6I :6I2.I83 BY ALISON S. DALE. concluded that the apparent vaso-constriction obtained by F r6 hli c h and

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1 6I :6I2.I83 A REVERSED ACTION OF THE CHORDA TYMPANI ON THE VENOUS OUTFLOW FROM THE SUBMAXILLARY GLAND. BY ALISON S. DALE. (From the Physiological Laboratory, Cambridcgel.) INTRODUCTORY. FROiHLICH and LOEWI [1906, 1908] claim to have shown that the chorda tympani nerve supplies the submaxillary gland with vaso-constrictor as well as vaso-dilator fibres. Their proof rests on the fact that, after inhalation of sufficient quantities of amyl nitrite, stimulation of the chorda diminishes the flow of blood from the submaxillary vein, instead of increasing it as in the normal animal. They conclude that vasoconstrictor fibres are present in the nerve, their action being normally masked by that of the more potent vaso-dilator ones, and that amyl nitrite, by a specific paralytic action on the vaso-dilator fibres, exposes the function of the vaso-constrictors. Doubt was thrown on these experiments by Bayliss [1908], who was unable to obtain the reversal of chorda action after amyl nitrite, and concluded that the apparent vaso-constriction obtained by F r6 hli c h and L o ewi was due to a spread of current into the surrounding voluntary muscles, causing them to contract and so to occlude the blood vessels. He mentioned another explanation, suggested to him by B arcroft, but did not test it experimentall,y. The suggestion was that, with the very low blood-pressure and dilated state of the blood vessels produced by amyl nitrite, stimulation of the chorda could not cause much further arterial dilatation, and might then cause an actual decrease of blood flow in the veins, owing to the abstraction of fluid from the blood to form saliva. Neither Fr6hlich and Loewi nor Bayliss had measured the saliva produced, Bayliss merely remarking that, on looking over his experimental results, he found B a r crof t's explanation insufficient. It was decided, therefore, to repeat the experiments, measuring the blood flow and the volume of the saliva, and to compare the decrease in the 1 The work here presented was carried out during tenure of the Bathurst Studentship of Newnham College, and the Michael Foster Studentship of the University of Cambridge.

2 450 A. S. DALE. volume of blood leaving the veins during chorda stimulation with that of the saliva secreted in the same period. METHODS. The animals used were cats. They were anaesthetized with chloroform and ether, and this was followed by injection of chloralose, the dose being 0.1 g. per kg. body weight. A cannula was placed in the trachea for the purpose of administering amyl nitrite, and also for artificial respiration when necessary. The left carotid artery was prepared for the registration of blood-pressure. The right submaxillary gland was next exposed, the submaxillary vein was found, and all other veins opening into the external jugular vein were tied. The chorda lingual nerve was prepared for stimulation in the usual way, and a fine glass cannula was inserted into the submaxillary duct. The cannula was connected by a short piece of rubber tubing to a horizontally fixed, graduated tube. A little water was injected into this tube to serve as an index of the flow of saliva. An intravenous injection of heparine was now given, the dose being about 0-01 g. per kg. body weight. The cannula was inserted into the carotid artery and connected to the manometer, and finally a cannula was tied into the jugular vein, and connected with a long graduated tube, fixed horizontally, for measuring the venous blood flow. The progress of the blood along the tube was continuously recorded by means of an electric signal writing on the drum, and another signal, writing immediately below, marked the beginning and end of chorda stimulation. The secretion of saliva often continued for a short time after stimulation had stopped. In such cases the moment when secretion stopped was recorded, and the time from the beginning of stimulation to this moment used in the calculations. The tube for recording the blood flow was divided into half-centimetres, and the signal was depressed as the blood passed each half-centimetre mark. The volume corresponding to a given length of tube had been determined beforehand by calibration with a standard pipette. This method is not free from error, but it was found to be sufficiently accurate for the purpose, and is, in any case, more exact than the method of counting drops used by Frohlich and Loewi. The amyl nitrite was administered intra-tracheally. RESULTS. The first point to be noted is that, in confirmation of Fr6hli ch and Loewi, it was always found possible to obtain the reversal of chorda

3 REVERSAL OF CHORDA ACTION. action on the venous outflow with administration of amyl nitrite. It is difficult to understand why it was never observed by Bayliss. An example of the effect of chorda stimulation on the venous flow before and during amyl nitrite action is given below. Exp. 4. Cat: 2 kg. (a) Before amyl nitrite. Venous flow before stimulation: c.c. per min. Venous flow during stimulation: 2-8 c.c. per min. (b) During amyl nitrite inhalation. Venous flow before stimulation: 1-5 c.c. per min. 451 Duration of stimulus: 24 sec. Venous flow during stimulation: c.c. Before stimulation the flow during 24 sec. was 0-6 c.c. The flow therefore decreased during stimulation by 0075 c.c. Volume of saliva secreted in response to this stimulus: c.c. It is evident from this example that, during the action of amyl nitrite, the effect of chorda stimulation on venous outflow is, indeed, reversed. A comparison, however, of the decrease in the volume of venous flow with the volume of saliva secreted in the same time shows that, in this case, the decrease in the flow can be accounted for by abstraction of fluid from the blood to form saliva. In order to prove the existence of vaso-constrictor fibres in the chorda tympani it would be necessary to show a decrease in the blood flow greater than the volume of saliva secreted. In all, six experiments were made in which amyl nitrite was used, and in these, twelve instances of reversal of the chorda effect were obtained. In two out of these twelve instances the decrease in blood flow was greater than could be accounted for by the saliva secreted. Both these cases come from the same experiment. In one (Table II, No. 7) the difference is so small as to be of doubtful significance, in view of the comparatively rough methods of measurement. In the other case (Table II, No. 5) the difference is larger; on the other hand, a third stimulation in this experiment (Table II, No. 6) produced a decrease which was less than the volume of saliva secreted. It would clearly not be justifiable to conclude from these two exceptions that the chorda contains vaso-constrictor fibres. If the action of amyl nitrite in producing this apparent reversal of chorda action is due, as suggested, to the fact that it has already produced an almost maximal arterial dilatation in the gland before the chorda is stimulated, it should be possible to obtain a similar reversal when the vaso-dilatation is produced by other means. Further experiments were, therefore, carried out in which the blood-pressure was

4 452 A. S. DALE. lowered by over-ventilation and by injection of histamine. It was found that reduction of the blood-pressure in either of these ways produced a reversal of the chorda effect on venous outflow. In one experiment another method of producing this reversal was found. The blood-pressure was very low, owing to a slight overdose of chloralose, and the cat had to be kept alive by artificial respiration. A first stimulation of the chorda produced an acceleration of the venous flow in the usual way. It was found, however, that the vaso-dilator action persisted long after the stimulation was ended and the salivary secretion had stopped. If the chorda was now re-stimulated, during this phase of persistent vasodilatation, the venous outflow was immediately diminished. Table I gives an example of each of the three types of experiment. TABLE I. Dura- Normal tion Calcurate of lated of flow stimu- normal Actual De- (c.c. per lation flow flow crease Saliva Example min.) (sec.) (c.c.) (c.c.) (c.c.) (c.c.) 1 Over-ventilation Histamine Post-stimulatory vaso-dilatation Examples 1 and 2 from Exp. 8. Example 3 from Exp. 9. In view of these results, it is impossible to conclude with Fr6hlich and Loewi that amyl nitrite exerts a specific paralytic action on the vaso-dilator fibres of the chorda tympani. As regards the true mechanism of the reversal effect, the results obtained give very strong support to the suggestion made by Barcroft and quoted by Bayliss. Ten experiments in all were performed, giving twenty-nine instances of reversal of chorda action. Of these twenty-nine instances there are only two in which the decrease in blood flow is definitely greater than can be accounted for by the saliva secreted. One of these has been mentioned already, and the other (Table II, No. 24) occurred in Exp. 9. (Restimulation during post-stimulatory vaso-dilatation.) Taking the results in general we may say that the decrease in blood flow during stimulation and the volume of saliva secreted are always of the same order of magnitude. In Table II these values for the whole series of experiments are given. It will be seen that, in the majority of cases, the volume of the saliva secreted is greater than the decrease in the volume of blood leaving the gland; such a difference is easily explained on the ground that a small vaso-dilatation is still produced by the stimulus.

5 REVERSAL OF CHORDA ACTION. 453 TABLE II. Decrease Decrease Decrease in in in venous venous venous flow Saliva flow Saliva flow Saliva No. (C.C.) (C.C.) No. (c.c.) (c.c.) No. (C.C.) (C.C.) Nos and No. 21 during amyl nitrite administration. Nos during over-ventilation. Nos and Nos. 28, 29 during action of histamine. Nos during post-stimulatory vaso-dilatation. Another point, mentioned by Fr6hlich and Loewi, is that the vasoconstrictor fibres are paralysed by atropine; that is, that the decreased venous outflow produced by chorda stimulation during the action of amyl nitrite does not occur when atropine is given as well. This observation was confirmed in the present series of experiments, but does not support Frohli ch and Lo ewi's theorv of vaso-constrictor fibres. It is well known that atropine, in moderate doses, paralyses the secretory action of the chorda, leaving the vaso-dilator action unaffected. It is obvious that, if the reversal of the chorda effect produced by amyl nitrite is dependent on secretion of saliva, paralysis of the secretion will eliminate the apparent vaso-constriction. Frohlich and Loewi also mention a case, that of a dog, in which the reversal effect was present at the beginning of the experiment without any nitrites being given. This result would be readily explained if the blood-pressure were very low, owing to an excess of anaesthetic or some other cause. While, then, the experiments here recorded do not eliminate the possibility that the chorda tympani may contain some vaso-constrictor fibres, it may be claimed that they furnish an alternative and adequate explanation of the retarded venous outflow, observed by Frohlich and Loewi, with chorda stimulation under amyl nitrite action. When the arterial tone is lowered in any way, so that the chorda no longer produces effective arterial dilatation, loss of fluid to the saliva reduces the volume of blood reaching the veins.

6 454 A. S. DALE. SUMMARY. 1. The fact, observed by Fr6hlich and Loewi, that stimulation of the chorda tympani during the action of amyl nitrite may retard the venous outflow from the submaxillary gland, is confirmed. 2. Lowering of the arterial tone by other means produces a similar effect. 3. The diminished venous outflow is accounted for by loss of fluid from the blood to the saliva. 4. The action of amyl nitrite affords no adequate evidence of vasoconstrictor fibres in the chorda tympani. In conclusion I wish to express my thanks to Prof. Bar cro ft for permission to carry out these experiments in his laboratory, and to Dr Anrep for his help throughout. REFERENCES. Bayliss (1908). J. Phy8iol. 37, 256. Frohlich and Loewi (1906). Zbl. Phy8iol. 20, 229. Frohlich and Loewi (1908). Arch. exp. Path. Pharmak. 59, 64.