Am J Physiol Regul Integr Comp Physiol 307: R1396–R1404, 2014. First published October 29, 2014; doi:10.1152/ajpregu.00373.2014.

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Central Control of Fluid and Electrolyte Homeostasis

Properly timed exposure to central ANG II prevents behavioral sensitization and changes in angiotensin receptor expression Jessica Santollo,1 Philip E. Whalen,1 Robert C. Speth,2,3 Stewart D. Clark,4 and Derek Daniels1 1

Department of Psychology, University at Buffalo, State University of New York (SUNY), Buffalo, New York; 2Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, Florida; 3 Department of Pharmacology and Physiology, College of Medicine, Georgetown University, Washington, DC; and 4 Department of Pharmacology and Toxicology, University at Buffalo, SUNY, Buffalo, New York Submitted 2 September 2014; accepted in final form 28 October 2014

Santollo J, Whalen PE, Speth RC, Clark SD, Daniels D. Properly timed exposure to central ANG II prevents behavioral sensitization and changes in angiotensin receptor expression. Am J Physiol Regul Integr Comp Physiol 307: R1396 –R1404, 2014. First published October 29, 2014; doi:10.1152/ajpregu.00373.2014.—Previous studies show that the angiotensin type 1 receptor (AT1R) is susceptible to rapid desensitization, but that more chronic treatments that stimulate ANG II lead to sensitization of several responses. It is unclear, however, if the processes of desensitization and sensitization interact. To test for differences in AT1R expression associated with single or repeated injections of ANG II, we measured AT1R mRNA in nuclei that control fluid intake of rats given ANG II either in a single injection or divided into three injections spaced 20 min apart. Rats given a single injection of ANG II had more AT1R mRNA in the subfornical organ (SFO) and the periventricular tissue surrounding the anteroventral third ventricle (AV3V) than did controls. The effect was not observed, however, when the same cumulative dose of ANG II was divided into multiple injections. Behavioral tests found that single daily injections of ANG II sensitized the dipsogenic response to ANG II, but a daily regimen of four injections did not cause sensitization. Analysis of 125I-Sar1-ANG II binding revealed a paradoxical decrease in binding in the caudal AV3V and dorsal median preoptic nucleus after 5 days of single daily injections of ANG II; however, this effect was absent in rats treated for 5 days with four daily ANG II injections. Taken together, these data suggest that a desensitizing treatment regimen prevents behavior- and receptor-level effects of repeated daily ANG II. AT1R; water intake; drinking microstructure; fluid balance

requires constant coordination of numerous systems ranging from the smooth muscle cells of vessel walls to highly complex behavior that is under the control of the central nervous system. The behavioral component of this response is critical, especially in the response to fluid deficit, because without medical intervention, drinking is the only effective way for animals to replace lost fluids. The dipsogenic and natriorexigenic properties of ANG II are critical components of this behavior (4, 17, 18). Additionally, studies of drinking behavior have served as excellent models of ANG II action and have the potential to reveal further principles that apply to other actions of ANG II and,

REGULATION OF BODY FLUID HOMEOSTASIS

Address for reprint requests and other correspondence: D. Daniels, Dept. of Psychology, Univ. at Buffalo, SUNY, Buffalo, NY 14260 United States (e-mail: [email protected]). R1396

more generally, to other motivated behaviors and natural reward systems. A particularly interesting feature of the behavioral responses to ANG II is that they vary dramatically depending on the timing of administration. Previous in vivo studies from our laboratory and in vitro studies by others demonstrate that the angiotensin type 1 receptor (AT1R) is susceptible to rapid desensitization if there is repeated exposure to ANG II within a short time frame (7, 28, 29, 32–35). Behaviorally, this decreases the dipsogenic potency of ANG II treatment (21, 32–35). Studies using repeated or chronic exposure to ANG II over a longer timeframe, in contrast, reveal an opposite, sensitizing effect of the treatment (3, 19). For example, single daily injections of either 10 or 100 ng ANG II for 7 days increase the water intake (19). Little is known, however, about the mechanisms of both ANG II desensitization and sensitization or whether there are any interactions between these opposite effects of ANG II. A better understanding of these phenomena has the potential to reveal important aspects of behavioral sensitization and desensitization (tolerance) that could improve our understanding of the control of drinking behavior and could provide a more complete understanding of the principles involved in other systems that involve sensitization (e.g., hypertension, substance abuse, pain, allergies, or asthma). The present experiments were designed to explore mechanisms that underlie the behavioral changes that occur after repeated injections of ANG II (acute or more chronic) and to test for interactions between two paradigms that induce the changes. Because previous work from our laboratory has demonstrated that AT1R is necessary for behavioral desensitization after repeated ANG II treatment (34), we tested the hypothesis that the effective treatment causes changes in levels of AT1R mRNA in areas of the brain that control fluid intake. In a separate series of experiments, we then tested the hypothesis that a treatment regimen that causes desensitization affects the sensitization of fluid intake. Finally, we used receptor autoradiography to test the hypothesis that AT1R binding in nuclei that control fluid intake would be altered by the treatments used in the behavioral studies. METHODS

Animals and housing. Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 225–250 g upon arrival at the facility were used in all experiments. Rats were individually housed in

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SENSITIZATION AND DESENSITIZATION TO ANGIOTENSIN II

stainless-steel wire-hanging cages with access to tap water and rat chow (Teklad 2018; Harlan Laboratories) in a temperature- and humidity-controlled colony room on a 12:12-h light-dark cycle (lights on 0700). All behavioral experiments occurred during the early hours of the light phase. All experimental protocols were approved by the Animal Care and Use Committee at the University of Buffalo, and the handling and care of the animals were in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. Surgery. All animals underwent stereotaxic surgery to implant a chronic cannula aimed at the right lateral ventricle following standard laboratory procedures. Briefly, rats were anesthetized with an injection of a ketamine (70 mg/kg im; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (5 mg/kg im; Akron, Decatur, IL). The rat’s head was shaved and secured into a stereotaxic frame. A small incision was made on top of the skull, a small hole was drilled, and a 26-gauge guide cannula was implanted using the following coordinates: 0.9 mm posterior and 1.4 mm lateral to bregma, and 2.8 mm ventral to the skull. The cannula was fixed to the skull with bone screws and dental cement. All rats received a single injection of carprofen (5 mg/kg sc; Pfizer Animal Health, New York, NY) after surgery to minimize pain. One week later and five days prior to experiments, accurate cannula placement was verified by measuring the drinking response to an injection of 10 ng ANG II. Only rats that drank at least 6 ml in 30 min after ANG II treatment were included in the study. cDNA synthesis and RT-PCR. Real-time PCR was used to quantify AT1R mRNA levels in samples from the periventricular tissue surrounding the anteroventral third ventricle (AV3V), subfornical organ (SFO), and paraventricular nucleus of the hypothalamus (PVN). DNA-free total RNA was purified using the E.Z.N.A. MicroElute Total RNA kit (Omega Bio-Tek, Norcross, GA), including a deoxyribonuclease step. Reverse transcription was performed with 500 ng of RNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR was carried out using SYBR Green gene master mix (Bio-Rad), according to the manufacturer’s instructions. GAPDH was used as an internal control for quantification of mRNA. The primer sequences used were AT1R sense, 5=-CGGCCTTCGGATAACATGA, antisense, 5=-CCTGTCACTCCACCTCAAAACA, GAPDH sense, 5=-AACGACCCCTTCATTGAC, and antisense, 5=-TCCACGACATACTCAGCAC. The primer sequence was selected on the basis of a previous report (16) and is specific for the type 1A AT1R. Fluid intake measures. Total intake was calculated by weighing the water bottles before and after each test period. Licking behavior was measured using a contact lickometer (designed and constructed by the Psychology Electronics Shop, University of Pennsylvania, Philadelphia, PA) that recorded individual licks to allow for analysis of drinking microstructure. The lickometer interfaced with a computer using an integrated USB digital I/O device (National Instruments, Austin, TX). Home cages were affixed with an electrically isolated metal plate with a 3.175-mm-wide opening, through which the rat needed to lick to reach the drinking spout, minimizing the possibility of nontongue contact with the spout. AT1R autoradiography. Brains were sectioned on a cryostat at a thickness of 20 ␮m from the level of the AV3V (⫹0.6 mm bregma) through the PVN (⫺2.15 mm bregma), with the first three of every six sections being mounted on gelatin-coated slides, stored with desiccant at 4°C for 2 h, and then stored at ⫺20°C for no more than 5 days. On the day of processing, slides were thawed at RT for 45 min. Slides were then preincubated at RT in buffer (150 mM NaCl, 5 mM EDTA, and 50 mM NaPO4 at pH 7.1–7.20) for 30 min. Slides were then incubated at RT for 2 h in either 500 pM 125I-labeled sarcosine1 ANG II (125I-Sar1ANG II; see below) or 500 pM 125I-Sar1ANG II with 3-␮M nonlabeled ANG II. After incubation, slides were dipped in distilled water twice, rinsed seven times in buffer for 1 min, and then dipped in distilled water twice more. Slides were then air-dried and exposed to X-ray film for 24 h at ⫺20°C. Film was digitized, and

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densitometry was measured in the AV3V, dorsal median preoptic nucleus (dMnPO), SFO, and PVN with ImageJ software (National Institutes of Health, Bethesda, MD) using background-corrected values. After 2.5 half-lives (to allow the decay of the 125I), slides were Nissl stained to confirm localization of AT1R binding. No attempts were made to isolate the contribution of AT2R or AT1BR to the observed binding because the brain areas examined do not express AT2R (22, 25), and the AT1AR is the dominant subtype expressed in forebrain (15). Materials. 125I-Sar1ANG II was prepared using the chloramine T procedure (8) with HPLC purification to theoretical specific activity (2175 Ci/mmol), as described previously (26). ANG II was obtained from Phoenix Pharmaceuticals (Burlingame, CA) or American Peptides (Sunnyvale, CA); PD123319 from Tocris (Bristol, UK); EDTA, and NaCl were obtained from VWR International (Radnor, PA). Data presentation and analysis. All data are presented as means ⫾ SE. Images showing autoradiography were generated using screen captures and were organized into panels using Adobe Photoshop and Illustrator. No contrast adjustments were made. Images of Nisslstained sections were captured using a Nikon Eclipse 80i microscope with an attached Nikon DS-Fi1 camera. Images were acquired using NIS-Elements software and were contrast adjusted (applied to the whole image, not to specific parts) using Adobe Photoshop, before being assembled into panels in Adobe Illustrator. Drinking microstructure analysis was processed in a MatLab (MathWorks, Natick, MA) software environment before being ported to Excel (Microsoft, Redmond, WA) for final analysis. A burst was defined as at least two licks with an interlick interval of no more than 1 s. Burst size was defined as the average number of licks within a burst. RT-PCR values were calculated using the ⌬⌬CT quantification method with GAPDH as the normalizing housekeeping gene. Statistical analyses were performed using Statistica (StatSoft, Tulsa, OK). Changes in AT1R mRNA were analyzed using one-way ANOVAs for each brain region. Water intake and drinking microstructures on days 1 and 5 were analyzed using two-factor mixed design ANOVAs (group ⫻ day). AT1R binding was analyzed using one-way ANOVAs for each brain region. Fisher’s post hoc tests were used throughout to determine individual group differences after significant main or interaction ANOVA effects. Experimental designs: experiment 1: does a desensitizing treatment regimen affect AT1R mRNA levels? This experiment was performed to evaluate changes in AT1R mRNA caused by doses and administration paradigms used previously to show desensitization (32–35) and to compare these effects to administration of the same amount of ANG II given in a single bolus. To this end, 24 rats were divided into one of three groups. Rats in group 1 (n ⫽ 8) received three intracerebroventricular injections of 1 ␮l TBS vehicle spaced 20 min apart. Rats in group 2 (n ⫽ 8) received three intracerebroventricular injections, spaced 20 min apart, of the following: 1 ␮l TBS vehicle, 900 ng ANG II dissolved in 1 ␮l TBS vehicle, and 1 ␮l TBS vehicle. Rats in group 3 (n ⫽ 8) received three intracerebroventricular injections of 300 ng ANG II dissolved in 1 ␮l TBS vehicle spaced 20 min apart. Food and water were removed before the first injection. Four hours and 20 min after the final injection, rats were anesthetized by a 90-s exposure to isoflurane and then decapitated. This time point was chosen on the basis of previous reports demonstrating that AT1R mRNA can change as early as 3 h after manipulation (11). Brains were immediately removed from the skull, flash frozen with 2-methyl-butane (SigmaAldrich, St. Louis, MO) and stored at ⫺80°C. The AV3V, SFO, and PVN regions of the brain were obtained by sectioning 300-␮m coronal sections on a cryostat and then taking four 1-mm punches from each brain region. Tissue punches were stored at ⫺80°C until processing for AT1R mRNA content by RT-PCR. Experiment 2: does a desensitizing treatment regimen affect water intake sensitization? Thirty four rats were housed in custom-designed stainless-steel hanging wire cages, which allowed for continuous measurement of drinking behavior. Rats received one of four treat-

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ment regimens: 1) a single daily intracerebroventricular injection of 1 ␮l TBS vehicle (n ⫽ 5); 2) a single daily intracerebroventricular injection of 10 ng ANG II (dissolved in 1 ␮l TBS; n ⫽ 9); 3) a single daily intracerebroventricular injection of 40 ng ANG II (n ⫽ 10); and 4) four daily intracerebroventricular injections of 10 ng separated by 20 min each (n ⫽ 10). The treatment paradigm used was chosen to mimic the timing of injections used previously to study desensitization (32–35), but using a lower dose of ANG II to more closely resemble the dose used in earlier studies showing sensitization (19). This occurred daily for five consecutive days. Food and water were removed prior to the start of the experiment. Immediately after the final injection of the day, water was returned, and intake was monitored for 30 min. Food was not available during the 30-min water intake test. Experiment 3: does a desensitizing treatment regimen influence AT1R binding? To test the hypothesis that changes in AT1R binding are associated with the behavioral effects shown by experiment 2, we performed radioligand autoradiography in rats given the same treatments as described in experiment 2. To this end, 14 rats were divided into one of three groups: 1) a single daily intracerebroventricular injection of vehicle (1 ␮l TBS; n ⫽ 4); 2) single daily intracerebroventricular injection of 40 ng ANG II (n ⫽ 5); and 3) four daily intracerebroventricular injections of 10 ng separated by 20 min each (n ⫽ 5). This treatment regimen was repeated daily for five consecutive days. On the morning of day 6 (24 h after the previous injection), rats were anesthetized by 90-s exposure to isoflurane and then decapitated. Brains were immediately removed from the skull, flash frozen with 2-methyl-butane, and stored at ⫺80°C until processing. RESULTS

Experiment 1: does a desensitizing treatment regimen affect AT1R mRNA levels? To evaluate changes in AT1R after doses of ANG II used previously to produce desensitization, we injected rats intracerebroventricularly with a treatment regimen of three 300-ng injections, each given 20 min apart (32–34) and compared them with rats given the same cumulative dose in a single intracerebroventricular bolus and rats given an injection of vehicle. ANG II treatment appeared to increase AT1R mRNA expression in a brain region-specific manner

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(Fig. 1). In the AV3V, rats given a 900-ng ANG II injection in a single bolus (n ⫽ 5) had greater levels of AT1R mRNA than was found in the brains of vehicle-treated rats (n ⫽ 6). Brains of rats given 900 ng of ANG II delivered in three 300-ng injections over 40 min (n ⫽ 7), however, had levels of AT1R mRNA that were not different from the controls (F2,15 ⫽ 4.23; P ⬍ 0.05; Fig. 1A). Similarly, in the SFO, treatment with 900 ng ANG II (n ⫽ 8) was associated with greater levels of AT1R mRNA than was observed in vehicle-treated rats (n ⫽ 8), but this effect of ANG II was absent when the ANG II was delivered in three 300-ng injections (n ⫽ 8; F2,21 ⫽ 3.55; P ⬍ 0.05; Fig. 1B). Finally, in the PVN, there was no change in AT1R mRNA as a result of any ANG II treatment (n ⫽ 6 – 8/group; F2,19 ⫽ 0.07; P ⫽ 0.93; Fig. 1C). A small number of samples were excluded from the analysis because of organic contamination, and the sample sizes provided above reflect this exclusion. Experiment 2: does a desensitization treatment regimen affect water intake sensitization? Repeated injections of ANG II in close temporal proximity desensitize the drinking response to ANG II (32–34), but single daily injections appear to cause sensitization (19). The results from experiment 1 show that the timing of ANG II administration can influence subsequent gene expression. On the basis of these results, we hypothesized that the timing of ANG II administration could influence the development of sensitization. To test this hypothesis, we administered ANG II to rats, using a dose shown previously to cause sensitization (32–35), but with a timing of administration more similar to our studies of desensitization (19). To this end, we gave rats daily injections of vehicle, 10 ng ANG II, 40 ng ANG II, or a total of 40 ng ANG II/day in four 10-ng injections with 20 min between each. Daily ANG II treatment significantly influenced drinking behavior across the testing period (Fig. 2). We found a main effect of group (F3,30 ⫽ 25.12; P ⬍ 0.05), a main effect of day (F1,30 ⫽ 17.67; P ⬍ 0.05), and a group ⫻ day interaction (F3,30 ⫽ 4.87; P ⬍ 0.05). Post hoc tests showed that rats that received a single daily

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Fig. 1. Timing of ANG II-associated changes in angiotensin typee 1 receptor (AT1R) gene expression. In the SFO and AV3V, a single injection of 900 ng ANG II increased AT1R gene expression. When ANG II was delivered in three 300-ng injections, spaced 20 min apart, there was no change in AT1R gene expression (A and B). There was no change in AT1R gene expression in the PVN, regardless of ANG II treatment (C). *Significantly greater than vehicle and repeated ANG II, P ⬍ 0.05. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00373.2014 • www.ajpregu.org

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8

II treatment. In support of this, when only the caudal sections of the AV3V were analyzed, a planned comparison determined that the group treated with a single daily injection of 40 ng of ANG II had significantly less binding than that found in the vehicle group or the group treated with four daily injections of 10 ng ANG II (F2,11 ⫽ 3.42; P ⫽ 0.069; Fig. 4A). In the dorsal portion of the MnPO (dMnPO), there was less AT1R binding in the group treated with a single daily injection of 40 ng of ANG II than there was in the other groups (F2,11 ⫽ 4.34; P ⬍ 0.05; Fig. 5A). There were no treatment effects in AT1R binding in the SFO (F2,11 ⫽ 0.14; P ⫽ 0.864; Fig. 6A) or PVN (F2,9 ⫽ 0.21; P ⫽ 0.816; Fig. 7A). The analysis of the PVN excluded two subjects because of tissue damage in this region.

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Fig. 2. Repeated ANG II treatment differentially influenced fluid intake. Single daily injections of either 10 or 40 ng of ANG II were associated with greater intake on day 5 than was observed after the injection on day 1. When rats were given four daily injections of 10 ng (with 20 min between each) every day for 5 days, there was no difference in water intake between days 1 and 5. *Significantly greater than day 1, P ⬍ 0.05.

The present results show, for the first time, that behavioral and molecular changes caused by single daily injections with ANG II do not occur when the same cumulative amount of ANG II is given in multiple daily injections. Previous studies show that repeated treatment of ANG II desensitizes the AT1R

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injection of 10 or 40 ng of ANG II increased their water intake from day 1 to day 5 (P ⬍ 0.05); however, this increase was absent in rats treated with four daily injections of 10 ng ANG II (P ⬎ 0.05). Minimal drinking was observed in rats treated with vehicle, regardless of day. To further investigate the nature of the sensitization of water intake, burst analysis was conducted on the drinking patterns from rats in the groups that were treated with single daily injections of 10 and 40 ng of ANG II. Because of technical errors, data from three rats were lost and, therefore, excluded from the burst analysis. There was no effect of group, F1,14 ⫽ 0.06, day, F1,14 ⫽ 1.35, or an interaction between group and day, F1,14 ⫽ 0.06, all P values ⬎0.263 (Fig. 3A) on the number of bursts within the 30-min test period. A main effect of day, F1,14 ⫽ 10.88; P ⬍ 0.05, on burst size during the 30-min test period was detected (Fig. 3B). Burst size was significantly greater on day 5 compared with day 1. There was no effect of group, F1,14 ⫽ 0.59 or an interaction of group and day, F1,14 ⫽ 1.27, on burst size, all P values ⬎0.277. Experiment 3: does a desensitization regimen influence AT1R binding? Given the acute changes in mRNA found in experiment 1, we hypothesized that differences in AT1R binding were responsible for the behavioral effects of repeated injections of ANG II. To test this hypothesis, rats were given single daily injections of vehicle, of 40 ng of ANG II, or a daily treatment regimen of four 10-ng injections given 20 min apart. After 5 days of this treatment, brains were removed, and AT1R binding was evaluated using autoradiography. AT1R binding differed as a function of the treatment in distinct brain regions (Figs. 4 –7). We did not find a change in AT1R binding across the entire AV3V (F2,11 ⫽ 1.63; P ⫽ 0.348; Fig. 4A). Because the AV3V is composed of multiple nuclei, it is possible that specific regions within the AV3V respond differently to ANG

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Fig. 3. Drinking microstructural analysis. There was no significant change in burst number between day 1 and day 5 (A) in rats given single daily injections of ANG II. There was, however, a significant increase in burst size between day 1 and day 5 (B). *Significantly greater than day 1, P ⬍ 0.05.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00373.2014 • www.ajpregu.org

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40 ng Fig. 4. Effect of repeated injections of ANG II on AT1R binding in the anteroventral third ventricle (AV3V). Densitometric analysis of the entire AV3V region did not detect any treatment-associated changes in AT1R binding. A: when the caudal AV3V was analyzed separately, a decrease in AT1R binding was observed in rats treated daily with 40 ng of ANG II that was attenuated when rats were given the same cumulative dose of ANG II in four daily injections with 20 min between each injection. Representative autoradiograms of vehicle, single daily injections, and four daily injection treatments are shown in B–D, respectively. E: representative Nissl staining of a brain section from the vehicle treatment group is shown. 3V, third ventricle; LV, left ventricle; ac, anterior commissure. *Significantly less than Vehicle and 4 ⫻ 10 ng ANG II, P ⬍ 0.05.

as quickly as 1 min in vitro and reduces the dipsogenic potency of ANG II (7, 21, 28, 29, 32–34). On the basis of these findings, we hypothesized that repeated ANG II treatment would influence AT1R gene expression within nuclei involved in the control of fluid intake. Indeed, we found that a single injection of ANG II increased AT1R mRNA, but when the same amount of ANG II was delivered in three bouts, spaced 20 min apart, there was no change in AT1R mRNA. Guided by the finding that the timing was critical for the observed difference in AT1R mRNA, we performed a separate set of experiments to test the hypothesis that different timed injections would cause differences in the sensitizing effect of daily ANG II. These experiments used a lower dose of ANG II, to more closely resemble previous studies of water intake sensitization

(19) but manipulated the timing of the injections to test the relevant hypothesis. Although rats treated with a single daily injections of 10 or 40 ng ANG II showed the expected sensitization of water intake (19), rats given 40 ng ANG II in four 10-ng injections each day showed no similar sensitization. Finally, because a change in receptor binding is one possible mechanism underlying these behavioral differences (12, 37), AT1R binding was analyzed in nuclei involved in controlling fluid intake. Paradoxically, we found that rats treated with a single injection of 40 ng for 5 days (a treatment that produced reliable sensitization of water intake) had an apparent decrease in AT1R binding in the caudal AV3V and dMnPO. Although the decreased binding was the opposite of what was predicted from the increased behavioral response after the same treatment, the results are consistent with the behavioral studies. Although not directly comparable because of the different doses used, the results from experiments 2 and 3 are consistent with the results of experiment 1 in that the observed effect was dependent on the timing of the injections. Accordingly, these studies provide convergent evidence suggesting that the changes in mRNA, behavior, and binding associated with sensitizing treatments are all prevented by repeated administration of ANG II within a short timeframe. We first hypothesized that the timing of ANG II treatment would influence AT1R gene expression within nuclei involved in the control of fluid intake because previous studies show that the timing of ANG II treatment can influence drinking behavior. In vitro and in vivo studies show that AT1R is susceptible to rapid desensitization (7, 28, 29, 32–34) and AT1R is critical for the behavioral desensitization that is caused by repeated injections of ANG II (34). Accordingly, we tested the hypothesis that repeated ANG II treatment would influence AT1R gene expression in brain regions that are involved in fluid intake. Indeed, when rats received a single dose of ANG II, there was a significant increase in AT1R mRNA in both the AV3V and SFO, but not the PVN, and the effect was absent when ANG II treatment was delivered using the repeatedtreatment regimen time frame. This suggests that the effect of ANG II on expression of its receptor is anatomically selective and is influenced by the timing of the exposure. When considering the present experiments showing changes in mRNA, it is important to note that the expression of mRNA does not always correspond with the expression of the protein that it encodes. For example, estradiol can alter AT1R protein levels by posttranscriptional actions (14). Additionally, in the kidney, losartan increases AT1R mRNA, but decreases AT1R binding (36). Moreover, recent studies from our laboratory found that repeated injections of large doses of ANG II (the doses used in experiment 1 here) decreased radioligand binding (27), whereas the repeated injections here produced no difference in mRNA levels. As such, a change in mRNA should be interpeted with caution, and these examples highlight the importance of measuring levels of the relevant protein whenever possible. Because we observed region-specific differences in AT1R mRNA after acute bolus or timed ANG II injections, we next hypothesized that the timing of ANG II delivery would prevent the enhancement of water intake observed after single daily injections of ANG II. This intake sensitization has been studied using a variety of paradigms, including chronic central ANG II infusions, repeated bouts of deprivation with partial rehydra-

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00373.2014 • www.ajpregu.org

SENSITIZATION AND DESENSITIZATION TO ANGIOTENSIN II

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tion, and repeated injections of mineralocorticoids or diuretics, alone or in combination with ACE inhibitors (1, 3, 6, 9, 10, 20, 23, 24). Although the focus of these studies has been on the sensitization of sodium intake, both repeated daily injections and chronic exposure to ANG II sensitize the dipsogenic response (3, 19). Because our previous studies found that the desensitizing effect of acute repeated injections of ANG II was limited to water intake, without an effect on saline intake, we focused exclusively on a sensitizing treatment already known to enhance water intake (34). Indeed, whereas rats treated with a single daily injection of either 10 or 40 ng of ANG II increased their water intake across the 5-day testing period, rats given four daily injections of 10 ng of ANG II showed no sensitization of water intake. The inclusion of single injections of both 10 ng and 40 ng was important because each provides a direct respective comparison with the final injection of ANG II used in the desensitizing treatment regimen or with the cumulative total dose of ANG II provided. Given these comparisons, the results of this study demonstrate that properly timed administration of ANG II does not lead to behavioral sensitization of water intake and suggests this treatment regimen can prevent the behavioral sensitization of water intake that results from daily ANG II treatment.

B

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Fig. 5. Effect of repeated injections of ANG II on AT1R radioligand binding in the dorsal median preoptic nucleus (dMnPO). A: Densitometry analysis of the dMnPO revealed a decrease in binding when rats were given daily injections of 40 ng ANG II, but this decrease was not observed when rats were given the same cumulative dose of ANG II spread across four daily injections of 10 ng. Representative autoradiograms of vehicle, oncedaily injections, and four daily injection treatments are shown in B–D, respectively. E: representative Nissl staining of a brain section from the vehicletreatment group is shown. The dotted outline indicates the area included in the analysis. *Significantly less than vehicle and repeated ANG II, P ⬍ 0.05.

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To further understand the nature of the increase in water intake in this paradigm, we analyzed drinking microstructure. Analysis of drinking microstructure revealed that the sensitization of water intake, observed in the group treated with a single daily injection of 10 and 40 ng of ANG II, was mediated by a change in burst size. There were no significant changes in burst number. On the basis of previous studies (5), this suggests that the increased water intake is the result of changes in the orosensory value of the fluid and not the result of a change in postingestive feedback. To the best of our knowledge, this is the first examination of drinking microstructure across fluid sensitization. It is unclear whether the enhancement of intake in other sensitization paradigms is also the result of changes in orosensory properties vs. satiety signals. Therefore, future studies will be necessary to determine whether this is a general mechanism underlying sensitization of water intake or specific to daily repeated injections of ANG II. It is also important to recognize that the changes observed in the present study are occurring in a whole animal model, and, therefore, require extensive additional research to test for roles of confounding variables. Indeed, the treatments used here could have affected many things, including, but not limited to, vasopressin secretion, body temperature, and blood pressure. Because many of

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Fig. 6. Effect of repeated injections of ANG II on AT1R radioligand binding in the subfornical organ (SFO). Densitometric analysis of the SFO revealed no treatment effects on AT1R binding (A). Representative autoradiograms of vehicle, single daily injections, and four daily injection treatments are shown in B–D, respectively. E: representative Nissl staining of a brain section from the vehicle treatment group is shown.

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Fig. 7. Effect of repeated injections of ANG II on AT1R radioligand binding in the paraventricular nucleus (PVN). A: densitometric analysis of the SFO revealed no treatment effects on AT1R binding. Representative autoradiograms of vehicle, single-daily injections, and four daily injection treatments are shown in B–D, respectively. E: representative Nissl staining of a brain section from the vehicle treatment group is shown.

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these can directly or indirectly affect fluid intake, understanding the direct link to the observed changes requires further research. Although the precise link between the treatment and the response remains unclear, the present studies revealed the striking finding that once-daily ANG II treatment increased water intake accompanied by a paradoxical decrease in AT1R binding in the caudal AV3V and dMnPO. Determining the precise anatomical location of these changes is difficult with the techniques employed. Indeed, the changes that we refer to as occurring in sections of the caudal AV3V and dMnPO may reflect a more generalized change in the MnPO because there is likely ventral MnPO in the sections considered caudal AV3V. Although the precise anatomical locus of the change is difficult to clarify from our data, it is clear that the differences in binding depended on the timing of the injections. Specifically, whereas a single daily injection of ANG II decreased AT1R binding in these brain areas, this did not occur when the same cumulative amount of ANG II was split into four injections. Moreover, the observed differences were not observed in the SFO or PVN. The changes in the more ventral regions of the lamina terminalis are consistent with previous studies showing that the AV3V is particularly important in the behavioral desensitization caused by acute repeated injections of ANG II (32). Although the direction of the change in behavior may have predicted an increase in binding, the decreased binding observed in the present report is consistent with other reports. Indeed, Moellenhoff et al. (19) reported a decrease in c-Fos in the MnPO after daily injections of ANG II that is consistent with the direction of the change in binding observed here. In that study, however, changes in c-Fos were also observed in the SFO, PVN, and SON (19), where we did not find changes in AT1R binding. Moreover, two studies using repeated mineralocorticoid treatment found increased AT1R binding that was associated with the sensitization of saline intake (12, 37), suggesting that the observed sensitization can occur by different underlying mechanisms. Nevertheless, and perhaps more important, the efficacy of the treatments to affect behavior corresponded to the observed changes in binding. Indeed, we consistently found differences in behavior, AT1R mRNA, and AT1 binding in the rats given a sensitizing course

of treatment or single injection of ANG II, and these changes were each not present after the desensitizing treatment regimen, given either in a single day or across days. The present studies are limited, however, in that the doses used to study mRNA were different from those used to study behavior and receptor binding. Nevertheless, the changes in AT1R binding appear to be closely associated with, if not causing, the changes in behavior observed previously (32–35). The most parsimonious explanation is that ANG II binding to the AT1R directly causes the changes in radioligand binding observed here and that this change in receptor availability or affinity makes the animal less responsive to the dipsogenic effects of ANG II. Other less direct mechanisms are possible and could be suggested on the basis of previous studies on the mechanism(s) underlying sensitization of water and saline intakes after different means to produce the sensitization. For instance, the requirement for altered circulating hormones in the intake sensitization after repeated bouts of sodium depletion were ruled out by demonstrating that there are no lasting changes in circulating ANG II or aldosterone (24). Although our studies suggest a role for AT1R that is supported by other studies [e.g., AT1R blockade prevents furosemide/captoprilinduced sensitization (20)], our studies do not address other systems that may be critical for the observed behavioral changes. Yet other studies have found that mineralocorticoid receptor (MR) and NMDA receptors are associated with the intake changes observed after sensitizing treatments (9, 23), making these important targets for future investigations of the neurochemical systems involved in the interactions between sensitizing and desensitizing treatments. With respect to the potential interactions involving mineralocorticoids, our finding that AT1R binding in the MnPO was affected by the treatments used here may relate to previous studies showing that DOCA causes increased spontaneous activity, increased firing rates, and prolongs the effect of ANG II on neurons in the MnPO (30). Studies of sodium appetite found that the sensitizing effect of repeated treatments with furosemide is prevented by pretreatment with an MR antagonist (23). Consideration of a contribution by NMDA receptors is also important. A recent study showed that an NMDA receptor antagonist prevented the sensitization of water and saline intake after repeated treat-

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SENSITIZATION AND DESENSITIZATION TO ANGIOTENSIN II

ments with furosemide and captopril (9). Accordingly, future studies testing whether these changes occur after daily ANG II treatment could provide important insight into understanding the mechanisms underlying the behavioral sensitization of water intake. From a logical perspective, the critical question raised by the present experiments is how an increased behavioral response to ANG II is associated with a decrease in AT1R binding? We propose four nonmutually exclusive answers to this perplexing question. First, it is possible that the observed sensitization of water intake is independent of the changes in AT1R, but it is more directly linked to a learning effect. Specifically, during the days of the treatments, the rats may strengthen the association between the act of drinking water and the alleviation of the ANG II-induced drive to drink. Accordingly, this stronger association leads to increased intake. Indeed, previous studies described above showing a role for NMDA receptors in a different type of drinking sensitization is consistent with a role of learning (9). Second, it is possible that the intake sensitization is secondary to more primary changes in blood pressure. ANG II treatment induces a pressor response and increased blood pressure inhibits fluid intake (13, 31). If repeated ANG II treatment has a primary effect that desensitizes the pressor response (consistent with the decreased binding), this could disinhibit the effect of increased blood pressure on fluid intake. Previous studies using different models of sensitization are, however, inconsistent with this explanation. Specifically, 8 wk of DOCA treatment caused a sensitized drinking and pressor response to either central or peripheral ANG II (37); however, this study also showed an increase in AT1R binding, suggesting that the responsible mechanisms are quite different. Third, the decreased binding that was associated with increased behavior in the present studies may reflect an enhanced efficiency of the receptor. This could be a result of neural plasticity [consistent with the above-mentioned requirement for NMDA receptors (9)] that perhaps involves changes in specific signaling molecules downstream from the AT1R. Fourth, although the focus of the present and of previous studies has been on the apparent sensitization of behavior, which might predict an increase in a critical receptor, it is possible that this perspective is flawed and that the increased behavior is not because of sensitization of the behavior itself, but is instead due to a developed tolerance to the satiating effect of the consumed fluid. In this case, the reduction in receptor binding would be in the same direction as the reduced satiating potency of the consumed fluid. This possibility, however, requires that the AT1R be reconsidered as a critical part of drinking termination, rather than an integral part of the onset of drinking. This seems unlikely, and the present drinking microstructure analysis suggests that a change in the orosensory properties of the fluid, not a change in satiety, was involved in the increased drinking. Nevertheless, these possibilities offer reasonable starting points that can be used to direct future studies. Perspectives and Significance The results here provide the first studies suggesting that properly timed application of ANG II can prevent the sensitizing effect normally observed after daily injections of ANG II. Whether or not these results apply to chronically elevated ANG II or any of the sensitization that may underlie fluid

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balance disorders, such as hypertension remains an avenue for future research. Nevertheless, the studies open the door to the exciting, although perhaps remote, possibility that a paradigm shift in the treatment of hypertension is worth some consideration. Indeed, current strategies that target the renin-angiotensin system to control hypertension all seek to decrease ANG II activity, and an increase in ANG II would be contraindicated. Our studies show that the timing of ANG II produces marked differences in the response to the treatment, suggesting that proper timing of ANG II may prevent some of the consequences of elevations of ANG II. Accordingly, it is tempting to speculate that future views of the anti-ANG II strategy could be viewed similarly as the current perspective on the treatment of congestive cardiomyopathy. Indeed, several decades ago, treatment of chronic heart failure focused on maintaining or increasing adrenergic tone, but now the opposite approach, using cardio-selective ␤-blocking agents, is preferred and viewed as far more effective (2). In this respect, a new approach, which was the exact opposite of the previous approach and which was formerly contraindicated, was found to be far more effective. Perhaps fanciful, but it is possible that these studies provide the groundwork for a similar reversal in the treatment of hypertension by drugs targeting the renin-angiotensin system. ACKNOWLEDGMENTS We would like to thank Dr. Peter Vento, Dr. Duncan MacLaren, and Anikó Marshall for technical assistance. GRANTS This work was supported by National Institutes of Health (NIH) Grants HL-091911 to D. Daniels, DK-098841 to J. Santollo, HL-113905 to R. C. Speth, and DA-0124754 to S. D. Clark. Support was also provided by a Pilot Award from the Translational Technologies Component of the Georgetown, Howard Universities Center for Clinical and Translational Science UL1TR000101 to R. C. Speth. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: J.S., S.D.C., and D.D. conception and design of research; J.S. and P.E.W. performed experiments; J.S. and D.D. analyzed data; J.S., R.C.S., S.D.C., and D.D. interpreted results of experiments; J.S. and D.D. prepared figures; J.S. and D.D. drafted manuscript; J.S., P.E.W., R.C.S., S.D.C., and D.D. edited and revised manuscript; J.S., P.E.W., R.C.S., S.D.C., and D.D. approved final version of manuscript. REFERENCES 1. Acerbo MJ, Johnson AK. Behavioral cross-sensitization between DOCA-induced sodium appetite and cocaine-induced locomotor behavior. Pharmacol Biochem Behav 98: 440 –448, 2011. 2. Bristow MR. ␤-adrenergic receptor blockade in chronic heart failure. Circulation 101: 558 –569, 2000. 3. Bryant RW, Epstein AN, Fitzsimons JT, Fluharty SJ. Arousal of a specific and persistent sodium appetite in the rat with continuous intracerebroventricular infusion of angiotensin II. J Physiol 301: 365–382, 1980. 4. Daniels DF, Fluharty SJ. Neuroendocrinology of body fluid homeostasis. In: Hormones, Brain and Behavior, edited by Pfaff DW, Arnold AP, Fahrbach SE, and Etgen AM. San Diego, CA: Academic Press, 2009, p. 259 –288. 5. Davis JD. The microstructure of ingestive behavior. Ann NY Acad Sci 575: 106 –119; discussion 120 –101, 1989. 6. De Luca LA Jr, Pereira-Derderian DT, Vendramini RC, David RB, and Menani JV. Water deprivation-induced sodium appetite. Physiol Behav 100: 535–544, 2010.

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22. Rowe BP, Grove KL, Saylor DL, Speth RC. Angiotensin II receptor subtypes in the rat brain. Eur J Pharmacol 186: 339 –342, 1990. 23. Sakai RR, Fine WB, Epstein AN, Frankmann SP. Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behav Neurosci 101: 724 –731, 1987. 24. Sakai RR, Frankmann SP, Fine WB, Epstein AN. Prior episodes of sodium depletion increase the need-free sodium intake of the rat. Behav Neurosci 103: 186 –192, 1989. 25. Song K, Allen AM, Paxinos G, Mendelsohn FA. Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J Comp Neurol 316: 467–484, 1992. 26. Speth RC, Harding JW. Radiolabeling of angiotensin peptides. Methods Mol Med 51: 275–295, 2001. 27. Speth RC, Vento PJ, Carrera EJ, Gonzalez-Reily L, Linares A, Santos K, Swindle JD, Daniels D. Acute repeated intracerebroventricular injections of angiotensin II reduce agonist and antagonist radioligand binding in the paraventricular nucleus of the hypothalamus and median preoptic nucleus in the rat brain. Brain Res 1583: 132–140, 2014. 28. Thomas WG. Regulation of angiotensin II type 1 (AT1) receptor function. Regul Pept 79: 9 –23, 1999. 29. Thomas WG, Thekkumkara TJ, Baker KM. Cardiac effects of AII. AT1A receptor signaling, desensitization, and internalization. Adv Exp Med Biol 396: 59 –69, 1996. 30. Thornton SN, Nicolaidis S. Long-term mineralocorticoid-induced changes in rat neuron properties plus interaction of aldosterone and ANG II. Am J Physiol Regul Integr Comp Physiol 266: R564 –R571, 1994. 31. Thunhorst RL, Lewis SJ, Johnson AK. Role of arteria baroreceptor input on thirst and urinary responses to intracerebroventricular angiotensin II. Am J Physiol Regul Integr Comp Physiol 265: R591–R595, 1993. 32. Vento PJ, Daniels D. The anteroventral third ventricle region is critical for the behavioral desensitization caused by repeated injections of angiotensin II. Behav Brain Res 258: 27–33, 2014. 33. Vento PJ, Daniels D. Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II. Exp Physiol 97: 1305–1314, 2012. 34. Vento PJ, Daniels D. Repeated administration of angiotensin II reduces its dipsogenic effect without affecting saline intake. Exp Physiol 95: 736 –745, 2010. 35. Vento PJ, Myers KP, Daniels D. Investigation into the specificity of angiotensin II-induced behavioral desensitization. Physiol Behav 105: 1076 –1081, 2012. 36. Wang DH, Du Y, Zhao H, Granger JP, Speth RC, Dipette DJ. Regulation of angiotensin type 1 receptor and its gene expression: role in renal growth. J Am Soc Nephrol 8: 193–198, 1997. 37. Wilson KM, Sumners C, Hathaway S, Fregly MJ. Mineralocorticoids modulate central angiotensin II receptors in rats. Brain Res 382: 87–96, 1986.

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