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Estrogen attenuates the drinking response induced by activation of angiotensinergic pathways from the lateral hypothalamic area to the subfornical organ in female rats
Behavioural Brain Research 122 (2001) 33 – 41 www.elsevier.com/locate/bbr
Research report
Estrogen attenuates the drinking response induced by activation of angiotensinergic pathways from the lateral hypothalamic area to the subfornical organ in female rats Shigeko Fujisawa a, Junichi Tanaka a,b,*, Masahiko Nomura a b
a Department of Physiology, Saitama Medical School, Iruma-gun, Saitama 350 -0495, Japan Department of Human De6elopment, Naruto Uni6ersity of Education, Naruto-cho, Naruto, Tokushima 772 -8502, Japan
Received 26 September 2000; received in revised form 2 January 2001; accepted 2 January 2001
Abstract The present study was carried out to investigate whether estrogen modulates the drinking response induced by activation of angiotensinergic neural pathways from the lateral hypothalamic area (LHA) to the subfornical organ (SFO) in the female rats. Microinjection of ANG II (10 − 10 M, 0.2 ml) into the LHA caused drinking in 17 out of 26 ovariectomized (OVX) female rats that were treated with propylene glycol (PG) vehicle and in 18 out of 28 OVX female rats that were treated with estrogen benzoate (EB). In both groups, previous injections of the ANG II antagonist saralasin (Sar, 10 − 10 M, 0.2 ml) into the SFO significantly attenuated the water intake caused by the ANG II injection, suggesting that the ANG II-induced drinking response may be mediated by the angiotensinergic LHA projections to the SFO. Injections of ANG II (10 − 10 M, 0.2 ml) into the SFO elicited drinking in all the animals that demonstrated the drinking response to ANG II injected into the LHA. The amount of water intake caused by either the injection of ANG II into the LHA or the SFO was significantly greater in the PG-treated than in the EB-treated animals. These results suggest that the circulating estrogen may act to attenuate the dipsogenic response induced by activation of the angiotensinergic pathways from the LHA to the SFO. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Drinking; Lateral hypothalamic area; Subfornical organ; Estrogen; Angiotensin II; Saralasin; Rat
1. Introduction It is well known that the subfornical organ (SFO) is a circumventricular organ that lacks a normal blood – brain barrier [32], and at which circulating angiotensin II (ANG II) directly produces the activation of neuronal circuits that elicit increases in drinking [8,15,16,26,30,41,46,57], arterial pressure [15,16,26,30,31,41] and vasopressin secretion [18,24]. Neuroanatomical tracing studies have revealed that the SFO innervated by angiotensinergic nerve terminals derived from the lateral hypothalamic area (LHA) and zona incerta [27,28], sites thought to be closely involved in the dipsogenic response [13,37,38]. Previous electro* Corresponding author. Tel./fax: +81-88-6876243. E-mail address: [email protected] (J. Tanaka).
physiological observations have shown that the LHA neurons with ascending projections to the SFO are highly sensitive to ANG II [54], and that electrical [21,55,59] or chemical (ANG II) [60] stimulation of the LHA activates SFO neurons through ANG II receptors. The angiotensinergic neural circuits from the LHA to the SFO have been shown to be implicated in the control of drinking behavior [51]. It has been demonstrated that SFO neurons contain both ANG II AT1 receptors [3,33,42,47,64,67] and estrogen receptors [22,40,43,45,65]. Previous studies have shown that estrogen reduces ANG II binding to AT1 receptors [22] and the number of SFO neurons that express AT1 receptors [43]. We have recently observed that the level of circulating estrogen alters the spontaneous firing rate of SFO neurons [48,49] and their responsiveness to either ANG II applied ion-
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tophoretically [48,49] or systemically [48] and the angiotensinergic neural inputs from the LHA [49]. The objectives of current study were to clarify whether estrogen modulates the drinking response induced by activation of the angiotensinergic efferent pathways from the LHA to the SFO. We compared the amount of water intake caused by ANG II injected directly into the LHA or the SFO between ovariectomized (OVX) female rats that were treated with propylene glycol (PG) vehicle and OVX female rats that were treated with estrogen benzoate (EB). We also investigated the effects of pretreatment with saralasin (Sar) in the SFO on the drinking responses caused by the ANG II injection into the LHA to determine the responses were mediated by the angiotensinergic pathways. 2. Materials and methods
2.1. Animals Female Wistar rats (n =63) weighing 240 – 330 g were used for the experiments. These animals were housed under controlled conditions in a room at 23 – 25°C with a 12 h light/dark cycle (light at 7:00 – 19:00). Water and food were available ad libitum to all the animals.
2.2. Surgery The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and bilaterally OVX. The rats were then placed in a stereotaxic frame. The dorsal surface of the skull was exposed by midline incision. Twenty-six-gauge stainless steel cannulae were stereotaxically lowered into the LHA and SFO. The cannulas were embedded in dental acrylic anchored by small jeweler’s screws fixed in the skull. Procaine penicillin (100 000 IU, i.m.) was injected after surgery. The 26-gauge cannula served as a guide for a 33-gauge stainless steel injector cannula, which was inserted just before injections. The tips of injectors and guide cannulas were flush during insertion. Each guide cannula was filled with an obturator of the same gauge as the injector cannula when the animals were not being tested. The animals were given 1 week to regain body weight to pre-surgical levels and to reestablish a normal pattern of 24 h food and water intake before treatment was begun.
2.3. Steroid treatment The OVX animals were divided into two groups and received either a subcutaneous vehicle injection of PG (100 ml; Sigma; n= 31) or a subcutaneous injection of EB (10 mg dissolved in PG; Sigma; n =32) on seven consecutive days.
2.4. Peptide ANG II (Asp1-Ile5-ANG II) and its specific antagonist, Sar (Sar1-Val5-ANG II) were obtained from SIGMA and Peptide Institute, respectively. These peptides were dissolved in isotonic saline and frozen in aliquots. Aliquots were thawed immediately before being used.
2.5. Intracranial injections The third day after the start of PG- or EB-treatment, each rat was removed from its home cage, the obturator was removed. The injectors, filled with injectate and connected to two remote 0.5 ml gas chromatography syringes (Hamilton) via 1.0 m of polyethylene tubing, were inserted into the implanted guide cannulas. The injectate within the tip of the injector was separated from the tip of the cannula, and therefore from the rat, by a 0.02 ml air bubble. The injector tip terminated flush with the intracranial tip of the guide cannula. The rat was then placed in the metabolism cage. Immediately following placement of the rat in the cage, ANG II or saline vehicle was injected into the LHA. The latency to the onset of drinking was recorded, and water intake was then monitored for 15 min following the injection. Each rat was given only one intracranial injection per day. Two days after the ANG II injection, the effect of pretreatment with Sar or saline in the region of the SFO on the drinking response to the ANG II injection was tested. The antagonist or vehicle was injected 30 s before the ANG II injection. Two days after the Sar or saline treatment, the effects of microinjection of ANG II or saline into the SFO were examined. In the previous study, we have demonstrated that microinjection of ANG II into the LHA in a dose of 10 − 10 M elicits a robust drinking response and that the response is attenuated by the Sar treatment with a dose of 10 − 10 M in the SFO [51]. In this study, injections of ANG II and Sar were thus administered in a dose of 10 − 10 M. Because it is crucial to minimize diffusion of injectate in neuroanatomical localization experiments, all injections of the drug solutions or vehicle were given in a volume of 0.2 ml. The injections were achieved at a rate of 0.02 ml/s.
2.6. Histology At the completion of each experiment, injections of isotonic saline (0.2 ml) containing 2% Pontamine sky blue dye were made in order to confirm more precisely the location of cannula tips and to measure the spread of the injected solution. Each animal was sacrificed with an overdose of sodium pentobarbital and perfused through the heart with isotonic saline to clear blood, which was followed by 10% formalin saline for fixation.
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Brains were removed and stored in formalin saline before being cut on a freezing microtome at 50 mm in transverse sections. Sections were mounted on glass slides and stained with neutral red. The tip positions of cannulas were determined by examination of the slides with a light microscope. The stereotaxic coordinates for marking sites were determined according to the atlas of Paxinos and Watson [39].
2.7. Statistics All values are reported as the mean and standard deviation of the mean. Drinking response data were analyzed with a two-way analysis of variance. A probability of less than 0.05 was required for significance.
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3. Results
3.1. Drinking induced by angiotensin II injected into the lateral hypothalamic area Histological analysis of the brains of the rats showed that the cannula tips of 5 (2 responsive and 3 unresponsive to ANG II) PG-treated and 4 (2 responsive and 2 unresponsive to ANG II) EB-treated rats were located in the surrounding region of the LHA (Fig. 1(A)). The sites approximately 0.2 –0.4 mm away from the center of the cannula tip were stained with Pontamine sky blue dye. The data from these nine animals were not included in the further analysis.
Fig. 1. The location of the cannula tips. (A) Open and closed symbols on schematic transverse section depict the loci of the cannula tips in the PG- and EB-treated OVX female rats, respectively. (A) Circles and triangles indicate the loci of the cannula in the LHA and its surrounding region where microinjected ANG II elicited drinking and was without effect, respectively. Squares indicate the tip site of cannula that was used for injection of saline vehicle. (B) Circles and triangles indicate the tip sites of cannulas that were utilized for microinjection of Sar or ANG II and for microinjection of saline vehicle, respectively. Squares indicate cannula tips in the surrounding region where microinjected ANG II was without effect. The location of cannula tips in the third ventricle was not shown. AH – anterior hypothalamic area, DM – dorsomedial hypothalamic nucleus, LH – lateral hypothalamic area, PT – paratenial thalamic nucleus, SFO – subfornical organ, SM – stria medullaris of the thalamus, TS – triangular septal nucleus, VHC – ventral hippocampal commissure, VMH – ventromedial hypothalamic nucleus, ZI – zona incerta, and 3V – third ventricle.
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responded to ANG II (Fig. 1(A)). Although the amount of water intake was significantly greater in the PG-treated than in the EB-treated animals (F(1,45)= 12.520, PB 0.05), when the data from all the animals were analyzed, the data from the 12 animals that did not demonstrate the response based on the criterion mentioned above were excluded from the further analysis. Microinjections of saline vehicle into the LHA did not cause a marked water intake in both groups (3 PG-treated and 4 EB-treated animals; water volumes were less than 0.3 ml; Fig. 2).
3.2. Attenuated drinking by the saralasin treatment in the subfornical organ
Fig. 2. Total water intake (in 15 min) in response to microinjection of ANG II (10 − 10 M) or vehicle (Saline) into the LHA in OVX female rats that were treated with either PG (OVX + PG, open histogram bars) or EB (OVX+EB, closed histogram bars). Total water intake was significantly lower in the EB-treated than in the PG-treated animals. Each histogram bar and its vertical bar in this and subsequent figures represent the mean and standard deviation of the mean. (*) PB 0.01 compared with those of ANG II in OVX + PG.
The rats having the cannula tips in the LHA received the intracranial injection of either ANG II or saline. In our previous investigations, we have found that microinjection of ANG II into the LHA in the same dose as this study causes a robust drinking response (water volume, more than 3.0 ml for 15 min after the injection) and similar injections of saline vehicle often produce a weak response (water volume, less than 0.5 ml) [51]. To assess more precisely the drinking response induced by ANG II injected into the LHA and the effects of inactivation of the SFO on the response, a minimum of 1.0 ml water intake was classified as an efficacy of ANG II. Seventeen out of 23 PG-treated animals tested showed drinking in response to the ANG II injection and six were without effect (water volume, 0.590.3 ml, ranging 0.3 –0.7 ml). Of the 24 EB-treated animals having the cannula tips in the LHA, 18 exhibited drinking in response to the ANG II injection, while the remaining six animals were unresponsive (water volume, 0.39 0.2 ml, ranging 0.1 – 0.5 ml). The amount of water intake was significantly greater in the PG-treated (water volume, 3.790.6 ml, ranging 1.8 – 5.2 ml) than in the EB-treated (water volume, 2.390.4 ml, ranging 1.4 – 5.0 ml) animals (F(1,33) =31.995, P B0.01; Fig. 2). There was no significant difference in the latency of drinking response between the PG-treated (32911 s, ranging 3–127 s) and EB-treated (379 12 s, ranging 5 – 105 s) animals. No apparent topographical differences were found between the PG- and EB-treated animals in the positions of cannula tips of animals
To determine if the drinking response resulting from the ANG II injection into the LHA was mediated by the angiotensinergic LHA projections to the SFO, the effects of inactivation of ANG II-sensitive SFO neurons following previous injections of the Sar on the response were investigated in the animals which demonstrated the response to ANG II. Histological observations from serial sections showed that the14 PG-treated and 14 EB-treated animals which displayed the ANG II-induced drinking had the cannula placement either just above or within the SFO, and the remaining seven animals had cannula greater than 0.2 mm away from the main body of the SFO (Fig. 1(B); n= 4) or the third ventricle (n= 3). Since we have previously observed that the pretreatment with Sar in the ventral hippocampal commissure or the third ventricle has no significant influence on the water intake induced by injections of ANG II into the LHA [51], the data from seven animals were not included in the analysis. In fact, the drinking response induced by the ANG II injections into the LHA was not affected by previous injections of Sar into the surrounding sites of the SFO (mean water volume; the PG-treated animals, 3.6 ml in the pretreatment vs. 3.5 ml in the Sar treatment, n= 2; the 2 EB-treated animals, 2.0 ml in the pretreatment vs. 2.2 ml in the Sar treatment, n= 2). The animals having good cannulae (n=28) received either pretreatment with Sar or saline vehicle. In both groups (10 PG-treated and 10 EB-treated animals), previous injections of Sar significantly attenuated the water intake in response to ANG II injected into the LHA (F(1,9)=49.005, PB0.001 for the PGtreated animals; F(1,9)= 52.946, PB 0.001 for the EBtreated animals; Fig. 3). No complete blockage of the drinking response by the pretreatment was found in any animals tested. There was no significant difference between the PG-treated (529 15% decrease compared with the pretreatment) and EB-treated (489 17% decrease compared with the pretreatment) animals in the amount of the attenuation of total water intake by the Sar treatment. Previous injections of saline vehicle, on
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Fig. 3. Effects of pretreatment with Sar (10 − 10 M) or vehicle (Saline) in the SFO on water intake induced by ANG II (10 − 10 M) injection into the LHA in the PG-treated (OVX +PG, open histogram bars) and EB-treated (OVX + EB, closed histogram bars) animals. In both groups of female rats, the total water intake to ANG II injected into the LHA was significantly diminished by previous injection of Sar, but not by saline, in the SFO. (*) P B0.001 compared with those of ANG II.
the other hand, had no significant influence on the drinking response in both groups (4 PG-treated and 4 EB-treated animals; Fig. 3). Pretreatment with Sar in the SFO did not cause significant differences in the latencies to drinking in both groups (the PG-treated animals, 34913 s in the pretreatment vs. 389 15 s in the Sar treatment; the EB-treated animals, 389 16 s in the pretreatment vs. 429 17 s in the Sar treatment).
4. Discussion The present data indicate that, in both groups of female rats, the drinking response induced by the injection of ANG II into the LHA is significantly reduced by the previous injection of Sar into the SFO, and are consistent with the previous behavioral observations in
3.3. Drinking induced by angiotensin II injected into the subfornical organ The animals having good cannula placement in the SFO region received either an injection of ANG II or saline into the SFO. ANG II injected directly into the SFO elicited drinking in all the PG-treated (n = 10) and EB-treated (n=10) animals tested, which demonstrated the attenuation of the ANG II-induced response following the Sar treatment in the SFO (Fig. 4). The amount of water intake was significantly greater in the PG-treated (water volume 5.091.0 ml, ranging 2.6 –7.9 ml) than in the EB-treated (water volume 3.49 0.6 ml, ranging 2.1 –6.3 ml) animals (F(1,18) = 27.872, PB 0.01; Fig. 4). There was no significant difference in the latency of drinking response between the PG-treated (279 14 s, ranging 4 – 87 s) and EB-treated (33915 s, ranging 3–63 s) animals. Saline vehicle injected into the SFO did not cause a marked water ingestion in both groups (3 PG-treated and 4 EB-treated animals; water volume were less than 0.3 ml; Fig. 4).
Fig. 4. Total water intake (in 15 min) in response to microinjection of ANG II (10 − 10 M) or vehicle (Saline) into the SFO in the PG-treated (OVX+ PG, open histogram bars) and EB-treated (OVX + EB, closed histogram bars) animals that demonstrated the drinking response to ANG II injected into the LHA. Total water intake was significantly lower in the EB-treated than in the PG-treated animals. (*) PB 0.01 compared with those of ANG II in OVX + PG.
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male rats [51]. The histological analysis revealed roughly that the cannula tips in the animals which demonstrated the ANG II-induced drinking are positioned in the LHA sites where are innervated with ANG II-immunoreactive nerve terminals [27,28] and ANG II-sensitive neurons projecting to the SFO exist [54]. The Pontamine sky blue injected through the cannulae spread over only a limited range. In addition, we have previously confirmed that pretreatment with Sar in same dose as the present study in the third ventricle or ventral hippocampal commissure does not cause a marked change in the water intake induced by injections of ANG II into the LHA [51]. Therefore, it is suggested that the drinking response observed in this study might be mediated, at least in part, by the angiotensinergic neural pathways to the SFO, which are activated by ANG II. Since the Sar injection into the SFO is not able to abolish completely the ANG II-induced drinking response and to elicit any significant changes in the response latency, it is possible that other neural circuits that are activated by ANG II may be also involved in the drinking response. The present study provides the first demonstration that estrogen attenuates the drinking response induced by activation of both LHA and SFO neurons sensitive to ANG II. It has been known that estrogen attenuates the drinking induced by systemic or intraventricular injections of ANG II. Intracerebroventricular administrations of estrogen have been shown to diminish the increased drinking and the increase in arterial pressure induced by ANG II [19,20]. In addition, ANG II-induced drinking has been shown to be attenuated by estrogen treatment [9 – 12,23]. The present data strongly suggest that the angiotensinergic systems in both the LHA and the SFO may be involved in the attenuation in ingestive behaviors observed in the previous studies and in female rats during estrus cycle [9,63]. The SFO is a logical structure for circulating ANG II to elicit drinking behavior [8,15,16,26,30,41,46,57]. Thus, it is possible to speculate that estrogen acting at the SFO may attenuate the drinking response caused by systemic injections of ANG II. On the other hand, it might be expected that the drinking response induced by intracerebral administrations of ANG II might be diminished through the actions of estrogen on both LHA and SFO neurons sensitive to ANG II. The findings in which the amount of water intake induced by the ANG II injection into the LHA, that is reduced by previous injections of Sar into the SFO, is significantly lower in the EB-treated than in the PGtreated OVX animals offer the proposition that the activity of angiotensinergic LHA efferents to the SFO may be modified by the circulating level of estrogen. In addition, that the amount of water intake caused by the injection of ANG II into the SFO is significantly lower in the EB-treated than in the PG-treated animals sug-
gests the possibility that the estrogen-induced suppressive action of drinking responses may occur at the terminal sites of the angiotensinergic pathways from the LHA. This speculation may be supported by recent electrophysiological observations showing that the responsiveness of SFO neurons to either LHA stimulation or ANG II applied iontophoretically or systemically is much greater in the PG-treated than in EB-treated OVX female rats [48,49]. Thus, it is possible that the estrogen-induced decreased sensitivity of SFO neurons to ANG II released from the nerve terminals arising from the LHA neurons may attribute to the attenuation of water ingestion in the EB-treated animals. It remains, on the other hand, the possibility that estrogen acts at the LHA to alter the responsiveness of SFO projecting angiotensinergic neurons to ANG II, thereby causing reduced water intake. There are some possible mechanisms to explain the decreased sensitivity of SFO and/or LHA neurons to ANG II caused by estrogen. One explanation is that circulating estrogen may cause changes in the membrane conductance of these neurons. It has been reported that estrogen alters the sensitivity of neurons to neurotransmitters [1,66] by modifying membrane ionic conductance [14,25,34,35,44]. A related possibility is that estrogen may regulate the activity and/or number of ANG II AT1 receptors in these neurons. Indeed, estrogen decreased ANG II binding to AT1 receptors in the SFO [22]. In addition, it has been demonstrated that AT1 and estrogen receptors are localized to the same neurons in the SFO and increases in the circulating level of estrogen reduce the expression of both the AT1 and estrogen receptors in these SFO neurons [43]. Thus, it is suggested that estrogen may alter the sensitivity to ANG II (i.e. the angiotensinergic LHA inputs) by down regulating the number of AT1 receptors. A third possibility is that estrogen may modulate the synthesis of ANG II in SFO projecting angiotensinergic LHA neurons. It has been shown that estrogen alters the synthesis of neurotransmitters in the hypothalamus [2]. Fourth possibility is that synaptic morphological changes upon SFO neurons caused by estrogen may elicit the suppressed sensitivity to ANG II. In fact, it has been reported that estrogen causes changes in synaptic morphology [5]. Further studies are necessary to clarify the precise mechanism of decreased sensitivity of neurons to ANG II. Experimental observations in several lines have indicated that neural interactions between the SFO and the anteroventral third ventricle (AV3V) region as well as the hypothalamic paraventricular nucleus (PVN) are important for mediating a dipsogenic response [4,6,8,15,16,26 –28,36,50,52,54,55,57,58,62]. It has been shown that lesions of either the AV3V region [16] or the PVN [15] reduce the robust drinking induced by injections of ANG II into the SFO. Electrophysiological
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investigations have demonstrated that SFO neurons projecting directly to the AV3V region [56,58,62] or the PVN [52,53,55,60] are highly sensitive to ANG II, and electrical or chemical (ANG II) stimulation of the LHA produces an increased neuronal firing of these SFO neurons through ANG II receptors [55,59,60]. These findings and our data lead to the proposition that the angiotensinergic LHA efferent projections to the SFO may activate SFO neurons projecting to the AV3V region or the PVN to cause drinking, and imply that circulating estrogen may decrease the drinking response caused by ANG II through altering the sensitivity of these neural circuits to ANG II. The LHA has been shown to be implicated in the neural regulation of fluid intake in response to a peripheral osmotic challenge [13,37,38]. The LHA receives direct efferent projections from the medial preoptic area and AV3V region [4,6], two sites where osmosensitive neurons exist [7,17,29]. Osmotic stimulation of the two sites following injections of hypertonic saline alters the activity of LHA neurons [37,38]. In addition, the excitability of LHA neurons projecting directly to the SFO is modified by changes in plasma osmolality [61]. These findings raise the hypothesis that the angiotensinergic circuits from the LHA to the SFO may serve an integrative role in the modulation of drinking in response to changes in the effective body fluid osmolality. Thus, the present data imply that estrogen may be involved in the control of water intake in response to changes in plasma osmolality through its modulatory action on the angiotensinergic LHA efferents to the SFO. In conclusion, the present data provide the first demonstration that estrogen modulates the dipsogenic response induced by the angiotensinergic neural inputs from the LHA to the SFO. Acknowledgements The authors wish to thank Dr. Koji Hori for his advice and encouragement, and gratefully acknowledge the excellent technical assistance of Mr. Takefumi Okumura. References [1] Arnauld B, Dufy B, Pestre M, Vincent JD. Effects of estrogens on the responses of caudate neurons to microiontophoretically applied dopamine. Neurosci Lett 1981;21:325–31. [2] Axelson JF, Shannon W, van Leeuwen FW. Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus. Neurosci Lett 1992;136:5– 9. [3] Bunnemann B, Iwai N, Metzger R, Fuxe K, Inagami T, Ganten D. The distribution of angiotensin II AT1-receptors subtype mRNA in the rat brain. Neurosci Lett 1992;142:155–8.
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[48] Tanaka J, Hayashi Y, Miyakubo H, Sakamaki K, Okumura T, Nomura S. Estrogen decreases the responsiveness of subfornical organ neurons projecting to the hypothalamic paraventricular nucleus to angiotensin II in female rats. Neurosci Lett, in press. [49] Tanaka J, Miyakubo H, Nomura M. Estrogen decreases the responsiveness of subfornical organ neurons to angiotensinergic neural inputs from the lateral hypothalamic area in the female rat. Exp Neurol submitted. [50] Tanaka J, Hayashi Y, Nomura S, Miyakubo H, Okumura T, Sakamaki K. Angiotensinergic and noradrenergic mechanisms in the hypothalamic paraventricular nucleus participate in the drinking response induced by activation of the subfornical organ in rats. Behav Brain Res 2001;118:117– 22. [51] Tanaka J, Hori K, Nomura M. Dipsogenic response induced by angiotensinergic pathways from the lateral hypothalamic area to the subfornical organ in rats. Behav Brain Res 2001;118:111–6. [52] Tanaka J, Kaba H, Saito H, Seto K. Electrophysiological evidence that circulating angiotensin II-sensitive neurons in the subfornical organ alter the activity of hypothalamic paraventricular neurohypophyseal neurons in the rat. Brain Res 1985;342:361– 5. [53] Tanaka J, Kaba H, Saito H, Seto K. Subfornical organ neurons with efferent projections to the hypothalamic paraventricular nucleus: an electrophysiological study in the rat. Brain Res 1985;346:151– 4. [54] Tanaka J, Kaba H, Saito H, Seto K. Angiotensin II-sensitive neurons in the rat lateral hypothalamic area with efferent projections to the subfornical organ. Exp Neurol 1986;94:791–5. [55] Tanaka J, Kaba H, Saito H, Seto K. Lateral hypothalamic area stimulation excites neurons in the region of the subfornical organ with efferent projections to the hypothalamic paraventricular nucleus in the rat. Brain Res 1986;379:200– 3. [56] Tanaka J, Nojima K, Yamamuro Y, Saito H, Nomura M. Responses of subfornical organ neurons projecting to the hypothalamic paraventricular nucleus to hemorrhage. Brain Res 1993;608:141– 4. [57] Tanaka J, Nomura M. Involvement of neurons sensitive to angiotensin II in the median preoptic nucleus in the drinking response induced by angiotensin II activation of the subfornical organ. Exp Neurol 1993;119:235– 9. [58] Tanaka J, Saito H, Kaba H. Subfornical organ and hypothalamic paraventricular nucleus connections with median preoptic nucleus neurons: an electrophysiological study in the rat. Exp Brain Res 1987;68:579– 85. [59] Tanaka J, Saito H, Kaba H, Nojima K, Seto K. Lateral hypothalamic region excites the activity of vasopressin neurons in the supraoptic nucleus through subfornical organ. Exp Neurol 1987;97:212– 8. [60] Tanaka J, Seto K. Lateral hypothalamic area and paraventricular nucleus connections with subfornical organ neurons: an electrophysiological study in the rat. Neurosci Res 1988;6:45–52. [61] Tanaka J, Seto K. Neurons in the lateral hypothalamic area and zona incerta with ascending projections to the subfornical organ area in the rat. Brain Res 1988;456:397– 400. [62] Tanaka J, Yamamuro Y, Saito H, Nomura M. Differences in electrophysiological properties of angiotensinergic pathways from the subfornical organ to the median preoptic nucleus between normotensive Wistar-Kyoto and spontaneously hypertensive rats. Exp Neurol 1995;134:192– 8. [63] Tarttelin MF, Gorski RA. Variations in food and water intake in the normal and acyclic female rat. Physiol Behav 1971;7:847– 52. [64] Tsutsumi K, Saavedra JM. Characterization and development of angiotensin II receptor subtype (AT1 and AT2) in rat brain. Am J Physiol 1991;261:R209– 16. [65] Voisin DL, Simonian SX, Herbison AE. Identification of estrogen receptor-containing neurons projecting to the rat supraoptic nucleus. Neurosci 1997;7:215– 28.
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Research report
Estrogen attenuates the drinking response induced by activation of angiotensinergic pathways from the lateral hypothalamic area to the subfornical organ in female rats Shigeko Fujisawa a, Junichi Tanaka a,b,*, Masahiko Nomura a b
a Department of Physiology, Saitama Medical School, Iruma-gun, Saitama 350 -0495, Japan Department of Human De6elopment, Naruto Uni6ersity of Education, Naruto-cho, Naruto, Tokushima 772 -8502, Japan
Received 26 September 2000; received in revised form 2 January 2001; accepted 2 January 2001
Abstract The present study was carried out to investigate whether estrogen modulates the drinking response induced by activation of angiotensinergic neural pathways from the lateral hypothalamic area (LHA) to the subfornical organ (SFO) in the female rats. Microinjection of ANG II (10 − 10 M, 0.2 ml) into the LHA caused drinking in 17 out of 26 ovariectomized (OVX) female rats that were treated with propylene glycol (PG) vehicle and in 18 out of 28 OVX female rats that were treated with estrogen benzoate (EB). In both groups, previous injections of the ANG II antagonist saralasin (Sar, 10 − 10 M, 0.2 ml) into the SFO significantly attenuated the water intake caused by the ANG II injection, suggesting that the ANG II-induced drinking response may be mediated by the angiotensinergic LHA projections to the SFO. Injections of ANG II (10 − 10 M, 0.2 ml) into the SFO elicited drinking in all the animals that demonstrated the drinking response to ANG II injected into the LHA. The amount of water intake caused by either the injection of ANG II into the LHA or the SFO was significantly greater in the PG-treated than in the EB-treated animals. These results suggest that the circulating estrogen may act to attenuate the dipsogenic response induced by activation of the angiotensinergic pathways from the LHA to the SFO. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Drinking; Lateral hypothalamic area; Subfornical organ; Estrogen; Angiotensin II; Saralasin; Rat
1. Introduction It is well known that the subfornical organ (SFO) is a circumventricular organ that lacks a normal blood – brain barrier [32], and at which circulating angiotensin II (ANG II) directly produces the activation of neuronal circuits that elicit increases in drinking [8,15,16,26,30,41,46,57], arterial pressure [15,16,26,30,31,41] and vasopressin secretion [18,24]. Neuroanatomical tracing studies have revealed that the SFO innervated by angiotensinergic nerve terminals derived from the lateral hypothalamic area (LHA) and zona incerta [27,28], sites thought to be closely involved in the dipsogenic response [13,37,38]. Previous electro* Corresponding author. Tel./fax: +81-88-6876243. E-mail address: [email protected] (J. Tanaka).
physiological observations have shown that the LHA neurons with ascending projections to the SFO are highly sensitive to ANG II [54], and that electrical [21,55,59] or chemical (ANG II) [60] stimulation of the LHA activates SFO neurons through ANG II receptors. The angiotensinergic neural circuits from the LHA to the SFO have been shown to be implicated in the control of drinking behavior [51]. It has been demonstrated that SFO neurons contain both ANG II AT1 receptors [3,33,42,47,64,67] and estrogen receptors [22,40,43,45,65]. Previous studies have shown that estrogen reduces ANG II binding to AT1 receptors [22] and the number of SFO neurons that express AT1 receptors [43]. We have recently observed that the level of circulating estrogen alters the spontaneous firing rate of SFO neurons [48,49] and their responsiveness to either ANG II applied ion-
0166-4328/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 1 7 6 - 0
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tophoretically [48,49] or systemically [48] and the angiotensinergic neural inputs from the LHA [49]. The objectives of current study were to clarify whether estrogen modulates the drinking response induced by activation of the angiotensinergic efferent pathways from the LHA to the SFO. We compared the amount of water intake caused by ANG II injected directly into the LHA or the SFO between ovariectomized (OVX) female rats that were treated with propylene glycol (PG) vehicle and OVX female rats that were treated with estrogen benzoate (EB). We also investigated the effects of pretreatment with saralasin (Sar) in the SFO on the drinking responses caused by the ANG II injection into the LHA to determine the responses were mediated by the angiotensinergic pathways. 2. Materials and methods
2.1. Animals Female Wistar rats (n =63) weighing 240 – 330 g were used for the experiments. These animals were housed under controlled conditions in a room at 23 – 25°C with a 12 h light/dark cycle (light at 7:00 – 19:00). Water and food were available ad libitum to all the animals.
2.2. Surgery The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and bilaterally OVX. The rats were then placed in a stereotaxic frame. The dorsal surface of the skull was exposed by midline incision. Twenty-six-gauge stainless steel cannulae were stereotaxically lowered into the LHA and SFO. The cannulas were embedded in dental acrylic anchored by small jeweler’s screws fixed in the skull. Procaine penicillin (100 000 IU, i.m.) was injected after surgery. The 26-gauge cannula served as a guide for a 33-gauge stainless steel injector cannula, which was inserted just before injections. The tips of injectors and guide cannulas were flush during insertion. Each guide cannula was filled with an obturator of the same gauge as the injector cannula when the animals were not being tested. The animals were given 1 week to regain body weight to pre-surgical levels and to reestablish a normal pattern of 24 h food and water intake before treatment was begun.
2.3. Steroid treatment The OVX animals were divided into two groups and received either a subcutaneous vehicle injection of PG (100 ml; Sigma; n= 31) or a subcutaneous injection of EB (10 mg dissolved in PG; Sigma; n =32) on seven consecutive days.
2.4. Peptide ANG II (Asp1-Ile5-ANG II) and its specific antagonist, Sar (Sar1-Val5-ANG II) were obtained from SIGMA and Peptide Institute, respectively. These peptides were dissolved in isotonic saline and frozen in aliquots. Aliquots were thawed immediately before being used.
2.5. Intracranial injections The third day after the start of PG- or EB-treatment, each rat was removed from its home cage, the obturator was removed. The injectors, filled with injectate and connected to two remote 0.5 ml gas chromatography syringes (Hamilton) via 1.0 m of polyethylene tubing, were inserted into the implanted guide cannulas. The injectate within the tip of the injector was separated from the tip of the cannula, and therefore from the rat, by a 0.02 ml air bubble. The injector tip terminated flush with the intracranial tip of the guide cannula. The rat was then placed in the metabolism cage. Immediately following placement of the rat in the cage, ANG II or saline vehicle was injected into the LHA. The latency to the onset of drinking was recorded, and water intake was then monitored for 15 min following the injection. Each rat was given only one intracranial injection per day. Two days after the ANG II injection, the effect of pretreatment with Sar or saline in the region of the SFO on the drinking response to the ANG II injection was tested. The antagonist or vehicle was injected 30 s before the ANG II injection. Two days after the Sar or saline treatment, the effects of microinjection of ANG II or saline into the SFO were examined. In the previous study, we have demonstrated that microinjection of ANG II into the LHA in a dose of 10 − 10 M elicits a robust drinking response and that the response is attenuated by the Sar treatment with a dose of 10 − 10 M in the SFO [51]. In this study, injections of ANG II and Sar were thus administered in a dose of 10 − 10 M. Because it is crucial to minimize diffusion of injectate in neuroanatomical localization experiments, all injections of the drug solutions or vehicle were given in a volume of 0.2 ml. The injections were achieved at a rate of 0.02 ml/s.
2.6. Histology At the completion of each experiment, injections of isotonic saline (0.2 ml) containing 2% Pontamine sky blue dye were made in order to confirm more precisely the location of cannula tips and to measure the spread of the injected solution. Each animal was sacrificed with an overdose of sodium pentobarbital and perfused through the heart with isotonic saline to clear blood, which was followed by 10% formalin saline for fixation.
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Brains were removed and stored in formalin saline before being cut on a freezing microtome at 50 mm in transverse sections. Sections were mounted on glass slides and stained with neutral red. The tip positions of cannulas were determined by examination of the slides with a light microscope. The stereotaxic coordinates for marking sites were determined according to the atlas of Paxinos and Watson [39].
2.7. Statistics All values are reported as the mean and standard deviation of the mean. Drinking response data were analyzed with a two-way analysis of variance. A probability of less than 0.05 was required for significance.
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3. Results
3.1. Drinking induced by angiotensin II injected into the lateral hypothalamic area Histological analysis of the brains of the rats showed that the cannula tips of 5 (2 responsive and 3 unresponsive to ANG II) PG-treated and 4 (2 responsive and 2 unresponsive to ANG II) EB-treated rats were located in the surrounding region of the LHA (Fig. 1(A)). The sites approximately 0.2 –0.4 mm away from the center of the cannula tip were stained with Pontamine sky blue dye. The data from these nine animals were not included in the further analysis.
Fig. 1. The location of the cannula tips. (A) Open and closed symbols on schematic transverse section depict the loci of the cannula tips in the PG- and EB-treated OVX female rats, respectively. (A) Circles and triangles indicate the loci of the cannula in the LHA and its surrounding region where microinjected ANG II elicited drinking and was without effect, respectively. Squares indicate the tip site of cannula that was used for injection of saline vehicle. (B) Circles and triangles indicate the tip sites of cannulas that were utilized for microinjection of Sar or ANG II and for microinjection of saline vehicle, respectively. Squares indicate cannula tips in the surrounding region where microinjected ANG II was without effect. The location of cannula tips in the third ventricle was not shown. AH – anterior hypothalamic area, DM – dorsomedial hypothalamic nucleus, LH – lateral hypothalamic area, PT – paratenial thalamic nucleus, SFO – subfornical organ, SM – stria medullaris of the thalamus, TS – triangular septal nucleus, VHC – ventral hippocampal commissure, VMH – ventromedial hypothalamic nucleus, ZI – zona incerta, and 3V – third ventricle.
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responded to ANG II (Fig. 1(A)). Although the amount of water intake was significantly greater in the PG-treated than in the EB-treated animals (F(1,45)= 12.520, PB 0.05), when the data from all the animals were analyzed, the data from the 12 animals that did not demonstrate the response based on the criterion mentioned above were excluded from the further analysis. Microinjections of saline vehicle into the LHA did not cause a marked water intake in both groups (3 PG-treated and 4 EB-treated animals; water volumes were less than 0.3 ml; Fig. 2).
3.2. Attenuated drinking by the saralasin treatment in the subfornical organ
Fig. 2. Total water intake (in 15 min) in response to microinjection of ANG II (10 − 10 M) or vehicle (Saline) into the LHA in OVX female rats that were treated with either PG (OVX + PG, open histogram bars) or EB (OVX+EB, closed histogram bars). Total water intake was significantly lower in the EB-treated than in the PG-treated animals. Each histogram bar and its vertical bar in this and subsequent figures represent the mean and standard deviation of the mean. (*) PB 0.01 compared with those of ANG II in OVX + PG.
The rats having the cannula tips in the LHA received the intracranial injection of either ANG II or saline. In our previous investigations, we have found that microinjection of ANG II into the LHA in the same dose as this study causes a robust drinking response (water volume, more than 3.0 ml for 15 min after the injection) and similar injections of saline vehicle often produce a weak response (water volume, less than 0.5 ml) [51]. To assess more precisely the drinking response induced by ANG II injected into the LHA and the effects of inactivation of the SFO on the response, a minimum of 1.0 ml water intake was classified as an efficacy of ANG II. Seventeen out of 23 PG-treated animals tested showed drinking in response to the ANG II injection and six were without effect (water volume, 0.590.3 ml, ranging 0.3 –0.7 ml). Of the 24 EB-treated animals having the cannula tips in the LHA, 18 exhibited drinking in response to the ANG II injection, while the remaining six animals were unresponsive (water volume, 0.39 0.2 ml, ranging 0.1 – 0.5 ml). The amount of water intake was significantly greater in the PG-treated (water volume, 3.790.6 ml, ranging 1.8 – 5.2 ml) than in the EB-treated (water volume, 2.390.4 ml, ranging 1.4 – 5.0 ml) animals (F(1,33) =31.995, P B0.01; Fig. 2). There was no significant difference in the latency of drinking response between the PG-treated (32911 s, ranging 3–127 s) and EB-treated (379 12 s, ranging 5 – 105 s) animals. No apparent topographical differences were found between the PG- and EB-treated animals in the positions of cannula tips of animals
To determine if the drinking response resulting from the ANG II injection into the LHA was mediated by the angiotensinergic LHA projections to the SFO, the effects of inactivation of ANG II-sensitive SFO neurons following previous injections of the Sar on the response were investigated in the animals which demonstrated the response to ANG II. Histological observations from serial sections showed that the14 PG-treated and 14 EB-treated animals which displayed the ANG II-induced drinking had the cannula placement either just above or within the SFO, and the remaining seven animals had cannula greater than 0.2 mm away from the main body of the SFO (Fig. 1(B); n= 4) or the third ventricle (n= 3). Since we have previously observed that the pretreatment with Sar in the ventral hippocampal commissure or the third ventricle has no significant influence on the water intake induced by injections of ANG II into the LHA [51], the data from seven animals were not included in the analysis. In fact, the drinking response induced by the ANG II injections into the LHA was not affected by previous injections of Sar into the surrounding sites of the SFO (mean water volume; the PG-treated animals, 3.6 ml in the pretreatment vs. 3.5 ml in the Sar treatment, n= 2; the 2 EB-treated animals, 2.0 ml in the pretreatment vs. 2.2 ml in the Sar treatment, n= 2). The animals having good cannulae (n=28) received either pretreatment with Sar or saline vehicle. In both groups (10 PG-treated and 10 EB-treated animals), previous injections of Sar significantly attenuated the water intake in response to ANG II injected into the LHA (F(1,9)=49.005, PB0.001 for the PGtreated animals; F(1,9)= 52.946, PB 0.001 for the EBtreated animals; Fig. 3). No complete blockage of the drinking response by the pretreatment was found in any animals tested. There was no significant difference between the PG-treated (529 15% decrease compared with the pretreatment) and EB-treated (489 17% decrease compared with the pretreatment) animals in the amount of the attenuation of total water intake by the Sar treatment. Previous injections of saline vehicle, on
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Fig. 3. Effects of pretreatment with Sar (10 − 10 M) or vehicle (Saline) in the SFO on water intake induced by ANG II (10 − 10 M) injection into the LHA in the PG-treated (OVX +PG, open histogram bars) and EB-treated (OVX + EB, closed histogram bars) animals. In both groups of female rats, the total water intake to ANG II injected into the LHA was significantly diminished by previous injection of Sar, but not by saline, in the SFO. (*) P B0.001 compared with those of ANG II.
the other hand, had no significant influence on the drinking response in both groups (4 PG-treated and 4 EB-treated animals; Fig. 3). Pretreatment with Sar in the SFO did not cause significant differences in the latencies to drinking in both groups (the PG-treated animals, 34913 s in the pretreatment vs. 389 15 s in the Sar treatment; the EB-treated animals, 389 16 s in the pretreatment vs. 429 17 s in the Sar treatment).
4. Discussion The present data indicate that, in both groups of female rats, the drinking response induced by the injection of ANG II into the LHA is significantly reduced by the previous injection of Sar into the SFO, and are consistent with the previous behavioral observations in
3.3. Drinking induced by angiotensin II injected into the subfornical organ The animals having good cannula placement in the SFO region received either an injection of ANG II or saline into the SFO. ANG II injected directly into the SFO elicited drinking in all the PG-treated (n = 10) and EB-treated (n=10) animals tested, which demonstrated the attenuation of the ANG II-induced response following the Sar treatment in the SFO (Fig. 4). The amount of water intake was significantly greater in the PG-treated (water volume 5.091.0 ml, ranging 2.6 –7.9 ml) than in the EB-treated (water volume 3.49 0.6 ml, ranging 2.1 –6.3 ml) animals (F(1,18) = 27.872, PB 0.01; Fig. 4). There was no significant difference in the latency of drinking response between the PG-treated (279 14 s, ranging 4 – 87 s) and EB-treated (33915 s, ranging 3–63 s) animals. Saline vehicle injected into the SFO did not cause a marked water ingestion in both groups (3 PG-treated and 4 EB-treated animals; water volume were less than 0.3 ml; Fig. 4).
Fig. 4. Total water intake (in 15 min) in response to microinjection of ANG II (10 − 10 M) or vehicle (Saline) into the SFO in the PG-treated (OVX+ PG, open histogram bars) and EB-treated (OVX + EB, closed histogram bars) animals that demonstrated the drinking response to ANG II injected into the LHA. Total water intake was significantly lower in the EB-treated than in the PG-treated animals. (*) PB 0.01 compared with those of ANG II in OVX + PG.
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male rats [51]. The histological analysis revealed roughly that the cannula tips in the animals which demonstrated the ANG II-induced drinking are positioned in the LHA sites where are innervated with ANG II-immunoreactive nerve terminals [27,28] and ANG II-sensitive neurons projecting to the SFO exist [54]. The Pontamine sky blue injected through the cannulae spread over only a limited range. In addition, we have previously confirmed that pretreatment with Sar in same dose as the present study in the third ventricle or ventral hippocampal commissure does not cause a marked change in the water intake induced by injections of ANG II into the LHA [51]. Therefore, it is suggested that the drinking response observed in this study might be mediated, at least in part, by the angiotensinergic neural pathways to the SFO, which are activated by ANG II. Since the Sar injection into the SFO is not able to abolish completely the ANG II-induced drinking response and to elicit any significant changes in the response latency, it is possible that other neural circuits that are activated by ANG II may be also involved in the drinking response. The present study provides the first demonstration that estrogen attenuates the drinking response induced by activation of both LHA and SFO neurons sensitive to ANG II. It has been known that estrogen attenuates the drinking induced by systemic or intraventricular injections of ANG II. Intracerebroventricular administrations of estrogen have been shown to diminish the increased drinking and the increase in arterial pressure induced by ANG II [19,20]. In addition, ANG II-induced drinking has been shown to be attenuated by estrogen treatment [9 – 12,23]. The present data strongly suggest that the angiotensinergic systems in both the LHA and the SFO may be involved in the attenuation in ingestive behaviors observed in the previous studies and in female rats during estrus cycle [9,63]. The SFO is a logical structure for circulating ANG II to elicit drinking behavior [8,15,16,26,30,41,46,57]. Thus, it is possible to speculate that estrogen acting at the SFO may attenuate the drinking response caused by systemic injections of ANG II. On the other hand, it might be expected that the drinking response induced by intracerebral administrations of ANG II might be diminished through the actions of estrogen on both LHA and SFO neurons sensitive to ANG II. The findings in which the amount of water intake induced by the ANG II injection into the LHA, that is reduced by previous injections of Sar into the SFO, is significantly lower in the EB-treated than in the PGtreated OVX animals offer the proposition that the activity of angiotensinergic LHA efferents to the SFO may be modified by the circulating level of estrogen. In addition, that the amount of water intake caused by the injection of ANG II into the SFO is significantly lower in the EB-treated than in the PG-treated animals sug-
gests the possibility that the estrogen-induced suppressive action of drinking responses may occur at the terminal sites of the angiotensinergic pathways from the LHA. This speculation may be supported by recent electrophysiological observations showing that the responsiveness of SFO neurons to either LHA stimulation or ANG II applied iontophoretically or systemically is much greater in the PG-treated than in EB-treated OVX female rats [48,49]. Thus, it is possible that the estrogen-induced decreased sensitivity of SFO neurons to ANG II released from the nerve terminals arising from the LHA neurons may attribute to the attenuation of water ingestion in the EB-treated animals. It remains, on the other hand, the possibility that estrogen acts at the LHA to alter the responsiveness of SFO projecting angiotensinergic neurons to ANG II, thereby causing reduced water intake. There are some possible mechanisms to explain the decreased sensitivity of SFO and/or LHA neurons to ANG II caused by estrogen. One explanation is that circulating estrogen may cause changes in the membrane conductance of these neurons. It has been reported that estrogen alters the sensitivity of neurons to neurotransmitters [1,66] by modifying membrane ionic conductance [14,25,34,35,44]. A related possibility is that estrogen may regulate the activity and/or number of ANG II AT1 receptors in these neurons. Indeed, estrogen decreased ANG II binding to AT1 receptors in the SFO [22]. In addition, it has been demonstrated that AT1 and estrogen receptors are localized to the same neurons in the SFO and increases in the circulating level of estrogen reduce the expression of both the AT1 and estrogen receptors in these SFO neurons [43]. Thus, it is suggested that estrogen may alter the sensitivity to ANG II (i.e. the angiotensinergic LHA inputs) by down regulating the number of AT1 receptors. A third possibility is that estrogen may modulate the synthesis of ANG II in SFO projecting angiotensinergic LHA neurons. It has been shown that estrogen alters the synthesis of neurotransmitters in the hypothalamus [2]. Fourth possibility is that synaptic morphological changes upon SFO neurons caused by estrogen may elicit the suppressed sensitivity to ANG II. In fact, it has been reported that estrogen causes changes in synaptic morphology [5]. Further studies are necessary to clarify the precise mechanism of decreased sensitivity of neurons to ANG II. Experimental observations in several lines have indicated that neural interactions between the SFO and the anteroventral third ventricle (AV3V) region as well as the hypothalamic paraventricular nucleus (PVN) are important for mediating a dipsogenic response [4,6,8,15,16,26 –28,36,50,52,54,55,57,58,62]. It has been shown that lesions of either the AV3V region [16] or the PVN [15] reduce the robust drinking induced by injections of ANG II into the SFO. Electrophysiological
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investigations have demonstrated that SFO neurons projecting directly to the AV3V region [56,58,62] or the PVN [52,53,55,60] are highly sensitive to ANG II, and electrical or chemical (ANG II) stimulation of the LHA produces an increased neuronal firing of these SFO neurons through ANG II receptors [55,59,60]. These findings and our data lead to the proposition that the angiotensinergic LHA efferent projections to the SFO may activate SFO neurons projecting to the AV3V region or the PVN to cause drinking, and imply that circulating estrogen may decrease the drinking response caused by ANG II through altering the sensitivity of these neural circuits to ANG II. The LHA has been shown to be implicated in the neural regulation of fluid intake in response to a peripheral osmotic challenge [13,37,38]. The LHA receives direct efferent projections from the medial preoptic area and AV3V region [4,6], two sites where osmosensitive neurons exist [7,17,29]. Osmotic stimulation of the two sites following injections of hypertonic saline alters the activity of LHA neurons [37,38]. In addition, the excitability of LHA neurons projecting directly to the SFO is modified by changes in plasma osmolality [61]. These findings raise the hypothesis that the angiotensinergic circuits from the LHA to the SFO may serve an integrative role in the modulation of drinking in response to changes in the effective body fluid osmolality. Thus, the present data imply that estrogen may be involved in the control of water intake in response to changes in plasma osmolality through its modulatory action on the angiotensinergic LHA efferents to the SFO. In conclusion, the present data provide the first demonstration that estrogen modulates the dipsogenic response induced by the angiotensinergic neural inputs from the LHA to the SFO. Acknowledgements The authors wish to thank Dr. Koji Hori for his advice and encouragement, and gratefully acknowledge the excellent technical assistance of Mr. Takefumi Okumura. References [1] Arnauld B, Dufy B, Pestre M, Vincent JD. Effects of estrogens on the responses of caudate neurons to microiontophoretically applied dopamine. Neurosci Lett 1981;21:325–31. [2] Axelson JF, Shannon W, van Leeuwen FW. Immunocytochemical localization of estrogen receptors within neurotensin cells in the rostral preoptic area of the rat hypothalamus. Neurosci Lett 1992;136:5– 9. [3] Bunnemann B, Iwai N, Metzger R, Fuxe K, Inagami T, Ganten D. The distribution of angiotensin II AT1-receptors subtype mRNA in the rat brain. Neurosci Lett 1992;142:155–8.
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