cannula, but rarely when the needle was extended beyond the tip of days. It is therefore thought that an unknown pyrogenic factor

Transcription

1 J. Phy8iol. (1970), 207, pp With 6 text-figure8 Printed in Great Britain PERFUSION FROM CEREBRAL VENTRICLE TO CISTERNA MAGNA IN THE UNANAESTHETIZED CAT. EFFECT OF CALCIUM ON BODY TEMPERATURE BY W. FELDBERG, R. D. MYERS* AND W. L. VEALEt From the National Institute for Medical Research, Mill Hill, London N. W. 7 (Received 10 September 1969) SUMMARY 1. A method is described for the perfusion of the liquor space from a lateral cerebral ventricle to the cisterna magna in the unanaesthetized cat. Perfusions were carried out for min using various physiological salt solutions whilst rectal temperature was recorded. 2. When the salt solution used contained calcium in the physiological concentration, rectal temperature remained unchanged, but when it contained no calcium an intense hyperthermia developed during the perfusion. The finding that calcium must be present in the perfusion fluid for preventing temperature from rising may lead to a new understanding of the working of the 'set-point' in the control of body temperature and of the mechanism of action of pyrogens. 3. Independent of the nature of the perfusion fluid a long-lasting late rise in temperature developed after a perfusion. This happened regularly when the infusion needle was inserted only into the hub of the ventricular cannula, but rarely when the needle was extended beyond the tip of the cannula shaft and the cannula had been flushed out during the preceding days. It is therefore thought that an unknown pyrogenic factor present in the lumen of the cannula and washed into the ventricle with the perfusion fluid is responsible for this effect. 4. The effluent collected from the cisterna contracted the fundus strip of the rat stomach. As the contractions were little affected by BOL, they are attributed mainly to an action of prostaglandin E1 and not to an action of 5-HT. There was no difference in the biological activity of the effluent whether the perfusion fluid contained calcium or not. * Supported in part by U.S.A. Office of Naval Research Contract N A , National Science Foundation Grant GB 7906 and by a grant from the Wallace Laboratories. Permanent address: Laboratory of Neuropsychology, Purdue University, Lafayette, Indiana, U.S.A. t David Ross Fellow of the Purdue Research Foundation. Permanent address: Laboratory of Neuropsychology, Purdue University, Lafayette, Indiana, U.S.A.

2 404 W. FELDBERG, R. D. MYERS AND W. L. VEALE INTRODUCTION Recently, methods have been developed to perfuse the liquor space of the brain from a lateral ventricle to the cisterna magna in unanaesthetized animals. This was first achieved in goats by Pappenheimer, Heisey, Jordan & Downer (1962), who implanted two guide tubes chronically in such a way that the tip of one rested above a lateral ventricle and the other above the cisterna magna; for perfusion, the ventricle and cisterna were each punctured with a sharp needle inserted through the guide cannulae. The same procedure has since been used to perfuse the cerebral ventricle in unanaesthetized dogs (Ashcroft, Dow & Moir, 1968), rabbits (Dow & Moir, 1969) and sheep (Barton, Bligh & Sharman, 1969). In the present experiments a similar method is described, for the perfusion in unanaesthetized cats. Using this method the effects of various perfusion fluids on body temperature have been examined. METHODS Cats of either sex, weighing between 2-8 and 3-5 kg, were used. In an aseptic operation under pentobarbitone sodium (36 mg/kg) anaesthesia, one Collison cannula was permanently implanted into the left lateral ventricle, and another, the cisternal cannula, was positioned and permanently fixed to the back of the skull so that its opening rested above the atlanto-occipital membrane. The cisternal cannula did not pierce the membrane, but served as a guide through which a hypodermic needle could be lowered and inserted through the membrane into the cisterna. This was done, after recovery from the operation, each time a perfusion of the cerebral ventricles was carried out. For cannulation of the lateral ventricle the cannula was screwed into the skull at a point 3 mm lateral to the mid line and 7 mm posterior to the coronal suture. The stainless-steel shaft of the cannula was 12 mm long and open ended. The length of the cisternal guide cannula was 32 mm, and that of its stainless steel shaft 18 mm. To fix the cannula in the position shown diagrammatically in Fig. 1, a mid line incision about 3 cm long was made at the back of the neck, and the underlying muscles were separated and retracted. The interparietal and the supra-occipital bones were scraped clean and two small stainless steel screws were inserted into each of these bones on either side of the mid line. From the lower end of the supra-occipital bone which forms the upper border of the foramen magnum, a small semicircular piece was removed in the mid line with nibbling forceps, and the cannula was positioned so that its tip passed through the semicircular opening and rested about 1 mm above the visible atlanto-occipital membrane. The cannula was then fixed to the bone with acrylic dental cement anchored by the four screws, and penicillin was sprinkled into the wound before the neck muscles and the skin were sutured. After the operation, the cats were given an intramuscular injection of 100 mg chloromycetin, daily for 3 days, and then used for perfusion of the ventricles which was carried out not more than twice a week. For each perfusion, the cat was removed from its cage, placed upon an operating table and observed, usually for half-an-hour, before the cisterna was punctured by a hypodermic needle inserted through the cisternal guide cannula. During this time the

3 CALCIUM AND BODY TEMPERATURE 405 cat was restrained as little as possible. The hypodermic needle used for puncturing the cisterna was of 25 gauge and 5 cm long; its hub had been removed and the free end was connected by a 30 cm length of poly6thylene tubing to a 1 ml. syringe; the needle, tubing and syringe were filled with 0 9 % (w/v) saline solution. When the tip of the needle had entered the cisterna and the syringe was removed, drops of clear fluid flowed from the tubing at a rate dependent upon the level at which the open end was held. It was kept approximately at the level of the cisterna. Once outflow was established, infusion was begun by inserting a hypodermic needle through the rubber diaphragm of the ventricular cannula. The needle was connected to a slow infusion pump by a 60 cm length of nylon tubing and inserted either into the hub or through the entire shaft, 1 mm beyond its tip, so that the cannula served as a guide I.P. Ar Fig. 1. Diagram to show position of the cisternal guide cannula with its tip resting about 1 mm above the atlanto-occipital membrane (M). The area between the skull and the interrupted line represents the acrylic cement by which the cannula is permanently fixed and anchored by four small screws (two being shown) to the back of the skull. I.p.: interparietal bone; S.o.: supra-occipital bone; C: occipital condyle; A: atlas. in the same way as the cisternal cannula. Perfusion was at a constant rate of 0.1 ml./ min and was usually continued for 30 min. A volume of about 3-5 ml. effluent was collected during this time. The sample of effluent was kept on ice and assayed against 5-HT on the rat stomach fundus strip according to the method described by Vane (1957). The fluid used for perfusion was either 0 9 % NaCl solution, or a solution containing 8-7 g NaCl, g KCI and 0-75 ml. 1 M-CaCl2 (equal g) in 1000 g, or the artificial c.s.f. of Merlis (1940). Its composition was (g/l.): NaCl 8 1; KCI 0-25; CaCl2 0 14; MgCl2 0 11; NaHCO3 1 76; Na2HPO4 0 07; urea 0-13, and glucose The NaCl solution was pyrogen-free and the other salt solutions were made up with

4 406 W. FELDBERG, R. D. MYERS AND W. L. VEALE pyrogen-free distilled water. The glassware as well as the nylon tubing attached to the perfusion pump were also pyrogen-free. In some experiments pooled c.s.f. collected from the cisterna of three unanaesthetized cats with implanted cisternal cannulae was used as the perfusion fluid. From each cat 1-2 ml. c.s.f. was removed with a hypodermic needle introduced through the guide cannula into the cisterna. Either the hypodermic needle had its hub removed and the outflowing drops from a tubing attached to the free end of the needle were collected, or the intact hypodermic needle was used to withdraw c.s.f. into a syringe. Spread of perfu8ate in the liquor space8. In order to find out which parts of the cerebral ventricles and of the subarachnoid space were reached by the fluid perfused from a cannulated lateral ventricle to cisterna, a bromophenol blue solution was used for the perfusion. From the insoluble acid obtained commercially (British Drug Houses Ltd.) in powder form, the water soluble sodium salt of bromophenol blue was prepared as described by Feldberg & Fleischhauer (1960), and a % solution was made up in artificial c.s.f. When this solution was perfused from the lateral ventricle to cisterna at a rate of 0 05 ml./min the dye appeared in the cisternal outflow tube after about 3 min. Perfusion was continued for another 27 min and was followed by perfusion with artificial c.s.f. for a few minutes whilst the cat was anaesthetized by an intraperitoneal injection of pentobarbitone sodium (36 mg/kg). A solution of 10% formaldehyde was then perfused through the head of the cat from the cannulated aorta and after removal of the brain and spinal cord, the spread of the perfusion fluid in the subarachnoid space was ascertained from the staining of the pia and surface of the brain and cord. The ventricles were then opened to find out which parts of their walls had become stained. Rectal temperature. A thermistor probe (Yellow Springs Instrument) was inserted about 10 cm into the rectum and held in position by adhesive tape affixed to the protruding end of the probe and gently wrapped around the base of the tail. Temperature was monitored continuously by a Kent multichannel recorder. The figures reproduced in this paper are plotted directly from the tracings obtained in this way. RESULTS When the cats were removed from their cages in which they had usually been lying curled up and asleep, some cats became playful, others tried to move about and again others were somewhat agitated. In several cats there was some shivering and rectal temperature rose about half a degree. The insertion of the needle into the cisterna and the perfusion itself produced no signs of discomfort. Whether body temperature was affected by the perfusion depended not only on the composition of the perfusion fluid but also on the method used for inserting the inflow needle. Fig. 2 shows the results obtained with the inflow needle inserted into the hub of the ventricular cannula. On perfusion with natural or artificial c.s.f. or with a salt solution NaCl, KCI and CaCl2 in physiological concentrations, no change in temperature occurred during the half hour perfusion, or there was a slight rise only. This is shown in records A to C. However, when a 0 9 % NaCl solution was used for the perfusion the cat began to shiver vigorously within a few minutes, and the ear vessels became constricted shortly after. Temperature rose

5 CALCIUM AND BODY TEMPERATURE 407 steeply and continued to do so during the entire period of perfusion. The rise often attained a rate of 0-10 C/min and at the end of a 30 min perfusion, temperature often exceeded 41 and sometimes even 42 C. Fig. 2 D illustrates the intense hyperthermia produced by a 30 min perfusion with 09 % NaCl solution. During the last minutes of such a perfusion AC 39 _ I U I I _ -C 40> 39~~ ~~~~~~ Hours Fig. 2. Records of rectal temperature from two cats (cat record A and C, 3*2 kg, cat record B and D, 3 0 kg). At the arrows removal of cats from cage. Between the interrupted lines perfusion from hub of ventricular cannula to cisterna with natural c.s.f. (A), artificial c.s.f. (B), salt solution containing NaCl, KCl or CaCl2 (C), and 0 9 % NaCl solution (D). the cats became lively and alert, the ear vessels again dilated, the respiratory rate increased to over 150/min and there were sometimes short periods of panting. After the perfusion, temperature continued to rise for a few minutes, then fell steeply whilst the rate of respiration declined and shivering decreased; usually shivering stopped within 15 min. Apart from the rise which occurred during the perfusion with 0-9 %

6 408 W. FELDBERG, R. D. MYERS AND W. L. VEALE NaCl solution, late after-rises were recorded in all experiments of Fig. 2 regardless of the perfusion fluid. As seen from the four records, temperature began to increase 1-1l hr after the end of the perfusion, continued to rise for 1-2 hr and remained high for several hours. The extent of this hyperthermia which was caused by shivering and skin vasoconstriction varied greatly, even in the same cat on repeated perfusion. Particularly oc A 42 I g ' I 2II I I I I 41 - i i 39 I Igi I 1 39 Hdou rs Fig. 3. Records of rectal temperature from same cat as record A and C of Fig. 2. At the arrows removal of cat from cage. Perfusion from hub of ventricular cannula to cisterna with artificial c.s.f. (between the first two dotted lines of record A and between the interrupted lines of record B) and with 0-9 %/ NaCl solution (between the third and fourth interrupted line of record A). large rises are show-n on records A and B of Fig. 3 which were obtained from the same cat as records A and C of Fig. 2. In the experiment of record A, Fig. 3, artificial c.s.f. was first perfused for 30 min. This produced a small rise which was insignificant in comparison with the rise to over 42 C during the subsequent perfusion, also for 30 min, with 0 9 %/ NaCl solution. After the end of this second perfusion, temperature fell steeply, but an hour later, before it had returned to the pre-perfusion level, it rose

7 CALCIUM AND BODY TEMPERATURE 409 again and remained near 420 C for the next few hours; after another 18 hr it had however returned to the pre-perfusion level. Record B of Fig. 3 was obtained on another day. This time there was no rise but a slight fall in temperature during the 30 min perfusion with artificial c.s.f., but afterwards temperature rose again to 410 C after the usual latency. The after-rise occurred in all experiments in which the inflow needle was inserted into the hub of the ventricular cannula; it was thought to be due to a pyrogenic factor present in the cannula and washed into the ventricle oc \ Hours Fig. 4. Records of rectal temperature from two cats (cat record A, 3-2 kg; cat record B, 3 0 kg). At the arrows removal of cats from cage. Between the interrupted lines perfusion from infusion needle, inserted through cannula directly into the left lateral ventricle, to cisterna with artificial c.s.f. (record A) and with 0.9% NaCl solution (record B). by the perfusion fluid. Usually, though not always, this rise did not occur when the inflow needle was inserted through the entire length of the shaft directly into the ventricle, and when, as a further precaution, the hub and shaft were washed out with 0-2 ml. artificial c.s.f. 48 and 24 hr before the perfusion. Results obtained with this procedure are shown in Fig. 4. On perfusion with artificial c.s.f. temperature remained virtually unchanged, not only during the perfusion but also during the subsequent 5 hr (record A). On perfusion with 0 9 % NaCl solution temperature rose to C during the 40 min perfusion and returned to the pre-perfusion level in 1 hr but did not rise again in the following 3 hr (record B). In several experiments an intraventricular injection ofas little as 0 15 ml. artificial c.s.f. into the hub of the cannula produced a long-lasting rise after a latency of about 12 hr. This result provides additional evidence for the presence within the cannula of a pyrogen which is washed into the ventricle.

8 410 W. FELDBERG, R. D. MYERS AND W. L. VEALE Since intense hyperthermia was produced during the perfusion with an isotonic solution of NaCl, whereas perfusion with a solution containing also KCI and CaCl2 in physiological concentrations did not affect temperature, the question arose as to which of the two cations was responsible for the suppression of the hyperthermic response. From the results of the two experiments of Fig. 5, it is evident that calcium exerted this effect. In both experiments perfusion was carried out with the inflow needle inserted through the shaft of the cannula and its tip resting in the ventricle. C A i 41 I J Hours Fig. 5. Records of rectal temperature from a 2-8 kg cat. At the arrows, removal of cat from cage. Between the interrupted lines perfusion from infusion needle, ilserted through cannula directly into the left lateral ventricle, to cisterna with a salt solution containing NaCl and KCI (record A) or NaCl and CaCl2 (record B). When the perfusion fluid contained 0-87 % NaCl and % KCI, temperature rose during the perfusion (record A), and the hyperthermia was indistinguishable from that obtained with perfusion of NaCl alone. On the other hand, when the perfusion fluid contained 0-87 % NaCl and % CaCl2, no hyperthermia occurred during the perfusion (record B). Staining of brain and spinal cord after perfusion with bromophenol blue. From the staining of the pia and surface of the brain and spinal cord after a 30 min perfusion with bromophenol blue it was evident that some of the perfusion fluid had passed from the cisterna into the subarachnoid space surrounding the brain stem, and as far caudally as the thoracic cord. However, it had not entered the fluid space surrounding the cerebrum.

9 CALCIUM AND BODY TEMPERATURE 411 After the perfusion with bromophenol blue the brain stem was deeply stained as far as the optic chiasma on its ventral surface and as far as the corpora quadrigemina on its dorsal surface. The cervical cord was deeply stained both on its dorsal and ventral surfaces. The staining was less intense in the upper thoracic cord; it faded and gradually disappeared in the lower thoracic cord. Vermis and some patches of the ventral surface of the cerebellum were deeply stained but neither the dorsal, the lateral nor the ventral surface of the cerebrum was stained. There was also no staining on the surface of the olfactory bulbs which showed that the perfusate TABLE 1. Contractions expressed as ng/ml. 5-HT, produced by cisternal effluent on fundus strip of rat stomach Perfusion fluid NaCl Cat no. NaCl NaCl KCI NaCl CaCi2 KCI CaCl2 Artificial c.s.f. Natural c.s.f * * 0* * ' * * *4 * Same samples as in Fig. 6. had not passed anteriorly along the mid line of the base of the brain. When the cerebral ventricles were opened the walls of the fourth, third and of both lateral ventricles were found to be deeply stained. Assay of perfusate. The effluent collected from the cisterna contracted the fundus strip of the rat stomach regardless of what fluid had been used for the perfusions. The contractions were assayed against those produced by 5-HT, and the 5-HT equivalents of 1 ml. effluent obtained from twentyone separate perfusions in five cats are given in Table 1. The 5-HT values varied between 0 4 and 2 ng/ml., even when perfusions were repeated on the same cat with the same kind of perfusion fluid. For instance, in cat No. 1, the values obtained from three perfusions carried out on different days with a 0 9 % NaCl solution were 2, 1 and 0-6 ng/ml., and in cat 2, the values obtained from four separate perfusions with artificial c.s.f. varied between 0 4 and 0 9 ng/ml. Values of over 1 ng/ml. were obtained only when the perfusion fluid contained no calcium, and the mean value from

10 412 W. FELDBERG, R. D. MYERS AND W. L. VEALE eight such perfusions was 1 1 ng/ml. as compared to the mean value of 0-6 ng/ml. from the other thirteen experiments of Table 1 in which the perfusions were carried out with solutions containing calcium. The contractions produced by the effluent were not due, or were due only in part, to 5-HT since they were not affected, or only somewhat reduced after treatment of the fundus strip with BOL. This is illustrated in Fig Fig. 6. Fundus strip of rat stomach suspended in 5 ml. Krebs solution. Between the panels 10 ng BOL added to and kept in the bath for 10 min. Responses to 0 5 ng 5-HT at 2, 5 and 8, to effluent from cat No. 4 of Table 1, 0 5 ml. at 1, 0-25 ml. at 3, and 0 3 ml. at 6, and to 0 5 ml. effluent from cat 1 of Table 1 at 4 and 7. for effluent obtained from two of the experiments given in Table 1. The first (at 1) and the third (at 3) contractions were produced by 0*5 and 0-25 ml. respectively of effluent collected from cat No. 4 during perfusion with a solution of NaCl and KCI; the second contraction (at 2) was produced by 0 5 ng 5-HT. After the fundus strip was treated with BOL, 0 5 ng 5-HT added to the bath no longer contracted the fundus strip (at 5 and 8) whereas the contraction produced by 0 3 ml. of the effluent (at 6), was a

11 CALCIUM AND BODY TEMPERATURE 413 little greater than that previously obtained with 025 ml. (at 3). Thus the contraction produced by this effluent was not reduced by BOL. On the other hand, the contraction produced by effluent collected from cat No. 1 during its second perfusion with artificial c.s.f. was somewhat reduced after BOL, as is evident from the contraction produced by 05 ml. before (at 4) and after treatment of the fundus strip with BOL (at 7). In a few experiments c.s.f. was removed from the cisterna and tested on the fundus strip. In each instance it produced a contraction and when assayed against 5-HT the effect of 1 ml. c.s.f. was found to correspond to that of 05-2 ng. Twice, samples of c.s.f. were removed from each of two cats of Table 1 (Nos. 2 and 4). When assayed against 5-HT the contractions of the two samples from cat No. 2 corresponded to 0 8 and 0 8 ng/ml. and from cat No. 4 to 05 and 0-8 ng/ml. Further samples were twice removed from each of two other cats and the corresponding values were 0 5 and 1-25 ng/ml. for one cat and 2 and 2 ng/ml. for the other. The contractions produced by the c.s.f., like those produced by perfusate, were not due to 5-HT since they were not abolished by treatment of the fundus strip with BOL or at most were somewhat reduced. DISCUSSION In all perfusion methods in which fluid delivered into a lateral ventricle is collected from a cannulated cisterna, the perfusion is not that of a closed fluid system because the cisternal cannula is in open contact with the entire subarachnoid space. Thus, the effluent will have an admixture of cerebrospinal fluid, a fact which has to be taken into account when determining the presence in the effluent of substances which occur naturally in the c.s.f. On the other hand, some of the perfusion fluid will not be collected in the effluent but will escape into the subarachnoid space. Therefore, when these methods are used for the perfusion of drugs through the cerebral ventricles, their actions may not be limited to structures in the ventricular walls and to those in the medulla lying in the direct pathway from the foramina of Luschka to the opening of the cisternal cannula. From the staining observed when the dye bromophenol blue was added to the perfusion fluid, it became evident that drugs perfused in this way may spread through the subarachnoid space caudally to the lower thoracic cord and cranially along the ventral surface of the brain as far as the optic chiasma. These are the disadvantages inherent in a perfusion from ventricles to cisterna in contrast to a perfusion from ventricles to aqueduct in which the fluid system is closed, and drugs can be perfused in such a way as to limit their actions even to parts of the ventricular cavities (Carmichael, Feldberg & Fleischhauer, 1964). However, such perfusions have thus far been

12 414 W. FELDBERG, R. D. MYERS AND W. L. VEALE possible only in anaesthetized cats whereas the method described in the present paper has the advantage that the perfusion can be carried out without the use of an anaesthetic. The finding that a physiological salt solution which contains calcium chloride can be perfused through the cerebral ventricles without affecting body temperature, whereas intense hyperthermia develops during a perfusion with a fluid that lacks calcium but contains either sodium chloride alone or sodium and potassium chloride, is of interest to the problem of the function of the 'set point' and of the mechanism of action of pyrogens. In homoeothermic animals body temperature remains relatively constant throughout life in spite of the many nervous influences which act on the thermoregulatory system in the hypothalamus and tend to raise or lower temperature. The hypothalamus has therefore a 'built-in mechanism' which controls the level at which temperature is to be set and maintained. It is called the 'set-point' and the concept of a set-point is based on the work of Leibermeister (1875). The functioning of the set-point is usually explained by analogies with models taken from physics. An explanation more in physiological terms may be derived, however, from the hyperthermia obtained with the perfusion of a calcium-free salt solution. This hyperthermia emphasizes the role ofcalcium in preventing temperature from rising to fever level and suggests a correct level of calcium or its permeability in the hypothalamus as the physiological basis of this set-point. According to this view any factor whichwould lower the level of calcium or prevent its actionwithin this structure should result in a higher set-point being maintained, and this may also be the mechanism by which pyrogens act. At present, however, with only one fact so far possible, namely that the fluid bathing the hypothalamus must contain calcium in order to prevent pyrexia, it is not possible to explain in detail the working of the set-point in terms of ionic changes, particularly of calcium ions, or to explain how pyrogen produces an ionic change, similar in its effect to that brought about by lack of calcium ions in the perfusion fluid. Yet this new approach may be a more rewarding one to pursue than to fall back on models taken from physics. On perfusion with calcium-free solution there was not only a rise in temperature but also an increase in the rate of respiration, and the cats became lively and more alert. In this connexion it is interesting to note that calcium-free solutions perfused through the lower spinal subarachnoid space of anaesthetized dogs were found by Merlis (1940) to augment the spinal flexor reflex, toi increase muscle tone and to elicit spontaneous twitching of the muscles of the lower half of the body. The long-lasting late rise in temperature which occurred following a perfusion did not depend on the nature of the perfusion fluid and is attributed to an unknown pyrogen. As this late rise usually did not occur when the

13 CALCIUM AND BODY TEMPERATURE 415 injection needle was inserted through the whole length of the shaft of the cannula and 1 mm beyond its tip, instead of into the hub of the cannula, and when as a further precaution hub and shaft were flushed out 48 and 24 hr before a perfusion the pyrogen appears to originate in the fluid within the cannula. However, the possibility cannot be excluded that a pyrogenic factor is also released during the injection from the tissue surrounding the tip of the cannula. This factor could be responsible for the rise in temperature which sometimes occurred in spite of all precautions taken. The finding that a late rise in temperature may also occur with an injection of as little as 0*15 ml. fluid into the ventricular cannula has to be taken into account when the effects of intraventricular injections of drugs on temperature are studied and when these injections lead to a late longlasting rise. The contractions produced by cisternal effluent on the fundus strip of the rat stomach can only to a small degree be due to an action of 5-HT as they were not abolished by BOL; at most they were somwhat reduced. In previous experiments (Feldberg & Myers, 1966) in which the cerebral ventricles were perfused in the anaesthetized cat, the effluent collected from the aqueduct produced contractions of the fundus strip which also were reduced but not abolished by BOL; the BOL resistant part was attributed to the effect of prostaglandin-like substances. From recent findings we may conclude that it is prostaglandin E1 which is responsible since this prostaglandin was identified in the perfusate from the cerebral ventricles of dogs (S. W. Holmes, Personal communication). In the present experiments c.s.f. removed from the cisterna without any perfusion exerted similar activity on the fundus strip to the effluent so that part of its activity may have been due to admixture with c.s.f. However, the main activity must have been added to the perfusing fluid on its passage through the ventricles, since the activity in the effluent was as strong as in c.s.f. and, as just mentioned, is also present in perfusate collected from the aqueduct in anaesthetized cats in which no admixture with c.s.f. from the subarachnoid space takes place. Although perfusion with a calcium-free solution produced intense hyperthermia, the pharmacological activity of perfusate collected during this condition was not significantly greater than that of perfusate collected during perfusion with a calcium containing solution. This, however, would be expected if, as we assume, the hyperthermia is due to an action of the calcium-free solution on the cells in the hypothalamus, and not on the monoaminergic neurons which release 5-HT and innervate these cells. Yet the evidence is not conclusive because any contribution made by the anterior hypothalamus to the total biological activity of the perfusate would represent too small a fraction to be detectable by the methods used. 14 PHY 207

14 416 W. FELDBERG, R. D. MYERS AND W. L. VEALE REFERENCES ASHCROFT, G. W., Dow, R. C. & Mom, A. T. B. (1968). The active transport of 5- hydroxyindol-3-acetic acid and 3 methoxy 4 hydroxyphenylacetic acid from a recirculatory perfusion system of the cerebral ventricles of the unanaesthetized dog. J. Physiol. 199, BARTON, A. J., BLIGH, J. & SHARMAN, D. F. (1969). Improved techniques for the chronic cannulation of the lateral cerebral ventricle and the cisterna magna of the Welsh Mountain sheep. J. Phy8iol. 200, 25-27P. CARMICEAEL, E. A., FELDBERG, W. & FLEISCHHAUER, K. (1964). Methods for perfusing different parts of the cat's cerebral ventricles with drugs. J. Physiol. 173, Dow, R. C. & Mom, A. T. B. (1969). Perfusion of the cerebral ventricular system in the conscious rabbit. Br. J. Pharmac. Chemother. 36, P. FELDBERG, W. & FLEISCHHAUER, K. (1960). Penetration of bromophenol blue from the perfused cerebral ventricles into the brain tissue. J. Phy8iol. 150, FELDBERG, W. & MYERS, R. D. (1966). Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles. J. Phy8iol. 184, LIEBERMEISTER, C. (1875). Handbuch der Pathologie und Therapie des Fieber8. Leipzig: F. C. W. Vogel. MERLIS, J. K. (1940). The effect of changes in the calcium content of the cerebrospinal fluid on spinal reflex activity in the dog. Am. J. Phy8iol. 131, PAPPENHEIMER, J. R., HEISEY, S. R., JORDAN, E. F. & DowNER, J. DE C. (1962). Perfusion of the cerebral ventricular system in unanaesthetized goats. Am. J. Phy8iol. 203, VANE, J. R. (1957). A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. Chemother. 12,