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Sensitivity to angiotensin II of neurons in the circumventricular organs of polydipsic inbred mice
Brain Research, 524 (1990) 181-186 Elsevier BRES 15738
181
Research Reports
Sensitivity to angiotensin II of neurons in the circumventricular organs of polydipsic inbred mice Yukio Hattori* and Kiyomi Koizumi Department of Physiology, State University of New York, Health Science Centerat Brooklyn, Brooklyn, NY (U.S.A.) (Accepted 6 February 1990) Key words: Genetic polydipsia; AV3V; Subfornical organ; Water intake; Hypothalamic slice; Central angiotensin system; Neuron activity; Angiotensin II
The polydipsic inbred mice, STR/N, are known to possess an extremely strong appetite for drinking but no abnormality in the vasopressin system and renal functions. In brain slice preparations the sensitivity of neurons in the anteroventral region of the third ventricle (AV3V) and the subfornical organ (SFO) to angiotensin II (ANG If) was investigated using extracellular recordings in the STR/N and its control, Swiss/Webster (S/W) mice. In the AV3V, less proportion of neurons (15 out of 168; 9%) of the STR/N than that in the S/W (49/206; 24%) was excited by ANG II added to the medium. In the SFO, a proportion of neurons excited by ANG II was again lower in the STR/N (27/104; 26%) than in the S/W mice (64/118; 54%). The threshold concentration of ANG II for excitation of the AV3V and SFO neurons was, however, similar for both strains, 10 -9 M or less. Only one neuron in the SFO of a S/W mouse was inhibited by ANG II application. The excitatory effect of ANG II on AV3V and SFO neurons of both strains of mice persisted under synaptic blockade and was reversibly antagonized by an ANG II antagonist, saralasin. Such differences in sensitivity to ANG II of neurons in the SFO and AV3V, the regions thought to be involved in drinking behavior, suggest the involvement, at least in part, of the central angiotensin system in the polydipsia of the STR/N mice. INTRODUCTION In the late 1950s, the inbred strain of mice, STR/N, was found to exhibit an extreme polydipsia (daily water intake up to 120 ml) and polyuria with low specific gravity and electrolyte concentrations 31. A hybridization study 3° suggested that the primary polydipsia of this strain of mice had a recessive genetic character. The polydipsic mice were characterized as follows32: (1) there was no abnormality in the osmolarity, sodium and other ionic concentrations in the plasma; (2) under water restriction urine with reduced volume and increased specific gravity was produced, and normal response was elicited by exogenous vasopressin; (3) histologically, there was no abnormality in neurosecretory cells in the supraoptic and paraventricular nuclei in the hypothalamus; (4) the STR/N mice survived well even when the water intake was restricted. These findings suggest that in the polydipsic mice central, but not peripheral, mechanisms play a key role as motivating factors in the polydipsia which is not prerequisite for their survival; such mechanisms, however, remain to be clarified. Many studies on drinking behavior have been made in relation to brain angiotensin system 25,27. Involvement of
circumventricular organs, such as the anteroventral region of the third ventricle (AV3V) and subfornical organ (SFO), in the angiotensin system has been suggested 3' 10,33. There is good histological evidence that the AV3V and SFO project directly or indirectly to the supraoptic nucleus 2'18'19. Electrophysiological studies suggest that efferent fibers from the SFO innervate neurosecretory cells in the supraoptic and paraventricular nuclei 22'z9, and that angiotensin II ( A N G II) may be a neurotransmitter for this projection 9. Mechanisms of polydipsia of the STR/N mice appear to involve certain brain regions or neuronal network other than the vasopressinergic neurons, since presence of vasopressin has been demonstrated in neurosecretory cells of the supraoptic and paraventricular nuclei in the polydipsic mice 31. Our recent studies on the drinking behavior of the STR/N 13'14 have revealed that an angiotensin I-converting enzyme inhibitor, captopril, injected subcutaneously reduces spontaneous drinking in the STR/N mice, and that an A N G II antagonist, saralasin, injected intracerebroventricularly causes a similar reduction in drinking only in the polydipsic mice. These findings have led us to speculate that some neuronal mechanisms relating to the angiotensin system are involved in the
* Present address: Department of Physiology, Okayama University Medical School, Okayama 700, Japan. Correspondence: K. Koizumi, Department of Physiology, Box 31, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 polydipsia of the STR/N
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MATERIALS AND METHODS
Animals Polydipsic inbred strain of mice, STR/N (see Acknowledgements) and their controls, Swiss/Webster (S/W), at 6-12 months of age were used. They were individually housed in plastic cages in a room maintained at 23 + 1 °C and 12/12 h light-dark cycle (lights on at 07:00 h). Tap water and dry food (standard laboratory chow) were given ad libitum.
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The experimental procedures were essentially the same as described by others 8'2°'21. Mice were stunned by a blow on the neck and decapitated, and brains were quickly removed. After cooling the brain in an incubation medium at 4 °C for approximately 1 min, the meninges were removed under microscopic observation. Coronal hypothalamic slices, 350-450/~m in thickness, containing either the AV3V or the SFO alone were cut in the cooled medium with the vibratome (Tokai Irika Kyushu Co.). The slices were placed in the incubation medium at room temperature and left for at least 1 h. Before transferring to a recording chamber, each slice was carefully trimmed with a microsurgery knife so that recordings were performed in a piece of tissue containing either the AV3V or the SFO with a total area less than 2 × 2 mm. The incubation medium was a modified Yamamoto's solution 8 which contained (in mM): NaCI, 124; KCI, 5; KHzPO 4, 1.24; MgSO4, 1.3; CaCI 2, 2.1; NaHCO3, 20; and glucose, 10. In a perfusing medium, Ca 2+ concentration was lowered to 0.75 mM to increase spontaneous activity of AV3V or SFO neurons z°21. When required, a low Ca 2÷ (0.5 mM) and high Mg 2÷ (9 mM) solution was used to block synaptic transmission8'2°'2~. The solutions were oxygenated with a gas mixture of 95% 02 and 5% CO2 throughout the experiments.
Recording The trimmed slice was placed on a sylgard mat glued to the bottom of the recording chamber which had a volume of 0.8 ml and held in place with a nylon net and platinum weights. The recording chamber was perfused at a rate of 2-3 ml/min using a peristaltic pump and excess medium was removed by suction, by which the perfusion medium in the chamber could be exchanged in 45 s. The temperature of the perfusing medium was kept at 35 + 0.5 °C. Angiotensin II (ANG II; Sigma) of varied concentrations was applied to the slice by perfusing from separate containers. When required, an A N G II antagonist, [Sarl-Ala8]-angiotensin II (saralasin; Sigma), at a concentration of 3-10 times that of ANG II was perfused 5 min or more prior to ANG II application. Extracellular recordings were obtained from neurons within the AV3V or SFO using glass micropipettes filled with 0.5 M sodium acetate and 2% Chicago Sky Blue (Sigma). The micropipette with a DC resistance of 20-35 MI2 was inserted into the slice under microscopic observation. Location of the AV3V or the SFO was clearly visible through transmitted light. In the AV3V, we recorded mainly in the periventricular region, which contained the organum vasculosum of the lamina terminalis, preoptic suprachiasmatic nucleus, periventricular preoptic nucleus and median preoptic nucleus. Using conventional recording methods, action potentials were displayed on a storage oscilloscope and stored on magnetic tape for further analysis. A window discriminator and an integrator were used for the continuous observation of the firing pattern of neurons. At the end of recording, a constant cathodal current of 5/~A was passed
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Fig. 1. Responses of AV3V (A) and SFO (B) neurons to application of ANG II at 10 -7 M in polydipsic STR/N mice. Each illustration shows rate-meter records and oscilloscope traces obtained at the time indicated by arrows. Open bars indicate the periods of ANG II application.
through the tip of the electrode for 3-5 min to deposit a blue spot from which the recording sites could be determined histologically.
Assessment of neuronal responses The responses of neurons in the AV3V or SFO to application of ANG II were assessed by observing the change in firing rates. Neurons were classified as having been excited or inhibited if their firing rates during a 1-min period of the maximal response to ANG II were increased or decreased by more than 20% compared to the average rate during a 5-min control period.
RESULTS
Effects o f A N G H on the A V 3 V and S F O neurons In t h e A V 3 V , e x t r a c e l l u l a r r e c o r d i n g s f r o m 168 n e u r o n s o f t h e S T R / N a n d 206 n e u r o n s o f t h e S / W m i c e w e r e m a d e f r o m t r i m m e d slice p r e p a r a t i o n s . T h e f i r i n g r a t e s ( m e a n + S . E . M . ) o f s p o n t a n e o u s l y a c t i v e A V 3 V cells o f t h e S T R / N a n d S / W w e r e 4.83 + 0.35 a n d 4.74 + 0.33
TABLE I
Responses of AV3V and SFO neurons of STR/N and S/W mice to application orANG H Region and strain
Total
Number of neurons (%) Excited
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15 (9)* 49 (24)
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153 (91) 157 (76)
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104 118
27 (26)* 64 (54)
0 (0) 1 (1)
77 (74) 53 (45)
*P < 0.001 vs S/W mice.
183 spikes/s, respectively. In the S F O , the m e a n firing rates ( m e a n + S . E . M . ) of 104 neurons of the STR/N and 118 o f the S/W were 4.52 + 0.42 and 4.31 + 0.38 spikes/s, respectively. T h e a m p l i t u d e of spikes r e c o r d e d from the A V 3 V and S F O neurons ranged from 0.5 to 5 mV. Most of the A V 3 V and S F O neurons showed continuous or irregular firing patterns. N o difference in the firing patterns of A V 3 V or S F O neurons was d e t e c t e d between the S T R / N and S/W strains. T h e application of A N G II at 10 -7 M to A V 3 V neurons excited some cells in both the STR/N and S/W. A n e x a m p l e taken from an STR/N mouse is shown in Fig. 1A. T h e p a t t e r n o f the response was similar in the S/W mice. The S F O neurons also were excited by A N G II at 10 -7 M in b o t h strains. A n e x a m p l e from the STR/N is shown in Fig. l B . A g a i n responses of S F O neurons to A N G II were indistinguishable b e t w e e n the two strains. O n l y one S F O cell of a S/W mouse and none of the A V 3 V cells were inhibited by A N G II. In the responsive neurons to A N G II in the A V 3 V and S F O , the firing rate r e a c h e d a m a x i m u m 2 - 3 min after the application of A N G II and recovered to pret r e a t m e n t level in 5-15 min after washing out the p e p t i d e with n o r m a l m e d i u m . The responses to A N G II were
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Proportions of AV3V and SFO neurons responsive to ANG H The responses to A N G II (10 -7 M) of A V 3 V and S F O neurons of the STR/N and S/W mice are s u m m a r i z e d in Table I. In the AV3V, 15 out of 168 neurons ( 9 % ) were excited by A N G II in the STR/N, while 49 out of 206 neurons (24%) were excited by the p e p t i d e in the S/W. Thus the p r o p o r t i o n of neurons excited by A N G II was significantly lower in the STR/N than in the S/W (X2 = 13.37, P < 0.001 at df = 1). In the S F O , 27 out of 104 neurons (26%) of the STR/N and 64 out of 118 neurons (54%) of the S/W were excited by A N G II. A g a i n , the p r o p o r t i o n of S F O cells excited by A N G II was significantly lower in the S T R / N than in the S/W (Xz = 17.12, P < 0.001 at df = 1).
Responses to ANG H under synaptic blockade Responsiveness of A V 3 V and S F O neurons to A N G II was e x a m i n e d u n d e r synaptic b l o c k a d e using a low Ca 2÷ and high Mg 2÷ perfusing m e d i u m , in which the spontaneous activity of most neurons tested was depressed. A l l of the A V 3 V (STR/N, n = 2; S/W, n = 5) and S F O (STR/N, n = 2; S/W, n = 4) n e u r o n s which had been
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Fig. 3. Effects of an ANG II antagonist saralasin on the excitatory responses of an AV3V neuron produced by ANG II. In the neuron of an STR/N mouse which had been excited by ANG II 10 -7 M (upper), saralasin 10 -6 M completely blocked the effects of ANG II 10 -7 M (middle) and after washing out saralasin the neuron was excited again by ANG II (lower). Open and solid bars indicate the periods during which ANG II and saralasin, respectively, were applied.
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Effects of an A N G II antagonist, saralasin, on A N G II-elicited excitatory responses were e x a m i n e d in A V 3 V (STR/N, n = 2; S/W, n = 3) and S F O (STR/N, n = 2; S/W, n = 2) neurons. Saralasin (3 × 10-7-10 -6 M) reversibly b l o c k e d the excitatory effects of A N G II (10 -7 M). In all neurons tested no difference was o b s e r v e d between the two strains of mice. Figure 3 shows the response of an A V 3 V n e u r o n of the STR/N.
Responses of A V 3 V and SFO neurons to different concentrations of A N G H
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To examine the d o s e - r e s p o n s e relationships, the activities of A V 3 V (STR/N, n = 8; S/W, n = 10) and S F O (STR/N, n = 7; S/W, n = 8) n e u r o n s were studied by giving different concentrations of A N G II, ranging from 10 -9 t o 10 -6 M. Figure 4 shows examples of A V 3 V neurons in each strain of mice. T h e increase in firing rate of the A V 3 V and S F O cells after application of A N G II was plotted against the concentration of the p e p t i d e , as shown in Fig. 5. A s A N G II concentration increased, the firing rate increased in a d o s e - d e p e n d e n t manner. The threshold concentration of A N G II for causing the
185 excitation was 10-9 M or less. In the AV3V and SFO, there were no differences between the two strains of mice in the values of threshold or patterns of dose-response relationships. DISCUSSION Earlier studies 4-6'11"2s have shown the effects of A N G II on neurons in the circumventricular organs, as well as in other regions of the central nervous system. A few reports, however, have been published on the effects of A N G II applied to the perfusing medium in brain slice preparations. For example, Okuya et al. 2° have found that in the rat A N G II excites neurons in the AV3V and SFO in vitro. The results presented here on the STR/N and S/W mice also demonstrated that some neurons in the AV3V and SFO were excited by A N G II applied to the medium, and only one SFO neuron of the control strain showed the inhibitory response to A N G II. The excitatory responses to A N G II observed in the present study indicated that AV3V and SFO neurons were themselves sensitive to A N G II in the absence of synaptic drive from neurons nearby, since the response persisted under the low Ca 2+ and high Mg 2+ medium, which was shown to block almost completely the synaptic transmission s . No difference between the two strains of mice was found in dose-response relationships and the threshold concentration of A N G II (10 -9 M or less) for the excitation of both AV3V and SFO neurons. The threshold value was somewhat higher than that reported for rats, which was less than 10-1° M 2°. There is no apparent reason for the difference; it may reflect possible variance of A N G II concentration in blood or cerebrospinal fluid between the two species, although no data on A N G II concentration of mice blood or cerebrospinal fluid are available. Palovcik and Phillips 23 have reported that both excitatory and inhibitory responses of hippocampal neurons to A N G II are blocked by a specific A N G II antagonist, saralasin. In the circumventricular organs, the present results also indicate that the excitatory response caused by A N G II was blocked by saralasin. In this study, however, it is impossible to know whether the inhibitory response to A N G II was blocked by the A N G II antagonist, since only one neuron in the SFO of control strain of mice was inhibited by A N G II. The A N G II-immunoreactive cell bodies or fibers 16A7 and A N G II binding sites 26"36 have been reported to be found in the SFO and AV3V. Further, it has been suggested that blood-borne A N G II acts mainly on specific A N G II receptors located in the SFO, and that the AV3V is a probable site for sensing cerebrospinal fluid-borne A N G
II 20'24. Thus, it is likely that the responses of AV3V and SFO neurons to A N G II observed in this study are due to mediation through specific receptors for the peptide. It is somewhat puzzling to find that in both the AV3V and SFO proportions of neurons responsive to A N G II in the polydipsic STR/N mice were less than those in control S/W mice. Recent studies by Koizumi 12 and Koizumi et al. TM and our unpublished observations have indicated that in satiated STR/N mice no marked drinking response was induced by intracerebroventricularly injected A N G II, although an appreciable reduction in drinking is produced by saralasin given through the same route. These findings suggest that angiotensin system functions feebly in the brain of the polydipsic mice. The lowered responsiveness to A N G II to initiate drinking of this special strain of mice may be due to the decrease in proportion of AV3V and SFO neurons responsive to A N G II. Another possible explanation may be that the lowered responsiveness of drinking to A N G II in the STR/N mice results from the down-regulation of A N G II receptors by overproduction of A N G II. The evaluation of this possibility, however, requires direct measurement of A N G II concentration and A N G II receptor binding in this special strain of mice. The functional significance of the circumventricular organs including the AV3V and SFO in drinking behavior has been shown in several species of animals 33, though no such study has been made on the STR/N mice. In addition to A N G II, it has been shown that the central opioid system is involved in the mechanisms of drinking 1' 7,15 and that endogenous opioid peptides inhibit the central action of angiotensin 34'35. Taken together, it is likely that the angiotensin- and opioid-systems contribute to the polydipsia of STR/N mice, in which lowered sensitivity of neurons in the AV3V and SFO may play a role. In conclusion, neurons in the AV3V and SFO of the polydipsic mice, STR/N, and the control strain of mice, S/W, were found to be directly excited by A N G II through specific A N G II receptors. The proportion of neurons excited by A N G II was lower in the STR/N than in S/W mice. It is likely that the sensitivity of AV3V and SFO neurons is decreased in the polydipsic mice, and this change may be one of the contributing factors in the polydipsia, though its exact mechanism is still unclear at present time.
Acknowledgements. This work was supported by a grant from the National Institute of Health, USPHS, NS-00847. We thank Dr. E. Silverstein of the Department of Medicine, State University of New York, Health Science Center at Brooklyn, for supply of STR/N mice. They were originally obtained from the National Institute of Health, Bethesda, MD.
186
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nervous system, Neuroendocrinology, 40 (1985) 2-24. 18 Lind, R.W., Van Hoesen, G.W. and Johnson, A.K., An HRP study of the connections of the subfornical organ of the rat, J. Comp. Neurol., 210 (1982) 265-277. 19 Miselis, R.R., Shapiro, R.E. and Hand, P.J., Subfornical organ efferents to neural systems for control of body water, Science, 205 (1979) 1022-1025. 20 Okuya, S., Inenaga, K., Kaneko, T. and Yamashita, H., Angiotensin II sensitive neurons in the supraoptic nucleus, subfornical organ and anteroventral third ventricle of rats in vitro, Brain Research, 402 (1987) 58-67. 21 Okuya, S. and Yamashita, H., Effects of atrial natriuretic polypeptide on rat hypothalamic neurones in vitro, J. Physiol. (Lond.), 389 (1987) 717-728. 22 Osaka, T., Yamashita, H. and Koizumi, K., Inhibition of paraventricular neurons by subfornical organ and AV3V in cats, Am. J. Physiol., 255 (1988) R961-R967. 23 Palovcik, R.A. and Phillips, M.I., Saralasin increases activity of hippocampal neurons inhibited by angiotensin II, Brain Research, 323 (1984) 345-348. 24 Phillips, M.I., Angiotensin in the brain, Neuroendocrinology, 25 (1978) 354-377. 25 Phillips, M.I., Functions of angiotensin in the central nervous system, Annu. Rev. Physiol., 49 (1987) 413-435. 26 Plunkett, L.M., Shigematsu, K., Kurihara, M. and Saavedra, J.M., Localization of angiotensin II receptors along the anteroventral third ventricle area of the rat brain, Brain Research, 405 (1987) 205-212. 27 Reid, I.A., Actions of angiotensin II on the brain: mechanisms and physiologic role, Am. J. Physiol., 246 (1984) F533-F543. 28 Sayer, R.J., Hubbard, J.I. and Sirett, N.E., Rat organum vasculosum laminae terminalis in vitro: responses to transmitters, Am. J. Physiol., 247 (1984) R374-R379. 29 Sgro, S., Ferguson, A.V. and Renaud, L.P., Subfornical organsupraoptic nucleus connections: an electrophysiologic study in the rat, Brain Research, 303 (1984) 7-13. 30 Silverstein, E., Effect of hybridization on the primary polydipsic trait of an inbred strain of mice, Nature (Lond.), 191 (1961) 523. 31 Silverstein, E., Sokoloff, L., Mickelsen, O. and Jay Jr., G.E., Polyuria, polydipsia and hydronephrosis in inbred strain of mice, Fed. Proc., 17 (1958) 457. 32 Silverstein, E., Sokoloff, L., Mickelsen, O. and Jay Jr., G.E., Primary polydipsia and hydronephrosis in an inbred strain of mice, Am. J. Pathol., 38 (1961) 143-159. 33 Simpson, J.B., The circumventricular organs and the central actions of angiotensin, Neuroendocrinology, 32 (1981) 248-256. 34 Summy-Long, J.Y., Keil. L.C., Deen, K., Rosella, L. and Severs, W.B., Endogenous opioid peptide inhibition of the central actions of angiotensin, J. Pharmacol. Exp. Ther., 217 (1981) 619-629. 35 Summy-Long, J.Y., Keil, L.C., Sells, G., Kirby, A., Chee, O. and Severs, W.B., Cerebroventricular sites for enkephalin inhibition of the central actions of angiotensin, Am. J. Physiol., 244 (1983) R522-R529. 36 Van Houten, M., Schiffrin, E.L., Mann, J.F.E., Posner, B.I. and Boucher, R., Radioautographic localization of specific binding sites for blood-borne angiotensin II in the rat brain, Brain Research, 186 (1980) 480-485.
181
Research Reports
Sensitivity to angiotensin II of neurons in the circumventricular organs of polydipsic inbred mice Yukio Hattori* and Kiyomi Koizumi Department of Physiology, State University of New York, Health Science Centerat Brooklyn, Brooklyn, NY (U.S.A.) (Accepted 6 February 1990) Key words: Genetic polydipsia; AV3V; Subfornical organ; Water intake; Hypothalamic slice; Central angiotensin system; Neuron activity; Angiotensin II
The polydipsic inbred mice, STR/N, are known to possess an extremely strong appetite for drinking but no abnormality in the vasopressin system and renal functions. In brain slice preparations the sensitivity of neurons in the anteroventral region of the third ventricle (AV3V) and the subfornical organ (SFO) to angiotensin II (ANG If) was investigated using extracellular recordings in the STR/N and its control, Swiss/Webster (S/W) mice. In the AV3V, less proportion of neurons (15 out of 168; 9%) of the STR/N than that in the S/W (49/206; 24%) was excited by ANG II added to the medium. In the SFO, a proportion of neurons excited by ANG II was again lower in the STR/N (27/104; 26%) than in the S/W mice (64/118; 54%). The threshold concentration of ANG II for excitation of the AV3V and SFO neurons was, however, similar for both strains, 10 -9 M or less. Only one neuron in the SFO of a S/W mouse was inhibited by ANG II application. The excitatory effect of ANG II on AV3V and SFO neurons of both strains of mice persisted under synaptic blockade and was reversibly antagonized by an ANG II antagonist, saralasin. Such differences in sensitivity to ANG II of neurons in the SFO and AV3V, the regions thought to be involved in drinking behavior, suggest the involvement, at least in part, of the central angiotensin system in the polydipsia of the STR/N mice. INTRODUCTION In the late 1950s, the inbred strain of mice, STR/N, was found to exhibit an extreme polydipsia (daily water intake up to 120 ml) and polyuria with low specific gravity and electrolyte concentrations 31. A hybridization study 3° suggested that the primary polydipsia of this strain of mice had a recessive genetic character. The polydipsic mice were characterized as follows32: (1) there was no abnormality in the osmolarity, sodium and other ionic concentrations in the plasma; (2) under water restriction urine with reduced volume and increased specific gravity was produced, and normal response was elicited by exogenous vasopressin; (3) histologically, there was no abnormality in neurosecretory cells in the supraoptic and paraventricular nuclei in the hypothalamus; (4) the STR/N mice survived well even when the water intake was restricted. These findings suggest that in the polydipsic mice central, but not peripheral, mechanisms play a key role as motivating factors in the polydipsia which is not prerequisite for their survival; such mechanisms, however, remain to be clarified. Many studies on drinking behavior have been made in relation to brain angiotensin system 25,27. Involvement of
circumventricular organs, such as the anteroventral region of the third ventricle (AV3V) and subfornical organ (SFO), in the angiotensin system has been suggested 3' 10,33. There is good histological evidence that the AV3V and SFO project directly or indirectly to the supraoptic nucleus 2'18'19. Electrophysiological studies suggest that efferent fibers from the SFO innervate neurosecretory cells in the supraoptic and paraventricular nuclei 22'z9, and that angiotensin II ( A N G II) may be a neurotransmitter for this projection 9. Mechanisms of polydipsia of the STR/N mice appear to involve certain brain regions or neuronal network other than the vasopressinergic neurons, since presence of vasopressin has been demonstrated in neurosecretory cells of the supraoptic and paraventricular nuclei in the polydipsic mice 31. Our recent studies on the drinking behavior of the STR/N 13'14 have revealed that an angiotensin I-converting enzyme inhibitor, captopril, injected subcutaneously reduces spontaneous drinking in the STR/N mice, and that an A N G II antagonist, saralasin, injected intracerebroventricularly causes a similar reduction in drinking only in the polydipsic mice. These findings have led us to speculate that some neuronal mechanisms relating to the angiotensin system are involved in the
* Present address: Department of Physiology, Okayama University Medical School, Okayama 700, Japan. Correspondence: K. Koizumi, Department of Physiology, Box 31, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
182 polydipsia of the STR/N
m i c e , in w h i c h s e n s i t i v i t y t o
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MATERIALS AND METHODS
Animals Polydipsic inbred strain of mice, STR/N (see Acknowledgements) and their controls, Swiss/Webster (S/W), at 6-12 months of age were used. They were individually housed in plastic cages in a room maintained at 23 + 1 °C and 12/12 h light-dark cycle (lights on at 07:00 h). Tap water and dry food (standard laboratory chow) were given ad libitum.
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The experimental procedures were essentially the same as described by others 8'2°'21. Mice were stunned by a blow on the neck and decapitated, and brains were quickly removed. After cooling the brain in an incubation medium at 4 °C for approximately 1 min, the meninges were removed under microscopic observation. Coronal hypothalamic slices, 350-450/~m in thickness, containing either the AV3V or the SFO alone were cut in the cooled medium with the vibratome (Tokai Irika Kyushu Co.). The slices were placed in the incubation medium at room temperature and left for at least 1 h. Before transferring to a recording chamber, each slice was carefully trimmed with a microsurgery knife so that recordings were performed in a piece of tissue containing either the AV3V or the SFO with a total area less than 2 × 2 mm. The incubation medium was a modified Yamamoto's solution 8 which contained (in mM): NaCI, 124; KCI, 5; KHzPO 4, 1.24; MgSO4, 1.3; CaCI 2, 2.1; NaHCO3, 20; and glucose, 10. In a perfusing medium, Ca 2+ concentration was lowered to 0.75 mM to increase spontaneous activity of AV3V or SFO neurons z°21. When required, a low Ca 2÷ (0.5 mM) and high Mg 2÷ (9 mM) solution was used to block synaptic transmission8'2°'2~. The solutions were oxygenated with a gas mixture of 95% 02 and 5% CO2 throughout the experiments.
Recording The trimmed slice was placed on a sylgard mat glued to the bottom of the recording chamber which had a volume of 0.8 ml and held in place with a nylon net and platinum weights. The recording chamber was perfused at a rate of 2-3 ml/min using a peristaltic pump and excess medium was removed by suction, by which the perfusion medium in the chamber could be exchanged in 45 s. The temperature of the perfusing medium was kept at 35 + 0.5 °C. Angiotensin II (ANG II; Sigma) of varied concentrations was applied to the slice by perfusing from separate containers. When required, an A N G II antagonist, [Sarl-Ala8]-angiotensin II (saralasin; Sigma), at a concentration of 3-10 times that of ANG II was perfused 5 min or more prior to ANG II application. Extracellular recordings were obtained from neurons within the AV3V or SFO using glass micropipettes filled with 0.5 M sodium acetate and 2% Chicago Sky Blue (Sigma). The micropipette with a DC resistance of 20-35 MI2 was inserted into the slice under microscopic observation. Location of the AV3V or the SFO was clearly visible through transmitted light. In the AV3V, we recorded mainly in the periventricular region, which contained the organum vasculosum of the lamina terminalis, preoptic suprachiasmatic nucleus, periventricular preoptic nucleus and median preoptic nucleus. Using conventional recording methods, action potentials were displayed on a storage oscilloscope and stored on magnetic tape for further analysis. A window discriminator and an integrator were used for the continuous observation of the firing pattern of neurons. At the end of recording, a constant cathodal current of 5/~A was passed
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Fig. 1. Responses of AV3V (A) and SFO (B) neurons to application of ANG II at 10 -7 M in polydipsic STR/N mice. Each illustration shows rate-meter records and oscilloscope traces obtained at the time indicated by arrows. Open bars indicate the periods of ANG II application.
through the tip of the electrode for 3-5 min to deposit a blue spot from which the recording sites could be determined histologically.
Assessment of neuronal responses The responses of neurons in the AV3V or SFO to application of ANG II were assessed by observing the change in firing rates. Neurons were classified as having been excited or inhibited if their firing rates during a 1-min period of the maximal response to ANG II were increased or decreased by more than 20% compared to the average rate during a 5-min control period.
RESULTS
Effects o f A N G H on the A V 3 V and S F O neurons In t h e A V 3 V , e x t r a c e l l u l a r r e c o r d i n g s f r o m 168 n e u r o n s o f t h e S T R / N a n d 206 n e u r o n s o f t h e S / W m i c e w e r e m a d e f r o m t r i m m e d slice p r e p a r a t i o n s . T h e f i r i n g r a t e s ( m e a n + S . E . M . ) o f s p o n t a n e o u s l y a c t i v e A V 3 V cells o f t h e S T R / N a n d S / W w e r e 4.83 + 0.35 a n d 4.74 + 0.33
TABLE I
Responses of AV3V and SFO neurons of STR/N and S/W mice to application orANG H Region and strain
Total
Number of neurons (%) Excited
Inhibited
Unresponsive
AV3V STR/N S/W
168 206
15 (9)* 49 (24)
0 (0) 0 (0)
153 (91) 157 (76)
SFO STR/N S/W
104 118
27 (26)* 64 (54)
0 (0) 1 (1)
77 (74) 53 (45)
*P < 0.001 vs S/W mice.
183 spikes/s, respectively. In the S F O , the m e a n firing rates ( m e a n + S . E . M . ) of 104 neurons of the STR/N and 118 o f the S/W were 4.52 + 0.42 and 4.31 + 0.38 spikes/s, respectively. T h e a m p l i t u d e of spikes r e c o r d e d from the A V 3 V and S F O neurons ranged from 0.5 to 5 mV. Most of the A V 3 V and S F O neurons showed continuous or irregular firing patterns. N o difference in the firing patterns of A V 3 V or S F O neurons was d e t e c t e d between the S T R / N and S/W strains. T h e application of A N G II at 10 -7 M to A V 3 V neurons excited some cells in both the STR/N and S/W. A n e x a m p l e taken from an STR/N mouse is shown in Fig. 1A. T h e p a t t e r n o f the response was similar in the S/W mice. The S F O neurons also were excited by A N G II at 10 -7 M in b o t h strains. A n e x a m p l e from the STR/N is shown in Fig. l B . A g a i n responses of S F O neurons to A N G II were indistinguishable b e t w e e n the two strains. O n l y one S F O cell of a S/W mouse and none of the A V 3 V cells were inhibited by A N G II. In the responsive neurons to A N G II in the A V 3 V and S F O , the firing rate r e a c h e d a m a x i m u m 2 - 3 min after the application of A N G II and recovered to pret r e a t m e n t level in 5-15 min after washing out the p e p t i d e with n o r m a l m e d i u m . The responses to A N G II were
Before
reversible and r e p r o d u c i b l e with r e p e a t e d applications of A N G II.
Proportions of AV3V and SFO neurons responsive to ANG H The responses to A N G II (10 -7 M) of A V 3 V and S F O neurons of the STR/N and S/W mice are s u m m a r i z e d in Table I. In the AV3V, 15 out of 168 neurons ( 9 % ) were excited by A N G II in the STR/N, while 49 out of 206 neurons (24%) were excited by the p e p t i d e in the S/W. Thus the p r o p o r t i o n of neurons excited by A N G II was significantly lower in the STR/N than in the S/W (X2 = 13.37, P < 0.001 at df = 1). In the S F O , 27 out of 104 neurons (26%) of the STR/N and 64 out of 118 neurons (54%) of the S/W were excited by A N G II. A g a i n , the p r o p o r t i o n of S F O cells excited by A N G II was significantly lower in the S T R / N than in the S/W (Xz = 17.12, P < 0.001 at df = 1).
Responses to ANG H under synaptic blockade Responsiveness of A V 3 V and S F O neurons to A N G II was e x a m i n e d u n d e r synaptic b l o c k a d e using a low Ca 2÷ and high Mg 2÷ perfusing m e d i u m , in which the spontaneous activity of most neurons tested was depressed. A l l of the A V 3 V (STR/N, n = 2; S/W, n = 5) and S F O (STR/N, n = 2; S/W, n = 4) n e u r o n s which had been
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Fig. 3. Effects of an ANG II antagonist saralasin on the excitatory responses of an AV3V neuron produced by ANG II. In the neuron of an STR/N mouse which had been excited by ANG II 10 -7 M (upper), saralasin 10 -6 M completely blocked the effects of ANG II 10 -7 M (middle) and after washing out saralasin the neuron was excited again by ANG II (lower). Open and solid bars indicate the periods during which ANG II and saralasin, respectively, were applied.
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.5 rain Fig. 4. Responses of AV3V neurons of STR/N (A) and S/W (B) mice to successive application of different concentrations of ANG II. Open bars indicate the periods during which the peptide was applied. excited by A N G II (10 -7 M) in the normal m e d i u m were also excited by the p e p t i d e at the same concentration in the low Ca 2÷ and high Mg 2÷ medium. A n example from the STR/N is shown in Fig. 2. No difference between the two strains of mice was observed.
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Effects of an A N G II antagonist, saralasin, on A N G II-elicited excitatory responses were e x a m i n e d in A V 3 V (STR/N, n = 2; S/W, n = 3) and S F O (STR/N, n = 2; S/W, n = 2) neurons. Saralasin (3 × 10-7-10 -6 M) reversibly b l o c k e d the excitatory effects of A N G II (10 -7 M). In all neurons tested no difference was o b s e r v e d between the two strains of mice. Figure 3 shows the response of an A V 3 V n e u r o n of the STR/N.
Responses of A V 3 V and SFO neurons to different concentrations of A N G H
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To examine the d o s e - r e s p o n s e relationships, the activities of A V 3 V (STR/N, n = 8; S/W, n = 10) and S F O (STR/N, n = 7; S/W, n = 8) n e u r o n s were studied by giving different concentrations of A N G II, ranging from 10 -9 t o 10 -6 M. Figure 4 shows examples of A V 3 V neurons in each strain of mice. T h e increase in firing rate of the A V 3 V and S F O cells after application of A N G II was plotted against the concentration of the p e p t i d e , as shown in Fig. 5. A s A N G II concentration increased, the firing rate increased in a d o s e - d e p e n d e n t manner. The threshold concentration of A N G II for causing the
185 excitation was 10-9 M or less. In the AV3V and SFO, there were no differences between the two strains of mice in the values of threshold or patterns of dose-response relationships. DISCUSSION Earlier studies 4-6'11"2s have shown the effects of A N G II on neurons in the circumventricular organs, as well as in other regions of the central nervous system. A few reports, however, have been published on the effects of A N G II applied to the perfusing medium in brain slice preparations. For example, Okuya et al. 2° have found that in the rat A N G II excites neurons in the AV3V and SFO in vitro. The results presented here on the STR/N and S/W mice also demonstrated that some neurons in the AV3V and SFO were excited by A N G II applied to the medium, and only one SFO neuron of the control strain showed the inhibitory response to A N G II. The excitatory responses to A N G II observed in the present study indicated that AV3V and SFO neurons were themselves sensitive to A N G II in the absence of synaptic drive from neurons nearby, since the response persisted under the low Ca 2+ and high Mg 2+ medium, which was shown to block almost completely the synaptic transmission s . No difference between the two strains of mice was found in dose-response relationships and the threshold concentration of A N G II (10 -9 M or less) for the excitation of both AV3V and SFO neurons. The threshold value was somewhat higher than that reported for rats, which was less than 10-1° M 2°. There is no apparent reason for the difference; it may reflect possible variance of A N G II concentration in blood or cerebrospinal fluid between the two species, although no data on A N G II concentration of mice blood or cerebrospinal fluid are available. Palovcik and Phillips 23 have reported that both excitatory and inhibitory responses of hippocampal neurons to A N G II are blocked by a specific A N G II antagonist, saralasin. In the circumventricular organs, the present results also indicate that the excitatory response caused by A N G II was blocked by saralasin. In this study, however, it is impossible to know whether the inhibitory response to A N G II was blocked by the A N G II antagonist, since only one neuron in the SFO of control strain of mice was inhibited by A N G II. The A N G II-immunoreactive cell bodies or fibers 16A7 and A N G II binding sites 26"36 have been reported to be found in the SFO and AV3V. Further, it has been suggested that blood-borne A N G II acts mainly on specific A N G II receptors located in the SFO, and that the AV3V is a probable site for sensing cerebrospinal fluid-borne A N G
II 20'24. Thus, it is likely that the responses of AV3V and SFO neurons to A N G II observed in this study are due to mediation through specific receptors for the peptide. It is somewhat puzzling to find that in both the AV3V and SFO proportions of neurons responsive to A N G II in the polydipsic STR/N mice were less than those in control S/W mice. Recent studies by Koizumi 12 and Koizumi et al. TM and our unpublished observations have indicated that in satiated STR/N mice no marked drinking response was induced by intracerebroventricularly injected A N G II, although an appreciable reduction in drinking is produced by saralasin given through the same route. These findings suggest that angiotensin system functions feebly in the brain of the polydipsic mice. The lowered responsiveness to A N G II to initiate drinking of this special strain of mice may be due to the decrease in proportion of AV3V and SFO neurons responsive to A N G II. Another possible explanation may be that the lowered responsiveness of drinking to A N G II in the STR/N mice results from the down-regulation of A N G II receptors by overproduction of A N G II. The evaluation of this possibility, however, requires direct measurement of A N G II concentration and A N G II receptor binding in this special strain of mice. The functional significance of the circumventricular organs including the AV3V and SFO in drinking behavior has been shown in several species of animals 33, though no such study has been made on the STR/N mice. In addition to A N G II, it has been shown that the central opioid system is involved in the mechanisms of drinking 1' 7,15 and that endogenous opioid peptides inhibit the central action of angiotensin 34'35. Taken together, it is likely that the angiotensin- and opioid-systems contribute to the polydipsia of STR/N mice, in which lowered sensitivity of neurons in the AV3V and SFO may play a role. In conclusion, neurons in the AV3V and SFO of the polydipsic mice, STR/N, and the control strain of mice, S/W, were found to be directly excited by A N G II through specific A N G II receptors. The proportion of neurons excited by A N G II was lower in the STR/N than in S/W mice. It is likely that the sensitivity of AV3V and SFO neurons is decreased in the polydipsic mice, and this change may be one of the contributing factors in the polydipsia, though its exact mechanism is still unclear at present time.
Acknowledgements. This work was supported by a grant from the National Institute of Health, USPHS, NS-00847. We thank Dr. E. Silverstein of the Department of Medicine, State University of New York, Health Science Center at Brooklyn, for supply of STR/N mice. They were originally obtained from the National Institute of Health, Bethesda, MD.
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