Reduction of voluntary alcohol consumption in the rat by transplantation of hypothalamic grafts

Brain Research, 632 (1993) 287-295 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

287

BRES 19593

Reduction of voluntary alcohol consumption in the rat by transplantation of hypothalamic grafts A.J. Lan§a

*, L . A . G r u p p ,

Y. Israel

Addiction Research Foundation and Departments of Pharmacology and Medicine, Unit,ersity of Toronto, Toronto, Ont., M5S 1A1 Canada

(Accepted 17 August 1993)

Key words: Hypothalamic graft; Alcohol consumption; Angiotensin II immunoreactivity; Subfornical organ; Paraventricular nucleus; Rapp SS/Jr rat

Stimulation of the peripheral renin-angiotensin system has been shown previously to decrease the voluntary intake of ethanol in the rat. The existence of a separate brain renin-angiotensin system, independent from that of the periphery, has been widely demonstrated. The brain renin-angiotensin system plays an important role in the regulation of water and electrolyte balance and neuroendocrine function. However, the role played by this system in the regulation of voluntary alcohol consumption has not yet been studied. The goal of the present work was to assess the feasibility of decreasing the voluntary alcohol intake in a strain of rats (Rapp SS/Jr rats) that have a genetic deficiency responsible for a low activity of the renin-angiotensin system and elevated alcohol intake. Adult Rapp SS/Jr rats received intraventricular transplants of fetal hypothalamic grafts (from normal donors), known to contain angiotensin-immunoreactive cell bodies. Our studies revealed that angiotensin-immunoreactivity in the cell bodies and fibres in the paraventricular, supraoptic and suprachiasmatic nuclei of the hypothalamus in Rapp SS/Jr rats was markedly reduced. Animals that had surviving grafts containing angiotensin-immunoreactive cell bodies in the dorsal third ventricle--but not in the ventral third ventricle, in the lateral ventricles, or sham operated animals--had a 40% decrease of their voluntary alcohol intake, when compared to their intake before surgery, or to the control group. However, water consumption was not reduced in both the sham and transplanted animals. The reduction of alcohol intake obtained after transplantation of angiotensin-producing cells suggests that the brain renin-angiotensin system may play a modulatory role in the regulation of voluntary alcohol intake, although they do not exclude the participation of other neurotransmitters in the modulation of alcohol consumption.

INTRODUCTION

a t e d t h r o u g h d i f f e r e n t s u b t y p e s of d o p a m i n e r e c e p tors33,61, 65.

T r a n s p l a n t a t i o n o f fetal n e r v o u s tissue has p r o v i d e d v a l u a b l e i n f o r m a t i o n on the m e c h a n i s m s g o v e r n i n g dev e l o p m e n t a n d r e g e n e r a t i o n in t h e m a m m a l i a n c e n t r a l n e r v o u s system 24,35,47,62. N e u r a l t r a n s p l a n t a t i o n has

N e u r a l t r a n s p l a n t a t i o n has also b e e n successfully u s e d to c o r r e c t situations c a u s e d by g e n e t i c deficiencies. T r a n s p l a n t a t i o n o f fetal v a s o p r e s s i n - p r o d u c i n g n e u r o n s into t h e t h i r d ventricle o f rats h o m o z y g o u s for d i a b e t e s insipidus ( B r a t t l e b o r o rats), which a r e u n a b l e to p r o d u c e vasopressin, has also m e t with c o n s i d e r a b l e success 15'68. V a s o p r e s s i n - c o n t a i n i n g grafts of fetal hy-

b e e n p a r t i c u l a r l y successful in t r e a t i n g f u n c t i o n a l deficiencies c a u s e d by i r r e v e r s i b l e d a m a g e o f t h e c e n t r a l n e r v o u s s y s t e m - - a s s e e n in d i s o r d e r s such as P a r k i n son's a n d H u n t i n g t o n ' s d i s e a s e 29,39. T h e i m p l a n t a t i o n o f fetal d o p a m i n e - p r o d u c i n g m e s e n c e p h a l i c grafts into the d o p a m i n e - d e p l e t e d c a u d a t e - p u t a m e n o f p a r k i n s o nian rodents and humans ameliorates motor impairments, i n c l u d i n g rigidity, t r e m o r a n d h y p o k i n e s i a 2,4°. R e c e n t studies have shown t h a t t h e b e h a v i o u r a l effects p r o d u c e d by t h e d o p a m i n e - p r o d u c i n g grafts a r e m e d i -

* Corresponding author. Fax: (1) (416) 978-4811. SSDI 0006-8993(93)E 1182-3

p o t h a l a m u s f r o m n o r m a l d o n o r s survived t r a n s p l a n t a tion into the t h i r d v e n t r i c l e o f B r a t t l e b o r o rats a n d w e r e able to c o r r e c t t h e d i a b e t e s insipidus s y n d r o m e by r e d u c i n g w a t e r i n t a k e a n d raising u r i n e o s m o l a l i t y to values i d e n t i c a l to t h e o n e s s e e n in B r a t t l e b o r o rats t h a t have b e e n c h r o n i c a l l y t r e a t e d with v a s o p r e s sin 15'68'69. R e s t o r a t i o n o f c i r c a d i a n rhythms, t h r o u g h

288 the implantation of the suprachiasmatic nucleus of the hypothalamus, has been demonstrated by using strains of hamsters that have different circadian patterns 5~'~7. The predisposition to develop alcoholism and the effects of ethanol on the nervous system are, in part, genetically determined 1'4'66. Chronic alcohol consumption frequently leads to hypertension in humans 4~'43, and previous reports have suggested the renin-angiotensin system as a plausible mediator of this effect 42'43'77. Alcohol consumption, either chronic or acute, produces an elevation in activity of the plasma renin-angiotensin system in humans, as well as in rats 2~'41"42"54"8°. However, the functional significance of this effect was unknown until Grupp and collaborators showed that the renin-angiotensin system can also modulate alcohol consumption 22. Manipulations which increase activity in the R-A system produce a decrease in alcohol intake, while reductions in the activity of this system lead to an increase in alcohol intake 1~'22'27. These findings suggest that there is a functional and reciprocal relationship between alcohol intake and the R-A system. The existence of a separate brain renin-angiotensin system, independent from that of the periphery, has been established by biochemical ~2'25, immunohistochemica13,m, ll,13,36,37,3s,s8, n e u r o a n a t o m i c a 1 2 3 , 3 2 , 3 6 , 3 8 , 53, pharmacological 16'17'31"45'64'7°'7e, as well as in situ hybridization techniques s'74. In the diencephalon, the subfornical organ (SFO) is particularly rich in angiotensin-immunoreactive (AnglI-IR) cell bodies and fibers 32"37"3~'53. The SFO is a circumventricular organ and lacks an effective blood-brain barrier, allowing it to be a particularly effective sensor of the circulating levels of angiotensin and to play a key role in the regulation of angiotensin-mediated effects 51'52'67. The SFO projects heavily to the paraventricular and the supraoptic nuclei of the hypothalamus 37'46. These subfornical-hypothalamic projections are excitatory and approximately 50% of their fibers are angiotensin-imm u n o r e a e t i v e g , 2 3 , 3 2 , 36,38.53,60.

The medioventral hypothalamus contains angiotensin-producing neuronal cell bodies that are located in the magnocellular part of the supraoptic (SON) and paraventricular (PVN) nuclei, as well as in the parvocellular portion of the PVN and suprachiasmatic ( S C h N ) n u c l e i 26'34"36'38. The brain R-A system plays an important role in the regulation of water and electrolyte balance and neuroendocrine function ~4. However, its role in the regulation of voluntary alcohol consumption has not yet been adequately investigated. The goal of the present work was to study this possible role by implanting fetal hypothalamic tissue containing angiotensin-producing neurons, from normal rat

donors, into the third ventricle of alcohol-drinking Rapp S S / J r (salt-sensitive) rats. This strain of inbred rats 5s has a genetic deficiency of the R-A system 7'4'I and shows an elevated alcohol consumption 21'4~. MATERIALS

AND METHODS

Animals The study was conducted on thirty-one adult male Rapp S S / J r rats (Harlan Sprague-Dawley) weighing 200-250 g at the beginning of the experiment. The animals were individually housed under standard conditions and were kept on a 12-h light/dark cycle. All the experimental procedures described in the present report were approved by the Animal Care Committee of the University of Toronto. Animals submitted to surgery were previously anesthetized with sodium pentobarbital (55 m g / k g , i.p.).

Drinking procedure The animals were individually caged and had free access to food 24 h a day. The 24-h two-bottle choice procedure was used, which offered the animals a choice between an alcohol solution (3% or 6% weight/volume) and water. The location of the tubes was alternated daily. Before transplantation the animals were offered a choice between 3% (w/v) alcohol solution and water (phase 1, 22 days), followed by a phase of 6% (w/v) alcohol vs water (phase 2, 22 days). In the third phase (10 days) the animals were presented with water in both tubes. The animals were then submitted to the transplantation procedure and allowed a recovery period of at least 1 week. After recovery the animals were reassessed for drinking behavior beginning with water only (phase 4, 23 days) followed by 3% alcohol vs. water (phase 5, 14 days) and finally by 6% alcohol vs. water (phase 6, 19 days). During all phases of the experiment the body weight of the animals was recorded daily, and the amounts of ethanol and water drank every day were expressed in milliliters per kilogram of body weight. The data obtained in the drinking studies were statistically analysed by A N O V A and post-hoc two-sample t-test.

Transplantation procedure Of the initial thirty-one adult male Rapp S S / J r rats 20 received fetal hypothalamic grafts and 11 were sham operated controls. Timed pregnant Sprague-Dawley rats were anesthetized with pentobarbital (50 m g / k g , i.p.). Graft tissue (1 m m 3) from the medioventral hypothalamic primordium of normal Sprague-Dawley fetuses on day 16 to 19 of gestation (E16-E19; c r o w n - r u m p length 16-29 m m ) 68 was isolated under sterile conditions by microdissection 35 in Hank's balanced salt solution, calcium- and magnesium-free (HBSS). Immediately after dissection the grafts in HBSS (total volume of 30 /zl) were stereotaxically injected into the third ventricle or the lateral ventricle of the host. Injection coordinates for the implantation of the solid hypothalamic fetal grafts (E16-E19) were: third ventricle: AP = 2 m m posterior to bregma, 0 m m lateral and 9.5 m m ventral to the skull surface; Lateral ventricle: AP = 0.8 m m posterior to bregma, 1.5 mm lateral and 4.5 mm ventral to the skull surface, with the incisor bar set at 3.3 m m below zero, according to Paxinos and Watson 5°. The sham operated animals received a 3 0 / z l injection of HBSS, without tissue, into the third ventricle using the same coordinates as reported above.

Histological and immunocytochemical procedures At the end of the e x p e r i m e n t - - a f t e r the behavioural studies were concluded (i.e. starting at 3 m o n t h s after the transplantation procedure, and up to 6 m o n t h s after t r a n s p l a n t a t i o n - - t h e animals were stereotaxically injected with amastatin or bestatin (100 nmol in 10 /xl), respectively a m i n o p e p t i d a s e A and a m i n o p e p t i d a s e B inhibitors 31"73'sl, into the lateral ventricle (AP = 0.8 m m posterior to bregma, L = 1.5 mm, DV = 4.5 mm) 15 rain before being intracar-

289 dially perfused with a fixative solution. Amastatin and bestatin were both equally successful in enhancing the visualization of angiotensin immunoreactivity by preventing its enzymatic degradation. The animals were perfused with 100 ml of saline, followed by 400 ml of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.25). Immediately after perfusion the brains were removed from the skull and immersion fixed in the same fixative containing 10% sucrose for 12-24 h at 4°C. Coronal serial sections (28 ,o,m thick) were cut on a cryostat. The sections were subsequently processed for immunocytochemistry using antibodies against angiotensin II (AnglI-Ab) and against arginine-vasopressin (AVP-Ab). Free-foating sections were pre-incubated for 1 h in phosphate buffer saline (PBS; 0.1 M pH7.25) with 2% normal goat serum (to reduce the background) and 0.3% Triton X-100 (to improve antibody penetration). Alternate sections were then incubated for 12-24 h at 4°C in a primary antibody solution containing either antibodies against angiotensin II (AII-Ab; 1/500) or against arginine-vasopressin (AVP-Ab; 1/2000). Both primary antisera were raised in rabbit. The indirect immunofluorescence technique was followed using secondary antibodies (goat anti-rabbit) labelled with fluorescein isothiocyanate (FITC) (1/80) or tetramethylrodamine isothiocyanate (TRITC) (1/40). The sections were photographed using a Zeiss Axioscope epifluorescence microscope. The antibody against Ang II (Ganten/DE) was graciously provided by Dr. D. Ganten, German Institute for High Blood Pressure Research, Heidelberg, Germany, and its specificity has been detailed elsewhere 3'38'53. The antibody against AVP was purchased from Accurate Chem. and Sci. Corp. (USA). The FITC- and TRITC-labelled antibodies were purchased from Sigma Chem. Comp. (USA). Following the immunocytochemical studies the sections were stained with Cresyl violet and compared to the results obtained with the immunocytochemistry. At the end of the experiment the data obtained from the drinking studies were statistically analyzed and were correlated, on an individual basis, with the results from the histological and the immunocytochemical studies.

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RESULTS Of the 20 animals that received hypothalamic grafts 4 animals died prior to completion of the experiment and their data will not be reported. Two of these animals died within less than twelve h after surgery and the cause of death was a surgery-related hemorrhage. The other two animals died during the post-surgery drinking studies. Post-mortem studies revealed that pneumonia was the cause of death. The histological and immunocytochemical studies of the remaining sixteen animals showed that eight had grafts located in the dorsal third ventricle (Transplant group) which were found to be viable as shown by histology. Four animals had surviving grafts located in the lateral ventricle or in the ventral portion of the third ventricle. The remaining four animals either had transplants which showed extensive signs of degeneration, containing abundant lymphocytes and macrophages (n = 2), or the transplant was not present (n = 2). Of the 11 animals that received the sham operation (Sham group) two died before completion of the experiment, and only the data from the remaining animals (n = 9) will be reported. The cause of death was not related to the surgical manipulation.

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Fig. 1. Drinking behaviour of the transplanted (e) (n = 8) and sham operated animals (O) (n = 9). Each point represents the group mean values on each test in two consecutive days, and vertical bars indicate standard errors of the mean. (A) Drinking of the 3% (w/v) alcohol solution before (phase 1) and after transplantation (phase 5). (B) Drinking of the 6% (w/v) alcohol solution before (phase 2) and after transplantation (phase 6). (C) Water drinking during the phases of water in both tubes before (phase 3) and after transplantation (phase 4).

Drinking studies Fig. 1A indicates that, following the grafting procedure, animals that received the graft in the dorsal third ventricle (Transplant group) showed a reduction in the consumption of the 3% (w/v) ethanol solution while the Sham group showed a tendency to increase its alcohol intake. A two-way ANOVA revealed a significant group X phase interaction (F1,~5 = 20.13, P = 0.0004) indicating that the two groups did differ in the changes in alcohol consumption resulting from surgery. Following surgery the Transplant group drank 40% less alcohol compared to its pre-surgery intake ( t 7 = 7.0,

290 P < 0.001) and 34% less alcohol than the post-surgery consumption of the Sham group (tt5 = 2.27, P < 0.05). Figure 1B illustrates the consumption of the 6% (w/v) alcohol solution in both the Sham and Trans-

plant groups. A two way A N O V A revealed significant effects of phase (F~.15 = 11.32, P = 0.004) and group × phase interaction (Fj.~5 = 6.11, P = 0.003) indicating that the significant increase in alcohol consumption following surgery occurred only in the Sham operated group and not in the Transplant group. Post hoc tests revealed that following surgery the Transplant group drank significantly less alcohol than the Sham group (t15 = 2.26, P <0.05) and that only the Sham group significantly increased its alcohol intake after surgery (t~ = 3.3, P < 0.01). Taken together these findings indicate that the grafts of fetal hypothalamus located in the dorsal third ventricle do significantly reduce alcohol-seeking behaviour. Finally, Fig. 1C shows that water intake in both the sham-operated and transplanted animals that received the hypothalamic graft in the dorsal third ventricle was not reduced after surgery, and was identical in the two groups. This suggests that the reduction in alcohol intake observed in the Transplant group is a result of the interaction between the graft and alcohol intake per se and not the result of a non specific decrease in the consumption of all fluids. The body weights of all the animals used in this experiment were recorded daily, and no significant differences between groups were found at any phase of our study.

Histological and immunocytochernical studies The h&tological studies of the Nissi stained sections

Fig. 2. Nissl staining of host brain sections showing the surviving hypothalamic transplants (E16-E19) in animals sacrificed 6 months after transplantation. A: hypothalamic transplant (T) located in the dorsal portion of the third ventricle (IIIV) attached to the subfornical organ (SFO) of the host's brain. An extensive interface (arrows) can be seen between the transplant and the SFO. B: higher magnification of the previous figure showing that no well defined histological barrier is seen between the SFO and the transplant (T). C: hypothalamic transplant showing large-sized magnocellular neurons (arrows) and small-sized parvocellular neurons (arrowheads). Scale bars: A = 250 ~zm;B = 100 ~m; C = 50 p.m.

of the surviving transplants present in the dorsal third ventricle (n = 8), ventral third ventricle or lateral ventricle (n = 4) did not show any signs of neuronal degeneration or invasion by proliferating lymphocytes or macrophages, even after long survival times of up to 6 months post-transplantation. A broad interface was seen between the transplant and the host brain, particularly when the transplants were adjacent to the subfornical organ (Fig. 2A). A higher magnification of the same transplant (Fig. 2B) shows that the degree of integration between the two structures occurs to such an extent that no obvious boundaries are seen between them. The morphological characteristics of the neuronal population present in all the surviving grafts showed that two main types of neurons are present: (a) large-sized neurons, identical to the magnocellular neurons of the PVN and SON (Fig. 2C, arrows); and (b) small-sized neurons identical to the parvocellular population of the PVN and SChN (Fig. 2C, arrowheads). The immunocytochernical studies demonstrated the existence of angiotensin-immunoreactive cell bodies in the hypothalamic grafts located in the third as well as

291 Alternate sections were immunostained with antibodies against arginine-vasopressin (AVP) (Fig. 4). AVP-immunoreactivity was particularly intense in the magnocellular part of the PVN, SON and accessory magnocellular neurons in the host brains of Rapp S S / J r rats. A weaker AVP-immunoreactivity was also seen in the SChN. AVP-immunoreactive cell bodies and abundant fibers were also seen in the hypothalamic transplants located in the third ventricle. DISCUSSION

Experimental model Fig. 3. Immunocytochemical demonstration of angiotensin-immunoreactive cell bodies (arrows) in a hypothalamic transplant located in the third ventricle of the host brain. The AnglI-containing cell bodies are shown after incubation in primary antibody raised against AnglI, and after incubation in a TRITC-labelled second antibody. Scale bar = 50/zm.

in the lateral ventricles. Fig. 3 shows a transplant located in the third ventricle, containing angiotensinimmunoreactive cell bodies (Fig. 3, arrows). These cell bodies belonged to magnocellular, as well as parvocellular neurons. In the host brain of Rapp S S / J r rats treated with aminopeptidase inhibitors only occasionally a few and weakly AnglI-IR cell bodies and fibres were seen in the PVN, SON or SChN of the hypothalamus. In the subfornical organ weak angiotensin-immunoreactive cell bodies were located in the periphery, in animals treated with aminopeptidase inhibitors.

The Rapp S S / J r rat is a line of inbred rats initially selected for its susceptibility ( S S / J r ) or resistance ( S R / J r ) to the hypertensive effects of a high salt diet 58. This line of animals was selected from an outbred population of salt-sensitive and salt-resistant rats from the original Sprague-Dawley stock 5'6. The Rapp S S / J r rat has a structural alteration of the renin gene 7, responsible for lower plasma renin activity than the control Rapp S R / J r (salt-resistant) rats 3°'59, and also shows an increased alcohol consumption 2L48. The association of low basal levels of plasma renin activity with elevated alcohol intake is also present in a genetically selected rat line which prefers alcohol (i.e. the alcohol-preferring P rat) ~8. This association suggests that genetically inherited differences in the activity of the renin-angiotensin system might play a role in the regulation of voluntary alcohol intake. In our work we studied the brain renin-angiotensin system of the Rapp S S / J r rat using immunocytochemistry, with antibodies against angiotensin II, in order to establish if the low basal levels of activity of the plasma renin-angiotensin system was also expressed in the brain. At the telencephalic level we have studied the distribution patterns of angiotensin II and vasopressin immunoreactivity in the subfornical organ (SFO) and the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SChN) nuclei of the hypothalamus. These structures were selected for the role they have been shown to play in the regulation of alcohol cons u m p t i o n - - S F O 2 ° - - a n d in the neuroanatomical circuitry underlying the central actions of angiotensin-SFO, PVN, SON, SChN 14'32'36'38,46,67.

Histological and behavioural studies Fig. 4. Immunocytochemical demonstration of vasopressin-immunoreactive cell bodies (arrows) and fibers in the paraventricular nucleus of the host brain. A hypothalamictransplant (T) is located in the third ventricle (IIIV). Vasopressin-immunoreactive cell bodies and fibers are shown after incubation in primary antibody raised against AVP, and a TRITC-labelled second antibody. Scale bar = 100/~m.

Weak AnglI-immunoreactive cell bodies were seen along the marginal part of the subfornical organ in the Rapp S S / J r rat brain. The distribution pattern of AnglI-immunoreactive neuronal somata in the SFO closely resembles the one previously reported by Lind and colleagues 38 in the normal Sprague-Dawley rat.

292

However, a striking decrease in the number of AnglI-IR cell bodies in the hypothalamic nuclei was found, in comparison to the normal rat 3<3s. The distribution of vasopressin-immunoreactivity in the Rapp S S / J r hypothalamus here reported is identical to the one described for the normal rat by Swanson and Sawchenko 75, and contrasts with the scarcity in AnglIimmunoreactivity in the same hypothalamic nuclei. These results suggest that in the Rapp S S / J r rat the genetic deficiency responsible for the reduction of angiotensin-immunoreactive material in the neuronal population of the hypothalamic nuclei is selective for that peptidergic family, and not the consequence of a general failure of the peptide production mechanisms of those neurons. This interpretation is supported by the results obtained by Lind and collaborators 36 in the Brattleboro rat, in which the absence of vasopressinimmunoreactivity in the PVN, SON and SChN contrasts with the presence of angiotensin-immunoreactivity in the same cells. Previously reported immunocytochemical studies have shown that at the hypothalamic level AlI-immunoreactive cell bodies were most abundant in the magnocellular divisions of the PVN and SON. AlI-immunoreactive cell bodies were also present in the parvocellular division of the PVN and in the SCHN3'1°'13'26'32'34'36'38. The paraventricular, supraoptic and suprachiasmatic nuclei are particularly well known for the production of vasopressin and oxytocin by their neurons (for a review see ref. 79). Angiotensin and vasopressin have been shown to coexist in the same neuronal population of the rat hypothalamus, although oxytocin-producing cells do not contain AnglI-IR material 26,34,36,38. The hypothalamic neurons of the PVN, SON and SChN not only produce angiotensin, vasopressin and oxytocin, but also contain a wide variety of neuroactive substances--namely, dynorphin, leucine-enkephalin, methionine-enkephalin, neurotensin and cholecystokinin 44'vS'Ts. However, it has been shown that neither oxytocin-related peptides ss nor vasopressin mediate the inhibition of alcohol drinking by the renin-angiotensin system 63. Of the other neuropeptides present in these hypothalamic neurons only the endogenous reninangiotensin system has, so far, been implicated in the inhibition of voluntary alcohol intake 1~. Peripheral administration of cholecystokinin (CCK) has been shown to reduce alcohol intake 76 but the functional role of the endogenous CCK system in the regulation of alcohol intake has still to be determined. Even though the present results do not exclude the role played by other neurotransmitters normally produced by the graft in the regulation of alcohol intake, they suggest that the

modulatory role they play on alcohol intake is consistent with an angiotensinergic regulation. The location of the transplants seems to be crucial for the production of behavioural effects. The animals (n = 8) that showed behavioural effects, i.e., reduction of the voluntary intake of ethanol, had surviving grafts located in the dorsal portion of the third ventricle--adjacent to the subfornical organ. Thc other animals (n = 4) that had surviving grafts located in the lateral ventricles or in the ventral portion of the third ventricle did not reduce their alcohol drinking. Thus, the location of the graft in the vicinity of the SFO appears to be critical for the grafts to produce a change in behaviour. These results strongly suggest that the modulation of the brain renin-angiotensin system by the hypothalamic grafts is more efficiently achieved through the SFO, due to the lack of blood-brain barrier in this circumventricular organ, its role as a sensor for angiotensin and its neuroanatomical connections ~<32' 36 38,46,52,60,67,70. A similar situation, demonstrating the importance of the transplant location in the production of specific behavioural effects has been reported by Sladek et al. 15'6s. These authors reported that when surviving grafts containing vasopressin-producing cells were located in the lateral ventricles they failed to produce any behavioural effects in the host animal, while identical grafts located in the third ventricle successfully produced behavioural effects in the recipients ~s'6s. It has also been shown that the intracerebroventricular administration of angiotensin produces different responses in noradrenaline release 7~, water drinking and vasopressin release 67 depending on the injection site. Injection of All in the ventral third of the third ventricle strongly elicits water drinking but not AVP secretion, while All injection adjacent to the subfornical organ elicits both water drinking, and vasopressin release. These results are in agreement with our findings that the transplants adjacent to the subfornical organ, but not the ones located on the ventral part of the third ventricle or in the lateral ventricles, were more successfull in reducing alcohol intake, therefore stressing the interpretation that the specificity of the effects depends on the activation of well defined neuroanatomical structures. Finally, our results are also in agreement with the results previously reported by Grupp and colleagues 20,22, indicating that the subfornical organ is a primary target of the angiotensin II-mediated regulation of alcohol intake. Our results show that fetal grafts from the medioventral hypothalamus of normal rats, implanted in the dorsal portion of the third ventricle, significantly reduced the voluntary alcohol intake in the host ani-

293 mals (Rapp S S / J r rats). However, the transplants did not reduce water consumption, suggesting that the reduction in alcohol intake observed in the animals that received the graft in the dorsal third ventricle is a result of a specific interaction between the graft and alcohol intake per se and not the result of a non specific decrease in the consumption of all fluids in general, or simply the result of the presence of grafted material into the ventricular cavities. The transplants were viable for at least six months after surgery and contained angiotensin-immunoreactive cell bodies. An extensive histological integration was developed between graft and host brain. Our data suggest that the brain renin-angiotensin system may play a modulatory role in the regulation of alcohol intake, but do not exclude the participation of other neurotransmitters in the modulation of alcohol consumption. These results constitute the first demonstration of a long lasting effect of brain cell grafts on self-administration of an addictive substance. Acknowledgements. This research was supported by the Addiction Research Foundation, Toronto, Ont., Canada. We thank A. Ganstal and E. Karkouti for their technical assistance and Dr. S.J. Mihic for his help with statistical analysis.

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