Actions of angiotensin on area postrema of the rat

216

Brain Research, 380 (1986) 216-228 Elsevier

BRE 11921

Actions of Angiotensin on Area Postrema of the Rat W.E. WATSON Department of Physiology, University Medical School, Edinburgh ( U. K.) (Accepted December 31st, 1985) Key words: angiotensin II - - area postrema - - sodium intake - - sodium balance

Previous studies have reported that rats drink more saline after area postrema has been removed. The results presented here indicate that prolonged administration of angiotensin II into area postrema of unrestrained rats at 4 pmol/h also caused them to drink more saline. They drank more when angiotensin was released in the anterolateral part of the organ than when it was released anteromedially. Diurnal variation of drinking was not disordered. Dose-response curves showed that rats lacking area postrema drank more saline in response to systemic angiotensin than sham operated animals. The very large 'spontaneous" consumption of saline by rats lacking area postrema was not diminished by saralasin, an angiotensin antagonist. It is concluded that area postrema is a site where systemic angiotensin can act to promote sodium consumption: and that although removing area postrema increases the sensitivity of the drinking response to systemic angiotensin, this enhanced sensitivity is not the cause of the increased sodium consumption. INTRODUCTION A r e a p o s t r e m a in the rat is an unpaired organ lying superficially on the back of the medulla at the caudal end of the fourth ventricle, overlying the rostral end of the central canal of the spinal cord. Laterally it is related to the caudal part of the nucleus of the tractus solitarius. The caudal a t t a c h m e n t of the tela choroidea to area p o s t r e m a ' s rostral end separates two surfaces of area p o s t r e m a each b a t h e d with cerebrospinal fluid (CSF)54: a ventrorostral surface b a t h e d with fluid of the fourth ventricle and a dorsal surface bathed with fluid of cisterna magna. The rat lacks a differentiated area s u b p o s t r e m a 26'32'35. The extracellular space of a r e a p o s t r e m a includes large perivascular clefts 37. Materials of high molecular weight can gain access to this extracellular fluid by 3 routes: from plasma through the f e n e s t r a t e d e n d o t h e l i u m of a dense network of wide capillaries17'18'42'54'6°'67; from the C S F of the fourth ventricle by passing between e p e n d y m a l cells 25; and from the C S F of cisterna magna by passing mainly along wide perivascular spaces 67. Passage of materials between the extracellular space of area p o s t r e m a and that of adjacent brain might be i m p e d e d 4°.

This p a p e r is c o n c e r n e d with the actions of angiotensin administered directly into the extracellular space of area postrema. The organ contains sites, p r e s u m a b l y receptors, which bind the peptide with high affinity 2~'31'6s. A s well as capillaries and glia 36'6°, area p o s t r e m a contains nerve cell bodies 4"5'43'47"4s" 52,6s which are activated by some locally applied peptides or acetylcholine 6't2 but not by angiotensin. A r e a p o s t r e m a also contains nerve processes originating from the nucleus of the tractus solitarius, from the vagus and glossopharyngeal nerve and from the h y p o t h a l a m u s 3°'43'4s. In some species, but not in the rat 29'72, area p o s t r e m a mediates central cardiovascular effects of angiotensin l°'2a'33,34's7'ss. The ability of angiotensin to enter the brain from plasma except at sites having fenestrated capillaries is slight 56'61"63'69. Angiotensin administered intravenously or intracerebroventricularly has a well described role in acting on structures surrounding the rostral end of the third ventricle to cause drinking 21 or salt hunger<11: especially on the subfornical organ j'2°, o r g a n u m vasculosum of the lamina terminals 5° and the septum and preoptic nuclei 62. A r e a p o s t r e m a has a n u m b e r of structural similarities to the subfornical organ, and is also closely related to a choroid plexus. Removing

Correspondence: W.E. Watson, Department of Physiology, University Medical School, Teviot Place, Edinburgh EH8 9AG, U.K. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

217 area postrema makes rats drink much more saline 15'7°. The study reported here has 3 parts. The first describes the effect upon drinking of allowing angiotensin to diffuse into area postrema, explores and renders improbable the possibility that changes result from altered renal function. The second seeks an altered sensitivity of rats to injected angiotensin after area postrema has been removed, as significant differences in response to single doses of angiotensin have sometimes, but not always, been observed 19'29. The third describes the effect of an angiotensin antagonist upon the increased drinking seen in such rats. MATERIALS AND METHODS Drugs Angiotensin II and Sarl-AlaS-angiotensin II (saralasin) were purchased from Sigma. Whether given into area postrema or into the CSF of the fourth ventricle, angiotensin was allowed to diffuse down a glass capillary from a small reservoir flask. The technique is a modification of that of Dames et a1.16. A bulb of about 1 mm diameter was blown in the end of thin-walled glass tubing (o.d. approx. 2501~m). The bulb was packed with powdered angiotensin II and usually finally contained between 100 and 300/~g of dry drug. A capillary (o.d. approx. 120~m, i.d. approx. 100~m) was sealed into the neck of the flask with methacrylate cement (Fig. la). The flask was filled with pyrogen-free distilled water previously saturated with angiotensin II. Flasks were prepared in batches of 20-30, made from the same capillary glass, and randomly assigned either for implanting or for calibration. If a flask filled with dry angiotensin powder was not required immediately, the end of the capillary was sealed in a small flame and the flask stored at -20 °C. Nearly always the calibration of the rate of release of angiotensin was not measured on an implanted flask, but on a similar flask prepared from the same batch of tubing. After being washed in the manner described above, a flask for calibration was transferred to a 5 I~1 drop of water placed on a fragment of glass coverslip which had previously been thoroughly washed in water, alcohol and dried. The coverslip, droplet and flask were placed in a vaseline-sealed Petri dish saturated with water vapour for between 1

and 7 days at 37 °C. The flask was then removed and the coverslip gently and carefully dried in air. The coverslip was weighed 3 times on a microbalance, thoroughly washed in water and alcohol, dried in air and again weighed 3 times. The quantity of angiotensin was determined by subtraction. The rate of release in vitro for 54 flasks was 2.2 _+ 0.2/~g/day (mean _+ S.E.M.). On 19 occasions the rate of discharge from the same flask was compared over two consecutive 5-day periods. The rate in the first period was 2.1 _+ 0.21tg/day, and in the second 1.9 + 0.2/~g/day. Bulbs which contained no angiotensin and treated similarly showed a 'rate of release' o f - 0 . 1 _+ 0.1 gg/day. In other studies angiotensin or saralasin was injected subcutaneously over the belly wall to study short-term drinking behaviour. Animals Male Sprague-Dawley rats were reared in litters of 8-12, transferred to individual metabolism cages when aged 4 weeks, and maintained at 21-23 °C with a light-dark cycle of 12/12 h. They all received food pellets (Rat and Mouse No. 1 modified, expanded maintenance diet, Na 97.4 pmol/g, K 179 pmol/g) and distilled water ad libitum. As each rat reached a weight of 197 g it was assigned to one of 15 groups. The number of rats in each group is indicated in brackets after its primary definition. Rats were assigned to groups as described previously 7°, and retrospective analysis of the composition of each group indicates no weighting of any by a significantly large or small number drawn from any one litter. Rats of group N (10) were normal and not operated on at all. Group A P T - A (10) received angiotensin II into area postrema: those of A P T - S O (10) had an identical operation, but the implanted flask contained no angiotensin. Animals of group A P T - A NaK (40) were treated in the same manner as those of APT-A, but had in addition continuous access to solutions of sodium chloride (150 raM) and potassium chloride (150 raM). Rats of A P T - S O - N a K (10) resembled those of APT-SO but also had access to these additional solutions. VEN-A (10) received angiotensin into the CSF of the fourth ventricle: VENSO (10) had a flask similarly implanted which lacked angiotensin. Groups VEN-A-NaK (10) and VENSO-NaK (10) resembled V E N - A and VEN-SO, re-

218 spectively, but had, in addition, continuous access to the solutions of sodium chloride and potassium chloride. JT-A-NaK (10) received angiotensin into the medulla adjacent to area postrema. A r e a postrema was removed from rats of A P T (10), as described previously 7°, but only exposed in those of SO (10). Groups A P T - N a K (20) and S O - N A K (10) were identical with groups A P T and SO, respectively, but had continuous access to the additional solutions. SO-PFN a K (8) had area postrema exposed, and free access to additional solutions, but was additionally pair fed: each rat receiving on each postoperative day only the quantity of food eaten by a matched rat in A P T - N a K . The animals were drawn from 22 litters over a period of 19 months. The forty rats of group A P T - A - N a K contained an initial batch of 10 which were individually matched with those of A P T - S O - N a K , and a later batch of 30. Fourteen months separated the observations made on the two batches, but no significant difference between early and late observations was found. Eight rats were used in a preliminary study: 4 in a group equivalent to A P T - A - N a K and 4 equivalent to A P T - S O - N a K . As this preliminary study was used to design the major investigation, and to determine the statistical procedures to be used, the results from these eight rats are not included in the data presented: although they agree well.

Operation The dorsal aspect of the medulla was exposed under pentobarbitone anaesthesia (12 mg in 0.2 ml). After dividing the posterior atlanto-occipital membrane, dura and arachnoid, the area postrema was easily accessible at the level of foramen magnum (Fig. 1B). In those animals in which the area postrema was to be removed or merely exposed, the procedure was as before 7°. When a flask with or without angiotensin was to be introduced into area postrema a tear about 200~m long was made in the pia over the left dorsal column nuclei 500~tm behind the posterior angle of area postrema. The tip of the capillary was inserted through the tear and advanced into area postrema. The capillary lay sufficiently superficial for its tip to be clearly visible through a dissecting microscope. While an attempt was usually made to direct the tip toward a particular part of area postrema, insertion was only attempted once: to avoid the addi-

tional damage that would otherwise inevitably result from repeated insertion and withdrawal. The flask was gently supported in position for 3 - 5 min. It had then no tendency to slip out and tissue glue was not required. Bleeding around the capillary or at its tip was seldom a problem despite the vascularity of area postrema. While observing the bulb and the capillary tip the head was alternately fully flexed and extended several times to ensure that the bulb remained in position, and could not become trapped beneath the arch of the atlas. The final position of the tip was recorded as being anterolateral, anteromedial or posteromedial within the area: or as lying against the area in group JT-A-NaK. When a flask with or without angiotensin was to be inserted into the fourth ventricle, area postrema was exposed in the same way. The tela choroidea was gently torn on the right side in the angle between the rostral end of area postrema and the medulla; the small leash of vessels lying sagittally and passing between area postrema and the caudal end of the choroid plexus was not torn, and the posterior end of the choroid plexus and the posterior choroidal vein were left intact. The capillary was directed gently through the tear ventral to the choroid plexus, and the bulb of the flask rested on the dorsal column nuclei.

Daily estimations Each rat was weighed; food, water, sodium chloride and potassium chloride consumptions were measured daily as described previously 7°. The daily excretion of sodium and potassium, and the daily sodium and potassium balances were measured as previously described 7°, in groups A P T - A , APT-SO, V E N - A and VEN-SO. In addition to daily measurement, electrical recordings were frequently made of lapping, allowing a record of the pattern of drinking to be made. A current of less than 0.1/~A flowed when a rat made contact with a drinking spout. Preliminary experiments indicated that switching the recording system either on or off was without effect on the volume a rat drank daily or in a 2-h test period. During periods of observation, less than 1% of contacts with the spout were unassociated with drinking; to reduce even these as far as possible, the recording system disregarded isolated single contacts. The number of laps from each of 3 spouts was recorded every 5 rain for periods ex-

219 tending from 2 h to several days.

Drinking trials Most of these lasted 2 h; the procedure differed in different series. In the first series normally sated rats of groups APT-NaK, SO-NaK, APT, SO and SO-PF-NaK were placed individually in a clean cage without food for 2 h during the light phase of the light-dark cycle, when they normally drank little. For this 2 h they had access to water only. On entering the cage each rat was injected subcutaneously with either angiotensin II or an equal volume of 150 mM sodium chloride. Each rat had 14 or 16 trials with 7 or 8 different doses of angiotensin II, ranging from 5 to 640 ktg: each matched with a saline control. The order of dose, and the ordering of saline and angiotensin administration was random for each pair of rats; but each rat in SONaK, SO-PF-NaK or in SO experienced the same sequence of trials as its paired rat in APT-NaK or in APT. Each rat had two familiarization trials in which it received saline before beginning the tests. The second series was identical with the first: except rats had access to water, 150 mM sodium chloride and 150 mM potassium chloride in the 2-h test period. The third series was designed to find the dose of subcutaneous saralasin that could prevent or reduce drinking induced by subcutaneous angiotensin. At each trial a rat of group N was injected twice: once 10 min before entering the cage with either saline or saralasin (0.35 ktg/g or 0.7 Itg/g) and once on entering the cage with angiotensin II (0.5 Bg/g). Lapping was recorded in this series in case the effect of saralasin was of shorter duration than that of angiotensin. Each rat received 4 trials: two with saline, and one with each dose of saralasin. In the fourth series rats of APT and SO received saralasin (0,7/~g/g) subcutaneously 10 min before the start of drinking trials, at which water, sodium chloride and potassium chloride were available. The trials were the only periods in which the rats had access to the additional solutions. The fifth series was designed to see whether saralasin would reduce the volume of sodium chloride drunk by rats which had continuous access to it: as mechanisms causing short-term drinking may differ from those regulating consumption of saline when it is continuously present, and because short-term

drinking is associated with hormonal changes 39. Rats of group APT-NaK remained in their own cages for the trials. The trial began at 20.00 h at the onset of darkness, and lasted for 2 h. At its commencement each rat received either saline subcutaneously or saralasin (0.7/zg/g). Nine trials, 3 with saline, 3 with saralasin and 3 with neither, were conducted on each rat. Lapping was recorded during these trials to exclude a hypothetical reduction produced early by saralasin, counterbalanced by an increased consumption in the later part of the trial. The rats had access as usual to food, water, sodium chloride and potassium chloride.

Cerebroventricular perfusion In the sixth series a cannula was placed in the lateral cerebral ventricle of 6 rats of group APT-NaK under pentobarbitone anaesthesia (12 mg in 0.2 ml), and connected to an interscapular osmotic minipump, which delivered saralasin dissolved in artificial CSF at 1 tzl/h (12 /~g/h: approximately 0.36 ktg/g/h) for 7 days. In addition to measuring the volumes of water, sodium chloride and potassium chloride drunk daily, lapping was continuously recorded. At the onset of darkness on the third and fifth days each rat received additional subcutaneous saralasin (0.7tzg/g). The position of the cannula was confirmed postmortem from the distribution of perfused dye.

Histology The rat was fixed by retrograde aortic perfusion, and serial sections were cut transversely through the medulla. The area of area postrema and of the adjacent parts of the nucleus of the tractus solitarius were measured with a projection microscope at intervals of 100/~m for the full rostrocaudal extent of area postrema. A characteristic lesion resulting from inserting a capillary is shown in Fig. lc.

Statistical analysis Two tests were used to determine the statistical significance of differences between groups: Student's t-test, and Wilcoxon's signed-rank test. Each rat in each test group was individually matched preoperatively, and hence prospectively, with a rat in each control group: for example, each rat in APT-A-NaK was matched with a rat in APT-SO-NaK, VEN-A-NaK and VEN-SO-NaK, and each in APT with one in

220 p e r i o d , or for such limited part of the p o s t o p e r a t i v e p e r i o d as the preliminary study h a d indicated to be a p p r o p r i a t e . The calculated a r e a was c o m p a r e d with that obtained from the p a i r e d rat in another group. The d a t a from all (usually 10) similar pairs was then analyzed. The first figure given in the text results from applying S t u d e n t ' s t-test, and the second from W i l c o x o n ' s signed-rank test. T h e results of the first two series of drinking trials were analyzed similarly: by comparing the area b e n e a t h d o s e - r e s p o n s e curves. The significance of the effects of saralasin u p o n drinking, m e a s u r e d in the remaining drinking trials, was calculated by applying Student's t-test to the data o b t a i n e d in periods following saralasin and in periods following saline. RESULTS

Implanting angiotensin A preliminary study indicated that the results should be analyzed for 3 periods: days 1 - 7 , days 8 - 1 4 , and thereafter.

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Fig. 2. Volume of saline drunk daily, against time in days. Operation on day 0. Bars represent standard deviation. Top: Q, group APT-A-NaK (40 rats); ©, group APT-SO-NaK (10 rats). Bottom: @, group VEN-A-NaK (10 rats): ©, group VEN-SO-NaK (10 rats).

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Fig. 3. Solution preference, calculated as the percentage of total fluid intake taken as each of 3 solutions: sodium chloride, potassium chloride and water. The graph shown is part of a larger graph in which each axis starts at 0 and ends at 100. As the 3 coordinates of any point within the triangle add up to 100, the solution preferences, being calculated as percentages can be represented by a point. The 3 axes are water intake, sodium chloride intake and potassium chloride intake, each expressed as a percentage of the total fluid intake. Q, mean value for 5 preoperative days combined; O, mean value for each of 4 postoperative days. The line joins consecutive days, starting at the solid symbol. Left graph: group APT-A-NaK (40 rats). Right graph: group VEN-A-NaK (10 rats).

First 7postoperative days In the first postoperative days, rats of group A P T A - N a K d r a n k m o r e saline than those of A P T - S O N a K ( P < 0.01; P < 0.01), V E N - A - N a K (P < 0.01; P < 0.05), V E N - S O - N a K ( P < 0.01; P < 0,01) or JTA - N a K ( P < 0.01; P < 0.01). The greatest increase was seen on the third day (Fig. 2). Rats of V E N - A N a K d r a n k m o r e saline than V E N - S O - N a K ( P < 0.05; P < 0.01); the increase was smaller and briefer (Fig. 2). In this p e r i o d groups A P T - A , A P T - S O , VEN-A, VEN-SO, APT-A-NaK, APT-SO-NaK, V E N - A - N a K , V E N - S O - N a K and J T - A - N a K drank less water than N (P < 0.05; P < 0.05); for water there was no significant difference b e t w e e n the operated groups. All rats d r a n k very little potassium chloride at any time (mean 0.21 ml daily) and this was unchanged ( P > 0.05; P > 0.05 for all comparisons). The totalfluid intake of A P T - A - N a K increased m o r e than that of A P T - S O - N a K ( P < 0.01; P < 0.01), V E N - A - N a K ( P < 0.05; P < 0.05) and V E N - S O N a K ( P < 0.01; P < 0.01). W h e n the intakes of each of the 3 fluids is expressed individually as a percentage of total fluid intake, a large increase can be seen in the sodium preference of A P T - A - N a K ( P < 0.01;,P < 0.01), and a smaller increase in group V E N - A NaK (P < 0.1; P < 0.05) (Fig. 3). Of the very large volume of saline d r u n k daily by 10 unselected rats of A P T - A - N a K , lapping records showed that 85% was

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Fig. 4. Distribution of drinking within a 24-h day. Polar coordinates. Midnight (24 h) is at top of graph, and hours increase clockwise: midday (12 h) at bottom of graph. The oblique line from 20.00 to 08.00 h separates light from dark period. Each sector of circumference is 1 h. The radius is the volume drunk in that hour of the 24-h day. The top graph shows combined mean values derived from 10 rats of APT-A-NaK measured on the 3 days preceding operation. The rats were consecutively numbered within the group, but otherwise unselected. The bottom graph shows combined mean values derived from the same rats measured in the second, third and fourth postoperative days.

consumed in the 12 h of darkness, a figure not significantly different from that found preoperatively, 82% (P > 0.05; P > 0.05) (Fig. 4). The greatest effect of angiotensin in group A P T A - N a K was seen when the capillary tip lay in the anterolateral zone of a r e a postrema, than when it lay anteromedially ( P < 0.05; no Wilcoxon) (Fig. 5). D a t a from the 4 rats in which the tip was posteromedial is not shown; their mean values were less than those of the a n t e r o m e d i a l group, but not significantly less. The amount of sodium c o n s u m e d was greater when the capillary tip lay anterolaterally in A P T - A N a K than in J T - A - N a K ( P < 0.01; no Wilcoxon). Rats in all o p e r a t e d groups lost weight on the first one or two p o s t o p e r a t i v e days. G r o u p s in which the capillary lay in a r e a p o s t r e m a lost m o r e than those in which it lay in the ventricle ( P < 0.05; P < 0.05). The differences b e t w e e n A P T - A , A P T - A - N a K , A P T - S O and A P T - S O - N a K were not significant ( P > 0.05;

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Between the eighth and fourteenth postoperative days the sodium consumption was significantly greater in APT-A-NaK and APT-SO-NaK than in VENA-NaK and VEN-SO-NaK (P < 0.05; P < 0.05 for all combinations) (Fig. 2). In both APT-A-NaK and APT-SO-NaK the increased intake diminished towards the end of the period. No significant difference was found between operated groups in the volume of water, or the volume of potassium chloride consumed.

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Fig. 5. Volume of saline drunk daily, against time in days. Operation on day 0. Bars represent standard deviation. 0 , group APT-A-NaK: capillary tip anterolateral (23 rats); ©, group APT-A-NaK: capillary tip anteromedial (13 rats). Values for 4 rats of group APT-A-NaK not shown as tip lay posteromedially.

P > 0.05 for all combinations). The differences between VEN-A, VEN-SO and VEN-A-NaK were not significant (P > 0.05; P > 0.05 for all combinations): rats of VEN-SO-NaK lost marginally less weight than VEN-A (P < 0.05; P > 0.05). All operated groups ate less than group N on the first two postoperative days (P < 0.05; P < 0.05). Although groups APT-A, APT-SO, APT-A-NaK and APT-SO-NaK ate less than the other groups, the difference lacked statistical significance. Correlation of summed postoperative daily food intake with body weight determined both for individual pairs of rats and for the daily means of groups indicated no significant difference between any operated groups.

Sodium and potassium balances Sodium and potassium balances were significantly reduced on the first two postoperative days in groups APT-A, APT-SO, VEN-A and VEN-SO, both being negative in nearly all rats on the first day (P < 0.01; P < 0.01). There was otherwise no significant difference between the operated groups, whether measured over the first 7 postoperative days alone or over the 25-day postoperative period studied. Groups which ate less and gained weight rather less rapidly had lower balances, but the differences lacked statistical significance.

Histology The glass capillary produced a cylindrical zone of damaged, degenerating or reacting cells about 250 ktm in diameter (Fig. lc). Commonly the surrounding tissue was distorted. The measured area of area postrema was diminished to 72 _ 12% (mean _ S.E.M.) and no significant differences were found between APT-A, APT-SO, APT-A-NaK and APT-SO-NaK. The area of the nucleus of the tractus solitarius was not significantly diminished. Lesions in group JT-ANaK were all outside area postrema; in this group the area of the nucleus of the tractus solitarius was reduced to 63 _ 14%, the reduction being confined to the side of insertion. The area of area postrema was not significantly reduced in this group. No damage to vagal or hypoglossal nuclei was seen. One early rat of group VEN-A was found to have severe gliosis of the rostral part of one hypoglossal nucleus, being entirely confined to the nucleus, and affecting nearly all of it. The cause of the change is unknown; the adjacent brainstem showed no evidence of damage from an inserted capillary, or vascular damage. Data from this rat, although unexceptional, was not included in group VEN-A. Another rat was substituted. The substitution was without statistical significance.

Drinking responses In series 1 and 2 the drinking response to the administration of various doses of angiotensin was studied, each having a control response to the administration of the same volume of saline. The volumes shown in Fig. 6 indicate the difference in volume drunk. This approach was used, as rats lacking area postrema normally drank either me/re water, when this alone was available, or more saline than rats which had been sham operated. In series 1 angioten-

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Fig. 7. Volume of saline drunk in consecutive 5-rain intervals within first 2 h of dark phase. Mean values for 10 consecutive rats of Group APT-NaK: 3 tests on each rat with each solution. Top: drinking profile if rat neither handled nor injected. Middle: drinking profile if rat injected with saline. Bottom: drinking profile if rat injected with saralasin.

sin caused rats to drink more water; this increase was greater in A P T than in SO rats (P < 0.05; P < 0.05) (Fig. 6). Similarly rats of A P T - N a K drank more than those of SO-NaK. In series 2 angiotensin caused rats of groups A P T - N a K , SO-NaK, SO and SO-PF-NaK to drink more saline. The increase was greater in A P T - N a K than in SO-NaK (P < 0.05; P < 0.05) (Fig. 6). Group SO-P-NaK did not differ significantly from SO-NaK (P > 0.05; P > 0.05). Group A P T drank so much saline in every test, 33.1 + 4.6 ml (mean + S.E.M.) whether injected with angiotensin or saline, that no effect of injected angiotensin could be discerned. Records of lapping indicate that over 80% of the saline was drunk in the first 30 rain. In series 2, when saline was available, the volume of water drunk was small, not increased by angiotensin and not significantly different between groups. The third series showed that subcutaneous injection of 0.35 ug/g saralasin reduced the saline consumption induced in rats of group N by subcutaneous injection of 0.5 ~g/g angiotensin II from 14.7 + 2.6 ml to 3.2 + 1.1 ml. Increasing the dose of saralasin to 0.7/~g/g reduced the consumption to 3.6 _+ 1.0 ml. In series 4, rats of group A P T drank 33.2 _+ 5.6 ml saline in 2 h. Most of this was consumed in the first 30 min. After 0.7/~g/g saralasin they drank 36.4 _+ 7.0 ml. The difference lacks significance (P > 0.05). Saralasin had no effect on the distribution of lapping within the 2-h period. In series 5, rats of group A P T - N a K drank 10.0 _+ 1.9 ml saline in the first 2 h of darkness. After receiving 0.7/~g/g saralasin they drank 14.8 -+ 6.8 ml. The difference is not significant (P > 0.05). The distribution of lapping within the period was the same in rats which received saline and in rats which received angiotensin (Fig. 7), but both differed significantly within the first 30 min of this period from undisturbed animals. Handling and injecting did not significantly alter the total volume drunk within either the first hour, or in the whole 2-h period, but only the initial distribution of the drinking. Handling did not alter the distribution of drinking within the 2-h test period in series 1-4. Perfusing the cerebral ventricles with saralasin did not reduce the daily intake of saline in 6 rats of group A P T - N a K . Before perfusion the mean daily intake was 73.4 _+ 12.7 ml; during perfusion on days 2, 4 and 6 it was 96.8 + 12.1 ml (P > 0.05); on days 3 and 5 when the animal received 0.7 ~g/g saralasin subcuta-

224 neously as well, the volume of saline drunk in the first 2 h of darkness did not alter significantly (P > 0.05). While rats received saralasin intracerebroventricularly the proportion of the intake taken during the light phase of the day increased significantly (P < 0.05). DISCUSSION In the method used angiotensin diffused into area postrema from a small local reservoir. The rate of release in vitro was reasonably constant for at least 10 days. In this study it is assumed that the rate of release in vivo did not exceed that in vitro. This rate, 4 pmol/h is close to the rate of administration into the third ventricle or structures of its wall which results in drinking 22. Possibly the rate of release into area postrema was lower than this, especially when the adjacent tissue reacted to the implanted glass. No attempt was made to measure the rate of release in vivo using radiolabelled angiotensin, as it seemed that interpretation of any measured 'steady state' rate of excretion of isotope would be difficult: especially in the context of isotope reutilization following destruction of the peptide, a situation likely to change as the local tissues reacted. The rate of release in vitro was steady. It is likely to have been reasonably steady in vivo also, although possibly influenced by changes of temperature especially at the time of insertion and to a lesser extent diurnally after implantation. Although the flask had been warmed for several hours before implantation, it is likely that its temperature fell when it was transferred to the rat, and rose again on implantation. Even if it is assumed that the temperature increased by 20 °C on implantation the calculated discharge of angiotensin resulting from thermal expansion of the fluid and of the very small residual air bubble (less than 30 nl) would not exceed 200 ng: and would be diminished below that figure by the extent to which angiotensin-free water or CSF was drawn into the capillary during the preceding period of cooling, by the accompanying thermal expansion of the glass bubble, and by the reduction in concentration of angiotensin within the lower part of the capillary resulting from the establishment of a diffusion gradient during the period of preoperative equilibration. Furthermore, angiotensin discharged when the capillary is inserted could not be responsible for

the considerable sodium consumption found between the second and fourth postoperative days. It is most improbable that the far smaller (1 °C) diurnal change in body temperature 39 altered the rate of angiotensin release to a degree of biological significance: and most improbable that any such variation was responsible for the considerable difference in saline consumption between the light and dark phases of the day. The effect of angiotensin lasted about 6 days. At the end of this period the bulbs still contained most of their angiotensin, which retained biological activity. A bulb removed from an animal after 8 days elicited a response in another rat (unpublished observation). This is consistent with the reported stability of angiotensin in solution in CSF 49. The reason why drinking stopped after 6 or 7 days is not known; it could be a consequence of associated gliosis, of enhanced local destruction by peptidases, down regulation of local receptors, or a result of sodium loading for several days45. A similar transient effect of angiotensin accompanying sustained perfusion into the cerebral ventricles has been observed 49"64, but not explained. The distance that released angiotensin diffused is not known. When released into area postrema it is likely that diffusion was considerably limited by the closely woven network of wide, permeable capillaries: which would have acted as a complex sink for materials in higher concentration in the tissue than in the plasma. Horseradish peroxidase, released by diffusion into area postrema could not be detected histochemically more than 150/tm from the capillary tip with the exception of staining some nerve cell bodies of the nucleus of the tractus solitarius (unpublished observations) presumably by transport within the cell processes: in contrast, the enzyme could be detected 450 ktm from the capillary tip when it lay in the nucleus adjacent to area postrema; this increased diffusion was found only within brain outside area postrema, and not within the adjacent part of the area. The significant difference in drinking response found when the capillary tip lay in different parts of area postrema might also indicate that angiotensin diffused only a small distance: provided it is assumed that the differences resulted from variation of the site of release rather than from variation of the position of damage. Regional differences in the distribution of nerve cells 6 and in capillary organization 55 have

-

-

225 been described, and different regional densities of nerve processes penetrating from outside might be expected. It is possible that the reported failure of local angiotensin to activate neurones within area postrema 6 results from the receptors being on the postremal dendrites of neurones of the nucleus of the tractus solitarius: a possibility strengthened by the staining of such ipsilateral neurones when peroxidase diffuses into area postrema (unpublished observations). It is not likely that angiotensin entering area postrema is acting by escaping into the CSF, as its effect in group VEN-A-NaK was less and more transient than in group APT-A-NaK. Conversely it is possible that the effects of angiotensin in group VEN-A-NaK are not mediated by area postrema. Area postrema is not downstream from the fourth ventricle, as the rat lacks a foramen of Magendie; probably the large choroid plexus of the fourth ventricle makes CSF flow rostrally and laterally. The cilia of the floor of the fourth ventricle also beat in this direction 71. Angiotensin released in the fourth ventricle could probably reach periventricular structures of the third ventricle nearly as easily as area postrema, through the to-andfro movements of CSF accompanying breathing, pulsations and movement. From the eighth to the fourteenth postoperative day rats with a capillary in area postrema drank more saline whether the flask contained angiotensin or not; rats with a capillary in the fourth ventricle did not. This late increase almost certainly resulted from tissue damage. Removing area postrema increases the volume of saline drunk considerably 15'7°. If this interpretation is correct it indicates that small degrees of damage confined to area postrema can cause rats to drink more. This contrast with the findings of Contreras and Stetson 15who indicated that almost complete removal was necessary for increased drinking. As their studies took place about 3 weeks after the lesion, earlier and transient changes would not have been seen. The greater weight loss and lesser food intake seen in groups having the capillary in area postrema is similarly likely to be a result of damage. These changes too are much greater and more persistent after area postrema has been removed 3'13A4'7°. As angiotensin did not reduce the sodium balance, the increased consumption of sodium chloride could not have resulted from measurable sodium loss. Removing area postrema similarly is without direct ef-

fect on sodium balance 7°, although it can be disordered if the medullary lesion is more extensive 66. Natriuresis was also absent in group VEN-A. Increased renal sodium loss can follow the perfusion of the lateral cerebral ventricle with greater doses of angiotensin 27. It therefore appears that the primary act of angiotensin on area postrema is the direct promotion of sodium intake. The persisting diurnal variation of saline drinking suggests that the administered angiotensin did not grossly disrupt the normal drinking behaviour. The drinking response to systemic angiotensin can in the rat be reduced or abolished by lesions of the subfornical organ 1,49, or of its efferent connections 20'38'44'46. If the same is true of saline consumption then the action of area postrema would have to be expressed either through this part of brain or through situations of convergence of information from these two sites. In no group at any time was the consumption of potassium chloride significantly altered. It is likely that the concentration of angiotensin II normally present in the extracellular fluid of area postrema is largely determined by plasma constituents; whether the plasma angiotensin II concentration is the determining factor, or another component of the systemic renin-angiotensin system, is unknown. Brain contains and can synthesize all necessary constituents of this system 51'53 including an angiotensinogen but whether area postrema can do so has not been determined. The large amount of converting enzyme demonstrable histochemically on the luminal surface of the adjacent choroid plexus 2'7 might not influence area postrema as the tenuous capillary connections between choroid plexus and area postrema 4t seem too slender to provide a significant flow to the organ. It is necessary to attempt to reconcile the enhanced consumption of sodium produced by angiotensin with that found after removing area postrema; for it might be expected that removing an area that can promote the ingestion of sodium chloride would diminish rather than increase the amount consumed. As a working hypothesis it seemed possible that removing area postrema might increase the sensitivity of receptors to angiotensin elsewhere in the brain: especially as the saline consumption increased gradually and after a latency7°. The finding that rats lacking area postrema drank more water when challenged with a single

226 dose of angiotensin a9 was consistent with this hypothesis; but H a y w o o d 29 found no significant increase in the volume drunk. The d o s e - r e s p o n s e curves given h e r e show that rats lacking area p o s t r e m a d r a n k m o r e w a t e r when challenged with angiotensin if saline was not available, but p r e f e r r e d to drink m o r e saline. The threshold dose of subcutaneous angiotensin n e e d e d to initiate drinking was rather higher - - as might be exp e c t e d - - than that found after intravenous injection 23. The difference b e t w e e n groups A P T - N a K and S O - N a K was significant but small. E v e n very large doses of angiotensin did not cause rats of group SON a K or A P T - N a K to drink as much saline as those in group A P T . A s the increased sensitivity of rats to systemic angiotensin after removing area p o s t r e m a was consistent with the hypothesis, although disappointingly small, the effects of an angiotensin antagonist Sarl-Ala8-angiotensin II upon the drinking of rats in group A P T - N a K was studied. Saralasin was given subcutaneously in a dose shown to diminish considerably drinking induced by systemic angiotensin (series 3), but it failed to reduce the avid drinking of saline seen in rats after removing area postrema, either when saline was present for short periods only (group A P T ) or continuously (group A P T - N a K ) . R a t h e r , in b o t h circumstances the volume drunk increased, but not significantly.

REFERENCES 1 Abdelaal, A.E., Assaf, S.Y., Kucharczyk, J. and Mogenson, G.J., Effect of ablation of the subfornical organ on water intake elicited by systemically administered angiotensin II, Can. J. Physiol. Pharmacol., 52 (1974) 362-363. 2 Arregui, A. and Iversen, L.L., Angiotensin-converting enzyme: presence of high activity in choroid plexus of mammalian brain, Eur. J. Pharmacol., 52 (1978) 147-150. 3 Berger, B.D., Wise, C.D. and Stein, L., Area postrema damage and bait shyness, J. Comp. Physiol. Psychol., 82 (1973) 475-479. 4 Brizzee, K.R. and Klara, P.M., The structure of the mammalian area postrema, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 2944-2948. 5 Brizzee, K.R., Palazzo, M.C., Klara, P.M. and Hofer, H., Effects of systemic administration of 6-hydroxydopamine on the circumventricular organs in non-human primates. I. Area postrema, Cell Tissue Res., 187 (1978) 115-127. 6 Brooks, M.J., Hubbard, J.I. and Sirrett, N.E., Extracellular recording in rat area postrema in vitro and the effects of cholinergic drugs, serotonin and angiotensin II, Brain Research, 261 (1983) 85-90.

While the value of ascribing a cause to an insignificant observation is dubious, the increase might have resulted from the known w e a k agonist action of the drug. Saralasin even failed to r e d u c e the increased drinking when it was infused into the lateral c e r e b r a l ventricles at a rate known to antagonize effectively some central actions of angiotensin 8'22, either with or without additional systemic saralasin. These findings do not firmly p r o v e that angiotensin is not involved at all in the saline consumption of rats of group A P T N a K but any such action would have to be on receptors which are not blocked by saralasin, or which are at sites not accessible to intracerebroventricular or systemic antagonist. It would a p p e a r to be necessary to conclude that angiotensin by acting within a r e a p o s t r e m a can cause rats to drink m o r e saline: that the sensitivity to systemic angiotensin increases after area p o s t r e m a has been r e m o v e d , but not by much: and that this increased sensitivity is not the cause of the increased consumption of saline found in these rats. ACKNOWLEDGEMENTS I am grateful to the Medical R e s e a r c h Council for support, to Mrs. K. G r a n t for expert histological assistance, and to the Faculty A n i m a l A r e a for the facilities provided.

7 Brownfield, M.S., Reid, I.A., Ganten, D. and Ganong, W.F., Differential distribution of immunoreactive angiotensin and angiotensin-converting enzyme in rat brain, Neuroscience, 7 (1982) 1759-1769. 8 Bruner, C.A., Weaver, J.M. and Fink, G.D., Will chronic intracerebroventricular saralasin infusion produce selective blockade of brain angiotensin II receptors in the rat, J. Pharmacol. Exp. Ther., 226 (1983) 13-18. 9 Bryant, R.W., Epstein, A.N., Fitzsimmons, J.T. and Fluharty, S.J., Arousal of a specific and persistent sodium appetite in the rat with continuous intracerebroventricular infusion of angiotensin II, J. Physiol, (London), 301 (1980) 365-382. 10 Buckley, J.P. and Jandhyala, B.S., Central cardiovascular effects of angiotensin, Life Sci., 20 (1977) 1485-1494. 11 Buggy, J. and Fisher, A.E., Water and sodium intake: evidence for a dual central control for angiotensin, Nature (London), 250 (1975) 733-735. 12 Carpenter, D.O., Briggs, D.B. and Strominger, N., Behavioural and electrophysiological studies of peptide-induced emesis in dogs, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 2952-2954. 13 Coil, J.D. and Norgren, R., Taste aversions conditioned

227 with intravenous copper sulfate: attenuation by ablation of area postrema, Brain Research, 212 (1981) 425-433. 14 Contreras, R.J., Foxe, E. and Drugovich, M.L., Area postrema lesions produce feeding deficits in the rat: effect of preoperative dieting and 2 deoxyglucose, Physiol. Behav., 29 (1982) 875-884. 15 Contreras, R.J. and Stetson, P.W., Changes in salt intake after lesions of the area postrema and nucleus of the solitary tract in rats, Brain Research, 211 (1981) 355-366. 16 Dames, W., Joo, F. and Wolff, J.R., A method for localized and longlasting microapplication of drugs into nervous tissue of freely moving animals, Exp. Brain Res., 36 (1979) 259-264. 17 Dempsey, E.W., Neural and vascular ultrastructures of the area postrema in the rat, J. Comp. Neurol., 150 (1973) 177-200. 18 Dermietzel, R. and Liebstein, A.G., The microvascular pattern and perivascular linings of the area postrema. A combined freeze etching and ultrathin section study, Cell Tissue Res., 186 (1978) 97-110. 19 Edwards, G.L. and Ritter, R.C., Area postrema lesions increase drinking to angiotensin and extracellular dehydration, Physiol. Behav., 29 (1982) 943-947. 20 Eng, R. and Miselis, R.R., Polydipsia and abolition of angiotensin-induced drinking after transection of subfornical organ efferent projections in the rat, Brain Research, 225 (1981) 200-206. 21 Fitzsimmons, J.T., The role of a renal thirst factor in drinking induced by extracellular stimuli, J. Physiol. (London), 201 (1969) 349-368. 22 Fitzsimmons, J.T., Epstein, A.N. and Johnson, A.K., Peptide antagonists of the renin-angiotensin system in characterization of receptors for angiotensin-induced drinking, Brain Research, 153 (1978) 319-331. 23 Fitzsimmons, J.T. and Simons, B.J., The effect on drinking in the rat of intravenous infusion of angiotensin, given alone or in combination with other stimuli of thirst, J. Phys.. iol. (London), 203 (1969) 45-57. 24 Gildenberg, P.L., Ferrario, C.M. and McCubbin, J.W., Two sites of cardiovascular action of angiotensin II in the brain of the dog, Clin. Sci., 44 (1973) 417-420. 25 Gotow, T. and Hashimoto, P.H., Fine structure of the ependyma and intercellular junctions in the area postrema of the rat, Cell Tissue Res., 201 (1979) 207-225. 26 Gwyn, D.G. and Wolstencroft, J.H., Cholinesterase in the area subpostrema, J. Comp. Neurol., 133 (1968) 289-308. 27 Halperin, E.S., Summy-Long, J.Y., Keil, L.C. and Severs, W.B., Aspects of salt/water balance after cerebroventricular infusion of angiotensin II, Brain Research, 205 (1981) 219-221. 28 Harding, J.W., Stone, L.P. and Wright, J.W., The distribution of angiotensin II binding sites in rodent brain, Brain Research, 205 (1981) 265-274. 29 Haywood, J.R., Fink, G.D., Buggy, J., Phillips, M.I. and Brody, M.J., The area postrema plays no role in the pressor action of angiotensin in the rat, Am. J. Physiol., 239 (1980) H108-Hl13. 30 Hosoya, Y. and Matsushita, M., A direct projection from the hypothalamus to the area postrema in the rat, as demonstrated by the HRP and autoradiographic methods, Brain Research, 214 (1981) 144-149. 31 van Houten, M., Schiffren, E.L., Mann, J.F.E., Posner, B.I. and Boucher, R., Radio autographic localization of specific binding sites for blood-borne angiotensin II in rat

brain, Brain Research, 186 (1980) 480-485. 32 Joo, F. and Csillik, B., Topographical correlation between the haemato-encephalitic barrier and the cholinesterase activity of brain capillaries, Exp. Brain Res., 1 (1966) 147-151. 33 Joy, M.D., The intramedullary connections of the area postrema involved in central cardiovascular response to angiotensin II, Clin. Sci., 41 (1971) 89-100. 34 Joy, M.D. and Lowe, R.D., Evidence that the area postrema mediates the central cardiovascular response to angiotensin II, Nature (London), 228 (1970) 1303-1304. 35 Karcsu, S. and Toth, L., Fine structural localization of acetyl cholinesterase in capillaries surrounding the area postrema, Brain Research, 95 (1975) 137-141. 36 King, L.S., Cellular morphology in area postrema, J. Comp. Neurol., 66 (1937) 1-21. 37 Klara, P.M. and Brizzee, K.R., The ultrastructural morphology of the squirrel monkey area postrema, Cell Tissue Res., 160 (1975) 315-326. 38 Knepel, W., Nutto, D. and Meyer, D.K., Effect of transection of subfornical organ efferent projections on vasopressin release induced by angiopressin or noprenaline in the rat, Brain Research, 248 (1982) 180-184. 39 Krieger, D.T., Food and water restriction shifts corticosterone, temperature, activity and brain amine periodicity, Endocrinology, 95 (1974) 1195-1201. 40 Krisch, B., Leonhardt, H. and Buchheim, W., The functional and structural border between the csf- and blood-milieu in the circumventricular organs (organum vasculosum laminae terminalis, subfornical organ, area postrema) of the rat, Cell Tissue Res., 195 (1978) 485-497 and correction Cell Tissue Res., 200 (1978) 335. 41 Kroidl, R., Die arterielle und ven6se Versorgung der Area postrema der Ratte, Z. Zellforsch. Mikroskop. Anat., 89 (1968) 430-452. 42 Leonhardt, H., Uber die Blutkapillaren und perivaskulfiren Strukturen der Area postrema des Kaninchens und tiber ihr Verhalten im Pentamethylentetrayol (cardiazol) Krampf, Z. Zellforsch. Mikroskop. Anat., 76 (1967) 511-524. 43 Leslie, R.A. and Gwyn, D.G., Neuronal connections of the area postrema, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 2941-2943. 44 Lind, R.W. and Johnson, A.K., Central and peripheral mechanisms mediating angiotensin-induced thirst, Exp. Brain Res., Suppl. 4 (1982) 353-364. 45 Mann, J.F.E., Schriffen, E.L., Schiller, P.W., Rascher, W., Boucher, R. and Genest, J., Central actions and brain receptor binding of angiotensin II: influence of sodium intake, Hypertension, 2 (1980) 437-443. 46 Miselis, R.R., The efferent projections of the subfornical organ of the rat: a circumventricular organ with a neural network subserving water balance, Brain Research, 230 (1981) 1-23. 47 Morest, D.K., A study of the structure of the area postrema with Golgi methods, Am. J. Anat., 107 (1960) 291-303. 48 Morest, D.K., Experimental study of the projection of the nucleus of the tractus solitarius and the area postrema in the cat, J. Comp. Neurol., 130 (1967) 277-300. 49 di Nicolantonio, F., Mendelsohn, F.A.O., Hutchinson, J.S., Takata, Y. and Doyle, A.E., Dissociation of dipsogenic and pressor responses to chronic central angiotensin II in rats, Am. J. Physiol., 242 (1982) R498-R504. 50 Phillips, M.I., Angiotensin in the brain, Neuroendocrinolo-

228

gy, 25 (1978) 354-377. 51 Phillips, M.I., New evidence for brain angiotensin and for its role in hypertension, Fed. Proc. Fed. Am. Soc. Exp. Biol., 42 (1983) 2667-2672. 52 Pickel, V.M. and Armstrong, D.M., Ultrastructural localization of monoamines and peptides in rat area postrema, Fed. Proc. Fed. Am. Soc. Exp. Biol., 43 (1984) 2949-2951. 53 Raizada, M.K., Phillips, M.I. and Gerndt, J., Primary culture from fetal brain incorporates 3H-isoleucine and 3H-valine into immunoprecipitable angiotensin II, Neuroendocrinology, 36 (1982) 64-67. 54 Rohrscheider, I., Schinko, I. and Wetzstein, R., Der Feinbau der Area postrema der Maus, Z. Zellforsch. Mikroskop. Anat., 123 (1972) 251-276. 55 Roth, G.J. and Yamomoto, W.S., The microcirculation of the area postrema in the rat, J. Comp. Neurol., 133 (I968) 329-340. 56 Schelling, P., Hutchinson, J.S., Ganten, U., Sponer, G. and Ganten, D., Impermeability of the blood-cerebrospinal fluid barrier for angiotensin II in rats, Clin. Sci. Mol. Med., Suppl. 3, 51 (1976) 399s-402s. 57 Scroop, G.C., Katic, F.P., Brown, M.J., Cain, M.D. and Zeegers, P.J., Evidence for a significant contribution from central effects of angiotensin in the development of acute renal hypertension in the greyhound, Clin. Sci. Mol. Med., 48 (1975) 115-119. 58 Scroop, G.C., Katic, F., Joy, M.D. and Lowe, R.D., Importance of central vasomotor effects in angiotensin-induced hypertension, Brit. Med. J., 1 (1971) 324-326. 59 Sernia, C. and Mowchanuk, M.D., Brain angiotensinogen: in vitro synthesis and chromatographic characterization, Brain Research, 259 (1983) 275-283. 60 Shimizu, N. and Ishii, S., Fine structure of the area postrema of the rabbit brain, Z. Zellforsch. Mikroskop. Anat., 64 (1964) 462-473. 61 Shrager, E.E., Osborne, M.J., Johnson, A.K. and Epstein, A.N., Entry of angiotensin into cerebral ventricles and circumventricular structures. In D.S. Davies and J.L. Reid (Eds.), Central Actions of Drugs in Blood Pressure Regula-

tion, Univ. Park Press, Baltimore, MD, 1975, pp. 321-346. 62 Simmonet, G., Bioulac, B., Rodriquez, F. and Vincent, J.D., Evidence of a direct action of angiotensin II on neutones in the septum and in the medial preoptic area, Pharmacol. Biochem. Behav., 13 (1980) 359-363. 63 Simpson, J.B., The circumventricular organs and the central actions of angiotensin, 'Neuroendocrinology, 32 (1981) 248-256. 64 Singh, R., Husain, A., Ferrario, C.M. and Speth, R., Rat brain angiotensin II receptors: effects of intracerebroventricular angiotensin II infusion, Brain Research, 303 (1984) 133-139. 65 Sirrett, N.E., McLean, A.S., Bray, J.J. and Hubbard, J.I., Distribution of angiotensin II receptors in rat brain, Brain Research, 122 (1977) 299-312. 66 Snyder, R.L. and Sutin, J., Effect of lesions of the medulla oblongata on electrolyte and water metabolism in the rat, Exp. Neurol., 4 (1961) 424-435. 67 Torack, R.M. and Finke, E.H., Evidence for a sequestration of function within area postrema based on scanning electron microscopy and penetration of horseradish peroxidase, Z. Zellforsch. Mikroskop. anat., 118 (1971)85-96. 68 Torack, R.M., Stranahan, P. and Hartman, B.K., The role of norepineptrine in the function of area postrema. I. Immunofluorescent localization of dopamine-/3-hydroxylase and electron microscopy, Brain Research, 61 (1973) 235-252. 69 Volicer, L. and Loew, C., Penetration of angiotcnsin into the brain, Neuropharmacology, 10 (1971) 631-636. 70 Watson, W.E., The effect of removing area postrema on the sodium and potassium balances and consumptions in the rat, Brain Research, 359 (1985) 224-232. 71 Worthington, W.C. and Cathcart, R.S., Ependymal cilia: distribution and activity in the adult human brain, Science, 139 (1963) 221-222. 72 Zandberg, P., Palkovits, M. and de Jong, W., The area postrema and control of arterial blood pressure; absence of hypertension after excision of the area postrema in rats, Pliigers Arch,, 372 (1977) 169-173.