Impaired drinking to angiotensin II after subdiaphragmatic vagotomy in rats

Physiology & Behavior, Vol. 24, pp. 1177-1180. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.

Impaired Drinking to Angiotensin II After Subdiaphragmatic Vagotomy in Rats NEIL ROWLAND

Departments of Psychology and Psychiatry, University of Pittsburgh, PA 15260 R e c e i v e d 18 J a n u a r y 1980 ROWLAND, N. Impaired drinking to angiotensin H after subdiaphragmatic vagotomy in rats. PHYSIOL. BEHAV. 24(6) 1177-1180, 1980.--Rats received total bilateral subdiaphragmatic vagotomy and, one month later, were fitted with chronic intravenous or intracerebroventricular cannulas. The vagotomized rats showed much reduced drinking compared with controls during intravenous infusion of angiotensin II. Their drinking to intracerebroventricularly administered angiotensin II was, however, less affected. The possible role of the vagus nerve in the mediation of angiotensin and other types of drinking is discussed.

Vagotomy

Angiotensin II

Drinking

RATS with subdiaphragmatic vagotomy (VAGX) exhibit disordered drinking. They drink less than normal rats under ad libitum conditions or after water deprivation, and exhibit reduced and long latency drinking after IP or intravenous (IV) injections of hypertonic NaC1 [7-12]. They also show impairments in h y p o v o l e m i a - and i s o p r o t e r e n o l - e l i c i t e d drinking [2,8]. Collectively, these data suggest that peripheral mechanisms play either a sensory and/or a tonic facilitatory role in drinking, at least under the conditions so far tested. However, in view of our recent re-evaluation of the so-called regulatory drinking deficits after various brain lesions [19], deficits which bear some resemblance to the disorders shown after VAGX, we thought it necessary to investigate whether V A G X rats are able to show rapid drinking responses to any thirst stimulus and in particular to one that acts in the brain. In these experiments we have tested VAGX rats for drinking to the dipsogen angiotension II (AII) [22] given IV or intracerebroventricularly (ICVT). METHOD

Animals and Housing Thirty-six male rats (Sprague Dawley and Long Evans, Zivic Miller, Pittsburgh) of initial weights 230-300 g were housed individually in hanging wire cages. Tap water was available at all times and, unless indicated, chow pellets were offered ad libitum on the cage floor. Lights were on from 0700-1900 hr, and testing was performed between 1000 and 1300 hr.

Surgery All surgery was performed using Equithesin (2.5 ml/kg) anesthetic. Bilateral subdiaphragmatic vagotomy was performed using procedures described before (e.g. [ 11,20]); this method severs the gastric, pancreatic and hepatic branches of the vagus. Control rats were laparotomized. These control rats fully recovered feeding in 1-2 days, while VAGX rats required 2-12 days for food intake to reach 50% control

levels, and longer for further recovery; the most anorexic were offered palatable moist foods to promote refeeding. The experiments here described were performed on three different batches of rats over the course of one year, and the results each time were identical. In one batch, three rats received section to only the gastric branches of the vagi. Four to six weeks postoperatively, all rats were implanted with indwelling jugular vein catheters [14]; one batch of rats were also implanted with 23 ga stainless steel cannula aimed to end 0.4 mm above the right lateral cerebral ventricle. These implants were secured to the skull with screws and dental cement and four days' recovery was allowed before testing commenced. The tests were hence completed by about 2 months post VAGX, a time at which any disputed recovery of function would not have occurred [2,7]. At the end of behavioral tests, the completeness of vagotomy was assessed by indirect methods (see below), and the ICVT cannula placement was verified by injecting 1/xl green dye and observing that it had obtained access to the ventricular system.

Infusions and Injections IV infusions were performed in cages described before [14]. The animals were housed overnight before the test in these cages with food and water available. Next morning, fresh water was provided in a graduated burette, and the rats connected to the infusion apparatus. Within 10 min the animals had settled, and IV infusion was begun. Angiotensin II (Asp 1, Ileu 5 AII, Schwartz Mann) was infused at the rate of 64 ng/10/xl/min for a period of one hour, and water intake (with food present) was recorded after 20, 40 and 60 min. Two days later, the infusion was 256 ng/10/zl/min AII. Another 2 days later, 2M NaCI was infused at the rate of 1.5 ml/45 min (total 3 meq N a ÷ per rat), and water intake (in the absence of food) measured after 1, 3 and 6 hr. F o r the ICVT drinking tests, the animal was removed from the home cage, gently held in a towel, and the wire obturator removed from the cannula. A 30 ga injector can-

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/061177-04502.00/0

1178

ROWLAND TABLE 1 DRINKING RESPONSES OF VAGOTOMIZEDAND CONTROLRATS TO INTRAVENOUS(IV) AND INTRACEREBROVENTRICULAR(ICVT) THIRST STIMULI Vagotomy No. drinking Intake

Treatment

Control No. drinking Intake

IV angiotensin II 64 ng/rat/min

20 min 60 min

4/18 5/18"

0.46 _+ 0.24 0.66 _+ 0.26t

4/10 8/11

1.50 _+ 0.63 2.43 _* 0.57

IV angiotensin 1I 256 ng/ral/min

20 min 60 min

7/18" 11/18

0.51 -+ 0.17t 2.31 _+ 0.63t

9/10 9/10

3.55 ~ 0.72 6.10 __+_0.99

IV NaC1, 3 meq

1 hr 6 hr

9/17 9/17

3.16 _+ 1.00 3.18 _+ 1.00

8/9 8/9

4.12 ± 1.19 5.09 ___ 1.10

0 (Vehicle) 10 13 tool 5x 10-1:~mol 10 ,2 tool 5x10 r~ mol

13/27 6/9 8/9 13/18 11/18

1.75 _+ 0.48 1.78 _+ 0.86 1.96 -+ 0.88 3.41 _+ 0.735 5.92 _+ 1.675

5/15 5/5 4/5 10/10 8/10

0.78 ___0.35 1.94 +_ 0.53 3.96 ± 1.50:~ 5.11 +__ 1.19§ 6.04 ~ 1.94¢

ICVT antiotensin II (30 min intakes)

Intakes are shown as Mean _+ SEM at times indicated. Food was present except during the NaCI infusions. Each animal was used only once per dose in the infusion study, but in the ICVT study rats were used repeatedly and data are averaged. The mean water intakes include the non-responders. *p<0.05 fraction drinking less than controls (chi square test). +p<0.01 less than controls (2-tailed t-tests). $p<0.10, §p<0.01 above vehicle baseline (2-tailed t-tests).

nula, 0.6 mm longer than the implanted guide, was introduced and 1 p.l was injected from a remote microdrive and 25 /~! syringe. Various doses of A l l (Asp 1 Ileu 5) or the 0.9% NaCI vehicle were injected on different days, and both the latency to drink and the water intake after 30 min were recorded.

Verification of Vagotomy Once elective feeding had recovered, all but three of the VAGX rats exhibited rates of body weight increase which were below the control range. Five VAGX and two control rats were tested for meal patterns on a liquid diet comprised of sugar and dried milk [20]; the mean meal size of the controls was 5.7 ml and that of the VAGX rats 3.7 ml, U(5,2)=0; p=0.047. After completion of the experiments, and when the rats were again eating chow, they were fasted for 3 hr, anesthetized and the stomachs removed and weighed. Controls' stomachs weighed a mean of 4.0 g (0.98% body weight) while those of VAGX weighed 6.1 g (1.62% body weight; p<0.01). The stomachs of the rats with gastric vagotomy were also enlarged (6.5 g). These criteria of aphagia, impaired weight gain, small liquid meals, and gastric retention of solid food are all indicative of complete VAGX [12, 13, 20]. Notice that none of these tests verifies the section of pancreatic or hepatic branches. However, selective hepatic branch VAGX does not interfere with osmotic drinking [10]. Each rat in the VAGX population was outside the control range on at least one of the above tests, and we did not eliminate any animals. However, if marginal cases, such as the three which gained weight rapidly had been eliminated, then the results we are about to describe might have been even more clear-cut. RESULTS VAGX rats showed a profound impairment in drinking to

IV infusions of A l l at both 64 and 256 ng/min (Table 1). Both these doses induced drinking in all but one control rat. The effect is particularly well illustrated by referring to the 20 min point with 256 ng/min: all but one control had ingested more than 2.0 ml, but no VAGX rat had ingested more than 2 ml, and 11/18 had not drunk at all. The deficit thus appears to be both one of latency to drink and amount ingested since, when the infusion was continued the VAGX rats remained behind the controls. The three rats with gastric vagotomy drank a mean of 1.47 and 3.77 ml/hr after the 64 and 256 ng/min doses, respectively, values which are intermediate between those of the VAGX and control groups shown in Table 1. The drinking deficit was not so apparent following ICVT injections of AII (Table 1). Only one VAGX rat failed to drink after every injection. Further, the amounts drunk were not statistically different between VAGX and control groups. The latencies to drink were also not markedly different: at 5 x 10-12 mol AII the median latency to drink by controls was 80 sec, and the median latency for the VAGX group was 100 sec (including only the drinkers) or 180 sec (including five negative responses). The nonsignificant tendency for the VAGX rats to drink less than controls may reflect a small insensitivity; however, because the VAGX rats drank more after vehicle injections than did the controls, it was not possible to test this insensitivity using still lower doses of All. The drinking responses to IV NaC1 were attenuated in the VAGX group although, because of within group variability and one non-responding control, these differences were not statictically significant. After 1 hr, only 9 out of 17 VAGX rats had drunk, and none of the remaining eight drank in the remainder of the 6 hr test. In contrast, 9 out of 10 controls had drunk after 1 hr. Five of the VAGX rats drank amounts comparable to controls (mean 8.9 ml) after NaCI infusion. If these animals are excluded from analysis, the mean intake of

V A G O T O M Y IMPAIRS A N G I O T E N S I N D R I N K I N G the VAGX group was 0.81 ml after NaCI infusion, and only 1.48 ml after 1 hr of A I I infusion at 256 ng/min. All of these five rats drank substantial amounts to AII; there were, however, other V A G X rats which also drank some during IV AII but which drank little or nothing after IV NaC1. The three rats with gastric vagotomy drank 5.6 ml after NaC1 infusion, and did not differ from controls. DISCUSSION These results indicate that VAGX rats have profound drinking deficits to IV administrations of AII. We used a low (64) and high (256 ng/min) dose of AII; the former dose is the lowest which in our hands produces drinking in more than 90% of unselected control rats. Notice, however, this dose is about 5 × the threshold dose which has been found in rats specially selected for bold diurnal drinking [22]. In preliminary experiments not here reported the increase in arterial blood pressure to both IV A l l infusions was similar in normal and VAGX rats. However, this bears more careful scrutiny since access of AII to its postulated thirst receptors in the brain may be faciliated by high blood pressure; even a slightly smaller pressor response therefore might reduce the amount of circulating A l l which reaches receptors inside the blood brain barrier (BBB). This argument does not hold, of course, if all of the critical receptors are in an area devoid of a BBB, such as the subfornical organ [22]. In contrast to the profound drinking deficits after IV A l l , the drinking observed after ICVT A l l was relatively normal both in terms of amount drunk and latency. Kraly has shown that V A G X rats clear water from their stomachs more rapidly than do controls, and this could produce a premature satiety signal [7,9]. This mechanism can account for reduced drinking to A l l , or other thirst stimuli, but cannot account for latency changes. The present demonstration that AII ICVT elicits rapid drinking in most, but not all, VAGX rats shows there is no general problem in rapid initiation of responses (in this regard, V A G X also show normal responses to thermal pain [11]). Also there is no apparent relation between the drinking response to IV and ICVT AII: the rats with ICVT cannulas were also tested IV and those which drank well after the central injections showed profound deficits to IV AII. (Conversely, the one V A G X rat which consistently did not drink to ICVT injections drank quite well to 256 (but not 64) ng/min AII IV). Our VAGX rats drank less than controls after IV NaC1, but the effect was not statistically reliable. Possibly with a

1179 more rigorous procedure for verifying completeness of VAGX we would have obtained the robust deficits reported by others [2, 7-12]. Nevertheless, the fact that our AII drinking deficits were significant while those to NaCI were not implies that the drinking deficit to A l l is at least as great as any osmotic impairment. This is of interest because the osmotic thirst deficits have been attributed to the loss of signals from visceral osmoreceptors after VAGX [7,11]. However, the fact that even greater thirst deficits are seen after a dipsogen which acts in the brain (AII), as well as to volume depletion and isoproterenol [2,8], suggests we should still entertain alternative explanations. One possibility is that central osmoreceptors do underlie the drinking response, and the VAGX effect to NaCI and A l l are of a similar nature, for example a loss of tonic vagal excitation to brain regions involved in ingestion. Interestingly, ingestive behaviors elicited by electrical stimulation of the brain are disrupted by V A G X [3,16]. Since hepatic osmoreceptors [1, 4, 5, 18] are likely candidates for peripheral osmoreception, it would be expected that hepatic vagotomy would interfere with saltinduced drinking. This expectation has not been borne out [10]. The rats with gastric vagotomy in the present study showed normal responses to NaCl infusion. We must thus consider some less specific effect of VAGX on ingestion (e.g. [6]). Finally, it has been proposed that vagai afferents may reach the zona incerta region [11, 15, 17, 18], and it is of interest that animals with zona incerta lesions show drinking deficits to both NaCl and AII [21,23]. Like VAGX rats, the animals with zona incerta lesions show lowered water to food ratios [23]; they do, however, increase their drinking when salt is added to their food [21]. We have found the same effect in VAGX rats: water to food ratios were 1.4 ml/g on regular chow and 2.1 ml/g with 3% NaCl-supplemented chow; corresponding control values were 1.8 and 3.0 ml/g ( N ' s = 3 unpublished experiments, 1978). Thus we find a similar spectrum of deficits in VAGX and zona incerta lesioned rats, with the exception of drinking after ICVT AII [21,23]. The parallels between the zona incerta lesioned and VAGX preparations are interesting, and are both similar to deficits in quinine-drinking intact rats [19]. We have suggested that many of these thirst deficits might be a function of our testing paradigms rather than, or in addition to, and specific neural damage. The present demonstration of impaired drinking to IV AII in VAGX seems to demand a reconsideration of the meaning of their osmotic deficits.

REFERENCES 1. Adachi, A., A. Niijima and H. L. Jacobs. An heptic osmoreceptor mechanism in the rat: electrophysiological and behavioral studies. Am. J. Physiol. 213: 1043-1049, 1976. 2. Anika, S. M., T. R. Houpt and K. A. Houpt. Recovery from disordered drinking by vagotomized rats. Physiol. Behav. 22: 605-607, 1979. 3. Ball, G. G. Vagotomy; Effect on electrically elicited eating and self-stimulation in the lateral hypothalamus. Science 184: 484485, 1974. 4. Glasby, M. A. and D. J. Ramsay. Hepatic osmoreceptors? J. Physiol. 243: 765-776, 1974. 5. Haberich, F. J. Osmoreception in the portal circulation. Fedn. Proc. 27: 1137-1141, 1968.

6. King, B. M., R. C. Carpenter, B. A. Stamoutsos, L. A. Frohman and S. P. Grossman. Hyperphagia and obesity following ventromedial hypothalamic lesions in rats with subdiaphragmatic vagotomy. Physiol. Behav. 20: 643-651, 1978. 7. Kraly, F. S. Abdominal vagotomy inhibits osmotically-induced drinking in the rat. J. comp. physiol. Psychol. 92: 999-1013, 1978. 8. Kraly, F. S., J. Gibbs and G. P. Smith. Disordered drinking after abdominal vagotomy in rats. Nature 258: 226--228, 1975. 9. Kraly, F. S., G. P. Smith and W. J. Carty. Abdominal vagotomy disrupts food-related drinking in the rat. J. comp. physiol. Psychol. 92: 196-203, 1978.

1180 10. Martin, J. R., P. J. Geiselman and D. Novin. Vagal mediation of a visceral drinking system in the rat. Neurosci. Abstr. 3: 505, 1977. 11. Martin, J. R., P. J. Geiselman and D. Novin. Drinking to intracellular dehydration following vagotomy in rats. Physiol. Behav. 23: 527-537, 1979. 12. Martin, J. R., R. C. Rogers, D. Novin and D. A. VanderWeele. Excessive gastric retention by vagotomized rats and rabbits given a solid diet. Bull. Psychon. Soc. 10: 291-294, 1977. 13. Mordes, J. P., M. El Lozy, M. G. Herrera and W. Silen. Effects of vagotomy with and without pyloroplasty on weight and food intake in rats. Am. J. Physiol. 236: R61-R66, 1979. 14. Nicolaidis, S., N. Rowland, M. -J. Meile, P. Marfaing-Jallat and A. Pesez. A flexible technique for long term infusions in unrestrained rats. Pharmac. Biochem. Behav. 2: 131-136, 1974. 15. Norgren, R. and C. M. Leonard. Ascending central gustatory pathways. J. comp. Neurol. 150: 217-237, 1973. 16. Powley, T. L. and C. A. Opsahl. Autonomic components of the hypothalamic feeding syndromes. In: Hunger: Basic Mechanisms and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 313-326. 17. Ricardo, J. A. and G. T. Koh. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Res. 153: 1-26, 1978.

ROWLAND 18. Rogers, R. C., D. Novin and L. L. Butcher. Hepatic sodium and osmoreceptors activate neurons in the ventrobasal thalamus. Brain Res. 168: 398-403, 1979. 19. Rowland, N. Regulatory drinking: Do the physiological substrates have an ecological niche? Biobehav. Rev. 1: 261-272, 1977. 20. Rowland, N. Meal patterns in rats with nigrostriatal dopaminedepleting lesions, subdiaphragmatic vagotomy, and their combination. Brain Res. Bull. 3: 8%91, 1978. 21. Rowland, N., S. P. Grossman and L. Grossman. Zona incerta lesions: Regulatory drinking deficits to intravenous NaC1, angiotensin, but not to salt in the food. Physiol. Behav. 23: 745-750, 1979. 22. Simpson, J. B., A. N. Epstein and J. S. Camardo. Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of the rat. J. comp. physiol. Psychol. 92: 581-608, 1978. 23. Walsh, L. L. and S. P. Grossman. Dissociation of responses to extracellular thirst stimuli following zona incerta lesions. Pharmac'. Biochem. Behav. 8: 40%415, 1978.