1 hr. A further decrease then followed, reaching a minimum at 5 hr.10 hr after mating the peptidase activity was normal, and this was followedby another depression at 12 hr. No such obvious pattern was found in theactivity of the particulate peptidase. The depression in supernatantpeptidase activity can be correlated with the gonadotrophin release notedafter mating in rabbits by other workers (Hilliard, Hayward & Sawyer,1964).

Preliminary work with ethinyl oestradiol showed that the peptidases areaffected by at least one antiovulatory compound (Firth & Hooper, 1968).This aspect has been looked at in more detail where ethinyl oestradiol,chlormadinone acetate, norethindrone and oestrone were injected intra-muscularly 24 hr before mating, and the rabbits were kiRled either - hror 24 hr later. The doses chosen were those known to have antiovulatoryeffects. The peptidase activity in the two fractions was measured and anincrease in supernatant peptidase activity was seen in all cases; no changein particulate peptidase activity was found.

Several workers have suggested that the hypothalamus is the site ofaction of certain antiovulatory compounds (e.g. Kanematsu & Sawyer,1965; Exley, Gellert, Harris & Nadler, 1968). The compounds may act byinhibiting the releasing factors and hence the release of the gonado-trophins. The rise in peptidase activity shown here lends further supportto the idea that these inhibitors may regulate the availability of thegonadotrophic hormone releasing factors.

b

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2PROCEEDINGS OF THEWe thank the Smith Kline and French Foundation for financial support and the

M.R.C. for a research grant to D.A.F.

REFERENCES

ExT-Y, D., GELLERT, R. J., HlARRIs, G. W. & NADLER, R. D. (1968). J. Phy8iol.195, 697-711.

FrRTH, D. A. & HOOPER, K. C. (1968). Biochem. J. 108, 510-511.HILLIARD, J., HIAYwAARD, J. N. & SAwYER, C. H. (1964). Endocrinology 75, 957-963.HOOPER, K. C. (1968). Biochem. J. 110, 151-153.KANEMATSU, S. & SAwYER, C. H. (1965). Endocrinology 76, 691-699.

Actions of LSD-25 and reserpine on a serotonergic synapseBY G. A. COTTRELL. Gatty Marine Laboratory and Physiology Department,St Andrews UniversityThere are excitatory synaptic connexions between the giant serotonin-

containing neurone of each metacerebral ganglion (see Cottrell & Osborne,1970) and neurones located laterally in each buccal ganglion of the snail,Helix pomatia. Each giant cell innervates ipsilateral and contralateralbuccal ganglia. Simultaneous intracellular recordings made from one of thegiant cells and an appropriate buccal ganglion cell, in isolated preparations,have shown the following: (1) Giant cell spikes result in small depolarizingpotentials (which sum) and then spike firing in each 'buccal cell'. (2) Theamplitude of successive small potentials increases during a short burst ofimpulses in a giant cell. (3) Hyperpolarization of the 'buccal cell' causes anincrease in the amplitude of the small potentials. They continue to in-crease in amplitude with increased hyperpolarization and are not lostwith repeated stimulation. (4) They occur with a constant delay of about100 msec after each spike. These data suggest that there is a direct synapticconnexion between each giant serotonin cell and the buccal ganglion cellsdescribed and that the small potentials are excitatory post-synapticpotentials (EPSPs). The 'buccal cells' are depolarized and fire spikes whenserotonin (10-7 g/ml. or greater) is applied.

Reserpine antagonizes transmission between the cells. Animals wereinjected with 1 mg of soluble reserpine over 3 days and transmissionbetween the cells examined as before. The initial response to a burst ofgiant cell impulses appeared normal, or even potentiated, but therefollowed a marked reduction in the amplitude of the EPSPs, comparedwith controls, during continued stimulation of a giant cell. An impairmentof transmission might be expected if serotonin is indeed used as the trans-mitter substance by the giant neurones, since it is known that reserpinedepletes the amine from these cells (G. A. Cottrell & N. N. Osborne, 1969;unpublished observations).

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PH YSIOLOGICAL SOCIETY, FEBRUARY 1970

The main effect of LSD was to increase the size of EPSPs obtained afterrepeated firing of a giant cell (40 or more impulses at 2.5/sec). The effectwas observed after perfusing 10- g/ml. LSD for 20 min but it did notreach a maximum until after 1 hr of perfusion when an increase of 100%in EPSP amplitude, compared with controls, was frequently observed.Another effect of LSD (10- g/ml.) was a reduction in the size of the firstfew EPSPs during a burst of spikes. It seems unlikely that the results canbe explained simply in terms of an increased sensitivity to serotonin, orincreased membrane resistance, of the post-synaptic cell. It is suggestedthat LSD may have a presynaptic action on the system.

REFERENCES

CoTTREi.u, G. A. & OSBORNE, N. N. (1970). Nature, Lond. 225, 470-472.

Studies of the effects of dimethothiazine on muscle spindle activityin the decerebrate catBY D. R. MAxwELL and K. F. RHODES. Research Laboratories, May andBaker Ltd., Dagenham, Essex

Dimethothiazine is a phenothiazine derivative which reduces therigidity of the cat decerebrated at the intercollicular level, whilst havinglittle activity on polysynaptic spinal reflexes in the cat, or sedative activityin rodents (Keary & Maxwell, 1967). We have studied the effects ofdimethothiazine on the discharge of afferent fibres from muscle spindlesin the soleus muscle of the decerebrate cat during stretching of the muscleunder controlled conditions. Three parameters were used to measurespindle sensitivity: (i) the resting frequency before stretch of soleus; (ii)the maximum frequency during the dynamic phase of stretch; and (iii)the frequency during maintained stretch.

Intravenous doses of dimethothiazine which reduce decerebrate rigidity(2-4 mg/kg) reduced the discharge of primary and secondary endings underall three of the above conditions (Fig. 1). Larger doses had little furthereffect on the discharge frequency. These effects on muscle spindle activitywere not correlated with changes in arterial pressure.The discharge frequencies recorded from de-efferented muscle spindles

in the soleus of the decerebrate cat were similar to those obtained in pre-parations with intact ventral roots after a large dose of dimethothiazine.The drug had little significant effect on the discharge frequency of

afferent fibres from muscle spindles in the soleus muscle of preparationswhere the ventral roots were cut.Matthews (1962) and Brown & Matthews (1966) have shown that the

fusimotor fibres may be differentiated into two types: static and dynamic.b-2

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That dimethothiazine reduced the discharge of afferent fibres fromsecondary endings of muscle spindles indicates that the drug is affectingstatic fusimotor activity. There is also evidence that the drug may beaffecting dynamic fusimotor fibre activity.The evidence suggests that dimethothiazine reduces decerebrate rigidity

by decreasing fusimotor activity whilst having little direct depressanteffect on the muscle spindle itself.

Stretch Maintain tension280 _1

Wi 2400

. 200 -

E

160 -

120

80.- 80t

40

1 2 3 4 5Time (sec)

Fig. 1. Decerebrate cat (1-9 kg). Dorsal roots (L6, L7, SI) cut. Effect ofdimethothiazine on discharge of primary spindle ending (conductionvelocity 72 m/sec) from soleus undergoing 2 cm stretch at 20 mm/sec.0 Control. 0 Dimethothiazine 4 mg/kg i.v.

REFERENCESBROWN, M. C. & MATTrHEws, P. B. C. (1966). Control and Innervation of Skeletal

Mu8cle, ed. ANDREW, B. L., p. 18. Dundee: Thomson and Co.KEARY, E. M. & MAXWELL, D. R. (1967). Br. J. Pharmac. Chemother. 30, 400-416.MATTHEWS, P. B. C. (1962). Q. Jl exp. Phy8iol. 47, 324-333.

An inhibitory effect of prostaglandin E2 on neuromuscular trans -mission in the guinea-pig vas deferensBY N. AMBACHE and M. ABoo ZAR. Medical Research Council, Depart-ment of Physiology, Royal College of Surgeons of England, Lincoln's InnFields, London, W.C. 2The vas was suspended at 350 C in a 2 ml. bath with built-in vertical

platinum electrodes, in Krebs-Henseleit solution gassed with 5% C02-95% 02. An electronic stimulator with an output of 500 mA was employed




for electrical stimulation. Longitudinal contractions were elicited once aminute by trains of 5-50 pulses of supramaximal voltage (each pulse of1 msec width) at 50 Hz. The neurogenic origin of these contractions wasconfirmed by their complete extinction by tetrodotoxin (10-7 g/ml.); andthe fact that they are due to stimulation of post-ganglionic motor neuronesby the inability ofhexamethonium (10- g/ml.) to reduce these contractions.PGE2 but not F2X inhibited these contractions when elicited by 5-10 but

not by 50 pulses/train (Fig. 1; 10 pulses/train). Detectable inhibition was

Noradrenafine5 aig/mI .

PGE2 5 ng'm.

Fig. I

obtained with 5 x 10-12 g/ml. and almost total inhibition with 2-5 ng/ml.of PGE2. This effect of PGE2 cannot be attributed either to a non-specificdepression of the smooth muscle or to a specific antagonism against nor-adrenaline or acetylcholine, since the contractions elicited by these twosubstances were potentiated by PGE2. Nor is it due to a cholinergic mech-anism (Euler & Hedquist, 1969), as it persists in atropine. It was notblocked by the PG-antagonist, SC-19220. When the relative potency ofPGE2 is compared with that of bretylium or of guanethidine on this pre-paration a striking disparity is noted. Birmingham & Wilson (1963) foundthat 4,tg/ml. of guanethidine or 8 jug/ml. of bretylium were needed toabolish electrically evoked contractions of the vas; in contrast, as little as

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2-5 ng/ml. of PGE2 produced almost the same result in the present experi-ments. This high order of activity is surprising, since PGE2 has not beenreported to possess any significant adrenergic neurone-blocking property;it might therefore indicate a regulatory role for prostaglandins in sometypes of humoral transmission.

REFERENCES

BIRMINGHEAM, A. T. & WILSON, A. B. (1963). Br. J. Pharmac. Chemother. 21, 569-580.

EULER, U. S. V. & HEDQVIST, P. (1969). Acta phy8iol. 8cand. 77, 510-512.

The effect of urethane on transmission along the dorsal columnsensory pathway in the ratBY A. ANGEL and J. UNWI.* Department of Physiology, The Universityof Sheffield, S10 2TN

Angel (1967b) showed that the cortical responses evoked by peripheralelectrical stimulation became diminished in size and increased in latencyafter administration of anaesthetic agents. Changes in blood pressure orrespiration do not explain this effect (Angel & Unwin, 1969).To determine where anaesthetic agents exert their effect, acute experi-

ments were performed to record single unit responses from neuroneslocated in the relay nuclei of the dorsal column pathway and from neuronesin the primary cortical receiving area. It was found that cells in the cuneatenucleus responding with the shortest latencies to forepaw stimulation(2.8-2.9 msec; mean earliest and latest latencies of forty-five cells) showedno reduction in their probability or latency of discharge to supramaximalstimuli, until the anaesthetic depth seriously impaired the animal'srespiratory effort. Cells in the ventrobasal thalamus responding withlatencies of 4*3-5-9 msec (means of twenty-eight cells) showed a pro-gressive increase in latency and a decrease in probability of discharge asthe anaesthetic dose was increased. Cells inl the cerebral cortex respondingto stimulation of a limited area of the contralateral body surface can bedivided into two quite distinct groups (Angel 1967a). The first grouprespond with latencies of 5-8 msec (means of seventy-eight cells) and do soduring the first negative wave of the cortical surface response. These cellsshow the same behaviour to increasing depth of anaesthesia as ventrobasalthalamic cells. The second group of cortical cells responded with latenciesof 10-14 msec (mean of nine cells) during the second negative wave of thecortical response and ceased to respond after small increases in anaes-thetic depth. The behaviour of one sensory cell of each type is shown in

* S.R.C. Scholar.

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PHYSIOLOGICAL SOCIET Y, FEBRUARY 1970

Fig. 1. These results indicate that anaesthetic agents exert their effectmainly by impeding transmission through the ventrobasal thalamus.

1-10_ . 0A

0

0.6

r6

0

.' L\40

0I I I I I 1

1.0 1-2 1X4 16 1f8 2-0.25% urethane (ml.)

Fig. 1. Examples of the effect of increasing the depth of anaesthesia on theprobability of discharge of four cells in the sensory pathway. A cell from thecuneate nucleus (0-0), from the ventrobasal thalamus (A-A), fromthe first (A-A) and second (@-@) groups of cortical cells. Ordinate,probability of response (if the cell responded twenty times to twenty con-secutive stimuli the probability was taken as unity). Abscissa, dose ofurethane expressed as ml. of a 25 % solution injected intraperitoneally.

REFERENCES

ANGEL, A. (1967a). J. Physiol. 191, 427-448.ANGEL, A. (1967b). J. Phy8iol. 192, 6-7P.ANGEL, A. & UNwiN, J. (1969). J. Physiol. 205, 25-26P.

The effects of cortisol on the carbohydrate metabolism ofhypophysectomized and of thyroidectomized calvesBY R. S. COMLTNE, A. V. EDWARDS and P. W. NATHANIELSZ. PhysiologicalLaboratory, University of CambridgeThe removal of the pituitary gland in the young calf appears to sensitize

the myocardial cells to the glycogenetic action of glucocorticoids; theadministration of cortisol in doses as small as 2 mg. kg-'. day-' raises thecardiac glycogen concentration of hypophysectomized animals but theeffect is reduced if the pituitary stalk is not completely removed at opera-tion (Fig. 1). Administration oflarger amounts ofcortisol (4 mg.kg-' . day-')

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to thyroidectomized calves also results in the accumulation of excessiveamounts of glycogen in the myocardium. The reduction in thyroidfunction which follows hypophysectomy may therefore provide a partialexplanation of this effect.

Since abnormally high myocardial glycogen concentrations occur afterpancreatectomy (Cruikshank, 1913), it may be significant that hyper-glycaemia and associated glycosuria were observed in several of the hypo-physectomized animals after varying intervals. In thyroidectomized calves,doses of either 2 or 4 mg cortisol kg-' . day-' produced hyperglyeaemia in

60

50 /

bo~~~~~~~~~~~o0~~~~~~~~~~~~~~~

to /

bO /be /

200 80

L. /

o /_

l0 CPO /000

_ 0 /~~~~000I0~~ 20 0 /~~~~0

0 10 20 30 40 so 60 70 80 00Age (days)

Fig. 1. The effect of cortisol on the cardiac glycogen concentration of thecalf. 0, normal values. o, control animals given cortisol (4 mg. kg-'.day-'). A, adrenalectomized, given cortisol (4 mg. kg-'. day-'). 0*hypophysectomized, stalk removed, given cortisol (2 mg. kg-'. day-').A, hypophysectomized, stalk remaining, given cortisol (2 mg. kg-'. day-').Horizontal bars show normal range of cardiac glycogen concentrationsbetween 7 and 500 days of age.




the majority of animals within 2-4 weeks. In the animals given the higherdose of cortisol the blood glucose concentration rose to values in excess of300 mg/100 ml. and neutral fat was present in the blood in amounts up to15%, v/v; glycosuria and ketonuria were also present.The diabetic syndrome induced by cortisol administration in thyroid-

ectomized calves was reversed by injections of thyroxine in doses as smallas 10 gg.kg-'.day-' for 5-7 days. This produced a blood thyroxine con-centration comparable to that normally found in animals of this age;hyperglycaemia did not recur after withdrawal of thyroxine although theadministration of cortisol was continued.Young calves on a milk diet appear to be especially susceptible to the

diabetic effect of cortisol in the absence of thyroxine. Weaning invariablyabolished the hyperglycaemia together with the associated metabolicchanges and the condition could not be produced in adult thyroidectomizedsheep even after the administration of excessive doses of cortisol (14mg. kg-'. day-') for prolonged periods.

REFERENCE

CRuSHANK, E. W. H. (1913). J. Phy8iol. 47, 1-14.

Effects of a surface active contraceptive agent (nonylphenoxy-polyethoxyethanol) on the vagina; a new functional approach toassessing the actions on the vagina of spermicidesBY FRANCES EDWARDS, J. H. KUGLER and R. J. LEVIN. Department ofPhysiology and Department ofHuman Biology and Anatomy, The University,Sheffield, S10 2TNThe standard methods for assessing the harmful effects of spermicides

on the vagina based on observing pathological changes in its gross orhistological structure after repeated instillation (Carleton & Florey, 1931;Holzaepfel, Warner, Buxton & Howard, 1958; Boyland, Roe & Mitchley,1966) are too insensitive to identify subtle changes in vaginal cell physi-ology. As the epithelium generates an electrical potential difference (p.d.)and a short-circuit current (s.c.c.) in vitro (Levin, 1968; Edwards & Levin,1970), we have explored the possibility of using changes in this activityto assess any immediate effects of spermicides on the vagina. The spermi-cide chosen, nonylphenoxypolyethoxyethanol (nonyl-p), is a surfaceactive agent marketed in creams, pessaries and foams in concentrationsranging from 2 to 12-5 %. We have tested this compound (2-2.5 %) on therat and hamster vagina in vivo and on rat vagina in vitro. In some cases,a double chamber was used in vitro that exposed one half of a vagina tocontrol buffer and the other to buffer containing nonyl-p.

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PROCEEDINGS OF THE

Instillation of nonyl-p into rat and hamster vagina in vivo caused aprecipitous fall of the transvaginal p.d. A similar effect was observed invitro with a concomitant fall in the tissue resistance when nonyl-p wasadded to the mucosal fluid. The s.c.c., however, was increased initially butthis was transient and it rapidly fell to low values with a time coursesimilar to that of the fall of the p.d. and resistance. These effects wereirreversible; washing out and replacing with fresh buffer did not restorenormal activity. Electron micrographs of vaginas exposed to nonyl-p for30 min did not show any consistent damage to intracellular organelles anddesmosomes were still intact.

Neither dimethyl sulphoxide, Tween 80 nor mannitol (osmotic control)affected the vagina like nonyl-p. Transuterine endometrial p.d. (C.Kyriakides and R. J. Levin, unpublished observations) and the glucosetransfer p.d. across the rat jejunum were also rapidly depressed by nonyl-p.Testing the action of nonyl-p on a human vagina by changes in theabdomino-vaginal p.d. in a conscious woman gave inconclusive results.Although the first intravaginal injection of a contraceptive foam containingnonyl-p affected the p.d., subsequent injections on two consecutive dayslater did not show clear-cut effects.

Because this new functional approach of assessing the immediate effectsof chemicals on the vagina is simple, rapid and quantitative it could becomea useful adjunct to the routine screening tests previously employed. Itsvalue is demonstrated by our preliminary results that show the spermicidenonyl-p to have a potent and hitherto unsuspected action on the bio-electric activity of the rat and hamster vagina. It is yet to be ascertainedhow important this action is in woman.

We would like to thank Coates and Cooper Ltd., Ortho Pharmaceutical Ltd. andW. J. Rendell Ltd., for supplying samples of nonylphenoxypolyethoxyethanol andother contraceptive chemicals.One of us (F.E.) is supported by the British Empire Cancer Campaign.

REFERENCES

BoAND, E., ROE, F. J. C. & MITCHLEY, B. C. V. (1966). Br. J. Cancer 20, 184.CARLETON, H. M. & FLOREY, H. (1931). J. Ob8tet. Gynaec. Br. Commonw. 38, 555.EDWARDS, F. & LEVLN, R. J. (1970). J. Phy8iol. (In the Press.)HOLZAEPFEL, J. H., WARNER, J. S., BUXTON, J. A. & HOWARD, J. A. (1958). J. Am.pharm. A88. (Sci. Edn.) 7, 423.

LEvIN, R. J. (1968). J. Phy8iol. 194, 94-95P.

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Efferent activity in the lateral line nerve of dogfishBY B. L. ROBERTS and I. J. RUSSELL. The Laboratory of the MarineBiological Association, Plymouth, and Department of Zoology, Universityof CambridgeThe lateral line organs of lower vertebrates are related in development

and evolution to the sense organs of the semicircular canals and inner ear(Pumphrey, 1950). It is now well established that the auditory receptorsreceive an inhibitory efferent supply from the olivo-cochlear bundle(Engstrom, 1958; Fex, 1962) and recently Russell (1968) has demon-strated the presence of an inhibitory efferent system to the lateral line ofthe toad, Xenopus. Although it is known from electron microscopic studiesthat the lateral line neuromasts of some fishes are doubly innervated(Flock, 1965), there has been little experimental work. We have nowrecorded efferent activity in the lateral line nerve of dogfish (ScyliorhinU8canicula L.).The fish were decerebrated under tricaine anaesthesia (MS 222, Sandoz)

and supported firmly along the body in a tank of circulating sea water.Recordings were made with bipolar platinum electrodes from filamentsof the lateral line branch of cranial nerve X, which was severed peri-pherally. Efferent activity, involving small axons of slow conductionvelocity (12 m/sec at 140 C), could be recorded from many of the filaments,particularly when the fish were stimulated mechanically. Although a fewunits discharged in time with respiratory movements, most were notspontaneously active. Very gentle touch anywhere on the head and gillregion evoked efferent discharges, but stronger stimulation was necessaryelsewhere on the body. Gentle ipsilateral, mechanical stimulation to thelateral line canal did not excite efferent activity but electrical stimulationof the nerve was followed by a synchronous efferent discharge.The efferent neurones are probably situated in the dorsal medial nucleus,

for a small lesion in the anterior part of the medulla oblongata abolishesefferent activity.

REFERENCES

ENGSTROM, H. (1958). Acta oto-lar. 49, 109-120.FEX, J. (1962). Acta phy8iol. 8cand. 55, suppl. 189.FLOCK, A. (1965). Acta oto-lar. suppl. 199.PumPHREY, R. J. (1950). Symp. Soc. exp. Biol. 4, 3-18RUSSELL, I. J. (1968). Nature, Lond. 219, 177-178.

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Documents

Haematology, The London Hospital Medical College, London, E. 1