The role of mesencephalic structures in thirst induced by centrally administered angiotensin II

Brain Research, 126 (1977)225-241

225

(~) Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

THE ROLE OF M E S E N C E P H A L I C S T R U C T U R E S IN T H I R S T I N D U C E D BY C E N T R A L L Y A D M I N I S T E R E D A N G I O T E N S I N II

JOHN KUCHARCZYK* and GORDON J. MOGENSON** Department of Physiology and Psychology, University of Western Ontario, London N6A 5Cl, Ont. (Canada)

(Accepted August 26th, 1976)

SUMMARY (1) In 27 animals microinjection of 25-100 ng of angiotensin II through chronic cannulae implanted in the preoptic region initiated drinking and in subsequent acute experiments influenced the spontaneous discharge rate of single neurons in the ipsilateral mesencephalon. Of 148 neurons for which recordings were made, 52 (35%) increased their frequency of spike potentials following administration of angiotensin II, 2 (1%) showed inhibition and 94 (64%) showed no change in firing rate. (2) In another series of 44 animals, unilateral or bilateral lesions of the midbrain ventral tegmentum or reticular formation were found to have little or no effect on water intake elicited by the microinjection of angiotensin II into the preoptic region. (3) In contrast to the effects oftegmental and reticular lesions, unilateral lesions located dorsally and laterally to the mammillary peduncle, in the area of passage of the medial forebrain bundle, significantly attenuated the dipsogenic response to either contralateral or ipsilateral injections of angiotensin II into the preoptic region. With bilateral lesions this effect was permanent. (4) Since the more caudal lesions were relatively ineffective in disrupting the elicited drinking, it is suggested that signals from angiotensin II receptors in the preoptic region are transmitted along pathways which diverge in the midbrain. (5) The possibility of a forebrain-hypothalamus-midbrain circuit mediating thirst initiated by activation of the renin-angiotensin system is discussed. INTRODUCTION Drinking initiated by the administration of angiotensin 1I (ANG II) to the preoptic region (POA) of the rat is attenuated by lesions of the midlateral hypothalamus3,19 * Present address: The Physiological Laboratory, Downing Street, Cambridge CB2 3EG, Great Britain. ** To whom reprint requests should be addressed.

226 suggesting that signals from A N G Ii receptors in the POA are transmitted to this area of the diencephalon. Since lesions of the midbrain tegmentum and central grey have been reported to cause adipsia and aphagiaS,2'~, 23 and electrical stimulation of the ventral tegmentum elicits drinking and feeding 3v,4'~, the signals associated with angiotensin-induced drinking may project caudally through the midlateral hypothalamus to the midbrain. This hypothesis was investigated in the present study using electrophysiological and lesioning techniques. METHODS Initially, as a guide to the placement of lesions in the midbrain, it was decided to administer A N G II through chronic cannulae implanted in the POA and to observe the effects of A N G II on the spontaneous discharge frequency of single neurons in the midbrain and upper brain stem in subsequent acute recording experiments.

Microelectrode recording experiments with angiotensin II Preliminary drinking tests. Thirty-eight male Wistar rats weighing 250-350 g were anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg, i.p.), preceded by atropine sulfate (5 nag) and implanted with a single 23-gauge stainless steel cannula into the POA using stereotaxic techniques described previously 3. Following postoperative recovery, rats were tested for the occurrence of drinking after the microinjection of 100 ng of synthetic angiotensin II amide (Hypertensin, Ciba) dissolved in l #1 distilled water 6 through the indwelling POA cannula. Water intake ~:~:0.5 ml) was then measured in the animal's home cage over the next 30 min Acute single unit recording. Only those animals which drank more than 5 ml of water during each of two preliminary tests with A N G lI were used in the recording experiments. In these animals (n -: 27) A N G lI (100 ng in 1 #l distilled water) was microinjected into the POA through the implanted cannuta and the effects of A N G I1 on the frequency of firing of neurons in the midbrain and upper brain stem was measured. Action potentials of midbrain and brain stem neurons were recorded extracellularly with stainless steel microelectrodes (1-5 ~m tip diameter. 0.5--5 M~2 initial DC resistance in saline) insulated with lnsul-X (Insul-X Product Corp.). Signals were led into a Grass P15 AC preamplifier with a bandpass of 300-3000 c/sec. Preamplifier output was displayed on Tektronix 565 and 549 oscilloscopes for continuous and single sweep registration and was also led to a Grass AM 7 audiomonitor. In order to investigate the effects of administrating ANG ll to the POA on midbrain and upper brain stem unit activity, a Biomac 1000 signal analyzer (Data Instruments Ltd.) was used to generate frequency-time histograms. No more than 10 intracranial microinjections were made per animal in any recording session, and an interval of 40 min or more elapsed between two successive injections. In addition. every fifth unit was routinely tested with distilled water 6. In 7 rats. the femoral artery was cannulated with a polyethylene catheter connected through a Statham 23 db pressure transducer to a Grass 7B polygraph in order to record the arterial blood

227 pressure. For the same group of animals (n = 7) cortical E E G recordings were obtained through 3 nichrome wires implanted in the skull overlying the occipital cortex and lead directly into a wide-band EEG preamplifier mounted in a Grass 7B polygraph. In selected control experiments, A N G II was microinjected into the POA and unit recordings were obtained from sites in the cerebral cortex, hippocampus, thalamus and the basal forebrain rostral to the POA. In other experiments, A N G II was administered through a cannula implanted in the cerebral cortex and the response of units in the midbrain and brain stem was measured. Finally, the frequency of firing of units in the midbrain and brain stem was recorded following the microinjection of 25, 50 and 100 and 200 ng o f A N G II (all dissolved in 1 ~ul distilled water) through an indwelling POA cannula. Lesion experiments In order to determine whether circumscribed critical areas for the drinking response to intracranial A N G II might be found within the larger area of the midbrain in which unit responses to A N G lI were obtained, the effects of unilateral and bilateral mesencephalic lesions on drinking elicited by A N G 11 were determined. Animals and surgery. Animals (n 44) were individually housed and maintained on Purina laboratory chow and tap water ad libitum, except as noted. Two 23-gauge stainless steel cannulae were implanted bilaterally into the POA of each animal using standard stereotaxic techniques and barbiturate anesthesia. During the same operation two monopolar electrodes made from size 00 insect pins and insulated except for 0.5 mm at the tip were also implanted bilaterally into various locations in the mesencephalon. Pre-lesion measurements. About one week after implantation of the cannulae and electrodes food pellets were removed from the cages and replaced with food cups containing a measured weight of a high-carbohydrate diet 4°. For the next 6 days food and water intakes and body weights were determined daily. This procedure was repeated at selected 6-day intervals to ensure that the animals were healthy and gaining in body weight. Beginning on the tenth postoperative day each animal was tested once daily for drinking following the unilateral administration of A N G I1 (100 ng in 1 ~1 distilled water) to the POA. Water intakes ( ~ 0 . 5 ml) were measured for the next 30 rain in the animal's home cage with food freely available. Only those animals which drank more than 5 ml of water in each of 4 consecutive tests with A N G II were used in the lesion experiments. Lesion and post-lesion measurements. Unilateral lesions were made in 36 rats under ether anesthesia by passing 1.5-3.0 mA direct current for 15-40 sec between the implanted monopolar electrode and an indifferent electrode clipped to the ipsi[ateral ear. Testing with A N G I I was resumed following complete postoperative recovery (2-14 days post-lesion) (see Fig. 5). A second lesion, contralateral to the first, was made in 17 of the animals 24-31 days after the first lesion and testing with AN(3 11 was continued for an additional 50-58 days in the same manner as after the first lesion.

228 At the end o f this period o f time the animals were sacrificed by an overdose o f sodium pentobarbital and were perfused intracardially with 70 ml of 10c',:i formalin solution. The brains were removed and fixed in formalin for 48 h after which frozen sections were cut coronally at 50 # m and stained with thionin. Statistical analysis, Student's t-test for correlated samples was used to c o m p a r e responses o f the different groups o f animals to A N G I[. RESU LTS Effect o f P O A administration o f A N G H on midbrain unit activity

Extracellular recordings were obtained f r o m 148 neurons in the midbrain and upper brain stem o f which 52 (35%) increased their rate o f discharge in response to the administration o f 100 ng o f A N G II to the ipsilateral POA. Two units (1%) showed inhibition and 94 (62%) others showed no change in their discharge frequency. The latencies to the onset o f responses ranged f r o m 25 sec to 7 min and the response durations ranged f r o m 20 sec to 6 rain 45 sec. There did not appear to be a relationship between the locus o f the neurons and the latencies or response durations following the administration o f A N G II. Table I summarizes the numbers o f units and the patterns o f responses and Fig. 1 shows the location o f iron deposits corresponding to the sites o f recording. TABLE I Numbers of units in various midbrain structures and the patterns of responseJollowing the administration o f lO0 ng angiotensin to the ipsilateralpreoptic region Sites of recording

Periventricular grey substance Bed nucleus posterior commissure Reticular formation Ventral tegmental nucleus Tsai lnterpeduncular nucleus Nucleus Darkschewitch Central tegmental nucleus Red nucleus Substantia nigra Interstitial nucleus of Cajal

Total numbers Numbersof of units units recorded responding

Patterns of response ..................... Activated Inhibited

16

4

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0

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Fig. 1. Frontal sections through the mesencephalon redrawn from the stereotaxic atlas of Pellegrino and Cushman as showing the sites of recordings and the types of unit responses observed following the unilateral microinjection of 100 ng of angiotensin 11 into the ipsilateral preoptic region. Solid triangles indicate sites of facilitated units and open circles mark sites where no effect was obtained.

230 When 100 ng of A N G II was administered to the POA, activation (n ; 18) or inhibition (n 2) of unit activity occurred concomitantly with desynchronization of cortical EEG and an increase in arterial blood pressure (mean change .... : 5 mm Hg, range -- -i 3-15 mm Hg). A typical response is shown in Fig. 2A. With the lowest dose of A N G I1 (25 ng) cortical EEG and blood pressure changes were not observed although 2 of 14 units increased their rate of discharge (Fig. 2B). PST histograms showing the response of a single neuron in the midbrain tegmentum to the administration of 25, 50, 100 and 200 ng of A N G 11 to the POA are presented in Fig, 3. The effects of microinjecting 100 ng of A N G II into the POA on the rate of

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Fig. 2. A: continuous polygraph tracings showing the changes in cortical EEG (top tracing), arterial blood pressure (middle tracing), and spike discharge of a neuron in the midbrain following the unilateral administration of 100 ng of angiotensin 11 to the ipsilateral preoptic region. Note that the blood pressure (trace 580) and EEG (trace 583) changes preceded the increase in unit discharge frequency (trace 583). B: continuous polygraph tracings showing cortical EEG, arterial blood pressure and discharge of another unit in the midbrain following the administration of 25 ng of angiotensin II. The time at which angiotensin was administered is indicated by arrows. Note that blood pressure and EEG did not change although the neuron increased its rate of discharge (traces 444 and 445). What appears to be an increase in neuronal discharge rate during and immediately following the microinjections is due to a large transient change in voltage which caused a temporary imbalance in the amplifier. This unstable period associated with the microinjection procedure, also seen in Fig. 3, lasted for 5 set or less. The time scale is in sec.

232

A

B

Fig. 3. Poststimulus time histograms showing the response of a single unit in the rnidbrain tegmentum to the unilateral administration of different doses of angiotensin II to the ipsilateral pre0ptic region: A, 25 rig; B, 50 ng; C, 100 ng; D, 200 rig. Time of administrations is indicated by arrows. No less than 40 min elapsed between successive injections. The order in which doses were administered was: 100 ng, 50 ng, 25 rig, 200 ng. to A N G II were as follows: thalamus, 0, 0, 8 ; caudate nucleus, 0, 0, 7; hippocampus, 2, 0, 8. In two other animals, microinjection of 100 ng of A N G II into the cerebral cortex did not affect the baseline discharge rate of 12 units in the midbrain for which extracellular recordings were made.

Effects of unilateral lesions of the midbrain on drinking to angiotensin 11 The lesions, illustrated in Figs. 4 and 5, were located within an area bounded rostrally by the mammillary peduncle and caudally by the anterior border of the inferior colliculus and between the midline and the lateral lemniscus (coordinates: 0.1-4.0 m m ventrodorsal, 0.4-2.0 lateral. 0.5-2.0 rostrocaudal). Three groups were formed on the basis of the locus of the lesions. The lesions in the first group (n = 13) were located in the region of the ventral tegmentum from which changes in the discharge rate of single neurons were recorded in response to A N G II administered to the preoptic region. The nuclear structures which were damaged or destroyed unilaterally by this type of lesion included the interpeduncular nucleus, the ventral tegmentaI nucleus of Tsai, the central tegmental nucleus, the red nucleus and the medial aspects of the substantia nigra. Fiber tracts which appeared to have been transected unilaterally by the ventral tegmentat lesions

233 included the habenulointerpeduncular tract, the mammillotegmental tract and the central tegmental tract. The medial lemniscus also received moderate to extensive damage. Unilateral ventral tegmental lesions had little or no effect on drinking elicited by A N G II administered to the POA (Fig. 6). There was an indication of a slight attenuation of elicited water intake when the lesion encroached on the neural tissue adjacent to the interpeduncular nucleus (n 5), however, the variability of this effect was great and the differences were not statistically significant. Seven of the animals with unilateral lesions in the ventral tegmental area showed varying durations of aphagia postoperatively (mean number of days of aphagia 2.8, range of 1-4 days). Of these 7 rats, 5 required intragastric feeding for from 2 to 4 days. The lesions also typically caused a small reduction in water intake for 1-3 days post-lesion but thereafter dairy water intakes returned to pre-lesion levels. A second group of lesions (n -- 15) was located in the reticular formation ventrolateral to the central grey. In 8 of the animals the lesions extended unilaterally into the central grey and produced damage to the bed nucleus of the posterior commissure, the nucleus of Darkschewitsch and the interstitial nucleus. In the remaining animals the lesions unilaterally damaged or destroyed the lateral tegmental nucleus, the substantia nigra, large areas of the midbrain reticular formation, the medial lemniscus and the habenulointerpeduncular tract. None of the unilateral lesions in the second group had a significant effect on drinking elicited by ANG II administered to the POA (Fig. 6). Four of the lesions which were located dorsal and lateral to the red nucleus (11 9) produced 1-5 days of aphagia and 3-9 days of hypodipsia postoperatively. Most of the animals with lesions of the reticular formation ventrolateral to the central grey were hyperactive, lost body weight in the immediate post-lesion period and were difficult to handle. Testing with ANG II was not resumed until 5-14 days postoperatively. In a third group the lesions (n = 8) destroyed the mammillary nuclei and the ventral tegmental nucleus of Tsai unilaterally, transected the medial lemniscus and the medial forebrain bundle and partially ablated the rostral part of the midbrain reticular formation (Fig. 5A, B). The common area of destruction produced by these lesions was dorsal and lateral to the mammillary peduncle, in the area of passage of fibers of the medial forebrain bundle (MFB). These unilateral lesions of the rostral midbrain caused a significant decrease of water intake elicited by A N G II administered through either preoptic cannula in the immediate post-lesion period in 7 of 8 animals (t - 2.44, P < 0.05 in comparison with pre-lesion intakes). The attenuation of elicited drinking was transient, however, and recovery was observed in all 7 animals by the sixth postoperative day (Fig. 6). In contrast to the effects of lesions located more caudally in the midbrain ventral tegmentum or in the area ventrolateral to the central grey, rats with lesions of the rostral midbrain showed no significant change in 24 h food and water intakes or in the rate of body weight increase. Effi~cts of bilateral lesions of the midbrain on drinking to angiotensin H Seventeen of the animals that received unilateral lesions were given a second lesion, contralateral to the first, through an indwelling electrode 26-32 days following the first lesion.

234 As shown in Fig. 6, the second lesion of the midbrain ventral tegmentum (n = 4) or of the area ventrolateral to the central grey (n -:- 6) did not significantly affect drinking elicited by A N G II. In the majority of cases, animals that received lesions of either of these two sites were hypophagic and hypodipsic in the immediate post-lesion period and could not be tested with A N G II for 7-10 days postoperatively. Three of the rats with lesions of the midbrain ventral tegmentum required 214 days

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Fig. 4. A: reconstructions of the individual unilateral lesions in the midbrain which did not affect drinking to angiotensin II. The lesions are superimposed on frontal sections from a rat stereotaxic atlas 35. The shaded area shows the region of overlap of the lesions. Numbers at the lower left of each figure are taken from the atlas and refei to the distance behind bregma. B: reconstructions of individual bilateral lesions of the rnidbrain which were not effective in disrupting water intake elicited by angiotensin 11. The numbers inside the lesions refer to the individual animals in which the lesions were made.

236

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Fig. 5. A: location of lesions in the v e n t ~ m e d i a l region of the r0stral midbrain marked on sect'ons of brain from a rat stereotaxic atlas 35. The portion shaded in black indicates the area common to all lesions and the black outline indicates the largest extent of all lesions. These lesions produced a significant attenuation of the drinking response elicited by angiotensin II (see text). B: representative photomicrograph showing the locus of the rostrat midbrain lesions. Horizontal bar at bottom equals 2 mm.

237

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Fig. 6. Water intakes of rats following (A) unilateral lesions of the midbrain ventrolateral to the central grey (open circles), of the paramedial dorsal and vential tegmentum (solid circles) and of the ventromedial area of the rostral midbrain (triangles) during the 30 min period following the unilateral microinjection of 100 ng of angiotensin II into the ipsilateral preoptic region, and (B) following a second, contralateral lesion. Results are expressed as mean i S.E.M. of consecutive ipsilateral and contralateral injections. Time of the lesions is indicated by arrows. o f intragastric feeding. Two o f these animals and two rats with extensive lesions in the reticular formation dorsolateral to red nucleus showed signs of spasticity of the hind limbs, loss of balance and rotational movements for 2-5 days post-lesion. All 7 animals in which a second lesion was made in the rostral midbrain showed a significant (t = 2.31, P < 0.05 in comparison with pre-lesion intakes) and chronic decrease in water intake elicited by A N G II for the entire 58-day period following the second lesion. During this time the animals drank less than 30°o of their pre-lesion intakes to A N G 11. In the final test with A N G II on day 58 the animals with rostral midbrain lesions drank significantly less than during the pre-lesion period (t -~ 2.23, P < 0.05). The changes in daily food and water intakes after the second lesion were minor, although 2 of 7 animals were hypodipsic for 7 and 11 days post-lesion, respectively. All rats had regained their pre-lesion b o d y weights by the fourth postoperative day.

238 DISCUSSION The results of this investigation indicate that signals from receptors for A N G il in the forebrain are transmitted to the mesencephalon. This interpretation was suggested initially by the electrophysiological results, in which it was found that microinjection of A N G II into the POA influenced the discharge rate of single neurons in the midbrain, and was subsequently confirmed by the lesion experiments in which ablation of the rostral midbrain disrupted drinking to A N G 1I. Extracellular recordings were used in this study as a guide for determining where to place lesions for the behavioral tests. Most of the units which responded to A N G II were located in an area just ventrolateral to the central grey, or in the paramedial dorsal or ventral tegmentum. Since lesions placed in either of these areas had little or no effect on drinking induced by A N G II, the changes in midbrain unit discharge frequency could be interpreted as a non-specific effect of A N G 1I. Alternatively, it is possible that the lesions placed posterior to the meso-diencephalic junction were not large enough to destroy a significant portion of the projection area in the midbrain fibers carrying information from A N G II receptors in the forebrain. This suggestion is supported by the results of anatomical studies which have shown fibers of the major limbic input into the midbrain, viz. the medial forebrain bundle (MFB,) to demonstrate considerable ipsilateral divergence and crossing-over at the level of the supramammillary decussationl,"4, z0,31 and as well by the observations that lesions of the more compact aspects of the MFB at the level of the midlateral hypothalamus 3,19 or in the rostral midbrain disrupt drinking induced by the microinjection of A N G I1 into the POA. A second important consideration when interpreting the electrophysiological data is whether the changes in the discharge of midbrain neurons reflect changes in midbrain thirst systems activated by A N G II or are secondary to blood pressure or EEG changes. The issue of the specificity of the unit responses is further complicated because desynchronization of EEG or increased discharge frequency of neurons is difficult to interpret in terms of specific behavioral arousal when the animal is anesthetized. The recorded latencies for the unit effects were generally the same or longer than those observed for the elicitation of drinking in the behavioral experiments and typically occurred before or with the same latency as the EEG and blood pressure changes. These results could be interpreted to indicate a non-specific effect of A N G II, since it has been shown that slight increases in arterial pressure can cause marked changes in the spontaneous activity of central neurons 15. However, two sets of observations suggest that the changes in neuronal firing frequency may have been mediated by a direct action of A N G II. First, at the lowest dose of A N G II (25 ng), facilitation of two neurons in the ventral tegmentum occurred in the absence of any detectable changes in E E G or blood pressure. Second, Findlay 7 has shown that POA neurons respond with a latency of 25 see or more following local application of A N G II by microiontophoresis so that the long latency responses of midbrain neurons may merely reflect slow binding of A N G II at its receptor sites. However, since Phillips and Felix z6 have shown very short latency responses in the subfornical organ to

239 A N G II microiontophoretically administered, it is not clear why binding of the peptide might be much slower in one area of the brain (e.g., POA) than in another (e.g., SFO). Considered together with previous work3, ln,2v the data reported here suggest that the POA, hypothalamus and midbrain are connected by a neural circuit through which signals for drinking induced by A N G II are directed. Other studies have shown that limbic forebrain-midbrain interactions may also participate in the neural control of agonistic z and emotional 12,18,28,38 behaviors, in the neural processing of sensory information 34, and in sleep 21,33. For agonistic and ingestive behaviors, it has been proposed that the primary role of limbic structures and the hypothalamus is to integrate the various endocrine, autonomic and skeletal efferent signals whereas the midbrain appears to be more involved in coordinating the motor patterns required to perform the appropriate behavior 25. According to such a view, 'command signals' from ANG II receptors in the forebrain could activate appropriate mesencephalic systems to initiate water intake. Although a rostrocaudally directed forebrain-hypothalamus midbrain circuit subserving ANG II drinking is suggested by the results of this study, other neural systems may also be involved. For example, it has been shown that in addition to the descending MFB, limbic structures project caudally by a more dorsal route which passes through the habenular nucleus and then continues ventrocaudally to the interpeduncular nucleus, dorsal and ventral tegmental nuclei and caudal central grey I/)4,30, 4:3. Although the present results indicate that the MFB is the major pathway for trailsmitring signals from A N G 1I receptors to the mesencephalon, the observation that the elicited drinking is not completely abolished following lesions of the midlateral hypothalamus 19,'-'7 or the rostral midbrain suggests the possibility that the dorsal pathway may also play a role in drinking to A N G II. It is also possible that water intake induced by A N G I1 may not be mediated exclusively by descending pathways. In addition to limbic forebrain projections to the mesencephalon there are ascending parallel fibers in the MFB which terminate in, or send collaterals to, the lateral hypothalamus, preoptic region, septum and the rhinencephalon l°,11,')4. Morgane "9 and others~, 3z have suggested that these rostral connections may be important in ingestive behaviors. Another alternative is that other limbic and hypothalamic structures may play an important modulatory role in the initiation of drinking by A N G lI by exerting descending influences directly, or indirectly, onto the midbrain motor systems. This possibility is suggested by anatomical data which has shown that the path neurons of the descending MFB, especially those in the lateral hypothalamus, demonstrate highly developed dendritic fields that intermingle extensively with adjacent neural populations so that activity in widespread forebrain and diencephalic fields may be conveyed to recipient midbrain structures 24. The finding that lesions of the ventromedial nucleus of the hypothalamus cause an increase in water intake elicited by the microinjection of A N G II into the POA 1'~ is consistent with this view. Finally, it should be noted that a definitive interpretation of the results presented here will depend on knowing the exact loci of the receptors for A N G lI. Since it has

240 been s h o w n that water intake may be initiated by the m i c r o i n j e c t i o n o f A N G !1 into the subfornical organ 39 or a n t e r i o r third ventricle '~6, these structures may co n t ai n receptors which are distinct f r o m those in the P O A . Recent studies "m,2v support this p r o p o s a l and further suggest that separate neural p a t h w a y s m ed i at e drinking elicited by microinjection o f A N G If into the P O A , subfornical o r g a n and third ventricle. ACKNOWLEDGEM ENTS Th e a u t h o r s express their a p p r e c i a t i o n to k. Brizio, A. M o k and B. W o o d s i d e for their technical assistance a n d to F. Calaresu, D. C o o k e , R. W ei ck and M. Evered for helpful suggestions in the p r e p a r a t i o n o f the manuscript. S u p p o r t e d by grants from the Medical Research C o u n c i l o f C a n a d a and the N a t i o n a l Research C o u n c i l o f C a n a d a .

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