findings have been communicated to the Physiological Society (Marley,

Transcription

1 J. Physiol. (1965), 180, pp With 14 text-figure8 Printed in Great Britain PHYSIOLOGY AND PHARMACOLOGY OF THE SPLANCHNIC- ADRENAL MEDULLARY JUNCTION BY E. MARLEY AD GWENDA I. PROUT From the Institute of Psychiatry, Maudsley Hospital, Denmark Hill, London, S.E. 5 (Received 10 July 1964) The adrenal medulla is the ancestral homologue of autonomic ganglia. The innervation of the medulla through a complex of nerves rather than a single trunk allows physiological dissection of synaptic behaviour. In previous experiments, synaptic function within the adrenal medulla of the cat has been studied by testing its response to nervous excitation and by investigating properties such as threshold, fatigue and susceptibility to ganglion-blocking drugs (Marley & Paton, 1961). New data are here presented on the threshold to nerve excitation, spatial and temporal recruitment, fatigue, the effect of drugs on these phenomena and the presence of functional units within the adrenal medulla. The preliminary findings have been communicated to the Physiological Society (Marley, 1961; Marley & Prout, 1963). METHODS Cats were anaesthetized with chloralose (80 mg/kg i.v.) after induction with ethyl chloride and ether. The vagi were cut in the neck and the animal was artificially respired. Carotid arterial blood pressure was recorded with a mercury manometer on a kymograph. Usually the splanchnic nerves of one side were stimulated. They were approached retroperitoneally through a lumbar incision and the adrenolumbar vessels were divided between ligatures. As described elsewhere (Marley & Prout, 1965), there are three or four upper splanchnic nerves to the adrenal medulla. These were identified and cut through and the sympathetic chain was removed from the first to the fourth lumbar sympathetic ganglia. The abdominal viscera including the adrenal gland not under test were removed, and the renal vessels were tied. Heparin (10 mg/kg i.v.) was injected. In several recovery experiments the adrenal glands were chronically denervated under aseptic conditions, the cats being anaesthetized with halothane (Fluothane) and oxygen by a modification of the method for anaesthetizing new-born animals (Marley & Payne, 1962). These cats were re-anaesthetized with chloralose days later and prepared as described. The splanchnic nerves were stimulated with platinum electrodes (cathode distal) using supramaximal rectangular (0-5 msec) pulses of 20 V at varying frequencies. The excitation frequency and number of stimuli were counted with a Racal digital frequency meter. For rapid excitation with up to ten stimuli, one stimulator provided the excitation frequency and the other a variable 'gate' during which a Stevens-Arnold 'millisec relay' was closed connecting the first stimulator to the electrodes; the stimuli were counted on an oscilloscope. In experiments to determine the stimulation frequency for optimal sympathin output, as well as those in which cocaine or phenoxybenzamine was injected into the cat, the excitation 31-2

2 484 E. MARLEY AND GWENDA I. PROUT frequencies were randomized with a Latin Square. For prolonged splanchnic nerve excitation the current to and from the electrodes was passed through 1,uF condensers to prevent electrode polarization (Garry & Gillespie, 1955). To excite synchronously two ipsilateral splanchnic nerves, one stimulator was connected into a junction box with connexions in parallel to the stimulating electrodes; stimuli were monitored on a double-beam oscilloscope. In two experiments direct excitation of the adrenal medulla was made with the bipolar stimulating electrode described by Bradley & Key (1958); the electrode was passed into the gland through a small incision on the ventral surface. Assay of sympathin. The sympathin released from the adrenal medulla was assayed by allowing the blood from the carotid artery or the adrenolumbar vein, or both, to superfuse the isolated rat stomach strip. In addition, blood from the adrenolumbar vein was collected and assayed on the blood pressure and uterus of the cat. Blood returned Carotid B A to jugular bloo vein F.V.C. Adrenal_.- Adrenal venous blood glandswater at To jugular Water C vein ( 14)Th Caarotid To jugular vein w qdrenalnblood blood Adrenalo lcarotid blood blood Fig. 1. Arrangement of carotid arterial and adrenal venous extracorporeal circulations in the cat for superfusing either a single rat-stomach strip (A) or strips in series (B). For details see text. Superfusion arrangement. The principle is that described by Vane (1958, 1964). The arrangement is illustrated in Fig. 1. For assay of large quantities of liberated sympathin, the blood was drawn from the carotid artery at ml./min through silicone rubber tubing by a roller-pump (Saxby, Siddiqi & Walker, 1960) driven by a Servomex motor controller. The blood was warmed in a water jacket at 400 C before superfusing the rat isolated stomach strip (Vane, 1957), then collected in a nylon organ bath and returned through silicone rubber tubing by gravity feed or through the roller-pump to the jugular vein. For assay of small amounts of sympathin, in addition to the carotid circuit an adrenal venous extracorporeal circuit was established through a cannula tied into the adrenolumbar vein, its fenestrated tip pointing medially and overlapping the adrenal gland (Marley, 1961). If the cannula was tied into the vein lateral to the gland, the vessel collapsed owing to suction exerted by the roller-pump, and blood flow ceased. Adrenal venous (3-5 ml./min) and carotid arterial blood (4-6 ml./min) were pumped separately through silicone rubber tubing by the roller-pump. The opening of the adrenolumbar vein into the inferior vena cava was left patent as otherwise the adrenal blood flow was unsatisfactory, and suction exerted by the roller-pump led to sustained sympathin discharge. Thus some vena caval blood mixed with adrenal blood was pumped from the cat.

3 ADRENAL MEDULLA 485 Tests were made to ensure that with the opening of the adrenolumbar vein into the vena cava patent, the sympathin released on excitation of the splanchnic nerve came solely from the adrenal medulla. This was shown by tying a cannula into the adrenolumbar vein between the gland and the vena cava, excluding the adrenal gland from the circulation, and superfusing the stomach strip with vena caval blood. No sympathin effect was obtained under conditions in which the blood from the adrenolumbar vein gave positive results. After the blood was pumped from the body and warmed in the water jacket, the blood was mixed before superfusing a single stomach strip (Fig. 1 A). Alternatively, the blood streams superfused two strips in series (Fig. 1 B); the upper strip received carotid, and the lower adrenal together with the carotid blood which had superfused the upper strip. Superfusion of the lower strip by arterial as well as venous blood was necessary, for the strip loses sensitivity in deoxygenated blood. The blood was returned to the jugular vein. With this arrangement the lower strip monitored adrenal medullary secretion, whereas the upper strip detected sympathin in the general circulation. The rat-stomach strip was twice as sensitive to adrenaline as to noradrenaline. The strip in the adrenal circuit responded to secretion equivalent to 1-5 ng adrenaline, whereas with strips superfused by carotid blood the equivalent of 50 ng adrenaline has to be liberated to be detected. The tone of the strips was recorded with identical auxotonic (Paton, 1957) or isotonic levers of 16, 18, 20 or 22 x magnification. Flatter stimulus-response slopes were obtained with auxotonic than with isotonic levers and this was advantageous when the strip was extremely sensitive and graded responses were required, because it provided a greater range of responses before maximal relaxation was produced. With mixed blood from the adrenal vein and the carotid artery as superfusate, the tone of the strip often dwindled and sensitivity declined; tone could be restored by administering oxygen to the cat (1-2 I./min). In the course of several experiments it became necessary to replenish the blood. This was done by giving up to 50 ml. heparinized blood from a donor cat or a solution of 6 % dextran in NaCl solution. To determine the threshold for sympathin release on nerve excitation only the adrenal venous extracorporeal circuit was established. Adrenal venous blood was drawn from the gland at ml./min by the roller-pump with the opening of the adrenolumbar vein into the inferior vena cava patent. The interval between excitation of the splanchnic nerve and the relaxation by the liberated sympathin of the rat-stomach strip was timed. On subsequent nerve excitation, blood was collected in chilled siliconed centrifuge tubes for 1 min, after having passed the water jacket, commencing 30 sec before the time determined for relaxation. The sympathin content of the plasma was then assayed by the in vitro methods. Assay in vitro. To collect blood (ca. 2 ml.) from the adrenal gland a polyethylene cannula was tied into the adrenolumbar vein with the tip just lateral to and pointing towards the gland. The opening of the adrenolumbar vein into the inferior vena cava was closed, and adrenal blood flowed into the cannula. The cannula had a side arm close to the insertion into the vein, so at the end of collection the residual blood in the polyethylene cannula could be blown into the collecting tube. The venous samples were collected for at least 5 min in chilled siliconed graduated centrifuge tubes containing 1 mg heparin and immediately centrifuged at 3000 rev/min for 10 min at 00 C. Plasma and cell volumes were drawn into siliconed pipettes and transferred to sealed siliconed bottles and kept on ice until assay. The plasma was assayed against noradrenaline on the arterial blood pressure in pithed rats (Shipley & Tilden, 1947) injected with hyoscine (2-0 mg I.M.), and against adrenaline on the electrically stimulated rat uterus pretreated with stilboestrol 100 jig I.M. (Harvey & Pennefather, 1962). The uterus was suspended in a 10 ml. organ bath containing the solution described by Gaddum, Peart & Vogt (1949). Oxygen was bubbled through the solution and its temperature was maintained at 28-30' C. The uterine contractions were recorded with a pendulum auxotonic lever. If samples were assayed from cats which had been injected with eserine, hyoscine was added to the bathing fluid to give a concentration of The

4 486 E. MARLEY AND GWEADA I. PRO UT amount of the two catechol amines in the plasma was determined from the adrenaline and noradrenaline equivalents assayed with the two methods according to the calculations devised by Marley & Paton (1961). RESULTS In previous experiments (Marley & Paton, 1961) it was found that on supramaximal stimulation at 1/sec of the first splanchnic nerve 900 shocks were required to detect an increase in the secretion of sympathin from the adrenal medulla. In the present experiments, in which the blood from the adrenolumbar vein was directly superfused over the rat-stomach strip, an increased secretion was observed with a much smaller number of stimuli. With supramaximal stimuli, that is under conditions of complete spatial recruitment, two or three shocks applied to the first or second, and five shocks applied to the third splanchnic nerve at a frequency of 1/sec were sufficient to elicit an increased sympathin secretion (Fig. 2). The fact that, on exciting the third nerve, five shocks instead of two or three were required presumably reflects the fact that the smaller nerve innervated fewer secretory cells. When the first or second splanchnic nerves were stimulated at a greater frequency (32 or 64/sec) to give optimal temporal recruitment and 200 shocks were applied, the threshold excitation intensity was 1-2 T. The relaxations of the superfused stomach strip produced by the blood on supramaximal stimulation of the first splanchnic nerve with two to three shocks at 1/sec were assayed against adrenaline. The amounts of sympathin secreted per shock were found to be equivalent to 5-10 ng adrenaline and, on exciting the second and third splanchnic nerves, the amounts were usually less. When the samples from the adrenal extracorporeal circuits were collected and assayed separately for adrenaline on the rat uterus and for noradrenaline on the rat blood pressure it was found that on supramaximal stimulation at 1/sec at least ten shocks were required to detect an increased secretion. The mean amount of adrenaline plus noradrenaline secreted was 9 1 ng/shock if ten but only 2 ng/shock if twenty shocks were applied. Control samples collected before or after exciting the splanchnic nerves were not inactive. The small amounts of amine present in these samples have been deducted from the values given for the increased secretion by stimulation. It was further observed that it made a difference whether the control blood was allowed simply to flow out from the adrenolumbar vein or was pumped through the extracorporeal circuit, when it then contained 3-5 times greater amounts of the amines, particularly of noradrenaline. As these control samples contain blood not only from the adrenal medulla but also from the vena cava, this increase in noradrenaline content suggests that the stress of the extracorporeal circuit results in a general

5 ADRENAL MEDULLA 487 activation of the sympathetic nervous system and that the noradrenaline is derived at least partly from post-ganglionic sympathetic nerve endings. After eserine (0-2 mg/kg i.v.), which allows accumulation of acetylcholine in the adrenal medulla, the amount of amines secreted per shock increased. In three such experiments with an adrenal extracorporeal circuit, ten and twenty shocks were applied to the first splanchnic nerve at 1/sec. The mean value per shock was 8&5 ng before and 205 ng after eserine when ten stimuli, and 3 and ng when twenty stimuli were A B C Ad Ad Ad S S S S 25 ng 10 ng 35 ng 1 sec 1sec 1 sec isec E F Mir LJ1 S S NAd Ad Ad Ad S 1 sec 1 sec 100 ng 50 ng 25 rng 50 ng 2 sec Fig. 2. Rat-stomach; strip responses to superfused blood from carotid artery and adrenolumbar vein of a 2-4 kg cat. At Ad or NAd, injections of adrenaline or noradrenaline into adrenal extracorporeal circulation; doses in ng stated below. At S, supramaxixnal excitation of left first (B and C) or second (D and F) splanchnic nerves; frequency and number of shocks stated below. Time marker in minutes in all the figures.

6 488 E. MARLEY AND GWENDA I. PROUT applied. In one experiment it was found that five shocks, which did not lead to a detectable secretion before eserine, produced secretion of 4 6 ng/ shock after this drug. Eserine also increased the resting secretion, which rose about fourfold. With cocaine (1 mg/kg I.v.), which competes for the noradrenaline store (Farrant, 1963) and should hinder reabsorption of the amine by the secretory cells, higher values for the amounts of amine secreted were obtained. For instance, in two experiments in which twenty shocks at 1/sec were applied, the mean secretion of 1-96 ng amine/shock rose to 7-8 ng/shock after the cocaine injection. In one of the two experiments, the resting secretion did not rise; in the other, it rose slightly only after cocaine injection. Spatial and temporal recruitment To determine the total amounts of amines secreted by a burst of 200 shocks, the carotid blood was superfused on the rat-stomach strip and assayed against noradrenaline. With different frequencies of supramaximal stimulation it was found that the optimal excitation rate was faster for the larger than for the smaller splanchnic nerves. It was 30-60/ sec for the first and second, and 16/sec for the fourth splanchnic nerve, and remained constant throughout an experiment. On applying 200 shocks to the first nerve the total output of amine on excitation at 16/sec was sometimes equal, sometimes less and sometimes more than on excitation at 128/sec. As supramaximal stimuli were used these variations presumably depended on the efficacy of temporal recruitment. Spatial and temporal recruitment were therefore examined. Spatial recruitment was tested by exciting the first splanchnic nerve with 200 shocks at constant frequency but varying voltage. With 1 or 2 V, increased secretion could be detected at all frequencies tested, i.e. 4, 8, 16, 32 and 64/sec, as illustrated in the experiments of Fig. 3A and in the control experiments of Fig. 5. At the lower frequencies, 4, 8 and sometimes 16/sec, the output rose linearly with increasing the stimulation intensity to a maximum at about 10 V. The linear slope indicated that the spatial recruitment was a graded phenomenon up to a stimulation intensity of 10 V, when recruitment was complete. Temporal recruitment was examined by giving 200 shocks at constant voltage but varying the frequency. As shown in Fig. 3B the stimulusresponse slope remained about the same with a stimulus intensity of 2 V when the frequency wasincreasedfrom 8 to 64/see, but with higher stimulus intensities, i.e. 3, 4, 5 and 10 V, this slope became steeper on increasing the frequency from 8 to 32/sec. The fact that this did not happen with the low stimulation intensity of 2 V shows that temporal summation depends on

7 ADRENAL MEDULLA 489 adequate spatial recruitment. In most experiments, temporal recruitment was pronounced at 16/sec. Such a result is illustrated in the experiment of Fig. 5A, by the three control stimulus-response curves obtained at 4, 8 and 16/sec excitation. In some experiments temporal recruitment was pronounced only when the frequency was increased to 32/sec. 12 A e 1 0d 9 C 8 o0 7 o6 5 b5 o~4 02 3~~~~~ o r4a5 10 0i250o50r751b0 12 -c~ B Voss oug adrenaline E u- ~9 8 c 7 0o l Excitation frequency (per sec) Fig. 3. Graphs of relaxation of rat-stomach strips superfused by carotid arterial blood during stimulation of left first splanchnic nerve with 200 shocks, showing spatial and temporal recruitment. Each value is the mean from two experiments in different cats. A, ordinate, relaxation (cm); ab.i8a, excitation voltage or, in e, calibrating doses of adrenaline. (a, b) Excitation at and 16/sec; linear increanse in sympathin secretion with increase in excitation voltage. (c, d) Excitation at 32 andl 64/sec; steep stimulus-response slopes, indicating efficacy of temporal recruitment. B, ordinate as in A; aobtia, excitation frequency. (V-V, 2 V; N-UE, 3V; 0-0O, 5V; El-U, 10 V.) Dependence of temporal recruitment on adequate spatial recruitment, since stimulus-response slope is flat at 2 V, but steep at 3 V or greater. The curves b and c in Fig. 3A demonstrate that temporal recruitment is a more efficient way than spatial recruitment for increasing the amount of sympathin secreted. Curve b was obtained at a frequency of 16/sec and shows the relatively small increase in sympathin secretion on increasing the intensity of stimulation from 2 to 10 V. In contrast curve c, obtained at a frequency of 32/sec, shows that an increase in the stimulation intensity

8 490 E. MARLEY AND GWENDA I. PROUT from 2 to 3 V results in the secretion of amounts of sympathin much greater than those secreted at 10 V with a frequency of 16/sec. The curves of Fig. 3B further suggest that with slow rates of firing in the first splanchnic nerve spatial summation is as important as temporal summation, but that with faster rates of firing temporal summation takes precedence. It proved difficult to examine spatial and temporal summation in the small third and fourth splanchnic nerves. Therefore, summation was *~ A f11 10 on 1 L /28 9 S _ Shocks/sec jug adrenaline o , ~~2~~ Shocks/sec Atg adrenaline Fig. 4. Graphs of relaxation of rat-stomach strip superfused by carotid blood from two cats, to show how partial division of the right first splanchnic nerve affects secretion on supramaximal nerve excitation. A, 3-4 kg cat: responses to 128 shocks before (open circles) and to 128 and 256 shocks after (closed circles) approximate hemisection of the splanchnic nerve. B, 2-8 kg cat: responses to 600 shocks before (open circles) and to 1200 and 2400 shocks after (closed circles) almost complete division of the splanchnic nerve. Ordinates: relaxation (cm). Abscissae: left, excitation frequencies; right, calibrating doses of adrenaline i.v.

9 ADRENAL MEDULLA 491 studied in the first splanchnic nerve after partial division. After approximate hemisection of this nerve, 128 supramaximal stimuli were applied to the nerve at various frequencies. In four of these experiments, maximal secretion was obtained as in the undivided nerve at a frequency of 64/sec, but in two maximum secretion was obtained at 32/sec, as shown in Fig. 4A. This result is at variance with the optimal stimulation frequencies of about 16/sec found in the third and fourth splanchnic nerves. With excitation at 32 or 64/sec, i.e. at frequencies in which temporal summation was most effective in the undivided nerve, hemisection had reduced the maximal secretion of sympathin to about one-quarter. Doubling the number of shocks increased the secretion only by a relatively small amount, so that the secretion remained lower than that of the undivided nerve (Fig. 4A). With stimulation of the almost completely divided nerve, output was drastically reduced and, as shown in Fig. 4B, there was no peak in secretion over the frequency range 16-64/sec even when the number of stimuli was increased fourfold to Thus, temporal recruitment is more effective the larger the number of secretory units involved, and even with complete spatial summation, increase in number of stimuli is a poor substitute for temporal recruitment. It has been shown that on application of supramaximal shocks, secretion became optimal at 30-60/sec and then declined with faster excitation. A similar result has been obtained by Rosenblueth (1932) and ascribed to successive shocks falling within the refractory period. However, this appears not to be the whole explanation, since, when only five or ten supramaximal shocks were applied to avoid temporal dispersion of impulses, some secretion occurred at excitation rates up to 600/sec. Effect of drugs on recruitment Drugs acting at the synpase. Hexamethonium. The threshold for sympathin secretion on exciting the first splanchnic nerve with 200 supramaximal shocks was raised two- to threefold after hexamethonium bromide. This is illustrated by the curves in Fig. 5A, obtained with stimulation frequencies of 4, 8 and 16/sec. When the sympathin secretion with suprathreshold stimulation obtained after hexamethonium is compared with that obtained with the same stimulation intensity before hexamethonium, it is seen that the amounts of sympathin secreted are reduced but that the stimulus-response slopes are roughly parallel, being flat and linear with slow and steep and non-linear with fast excitation. When there was recovery from the effect of hexamethonium, which lasted min, and the amount of sympathin secreted increased gradually, the slopes of the stimulus-responses remained roughly parallel, as is illustrated for a stimulation frequency of 16/sec in Fig. 5A. These observations are in

10 492 E. MARLEY AND GWENDA I. PRO UT accord with the view that hexamethonium acts by competitive blockade. The fact that after hexamethonium the slopes remained flat and linear with slow and steep and non-linear with fast excitation showed that in spite of the impairment of secretion, temporal recruitment was still more 10 4/sec 8/sec 8 L 6 L 1 6/sec h2 h' Wh, a3 &4 0 0 E 0 C) 4-0._ x B F8/sec h 2 -f h O ± ± /sec 8/sec 10_ 6 t e ~~~e ~~~c Volts,ug adrenaline Fig. 5. Graphs of relaxation of rat-stomach strip superfused by carotid blood from cats following stimulation of the first left splanchnic nerve with 200 shocks or adrenaline i.v. Effects of hexamethonium (two cats: A and B) and of eserine (one cat, C) on spatial and temporal recruitment. A and B: responses to stimulation at 4, 8, 16 and 32/sec before (c) and some time (h) during the first hour after an intravenous injection of hexamethonium (1 mg/kg). hl and h2 were obtained 1 and 2 hr after the injection, to show the recovery. C: response to stimulation at 4, 8 and 16/sec before (c) and after (e) eserine (0.2 mg/kg i.v.). Ordinates: relaxation (cm). Ab8ci88ae: excitation voltage or (for each last graph on the right) calibrating doses of adrenaline, i.v.

11 ADRENAL MEDULLA 493 efficacious than spatial recruitment. The hexamethonium blockade could be overcome with fast stimulation, as shown in Fig. 5B. At a frequency of 32/sec, the amounts of sympathin secreted with stimulation intensities of 5 and 10 V are the same before and after hexamethonium. Eserine. The threshold for sympathin secretion on exciting the first splanchnic nerve with 200 supramaximal shocks was lowered after eserine (Fig. 5 C) and the amounts of secreted sympathin increased. The stimulusresponse slopes remained roughly parallel to those obtained before eserine, indicating a proportionate relation between the amount of transmitter persisting at the synapse and the quantity of sympathin secreted. With slow excitation of 4/sec spatial recruitment was enhanced, whereas with 8/sec excitation the increase in efficacy of temporal recruitment was more obvious, since the stimulus-response slope corresponded to those found with faster (16, 32, 64/sec) excitation in the absence of eserine. When 200 shocks were applied at a frequency of 16 or 32/sec and an intensity of 10 V, secretion of sympathin continued after stimulation had ceased, since the superfused stomach strip remained relaxed for 5-10 min, whereas without eserine the strip regained tone as soon as stimulation ceased. This prolonged secretion of svmpathin was not evident on stimulation at a lower frequency and voltage. Thus, under the influence of eserine, only stimulation giving maximal or near-maximal spatial and temporal recruitment resulted in an activation of secretory cells, which continued after stimulation ceased. Drugs acting post-synaptically. Cocaine and phenoxybenzamine are supposed to act post-synaptically either by hindering reabsorption of amine after extrusion by the secretory cell or by preventing uptake of amine on adrenergic receptors within the adrenal gland. For testing the effect of these drugs on the release of sympathin, the method of superfusing the rat-stomach strip was not used, because cocaine enhances the effect of sympathin on this preparation and phenoxybenzamine may have the opposite effect. Instead the released sympathin was determined by taking blood from the adrenolumbar vein and assaying it on the rat uterus and rat blood pressure. After an intravenous injection of cocaine (1-4 mg/kg) the output of sympathin on stimulation of the first splanchnic nerve with 200 supramaximal shocks at 10/sec rose two- to elevenfold. A characteristic feature of the cocaine effect was that it developed gradually and took sometimes 60 min before the increased secretion of sympathin on stimulation of the splanchnic nerve had reached its maximum. In the experiment shown in Fig. 6 the stimulation of the splanchnic nerve had increased the secretion of sympathin from ca. 26 to 60 ng/min at 15 min and to 170 ng/min at 35 min after the cocaine injection. When stimulation was repeated after

12 494 E. MARLEY AND GWENDA I. PROUT another 20 min secretion amounted to 125 ng/min. This drop does not indicate a fall in catecholamine secretion, but is the result of collecting the blood over a longer period. The amounts of catecholamines secreted per shock during the four periods of stimulation were 0 5, 1F5, 5-5 and 5*5 ng. The cocaine had scarcely any effect on the resting secretion. The A B C D i1 mg/kg 0 5 ng cocaine 1.5 ng 5.5ng 55ng Minutes Fig. 6. Histogram of secretion of noradrenaline (black) and of adrenaline (white) elicited by 200 supramaximal shocks at 10/sec applied (at the arrows) to the left first splanchnic nerve in a 4-0 kg cat. Effect of a slow intravenous injection of cocaine (1 mg/kg) shown by comparing secretion on stimulation before (A) and at different times after (B, and D) cocaine. The figures under the arrows give the combined amounts of amines in ng secreted per shock for each stimulation period. Ordinates: amine secreted/min in ng. Abscissae: time in minutes. small rise in resting secretion seen in Fig. 6 is within the limit of experimental error. In some experiments, stimulation during the first 5-10 min after an intravenous injection of 2 or 4 mg/kg resulted in a diminished secretion of the catecholamine, probably due to the local anaesthetic

13 ADRENAL MEDULLA 495 action of cocaine. When stimulation was repeated in the following min the usual rise in catecholamine secretion was obtained. The effect of larger doses of cocaine (8 mg/kg) on the secretion of catecholamines was not examined, because they produced such a profound fall in arterial blood pressure that the blood flow from the adrenolumbar vein practically ceased. The effect of cocaine (1 mg/kg) on catecholamine secretion produced by 200 supramaximal shocks was not as great when the frequency of stimulation was 30/sec instead of 10/sec. In four experiments with stimulation at 30/sec the mean amount of amine secreted rose from 4X7 to 7X2 ng/shock 30 min after the cocaine injection. In assessing this increase it has to be stated that in control experiments in which the stimulation with 200 supramaximal shocks at 30/sec was repeated after 30 min without cocaine the secretion per shock declined sometimes as much as half. Phenoxybenzamine. The phenoxybenzamine bonds ionically on receptors, and can therefore be displaced by the catecholamines unless this bonding has become irreversible. To achieve this condition 2 hr were allowed to elapse after an intravenous injection of 10 mg/kg phenoxybenzamine before the effect of splanchnic-nerve excitation was tested. The injection did not affect the adrenal medullary secretion produced by stimulation of the first splanchnic nerve with 200 supramaximal shocks at either 10 or 30/sec. The mean secretion of amine in four cats on stimulation at 10/sec was 2*56 ng/shock after phenoxybenzamine compared with the mean secretion of 1-68 ng/shock in four control cats without phenoxybenzamine. The corresponding values on stimulation at 30/sec were 3-25 ng/shock and 4-42 ng/shock. Direct stimulation of the adrenal medulla In two experiments the secretion obtained on giving 50 shocks at the same stimulation intensity either to the splanchnic nerve or directly to the adrenal medulla through a bipolar electrode in its substance was compared. The secretion was assayed against adrenaline on the rat-stomach strip superfused in the one experiment by carotid arterial blood and in the other by carotid arterial and adrenal venous blood. In both experiments secretion was much greater on nerve excitation. Spatial recruitment was obtained on directly exciting the medulla for secretion progressively increased on raising the stimulus intensity at 30/sec from 1 to 2, 5 and 10 V. Temporal recruitment, which occurs on stimulation of the first splanchnic nerve, was not observed on directly stimulating the adrenal medulla. For instance, secretion on giving 50 shocks to the first splanchnic nerve at 10 V increased progressively with excitation at 5, 10, 30 and 60/sec and then declined at 120/sec. On directly

14 496 E. MARLEY AND GWENDA I. PRO UT exciting the adrenal medulla the secretion remained the same at these excitation frequencies and was smaller than that obtained with nerve stimulation at 5/sec. Thus, temporal recruitment appears to depend on spread of excitation through the medullary nerve plexus. Comparison of the secretion on excitation of the three splanchnic nerves Quantitative data on the secretion obtained on exciting the upper three splanchnic nerves to the adrenal medulla do not appear to be available. These three nerves were, therefore, excited on the same side and the blood taken from the adrenolumbar vein was assayed on the rat uterus and the rat blood pressure. The results are shown in Table 1, which give the mean TABLE 1. Mean amount of amine in ng secreted per shock at various excitation frequencies applied to the splancbnic nerves. 200 supramaximal stimuli given to the first and 256 to the second or third splanchnic nerve. Blood removed from the adrenolumbar vein and assayed on the rat uterus and blood pressure Splanchnic Excitation rate/sec nerve No. of, A_--_ Preparation excited expts Acute division of all splanchnic nerves First Second Third days after division of ipsilateral First nd, 3rd and 4th splanchnic nerves and removing the ipsilateral lumbar sympathetic trunk days after partial division of First the first splanchnic nerve amine secreted per shock at different frequencies on applying 200 supramaximal stimuli to the first and 256 supramaximal shocks to the second and third splanchnic nerves. At a high frequency of stimulation (30/sec to the first and 32/sec to the second and third nerves) the amounts of amine secreted per shock were 4*23 ng for the first and 1-22 and 0-85 ng for the second and third splanchnic nerves. This result is probably accounted for by the differences in the number of secretory cells innervated by the three nerves, for they diminish in size from above downwards. The results given in Table 1 also illustrate that the number of nerve fibres in the third splanchnic nerve is apparently too small to ensure significant temporal recruitment. On stimulation of the first and second splanchnic nerves, the amount of amine secreted at 30 or 32/sec was much greater than on stimulation at 10 or 16/sec, whereas there was only a small difference in the amount of amine secreted per shock on stimulation of the third nerve at 16 or 32/sec. Tests were also made in superfusion experiments to ascertain how the

15 ADRENAL MEDULLA 497 number of stimuli affected secretion. When stimuli were given as brief bursts to avoid fatigue (Fig. 7) there was an increase in the sympathin secreted which, when plotted, proved to be arithmetically and linearly related to the geometric increase in number of stimuli. A B Ad Ad Ad S S s 2Opug 10,ug 40,yg 1Osec 10sec 10sec Mll MmT Fig. 7. Rat-stomach strip, response to superfused blood from the carotid artery of a 4-2 kg cat, showing arithmetic increase in secretion of adrenal medullary hormones on geometric increase in the number of stimuli applied to the left second splanchnic nerve. A, calibrating doses of adrenaline i.v. (Ad). B, responses to excitation with 800, 200 and 400 supramaximal stimuli at 10/sec. Functional units within the adrenal medulla Young (1939) suggested on anatomical evidence that the ipsilateral splanchnic nerves innervate different portions of the adrenal medulla. On this assumption, the secretion on synchronous excitation of the first and second splanchnic nerves should considerably exceed that on exciting each nerve separately provided complete spatial and temporal recruitment was ensured. This was, in fact, observed, as shown in Fig. 8, which gives the sympathin activity of blood superfusing the rat-stomach strip after stimulation of the splanchnic nerves with 200 supramaximal stimuli (at C) and the activity of three doses of adrenaline (at A and B). The secretion on stimulating separately either the second or first splanchnic nerve was much less than on synchronous stimulation of both nerves, since the activity on stimulation of the second and first nerve corresponded to about 0-25 and 0 5,ug adrenaline, whereas that on synchronous stimulation of both nerves was equivalent to nearly 1 jtg adrenaline. The fact that the secretion on synchronous stimulation of both nerves was at least as great as the combined secretion on stimulating them separately is evidence 32 Physiol. 180

16 498 E. MARLEY AND GWENDA I. PROUT that occlusion of stimuli could only have been minimal. As supramaximal stimuli were given, the larger secretion obtained on synchronously exciting both nerves was not due to summation at the subliminal fringe. Ad Ad Ad S2 S1 S1 ±S2 0 54ug 1O0ug 075,ug S2.1 _ S 1 +S2 S21-8/sc Si +S2 '!1 + :3L 3JL/ Si +S2 Fig. 8. Rat-stomach strip; responses to superfused blood from the carotid artery of a 2-9 kg cat, showing separate innervation of secretory cells by the left first and second splanchnic nerves. A and B, calibrating doses of adrenaline (Ad) i.v. C, responses to supramaximal stimulation with 200 shocks (32/sec) applied either to the second (S 2) or to the first (S 1) left splanchnic nerve, or synchronously to both (Sl+S2). D, responses to prolonged stimulation of S2 (32/sec) until fatigue developed, and then superimposed stimulation of S 1 (32/sec). E, F, G, responses to stimulation at 8, 16 and 32/sec of S2 and then of both S1 and S2 together. Respective durations of supramaximal excitation given below traces. VL

17 ADRENAL MEDULLA 499 The results of the following experiments, although not providing evidence that the three splanchnic nerves innervate different portions of the adrenal medulla, at least show that they establish independent synapses with the medullary cells. When the second splanchnic nerve was excited until synaptic fatigue was complete so that sympathin secretion had ceased, then superimposed excitation of the first splanchnic nerve resulted in a vigorous sympathin discharge. This is shown in Fig. 8D. Another experimental procedure indicating independent synapses of the three splanchnic nerves was one of establishing secretion first with excitation of the second or third nerve and, whilst this excitation continued, to superimpose stimulation of the first splanchnic nerve. The results of such an experiment are illustrated in Fig. 8. The second nerve was excited with supramaximal stimuli at 8/sec (Fig. 8E), 16/sec (Fig. 8F), and 32/sec (Fig. 8G). When secretion was established, the first splanchnic nerve was excited as well with the same frequencies; this resulted in all three conditions in an increased secretion, which indicated activation of additional secretory cells. This experiment also illustrates an aspect mentioned before (p. 491), namely that the effectiveness of temporal recruitment depends on the number of secretory cells excited. On stimulation of the large first splanchnic nerve there was pronounced secretion of sympathin at all three frequencies, whereas on excitation of the smaller second splanchnic nerve the secretion was pronounced only at 32/sec, but negligible at 8 and 16/sec. Some overlapping in innervation by the three splanchnic nerves appears to take place. The evidence is derived from the results obtained on two series of experiments of partial chronic denervation of the medulla. In the first series, partial chronic denervation was produced by section of the second and third splanchnic nerves. Simeone (1938) had found that the secretion on stimulation of the first splanchnic nerve from such a partially denervated medulla was greater than that from the normally innervated contralateral medulla. He measured the secretion semiquantitatively by recording the contraction of the nictitating membrane. In the present experiments blood was removed from the adrenolumbar vein and assayed on the rat uterus and the rat blood pressure. The results which confirm those of Simeone are shown in Table 1. The values are the mean amine secreted per shock from fifteen control experiments in which the first splanchnic nerve was stimulated and from two experiments in which the nerve was stimulated fourteen days after dividing the remaining splanchnic nerves. At stimulation of 10/sec the secretion per shock increased from the control value of 1x8 to 3-89 ng in the denervation experiments; at 30/sec there was also an increase, but only from 4-23 to 5.43 ng. This increase implies that acetylcholine liberated at the pre- 32.2

18 500 E. MARLEY AND GWENDA I. PRO UT ganglionic terminals on exciting the first splanchnic nerve was acting on secretory cells sensitized by chronic denervation of the second and third nerves. In the second series of experiments the first splanchnic nerve was partially cut and days were allowed for degeneration. The mean amine secreted per shock on exciting the first splanchnic nerve was reduced, but much less than in those experiments in which the partially denervated first splanchnic nerve was stimulated immediately after hemisection. As shown in Fig. 4A, the secretion of the acutely hemisected nerve was about one-quarter that of normal. After chronic partial denervation, the reduction was about 50 % at 10/sec and about 25 % at 30/sec. This is shown in Table 1, where the mean secretions per shock from fifteen normal and from three partially denervated medullae are given for these frequencies of stimulation. Sustained excitation and fatigue Fatigue occurred earlier on sustained excitation of the smaller than of the larger splanchnic nerves, as was shown on the superfused rat-stomach strip by return of tone. It developed rapidly on exciting the third and fourth splanchnic nerves, but more slowly with the second and slowest with the first splanchnic nerve. Figure 9 illustrates the striking difference in the development of fatigue on stimulation of the fourth and first splanchnic nerves. On stimulation of the fourth nerve at 20/sec fatigue developed in 2 min (at A), whereas on stimulation of the first at 32/sec fatigue was only moderate after 60 mi (at B and (7). The difference is even more significant since the first splanchnic nerve was stimulated at the higher frequency and fatigue occurs more readily with fast than with slow excitation (Marley & Paton, 1961). On cessation of stimulation both nerves recovered within 5-8 min. Figure 10 shows a similar result on stimulation of the third and first splanchnic nerves. The histograms A and B are from experiments in different cats. The blood was removed from the adrenolumbar vein and assayed for adrenaline on the rat uterus and for noradrenaline on the rat blood pressure. Although the combined output of catecholamines was at least ten times greater on stimulation of the first splanchnic nerve, fatigue developed much more slowly than on stimulation of the third nerve. During the first 10 min of stimulation at 30/sec the amine secreted per shock was 0-2 ng on excitation of the first and ng on excitation of the third splanchnic nerve. On continued excitation of either nerve secretion dwindled until it was the same as that of the resting state. The fatigue which occurs on prolonged excitation of a sympathetic ganglion has been attributed by Perry (1953) to exhaustion of 'available'

19 ADRENAL MEDULLA 501 Min.77 C Ad Ad S4 S4 SI Nic * mg fig l.g Fig. 9. Rat-stomach strip; responses to superfused blood from the carotid artery of a 2*5 kg cat, showing the difference in the development of fatigue on supramaximal stimulation of the left fourth and first splanchnic nerves. A, at Ad calibrating doses of adrenaline i.v.; at S4 excitation of fourth splanchnic nerve (20/sec). Duration of excitation (shown above) was each time 5 min and was sufficient to produce fatigue. B and C, 70 min stimulation of first splanchnic nerve (S 1) at 32/sec. (20 min interval between the panels.) Before the end of stimulation, 0-25 mg nicotine (Nic) i.v. still releasing sympathin. z A *E , , S3 Si d 40 E 30 -E 20 I Minutes Minutes Histograms of secretion of adrenaline (upper records) and noradrenaline Fig. 10. (lower) in cats, to show difference in development of fatigue on supramaximal excitation at 30/sec of the third (S3) and first (S1) splanchnic nerves. Blood removed from the adrenolumbar vein and assayed for adrenaline on rat uterus and for noradrenaline on rat blood pressure. Solid bars, duration of excitation. A, 3*6 kg cat: excitation of S3 for 21 and then for 8 min. B, 2-8 kg cat: excitation of S 1 for 50 and then for 21 min. Ordinaot: adrenaline (Ad) and noradrenaline (Nad) in ng/min. Ab8ci8s8ae: time in minutes.

20 502 E. MARLEY AND GWENDA I. PROUT acetylcholine in the preganglionic nerve endings. A method of testing this theory would be to find out if the rate of onset of fatigue is dependent on the number of nerve fibres excited and thus on the amount of available acetylcholine. Therefore the effect of partially cutting the first splanchnic nerve on the onset of fatigue was studied. As shown in Fig. 11 the first splanchnic nerve did not fatigue with 25,000 shocks (at B), but after it had been partially divided fatigue occurred with 5000 stimuli (at C). A A Ad Ad Ad 2 0,ug 1 0,ug 4-0,ug C D Min Fig. 11. Rat-stomach strip; response to superfused blood from the carotid artery of a 4*2 kg cat, showing the effect of partial division of the first right splanchnic nerve on development of fatigue during prolonged supramaximal stimulation at 32/sec (durations shown above). Responses A to calibrating doses of adrenaline (Ad) i.v., B to 25,000 shocks, C to 5000 shocks applied above, and D to 25,000 shocks applied below the partial division. In B, C and D the locations of stimulating electrodes on the splanchuic nerve and in relation to the right suprarenal gland are shown below the tracings.

21 ADRENAL MEDULLA 503 further control was carried out by re-exciting the entire nerve between the hemisection and the adrenal gland. Again no fatigue occurred with 25,000 shocks (at D). The outcome of this experiment is in accord with the greater susceptibility to fatigue of the smaller splanchnic nerves. If onset of fatigue depends on exhaustion of 'available' acetylcholine then it should be more difficult to elicit after eserine. To demonstrate this it was first necessary to inject hyoscine into the cat to render the ratstomach strip insensitive to acetylcholine in the superfused blood. Otherwise an apparently opposite U.. result was obtained. For instance, in three A B _, Hyosc'ne Ad 1 0 g Eserine 2 mg kgd E02 mg kg 36 minf E} F Eserine t2:l ij Ad 0O4 mg kg Min 1-Oug Fig. 12. Rat-stomach strip; responses to superfused blood from carotid artery. Upper records, 4 kg cat, no hyoscine; lower, 2-6 kg cat with hyoscine, 2 mg/kg i.v. Effect of intravenous eserine on prolonged supramaximal excitation at 32/sec of left first splanchnic nerve; duration of excitation of splanchnic shown above traces. At Ad, 1.0,ug adrenaline i.v. B and D before and C, E and F after eserine i.v. in doses shown. Interval of 36 min between E and F. superfusion e-xperiments, with the injection of eserine alone into the cat, the strip relaxed at the beginning of excitation, but then quickly regained its resting tone much more rapidly than in experiments without eserine. One of these experiments is shown in Fig. 12B and C. The eserine in the blood was not the cause for the increased tone, since the strip relaxed well when adrenaline was subsequently injected or infused intravenously into the cat and the blood continued to superfuse the strip. The increased tone could be attributed to the muscle-stimulating action of the acetylcholine

22 504 E. MARLEY AND GWENDA I. PROUT released from the endings of the splanchnic nerve and protected from destruction by eserine. When this action of the released acetylcholine was prevented by repeating the experiments after administering hyoscine to the cat, the relaxation of the stomach strip by the superfused blood during splanchnic-nerve excitation was very prolonged and, as shown in Fig. 12 (at E and F), continued for some time after stimulation ceased. A similar observation had been made in the recruitment experiments (p. 493) for which short bursts of stimulation were used. This continued relaxation after stimulation indicates spontaneous activation of secretory cells due possibly to persistence of undestroyed acetylcholine in the adrenal medulla. This would imply a continuation for some time of the release of small amounts of acetylcholine after stimulation. The relaxation after stimulation may be a phenomenon similar to the asynchronous postganglionic activity of ganglia that develops in the presence of eserine after cessation of preganglionic excitation (Takeshige & Volle, 1962). Eserine not only delays fatigue but also greatly increases the amount of amine secreted on stimulation of the splanchnic nerves. This is shown in the experiment illustrated in Fig. 13 in which the blood from the adrenolumbar vein is assayed on the rat uterus and rat blood pressure. Secretion during the first 6 min corresponds to about 1700 ng/min amine, or 0-88 ng/ shock, whereas in the corresponding experiment of Fig. lob without eserine, about 555 ng/min amine or 0-31 ng/shock was secreted during the 13 min of maximal secretion. Thus, in the presence of eserine about three times the amount of amine are secreted per shock. Figure 13 also illustrates the continuation of secretion after stimulation. Secretion continued at a rate of ca. 200 ng/min during the next 10 min. The fatigue on prolonged excitation of the first splanchnic nerve has been ascribed to synaptic failure rather than to exhaustion of amine, because after such fatigue nicotine elicited vigorous sympathin discharge (Marley & Paton, 1961). Such an effect of nicotine is shown in the experiment shown in Fig. 9 C. The interpretation of this result, however, is open to the criticism that the first splanchnic nerve does not innervate all secretory cells. Therefore, the experiment was repeated by stimulating both the first and second splanchnic nerves, which innervate virtually all medullary cells (Young, 1939), until fatigue was established; the result was the same, i.e. intravenous injection of nicotine still elicited the secretion of considerable amounts of sympathin. In previous experiments (Biilbring & Burn, 1949; Marley & Paton, 1961) secretion of adrenaline seemed to fail before that of noradrenaline on stimulation of the first splanchnic nerve. In the present experiments, in which the blood from the adrenolumbar vein was assayed for adrenaline on the rat uterus and for noradrenaline on the rat blood pressure, no

23 ADRENAL MEDULLA 505 evideniee for such difference in the failure of secretion for the two amines was found, as slhow-n by the histograms of Fig. IOA and B. Secretion of botlh amines showed the same decline on prolonged stimulation. But there wras the following difference: on re-excitation after a pause of a few minutes, recovery of noradreinaline secretion developed appareiltly more rapidly tlhan that of adreinaliine. This w-as found in the abseince and presence of eserine and is slhown-n in thlie experiments of Figs. lob and 13, in wnn-hich the first splanclhniic nerve was re-excited 30 and 13 mini after a proloniged fatigue-producing stimutlation. 32/sec 32/sec Mlinutes Fig. 13. Histograml- of secretioin of noradrenialine (black) and adrenialine (white) on supramaximal excitationi at 32/sec of the left first splainchnic nierve in a 235 kg cat, showing the enihan-ced secretioni after ain intravenlous injection of eserine (0-2 mg/kg) given slowly before zero timne. Blood remox-ed froimi the adrenolumbar vein was assayed for adrenaline on rat uterus and for noradrenialine on rat blood pressure. Solid bars, dutration of exeitation. In the superfusion experiments of Figs. SD and 12 E the rapid relaxation of the superfused stomach strip on excitation of the first splanchnic nerve w-as followed by partial and transient, returin of muscle tone. This w-as frequently observed. It did not occur in superfusion experiments when cats were given a, slow initravenous infusion of either adrenaline or noradrenalinie or of both amines in equal amounts at a constant rate ( ag/min), no matter how sw-ift or great the initial relaxationl of the stomach strip. Therefore, the initial transient return of tone on splanichnic stim-ulation is a sign of diminished amine secretion. It may reflect a

24 506 E. MARLEY AND GWENDA I. PROUT reduced liberation of acetylcholine during this period as the result of the initial large release of acetylcholine, and later a partial recovery of the release. Or it may reflect a paralyzing action of the secretion by the large amounts of acetylcholine initially released and recovery from this paralysis as the release diminished. The fact that such transient return of tone was seen more often with 30 or 60 shocks/sec than with 8 shocks/sec favours perhaps the idea of a paralysing action of acetylcholine released in excessive Hexamethonium 1 mg/kg K.sK. K.s Mi A. Ad Hexamethonium 1-0,ag 1 mg/kg D E I 05,ccg 0-25 mg/kg 0-25 mg/kg Fig. 14. Rat-stomach strip; responses to superfused blood from carotid artery of two cats (upper record, 5 kg and lower record, 2-6 kg cat), showing effect of intravenous hexamethoniurm on secretion produced by supramaximal excitation of left first splanchnic nerve and by nicotine. At Ad, adrenaline i.v. in stated doses. B, responses to excitation of splanchnic nerve (30/sec) before and in C immediately after, and then, 15, 45 and 90 min after hexamethonium (1 mg/kg I.v.); durations of stimulations shown above (kymograph stopped for a few min at K.S.). E and F, responses to 0-25 mg/kg nicotine (Nic) i.v. before and after hexamethonium (1 mg/lkg i.v.). Ad Nic Nic amounts. Against this idea, however, is the finding that the return of tone was not more prominent in the experiments carried out after intravenous eserine (compare Figs. 12E and 8D). Finally, it was shown that an injection of acetylcholine into the superior mesenteric artery of an eviscerated cat during splanchnic stimulation, at the time the superfused stomach strip showed the return of tone, resulted in relaxation. This indicated that the secretory cells were not refractory to acetylcholine. There was no evidence that fatigue developed more readily after hexamethonium. Secretion due to stimulation of the first splanchnic nerve was

25 ADRENAL MEDULLA 507 considerably diminished and abbreviated (Fig. 14C) after intravenous hexamethonium (1 mg/kg), but the secretory effect of nicotine was equally affected (Fig. 14F). Therefore, the abbreviated secretion during stimulation of the splanchnic nerve, as shown by the rapid return of tone of the superfused rat-stomach strip, is fully accounted for by the normal waning liberation of acetylcholine at the preganglionic endings becoming progressively incapable of surmounting blockade. On recovery from the effect of hexamethonium, the secretion on splanchnic stimulation became not only progressively greater, but also more sustained (Fig. 14C). The same happened to the secretion elicited by nicotine. DISCUSSION The adrenal medulla has many properties of a sympathetic ganglion. Its innervation is by cholinergic fibres; splanchnic stimulation causes the release of acetylcholine in the medulla (Feldberg & Minz, 1933) and the secretion of the medullary hormones is prolonged by eserine (Feldberg, Minz & Tsudzimura, 1934). The action of acetylcholine on the medulla is mainly 'nicotine-like', as it is greatly reduced by nicotine (Feldberg et al. 1934). Methonium compounds of different chain lengths affect the medulla in the same way as they do sympathetic ganglia: hexamethonium (C6) is the most potent compound, whereas decamethonium (C 10) is ineffective (Marley & Paton, 1961). On chronic partial denervation supersensitivity develops as it does in autonomic ganglia (Simeone, 1938). The present experiments establish another kind of similarity with sympathetic ganglia in that spatial and temporal recruitments were found to occur. Each axon of the nerve plexus of the cat's adrenal medulla innervates a number of secretory cells, the secretory unit, and several neurones terminate on the secretory unit (Hillarp, 1946). The spatial and temporal recruitments found on stimulation of the splanchnic nerves are explained by this kind of innervation. The physiological importance of spatial and temporal recruitments would appear to vary according to the frequency of discharge in the splanchnic nerves. With excitation frequencies up to 8/sec, corresponding to those of physiological discharge in sympathetic vasomotor nerves (Folkow, 1952), it was found that spatial was at least as important as temporal recruitment. With higher frequencies, which may correspond to the nerve discharge in emergencies such as asphyxia or haemorrhage, temporal recruitment took precedence and was extremely efficient over a short period. Temporal recruitment was found to depend not only on adequate spatial recruitment, but also on the number of nerve fibres excited, for after partial division of the first splanchnic nerve, secretion was much

26 508 E. MARLEY AND GWENDA 1. PROUT reduced, particularly in the range in which temporal recruitment was most effective. Since with the higher frequencies of stimulation the gland becomes rapidly unresponsive (Marley & Paton, 1961), sustained or frequent sympathin discharge under normal conditions would depend on modulated activity in the splanchnic nerves. The greater efficacy of excitation at higher rates (30-60/sec) implies some kind of temporal summation of the depolarizing effect of acetylcholine at the synapse. Further, the persistence of acetylcholine at the synapse after eserine would not only account for the larger amounts of sympathin then secreted on stimulation of the splanchnic nerve, but also for the finding that in this condition temporal recruitment occurred at a slower rate of excitation than in the absence of an anticholinesterase. As a corollary, temporal recruitment was impaired by hexamethonium, which competes with acetylcholine at the synapse. By comparing the effects of excitation of the three splanchnic nerves it was possible to gauge how the number of preganglionic fibres and connexions with secretory cells determines secretion. The smaller the splanchnic nerve the higher its threshold, the slower the excitation rate for optimal secretion and the smaller the secretion. Apart from the number of fibres to the secretory cells, their diameter is important in determining secretion. If the smaller nerves contain mainly C fibres this would impose a lower limit for optimal excitation than for the larger nerves, which was in fact found. Even with frog myelinated nerve-muscle preparations, prejunctional failure occurred with excitation above 10/sec (Krnjevic6 & Miledi, 1958, 1959). Temporal dispersion of impulses may also be a limiting factor, at least in the first splanchnic nerve; conduction velocities vary from 75 m/sec for its A fibres to 1 m/sec in the C fibres (McLeod, 1958). Certain cells in the cat's stellate ganglion cease responding to excitation exceeding 40/sec, which was ascribed partly to impulse dispersion (Larrabee & Bronk, 1947). Another factor would be the refractory period of the synapse. If the properties of the adrenal medulla were to resemble those of autonomic ganglia, the refractory period of the neuro-glandular synapse would be msec (Bishop & Heinbecker, 1930) and its theoretical upper frequency response would be 30-50/sec. This, in fact, corresponded to the excitation range for optimal sympathin secretion of the first and second splanchnic nerves. Post-synaptic factors were found to affect secretion as well. The amine appearing in the adrenolumbar vein may represent the difference between the amount secreted and the quantity reabsorbed or taken up on receptors within the adrenal medulla. Paton (1960) suggested that in the adrenal medulla sympathin is the dominant intracellular cation, released when the membrane potential is reduced but later, when the events of excitation are

27 ADRENAL MEDULLA 509 over, partly sucked back, recovered, and returned to store. The results obtained with phenoxybenzamine and cocaine are relevant to this problem. The finding that secretion on stimulation of the splanchnic nerve was not enhanced by phenoxybenzamine, a competitive antagonist for the ac-receptor, would suggest that blockade of these receptors does not influence the uptake. On the other hand the finding that cocaine increased the output of amine on nerve stimulation suggests that normally an uptake of the released amine does occur and that this uptake is prevented by cocaine. As phenoxybenzamine does not have this action, it would appear that cocaine does not act on the membrane of the secretory cells but intracellularly, preventing replenishment of the store. The effect would appear to be more pronounced on the noradrenaline granules, since output of noradrenaline was found to be enhanced to a greater extent than that of adrenaline. The significance of the finding that cocaine was more effective in enhancing the output when stimulation was at the low frequency of 10/sec (in comparison to 30/sec) might imply that a relatively long time is essential for the uptake to be fully efficient. Finally, the delay of 30 min before cocaine was effective suggests slow equilibration with the noradrenaline store. Developmentally the adrenal medulla represents the condensation of chromaffine tissue from several segments and this would account for its multisegmental nerve supply. Young (1939) has suggested that the adrenal medulla was divided into anatomical units. With the physiological tests based on synaptic properties and used in the present experiments, the first and second splanchnic nerves were found to establish separate synaptic connexions with secretory cells. The secretion obtained on exciting synchronously the two nerves equalled the combined output on separate excitation, indicating that there was little subliminal fringe and that the nerves mainly innervated separate groups of secretory cells. Yet some secretory cells may be common to both nerves, as suggested by the hypersensitization obtained in chronic denervation experiments. Thus, the composite ancestry of the adrenal medulla is mirrored in its function. Elliott (1912, 1913) found that after prolonged stimulation of the splanchnic nerve the effectiveness of excitation was reduced, although medullary sympathin was not depleted. Unresponsiveness of the medulla after prolonged nerve excitation may be due to presynaptic, synaptic, or post-synaptic failure (depletion of amines) or to a combination of these factors. The fatigue obtained with prolonged stimulation at optimal or slower excitation rates of the first and second splanchnic nerves was most likely due to synaptic failure and could be due to impairment in the release of acetylcholine at the nerve terminals. Evidence in favour of the synapse as the site of failure was provided by the observations that fatigue de-

28 510 E. MARLEY AND GWENDA I. PRO UT veloped more readily under the following conditions: on stimulation with faster rather than with slow frequencies, on stimulation of the smaller rather than the larger splanchnic nerves, on repeated stimulation and on stimulation of the partly cut nerve. The rapid recovery from fatigue was also in favour of this view; but this occurred only when the intervals between the periods of stimulation were not too short. If they were < 5 min, secretion ultimately dwindled, although nicotine still caused secretion of the medullary amines. In contrast, after depleting the medulla of sympathin by repeated injections of acetylcholine Butterworth & Mann (1957) found that the sympathin was not replaced within 15 hr; it required 6-7 days. Further evidence linking fatigue with synaptic failure was the finding that fatigue was more difficult to elicit after eserine. SUMMARY 1. In cats anaesthetized with chloralose the secretion of the adrenal medullary catecholamines on stimulation of the first, second, third and fourth splanchnic nerves was studied. Secretion was measured either by the relaxation of the isolated rat-stomach strip superfused with blood from the carotid artery and/or from the adrenolumbar vein, or by assaying blood taken from the adrenolumbar vein for adrenaline on the rat uterus and for noradrenaline on the rat blood pressure. 2. With supramaximal stimuli at 1/sec it was possible to detect secretion of the medullary hormones with two stimuli applied to the first or second, and with five shocks applied to the third splanchnic nerve. With 200 stimuli at a frequency of 30-60/sec the threshold excitation intensity was 1-2 V. 3. With ten supramaximal shocks, secretion per stimulus corresponded to 5-10 ng amine. With 200 or 256 shocks at optimal excitation (30-60/sec), 3-5 ng amine was secreted per shock. With excitation for 10 min secretion declined to < 0 5 ng per stimulus; with even longer excitation secretion dwindled until it was the same as during the resting state. 4. Secretion depended on the size of the splanchnic nerve excited. On stimulation of the first splanchnic nerve the secretion was larger than that obtained with the second splanchnic nerve, which in turn exceeded that on stimulation of the third or fourth nerves. The optimal excitation rate was faster for the large than for the small splanchnic nerves. It was 30-60/sec for the first and second nerves and about 16/sec for the third and fourth. 5. Evidence was found for spatial and temporal recruitments. At slow rates of excitation, spatial recruitment was at least as important as temporal, but with faster rates of excitation temporal recruitment took

29 ADRENAL MEDULLA 511 precedence. The efficacy of temporal recruitment depended on the number of secretory cells involved. Spatial and temporal recruitment were diminished by hexamethonium and enhanced by eserine; stimulusresponse slopes remained parallel to those obtained before these drugs were given. 6. Cocaine increased the secretion obtained by stimulation of the first splanchnic nerve, but the effect on the output of noradrenaline was greater than on adrenaline. 7. Hexamethonium diminished the secretion of the medullary hormones produced by stimulation of the splanchnic nerves or by nicotine. 8. Stimulation through an electrode inserted into the medulla was much less effective in causing secretion than stimulation of the splanchnic nerves. 9. The three splanchnic nerves were found to end not through a plexus but separately and to innervate different groups of secretory cells with only a small overlap in innervation. 10. Fatigue depended on the number of secretory cells innervated and appeared to be of a synaptic nature. It developed more rapidly on sustained excitation of the smaller third and fourth than of the larger first and second splanchnic nerves. Further, it developed more rapidly on stimulation of the partly cut than of the entire nerve. 11. After eserine, secretion continued for some time after stimulation of the first splanchnic nerve had ended, and fatigue developed less readily than in the absence of the anticholinesterase. 12. On sustained stimulation of the first or second splanchnic nerves at 30-60/sec there was an initial vigorous secretion of the medullary hormones, then a transient period of diminished secretion, in turn followed by partial recovery of secretion. This happened also after eserine. These changes in the secretion of medullary hormones were attributed to corresponding changes in the release of acetylcholine from the splanchnic nerves during the sustained stimulation. This work was supported by the Medical Research Council. One of us (E. M.) is indebted to Professor G. V. R. Born and Dr J. R. Vane for providing facilities during the early part of the work. We are extremely grateful to Professor W. Feldberg, F.R.S., for his tremendous help in revising the script. We acknowledge gifts of halothane from Imperial Chemical Industries Ltd. Thanks are due to D. J. Allen Esq. for expert technical assistance. REFERENCES BIsHoP, G. H. & HEINBECKER, P. (1930). Differentiation of axon types in visceral nerves by means of the potential record. Amer. J. Physiol. 94, BRADLEY, P. B. & KEY, B. J. (1958). The effect of drugs on arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. clin. Neurophy8iol. 10,

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