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Effects of the alpha antagonists and agonists injected into the lateral hypothalamus on the water and sodium intake induced by angiotensin II injection into the subfornical organ
Brain Research Bulletin, Vol. 48, No. 5, pp. 521–525, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
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Effects of the alpha antagonists and agonists injected into the lateral hypothalamus on the water and sodium intake induced by angiotensin II injection into the subfornical organ Nara Adriane Arau´jo Almeida, Vagner Roberto Antunes, Wilson Abra˜o Saad and Luiz Antonio de Arruda Camargo* Department of Physiology, School of Dentistry, Paulista State University, UNESP, Araraquara, Brazil [Received 1 June 1998; Revised 14 January 1999; Accepted 20 January 1999] ABSTRACT: The subfornical organ (SFO) and the lateral hypothalamus (LH) have been shown to be important for the central action of angiotensin II (ANG II) on water and salt regulation. Several anatomical findings have demonstrated neural connections between the SFO and the LH. The present experiments were conducted to investigate the role of the a-adrenergic antagonists and agonists injected into the LH on the water and salt intake elicited by injections of ANG II into the SFO. Prazosin (an a1-adrenergic antagonist) injected into the LH increased the salt ingestion, whereas yohimbine (an a2-adrenergic antagonist) and propranolol (a b-adrenergic antagonist) antagonized the salt ingestion induced by administration of ANG II into the SFO. Previous administration of clonidine (an a2-adrenergic agonist) or noradrenaline into the LH increased, whereas pretreatment with phenylephrine decreased the sodium intake induced by injection of ANG II into the SFO. Previous treatment with prazosin and propranolol reduced the water intake induced by ANG II. Phenylephrine increased the dipsogenic responses produced by ANG II, whereas previous treatment with clonidine injected into the LH reduced the water intake induced by ANG II administration into the SFO. The LH involvement with SFO on the excitatory and inhibitory mechanisms related to water and sodium intake is suggested. © 1999 Elsevier Science Inc.
actions of ANG II are due at least in part to its interactions with neurotransmitters, especially the catecholamines [26], it has also been suggested that adrenergic neurotransmitters in several hypothalamic areas may participate in the ANG II regulation of hydromineral fluid intake in a process that involves a1-, a2-, and b-adrenoceptors [3,6,24]. The lateral hypothalamus (LH) is another area of the rat forebrain that plays an important role in the regulation of water and salt intake [11,21,24]. The extensive neural pathways from the SFO to the hypothalamus have been implicated in the regulation of fluid intake and cardiovascular regulation. Several lines of evidence indicate that fibers from the SFO converge on the nucleus medianus (NM) and also project to the supraoptic nucleus (SON), paraventricular nucleus (PVN) and throughout the lateral preoptic area (LPO)-LH [28]. The central part of the SFO, which binds circulating ANG II [29], also contains an ANG II-immunoreactive terminal field that appears to arise from cells in the LH [23]. Considering the importance of the SFO and LH in the hydromineral fluid intake of rats and the evidence of important interactions between both areas, we investigated the effect of previous LH treatment with prazosin, yohimbine, propranolol, phenylephrine, clonidine, or noradrenaline on the water and salt intake induced by administration of ANG II into the SFO.
KEY WORDS: Subfornical organ, Lateral hypothalamus, Adrenergic antagonists, Adrenergic agonists, Water intake, Sodium intake.
MATERIALS AND METHODS General Procedures Animals. Male Holtzman rats weighing 260 –300 g at the beginning of the experiments were housed in individual metabolic cages. Standard Purina pellets (Na1 content 5 nmols/100 g), tap water and 3% NaCl solution were available ad libitum unless otherwise noted. Temperature in the animal colony was maintained at approximately 23°C. The 12:12-h light– dark cycle began with lights on at 0800. All experiments were started between 1000 and 2000 P.M. at least 3 days after surgery. Surgery. After an acclimatization period of 7 days, the animals
INTRODUCTION The subfornical organ (SFO) is one of the neural circumventricular organs lacking a blood– brain barrier and is important for body fluid balance [28]. Blood-borne angiotensin II (ANG II) penetrates the fenestrated capillaries of the SFO and activates receptors [18]. The SFO neurons are capable of responding to changes in ANG II concentration in the blood and may mediate induction of water and sodium intake [14,19,30]. It has been suggested that the central
* Address for correspondence: Dr. Luiz Antonio de Arruda Camargo, Department of Physiology, School of Dentistry, Paulista State University, UNESP, 1680 Humaita´ Street, Araraquara, SP 14801-903, Brazil. Fax: 55-(16)-222-4823.
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FIG. 1. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing the site of injection into the subfornical organ (arrow).
FIG. 2. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing bilateral the sites of injection into the lateral hypothalamus (arrows).
were maintained under tribromoethanol (Aldrich) (20 mg/100 g b. wt., intraperitoneal [i.p.]) anesthesia throughout surgery. A stainless steel guide-cannula (10 3 0.7 mm) outside diameter [o.d.]) was stereotaxically implanted into the brain with its opening protruding into the top of the SFO (coordinates: AP 5 1.0 mm caudal to the bregma; V 5 4.8 mm from the dura mater; L 5 0.0 mm from the sagittal line). For LH cannulation the cannula (14 3 0.7 mm o.d.) was positioned bilaterally as follows: AP 5 1.5 mm posterior to the bregma; V 5 7.5 mm below the dura mater; L 5 1.5 mm from the sagittal midline. For cannula implantation the stereotaxic incisor bar was positioned 2.5 mm above the interaural line. The cannula was secured to the top of the skull with dental cement and fastened with two screws. The insertion of a closefitting stylet kept the lumen free of debris and clots. A prophylactic dose of penicillin (30,000 IU) was given intramuscularly (i.m.) presurgically. Intracerebral injection techniques. Bolus intracranial injections were made after gently removing the animal from its cage, replacing the stylet with an injector that protruded 0.2 mm and 1.0 mm beyond the tip of the guide-cannula for SFO and LH, respectively, and was connected by PE-10 tubing to a 10.0-ml microsyringe, and injecting a total volume of 0.2 ml over a period of 30 s. Stylet and injector were always wiped with cotton soaked with 70% alcohol. After the injection, the injector was removed and replaced with the stylet, and the animal was returned to its cage for observation of its behavior. Drugs. ANG II, yohimbine hydrochloride, propranolol hydrochloride, noradrenaline bitartarate, dl-isoproterenol hydrochloride (Sigma Chemical Co., St. Louis, MO, USA) phenylephrine hydrochloride and clonidine hydrochloride (RBI, Natick, MA, USA) and prazosin hydrochloride (Pfizer Inc., Groton, CT, USA) were dissolved in 0.9% saline (vehicle). Yohimbine and prazosin were sonicated for better suspension. Histology. At the end of the experiments, the animals were anesthetized with ether and given a 2-ml injection of fast green dye via the intracranial cannula, followed by perfusion with saline and buffered formalin. The brains were removed, fixed in 10% formalin, frozen to 225°C and cut into 20- to 30-mm section. The presence of absence of dye in the ventricles was observed at this time. Only animals in which the presence of dye restricted to the SFO was noted were used in this study. Figures 1 and 2 presents the site of the injection into the SFO and the LH, respectively. Statistical analysis. The results are reported as mean 6 SEM and were analyzed by one-way analysis of variance. Values were
considered to be statistically significant when p , 0.05. The Newman-Keuls post-hoc test was used to assess the difference between individual means. Experimental Protocol Water intake or NaCl 3% intake. Five days after brain surgery, different groups of animals for water or sodium intake were submitted to experimental sessions. Each animals was submitted to four or five experimental sessions at 3-day intervals. An a-adrenergic antagonist (prazosin, yohimbine, or propranolol) and the a-adrenergic agonists (noradrenaline, clonidine, or phenylephrine) were injected into the LH 20 min before injection of ANG II into the SFO. Recording of water or sodium intake started immediately after ANG II injection and continued for 4 h. Before the salt appetite test the next morning, the overnight water intake was measured, and graduate cylinders were replaced with glass burettes containing 3% NaCl. The burettes were calibrate to 0.1 ml and fitted with drinking sponts. The presence of sodium was announced to the drinking rats by sprinkling a few drops of the saline solution on their lips and whiskers. RESULTS Effects of Pretreatment with Prazosin, Yohimbine, or Propranolol into the LH on the Sodium and Water Intake Induced by ANG II Injected Into the SFO The sodium intake observed during a period of 4 h in the control experiment (0.15 M NaCl into the SFO) was 1.3 6 0.6 ml. Injection of ANG II (10 pmol) into the SFO produced an increase in sodium ingestion (7.3 6 0.6 ml/2 h). Previous injection of prazosin (20, 40, and 80 nmol) into the LH potentiated the effect of ANG II on 3% NaCl intake induced by ANG II [F(3,35) 5 5.24, p , 0.05] (Fig. 3). Pretreatment with yohimbine and propranolol (20, 40, and 80 nmol) into the LH reduced the sodium intake induced by ANG II [F(3,30) 5 14.55, p , 0.05 and [F(3,30) 5 3.32, p , 0.05, respectively] (Fig. 3). Injection of only prazosin, yohimbine, or propranolol into the LH produced no alterations in salt intake. Control rats (0.15 M NaCl into the SFO) consumed a mean amount of 2.5 6 0.7 ml of water over a period of 4 h. ANG II (10 pmol) injected into the SFO led to ingestion of 16.7 6 1.8 ml of water over the same period. Previous treatment with prazosin and propranolol (40 and 80 nmol) into the LH antagonized the dipso-
FLUID CONTROL BY SFO-LH PATHWAYS
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FIG. 3. Effect of pretreatment with prazosin, yohimbine, and propranolol into the lateral hypothalamus on sodium intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
FIG. 4. Effect of pretreatment with prazosin, yohimbine, and propranolol into the lateral hypothalamus on water intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
genesis induced by ANG II administration into the SFO [F(3,32) 5 6.52, p , 0.05 and F(3,28) 5 3.89, p , 0.05, respectively] (Fig. 4). No changes in the dipsogenic effect of ANG II were observed after previous treatment with yohimbine (10, 20, 40, and 80 nmol) (Fig. 4). Injection of only prazosin, yohimbine, or propranolol into the LH produced no alterations in water intake.
DISCUSSION
Effects of LH Pretreatment with Clonidine, Phenylephrine, or Noradrenaline into the LH on the Sodium and Water Intake Induced by ANG II Injected into the SFO The saline ingestion observed during 4 h in the control experiment (0.15 M NaCl) and ANG II (10 pmol) injection into the SFO is presented in Fig. 5. Previous treatment with clonidine (40 and 80 nmol) and noradrenaline (20, 40, and 80 nmol) elicits a potentiation in sodium intake induced by ANG II injection into the LH [F(3,35) 5 4.55, p , 0.01 and F(3,34) 5 5.7, p , 0.01, respectively], whereas phenylephrine (40 and 80 nmol) decreased this effect [F(3,33) 5 5.37, p , 0.01]. Figure 5 shows water intake after SFO injection of saline control and ANG II. Previous treatment with clonidine (20, 40, and 80 nmol) into the LH decreased the water intake induced by LH administration of ANG II [F(3,33 5 3.23, p , 0.05] into the SFO, whereas phenylephrine (40 and 80 nmol) elicits a potentiation in water intake induced by ANG II [F(3,35) 5 3.73, p , 0.05]. No changes in the dipsogenic effect of ANG II were observed after previous treatment with noradrenaline [F(3,31) 5 0.55, p , 0.05].
The present results show that yohimbine and propranolol injected into the LH reduces, whereas the a1-adrenoceptor antagonist prazosin increases the sodium ingestion induced by ANG II injected into the SFO. The results also show that prazosin and propranolol injected into the LH reduce the dipsogenesis induced by administration of ANG II into the SFO. Studies with adrenergic antagonists suggest that noradrenaline has a dual role in the central control of water and salt intake [9,10] induced by ANG II. Water intake induced by central ANG II is inhibited by a1-, a2- or b-antagonists, depending on the site of injection. Prazosin and phentolamine injected into the rostral hypothalamus attenuated the drinking response induced by intracerebroventricular (i.c.v.) ANG II [20]. An inhibitory effect of increased on the dipsogenic effect of ANG II injected into the medial preoptic area (MPOA) has been demonstrated by the previous administration of prazosin and propranolol into the same area [3]. Prazosin and yohimbine, but not propranolol, injected into the median preoptic nucleus (MnPO) reduced the water intake induced by ANG II [25]. The a2-adrenergic antagonist, yohimbine is reported to enhance the dipsogenic effect of ANG II or ANG III [6,15] and to induce water and salt intake [16,17] when injected systemically, but it is not clear if it affects those behaviors by a direct central action or by a peripheral action [13]. However, it has been proposed [12] that the organum vasculosum laminae terminalis (OVLT) is the circumventricular organ site for a peripheral action of salt appetite because infusions of ANG II into the OVLT, but not the SFO, elicit salt appetite and because lesions of the OVLT suppress salt appetite aroused either by a low dose of oral
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FIG. 5. Effect of pretreatment with clonidine, phenylephrine, and noradrenaline into the lateral hypothalamus on sodium intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
captopril or by sodium depletion, by other side, lesions of the SFO reduce salt appetite aroused by sodium depletion [27,30]. The present study results also show that the a2-adrenoceptor agonist clonidine and noradrenaline injected into the LH increased the salt intake induced by ANG II injected into the SFO, whereas, the a1-adrenoceptor phenylephrine, but not clonidine and noradrenaline, injected into the LH increased the dipsogenic effect induced by SFO administration of ANG II. A role of the forebrain noradrenergic systems in the mediation of ANG II-elicited drinking and blood pressure responses has also been suggested [4,5]. The synthesis and turnover of hypothalamic catecholamines increased after i.c.v. administration of ANG II [1,8]. A relationship between noradrenaline and ANG II facilitation of NaCl intake was first shown by Chiaraviglio and Taleisnik [7], who obtained 1% NaCl intake in normovolemic animals with noradrenaline injected into the third ventricle close to the columns of the fornix. Other authors reported the same kind of increase after noradrenaline was injected i.c.v. [2] or into the paraventricular nucleus (PVN) [22]. In summary, the present study show that injection of prazosin, clonidine or noradrenaline into the LH increases, whereas yohimbine, phenylephrine or propranolol reduces the salt intake response produced by angiotensinergic activation of the SFO. Phenylephrine increases the dipsogenic response produced by ANG II, whereas previous treatment with clonidine or propranolol into the LH reduces the dipsogenic responses produced by ANG II administration into the SFO. These observations show that both a-adrenoceptor subtypes of LH are important for the fluid intake responses induced angiotensinergic activation of the SFO in rats.
´ JO ALMEIDA ET AL. ARAU
FIG. 6. Effect of pretreatment with clonidine, phenylephrine, and noradrenaline into the lateral hypothalamus on water intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
They also suggest that the central a-adrenergic receptors have a dual (inhibitory and excitatory) effect on the sodium and water intake induced by ANG II in rats). ACKNOWLEDGEMENTS
The authors appreciate the technical assistance of Reginaldo C. Queiro´z, Silas P. Barbosa, Silvia Fo´glia and Silvana A. D. Malavolta. They also thank Ana V. Oliveira for animal care. Research supported by CNPq and FAPESP.
REFERENCES 1. Alpers, R. H.; Steel, M. R.; Ganong, W. F. Angiotensin II increases catecholamine synthesis in select hypothalamic nuclei. Soc. Neurosci. Abstr. 8:421; 1982. 2. Antunes-Rodrigues, J.; McCann, S. M. Water, sodium chloride, and food intake, produced by injections of cholinergic and adrenergic drugs into the third ventricle of the rat brain. Proc. Soc. Exp. Biol. Med. 133:1464 –1470; 1970. 3. Bastos R.; Saad, W. A.; Menani, J. V.; Renzi, A.; Silveira, J. E. N.; Camargo, L. A. A. Role of adrenergic pathway of the medial preoptic area in ANG II-induced water and renal excretion in rats. Brain Res. 636:81– 86; 1994. 4. Bellin, I. B.; Bhatnagar, R. K.; Johnson, A. K. Periventricular noradrenergic systems are critical for angiotensin-induced drinking and blood pressure responses. Brain Res. 403:105–112; 1987. 5. Bellin, I. B.; Landas, S. K.; Johnson, A. K. Selective catecholamine depletion of structures along the ventral lamina terminalis: Effects on experimentally-induced drinking and pressor responses. Brain Res. 456:9 –16; 1988.
FLUID CONTROL BY SFO-LH PATHWAYS 6. Chan, J. Y. H.; Pan, S.; Chan, S. H. H. Participation of noradrenergic neurotransmission in angiotensin II-induced dipsogenic behavior in the rat. Life Sci. 48:1293–1301; 1991. 7. Chiaraviglio, E.; Taleisnik, S. Water and salt intake induced by hypothalamic implants of cholinergic and adrenergic agents. Am. J. Physiol. 216:1148 –1422; 1969. 8. Cunningham, J. T.; Sullivan, M. J.; Edwards, G. L.; Farimpont, R.; Beltz, T. G.; Johnson, A. K. Dissociation of experimentally induced drinking behavior by ibotenate injection into the median preoptic nucleus. Brain Res. 554:153–158; 1991. 9. De Luca, L. A., Jr.; Camargo, L. A. A.; Menani, J. V.; Renzi, A.; Saad, W. A. On a possible dual role for central noradrenaline in the control of hydromineral fluid intake. Braz. J. Med. Biol. Res. 27:905–914; 1994. 10. Ferrari, A. C.; Camargo, L. A. A.; Saad, W. A.; Renzi, A.; De Luca, L. A., Jr.; Menani, J. V. Clonidine and phenylephrine injected into the lateral hypothalamus inhibit water intake in rats. Brain Res. 522:125– 130; 1990. 11. Ferrari, A. C.; Camargo, L. A. A.; Saad, W. A.; Renzi, A.; De Luca, L. A., Jr.; Menani, J. V. Role of the alpha1- and alpha2-adrenoceptors of the lateral hypothalamus in the dipsogenic response to central angiotensin II in rat. Brain Res. 560:291–296; 1991. 12. Fitts, D. A.; Masson, D. B. Preoptic angiotensin and salt appetite. Behav. Neurosci. 104:643– 650; 1990. 13. Fitts, D. A. Effects of lesions of the ventral median preoptic nucley of the subfornical organ on drinking and salt appetite after deoxycorticosterone acetate or yohimbine. Behav. Neurosci. 105:721–726; 1991. 14. Fitts, D. A. Angiotensin II receptors in SFO but not in OVLT mediate isoproterenol-induced thirst. Am. J. Physiol. 267:R7–R15; 1994. 15. Fregly, M. J.; Rowland, N. E.; Greenleaf, J. E. Effects of yohimbine and tolazoline on isoproterenol and angiotensin II-induced water intake in rats. Brain Res. Bull. 10:121–126; 1983. 16. Johnson, A. K.; Beltz, T. G.; Edwards, G. L. A rapid onset sodium appetite induced by yohimbine. Soc. Neurosci. Abstr. 15:984; 1989. 17. Johnson, A. K.; Beltz, T. G.; Edwards, G. L. The nature of sodium appetite induced by yohimbine. FASEB J. 4:A1195; 1990. 18. Johnson, A. K.; Edwards, G. E. Central projections of osmotic and hypovolaemic signals in homestasis thirst. In: Ramsay, D. J., Booth, D. A., eds. Thirst. London: Springer Verlag; 1991:149 –175.
525 19. Johnson, A. K.; Thunhorst, R. L. Sensory mechanisms in the behavioral control of body fluid balance: Thirst and salt appetite. Prog. Psychobiol. Physiol. Psychol. 16:145–175; 1995. 20. Jones, D. L. Hypothalamic alpha-adrenergic blockade modifies drinking and blood pressure responses to central angiotensin II in conscious rats. Can. J. Physiol. Pharmacol. 66:1270 –1277; 1988. 21. Leibowitz, S. F. Pattern of drinking and feeding produced by hypothalamic norepinephrine injection in the satiated rat. Physiol. Behav. 14:731–742; 1975. 22. Leibowitz, S. F. Neurochemical of the hypothalamus. In: Morgane, P. J.; Panksepp, J., eds. Behavioral studies of the hypothalamus, vol. 3. New York: Marcel Dekker; 1980:299 – 437. 23. Lind, W. A.; Swanson, L. W.; Ganten, D. Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. Neuroendocrinology 40:2–24; 1990. 24. Pereira da Silva, R. K. P.; Saad, W. A.; Renzi, A.; Menani, J. V.; Camargo, L. A. A. Effect of lateral hypothalamus lesions on the water and salt intake, and sodium and urine excretion induced by activation of the median preoptic nucleus in conscious rats. J. Auton. Nerv. Syst. 53:195–204; 1995. 25. Pereira da Silva, R. K. P.; Menani, J. V.; Saad, W. A.; Renzi, A.; Silveira, J. E. N.; Luiz, A. C.; Camargo, L. A. A. Role of the alpha1-, alpha2- and beta-adrenoceptors of the median preoptic area on the water intake, renal excretion, and arterial pressure induced by ANG II. Brain Res. 717:38 – 43; 1996. 26. Summers, C. Norepinephrine increases angiotensin II binding in rat brain synaptosomes. Brain Res. Bull. 28:411– 415; 1992. 27. Thunhorst, R. L.; Erlich, K. J.; Simpson, J. B. Subfornical organ participates in salt appetite. Behav. Neurosci. 104:637– 642; 1990. 28. Thrasher, T. N. Role of forebrain circumventricular organs in body fluid balance. Acta Physiol. Scand. 583 (suppl.):141–150; 1989. 29. Van Houtten, M.; Schiffrin, E. L.; Mann, J. F. E.; Posner, B. I.; Boucher, R. Radioautographic localization of specific binding sites for blood born angiotensin II in rat brain. Brain Res. 186:480 – 485; 1980. 30. Weisinger, R. S.; Denton, D. A.; Di Nicolantonio, R.; Hards, D. K.; McKinley, M. J.; Oldfield, B.; Osborne, P. G. Subfornical organ lesion decreases sodium appetite in sodium-depleted rat. Brain Res. 526:23– 30; 1990.
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Effects of the alpha antagonists and agonists injected into the lateral hypothalamus on the water and sodium intake induced by angiotensin II injection into the subfornical organ Nara Adriane Arau´jo Almeida, Vagner Roberto Antunes, Wilson Abra˜o Saad and Luiz Antonio de Arruda Camargo* Department of Physiology, School of Dentistry, Paulista State University, UNESP, Araraquara, Brazil [Received 1 June 1998; Revised 14 January 1999; Accepted 20 January 1999] ABSTRACT: The subfornical organ (SFO) and the lateral hypothalamus (LH) have been shown to be important for the central action of angiotensin II (ANG II) on water and salt regulation. Several anatomical findings have demonstrated neural connections between the SFO and the LH. The present experiments were conducted to investigate the role of the a-adrenergic antagonists and agonists injected into the LH on the water and salt intake elicited by injections of ANG II into the SFO. Prazosin (an a1-adrenergic antagonist) injected into the LH increased the salt ingestion, whereas yohimbine (an a2-adrenergic antagonist) and propranolol (a b-adrenergic antagonist) antagonized the salt ingestion induced by administration of ANG II into the SFO. Previous administration of clonidine (an a2-adrenergic agonist) or noradrenaline into the LH increased, whereas pretreatment with phenylephrine decreased the sodium intake induced by injection of ANG II into the SFO. Previous treatment with prazosin and propranolol reduced the water intake induced by ANG II. Phenylephrine increased the dipsogenic responses produced by ANG II, whereas previous treatment with clonidine injected into the LH reduced the water intake induced by ANG II administration into the SFO. The LH involvement with SFO on the excitatory and inhibitory mechanisms related to water and sodium intake is suggested. © 1999 Elsevier Science Inc.
actions of ANG II are due at least in part to its interactions with neurotransmitters, especially the catecholamines [26], it has also been suggested that adrenergic neurotransmitters in several hypothalamic areas may participate in the ANG II regulation of hydromineral fluid intake in a process that involves a1-, a2-, and b-adrenoceptors [3,6,24]. The lateral hypothalamus (LH) is another area of the rat forebrain that plays an important role in the regulation of water and salt intake [11,21,24]. The extensive neural pathways from the SFO to the hypothalamus have been implicated in the regulation of fluid intake and cardiovascular regulation. Several lines of evidence indicate that fibers from the SFO converge on the nucleus medianus (NM) and also project to the supraoptic nucleus (SON), paraventricular nucleus (PVN) and throughout the lateral preoptic area (LPO)-LH [28]. The central part of the SFO, which binds circulating ANG II [29], also contains an ANG II-immunoreactive terminal field that appears to arise from cells in the LH [23]. Considering the importance of the SFO and LH in the hydromineral fluid intake of rats and the evidence of important interactions between both areas, we investigated the effect of previous LH treatment with prazosin, yohimbine, propranolol, phenylephrine, clonidine, or noradrenaline on the water and salt intake induced by administration of ANG II into the SFO.
KEY WORDS: Subfornical organ, Lateral hypothalamus, Adrenergic antagonists, Adrenergic agonists, Water intake, Sodium intake.
MATERIALS AND METHODS General Procedures Animals. Male Holtzman rats weighing 260 –300 g at the beginning of the experiments were housed in individual metabolic cages. Standard Purina pellets (Na1 content 5 nmols/100 g), tap water and 3% NaCl solution were available ad libitum unless otherwise noted. Temperature in the animal colony was maintained at approximately 23°C. The 12:12-h light– dark cycle began with lights on at 0800. All experiments were started between 1000 and 2000 P.M. at least 3 days after surgery. Surgery. After an acclimatization period of 7 days, the animals
INTRODUCTION The subfornical organ (SFO) is one of the neural circumventricular organs lacking a blood– brain barrier and is important for body fluid balance [28]. Blood-borne angiotensin II (ANG II) penetrates the fenestrated capillaries of the SFO and activates receptors [18]. The SFO neurons are capable of responding to changes in ANG II concentration in the blood and may mediate induction of water and sodium intake [14,19,30]. It has been suggested that the central
* Address for correspondence: Dr. Luiz Antonio de Arruda Camargo, Department of Physiology, School of Dentistry, Paulista State University, UNESP, 1680 Humaita´ Street, Araraquara, SP 14801-903, Brazil. Fax: 55-(16)-222-4823.
521
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FIG. 1. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing the site of injection into the subfornical organ (arrow).
FIG. 2. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing bilateral the sites of injection into the lateral hypothalamus (arrows).
were maintained under tribromoethanol (Aldrich) (20 mg/100 g b. wt., intraperitoneal [i.p.]) anesthesia throughout surgery. A stainless steel guide-cannula (10 3 0.7 mm) outside diameter [o.d.]) was stereotaxically implanted into the brain with its opening protruding into the top of the SFO (coordinates: AP 5 1.0 mm caudal to the bregma; V 5 4.8 mm from the dura mater; L 5 0.0 mm from the sagittal line). For LH cannulation the cannula (14 3 0.7 mm o.d.) was positioned bilaterally as follows: AP 5 1.5 mm posterior to the bregma; V 5 7.5 mm below the dura mater; L 5 1.5 mm from the sagittal midline. For cannula implantation the stereotaxic incisor bar was positioned 2.5 mm above the interaural line. The cannula was secured to the top of the skull with dental cement and fastened with two screws. The insertion of a closefitting stylet kept the lumen free of debris and clots. A prophylactic dose of penicillin (30,000 IU) was given intramuscularly (i.m.) presurgically. Intracerebral injection techniques. Bolus intracranial injections were made after gently removing the animal from its cage, replacing the stylet with an injector that protruded 0.2 mm and 1.0 mm beyond the tip of the guide-cannula for SFO and LH, respectively, and was connected by PE-10 tubing to a 10.0-ml microsyringe, and injecting a total volume of 0.2 ml over a period of 30 s. Stylet and injector were always wiped with cotton soaked with 70% alcohol. After the injection, the injector was removed and replaced with the stylet, and the animal was returned to its cage for observation of its behavior. Drugs. ANG II, yohimbine hydrochloride, propranolol hydrochloride, noradrenaline bitartarate, dl-isoproterenol hydrochloride (Sigma Chemical Co., St. Louis, MO, USA) phenylephrine hydrochloride and clonidine hydrochloride (RBI, Natick, MA, USA) and prazosin hydrochloride (Pfizer Inc., Groton, CT, USA) were dissolved in 0.9% saline (vehicle). Yohimbine and prazosin were sonicated for better suspension. Histology. At the end of the experiments, the animals were anesthetized with ether and given a 2-ml injection of fast green dye via the intracranial cannula, followed by perfusion with saline and buffered formalin. The brains were removed, fixed in 10% formalin, frozen to 225°C and cut into 20- to 30-mm section. The presence of absence of dye in the ventricles was observed at this time. Only animals in which the presence of dye restricted to the SFO was noted were used in this study. Figures 1 and 2 presents the site of the injection into the SFO and the LH, respectively. Statistical analysis. The results are reported as mean 6 SEM and were analyzed by one-way analysis of variance. Values were
considered to be statistically significant when p , 0.05. The Newman-Keuls post-hoc test was used to assess the difference between individual means. Experimental Protocol Water intake or NaCl 3% intake. Five days after brain surgery, different groups of animals for water or sodium intake were submitted to experimental sessions. Each animals was submitted to four or five experimental sessions at 3-day intervals. An a-adrenergic antagonist (prazosin, yohimbine, or propranolol) and the a-adrenergic agonists (noradrenaline, clonidine, or phenylephrine) were injected into the LH 20 min before injection of ANG II into the SFO. Recording of water or sodium intake started immediately after ANG II injection and continued for 4 h. Before the salt appetite test the next morning, the overnight water intake was measured, and graduate cylinders were replaced with glass burettes containing 3% NaCl. The burettes were calibrate to 0.1 ml and fitted with drinking sponts. The presence of sodium was announced to the drinking rats by sprinkling a few drops of the saline solution on their lips and whiskers. RESULTS Effects of Pretreatment with Prazosin, Yohimbine, or Propranolol into the LH on the Sodium and Water Intake Induced by ANG II Injected Into the SFO The sodium intake observed during a period of 4 h in the control experiment (0.15 M NaCl into the SFO) was 1.3 6 0.6 ml. Injection of ANG II (10 pmol) into the SFO produced an increase in sodium ingestion (7.3 6 0.6 ml/2 h). Previous injection of prazosin (20, 40, and 80 nmol) into the LH potentiated the effect of ANG II on 3% NaCl intake induced by ANG II [F(3,35) 5 5.24, p , 0.05] (Fig. 3). Pretreatment with yohimbine and propranolol (20, 40, and 80 nmol) into the LH reduced the sodium intake induced by ANG II [F(3,30) 5 14.55, p , 0.05 and [F(3,30) 5 3.32, p , 0.05, respectively] (Fig. 3). Injection of only prazosin, yohimbine, or propranolol into the LH produced no alterations in salt intake. Control rats (0.15 M NaCl into the SFO) consumed a mean amount of 2.5 6 0.7 ml of water over a period of 4 h. ANG II (10 pmol) injected into the SFO led to ingestion of 16.7 6 1.8 ml of water over the same period. Previous treatment with prazosin and propranolol (40 and 80 nmol) into the LH antagonized the dipso-
FLUID CONTROL BY SFO-LH PATHWAYS
523
FIG. 3. Effect of pretreatment with prazosin, yohimbine, and propranolol into the lateral hypothalamus on sodium intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
FIG. 4. Effect of pretreatment with prazosin, yohimbine, and propranolol into the lateral hypothalamus on water intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
genesis induced by ANG II administration into the SFO [F(3,32) 5 6.52, p , 0.05 and F(3,28) 5 3.89, p , 0.05, respectively] (Fig. 4). No changes in the dipsogenic effect of ANG II were observed after previous treatment with yohimbine (10, 20, 40, and 80 nmol) (Fig. 4). Injection of only prazosin, yohimbine, or propranolol into the LH produced no alterations in water intake.
DISCUSSION
Effects of LH Pretreatment with Clonidine, Phenylephrine, or Noradrenaline into the LH on the Sodium and Water Intake Induced by ANG II Injected into the SFO The saline ingestion observed during 4 h in the control experiment (0.15 M NaCl) and ANG II (10 pmol) injection into the SFO is presented in Fig. 5. Previous treatment with clonidine (40 and 80 nmol) and noradrenaline (20, 40, and 80 nmol) elicits a potentiation in sodium intake induced by ANG II injection into the LH [F(3,35) 5 4.55, p , 0.01 and F(3,34) 5 5.7, p , 0.01, respectively], whereas phenylephrine (40 and 80 nmol) decreased this effect [F(3,33) 5 5.37, p , 0.01]. Figure 5 shows water intake after SFO injection of saline control and ANG II. Previous treatment with clonidine (20, 40, and 80 nmol) into the LH decreased the water intake induced by LH administration of ANG II [F(3,33 5 3.23, p , 0.05] into the SFO, whereas phenylephrine (40 and 80 nmol) elicits a potentiation in water intake induced by ANG II [F(3,35) 5 3.73, p , 0.05]. No changes in the dipsogenic effect of ANG II were observed after previous treatment with noradrenaline [F(3,31) 5 0.55, p , 0.05].
The present results show that yohimbine and propranolol injected into the LH reduces, whereas the a1-adrenoceptor antagonist prazosin increases the sodium ingestion induced by ANG II injected into the SFO. The results also show that prazosin and propranolol injected into the LH reduce the dipsogenesis induced by administration of ANG II into the SFO. Studies with adrenergic antagonists suggest that noradrenaline has a dual role in the central control of water and salt intake [9,10] induced by ANG II. Water intake induced by central ANG II is inhibited by a1-, a2- or b-antagonists, depending on the site of injection. Prazosin and phentolamine injected into the rostral hypothalamus attenuated the drinking response induced by intracerebroventricular (i.c.v.) ANG II [20]. An inhibitory effect of increased on the dipsogenic effect of ANG II injected into the medial preoptic area (MPOA) has been demonstrated by the previous administration of prazosin and propranolol into the same area [3]. Prazosin and yohimbine, but not propranolol, injected into the median preoptic nucleus (MnPO) reduced the water intake induced by ANG II [25]. The a2-adrenergic antagonist, yohimbine is reported to enhance the dipsogenic effect of ANG II or ANG III [6,15] and to induce water and salt intake [16,17] when injected systemically, but it is not clear if it affects those behaviors by a direct central action or by a peripheral action [13]. However, it has been proposed [12] that the organum vasculosum laminae terminalis (OVLT) is the circumventricular organ site for a peripheral action of salt appetite because infusions of ANG II into the OVLT, but not the SFO, elicit salt appetite and because lesions of the OVLT suppress salt appetite aroused either by a low dose of oral
524
FIG. 5. Effect of pretreatment with clonidine, phenylephrine, and noradrenaline into the lateral hypothalamus on sodium intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
captopril or by sodium depletion, by other side, lesions of the SFO reduce salt appetite aroused by sodium depletion [27,30]. The present study results also show that the a2-adrenoceptor agonist clonidine and noradrenaline injected into the LH increased the salt intake induced by ANG II injected into the SFO, whereas, the a1-adrenoceptor phenylephrine, but not clonidine and noradrenaline, injected into the LH increased the dipsogenic effect induced by SFO administration of ANG II. A role of the forebrain noradrenergic systems in the mediation of ANG II-elicited drinking and blood pressure responses has also been suggested [4,5]. The synthesis and turnover of hypothalamic catecholamines increased after i.c.v. administration of ANG II [1,8]. A relationship between noradrenaline and ANG II facilitation of NaCl intake was first shown by Chiaraviglio and Taleisnik [7], who obtained 1% NaCl intake in normovolemic animals with noradrenaline injected into the third ventricle close to the columns of the fornix. Other authors reported the same kind of increase after noradrenaline was injected i.c.v. [2] or into the paraventricular nucleus (PVN) [22]. In summary, the present study show that injection of prazosin, clonidine or noradrenaline into the LH increases, whereas yohimbine, phenylephrine or propranolol reduces the salt intake response produced by angiotensinergic activation of the SFO. Phenylephrine increases the dipsogenic response produced by ANG II, whereas previous treatment with clonidine or propranolol into the LH reduces the dipsogenic responses produced by ANG II administration into the SFO. These observations show that both a-adrenoceptor subtypes of LH are important for the fluid intake responses induced angiotensinergic activation of the SFO in rats.
´ JO ALMEIDA ET AL. ARAU
FIG. 6. Effect of pretreatment with clonidine, phenylephrine, and noradrenaline into the lateral hypothalamus on water intake induced by injection of angiotensin II (ANG II) into the subfornical organ. Data are reported as mean 6 SEM. The number of animals is indicate at the top of each column. * p , 0.05, compared with saline (control); 1 p , 0.05, compared with ANG II.
They also suggest that the central a-adrenergic receptors have a dual (inhibitory and excitatory) effect on the sodium and water intake induced by ANG II in rats). ACKNOWLEDGEMENTS
The authors appreciate the technical assistance of Reginaldo C. Queiro´z, Silas P. Barbosa, Silvia Fo´glia and Silvana A. D. Malavolta. They also thank Ana V. Oliveira for animal care. Research supported by CNPq and FAPESP.
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