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PHYSIOLOGICAL SOCIETY, JULY 1970

COMMUNICATIONS

The accessory reflex of the crayfish stretch receptorBY J. K. S. JANSEN, A. Ni., K. ORMSTAD and L. WALL0E. Institute ofPhysiology, University of OsloThe abdominal stretch receptors of the crayfish are innervated by two

efferents, the so called large and small accessory nerve fibres (Alexan-drowicz, 1967). Their function is to inhibit the receptors (Kuffler &Eyzaguirre, 1955; Burgen & Kuffler, 1957). The large (Eckert, 1961) andsmall (Jansen, Nja' & Wall0e, 1969) can be reflexly activated by a stretchreceptor input to the same or neighbouring abdominal segments.We have now obtained further information on these accessory reflexes

by intracellular recording and activation of the slowly adapting receptorneurone with intact reflex connexions. The receptor neurone gives a main-tained discharge to a transmembrane depolarizing current step. Thisreceptor input gives a reflex activation of a medium sized and a smallefferent spike. Both are tonically activated throughout the period ofreceptor activation. These two efferent spikes are functionally identifiedby their effect on the membrane potential of the slowly adapting receptor.They both induce hyperpolarizing IPSPs at the normal level of membranepotential. However, the IPSP of the medium-sized spikes is approximatelyfive times larger than that induced by the small spikes.

Accordingly, when activated by an intrasomatically applied trans-membrane current the response of the receptor with its reflex connexionsis dominated by the activity of the larger inhibitory fibre. Each such IPSPwill usually reset the prepotential of the receptor spikes to its initial value.Their inhibitory effect is therefore largely determined by their timingrelative to the receptor firing. The later an IPSP occurs the greater itsdelaying effect on the following receptor spike. On account of considerablevariability in the central delay of the reflex the timing of the IPSPsrelative to the receptor spikes is not fixed and the average delaying effectof the IPSP is approximately equal to that of an appearance in the middleof a receptor interval.The over-all effect of the accessory reflex is therefore to reduce the firing

frequency of the receptor in proportion to its excitatory drive. In otherwords, the accessory reflex appears to reduce the sensitivity of the slowlyadapting receptor over its entire working range.

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REFERENCES

ASLEXANDROWiCZ, J. S. (1967). Biol. Rev. 42, 288-326.BuRGEN, A. S. V. & KUFFLER, S. W. (1957). Nature, Lond. 180,1490-1491.ECKERT, R. 0. (1961). J. cell. comp. Physiol. 57, 149-162.JANSEN, J. K. S., NJk, A. & WALLoE, L. (1969). Acts phy8iol. 8Cand. 76, 29A-30A.KUFFLER, S. W. & EYzAQUImm, C. (1955). J. gen. Phy8iol. 39, 155-184.

Further evidence for right atrial receptors affecting heart rate

BY C. T. KAPPAGODA, R. J. LINDEN and H. M. SNOW. CardiovascularUnit, Department of Physiology, University of Leeds

Stretching the junction between the superior vena cava and the rightatrium has been shown to produce a reflex increase in heart rate (Kappa-goda, Linden & Snow, 1970). The efferent path for this reflex is solely inthe sympathetic nerves and the afferent path is at least partially in thevagus nerves; it was suggested that the receptors involved in this reflexwere the right atrial receptors. The purpose of this investigation was tolocate receptors which were stimulated by the balloons used to stretchthe junction between the superior vena cava and the right atrium, andwhich gave rise to trains of impulses in the right cervical vagus nerve.Dogs were anaesthetized with chloralose and the chest opened on the

right side. The superior vena cava was cannulated through the externaljugular vein with a cannula which incorporated two balloons. One of theballoons acted as an inflatable cuff which occluded the superior vena cavaabout 3 cm above its entrance into the right atrium. The blood accumu-lating in the superior vena cava was pumped away using a variable rollerpump and returned to the animal through a cannula in the left femoralvein. The second balloon which was situated at the tip of the cannula wasthen distended so as to stretch the junction between the superior vena cavaand the right atrium.

Action potentials were recorded from slips of the right vagus nerve inthe neck in eight animals. In five of these animals fibres in which theimpulse frequency increased with distension of the terminal balloon werefound, and the behaviour and location of the receptors determined.During control periods before and after distension of the terminal

balloon the mean impulse frequency was 6-9 (range 0-20) impulses/beat.The volume ofthe balloon was increased up to avolume of 12 ml. Distensionof the balloon to 10 ml. in five animals caused a mean increase of 14-9impulses/beat (range 4-45). In previous experiments in which the reflexincrease in heart rate was demonstrated (Kappagoda et al. 1970) theterminal balloon was distended to an average volume of 8 ml. (range 4-10 ml.) in order to stretch the junction between the superior vena cava andthe right atrium.

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The animals were subsequently killed and the precise origin of the dis-charge determined. All the receptor endings were found to be situated inthe subendocardial tissue in the intrapericardial portion of the superiorvena cava, i.e. in the area stretched by the balloon.From these results it is concluded that distension of the terminal balloon

stimulates the right atrial receptors and that they are the most likelyreceptors to be involved in the reflex increase in heart rate previouslyreported (Kappagoda et al. 1970).

This work is supported by grants from the British Heart Foundation, the MedicalResearch Council, and the Wellcome Trust.

REFERENCE

KAPPAGODA, C. T., LINDEN, R. J. & SNOW, H. M. (1970). J. Physiol. 208, 59P.

An estimate of calcium concentration changes during the con-traction of single muscle fibres

BY C. C. ASHLEY. Department of Zoology, Bristol

Recently the photoprotein aequorin has been used to follow rapidcalcium changes within single muscle fibres during contraction (Ridgway& Ashley, 1967; Ashley & Ridgway, 1968). It is of interest to know theprecise size of these calcium changes that lead to the production of tension.

Single barnacle fibres, after injection with a known amount of aequorin,were rapidly injected with 10 4tl. of solutions containing different stabilizedcalcium concentrations. The light emission from these fibres rose to asteady value that was maintained for some 2-5 sec. The results from theseexperiments indicate a straight-line relationship between the steady lightemission and calcium concentration on a log-log plot with a slope of 1*6-1 7 (Fig. 1 a). This result suggests that the connexion between calcium andlight emission over these ranges approaches a square-law relationship.The mixing of small volumes of aequorin (ca. 0-05-1 /M) with buffered

calcium (ca. 01-10 fSM) in vitro, in the presence of 5-15 mm magnesium andat ionic strengths from 0-05 to 0-2, also confirms the square-law relationship(Fig. 1 b). This relation also holds for unbuffered calcium concentrationsabove about 1 ,UM under similar conditions (Fig. 1 b). At lower unbufferedcalcium concentrations, the relationship appears to become linear and asimple explanation for this linearity is that during the purification pro-cedures inactive aequorin is formed which is still able to bind calcium.The results obtained suggest that in a high potassium contraction, where

the light emission rises some 100 times, the calcium should rise from about

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134P PROCEEDINGS OF THE07-1-Ox10-7 M to about 0*7-1*0 x10-6 M. In addition the square-lawrelation implies that over a range of tension from at least 50 g cm-2 to400 g cm-2 there are two calciums involved per tension generating site.

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Fig. 1. (a). A log-log plot of the steady light emission from aequorin-injected muscle fibres versus calcium concentration. Fibres had been soakedfor 24-48 hr in OCa saline at 0-5° C to reduce excitability. Assay of theaequorin in the fibres after a high calcium injection suggested that some

50-60% had been activated. Assumed apparent binding constant for EGTA= 7-6 x 106 M-1 at pH 7-1. Aequorin concentration ca. 0-8-1-0 fIM.

(b) A log-log plot of the maximum steady light emission from a mixingchamber versus the calcium concentration. Total volume of chamber, 50pl., 5 mm magnesium, ionic strength 0-05, pH 6-8-7-5, aequorin concen-

tration ca. 0-3 /tM. (Q), unbuffered calcium, slope, 1-90; (e), bufferedcalcium corrected for magnesium ions, slope, 1-95.

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

REFERENCES

ASHLEY, C. C. & RIDGWAY, E. B. (1968). Nature, Lond. 219, 1168-1169.RIDGWAY, E. B. & ASHLEY, C. C. (1967). Biochem. biophy8. Re&. Commun. 29,

229-234.

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Calcium influxes in perfused squid giant axons during voltageclamp

By E. ROJAS and R. E. TAYLOR. Laboratory of Cellulular Physiology,Faculty of Sciences, University of Chile and Laboratory of Biophysics,National Institutes of Health, Bethesda, Maryland, U.S.A.

Calcium influx measurements were performed with 45Ca on internallyperfused squid giant axons from Dosidicus gigas under conditions of mem-brane potential control. On raising the external calcium concentration inthe sea water from 7 7 to 84 7 mM, the resting calcium influx at 100 C wasincreased from 0-016 + 0 004 p-mole/cm2 .sec to 0-142 + 0 055 p-mole/cm2 . sec. Working with intact axons from Loligo forbesi at 220 C, Hodgkin& Keynes (1957) found calcium influxes of 0 37 p-mole/cm2 .sec from22 mM-Ca and 0*61 p-mole/cm2 .sec from 112 mm-Ca.When depolarizing voltage clamp pulses were applied across the axon

membrane the calcium influx became appreciably greater. When theabsolute value of the membrane potential during the pulse was about+ 40 mV, and the pulse duration was 50 jtsec, the extra influx of calciumincreased with the external [Ca] from 0-00008 to 0-0012 p-mole/cm2 pulse;it was not obviously affected by raising the ambient temperature to 200 C.This early extra influx of calcium was unaffected by the addition of 20 mm-tetraethylammonium to the fluoride perfusion medium, even though underthese conditions the outward potassium current was almost completelyeliminated. Working with longer (6000 Itsec) pulses in 7.7 mm-Ca, theaverage extra influx of 0-0027 p-mole/cm2 pulse was also unchanged by thepresence of tetraethylammonium ions. The extra influx of calcium duringconducted action potentials was determined by Hodgkin & Keynes (1957)as 0-0062 p-mole/cm2.impulse from a solution containing 10-7 mM-Ca and55 mM-Mg and 0-083 p-mole/cm2 .impulse from a solution containing 112mm-Ca and no magnesium.

In some double tracer experiments of the same kind, the influxes of22Na and 45Ca were measured simultaneously. The ratio of the extra sodiuminflux during the pulse to the extra calcium influx increased from 815 to1558 when the depolarizing pulse was lengthened from 50 to 150 ,usec. Thisobservation supports the suggestion of Baker, Hodgkin & Ridgway (1970)that there is an early extra influx of calcium which precedes the turningon of the sodium permeability.

REFERENCES

BAKER, P. F., HODGKIN, A. L. & RIDGWAY, E. B. (1970). J. Physiol. 208, 80P.HODGKIN, A. L. & KEYNEs, R. D. (1957). J. Physiol. 138, 253-281.

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An increase in potassium efflux in human red cells associated withreversing the sign of the membrane potentialBy D. COTTERaELL and R. WHITTAM. Department ofPhysiology, Universityof Leicester

Concentration gradients and membrane permeability determine passivemovements of Na and K across red cell membranes. Another relevantfactor is the membrane potential (p.d.) which is given by the C1 distributionratio, [Cl]i/[Cl]o (Lassen & Sten-Knudsen, 1968; Burton & Jay, 1969). Thisratio has been varied by replacing external Cl with non-penetrating anions,to see how the p.d. influences Na and K movements, which were measuredboth inwards and outwards using tracers.The Cl in Ringer (150 mM-NaCl; 10 mM-KCl) was replaced (10-90 %) by

citrate or EDTA without altering the osmotic pressure. To eliminate trans-port by the Na pump, ouabain was added when K influx and Na effluxwere measured. Ouabain-insensitive (passive) K influx was reduced from0*90 to 0-25 S-equiv . ml.-' hr-1 in cells incubated in low-Cl Ringer (10-30 mM-Cl), whereas ouabain-insensitive (passive) Na efflux was increasedfrom 1-5 to 3-5. Less extensive replacement of external Cl caused smallerchanges in the fluxes. In Ringer containing polyvalent anions instead ofCl, the external Na concentration ([Na]0) was inevitably higher, and Nainflux was raised. Na influx was proportional to [Na]o and independent ofthe nature of the anion. In contrast, there was a striking increase in Kefflux in low-Cl Ringer. Thus, K efflux was raised from 1-4 to 15 whenexternal C1 was replaced by EDTA. This effect was reversible, as K effluxreverted to the normal low value when cells were transferred to normal-ClRinger. Net movements were roughly in accord with the unidirectionalfluxes.To see how these ion movements were dependent onmembrane potential,

measurements were made of the Cl distribution ratio, and the p.d. wascalculated from the Nernst equation. In normal-Cl Ringer, the ratio was0-7 (p.d. -9 mV) but in low-Cl Ringer, with citrate or EDTA replacement,[Cl]1 was greater than [Cl]o and the ratio was maintained at about 3*0,equivalent to a p.d. of 30 mV, the cell interior being positive. Use of theNernst equation requires that Cl is passively distributed, and this pointwas checked by measuring the C1 and H ion distribution in both high andlow-Cl Ringer as a function of external pH. The Cl ratio was inverselyproportional to external pH, and approximately proportional to [H]o/[H]1,as previously found with normal-Cl Ringer by Harris & Maizels (1952).Red cells suspended in non-electrolyte solutions extensively lose K, in a

way probably related to a change in membrane p.d. (LaCelle & Rothstein,1966). Our results suggest further that Na and K movements independent

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of the Na pump depend on the sign and magnitude of the membranepotential, and that the main effect of reversing the sign of the potentialis a marked increase in K efflux and a decrease ofK influx.

REFERENCES

BURTON, A. C. & JAY, A. W. L. (1969). Biophy8. J. 9, 115-121.HARRIS, E. J. & MAIZELS, M. (1952). J. Physiol. 118, 40-53.LACELLE, P. L. & ROTHSTEIN, A. (1966). J. gen. Physiol. 50, 171-188.LASSEN, U. V. & STEN-KNUDSEN, 0. (1968). J. Phy8iol. 195, 681-696.

Membrane potential of perfused and isolated rat liverBY M. CLARET and E. CORABOEUF. Laboratoire de Physiologie comparee et dePhysiologie cellulaire associe an C.N.R.S. Faculty des Sciences, 91-ORSA Y,France

Membrane potentials of in situ liver cells decrease rapidly after asphyxia(Toida, Tamai & Takagisi, 1958; Limberger, 1963; Coraboeuf, Beigelman &Breton 1964) or cyanide injections (Coraboeuf & Claret 1965) suggestinggreat dependence on metabolic activity. Liver cell membrane potentialsalso depend on endocrine state, since they increase after thyroidectomyand decrease after adrenalectomy (Claret, Coraboeuf & Ehrhart, 1966).As it is difficult to study in situ the relation between membrane potential

and external ionic concentrations, experiments were performed on isolatedrat liver perfused with Tyrode at 380 C saturated with a mixture of 97%02 and 3 % CO2. Micro-electrodes were slowly lowered (12 it/sec) into thetissue to a depth of 1-3 mm. During this movement 30-100 cells werepenetrated. The membrane potential was less negative in perfused liver(-33 mV) than when the liver was in situ (-53 mV) but remained stablefor hours. When biliary salts were added to Tyrode solution, the perfusedliver secreted bile in almost normal quantity.A tenfold increase in [K]0 (5.6 to 56 mM) results in a depolarization of

only a few mV when the abnormal medium acts briefly (1 min). This is inagreement with previous results suggesting a small value for PK/(PCl + PNa)(Schanne & Coraboeuf, 1966; Caille & Schanne, 1967). After the K-richmedium has acted for a longer time (10 min) a tenfold change in [K]0results in a fast and much larger potential variation, suggesting an increasein relative PK under such conditions.A decrease in [Cl]o leads to different effects according to the substituting

anion. (a) With Br and methulylsphate the potential does not change (cf.Haylett & Jenkinson, 1969). (b) With NO3 and SCN a large and briefhyperpolarization by 30-50 mV occurs leading to membrane potentials ashigh as -85 mV. These results suggest that Br and methylsulphate are as

e-2

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PPRO7CEEDINGS OF THE

permeable as is Cl, whereas NO3 and SCN are more permeable than is Cl.(c) Acetate, acetylglycinate and pyroglutamate cause a brief depolariza-tion (2-12 mV) that is followed by a slow hyperpolarization (15-25 mV). Ifchloride is re-admitted after the hyperpolarization has developed, afurther hyperpolarization of 10-20 mV occurs before the potential returnsto the initial value. The response to a tenfold change in [K]o reaches35 mV during prolonged perfusion with acetate-Tyrode. (d) With benzene-sulphonate, a brief depolarization (8 mV) is followed by a large hyper-polarization (30-35 mV) during which the response to [K]o changes isincreased while the response to [Cl]o changes is suppressed.An increase in external pH leads to a K-loss and to a large hyperpolari-

zation during which the response to [Cl]o changes is suppressed while theresponse to [K]o changes is increased. This suggests that PK is relativelygreater in alkaline media.

These results indicate that the membrane potential of liver perfusedwith normal Tyrode reflects a high PcI/PK ratio, but the part played byPNa remains to be determined.

REFERENCES

CATLTT, J. P. & ScHANNE, 0. (1967). Proc. Can. Fed. Biol. Soc. 10, 55-60.CLARET, M., CORABOEUF, E. & EHRHART, J. C. (1966). C. r. S6anc. Soc. Biol. 160,

476-479.CORABOEuF, E. & CLARET, M. (1965). J. Phy8iol., Parid, 57, 593-594.CORABOEuF, E., BEIGELMAN, P. M. & BRETON, D. (1964). C. r. hebd. Seanc. Acad.

Sci., Pari8, 259, 2300-2302.HAYLETr, D. G. & JENKINSON, D. H. (1969). Nature, Lond. 224, 80-81.LIMBERGER, J. (1963). Z. Kreb8forech. 65, 590-599.SCHAM-TE, 0. & CORABOEUF, E. (1966). Nature, Lond. 210, 1390-1391.TOIDA, N., TAMAI, T. & TAKAGISI, T. (1958). Kywuhu J. med. Sci. 9, 163-176.

The inhibitory effect of potassium ions on insulin-stimulated sugartransport in rat soleus musclesBY T. CLAUSEN and P. G. KOHN. Institute of Physiology, Arhus University,DenmarkIn muscle and adipose tissue, uptake of the non-metabolized sugar

3-O-methylglucose occurs by a saturable and insulin-sensitive process.However, uptake studies reflect the net result of influx and efflux, and arecomplicated by the need for accurate determination of extracellular space.Since no net accumulation of sugar occurs, influx and efflux may beexpected to be symmetrical. In epidydimal fat pads, the efflux of 3-0-methylglucose (3-0-mg) was found to be accelerated by insulin and anumber of other factors which stimulate the uptake of 3-O-methylglucoseand glucose (Clausen, 1969). We have now obtained similar results with

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PH YSIOLOGICAL SOCIETY, JULY 1970

soleus and extensor digitorum longus muscles from young rats (60-70 g).This communication is concerned with the role of extracellular cations inthe response of soleus muscles to insulin.Chaudry & Gould (1969) demonstrated stimulation of glucose uptake

by insulin into soleus muscles. We have obtained similar results for

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Fig. 1. Effect of extracellular K+ (substitution for Na+) on the stimulationof 14C-3-O-methyl glucose efflux by (a) 100 m-u./ml., or (b) 1 m-u./ml.insulin. Dashed curve shows efflux in absence of insulin. All curves repre-sent mean values from three muscles. Insulin continually present afterthe arrow. K+ altered ten minutes before the addition of insulin.

both glucose and 3-0-mg uptake with a maximum stimulation of two- tothree-fold. The much greater sensitivity of efflux to insulin is illustratedin the figure. Most of this stimulation could be abolished by 5 mm phlor-rhizin. In buffers where LiCl replaced NaCl, stimulation of sugar effluxwas found, as previously shown for uptake in diaphragm and adiposetissue (Bhattacharya, 1964), but the response to insulin was unaffected.KCl replacement was without effect on efflux per se but abolished theinsulin response. Lower levels ofK+ caused graded reductions of the insulinresponse, the sensitivity to K+ being greater for low doses of insulin (Fig.1 a, b). A similar suppression of the insulin response was found usingrubidium, but caesium had only a slight effect. K+-substitution alsopartially suppressed the stimulation of efflux by hyperosmolarity, 2,4-dinitrophenol and muscular work. Thus K+-ions may not only interferewith the action of insulin, but possibly also exert a more direct effect onthe sugar transport system.

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REFERENCES

BHATTACHARYA, G. (1964). Biochim. biophys. acta 93, 644-646.CHAUDRY, I. H. & GOULD, M. K. (1969). Biochim. biophys. acda 177, 527-536.CLAUSEN, T. (1969). Biochim. biophy8. acda 183, 625-634.

Mineralocorticoid action on human and cat-pad eccrine sweatglandsBy K. J. COLLINS, K. G. FOSTER and JUDITH L. HUBBARD. M.R.C.Environmental Physiology Unit, London School of Hygiene and TropicalMedicine, Keppel Street, London, W.C. 1

D-Aldosterone produces sodium retention in human thermal eccrinesweat glands (Collins, 1966), but there have been no similar studies on theeccrine glands of the palm or hairless footpad of mammals. An investi-gation was therefore made of the effects of exogenously administered D-aldosterone on human palmar, forearm and cat-pad sweat electrolytesusing a ventilated capsule method and infra-red water vapour analysis(Foster, 1966). In six subjects, measurements of palm and forearm sweat[Na] and [K] were made before, and at intervals up to 3 days after D-aldosterone (10 /ug/kg). In control determinations [Na] was found to besignificantly lower and [K] significantly higher in palmar than in forearmsweat, the sum of the cation concentrations being equal. D-aldosteronereduced [Na] but had little effect on [K] in both forearm and palmarsweat.In the cat's pad, sweating was induced by stimulation of the internal

plantar nerve (10 V, 1-3 c/s). Intramuscular injection of D-aldosterone(10-100 /,tg/kg) had no action on [Na] or [K] in serial samples of sweatcollected in individual cats up to 10 hr, nor in groups of cats studied atintervals up to 50 hr after injection. Removal of 15% of the cat's bloodvolume produced an effect on urinary electrolytes similar to that withexogenous aldosterone, but no change in sweat electrolytes determined upto 24 hr after haemorrhage. With low stimulation frequencies (< 1 c/s),sweat [Na] is reduced and [K] increased. Aldosterone was again withouteffect on cat-pad sweat produced by low frequency stimulation.The sweat-serum osmolality ratio in the cat-pad secretion is equal to or

slightly greater than 1, that in human sweat is less than 1. Cat-pad sweatalso contains large quantities of bicarbonate (- 60 m-equiv/l.), noammonium and is alkaline, while human sweat usually contains no bicar-bonate, small quantities of ammonium ion ( 4 m-equivl/l.) and is usuallyacidic (Foster, 1966). These differences in composition and the absence ofmineralocorticoid action can be attributed to the lack of a well-differen-tiated duct segment in cat-pad eccrine glands. The cat-pad glands provide

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an experimentally useful preparation for the study of the eccrine secretaryprocess relatively unmodified by ductal reabsorption and cation exchange.

REFERENCES

COLLNS, K. J. (1966). Clin. Sci. 30, 207-221.FOSTER, K. G. (1966). J. Phy8iol. 184, 106-119.

Cortisol turnover in the sheep foetus immediately prior to par-turitionByR. S. CoMLINE, P. W. NATHANIELSZ, R. B. PAISEY and MARIANSILvER.Physiological Laboratory, Cambridge

Gestation in the sheep is prolonged after foetal hypophysectomy(Liggins & Kennedy, 1968) or adrenalectomy (Drost & Holm, 1968) and apronounced rise in foetal plasma cortisol occurs immediately before normalparturition in this species (Basset & Thorburn, 1969). Infusions of adreno-corticotrophin or glucocorticoids into the intact sheep foetus precipitateparturition irrespective of the stage of gestation (Liggins, 1968; 1969).The experiments reported here were designed to investigate the cortisol

levels in foetal and maternal plasma, and also cortisol turnover within thefoetal compartment of the conscious sheep. In each animal a catheter wasinserted through the lateral tarsal vein into the foetal inferior vena cava

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PROCEEDINGS OF THE

under sodium pentobarbitone anaesthesia. Experiments were begun 2-3days after the operation, and observations were continued until parturitionoccurred spontaneously 8-17 days later. Foetal cortisol turnover wasmeasured at intervals during this period. 18 ,uc 3H-cortisol in 2 ml. foetallamb plasma solution (0.9 % (w/v) NaCl containing 5 ml. plasma/100 ml.)was injected through the foetal venous catheter at zero time: foetal andmaternal blood samples were taken at approximately hourly intervalsbetween 150 and 360 min after the injection.

Fig. 1 shows the experimental results from four animals. An approxi-mately eight-fold increase in cortisol turnover occurred during the50 hr preceding normal vaginal delivery (mean = 88 17 + 18*41 4ag/hr,n = 4) compared with the values obtained earlier in gestation (mean =10*94 + 1-19 1ag/hr, n = 7). The more rapid turnover observed immediatelybefore birth is comparable with the rate of infusion of dexamethasonenecessary to induce premature parturition in sheep (Liggins, 1969) in whichthe minimum effective dose given to the foetus was equivalent to 50-100/ug cortisol/hr.

Preliminary observations suggest that very little cortisol escapes fromthe foetal to the maternal circulation under these conditions, either duringthe period of low utilization or at the critical stage just before birth. Thusthe rapid increase in cortisol turnover probably represents a true utilizationby the foetus or placenta or both.

REFERENCES

BASSETT, J. M. & THORBURN, G. D. (1969). J. Endocr. 44, 285-286.DROST, M. & HOLM, L. W. (1968). J. Endoer. 40, 293-296.LIGGINS, G. C. (1968). J. Endoer. 42, 323-329.LIGGINS, G. C. (1969). J. Endocr. 45, 515-523.LIGGrNS, G. C. &EKENNEDY, R. C. (1968). J. Endocr. 40, 371-381.

The response to cardioactive drugs of hearts maintained in organcultureBY K. WILDENTEAL. Strangeways Research Laboratory, Cambridge*To provide a system for studying the heart in vitro for a longer time than

is possible with conventional methods, a technique has been developed formaintaining intact hearts from 19- to 21-day foetal mice in organ culture.Under appropriate conditions the hearts beat spontaneously and rhythmi-cally for 2-4 weeks before dying (Wildenthal, 1970 a, b). To determine theinfluence of long-term culture on the pharmacological responsiveness of

* Present address: University of Texas Southwestern Medical School at Dallas;Dallas, Texas, U.S.A.

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the hearts, the effects of several cardioactive drugs on the rate of beatingwere examined.

Acetylcholine (10-8 to 104 M) caused the sudden onset of bradycardia;the response was dose-dependent and was reversed when the hearts werereturned to control medium. Hearts remained responsive to the drug at allstages of culture, until they died.

Adrenaline and noradrenaline in concentrations of 10-8 to 10-6M causedregular, dose-dependent increases in rate. Levels of 105 M and higher notonly caused an absolute increase in the number of contractions, but alsoled occasionally to the onset of irregular rhythms, including atrial flutteror fibrillation and ventricular bigeminy. Responsiveness to noradrenalineand adrenaline persisted throughout the life of the heart in culture. Incontrast, tyramine (10-6 to 10-3 M) induced tachycardia in hearts main-tained in culture for less than a day, but those cultured for longer than2 days failed to respond.Low doses of ouabain (10-8 to 10-6 M) had no effect on cardiac rate,

although they increased the vigour of contraction; high doses (104 M) oftencaused arrhythmias, particularly ventricular bigeminy and trigeminy. Inaddition to the effect on cardiac rhythm, high levels of ouabain shortenedsurvival in culture.

Triiodothyronine (10-7 to 10-5M) induced tachycardia and occasionalarrhythmias. Its effect on hearts in organ culture, like its action in vivo,was slow in onset; after 2 days' exposure to the hormone the rate of beatingwas not significantly increased, but by 4-8 days it was double the controlrate.The results indicate that mouse hearts maintained in organ culture for

prolonged periods retain normal responsiveness to a variety of cardio-active agents. The ability to respond to an indirectly acting sympatho-mimetic drug such as tyramine is lost after a few days, presumably becausecultured hearts are denervated and lose their noradrenaline stores. Drugsthat act directly on the cardiac cells remain active throughout the life ofthe heart, suggesting that neither damage to receptor sites nor functionaldedifferentiationn' of the myocardium occurs in organ culture.

The author wishes to thank Dame Honor Fell, F.R.S. for her valuable advice andhelp with this study. The work was supported in part by a grant from the UnitedStates Public Health Service.

REFERENCES

WILDENTHAL, K. (1970a). J. Physiol. 207, 33-34P.WILDENTHAT, K. (1970b). J. Molec. Cell. Cardiol. 1, 101-104.

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PROCEEDINGS OF THEEffect of muscle activity on denervation hypsersensivityBY ROSEMARY JONES and GERTA VRBOVk. Department of Physiology,Medical School, Birmingham 15

Following denervation the sensitivity of the muscle to its chemicaltransmitter increases (Brown, 1937). This increased sensitivity is usuallyexplained by the spread of the chemosensitive area outside the end-plateregion (Ginetzinsky & Shamarina, 1942; Axelsson & Thesleff, 1959;Miledi, 1960). These findings led to the suggestion that the nerve exertsa direct desensitizing influence on the extrajunctional area. However,another possibility is that this desensitization occurs as a result of theactivity of the muscle fibre.

If the latter suggestion was correct, then in the absence of the spon-taneous activity displayed by denervated muscle fibres, denervationhypersensitivity would become even greater. Fibrillation activity is thoughtto be initiated at the original end-plate, which acts as a pace-maker (Belmar& Eyzaguirre, 1966). In new-born animals the end-plate is not yet differen-tiated and therefore the possibility that such muscles would not fibrillatewas first examined. The sciatic nerve of 3-day-old rats was sectioned underether anaesthesia, and the rats allowed to recover. At different intervalsafter the operation, electrical activity was recorded from the soleus muscleusing fine concentric needle electrodes. No fibrillation potentials could berecorded from these muscles.Advantage was taken of this finding and the sensitivity to acetylcholine

of denervated muscles of young animals was compared to that of adultdenervated muscles. The soleus muscles were excised and mounted in abath containing oxygenated Krebs-Henseleit solution. Contractions inresponse to acetylcholine added to the bath were recorded. 3 days afterdenervation the sensitivities of both young and adult muscles increased.Later the hypersensitivity of the muscles from young animals continuedto increase. Adult muscles did not show a further increase, after the timewhen fibrillation commenced.A further, more direct, approach was used to study this problem in the

next series of experiments. In adult rats the sciatic nerve was sectionedbilaterally under ether anaesthesia. Two days later, the animals wereanaesthetized with Nembutal and the soleus muscle on one side was directlystimulated by square wave pulses at 40/sec for 1 see every 3 min for 1 hr.Both the soleus muscles were then excised and acetylcholine sensitivitywas tested as described. The stimulated muscles were less sensitive toacetylcholine than the unstimulated controls. Twitch and tetanic tensionsof the stimulated and control muscles were similar. Resting potentials ofstimulated and control muscle fibres recorded using micropipettes filledwith 3 M-KCl also showed no significant difference.

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PHHYSIOLOGICAL SOCIETY, JULY 1970

It is concluded that muscle activity can decrease the sensitivity of themuscle fibre to acetylcholine, and denervation hypersensitivity could bedue to relative inactivity.We wish to thank the Medical Research Council for supporting this work.

REFERENCES

AXELSSON, J. & THESLEFF, S. (1959). J. Physiot. 147, 177-193.BELMAR, J. & EYZAGURRE, C. (1966). J. Neurophy8iol. 29, 425-441.BROWN, G. L. (1937). J. Phy8iol. 89, 436-461.GINETZINSKY, A. G. & SHAMARINA, N. M. (1942). U8p. Sovrem. Biol. 15, 283-294.MIIXDI, R. (1960). J. Physio7/. 151, 1-23.

Excitability of human motoneurones during effortBYA.J.McComAs,R. E. P. SIcAandA. R. M. UPToN.Muscular DystrophyLaboratories, Newcastle upon Tyne General Hospital and Department ofApplied Electrophysiology, National Hospital for Nervous Diseases, QueenSquare, London, W.C. 1 and Maida Vale, W. 9The excitabilities of ac-motoneurones innervating various human muscles

have been studied at rest and during voluntary activity. The method hasbeen to stimulate the nerve to a muscle and to record the different typesof evoked muscle action potential with surface electrodes (cf. Magladery& McDougal, 1950). Thus, while the earliest potential (M wave) resultsfrom direct excitation of motor axons, subsequent potentials denote dis-charges from motoneurones. These late responses have been attributed tomonosynaptic activation of motoneurones by large afferent axons frommuscle spindles (H reflex) and to 'backfiring' from motoneurones followingantidromic invasion (F wave). These two responses can be distinguishedby raising the stimulus strength; the F wave persists when the stimulus issupramaximal for the M wave, whereas the H reflex disappears, due tocollision between antidromic and reflexly elicited impulses in the motoraxons.

In the present study we have found that, in relaxed subjects, responseswith the characteristics of F waves could be recorded from the abductorpollicis brevis (APB), first dorsal interosseus of the hand, abductor digitiminimi and tibialis anterior muscles. The amplitudes of these wavesfluctuated but never exceeded 6% of those of the maximal M waves; Hreflexes could not be elicited. During voluntary contraction the F wavesbecame greatly enlarged in all the muscles tested, indicating increasedexcitabilities of the corresponding motoneurones. The potentiation of theF wave was always much more marked in APB; furthermore, duringvoluntary contraction of this muscle, an H reflex could be induced. Thisincrease in the excitabilities of APB motoneurones could still be detected

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when the subject 'willed' a movement but did not actually contract themuscle.Although the amount ofF wave potentiation in APB varied in different

individuals, there was good correspondence on the two sides in all thetwenty-two normal subjects studied. Therefore the possibility was exploredthat differences between the potentiated F waves on the two sides mightoccur in patients with relatively mild unilateral lesions of descending path-ways to motoneurones. In all but one of fourteen such patients a significantreduction in F wave potentiation could be detected in the APB of theaffected limb, even if no weakness was demonstrable.

REFERENCE

MAGLADERY, J. W. & McDOUGAL, J. B. JR. (1950). Johns Hopkins Hosp. Bull. 86,265.

Energy balance during working contractions of frog muscleBY CLAUDDE GILBERT and M. J. KUSHMERICK. Department of Physiology,University College London, Gower Street, London, W.C. 1The total energy output (heat+ work) appears accountable in terms of

net phosphocreatine (PCr) hydrolysis in complete cycles of contractileactivity of frog skeletal muscle at 00 C (Wilkie, 1968). That this relation-ship may not hold at each moment during the contraction was stronglysuggested by the finding that chemical change was small in contractionswhere no mechanical work was performed (Kushmerick, Larson & Davies,1969). In these experiments the production of heat could only be inferred:but the conclusion was borne out by others in which the heat was measureddirectly (Gilbert, Kretzschmar, Wilkie & Woledge, 1970). It was foundthat very little break-down of PCr occurs during the first second or two ofan isometric tetanus, despite the fact that a considerable quantity of heathad been evolved.The aim of the present experiments was to confirm on muscles poisoned

with iodoacetate, and with concomitant heat measurements, the con-clusion that had been arrived at from experiments on DNFB poisonedmuscles, namely that when work is performed this is accompanied bychemical change.Frog muscles poisoned with IAA (0.5 mm, 45 min) and N2 at 00 C were

tetanized for 1l- sec. The Levin Wyman ergometer to which they wereattached was released at 0 1 sec, the speed having been adjusted previouslyto give optimal work performance. The chemical changes were estimatedby comparison with an unstimulated paired muscle. In some experimentsthe heat production was measured, using an integrating thermopile (Wilkie,1968); in others from the same batch of frogs the early chemical changes

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were examined using the 'hammer' quick freezing apparatus (Kretzschmar& Wilkie, 1969) to interrupt activity at the chosen moment. The followingresults were obtained:

(1) The PCr split was much greater than at the same moment in anisometric contraction. Six to seven kcal of work were associated with eachmole of PCr split.

(2) The PCr splitting and production of inorganic phosphate were equal.(3) The total energy output (heat + work) measured 60 see after the

contraction, amounted to 14 kcal/mole.(4) The mean change in ATP was a slight increase, but this was not

enough to be statistically significant.

REFERENCESGILBERT, CLAUDE, KRETZSCHMAR, K. M., WILKIE, D. R. & WOLEDGE, R. C. (1970).

J. Phyaiol. 207, 15-16P.KRETZSOEMAR, K. M. & WIxn , D. R. (1969). J. Phy8iol. 202, 66-67P.KuS~mEmRIcK, M. J., LARSON, R. E. & DAVIES, R. E. (1969). Proc. R. Soc. B 17,

293-313.WnImE, D. R. (1968). J. Phypiol. 195, 157-183.

The relationship between the external calcium concentration andthe contracture tension developed by auricular trabeculae isolatedfrom the heart of the frog, Rana pipiensBy R. A. CIHPMAN and J. TUNSTALL. Department of Physiology, Universityof LeicesterFrog auricular trabeculae (40-100 It diameter) develop contractures in

Ringer containing 20-200 mm additional KCl; tension rises, and falls onreturn to normal K Ringer with a tj of about 3 see (for method see Chap-man & Tunstall, 1969). The magnitude of these contractures, evoked byshort applications of perfusion fluids containing a constant high [K] showsa sigmoid dependence on the calcium concentration in these solutions([Ca]o), suggesting that a power relationship might exist between the[Ca]. and the isometric contracture tension (Cmax).LUttgau & Niedergerke (1958) proposed a model for frog cardiac muscle

in which Ca ions activating contraction first compete for a receptor mole-cule, R, within the membrane of the muscle fibres, i.e.Na2R + Ca = CaR + 2Na. Presumably the number of receptor molecules

is finite, so that the proportion of the complex, as CaR, (F) is given byequation (1).

F- [Ca]oKCa (1)[Na]2+[Ca]oKca(where Kca is the equilibrium constant.

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PPPROCEEDINGS OF THE-

If R has several identical anionic sites that can combine with Ca ions,and the tension developed during a contracture is related directly to theproportion of R molecules saturated with Ca2+, then equation (1) becomes

C a ( [C] Ca )n(2)Cmax = [Na]2 a)(2

where ax and w are proportionality constants and n is the number of Ca ionsin the complex Ca. R.At [Ca]o below 1 mm, the log Cmax is related to log [Ca]. by a straight

line of slope+2*58 (S.D. 0.28). A plot of 1I/tCmax against L/[Ca]o gives astraight line to provide a value of 2-27 mM (S.D. 1.58) for Kca when half theR complex is saturated with Ca2+. Reduction of the [Na] in the perfusingmedia does not alter the slope of line relating log Cmax to log [Ca]0, but thevalue of the affinity constant for Ca2+ obtained from the double reciprocalplot is reduced (in 81-6 % of the normal [Na] the affinity constant is 1-32MM (S.D. 0-82)).

If the force generated by the contractile apparatus is directly related tocalcium concentration in the sarcoplasm ([Ca],), as assumed by Ebashi,Endo & Ohtsuki (1969), then our results suggest that the [Ca], establishedduring a high-K contracture depends on [Ca]3. It seems likely that most ofthis ionic Ca originates from outside the muscle cells, because contrac-tures are immediately abolished by removal of external Ca2+, and frogcardiac muscle has no T-system and little sarcoplasmic reticulum (Staley &Benson, 1969). A possible hypothesis is that during depolarization thesarcolemma releases Ca ions into the sarcoplasm in proportion to [Ca]3 inour model by dissociation of the Ca3R complexes. If, on the other hand,during high-K contractures, [Cali depends directly on [Ca]o (R combiningwith a single Ca ion), then the degree of activation of the contractileprocess should depend on [Ca]3.

REFERENCES

CHAPMAN, R. A. & TUNSTALL, J. (1969). J. Phyaiol. 201, 9-11P.EBASHI, S., ENDO, M. & OHTSUKI, I. (1969). Q. Rev. Biophy8. 2, 351-384.LUTTGAU, H. C. & NIEDERGERKE, R. (1958). J. Physiol. 143, 486-505.STALEY, N. A. & BENSON, E. S. (1969). J. cell Biol. 38, 99-114.

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The passage of amino acids into the rat's brain

By GUADELUPE BANOS, P. M. DANIEL,* S. R. MOORHOUSE and 0. E.PRATT.* Department of Neuropathology, Institute of Psychiatry, De Cres-pigny Park, London, S.E. 5

The brain is able to synthesize some of the amino acids which it requiresbut must obtain others from the blood stream. Little is known of the wayin which amino acids enter the brain in the living animal. We have investi-gated the entry of 23 amino acids into the brain, using more than 500 ratsaged 8-9 weeks.Under ether anaesthesia we gave a continuous intravenous infusion

composed either of a single amino acid radioactively labelled with 14C,35S or 125I and of relatively high specific activity, or of a mixture of thelabelled material with different quantities of the same amino acid un-labelled. Small blood samples were obtained at regular intervals throughoutthe experiment. At the end of the infusion the vascular system was per-fused with Ringer solution. Finally, the brain was removed and assayedfor radioactivity. The rates of entry were usually constant throughout theexperimental period but varied over a wide range for the amino acidsstudied, which included most of those usually found in protein, as well asthyroxine and also two amino acids not normally found in animal tissue(2-aminoisobutyric acid and DL-2-aminoadipic acid). The rates of entry ofamino acids, calculated as the radioactivity entering the brain per minute(expressed as a fraction of the radioactivity in an equal weight of theanimal's blood plasma), were highest for L-phenylalanine (0.0569 min-')and L-leucine (0.0395 min') and lowest for L-thyroxine (0.00059 min') andthe non-biological amino acids (e.g. DL-2-aminoadipic acid, 0-0016 min-').For amino acids which had a high rate of entry the experiments were

repeated with a similar quantity of the radioactively-labelled amino acidwith which increasing amounts of the non-radioactive amino acid had beenincorporated, a constant and abnormally high concentration ofblood aminoacid of known specific activity being maintained. As the amount of non-radioactive amino acid used was increased the rate of its entry into thebrain, as indicated by the radioactive material found there at the end oftheexperiment, did not increase in proportion to the rise in concentration ofthe amino acid in the blood but tended eventually to remain constant inspite of increasing blood concentrations. This indicated that carriermediated transport processes, only able to handle limited amounts ofamino acid, rather than passive diffusion, play a major role in the entryof these substances into the brain.

* Assisted by grants from the Research Fund of the Bethlem. Royal and theMaudsley Hospitals. the Medical Research Council and Roche Products, Ltd.

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PROCEEDINGS OF THEElectrical connexions between cells at neural stages of theaxolotlBY ANNE E. WARNER.* Department of Biology, The Middlesex HospitalMedical School, London W 1P 6DB

During the development of the axolotl, Amblystoma mexicanum, thecells destined to form the nervous system can be identified soon aftergastrulation as an oval area of ectoderm, the neural plate (develop-mental stage 13, Harrison, quoted by Rugh, 1962). The neural plate thenfolds in and rolls up to form a tube, the ectoderm originally adjacent to theneural plate meeting at the mid line to form a sheet lying over the neuraltube (stages 14-21). In the present investigation the electrical properties ofpresumptive nerve cells and ectoderm have been compared at thesedevelopmental stages.Embryos were stripped of jelly, capsule and vitelline membrane and

cultured in Holtfreter's solution at pH 7-4 (Holtfreter, 1943). Membranepotentials and input resistances were measured with K-citrate filled micro-electrodes in surface cells of the neural plate and ectoderm.

Neural plate cells (10-20 ,z in diameter) had membrane potentialsranging from -10 to -60 mV (mean - 35-2 mV + 1.2 S.E.M., n = 128).Cells in the surrounding ectoderm (20-30 It in diameter) had potentialsfrom -12 to -56 mV (mean - 30'6 mV + 1-5 S.E.M., n = 59). The truemean values for the membrane potentials probably lie closer to the topend of the recorded ranges because the cells are small, and thereforesensitive to damage by the electrodes. Input resistances were measuredwith the current-passing and voltage-recording micro-electrodes 10-20 Iapart in the neural plate, so that on occasion the electrodes were in adjacentcells rather than the same cell, and came to 5-2 x 106 Q + 1*14 x 106 (mean+ S.E.M., n = 34). In the ectoderm the interelectrode distance was about25 It and the input resistance was 9 3 x 106 Q + 1*4 x 106 (mean + S.E.M.,n = 32).

Before complete closure of the neural tube (up to stage 20) currentinjected into one cell spread throughout the neural plate and ectoderm.Calculations using a solution of the cable equations for current spreadfrom a point source into a flat sheet (Woodbury & Crill, 1961; Noble,1962) suggested that the space constant, A, was about 1 mm. Neural platecells were also coupled to the underlying notochord, as reported bySheridan (1968) in the chick embryo.Once the neural tube had closed, current injected into an ectodermal

cell lying over the neural tube no longer spread down into the presumptivenervous tissue; the cells had become electrically uncoupled from each

* Supported by the Medical Research Council.

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other. At about the same time electrical contact between the ectodermalcells originally adjacent to the neural plate was established across themid line.

REFERENCES

HOLTFRETER, J. (1943). J. exp. Zool. 193, 251-323.NOBLE, D. (1962). Biophys. J. 2, 381-393.RUGH, R. (1962). Experimental Embryology, 3rd edn. Minnesota: Burgess Publishing

Co.SHERIDAN, J. S. (1968). J. cell Biol. 137, 650-659.WOODBURY, J. W. & CRILL, W. E. (1961). In Nervous Inhibition, ed. FLOREY, E.

pp. 124-135. New York: Pergamon.

Natriuresis in rabbits acutely deprived of waterBY R. 0. LAw. Department of Agricultural Science and Applied Biology,University of CambridgeSodium balance has been examined in rabbits deprived of water and

offered a relatively dry (11 % water) pellet diet containing sodium (47.8m-equiv/kg), since it has been suggested that renal conservation of sodiumis poor in this species, which normally keeps in sodium balance by matchingoutput with dietary intake (Cizek, 1961).Twenty-seven adult male rabbits were subjected to alternating 4-day

periods of water deprivation and rehydration (water freely available).Body weight, urinary volume, food and water intake, haematocrit andplasma protein concentration, plasma and urinary sodium, potassium andosmolality were measured daily. Each animal acted as its own control.During water deprivation haematocrit and plasma protein concentrationboth rose (range of increase 7-14 %), indicating a contraction of plasmavolume. Plasma sodium and osmolallty, however, remained constant.This is in direct contrast to what has been observed in man (Black,McCance & Young, 1944) and the dog (Elkington & Taffel, 1942). Theurinary output ofsodium and the urine osmolality both increased. Allowingfor the dietary intake (which declined sharply during dehydration) the totalnet loss ofsodium during four days' water deprivation was within the range4-7 m-equiv/kg body weight. The urinary output ofpotassium decreased inproportion to the decreased dietary intake of potassium, the plasma levelremaining constant.The rabbit therefore maintains constancy of plasma sodium during

dehydration, while at the same time there is a marked decrease in plasmavolume. Since the latter is generally regarded as a stimulus to reninrelease, and since Langford & Pickering (1965) have shown that large dosesof synthetic angiotensin are natriuretic in the rabbit, the possibility of arelationship between the dehydration-induced natriuresis and activation

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of the renin-angiotensin system was examined. Assay of arterial plasmasamples (Regoll & Vane, 1964) from control and dehydrated rabbits didnot, however, show any significant increase in the circulating level ofangiotensin during dehydration. Nor was there any increase in thegranulation of the juxtaglomerular cells in sections stained by Bowie'smethod. Rat blood pressure bio-assay indicated an increased renin levelin the plasma of dehydrated rabbits. Examination of 230 maculae densaein sections (H & E stained) from control and dehydrated animals showeda highly significant difference (P = < 0.001) between the mean heightsof the macula densa cells from the two groups. The height of the maculadensa cells of the control animals exceeded the height of the cells in theopposite wall of the same tubule by 6x74 + 0x42 itl (s.E.M.), whereas thefigure in dehydrated animals was 3-25 + 0 53 ,u. These figures suggest anelevated sodium concentration in the fluid bathing the maculae densaeof dehydrated animals (Reeves & Sommers, 1965).

REFERENCESBLAoK, D. A. K., MOCANCE, R. A. & YOUNG, W. F. (1944). J. Phy8iol. 102, 406-414.CiEK, L. B. (1961). Am. J. Phyaiol. 201, 557-566.ELKINGTON, J. B. & TAFFEL, M. (1942). J. din. Invest. 21, 787-794.LANGFORD, H. G. & PICKERING, G. W. (1965). J. Physiol. 177, 161-173.REEVES, G. & SOMMERS, S. C. (1965). Proc. Soc. exp. Biol. Med. 120, 324-326.REGOLI, D. & VANE, J. R. (1964). Br. J. Pharmac. Chemother. 23, 351-359.

Interactions of intracranially administered renin or angiotensinand other thirst stimuli on drinkingBY J. T. FITZsimOws. The Physiological Laboratory, University of CambridgeThe rat deprived of its endogenous supply of renin by nephrectomy

drinks less after some extracellular stimuli to thirst than normal animalssubjected to the same procedures (Fitzsimons, 1969). The smaller intakeafter nephrectomy is not attributable to anuria, for the response by non-nepbrectomized animals to extracellular stimuli is not diminished byureteric ligation. Single injections of renin (Nutritional BiochemicalsCorporation), valine5 angiotensin II aide (Hypertensin, CIBA) orisoleucine5 angiotensin II amide (generously given by Dr Irvine Page) intothe septum, pre-optic or anterior hypothalamic areas of the rat causedrinking of water (Epstein, Fitzsimons & Simons, 1969). The smallestdose found to produce a detectable response in water-replete animals is 5 ng.One of several possible ways in which the renin-angiotensin system may

intervene in normal thirst is by sensitizing the part of the central nervoussystem concerned in drinking to the action of other thirst stimuli. Thiswas tested by subjecting rats with chronic intracranial cannulae implanted

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in the angiotensin sensitive region to the combined action of a centralstimulus, consisting of renin or angiotensin, and of a peripheral stimulus tothirst. In one experiment a single injection of 1, 5 or 50 ng of angiotensinII dissolved in 1#1 of water was injected through the cranial cannula15 min after the intraperitoneal injection of 0 5 ml. 2 M-NaCl, a cellularstimulus to thirst. This amount of saline increases the osmolality of thebody fluids by 2-0-3-5 %. The rat was then allowed to drink. The combina-tion of angiotensin and hypertonic saline caused more drinking thanfollowing either stimulus presented alone. Intracranial angiotensin in thesame doses augmented the water intake of rats made thirsty by injecting2 ml. of 20% (w/w) of polyethylene glycol dissolved in 0.9% NaCl intra-peritoneally 3 hr previously. Polyethylene glycol is an extracellularstimulus to thirst since it causes gradual sequestration of extracellularfluid in the peritoneal cavity (Fitzsimons, 1961). Intracranial renin (10m-u.) caused additional drinking by rats injected 15 min previously with2*5 ml. 2 M-NaCl; intracranial renin also caused additional drinking in ratsthat had been deprived of water for 24 hr and then injected with reninjust before being allowed to rehydrate.

In all cases the combined stimulus produced more rapid drinking andlarger intakes of water than were produced by the component stimuli andthis was not attributable to differing effects on excretion. In general therewas simple addition of the effect of the central stimulus and of the peri-pheral stimulus when both stimuli were presented together. After verylarge stimuli there was some occlusion of response. When both stimuli werejust less than threshold, neither stimulus alone producing drinking, thecombined stimulus caused drinking.

REFERENCES

EPSTEIN, A. N., FITZSIMONS, J. T. & SIMoNs, BARBARA J. (1969). J. Physiol. 200,98-100P.

FITZSIMONS, J. T. (1961). J. Physiol. 159, 297-309.FITZSIMONS, J. T. (1969). J. Physiol. 201, 349-368.

Characteristics of the (Na+ + K+)-stimulated ATPase of rat jejunumBy MARIE MULLANEY CASSIDY. Department of Physiology, UniversityCollege, Dublin

Several specialized sodium transport mechanisms exist in small intestinalepithelium. These processes are involved in the maintenance of ionicgradients and cell volume, the promotion of volume flow across the tissueand the transepithelial active transport of other ions and organic solutes.It has been suggested that this functional diversity is primarily mediatedby the asymmetric distribution within the epithelium of the sodium trans-

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1PROCEEDINGS OF THEport mechanism common to all cells and identified with the presence of a(Nal + K+)-activated ATPase (Schultz, 1969). Rat intestinal epitheliumpossesses such an enzyme (Robinson, 1970) and it was considered of in-terest to examine the effects on 'transport' enzyme activity of severalfactors which are known to alter the sodium transporting characteristicsof this tissue.The preparation chosen for study was a relatively crude homogenate of

rat jejunal mucosa, handled in the cold. With fresh material, values of40*9 + 1*2 n-moles ATP/mg protein and (Na+ + K+) ATPase levels of88-5 + 17*5 n-moles P/min .mg protein were obtained. At 40 C there was aprogressive rise in transport enzyme activity with time as has been foundrecently by Robinson, 1970. A similar enhancement of activity occurredfollowing repeated freezing and thawing.

TABLE 1Enzyme

(Na+ + K+)ATPase activity(n-moles P/min.mg as % of

Incubation conditions protein) control

Control 370C 219.0±10-9 100270C 37.1+ 7.7 17

370C+1 mg/ml. phosphotidylserine 241.0 ±83 110370 C+0 1 mm ouabain 81*8±15.3 37370 C+0 1 mm scillaren 33.6±7*9 15370 C+ 1 mm ethacrynic acid 60-4 ± 3.3 28

Tissue used was a mucosal homogenate from the middle fifth of the combinedileo-jejunum of the rat, 'aged' by storage at 40 0 for 9 days. Values shown representmean + S.E. for seven to ten animals.

The effect of several factors on the activity of the aged preparation isshown in Table 1. A small increase was noted with phosphotidylserine,consistent with reports of a unique phospholipid requirement for thissystem (Fenster & Copenhaver, 1967; Wheeler & Whittam, 1970). Whencompared within a group of eight animals all other factors tested evoked ahighly significant depression of enzyme activity (P < 0.001).

These results indicate that the effect of these manipulative tools onsodium linked transport phenomena in rat jejunum must be mediated atleast in part by their effects on the (Na++ K+)-stimulated ATPase of thetissue.

REFERENCESFENSTER, L. J. & COPENHAVER, J. H. (1967). Biochim. biophy8. Acta 137, 406-408.ROBINSON, J. W. L. (1970). J. Phy8iol. 206, 41-60.SCHuTZ, S. C. (1969). In Biological Membranes, ed. DOWBEN, R. M. Boston,

Massachusetts: Little Brown & Co.WHEELER, K. P. & WH=Am, R. (1970). J. Phy8iol. 207, 303-328.

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PHYSIOLOGICAL SOCIETY, JULY 1970Receptive properties of centrifugal cells projecting to the pigeonretinaBY A. L. HOLDEN. Institute of Ophthalmology, Judd Street, London, W.C. 1Output cells in the isthmo-optic nucleus, the origin of centrifugal fibres

running to the retina, were identifiedby antidromic activation. Stimulatingelectrodes were advanced to the isthmo-optic tract; extracellular singleunit records were taken with micropipette electrodes. The pigeons wereanaesthetized with urethane.Entry to the nucleus was detected by the abrupt appearance of the

antidromically evoked field potential, the A-wave (Holden, 1968), and wasconfirmed by subsequent histology.

Receptive fields were plotted for ninety-four cells, with visual stimuliprojected in the lateral visual field. The field outlines were circular orelliptical and ranged in extent between 50 and 200. Within this area a 10spot of light produced a phasic burst of spikes at 'on' and 'off'. Thelatency of 'on' firing to a 10 spot of 1-4 log ft. Lamberts was typically50-65 mseo. Thus at each presentation a brisk centrifugal message passesto the retina. Spontaneous firing was not observed to alter markedly atdifferent levels of background illumination.

All cells responded vigorously to moving stimuli, often at a lowerthreshold than for stationary spots. 65% were classed as non-directionallyselective, responding equally to all directions of movement through theirreceptive fields. 5 % were classed as directionally selective, showing a clear'null' direction. This was in each case for posterior movement. 30% showeddirectional selectivity of an intermediate kind, responding in all directions,but giving a minimal response in one direction. The minimal direction wasagain for posterior movement.The topographical organization of the nucleus shown anatomically by

McGill, Powell & Cowan (1966) was repeatedly observed. In the plane ofentry used in these experiments the first cells encountered served loca-tions in the lower quadrants. Successive units deeper in each track servedmore anterior and superior locations in visual space.

REFERENCES

HOLDEN, A. L. (1968). J. Phy8iol. 197, 183-198.McGILL, J. I., POWER~, T. P. S. & CowS, W. M. (1966). J. Anat. 100, 5-33.

Topographic distribution of pigment in the human lensBY J. MELLERIO. Department of Physiological Optics, Institute of Ophthal-mology, Judd Street, London W. C. 1H 9QS

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PROCEEDINGS OF THE

On the polar and equatorial elasticities of the human crystallinelensBY R. F. FISHER. Department of Physiological Optics, Institute of Ophthal-mology, Judd Street, London W.C. 1H 9QS

Formation of isorhodopsin in isolated frog retinae by intensenanosecond flashesBY CH. BAUMANN and W. ERNST.* Kerckhoff Institut der Max-Planck-Gesellschaft, 635 Bad Nauheim, Germany

Isolated retinae of Rana esculenta were irradiated by 2 ns flashes of530 nm light from a 'Febetron 706' used with a cadmium sulphide platelet(Field Emission Corporation, U.S.A.). Spectra of apparent optical densitybefore and after the flashes and following a full bleach were obtained asdescribed by Baumann (1970) and were used to determine how muchrhodopsin the flashes bleached. Spectra after the flashes showed a slightshift to shorter wavelengths, presumably because of the photogenerationof isorhodopsin. The fraction of isorhodopsin was estimated by the methodof Bridges (1961).Two flash intensities were used. The weak one bleached 23-9% of the

rhodopsin present. If multiple absorptions are neglected (ca. 4 %), Poissonstatistics (Williams, 1964) show that the mean number ofphotons absorbedper molecule from a weak flash was 0 35 and hence 0 70 from a strong flash,since it was double the intensity. A figure of 0-86 for strong flashes wasindependently derived from data on frog retina and specifications ofplatelet performance. Both values were used to calculate the proportionsof zero, odd- and even-numbered absorptions from a strong flash, whichwere within experimental error of the fractions of unbleached rhodopsin,bleached rhodopsin and isorhodopsin, respectively (Table 1). Such agree-ment strongly suggests that the photosensitivity of the product formedfrom rhodopsin is close to that of rhodopsin at 530 nm and that all thematerial produced by even-numbered absorptions is isorhodopsin. Varyingthese assumptions leads to inconsistencies between the fraction ofrhodopsinbleached and the fraction of isorhodopsin. An alternative hypothesis isthat the observed wave-length shift does not result solely from the forma-tion of isorhodopsin. However, all explanations imply that the photo-product in existence during our 2 ns flash does not behave like any of thoseidentified below 00 C (Hubbard, Bownds & Yoshizawa, 1965).

* Present address: Department of Neurophysiology, Institute of Ophthalmology,Judd Street, London W.C. 1

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TABUS 1Fraction of

Fraction of Fraction of rhodopsinrhodopsin rhodopsin converted

Experiment unbleached bleached to isorhodopsin1 0*400 0*437 0*1632 0*454 0*394 0-1523 0*468 0-402 0-1304 0.399 0405 0-196

Mean 0-431 0 409 0-160

Mean numberof photons Fraction of odd- Fraction of even-absorbed per Fraction of zero numbered numberedmolecule absorptions absorptions absorptions

0-70 0-498 0 375 0*1270-86 0*424 0*410 0 166

REFERENCES

BAUMANN, CR. (1970). Vision Res. (in the Press).BRIDGES, C. D. B. (1961). Biochem. J. 79, 135-143.HUBBARD, R., BOWNDS, D. & YOSHIZAWA, T. (1965). Cold Spring Harb. Symp.

quant. Biol. 30, 301-315.WILLIAMS, T. P. (1964). J. gen. Physiol. 47, 679-689.

Retinal disparity and retinal dominance of binocular corticalneuronesBY COLIN BLAKEMORE* and JOHN D. PETTIGREW. The NeurosensoryLaboratory School of Optometry, University ofCalifornia, Berkeley, California94720, U.S.A.

Binocular neurones in the cat's visual cortex respond to similarlyorientated targets shown to either eye (Hubel & Wiesel, 1962), or betterstill to both (Barlow, Blakemore & Pettigrew, 1967). The two eyes arecommonly not equally effective in driving a binocular cell and more oftenthan not the contralateral eye is dominant (Hubel & Wiesel, 1962;Blakemore & Pettigrew, 1970).

It is also said that binocular neurones do not necessarily have theirreceptive fields on exactly corresponding retinal points. The horizontalretinal disparity of the centres of the pairs of receptive fields varies overseveral degrees and therefore different cells should respond to objects atdifferent distances from the eyes (Barlow et al. 1967; Nikara, Bishop &Pettigrew, 1968; Blakemore, 1970).

* Present address: The Physiological Laboratory, University of Cambridge,Cambridge CB2 3EG, England.

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PROCEEDINGS OF THE

1041

54.,

disparityar1 t

2~~~~~~~~~~~~~~~

03

~~~~~~~~~~iegntere3

r ~~~ ~~~~~~~~~~ru

+1°~~~~.disparity diparit

Fig. 1. In this isometric plot of retinal disparity as a function of eye domi-nance each block represents a single, reliably plotted binocular cortical cell.Dominance groups 2 and 3 are, respectively, strongly and mildly dominatedby the contralateral eye: groups 6 and 5 are, respectively, heavily andweakly dominated by the ipsilateral retina. Group 4 units are equally in-fluenced by both eyes. (Groups 1 and 7 are monocular).

The upper set of histograms shows the distributions of horizontal dis-parity of the receptive fields broken down according to dominance groups.The data from thirteen cats are pooled by superimposing the mean hori-zontal disparity for each animal. All five histograms are added together toproduce the histogram in the middle of the figure. The scale of disparityon this histogram is also appropriate to the other five, zero being the mean,more convergent disparities lying to the left and more divergent ones to theright.The total number of blocks in each of the five individual histograms is

added to produce the distribution of eye dominance on the right. This showsthe tendency for the contralateral eye to dominate more often than theipsilateral.The lower set of five histograms analyses the data in exactly the same

way for vertical disparity. The histograms are added to produce the totaldistribution at the bottom of the figure. Notice that the total range ofhorizontal disparity is about three imes that of vertical and that there isno tendency for units with extreme retinal disparities to belong to extremedominance groups.

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We considered the hypothesis that units in the extreme eye dominancegroups (see Fig. 1), heavily dominated by one of the eyes, have receptivefields with large retinal disparities compared with units of group 4. Oneunfortunate corollary of this hypothesis might be that the observedvariation in disparity is largely due to errors of measurement, because it isdifficult to locate accurately a weak receptive field in the non-dominanteye.We re-analysed 139 binocular neurones from thirteen paralysed cats,

anaesthetized with nitrous oxide (Barlow et al. 1967). The eyes were suturedto rings. We plotted the receptive fields, calculated their disparities andestablished the eye dominance groups. In Fig. 1, histograms, for each eyedominance group, show the distributions of horizontal and vertical dis-parity, relative to the over-all means. There is simply no evidence thatunits with extreme retinal disparity, either horizontal or vertical, belongmore often to extreme eye dominance groups.We thank Professor H. B. Barlow for help and for support from his U.S. Public

Health Service grant (No. NINDB 05215).

REFERENCESBARLOW, H. B., BLAXKMORE, C. & PETIGREW, J. D. (1967). J. Phy8iol. 193,327-342.BTLAKEMOEx, C. (1970). J. Phyeiol. 209, 155-178.BLAKEMORE, C. & PETTIGREw, J. D. (1970). Nature, Lond. 225, 426-429.HUIBEL, D. H. & WIESEL, T. N. (1962). J. Phy8iol. 160, 106-154.NIKARA, T., BISHOP, P. 0. & PEIGREW, J. D. (1968). Expl Brain Rem. 6, 353-372.

Body temperature in exerciseBY CAROLYN BARNES and C. T. M. DAVIES. Medical Research CouncilEnvironmental Physiology Unit, London School of Hygiene and TropicalMedicine, University of London, Keppel Street, London, W.C. 1

It is well known that the body temperature rises in exercise but themechanisms underlying this phenomenon are still a matter of somedebate (cf. Nielsen, 1938, Benzinger, 1959; Bradbury, Fox, Goldsmith &Hampton, 1964). It has been suggested that the increase of body tem-perature in exercise is more closely related to relative (i.e. as a % ofmaximum aerobic power; I'>mmax) rather than absolute work performed(Saltin & Hermansen, 1966), but independent of the muscle mass involved(Nielsen, 1969). The effects of habituation to work and acclimatizationheat on these latter relationships have not been reported.

In the present study, we have measured the sweat rates, body tem-peratures and oxygen intakes (rO2) of two healthy male subjects-anathlete and non-athlete-during exercise of 1 hr duration. The subjectsworked with the arms, the legs, the arms and legs combined on a stationary

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PROCEEDINGS OF THEbicycle ergometer and walked and ran uphill on a motor driven treadmillover a wide range of exercise intensities before and after a period ofhabituation to work, and acclimatization to heat. Body temperature (Tty)was measured with a thermistor in the external auditory canal close tothe tympanic membrane; sweat rates were calculated from changes inbody weights and fo, determined by the standard Douglas bag technique.A total of 72 1 hr experiments were performed.

Analysis of the results showed that during the first few occasions ofmeasurement in the sedentary (but not in the athletic subject), the relation-ship of Tty to tO, was curvilinear rather than linear (P < 0 001) and inboth subjects Tty was more closely related to relative, rather than absolutework load. However, during the period of habituation this relationshipbegan to change and following acclimatization the reverse was true.Throughout the period of the investigation in the sedentary subject Tt.for a given 'Y02 of 2 5 I/min (Tty 2.5) fell by 1.010 C. The sweat rate rose butthe directly measured 41i2max increased by only 5 %. In the athelete all thesefour parameters remained relatively constant. In both subjects, the changein exercise Tty was independent of the muscle mass involved in the work.

It would appear that the thermoregulatory system can be trainedindependently of maximum aerobic power and in subjects habituated andacclimatized to both work and heat, Tty related more closely to absoluteenergy expenditure. Further, the results suggest that man's temperatureregulation in exercise is more a resultant of his ability to dissipate ratherthan to produce heat.

REFERENCES

BENZINGER, T. H. (1959). Proc. natn. Acad. Sci. U.S.A. 45, 645-659.BRADBURY, P. A., Fox, R. H., GOLDSMITH, R. & HAMPTON, I. F. G. (1964). J.

Phy8iol. 171, 384-396.NIELSEN, B. (1969). Acta physiol. 8cand. supply. 323.NIELSEN, M. (1938). Skand. Arch. Physiol. 79, 193-230.SALTIN, B. & HERmANSEN, L. (1966). J. apple. Physiol. 21, 1757-1762.

Effect of pyrogen injected into unanaesthetized rabbits duringperfusion of the cerebral ventricles with solutions of different ioniccompositionBy W. FELDBERG and P. N. SAXENA. National Institute for MedicalResearch, Mill Hill, London N. W. 7

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Interaction between peripheral temperature receptors and centralneurones responding to brain temperatureBY R. F. HELLON. National Institute for Medical Research, London, N. W. 7The role of the hypothalamic temperature sensors in the regulation of

body temperature is not yet known. It has been shown that a temperaturechange in the hypothalamus is not a necessary accompaniment to vigorousthermoregulatory responses such as panting and shivering (Forster &Ferguson, 1952; Hammel et al. 1964). Hammel (1965) has suggested thatperipheral temperature sensors modulate the firing of the hypothalamicsensors so as to change their activity at the prevailing brain temperatureand so provide a signal to initiate the thermoregulatory response. Thishypothesis has been tested in experiments on rabbits and cats anaesthe-tized with urethane.Neurones were selected whose firing rate could be driven by imposed

changes in hypothalamic temperature (Thy) while the external air tempera-ture (Ta) was held at 250 C. Ta was then lowered to 100 C or raised to350 C and the sensitivity of the cell to Thy was retested. Out of twenty-twocells, none were affected by raising Ta to 350 C but four showed achange in their properties at low Ta. All four neurones were 'warm-sensitive'and were classed as Type A (Hellon, 1967). In two of them there was ashift in the line relating Thy and firing rate without any appreciable changein the slope of this line, so that a higher firing rate was associated with anygiven Thy. These results are similar to those which Hammel's model wouldpredict.The other two neurones behaved differently and showed changes in the

slope of this line at low Ta. In one, the slope was increased so that the cellwas more sensitive to changes in brain temperature. In the other, coolingthe environment caused a marked slowing of firing rate, coupled with aloss of sensitivity to Thy.

These results provide neurophysiological evidence for an influence of Taon the functioning ofsome units which are sensitive to Thy.

REFERENCES

FORSTER, R. E. & FERGUSON, T. B. (1952). Am. J. Physiol. 169, 255-269.HAMMEL, H. T. (1965). Neurones and temperature regulation. In Physiological

Controls and Regulations, ed. YAMAMATO, W. S. & BROBECK, J. R. Philadelphia:Saunders.

HAMMEL, H. T., JACKSON, D. C., STOLWIJK, J. A. J., HARDY, J. D. & STR0MME,S. B. (1964). J. appl. Physiol. 18, 1146-1154.

HELLON, R. F. (1967). J. Physiol. 193, 381-395.

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The role of 5-hydroxytryptamine in the behaviour of cats

BY ELIZABETH E. SHILLITO. A.R.C. Institute of Animal Physiology,Babraham, Cambridge

Animals treated with parachlorophenylalanine temporarily lose thecapacity to synthesize 5-hydroxytryptamine (5-HT) (Koe & Weissman,1966) and therefore provide useful information about the function of thisamine. Male rats treated with this compound showed an increase in sexualbehaviour (Shillito, 1970).In the present work groups of six kittens or adult cats were watched

while living freely in an observation room and then given p-chlorophenyl-alanine orally on two successive days at doses of 400 or 300 mg/kg. Twocats in the group were treated at any one time. After the behaviouralchanges had been studied, the cats were killed and the concentration of5-HT in the brain was determined fluorimetrically.The p-chlorophenylalanine reduced the cerebral concentrations of 5-HT

by up to 90 %, and there was no progressive lowering after the first dose.The behavioural changes induced by the drug showed 24 hr after thefirst dose and started with an increase in scratching particularly in themiddle of the back. The cats given 800 mg/kg were very ataxic, but thiswas reduced with 600 mg/kg. All animals became restless and miaowedloudly. The kittens and females and some males started to rub against eachother and to tread with their forelegs in a pattern similar to pro-oestrusbehaviour. Furthermore, two male kittens and seven adult males began tomount other males 72 hr after treatment. The treated cats preferentiallymounted other treated males or subordinate normal males.When 5-hydroxytryptophan 5 mg/kg was injected i.P. into cats treated

with p-chlorophenylalanine, all abnormal behaviour stopped within anhour or two. This suggests that the changes were the result of depletion ofcerebral 5-hydroxytryptamine. Loss of cerebral 5-HT has been shown tocause lack of sleep and hypersensitivity to pain. The present observationsof restlessness and scratching confirm these findings. In addition, it appearsthat 5-hydroxytryptamine is an inhibitor of sexual behaviour in cats andthat it has this role in both males and females.

REFERENCES

KOE, B. K. & WEISSMAN, A. (1966). J. Pharmac. exp. Ther. 154, 499-516.SHITTO, E. E. (1970). Br. J. Pharmac. Chemother. 38, 305-315.

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PH YSIOLOGICAL SOCIET Y, JULY 1970

Release of 5-hydroxytryptamine (5-HT) from septum and caudatenucleusBY R. B. HOLMAN and MARTHE VOGT. A.R.C. Institute of Animal Physio-logy, Babrahum, CambridgeThe anterior horn of one lateral cerebral ventricle of the cat was per-

fused with artificial cerebro-spinal fluid at a rate of 5 ml./hr and 25 minsamples of the effluent were examined for their content of 5-hydroxytrypt-amine (5-HT). The cats were anaesthetized with chloralose and the per-fusion technique was that described by Carmichael, Feldberg & Fleisch-hauer (1964). 5-HT assays were performed on the rat's stomach strip(Vane, 1957).With this same technique, previous experiments (Portig & Vogt, 1969)

had shown that a multitude of stimuli released acetylcholine into the per-fusate, and electrical stimulation of the substantia nigra released dopamineand its main metabolite homovanillic acid. None of these stimuli released5-hydroxytryptamine, whether or not an inhibitor of monoamine oxidasehad been given. In the present work, electrodes were placed stereotaxicallyinto the two most rostral nuclei of the raph6, the nucleus linearis rostralisand linearis intermedius (Brodal, Taber & Walberg, 1960). Stimulation wasusually continued for 15 min during collection of a 25 min sample, thefrequency varied from 0 5 to 60 per see and the strength from 2-4 V.Of twenty-five stimulations in which the position of the electrode has beenverified histologically to be in or just lateral to the intermediate nucleus,sixteen caused an increase in the 5-HT concentration in the perfusate of100 % and over (mean 160 %), and nine a smaller rise with a mean of 47 %.Release per stimulus was greater at low frequencies such as 0 5 or 1/sec,but, up to 20/sec and for a stimulation period of 15 min, total release usuallyincreased with frequency. When the nucleus linearis intermedius wasstimulated, evoked responses were seen at low frequency of stimulation inboth septum and caudate nucleus. Both structures contain 5-HT, andthey may well both contribute to its release under the conditions of theseexperiments. The results with the nucleus linearis rostralis are still incom-plete. Since the cells in the raph6 nuclei are known to contain 5-HT, therelease of this substance into the ventricular fluid is interpreted as anactivation of endings of the raphe cells in the caudate nucleus and septum.However, the possibility that the pathway has intermediate stations can-not be ruled out.

REFERENCES

BRODAL, A., TABER, E. & WALBERG, F. (1960). J. comp. Neurol. 114, 161.CARMICHAEL, E. A., FELDBERG, W. & FLEISCHHAUER, K. (1964). J. Physiol. 173,

354.PORTIG, P. J. & VOGT, M. (1969). J. Physiol. 204, 687.VANE, J. R. (1957). Br. J. Pharmac. Chemother. 12, 344.

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Action of adrenaline on sweat glands and sudomotor trans-missionBy ADELE E. U. EDISEN and D. P. C. LLOYD. The Rockefeller University,New YorkMany agents, cholinergic and adrenergic, stimulate sweat glands of the

cat's footpad. Many agents block sudomotor transmission. Of the manyadrenergic blockers of sudomotor transmission some (e.g. dihydroergot-amine) act without depolarizing sweat gland cells (Lloyd, 1968). Others(e.g. TM 10) depolarize as they block (Lloyd & Edisen, 1970). Block, bywhatever means, at junctional regions means failure of post-junctionalstructures to respond as otherwise they would to prejunctional nerveimpulses.

Adrenaline is interesting with respect to action at junctional regions,especially at the sudomotor junction and on the effector sweat gland cells.Although the junctions are considered unequivocally cholinergic, adren-aline undeniably produces sweating in man and cat, a fact occasionallyconsidered of no physiological significance. Whether or not that be so it isworth while observing what happens to sweat gland cells and sudomotortransmission when adrenaline in small doses is intravenously injected.

Figure 1, exemplifying the effect, was obtained by the use of standardrecording procedures (Lloyd, 1961) and by infrequent stimulation of thesudomotor nerves (time line at bottom = 1 min). At the beginning arecontrol responses, even in amplitude and with steady base line. Then 12 jtg/kg adrenaline chloride was injected. The base line rises indicating depo-larization of the sweat gland cells and the sudomotor responses dwindle

Fig. 1. o -

Fig. 1.

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precipitately to 6 % of control value. The base line later swings positiveindicating secretion (Lloyd, 1961) and then, within some 30 min, base lineand sudomotor responses return to control level by reason of waningaction and reabsorption (Lloyd, 1961). Thus adrenaline depolarizes sweatgland cells, causes secretion and blocks sudomotor transmission. It issimultaneously a stimulator and a blocking agent, as are some of itsantagonists. The threshold dose is less than 0 3 /ug/kg.

Supported in part by U.S.P.H.S. grant No. NB 02816 from N.I.N.D.S., U.S.P.H.S.

REFERENCES

LLOYD, D. P. C. (1961). Proc. natn. Acad. Sci. U.S.A. 47, 351-358.LLOYD, D. P. C. (1968). Proc natn. Acad. Sci. U.S.A. 60, 115-117.LLOYD, D. P. C. & EDISEN, A. E. U. (1970). Proc. natn. Acad. Sci. U.S.A. 66, 243.

The effect of acute hypocapnia on maternal placental blood flow inrabbitsBY B. LEDUC. Nuffield Institute for Medical Research, University of OxfordThe physiological control of maternal placental flow is believed to be of

immediate importance to the foetus, but is not well understood. Maternalhyperventilation has been reported to cause a fall in foetal Po2 and non-respiratory acidaemia in foetal guinea-pigs and sheep. A decrease inmaternal placental blood flow was proposed as one of the mechanismsinvolved (Dawes, 1968). This hypothesis has been tested in pregnantrabbits near term, under pentobarbitone anaesthesia.

Placental flow was measured by injection of isotope-labelled micro-spheres into the left ventricle (Duncan, 1969). One injection was givenwhile the rabbit was breathing room air and another during hyperventi-lation. Arterial blood gases were measured immediately before and aftereach microsphere injection. During hyperventilation the arterial Pco.decreased from a mean of 34-17 mm Hg. The Po2 was kept constant byadjusting the 02 content of the gas mixture during hyperventilation.In thirteen rabbits hypocapnia caused a highly significant fall in

placental blood flow, while myometrial and ovarian flows remained un-changed. This fall did not occur when CO2 was added to the gas mixture inorder to keep the arterial Pco. constant during hyperventilation. In fiverabbits, moderate bypercapnia (Pco, 50-55 mm Hg) caused a rise inarterial pressure with a passive increase in placental flow. Severe hyper-capnia (PCO2 75-90mm Hg) was associated with placental vasoconstriction.Data were also available from forty additional rabbits, in which there was ahighly significant correlation between maternal placental blood flow and

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arterial Pco2. There was no correlation with arterial pressure, arterial Po2(range 70-110 mm Hg) or foetal position along the uterine horn.These observations show that hypocapnia is associated with maternal

placental vasoconstriction in the rabbit. They support Duncan's conclusionthat maternal placental flow is regulated by mechanisms different fromthose which control flow to the uterus, in this species.

REFERENCES

DAwES, G. S. (1968). Foetal and Neonatal Physiology. Chicago: Year Book MedicalPublisher.

DUNCAN, S. (1969). J. Physiol. 204, 421-433.

Spinal cord and root potentialsBy R. G. BORLAND, B. H. C. MATTHEWS and A. N. NICHOLSON. RoyalAir Force Institute of Aviation Medicine, Farnborough, Hampshire, andPhysiological Laboratory, Cambridge

Depolarization of the central end of a cut dorsal root, evokedby impulsesentering the spinal cord through a neighbouring root, is accompanied bypotentials recorded from the cord dorsum (Gasser & Graham, 1933). Aroot may provide a source for current flow to depolarized terminals withinthe cord, but can also act as a local lead for potentials from the cord surfaceas would a moist thread.

Spread of the dorsum potential into the root is prevented if it is drawnthrough a central hole in a small chlorided silver disk. With this guard ringonly component V is recorded (Fig. 1 A, D). In A the record is complicatedby the dorsal-root reflex. Fig. 1B shows superimposed recordings from twoguard-ring electrodes on the same root to a distant earth. Both show fivecomponents (Lloyd & McIntyre, 1949). Components I-IV common toboth cancel out with differential recording. Only the difference, in com-ponent V, can be due to sources in the root between the electrodes.

Section of roots close to or at the cord abolishes sources in the root. Thepositive intermediary cord dorsum potential disappears only in the dener-vated segment; above and below this it remains full size. This makes itimprobable that it originates in secondary neurones; also component V isfar more resistant to hypoxia and anaesthesia than any other cordactivity.Our results are consistent with a hypothesis that depolarization of

primary fibres is coupled to activity of glial cells evoked by impulseactivity. Activation of glial cells is known to occur elsewhere (Kuffler,1967; Grossmann et al. 1969); this seems a more probable cause of com-ponent V of the dorsal root potential than the activity of secondaryneurones.

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B

D -_

Fig. 1. Dorsal-root potentials recorded from a decerebrate cat, A, and aspinal frog, B, C, D. Stimulus to adjacent root. A and D. Recordings fromtwo ring electrodes encircling the root. B. Superimposed records fromproximal and distal rings to a distant earth lead. C. Recorded differentiallybetween the two rings. Scale in D indicates 10 msec intervals.

REFERENCES

GASSER, H. S. & GRAHAM, H. T. (1933). Am. J. Physiol. 103, 303.GRossMAN, R. G., WHITESIDE, L. & HAMPTON, T. L. (1969). Brain Re8. 14, 401.KUFFLER, S. W. (1967). Proc. R. Soc. B 168, 1.LLOYD, D. P. C. & McINTmE, A. K. (1949). J. gen. Phy8iol. 32, 409.

The effect of anaesthesia on units in the thalamic reticular for-mationBY A. ANGEL and G. V. KNOX.* Department of Physiology, The University,Sheffield, S 10 2TNAngel & Dawson (1961, 1963) showed that in rats anaesthetized with

urethane the electrical response of thalamic units in the direct sensorypathway, to a test stimulus applied to a forepaw, was increased if a pre-ceding conditioning stimulus (pinching or trains of electrical stimuli) wasapplied anywhere on the body surface. Furthermore, these thalamic unitswere not fired directly by the conditioning stimulus unless it was appliedto the same body site as the test stimulus.

* S.R.C. scholar.f

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In the course of this work, other thalamic units, which could be dividedinto two distinct types, were found which were influenced by stimulationof any part of the body surface. One type possessed a low 'resting' dis-charge frequency and showed an increase in frequency to a conditioningstimulus, the other with a high 'resting' discharge frequency exhibited afrequency decrease.Angel (1964) reported a direct relationship between the discharge fre-

quency of these unspecific thalamic units in the nucleus reticularis andnucleus ventralis pars dorsomedialis (terminology of de Groot, 1959) andthe responsiveness of the ventrobasal thalamus.

Angel & Unwin (1970) investigated the effect of an anaesthetic agent(urethane) on transmission along the dorsal column sensory pathway in therat, and their results indicated that the anaesthetic's effect was exertedmainly by impeding transmission through the ventrobasal thalamus. Ittherefore became a possibility that the thalamic reticular nuclei were con-cerned in the anaesthetic's effect at the thalamic level.In order to test this possibility, acute experiments were performed in

anaesthetized rats. Recordings were made extracellularly from (a) 30units having a low spontaneous discharge and (b) 10 units having a highdischarge rate. Increasing the depth of anaesthesia produced two maineffects; it had opposite effects on the two types of unit, i.e. units with lowfrequencies were slowed and those with high 'resting' frequencies speededup. In both cases, however, the anaesthetic reduced the effect of a condi-tioning stimulus on the 'resting' discharge.

After a train of 34 shocks at 3 msec separation, the maximum responseoccurred at 200-400 msec after the last shock and the effect lasted for3-5 sec. Increasing the anaesthetic depth reduced the time of effect of thetrain of shocks but not the time of maximum response.

Experiments on curarized, artificially respired animals showed that theanaesthetic's effect was not due to any respiratory change.We conclude therefore, that this is further evidence to support the

hypothesis that the thalamic reticular nuclei are intimately concernedwith the control of transmission through the ventrobasal thalamus.We would like to express our indebtedness to the M.R.C. for financial assistance

REFERENCES

ANGEL, A. (1964). J. Physiol. 171, 42-60.ANGEL, A. & DAwsoN, G. D. (1961). J. Physiol. 156, 23-24P.ANGEL, A. & DAwsoN, G. D. (1963). J. Physiol. 166, 587-604.ANGEL, A. & UNWIN, J. (1970). J. Physiol. 208, 32P.DE GROOT, J. (1959). Verh. K. Akad. Wet. 52, 4-40.

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Micro-electrode recordings from the pigeon lateral geniculatenucleusBY P. E. KING-SMITH. University Laboratory of Physiology, Oxford,OX1 3PTThe responses illustrated in Fig. 1 were recorded with tungsten micro-

electrodes from the nucleus geniculatis lateralis pars ventralis (Karten &Hodos, 1967) of pigeons under urethane anaesthesia; the recording sitewas confirmed histologically.

&L msecL

a < b c

d ; e

4,

0 3

.0 I

100 200 300 400 500 600Spike amplitude (FV)

Fig. 1. (a to e) Tracings from photographic records. (a, b) Response toa 10 spot switched on and off respectively. (c) On response to 10 spotusing a faster sweep speed. (d, e) On and off responses to diffuse light.Calibration: 0 5 mV and 10 msec for all records. Photocell records undertraces a, b, d and e. (f) An amplitude histogram for the slow spikes inresponse to a wide range of spot intensities.

A vigorous on response and a weak off response are observed using smallspots of light (Fig. la, b, c). This response generally consists of slownegative-going 'spikes' of 2-3 msec duration, and amplitude up to about0-8 mV. These spikes are much broader than impulses recorded from singleunits in other parts of the pigeon visual system (e.g. optic tectum, Hamdi& Whitteridge, 1954) and they may sometimes be recorded simultaneouslywith those briefer impulses (Fig. 1 d, e).

f-2

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170P PROCEEDINGS OF THEAn amplitude histogram (Fig. if) for the slow spikes, in response to

stimuli over a 100: 1 range in intensity, demonstrates that only certainspike sizes may occur and that, in this respect, these spikes behave likesingle units. In some cases, the slow waves are regularly preceded by smallbrief 'prepotentials' (Fig. 1 c). These observations suggest that the slowspikes are the post-synaptic response to impulses in single optic nervefibres, but it is not clear whether one or more lateral geniculate cells areinvolved.The responses differ from the on responses of the frog lateral geniculate

nucleus (Muntz, 1962) in some respects. First, the response to large spotsof light (50 or more) is much briefer than the response to smaller spots(Fig. 1 a, d) indicating a process of lateral inhibition. Secondly, photopicspectral sensitivity curves are similar to those observed in the pigeontectum with maxima at 560-580 nm. Thirdly, the retinotopic organiza-tion is found to be precise; anterior and superior visual fields are respec-tively represented in the posterior and lateral regions of the nucleus.

REFERENCES

HAMDI, F. A. & WHITTERIDGE, D. (1954). Q. J1 exp. Phy8iol. 39, 111-119.KARTEN, H. J. & HODOS, W. (1967). A Stereotaxic Atla8 of the Brain of the Pigeon.

Baltimore: Johns Hopkins.MuNTz, W. R. A. (1962). J. Neurophy8iol. 25, 699-711.

Excitation of identified supraoptic neurones by the iontophoreticapplication of acetylcholineBY J. J. DREIFUss and J. S. KELLY.* Department of Physiology, Univer-sity of Geneva, Switzerland

In keeping with the earlier experiments of Pickford (1939) in the dog,rat hypothalamic neurones located in the region of the supraoptic nucleuscan be excited by intracarotid injections of cholinomimetic drugs orhypertonic saline solutions (Dyball & Koizumi, 1969). A more directapproach, however, seems necessary, since both these effects may be ageneral feature of hypothalamic cells (cf. Cross & Green, 1959; Bloom,Oliver & Salmoiraghi, 1963).

Figure 1 shows oscillographic records from a supraoptic neurone excitedby an iontophoretic application of acetylcholine (ACh) in a rat anaesthe-tized by a mixture of pentobarbitone and urethane. The extracellularrecording electrode (4 M-NaCl with second barrel containing 1 M-ACh)was positioned near the bifurcation of the anterior and middle cerebralarteries exposed by a transpharyngeal approach. This neurone was also

* Canadian MRC Scholar, on leave from McGill University, while a IBROUNESCO bursar.

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excited by intracarotid injections of 5 % NaCl solution (w/v), and wasidentified as belonging to the supraoptic nucleus by cancellation of anti-dromic action potentials evoked by bipolar electrodes placed across thepituitary stalk, by action potentials initiated either spontaneously or byan iontophoretic application of ACh. As shown in Fig. 1, the ACh excita-tion takes several seconds to develop as found elsewhere in the mammalian

Fig. 1. Effect of acetylcholine applied by iontophoresis on the excitbbilityof an identified neurone of the rat supraoptic nucleus. A and B are controlrecords showing discharges evoked by ACh released by currents of 7 x 10-9(A) and 14 x 10-9 A (B). Records C-D, and E-F, respectively, show the dis-charge evoked by similar amounts of ACh at approximately 2 and 60 minafter an injection of 40 mg of atropine into the ipsilateral carotid artery.Calibration marks show 3 see and 400 /%V.

central nervous system. In this particular experiment, it was possible todepress both spontaneous and ACh evoked firing by an intracarotidinjection of 40 mg of atropine. Since the response to ACh was only par-tially antagonized by an extremely large dose of atropine, the possibilityremains that ACh receptors of supraoptic neurones in the rat are nicotinicrather than muscarinic in nature (Burn, Truelove & Burn, 1945).

This work was supported by the Swiss National Foundation for Scientific Researchand the F. Hoffmann-La Roche Foundation.

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1PROCEEDINGS OF THE

REFERENCES

BLOOM, F. E., OLIVER, A. P. & SALMOIRAcHI, G. C. (1963). Int. J. Neuro-pharmacol.2, 181-193.

BURN, J. H., TRUELOVE, L. H. & BuRN, I. (1945). Br. med. J. 1, 403-406.CROSS, B. A. & GREEN, J. D. (1959). J. Physiol. 148, 554-569.DYBALL, R. E. J. & KOIzuMI, K. (1969). J. Phy8iol. 201, 711-722.PICEFORD, M. (1939). J. Phy8iol. 95, 226-238.

The effect of topically applied amino acids on primary afferentterminal excitability in the rat cuneate nucleusBy N. DAVIDSON and C. A. P. SOUTHWICK. Department of Physiology,University of AberdeenThere is no agreement on the existence of compounds which have effects

on primary afferent terminals and primary afferent depolarization (PAD)consistent with those of a transmitter at the mammalian axo-axonicsynapse. The present experiments were designed to investigate the actionof gamma-aminobutyric acid (GABA) and other amino acids at this site.

A B CC.S.F. GABA 10-2M C.S.F.

~ ~~~~~~~~~L ~~~~~00#20 msec

Fig. 1. The effect of topically applied GABA (10-2 M) on primary afferentterminal excitability as indicated by antidromic potential height. RecordA shows a control antidromic potential evoked in the ulnar nerve from thecuneate nucleus during topical application of C.5.F. After 5 min topicalapplication of GABA a large increase was obtained in antidromic potentialheight (record B). Fifteen minutes after removal of GABA the potentialhad returned to control height (record C). Negative-going potentials areupward in all records; arrows indicate stimulus time.

Amino acid solutions (10-2 M and pH 6.8) were topically applied to theexposed cuneate nucleus of the chloralose-urethane anaesthetized ratusing a technique described by Malcolm, Saraiva & Spear (1967). Allamino acids were dissolved in artificial cerebrospinal fluid (Merlis, 1940)which also served as control solution. Changes in primary afferent terminalexcitability in the cuneate nucleus were revealed by the antidromicexcitability testing technique (Wall, 1958). Antidromic potentials were

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recorded monophasically from the ipsilateral ulnar nerve; PAD wasevoked by a conditioning volley from the ipsilateral median nerve.

L-Aspartate affected neither primary afferent terminal excitability northe amount of PAD (conditioned antidromic response height/test heightx 100) evoked from the median nerve. Glycine depressed terminal excita-bility by as much as 50 % (P < 0.001), but did not significantly alterevoked PAD.Both GABA (Fig. 1) and L-glutamate increased primary afferent terminal

excitability significantly (P < 0.001). The amount of evoked PAD wasincreased by glutamate while GABA had a variable depressant action.The abolition of PAD by picrotoxin was unaffected by glutamate butreversed by GABA.The action of GABA reported here is directly contrary to that reported

by Curtis & Ryall (1966) but remarkably similar to that observed byEccles, Schmidt & Willis (1963) in the cat spinal cord. The data supportthe hypothesis that GABA depolarizes primary afferent terminals byacting at the same receptor site as the transmitter substance that producesPAD.

REFERENCES

Cunns, D. R. & RYALL, R. W. (1966). Expl Brain Res. 1, 195-204.ECCLES, J. C., SCHMIDT, R. & WmLs, W. D. (1963). J. Physiol. 168, 500-530.MALCOLM, J. L., SARAIVA, P. & SPEAR, P. J. (1967). Int. J. Neuro-pharmacol. 6,

509-527.MERLIS, J. K. (1940). Am. J. Phyeiol. 131, 67-72.WALL, P. D. (1958). J. Physiot. 142, 1-21.

Antidromic invasion of cerebellar Purkyne cells

By J. C. ECCLES, D. FABER AND HELENA TBOR1ifKovk. Department ofPhysiology, State University of New York, Buffalo, New York 14214

Field potentials produced by antidromic invasion of cat Purkyn6 cellshave been extensively used in investigating the inhibitory and excitatorysynaptic actions on these cells (Eccles, Llinas & Sasaki, 1966; Bloedel &Roberts, 1969). In order to discover possible misinterpretations by thelatter authors we have investigated synaptic actions upon these fieldpotentials at very fast sweep speeds, examining depth profiles in detail.Some hours after decerebration under brief ether anaesthesia the study ofunanaesthetized (UAN) Purkyn6 cells (PC) was followed by investigationsat progressively cumulative levels of anaesthetic (pentobarbitone or Suritali.v.) up to at least 70 mg/kg. The antidromic field potential was conditionedby a parallel fibre volley for a range of test intervals up to 500 msec,potential profiles being observed through the whole depth of the cere-

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bellar cortex for both the conditioned and unconditioned responses(Eccles et al. 1966).We interpret our findings on the basis of a model of the Purkyn6 cell

which, as tested by invasion of antidromic impulses, has two differentlevels of safety factor: the all-or-nothing axon-soma transmission alreadydemonstrated by the IS-SD delay of about 0415 msec (Eccles et al. 1966);antidromic invasion from the soma to the large dendrites, which beginsabout 0-15 msec later and which is graded, there being various levels ofsafety factor. Antidromic invasion of the more superficial dendrites (200 ,uto surface) apparently does not occur. A parallel fibre volley has twoactions on PCs, directly and powerfully exciting by the dendritic spinesynapses, and inhibiting by the basket and stellate cell synapses (Eccleset al. 1966). In the UAN PCs dendritic excitation initially is more powerfulthan the inhibition, dominance of synaptic excitation even resulting inenhancement of the antidromic invasion into the large dendrites for testintervals up to 100 msec. This explains the facilitation described by Bloedel& Roberts (1969) without having recourse to their strange hypothesis ofreversed action of the parallel fibre-basket cell-PC system. At longer testintervals (100-500 msec) excitation of the PCs declines relative to theirinhibition.Even light anaesthesia depresses both dendritic and soma excitability.

Facilitation of dendritic invasion is eliminated and excitatory synapticaction on the dendrites no longer prevents basket cell inhibition fromblocking axon-soma transmission in many PCs; hence the observedinhibition of the antidromic field potentials even at brief test intervals(Eccles et al. 1966; Bloedel & Roberts, 1969). All gradations of this domi-nance of inhibition over excitation can be observed as anaesthesia is in-creased. Very deep anaesthesia abolishes the inhibitory action, which pre-sumably is due to depression of the inhibitory interneurones. As a finalstage the depth profile of the antidromic potentials indicates that there iseven blockage of axon-soma transmission of all PCs.

Supported by the NINDS Grants 5 RO1 NS 08221 02 and to D.F. F02 NB40544-02.

REFERENCES

BLOEDEL, J. R. & ROBERTS, W. J. (1969). J. Neurophysiol. 32, 75-84.ECCLES, J. C., LLINAS, R. & SASAKI, K. (1966). J. Phyaiol. 182, 316-345.

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Individual mechanisms activated in descending spino-spinalactivity in the chloralose anaesthetized catBY V. C. ABRAHAMS. Department of Physiology, Queen's University atKingston, Ontario, Canada

Spino-spinal activity in the chloralose anaesthetized cat is mediated viasupraspinal structures (Alvord & Fuortes, 1954; Shimamura & Yamauchi,1967; Devanandan, Eccles, Lewis & Stenhouse, 1969;Abrahams& Falchetto,1969) utilizing the cerebral cortex (Adrian & Moruzzi, 1939; Ascher,Jassik-Gerschenfeld & Buser, 1963; Abrahams, 1970). When a forepaw orneck nerve is stimulated a discharge is recorded in the lumbosacral ventralroots with a latency of 15-25 msec lasting 10 or 15 msec (Alvord & Fuortes,1954). If lumbosacral reflex excitability is tested during the discharge bothmonosynaptic and polysynaptic reflexes are found to be greatly poten-tiated. The spino-bulbo-spinal reflex (Shimamura & Livingstone, 1963) isabolished prior to the appearance of the ventral root discharge, and remainsabolished for about 400 msec. Following the ventral root discharge, themonosynaptic reflex remains enhanced for about a further 60 msec, butpolysynaptic reflexes are now reduced or abolished. The differential effecton monosynaptic and polysynaptic reflexes makes it unlikely that at thistime there is a generalized increase in motoneurone excitability of the typepresent during the ventral root discharge.The selective potentiation of monosynaptic reflex excitability might be

due to a pre-synaptic mechanism. Experimental testing of this hypothesisproves it not to be correct. Rather than the period of pre-synaptic excita-bility that might be expected to occur, a period of pre-synaptic inhibitionof Gp 1 A fibres was found. The existence of presynaptic inhibition with apeak 40-60 msec after stimulation was confirmed by the finding that dorsal-root potentials are regularly recorded in lumbosacral dorsal rootletsfollowing cervical nerve stimulation.

Thus, at least four distinct events occur in the lumbosacral cord indescending spino-spinal activity: (1) a period of enhanced motoneuroneexcitability, presumably due to EPSP activity (Devanandan et al. 1969)and coinciding with the ventral root discharge, (2) a period of presynapticinhibition of Gp 1 A fibres, (3) a prolonged inhibition of spino-bulbo-spinalactivity by an unknown mechanism, and (4) a late and prolonged mono-synaptic reflex facilitation, also by an unknown mechanism. Each of theseevents may depend on the existence of separate systems, each with aseparate intracerebral course since discrete cortical lesions lead to loss ofone or another of the four events with little or no effect upon the others.Further, cortical lesions at some sites affect descending effects from oneparticular nerve without having effect on the response to stimulation ofanother nerve.

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PROCEEDINGS OF THE

These experiments suggest that there are a number of parallel pathwaysopen in the chloralose anaesthetized cat whereby effects can be exertedon the spinal cord. These mechanisms may represent some or all of theways by which sensory systems can affect posture.

Supported by the Medical Research Council of Canada.

REFERENCES

ABRAHAMS, V. C. (1970). J. Phy8iol. (in the Press).ABRAHAMS, V. C. & FALcHmTTo, S. (1969). J. Phy8iol. 203, 435-447.ADRIAN, E. D. & MoRuzzi, A. (1939). J. Physiol. 97, 153-199.ALVORD, E. C. & FuoRTEs, M. G. F. (1954). Am. J. Phy8iol. 176, 253-261.AsCHER, P., JASsiK-GERsCHENFELD, D. & BUSER, P. (1963). Electroenceph. clin.

Neurophyaiol. 15, 246-264.DEVANANDAN, M. S., ECCLES, R. M., LEWIs, D. M. & STENEousE, D. (1969). ExplBrain Res. 8, 163-176.

SHIMAMURA, M. & LIVINGSTONE, R. B. (1963). J. Neurophysiol. 26, 258-272.SHIMAMu-RA, M. & YAmAucri, T. (1967). Jap. J. Physiol. 17, 738-745.

Support for an autogenetic excitatory reflex action of the spindlesecondaries from the effect of gamma blockade by procaineBY G. J. McGRATH* and P. B. C. MATTHEWS. University Laboratory ofPhysiology, Oxford

Procaine applied to a muscle nerve paralyses the gamma efferents beforeit interferes with conduction in large afferent or large efferent nerve fibres.Performed in the decerebrate cat this abolishes or reduces the stretchreflex by removing pre-existing fusimotor activity, thus reducing thespindle afferent discharge elicited by stretch (Matthews & Rushworth,1957 a, b). Classically, the relevant afferents for eliciting a stretch reflexwere thought to be solely the I a fibres from the spindle primary endings,but recent experiments suggest that the group II fibres from the spindlesecondary endings also contribute excitation to it (Matthews, 1969). If so,the effect of gamma blockade in reducing the stretch reflex would be dueto reduction in both I a and II discharges, which will occur concurrentlyon removing fusimotor activity. This can be tested by observing the effectof procaine nerve block on the reflex response to vibration.

Vibration at 100-200 Hz can be used to drive the primary endings todischarge at the applied frequency, irrespective of whether or not thefusimotor fibres are active; the secondary endings, however, are not signi-ficantly excited (Matthews, 1966, 1969; Brown, Engberg & Matthews, 1967).When vibration is applied simultaneously with stretch gamma block shouldreduce secondary firing but not primary firing, which will remain locked

* M.R.C. Scholar.

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PH YSIOLOGICAL SOCIETY, JULY 1970

to the vibration frequency. Thus if the I a fibres were solely responsible forthe stretch reflex then the response to the combined stimuli of stretch andvibration should be unaffected by gamma block. But if the group IIafferents also contribute excitation to the stretch reflex then the responseto stretch plus vibration should be reduced by gamma block in parallelwith the reduction in the reflex response to stretch alone. Experiments onthe soleus muscle of the decerebrate cat have now shown that this secondprediction is the correct one, thus supporting an excitatory role for thesecondaries.As before, gamma blockade was inferred from a decrease in the stretch

reflex occurring at a time after the application of procaine when the amotor fibres remained unaffected, judged by 100/sec stimulation. At thetime of y block, I a conduction was considered to be normal, withoutWedensky inhibition, both because I a fibres are paralysed in parallel witha fibres (Matthews & Rushworth, 1957 b) and because some vibrationreflex persisted until a motor paralysis. Group II block would be expectedto occur between that of the ax and y efferents, but its precise time isimmaterial for the present conclusion that the stretch reflex cannot bereadily attributed to Ia activity alone. Thus the experiments fortify thehypothesis that the spindle group II fibres contribute excitation to thestretch reflex of the decerebrate cat.

REFERENCES

BROWN, M. C., ENGBERG, I. E. & MATTHEWS, P. B. C. (1967). J. Physiol. 192,773-800.

MATTHEWS, P. B. C. (1966). J. Phy8iol. 184, 450-472.MATTHEWS, P. B. C. (1969). J. Phy8iol. 204, 365-393.MATTHEWS, P. B. C. & RUSHWORTH, G. (1957a). J. Physiol. 135, 245-262.MATTHEWS, P. B. C. & RUSHWORTH, G. (1957b). J. Phy8iol. 135, 263-269.

Tetrodotoxin resistant action potentials in denervated rat skeletalmuscleBY P. REDFERN and S. THESLEFF. Department of Pharmacology, Universityof Lund, Lund, Sweden

Some effects of posterior pituitary extracts on nucleoli of supra-optic nerve cellsBY R. M. WATT. Department of Physiology, University of EdinburghNormal male albino rats, aged 3 months, have been injected intraventri-

cularly with crude soluble extracts of the following tissues:Extract AE: pituitary posterior lobes incubated for 90 min in a high

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potassium, low sodium medium to provoke octapeptide release (Douglas& Poisner, 1964).

Extract AC: pituitary posterior lobes incubated for 90 min in Lockesolution.Extract BE: parietal cortex incubated for 90 min in the high potassium,

low sodium solution.Extract BC: parietal cortex incubated for 90 min in Locke solution.

150 aJ Nucleolar dry mass

0 Nucleolar nucleic acid

100 -_ _ _ _ _ _ _ _ _ _ _

50

(AC) (AE) (BC) (BE)

Fig. 1. Supraoptic nucleolar dry mass and nucleic acid content 48 hr afterintraventricular injection of extract of posterior pituitary or parietalcortex.Forty to seventy nucleoli, from three to four animals, contributed to each

mean value. Means, ± standard error, are presented as a percentage ofnormal control values. 100% nucleolar dry mass = 6-67 +0-45 g 10-12;100% nucleolar nucleic acid = 1-52 + 007 g 10-12. Columns AC, AE, BC,BE show results from animals injected with extracts AC, AE, BC, BErespectively. Extracts are as defined in text. The apparent loss of mass inthe BC group is not significant (P > 0.05).

Ofthe four extracts tested, only that derived from 'octapeptide-depleted'pituitary posterior lobes (AE) caused a significant increase in supraopticnucleolar dry mass (P < 0.0005) and nucleic acid content (P < 0 01) inrecipient animals (Fig. 1).

Dehydration produces similar supraoptic nucleolar changes (Watt,1970), but is unlikely to explain the differences recorded here as serumspecific gravity in those animals receiving the 'active' extract. AE did notdiffer from that in animals receiving 'inactive' AC (1x0205 + 0 0001 and1x0204 + 0*001 respectively, P > 0.3).


Cells from the posterior part of the arcuate nucleus showed no significantnucleolar changes following the injection of extract AC or AE.

REFERENCES

DoUGLAs, W. W. & PoisNER, A. M. (1964). J. Physiol. 172, 1-18.WATT, R. M. (1970). Brain Res. (In the Press.)

Movements of the jaw resulting from the application of externalforcesBY E. G. WALSH. Department of Physiology, University of EdinburghThe control mechanisms of the jaw have available a high 'power: weight

ratio' and need to protect the tongue and teeth. They have been investi-gated with a force generator coupled to a clamp on the lower jaw througha mechanical linkage. The motion is recorded by an 'infinite resolution'potentiometer. The subject lies on a couch with his occiput supported in thehollow of a block of expanded polystyrene. A triangular harness attachedto a metal plate pressed on to the forehead provides for further head stabili-zation by pulling upwards on the scalp. With an abruptly changing force

100 msec4 -i1.0 Nm

lose tForce. or 0'1 rd

Position

Velocity

~~~~~~~~~~~~~~~~~~~~~~~~~~IStrap M. -

R. masseter_

R. temporalls

Fig. 1. Rapidity of braking action when jaw starts to close due to abruptreversal of force. Activity seen in strap muscles (surface e.m.g.) comes afterpeak velocity has been reached. G.W. ? Aet. 14.

the jaw moves at first in the direction of the force but the peak velocity isusually achieved in 10-20 msec; deceleration is more prolonged and theresponse may be critically damped or show a little overshoot (Fig. 1). Withadded inertia the system becomes clearly resonant and its biomechanicalproperties are reminiscent of the wrist (cf. Walsh, 1968, 1969, 1970a, b).

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80P PROCEEDINGS OF THEPositive velocity feed-back produces tremor at 10-25 Hz according to thestiffness with which the jaw is held.The early braking seen with abrupt forces may not be related to reflex

action for:(1) No relevant e.m.g. changes have been observed in the temporalis,

masseter, or strap muscles in the 'pre-braking' period.(2) The latency of the jaw jerk, 8-4 msec (Goodwill, 1968), would not

allow adequate time for mechanical events.(3) The time taken to reach peak velocity does not lengthen if the

strength of the force is reduced although the actual velocity varies widely.An invariant time, 'isochronism', would be expected if the braking effectwas due to elasticity.

Accordingly it appears likely that the resistance initially observed isrelated to the stiffness of the muscular springing.The apparatus was constructed by G. Wright, Esq. The work has been supported

by the Royal Society (Government Grant) and the fund administered by Scott-Moncrieff, Thomson and Shiells.

REFERENCES

GoODWILL, C. J. (1968). Ann. phys. Med. 9, 183-188.WALSH, E. G. (1968). J. Physiol. 198, 69-72P.WALSH, E. G. (1969). J. Physiol. 202, 109-11OP.WALSH, E. G. (1970a). J. Physiol. 207, 16-17P.WALSH, E. G. (1970b). J. Physiol. (In the Press.)

The influence of chronic administration of pentagastrin on therat pancreasBY J. A. BARROWMAN and P. D. MAYSTON. Department of Physiology,The London Hospital Medical College, London, E. 1

Gastro-intestinal hormones are known to exert a trophic influence ontissues of the digestive tract. Gastrin and pentagastrin exert such effectson the parietal cells of the stomach (Polacek & Ellison, 1963; Crean,Marshall & Rumsey, 1969). Chronic administration of pancreozymin torats causes an increase in weight of an enzyme concentration in the pan-creas (Rothman & Wells, 1967). Gastrin and pancreozymin have a commonterminal tetrapeptide sequence (Jorpes, 1968); this is contained in thesynthetic pentagastrin. We were therefore interested to know whetherchronic administration of pentagastrin would stimulate growth of thepancreas.Adult Wistar rats, on a free diet, were given pentagastrin, 2 mg/100 g

body weight. day, or histamine 6 mg base/100 g body weight. day, individed doses by subcutaneous injections for 11 days. The histamine in-

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jections were used to investigate whether any observed effects on thepancreas were secondary to increased gastric acid secretion. Control ratsreceived saline injections. Following the last injection, the rats werestarved for 18-21 hr, then killed and a homogenate of pancreatic tissue wasprepared for the assay of soluble protein (Lowry, Rosebrough, Lewis Farr& Randall, 1951), amylase (Dahlqvist, 1962), lipase and esterase (Morgan,Barrowman, Filipek-Wender & Borgstr6m, 1968).

Pancreatic water content was determined by ashing. Estimates ofacinar cell size were made by nuclear counts on histological preparations.The remainder of the pancreas was carefully dissected and weighed.

TABLE 1. Results of a course of subcutaneous injections of pentagastrin and hist-amine in male Wistar rats. Data are expressed as means (± 5s.E. where five or moreobservations were made). Figures in parentheses indicate number of observations.Weights are expressed in terms of body weight of the rats at the time of killing.Enzyme measurements in pancreatic homogenates are related to the amount ofsoluble protein extracted. NS = not significant. None of the data from histamineinjected rats was significantly different from the controls

Value of P(penta-

Control gastrinMeasurement on Hist- (saline vS.the pancreas amine injection) Pentagastrin control)

Weight (mg/bO g) 411 383+ 8 496+ 18 < 0 01(4) (14) (10)

Pancreatic acinus cell 2288 + 122 1629+ 42 < 0 01nuclear count (30 (6) (6)fields of microscope) 1924 1938 - -

(4) (4)Ash (% of tissue) 1.9 2-15 + 0 07 2-15 ± 0-16 NS

(4) (9) (6)Soluble protein content 18-5 18*9 + 0 7 18-0 + 0.5 NS(mg/100 mg wet wt (4) (16) (12)tissue)Amylase (units/mg) 64 64+4 56±6 NS

(4) (12) (8)Lipase (units/mg) 11-9 ± 1.0 10-6 + 0 7 8-7 + 0 9 NS

(8) (16) (8)Esterase (Qt-equiv/ 5.8 4-6 ± 0-6 3-0 + 0-6 NShr/mg) (4) (9) (5)Body weight increase 5-5 6-8 ± 0 7 5.9 + 141 NS

M°0 (4) (11) (7)Weight duodenal 118 98± 18 149 < 0-05segment (see text) (4) (8) (4)(mg/100 g)

Weight kidneys 938 874 + 57 829 ± 68 NS(mg/100 g) (4) (10) (6)

Weight spleen 504 401 + 110 500 + 223 NS(mg/100 g) (4) (10) (6)

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82P PROCEEDINGS OF THEA segment of duodenum, from pylorus to the common bile duct, was also

excised and weighed.The data (Table 1) show that chronic administration of pentagastrin to

rats results in an increase in pancreatic weight with a parallel increase inacinar cell size. Unlike pancreozymin, pentagastrin does not appear toincrease the enzyme concentration in the pancreas.

REFERENCES

CREAN, G. P., MARSHALL, M. W. & RumSEY, R. D. E. (1969). Gastroenterology 57,147-155.

DAHLQVIST, A. (1962). Scand. J. din. Lab. Invest. 14, 145-151.JORPEs, J. E. (1968). Gastroenterology 55, 157-164.Lowny, 0. H., ROSEBROUGH, N. J., LEWIS FARR, A. & RANDALL, R. J. (1951).

J. biol. Chem. 193, 265-275.MORGAN, R. G. H., BARROWMAN, J. A., FPEK-WENDER, H. & BORGSTRM, B.

(1968). Biochim. biophy8. Acta 167, 355-366.POLACEK, M. A. & EiT isoN, E. H. (1963). Surg. Forum 14, 313-315.RoTT~w, S. S. & WELLs, H. (1967). Am. J. Phyeiol. 213,215-218.

Uptake of 3H-gamma-aminobutyric acid (GABA) by rat retinaBY MARGARET GOODCHILD and M. J. NEAL. Department of Pharmacology,School of Pharmacy, University of London, 29-39, Brunswick Square,London, W.C.1The brain possesses an efficient uptake mechanism for GABA (Iversen

& Neal, 1968) which has been utilized to label the endogenous GABA poolsin the cat cerebral cortex with [3H]GABA. This has recently enabled us todemonstrate the release of both radioactive and endogenous GABA fromthe cat brain after stimulation of inhibitory pathways (Iversen, Mitchell,Neal & Srinivasan, 1970), thus supporting the suggestion that GABA maybe an inhibitory transmitter substance in the brain. GABA may also havean inhibitory function in the retina where it occurs mainly in the ganglioncell layer (Kuriyama, Sisken, Haber & Roberts, 1968). The present in-vestigation was undertaken to determine whether the retina also possessesan uptake mechanism for GABA.

Retinas were dissected and each was incubated with GABA-2, 3-3H(5 x 108 M) in 10 ml. of oxygenated Irebs bicarbonate Ringer. The tissuewas then collected by rapid filtration, washed with 5 ml. ofice-cold mediumand the total radioactivity was determined by liquid scintillation counting.There was a rapid accumulation of radioactivity in the tissue, resulting

in a tissue/medium ratio of almost 50: 1 after incubation for 30 min at250 C. Ion-exchange and paper chromatographic analyses indicated thatmore than 90% of the radioactivity accumulated in the tissue was presentas unchanged [3H]GABA. The uptake of [3H]GABA exhibited saturation

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kinetics over a range of external GABA concentrations from 2 x 106 M to10-4 M, with an apparent Km for GABA = 4-0 x 10-5 M and Vmax = 0-167,smoles/g. min. The uptake of [3H]GABA was temperature dependent,being markedly reduced at 00C and optimal at 250 C. Replacement ofsodium chloride in the incubating medium by choline chloride orby sucrosereduced the uptake of [3H]GABA to less than 8% of the control values. Alarge reduction in uptake also occurred when the tissue was incubatedwith ouabain (10-5 M) or with 2,4-dinitrophenol (10-3 M). The uptake of[3H]GABA (5 x 10-8 M) was not affected by the presence of glycine, L-glutamate, L-aspartate, L-alanine, L-proline, or L-histidine (10-3 M).The results show the existence in the retina of a highly efficient and

specific uptake mechanism of GABA. The process shows many of thecharacteristics of an active transport system.We are grateful to the S.K.F. Foundation for financial support.

REFERENCES

IVERSEN, L. L., MITCHELL, J. F., NEAL, M. J. & SRINIvASAN, V. (1970). Br. J.Pharmac. Chemother. 38, 452P.

IVERSEN, L. L. & NEAL, M. J. (1968). J. Neurochem. 15, 1141-1149.KURIYAMA, K., SIs:EN, B., HABER B. & ROBERTS, E. (1968). Brain Re8. 9, 165-168.

A respiratory pump controlled by phrenic nerve activityBy A. HuszczuK. University Laboratory of Physiology, Oxford, and theLaboratory ofNeurophysiology, Polish Academy of Sciences Medical ResearchCentre, Warsaw, Poland

Artificial ventilation may affect the rhythmicity of the respiratorycentres as represented by phrenic nerve activity. In addition someclassical vagal reflexes re-investigated under these conditions seem to beaffected by the procedure of artificial ventilation itself (Stein, 1965;Cohen, 1969; Bystrzycka & Huszczuk, 1969).To investigate to what extent an arbitrarily imposed frequency of

ventilation might be responsible for disturbances of the inherent rhythmi-city of respiratory centres, a ventilation pump driven by phrenic nerveactivity has been built. The activity of C3 phrenic root is integrated sothat the signal obtained is proportional to transpulmonary pressurechanges in spontaneously breathing animals. Fig. 1 illustrates this corre-lation for quiet and C02-stimulated breathing. The validity of this rela-tionship has been confirmed in experiments with thirty rabbits. Theconstruction of the pump is based on a commercially available servo-positioning system which drives the piston of a ventilating syringe via aHulse rotary-to-linear movement transformation device. This could bereplaced by a more reliable worm and nut with ball-bearing device. To

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make the mechanical parts as simple as possible the number of activevalves is limited to one that is opened in expiration.

Over sixty experiments in which paralysed and non-paralysed rabbitshave been ventilated by means of the pump have been successfully per-formed. The anaesthetic was pentobarbitone (30-50 mg/kg, i.v.), andgallamine triethiodide (20-40 mg/kg, i.v.) was used as muscle relaxant.

3 min 7% CO2 in air

TPP F 0 wwrcm- -5 ' V VX 'H20L -10A A A AA

INT. PHR.l o /fA/0 10 /i[f.U. i/| f/f fl/f/f/

1 sec

Fig. 1. Relation between transpulmonary pressure (TPP) changes andintegrated phrenic nerve activity (INT. PHR.) in a spontaneously breath-ing rabbit before and during inhalation of 7 % C02 in air. V, inspired tidalvolume.

Volume changes are smooth and show no oscillations, and the arterialblood gas tensions are kept at normal levels. With vagi intact the gainof the system may be initially settled within the range 60-150% of thenominal value. Under these conditions a correction process is observed,and after a few breaths the respiratory control system re-adjusts tidalvolume to an optimal value. The C02-response curve has the same form asin spontaneously breathing animals.

Supported by a grant from the Wellcome Foundation.

REFERENCES

BYsTRZYcKA, E. & HuszczuK, A. (1969). Proc. XI. Congr. Pol. Phy8. Soc., pp.45-46.

CoHEN, M. I. (1969). J. Neurophysiol. 32, 356-374.STEIN, J. F. (1965). Univ. of Oxford Thesis, Oxford.

Central adaptation ,in the salamander spinal cordBy VICTORIA R. STIRLING. Department of Physiology, University CollegeLondon, Gower Street, London W.C. 1One approach to the problem of how neurones become 'correctly' con-

nected to one another, and to peripheral end organs, has been to study thechanges in the nervous system following experimentally produced nervelesions. In the lower vertebrates results were obtained suggesting the

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possibility that the peripheral connexions of nerves may control theestablishment of appropriate central connexions (Weiss, 1955). This sug-gestion was based almost entirely on observations of behavioural recoveryof animals after various operations had been performed on them.

In the present experiments changes in simple, measurable, parameters ofspinal reflexes have been investigated in adult salamanders (Salamandramaculosa L.) after the alteration of the peripheral innervation fields ofselected nerves. The reflex relationships in decerebrate salamanders,between spinal segments 15, 16 and 17 (which control the hind limb) havebeen studied, with particular reference to the latency between stimulationof one segmental nerve and the appearance of reflex responses in the othertwo. This latency was found to depend primarily upon the size of thesensory field of the stimulated nerve, and to a lesser extent, on the motorfield size of the reflexly excited nerve. These fields and reflex latencies werebilaterally symmetrical in individual animals, but varied considerablybetween animals. The peripheral sensory (and motor) fields of the 15thand 17th segmental nerves could be markedly increased following theircollateral sprouting to innervate adjacent tissue which had been dener-vated by removing the 16th segmental nerve.Four or more weeks after the operation the reflex latencies between the

15th and 17th nerves were reduced by about 20% on the operated side,but were within the range found in normal animals on the contralateral(control) side. The reduced latency on the operated side (with increasedsensory field) was equal to, or less than, that found in normal animals withsimilar-sized peripheral sensory fields. In a few animals there was reflexcoupling between the 15th and 17th nerves on the operated side whereasthe control side showed no such coupling.Animals recovered normal reflex movements (withdrawal responses) on

the operated side, these eventually becoming indistinguishable from thoseon the unoperated sides, which were unaffected by the operation. Func-tionally, the 16th segmental nerve plays a very important part in thesereflex movements in normal animals; on the operated side the 15th and17th nerves had clearly taken over the role of the 16th nerve.One interpretation of the present findings is that the increase in the

peripheral field of a nerve causes new, appropriate, reflex connexions todevelop. The appropriateness of these connexions was reflected in thereduced latency of the intersegmental reflex response, and probably wasalso important in the observed recovery of normal reflex behaviour.

REFERENCE

WEISS, P. A. (1 955). In Analy8i of Development, ed. WImLIER, WEISS & HAMBURGER,pp. 346-401. Philadelphia, London: Saunders.

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Effect of tetanus toxin in the goldfishBY J. DIAMOND and JANE H. MELLANBY. Department of Physiology,University College London, Gower Street, London, W.C. 1, and Sir WilliamDunn School of Pathology, Oxford

It is usually considered that the convulsions and spastic paralysis pro-duced by tetanus toxin are due to the blocking of inhibition at neurones inthe central nervous system (see Eccles, 1964). Earlier suggestions that thisaction of tetanus toxin is a presynaptic one have recently been supportedby Curtis & de Groat (1968).The present study began as an attempt to identify the site of action

of tetanus toxin more precisely, using as an experimental preparation theMauthmer cells of goldfish, and the inhibitory systems acting on them.However, it was found that although tetanus toxin killed the goldfish, theydied with an apparently flaccid paralysis, in contrast to the convulsivesymptoms usually seen in mammals. This result suggested the possibilityof a peripheral rather than a central site of action of the toxin, in the fish.

Goldfish were kept alive by perfusing the gills with oxygenated waterfor 24-72 hr after complete paralysis had been caused by tetanus toxinapplied directly to the brain. They were then prepared for electrophysio-logical investigation (Furshpan & Furukawa, 1962; Diamond, 1968). Theresponses of the Mauthner cells to both orthodromic and antidromicexcitation were found to be quite normal. Inhibition of the cells was pro-duced in two ways: by activation of the collateral inhibitory system, andby excitation of the contralateral VIII nerve; the resulting hyperpolari-zation, and/or inhibitory conductance increase, also appeared normal.There was therefore no evidence for a central action of the toxin.The possibility of a primary peripheral site of action oftetanus toxin was

investigated by injecting sublethal doses of the toxin into the pectoral finmuscles, which produces a local paralysis of the musculature. A nerve-muscle preparation was isolated from such a paralysed fin and its propertiescompared with those of a control preparation isolated from the oppositefin of the same fish, which had been injected with the toxin mixed with aneutralizing dose of antitoxin. The control fin was not paralysed, andbehaved normally. Both the nerve and the muscle were stimulated elec-trically, and the tension produced in the muscle and the compound nerveaction potentials were recorded. The toxin-paralysed muscle no longerresponded to stimulation of its nerve. The compound action potential ofthe nerve appeared normal and the muscle still responded, though lessstrongly than the control one, when directly excited.

It appears that while the central nervous system of the goldfish may behighly insensitive to tetanus toxin, the major part of the lethal action in

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PHYSIOLOGICAL SOCIETY, JULY 1970 187Pthe goldfish must be ascribed to a peripheral effect, the blocking of neuro-muscular transmission. Experiments are in progress to attempt to deter-mine whether this action is pre- or post-synaptic.

REFERENCES

CURTIS, D. R. & DE GROAT, W. C. (1968). Brain Re8. 10, 208-212.DIAMOND, J. (1968). J. Phy8iol. 194, 669-723.ECCLES, J. C. (1964). The Physiology of Synap8e8, p. 191. Berlin: Springer-Verlag.FuRSHPAN, E. J. & FuIRUKAWA, T. (1962). J. Neurophy8ioi. 25, 732-771.

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