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J Physiol573.1 (2006) pp 251262 251

Oestrogen and weight loss decrease isoproterenol-inducedFos immunoreactivity and angiotensin type 1 mRNAin the subfornical organ of female rats

Eric G. Krause, Kathleen S. Curtis, Todd L. Stincic, Jason P. Markle and Robert J. Contreras

Department of Psychology, Program in Neuroscience, Florida State University, Tallahassee, FL 32303-1270, USA

Studies from our laboratory and others show that oestrogen reduces angiotensin II

(Ang II)-induced water intake by ovariectomized rats. Elimination of endogenous oestrogen

by ovariectomy causes weight gain that can be reversed or prevented by oestrogen replacement.

Changes in body weight modify cardiovascular responses to Ang II but whether such changes

have similar effects on central and behavioural responses to Ang II is unknown. The goal of

this study was to evaluate the contributions of oestrogen and weight loss to isoproterenol

(isoprenaline; Iso)-induced Fos immunoreactivity (IR) and to angiotensin type 1 (AT1) receptor

mRNA in forebrain regions implicated in the control of fluid balance. Isoproterenol significantly

increased Fos IR in the hypothalamic paraventricular and supraoptic nuclei, the subfornical

organ (SFO), and the organum vasculosum of the lamina terminalis, but had no effect on AT1

mRNA expression. However, both Iso-induced Fos IR and the AT1 mRNA were attenuated in

the SFO of the oestrogen and weight loss groups compared with that of the control group.

Consequently, we examined the effect of weight loss on Iso-induced water intake and plasma

renin activity (PRA) and found that weight loss decreased water intake after Iso, but had no

effect on PRA. Thus, we propose that weight loss decreases Ang II-elicited water intake in the

female rat by down-regulating the expression of the AT1 receptor.

(Resubmitted 2 February 2006; accepted after revision 13 March 2006; first published online 16 March 2006)

Corresponding author R. J. Contreras: Department of Psychology, Program in Neuroscience, Florida State University,

Tallahassee, FL 32303-1270, USA. Email: [email protected]

The reninangiotensin system (RAS) is critically involvedin the regulation of blood pressure and fluid balance.The effector peptide of the RAS, angiotensin II (Ang II),mediates compensatory responses to blood loss, sodiumdepletion and hypotension. Ang II influences peripheraland central mechanisms that maintain blood pressure andvolume. In the periphery, Ang II causes vasoconstrictionby acting on angiotensin type 1 (AT1) receptors onblood vessels. Centrally, Ang II binds to AT1 receptors incircumventricular organs (CVOs) to initiate changes insympathetic nerve activity and increase water and sodium

consumption (McKinleyet al. 2003).Interestingly, the RAS is influenced by sex hormones,

and this influence is particularly pronounced in termsof behavioural responses to RAS activation. Studies fromour laboratory and others show that oestrogen attenuatesRAS-induced water intake by ovariectomized rats (Fregly& Thrasher, 1978; Findlay et al. 1979; Jonklaas & Buggy,1984; Kisley et al. 1999; Krause et al. 2003; Tanaka et al.2003). In our previous study, the peripheral synthesis ofAng II, as measured by plasma renin activity (PRA), wassimilar in oestrogen- and vehicle-treated ovariectomized

rats (Krause et al. 2003), suggesting that oestrogen actscentrally to modify responses to circulating Ang II. Infact, peripheral administration of oestrogen decreasesneuronal activity in the forebrain CVO, the subfornicalorgan (SFO), during intracarotid administration of Ang II(Tanaka et al. 2001). The SFO contains AT1 receptorswhich bind circulating Ang II, thereby activating centralpathways that mediate water intake. In addition, oestrogenand AT1 receptors are co-localized within SFO neurons(Rosas-Arellano et al. 1999) and oestrogen treatmentreduces Ang II binding to AT1 receptors in the SFO (Kisley

et al. 1999). The reduced binding observed in the SFOis probably due to decreased AT1 receptors, as oestrogendown-regulates AT1 receptor expression in vitro (Gragasinet al. 2003; Imanishi et al. 2005). Thus, oestrogen mayattenuate the responsiveness of the SFO to circulatingAng II by decreasing AT1 receptor expression, which inturn, modifies behavioural responses to RAS activation.

In addition to sex hormones, changes in body weightinfluence the RAS. Recent research has demonstratedthat several components critical for the biosynthesis ofAng II are present in adipose tissue (Karlsson et al.

C 2006 The Authors. Journal compilation C 2006 The Physiological Society DOI: 10.1113/jphysiol.2006.106740

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252 E. G. Krause and others J Physiol573.1

1998) and circulating levels of these components areincreased in obese rodents and humans (Boustanyet al. 2004; Engeli et al. 2005). Specifically, circulatingangiotensinogen is elevated in obese rodents, producinghypertension that can be reversed with AT1 receptorantagonists (Boustany et al. 2004; Boustany et al. 2005).Circulating levels of angiotensinogen, renin, aldosterone

and angiotensin-converting enzyme were increased inobese post-menopausal women and a 5% reductionin body weight greatly attenuated these increases andthe associated hypertension (Engeli et al. 2005). Takentogether, these studies suggest that changes in bodyweight modify RAS control of cardiovascular function.However, whether body weight also influences the centralor behavioural responses to RAS activation has yet to bedetermined.

While there is good evidence that oestrogen influencesneural and behavioural responses to RAS activation,an alternative variable that has not been considered is

oestrogen effects on body weight. It is well established thatelimination of endogenous oestrogen by ovariectomy isassociated with a 1216% increase in body weight thatcan be reversed or prevented by oestrogen replacement(McElroy & Wade, 1987). In our previous studyinvestigating oestrogen effects on water intake (Krauseet al. 2003), oestrogen replacement in ovariectomizedrats produced a 35% weight loss. Therefore, it ispossible that the changes in body weight that accompanyovariectomy and hormone replacement contribute tooestrogen effects on RAS-elicited water intake. At present,few researchers examining oestrogen effects on the centraland behavioural responses to RAS activation consider thepotential confound of altered body weight.

The goal of the present study was to test thehypothesis thatthe weightloss thataccompanies oestrogenreplacement contributes to oestrogen effects on centraland behavioural responses to RAS activation. We firstinvestigated the effect of oestrogen or weight loss onneuronal activity by examining Fos immunoreactivity(IR)in forebrain regions implicated in the control of fluidbalance after systemic administration of isoproterenol(Iso). Isoproterenolis a-agonist thatincreases circulatinglevels of Ang II by decreasing blood pressure andactivatingrenal -receptors (Kirby et al. 1994), and commonly is

used to examine stimulated water intake (Rettig et al.1981; Stocker et al. 2000; Krause et al. 2003). Next, weevaluated the effect of oestrogen or weight loss on AT1receptor mRNA expression in the SFO and the hypo-thalamic paraventricular nucleus (PVN) using in situhybridization. Results from these studies indicate thatoestrogen and weight loss had similar effects on Fos IRand AT1 receptor mRNA expression. Accordingly, we alsoexamined the effect of weight loss on Iso-induced waterintake and PRA and found that weight loss attenuatedwater intake after Iso but had no effect on PRA. The results

of these studies lead to the conclusion that body weightloss contributes to oestrogen attenuation of Ang II-elicitedwater intake in the female rat.

Methods

Animals

Adult female Sprague Dawley rats weighing between250 and 350 g were individually housed in plastic cagesand given ad libitum access to Purina rodent chow (no.5001) and water except where noted. Rats were kept ina temperature-controlled room (2224C) on a 12 : 12 hlightdark cycle with lights on at 07.00 h. Experimentsexamining water intake used a within subjects design sothat each animal served as its own control. All procedureswere approved by the Institutional Animal Care and UseCommittee at Florida State University.

Ovariectomy and oestrogen replacement

Under sodium pentobarbital anaesthesia (50 mg (kg bodyweight)1, i.p.; Abbott Laboratories, Chicago, IL, USA),rats were bilaterally ovariectomized using a ventralapproach and given 1 week to recover. Rats then weregiven 17--oestradiol-3-benzoate (OeB; 10g (0.1 ml)1

sesame oil, s.c.; Fisher Scientific, Fair Lawn, NJ, USA)or the oil vehicle (oil; 0.1 ml, s.c.) on a schedule thatmimics the pattern of oestrogen fluctuations during theoestrous cycle. Specifically, rats were given injections ofOeB or oil on two consecutive days, and tested 48 h

following the second injection (i.e. on Day 4). Oestrogenreplacement using this schedule reliably elicits lordosis48 h after the second injection when progesterone also isgiven (McCarthyet al. 1991; Schumacher et al. 1991) andhas been used in other studies examining OeB effects oningestive behaviours (Kisleyet al. 1999; Krause et al. 2003;Curtis et al. 2004; Curtis et al. 2005).

c-Fos immunohistochemistry

Ovariectomized rats were treated with OeB or oil asdescribed and on Day 4 were injected with isoproterenol

(30g (kg body weight)1, s.c.; oil, n= 9; OeB, n= 8) orthe 0.15 m NaCl vehicle (saline; oil, n= 5; OeB, n= 5).In our previous studies, OeB treatment caused a 35%reduction of body weight; therefore, an additional groupof ovariectomized rats (weight loss) was treated with oilon Day 1 and 2, and restricted to 810 g of chow on Day 3to produce a 35% weight loss. Subsequently, weight lossrats were injected with Iso (n= 9) or saline (n= 5) onDay 4.

Ninety minutes after the injections, rats were deeplyanaesthetized with sodium pentobarbital (75 mg (kg body

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J Physiol573.1 Oestrogen and weight loss modulate subfornical organ responses to isoproterenol 253

weight)1, i.p.) and then perfused with 0.15m NaCl,followed by 4% paraformaldehyde in 0.1m phosphatebuffer. The brains were removed, placed into 30% sucrosefor 48 h, and then cut into 40m coronal sections using acryostat.

c-Fos immunocytochemistry was performed aspreviously described (Curtis et al. 2002). Briefly,free-floating sections were washed in 0.05 m Tris-NaCl,soaked in 0.05 m Tris-NaCl containing 0.5% Triton X-100and 10% normal goat serum for 1 h, then incubated for20 h with a rabbit polyclonal anti-c-Fos peptide antisera(Santa Cruz Biotechnology, Inc.) diluted 1 : 30 000 in 2%normal goat serum. Sections were washed in 2% normalgoat serum and incubated for 2 h at room temperaturewith a biotinylated goat anti-rabbit antibody (VectorLaboratories) diluted 1 : 300 in normal goat serum.Bound secondary antibody was amplified during a 1.5 hincubation in an avidinbiotin complex (ABC Elite Kit,Vector Laboratories). Antibody complexes were visualized

using nickel-intensified diaminobenzidine (Kirkegaardand Perry Laboratories, Gaithersberg, MD, USA) and thisreaction was terminated by washing the sections in 0.05 mTris-NaCl. Sections were mounted on microscope slidesand coverslipped.

Quantification of Fos immunoreactivity

Fos IR was examined in forebrain regions implicated influid balance including the hypothalamic paraventricular(PVN) and supraoptic (SON) nuclei, SFO, and theorganum vasculosum of the lamina terminalis (OVLT).

Serial sections (40m) were taken from the laminaterminalis to the caudal portion of the median eminence.The largest neurons within the areas of interest havediameters of 1319m (Kiss et al. 1991); therefore,every third section was processed for Fos IR to ensurethat neurons occurring in consecutive sections were notcounted twice. Using NIH Image software, the numbers ofFos-positive nucleiwere counted in representativesectionsfrom each of these areas, based on anatomical landmarksas described by Paxinos & Watson (1997).

Three sections were taken from the SON, matchedbetween subjects using the optic tracts as a landmark.

For the PVN, the three sections that contained the largestlateral magnocellular subnucleus were taken and matchedbetweensubjects. Inthe cases of the SON and PVN, countswere taken from one side. Two sections were taken fromthe highly vascularized central portion of the SFO, andwere matched between subjects. Two sections from theOVLT were matched between subjects using lateral anddorsal boundaries as described by Bisleyet al. (1996). Theaverage number of Fos-positive nuclei in each area fromeach animal was calculated and group means for each areain each experimental condition were determined.

In situ hybridization

The effect of Iso on AT1 mRNA expression was assesseda priori by dividing treatment groups into those treatedwith saline and those treated with Iso. Using a ttest, it wasdetermined that Iso did not affect AT1 mRNA expressionin the SFO (P= 0.51) or PVN (P= 0.85). Therefore,

results from drug conditions were pooled within groupsfor subsequent analysis. Ovariectomized rats were treatedwith OeB (n= 5) or oil (n= 6) on Day 1 and 2 andweight loss rats (n= 6) were food restricted on Day 3 asdescribed. On Day 4, rats were deeply anaesthetized withsodium pentobarbital (75 mg (kg body wt)1, i.p.) anddecapitated. Brains were quickly removed and flash frozenin dry-ice-cooled 2-methylbutane and stored at 80C.Frozen brains were sectioned at 20 m using a cryostat.Serial sections were taken from the lamina terminalis tothe caudal portion of the median eminence. Every thirdsectionwasthawmountedontoaslideandstoredat80Cuntil processing for in situ hybridization.

In situ hybridization was used to visualize AT1 receptormRNA expression using a riboprobe transcribed froma cDNA template (nucleotides 14072144) from thelaboratory of Dr Steven Fluharty (Kisleyet al. 1999). Thisprobe is complementary to a specific mRNA sequence andallows visualization of the product of the gene target inspecific anatomical locations.

For riboprobe synthesis, 10l of [35S]uridinetriphosphate and 2.0l 5 transcription buffer, 1.0lof 0.1 m dithiothreitol, 1.0l each of 10 mm adenosinetriphosphate, cytosine triphosphate and guanidinetriphosphate, 2.0l linearized plasmid (1gml1) DNA

0.5l RNAse inhibitor (40 units l1), and 1.5 l SP6RNA polymerase (15 units l1) was combined andincubated for 2 h at 37C. DNAse (1.0l: RNAse-free)was added and the mixture was incubated for 15 min atroom temperature. Radiolabelled probe was purified withmicrospin chromatography columns (Bio-Rad, Hercules,CA, USA).

Slides were removed from 80C storage and placedin 4% paraformaldehyde at room temperature for 1 h.The slides were then washed in 2 300 mm NaCl30 mmsodium citrate, pH 7.2, three times for 5 min each.Slides were then washed in deionized water for 1 min

and placed in 0.1m

triethanolamine (pH 8.0)aceticanhydride, 400 : 1 (v/v) on a stir plate, for 10 min.The final rinse was in 2 300 mm NaCl30 mm sodiumcitrate, pH 7.2 for 5 min, followed by dehydration throughgraded alcohols and air-drying for 30 min. A coverslipwith 60l of 75% formamide hybridization buffer (10%dextrane sulphate, 3 300 mm NaCl30 mm sodiumcitrate (pH 7.2), 50 mm Na2HPO4 (pH 7.4), 10mmdithiothreitol,1Denhardtssolution(Sigma-Aldrich,St.Louis, MO, USA), 100 g (ml)1 yeast tRNA, and 0.01 mdithiothreitol) was placed on each slide. Strength of probe



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in 60l of hybridization buffer was approximately 12million c.p.m. Slides were placed in a covered tray withfilter paper saturated with 75% formamide.

After overnight incubation at 55C, coverslips wereremoved and slides were placed at room temperaturein 2 300mm NaCl30 mm sodium citrate, pH 7.2, for5 min, followed by RNAse (200g (ml)1 i n 1 0 mm

Tris-HCl, pH 8.00.5m NaCl) at 37C for 30 minand then washed as follows: 2 300 mm NaCl30 mmsodium citrate, pH 7.2, at room temperature for 10 min,1 300 mm NaCl30 mm sodium citrate, pH 7.2, for10 min at room temperature; 0.5 300 mm NaCl30 mmsodium citrate, pH7.2, at 55C for 60 min; and0.5 300 mm NaCl30 mm sodium citrate, pH 7.2, for10 min at room temperature. The slides were dehydratedin graded ethanol solutions, air dried, placed in X-raycassettes, and apposed to Kodak XAR-5 film for 10 days.

In situ hybridization image and data analyses

Film was developed using a Mini-Medical automaticdeveloper (AFP Imaging Corporation Elmsford, NY, USA)and then analysed with Image J 1.24 (NIH, USA). Imageswere analysed in representative autoradiographs from theSFO and PVN, which were matched between animalsbased on anatomical landmarks as described by Paxinos& Watson (1997). Tissue background readings weresubtracted from grey scale values from these regions.Gray scale values were converted into optical density andsubsequently averaged, providing one mean value perregion, per animal. Slides contained sections 60 m apart

and counts were taken from the three consecutive sectionswith the greatest grey scale values, thereby generating thegreatest average AT1 mRNA per region per subject. Countstaken for the SFO always contained its highly vascularizedcentral portion and counts taken for the PVN consistentlycontained the lateral magnocellular subdivision.

Plasma renin activity

Aseparategroupofovariectomizedratswereanaesthetizedwith sodium pentobarbital (50 mg (kg body weight)1,i.p.) and implanted with a catheter (MRE-025 tubing;

Braintree Scientific, Braintree, MA, USA) into thefemoral vein. The catheter was filled with 0.15 m NaClcontaining 50 U ml1 heparin and the free end was guidedsubcutaneously to exit between the scapulae. Animalswere given 23 days to recover prior to oil treatment orweight loss as described. On Day 4, water bottles wereremovedandovariectomizedratsweregivensubcutaneousinjections of saline (weight loss, n= 8; oil, n= 9) orIso (weight loss, n= 9; oil, n= 12). Thirty minuteslater, rats were deeply anaesthetized with sodium pento-barbital (30 mg (kg body weight)1, i.v.) and decapitated.

Trunk blood was collected 812 s after anaesthesia wasadministered, immediately placed into chilled tubescontainingEDTA(Vacutainer;BectonDickinson,FranklinLakes, NJ, USA), and centrifuged at 1000 g for 15 minat 4C. The plasma was removed and stored at 80Cprior to radioimmunoassay for PRA using an AutomaticGamma Counter (Titertek Instruments, Inc., Huntsville,

AL, USA) and a commercially available kit (DiaSorin,Stillwater, MI, USA).

Water intake

This experiment was designed to complement ourprevious study investigating the effect of OeB on waterintake after Iso (Krause et al. 2003). Another group ofovariectomized rats (n= 8) were treated with weight lossor oil as described and, on Day 4, were given subcutaneousinjections of Iso or saline. Rats then were given waterin graduated cylinders and intakes were recorded after2 h. All rats were tested in four conditions with 1 weekbetween conditions. To control for order effects, the ratswere randomly assigned to one of two testing sequences:(1) weight lossIso, oilsaline, oilIso, weight losssaline;or (2) weight losssaline, oilIso, oilsaline, weightlossIso.

Statistics

All data are expressed as means s.e.m. Values morethan three s.d.s from the mean were considered outliers

and were excluded from further analysis (n= 1). AShapiro-Wilk test of normality showed that the ANOVAresiduals for the untransformed Fos IR data were notnormally distributed. Consequently, Fos IR data were logtransformed; ANOVA residuals from the analysis of thetransformed data met the assumption of normality perthe Shaprio-Wilk test. Subsequently, differences in Fos IRwere assessed with a 2-factor ANOVA with group (OeB,oil, weight loss) and drug (saline and Iso) as the factors.Main effects or interactions (P< 0.05) were analysed witha Scheffes test for multiple comparisons (SAS 9.1, Cary,NC, USA).

The remaining data were analysed using Statistica

(StatSoft, Tulsa, OK, USA). Differences in the percentagechange in body weight and AT1 mRNA expression wereassessed using a 1-factor analysis of variance (ANOVA)with group (oil, OeB, weight loss) as the factor. Plasmarenin activities were assessed with a 2-factor ANOVA withgroup (oil and weight loss) and drug (saline and Iso)as the factors. Differences in water intake were assessedwith a 2-factor repeated measures ANOVA with group (oiland weight loss) and drug (saline and Iso) as the withinsubject factors. Main effects or interactions (P< 0.05)were analysed with Student-Newman-Keuls tests.



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Table 1. Body weights on Day 1 and Day 4 after oil (n= 14), OeB (n= 13), or

wt loss (n= 21)

Day 1 Day 4 Absolute %

body wts (g) body wts (g) change (g) change

Oil 307 7.8 315 7.5 7.4 1.4 2.5 0.5

OeB 311 9.7 302 9.3 9.3 1.6 3.0 0.5

Wt loss 300 6.3 289 5.9 10 1.1 3.5 0.3

Significantly different from oil (P< 0.001).

Results

Body weights

Table 1 shows the average body weights of rats on Day 1and Day 4 of the oestrogen replacement protocol used forFos IR and in situ hybridization as well as the absolute andpercentage change in the body weights of these groupsacross the 4-day protocol. As expected, there was an effectof group (F (2, 45)= 57.7, P< 0.001) on the percentage

change in body weight, with OeB and weight loss groupslosing weight, while the oil group gained weight. Posthoc analyses revealed similar reductions in body weight(P= 0.36) in OeB and weight loss rats and both groupsweighed significantly less (P< 0.001) than oil-treatedrats.

Isoproterenol-induced Fos immunoreactivity

Figure 1 shows the average number of Fos-positive nucleiin four hypothalamic structures after administration ofsaline or Iso. Fos IR after injection of saline was low in all

areas and administration of Iso greatly increased Fos IR inall brain regions examined.As shown in Fig. 1, the PVN had the highest levels of

Fos IR after saline, which were similar in oil, OeB andweight loss groups. Isoproterenol significantly increasedFos IR in the PVN of all three groups (F (1,31)= 113,

200

150

100

50

0Saline/PVN Iso/PVN Saline/SON Iso/SON Saline/SFO Iso/SFO Saline/OVLT Iso/OVLT

Fos-positiv

enuclei

Oil

Oeb

Wt loss

*

*

Figure 1. Fos-positive nuclei after injection of 0.15 M NaCl (saline) or Iso in rats treated with oil, OeB, or

weight loss

Oilsaline, n = 5; OeBsaline, n = 5; wt losssaline, n = 5; oilIso, n = 9; OeBIso, n = 8, wt lossIso, n = 9.

Abbreviations: paraventricular nucleus (PVN), subfornical organ (SFO), supraoptic nucleus (SON), organum

vasculosum of the lamina terminalis (OVLT). Significantly different from oilIso; significantly different from

oilsaline (data shown as means S.E.M.).

P< 0.001). Although there was a tendency for Fos IR tobe greater in the weight loss group, this difference was notstatistically significant (P= 0.10). Figure 2 shows photo-micrographs of representative coronal sections throughthe PVN. The Fos IR observed after Iso in all three groupsoccurred mostly in the lateral magnocellular subnucleusof the PVN; however, labelling was more extensive in theweight loss group.

Isoproterenol significantly increased Fos IR in the SON

(F (1, 31)= 490, P< 0.001) and this increase was similarin the three groups. Interestingly, there was an interactionbetween drug and group (F(2, 31)= 6.46, P< 0.05)) andpost hocanalyses revealed that OeB rats had significantlygreater Fos IR (P< 0.05) after saline than did oil-treatedrats (see also Hartley et al. 2004), but were not different(P= 0.06) from weight loss rats. Figure 3 shows photo-micrographs of representative coronal sections throughthe SON, which illustrates that Fos IR after Iso wasdistributed throughout the SON in all groups.

Fos IR in the SFO after saline was similar in all groups(Fig. 1). As expected, Iso significantly increased Fos IR

in the SFO (F (1,33)= 114, P< 0.001). There was aninteraction between group and drug (F (2,33)= 4.97,P< 0.05) and post hoc analyses revealed that OeB(P< 0.05) and weight loss (P< 0.001) rats hadsignificantly less Fos IR in the SFO after Iso than didoil-treated rats. Wt loss and OeB-treated rats had similar



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C

B

D

A

PVN

lMG

250-m

Figure 2. Coronal sections of PVNCoronal sections (40 m) at 10 showing Fos IR in the

PVN of rats treated with: 0.15 M NaCl (A), oil and Iso (B),

OeB and Iso (C), wt loss and Iso (D). Abbreviation: lateral

magnocellular subnucleus (lMG). Scale bar, 250 m.

Fos IR in the SFO after Iso (P= 0.097), although Foslabelling tended to be less extensive in the weight lossgroup, as illustrated in Fig. 4, showing photomicrographsof representative coronal sections through the SFO.

Figure 3. Coronal sections of SON

Coronal sections (40 m) at 10 showing Fos IR in the SON of rats treated with: 0.15 M NaCl (A), oil and Iso (B),

OeB and Iso (C) and wt loss and Iso (D). Scale bar, 250 m.

Finally, Fos IR in the OVLT after saline was not differentin oil, OeB or weight loss groups (Fig. 1). There wasa significant effect of drug on Fos IR in the OVLT (F(1,32)= 84, P< 0.001) but no differences in Fos IR after



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A

C D

SFO

250-mB

Figure 4. Coronal sections of SFO

Coronal sections (40 m) at 10 showing Fos IR in the SFO of rats treated with: 0.15 M NaCl (A), oil and Iso (B),


Iso in the three groups. Figure 5 shows photomicrographsof representative coronal sections through the OVLT,which demonstrate that Fos IR was located in more lateralparts of the OVLT in all groups.

AB

CD

250-m

Figure 5. Coronal sections of OVLT

Coronal sections (40 m) at 5 showing Fos IR in the OVLT of rats treated with: 0.15 M NaCl (A), oil and Iso (B),


In situ hybridization for AT1 receptor mRNA

Figure 6 shows the average AT1 mRNA expression, asindicated by optical density, in the PVN and SFO. AT1



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0.04

0.035

0.03

0.025

0.02

0.015

0.01

0.005

0

PVN SFO

OpticalD

ensity(arbitraryunits) Oil

Oeb

Wt loss

* *

Figure 6. AT1 mRNA expression

AT1 mRNA expression in the PVN and SFO of rats treated with oil,

n = 6; OeB, n = 5; or wt loss, n = 6 (data shown as means S.E.M.)., significantly different from oil.

mRNA expression was similar in the PVN of oil, OeB andweight loss rats (F (2,14)= 0.1, P= 0.91); however, therewas an effect of group on AT1 receptor mRNA expression

in the SFO (F (2, 13)= 4.18, P< 0.05). Post hoc analysesrevealed that AT1 receptor mRNA expression in the SFOwas significantly less in OeB (P< 0.05) and weight loss(P< 0.05) rats than in oil-treated rats, whereas OeB andweightlossratshadsimilarAT1receptormRNAexpressionin the SFO (P= 0.58).

Plasma renin activity

Figure 7 shows the average plasma renin activity (PRA)in oil and weight loss rats after saline or Iso. Plasmarenin activity after saline was similar in oil and weightloss rats. There was a significant effect of drug on PRA

(F (1,34)= 98.7, P< 0.001, Fig. 7) with greater than afourfoldincreaseinPRA;however,thisincreasewassimilarin the two groups.

Isoproterenol-induced water intake

Figure 8 shows the average water intake by oil and weightloss rats after saline or Iso. There was an effect of drug

0

2

4

6

8

10

12

Saline Iso

PRA(ngml1h

1)

Oil

Wt loss

n=8 n=9 n=9 n=12

Figure 7. Plasma renin activity

Plasma renin activity (PRA) in oil and wt loss rats after 0.15 M NaCl or

Iso (data shown as means S.E.M.).

(F (1,7)= 40.8, P< 0.001), with water intake after Isosignificantly greater than that after saline. Interestingly,there was a significant interaction between drugand group(F (1,7)= 3.86, P< 0.05) and post hoc analyses revealedthat weight loss rats drank significantly less water after Isothan did oil-treated rats (P< 0.01). When intakes wereexpressed per 100 g body weight, similar differences were

found (data not shown).

Discussion

The goal of the present study was to evaluate the effects ofOeB and weight loss on hypothalamic structures criticalfor controlling behavioural and physiological responsesto increased circulating Ang II. Oestrogen and weight lossrats had decreasedlevels of Fos IRand of AT1 mRNAin theSFO when compared with the oil-treated rats. In addition,despite similar PRAs, weight loss rats consumed about halfthe amount of water as did oil-treated rats after Iso, intakes

that were very similar to those of OeB and oil-treated ratsin our previous study (Krause et al. 2003). Taken together,theseresultssuggestthatthereductionsinbodyweightthataccompany OeB replacement contribute to OeB effectson RAS-stimulated water intake. To our knowledge, thisstudy demonstrates for the first time that body weightloss affects central responses to endogenously generatedAng II, possibly via the AT1 receptor, and suggests specificbehavioural consequences related to that activation.

AngiotensinIIisamajorinfluenceinbodyfluidhomeo-stasis and the -agonist Iso greatly elevates circulatinglevels of this peptide (Stocker et al. 2000). Hypotension

and consequent increases in circulating Ang II activatebrain regions implicated in body fluid regulation (Oldfield& McKinley, 1994; Rowland et al. 1994). Therefore, itis not surprising that Iso elicited robust Fos IR in theSON, PVN, SFO and OVLT in the present study. TheFos IR in the SFO and OVLT after Iso is probably theresult of circulating Ang II binding to AT1 receptors intheseCVOs, as peripheral pre-treatment with AT1 receptor

0

1

2

3

4

5

6

7

8

9

Saline Iso

WaterIntake(ml) Oil

Wt loss *

Figure 8. Water intake

Water intake of oil and wt loss rats after injection of 0.15 M NaCl

(saline) or Iso (n = 8; within subjects design; data shown as

means S.E.M.). , significantly different from oilIso.



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antagonists greatly reduces Iso-elicited Fos IR in thesenuclei (Oldfield & Mckinley, 1994). On the other hand,hypotension-induced activation of the SON and PVN isbelieved to be largely dependent on baroreceptor signalsbecause elimination of baroreceptor input by sinoaorticdenervation reduces, but does not eliminate, Fos IR inthese nuclei after hypotension (Potts et al. 1997). It has

been hypothesized that RAS activation contributes tothis residual Fos IR because circulating Ang II activatesangiotensin-responsive neurons in the SFO and OVLT andthese neurons send excitatory projections to the SON andPVN (Ferguson & Renaud, 1986). Thus, a combinationof baroreceptor input and circulating Ang II probablycontribute to the Fos IR we observed in the SON and PVNafter hypotension.

Although we did not examine the phenotype ofFos-positive neurons in this study, insight can be gainedfrom the pattern of activation. After Iso, Fos IR in theOVLT was confined to the lateral margins, the region that

contains the greatest densityof AT1 receptors. Thispatternof Fos IR in the OVLT is suggestive of Ang II-mediatedthirstratherthanosmoticallydriventhirst,whichproducesFos IR in the dorsal cap (Oldfield et al. 1994). Similarly, thedistribution of Fos IR in the PVN provides informationabout the function of neurons activated by Iso. In allthree groups, Iso elicited robust Fos IR that was mostprevalent in the lateral magnocellular subnucleus of thePVN. Many neurons within this region are neurosecretoryneurons that release vasopressin into systemic circulationvia the posterior pituitary. Circulating vasopressin actson the kidney to promote water conservation and onperipheral vasculature to produce vasoconstriction, whichare compensatory responses to volume loss or hypo-tension. Vasopressin release is modulated by baroreceptorinput (Potts et al. 1997) as well as by Ang II (Lee et al.1995). Thus, the robust activation we observed in thelateral magnocellular subdivision of the PVN and in theneurosecretory neurons of the SON is consistent withIso-induced hypotension and stimulation of the RAS.

In the PVN, there was a tendency for Iso-induced FosIR to be elevated in the weight loss rats, although thedifference was not statistically significant (P= 0.10). It ispossible that this trend was due to stress caused by foodrestriction, because experimental procedures that limit

food availability elevate basal levels of plasma cortico-sterone (Kiss et al. 1994). Circulating Ang II activatesneurons in the PVN that project to the median eminence(Ferguson, 1988), including neurons that contain cortico-tropin releasing hormone. Thus, it is possible that foodrestriction combined with Iso may be sufficiently stressfulto activate corticotropin releasing hormone-containingneurons in thePVN of weight loss rats; however, additionalexperiments will be necessary to address this issue.

Inourstudy,OeBselectivelydecreasedFosIRintheSFOafter Iso. These results conflict with those of another study

in which central Ang II administration did not decreaseFos in the SFO of OeB-treated rats but did increase Fos inthePVN(Kisleyetal. 2000). It is likely that methodologicaldifferences explain why we did not see increased Fos IR inthe PVN of our OeB-treated rats but, interestingly, Kisleyand colleagues had predicted that OeB would attenuateFos IR in the SFO after Ang II because their previous

work showed that OeB decreased Ang II binding to AT1receptors in the SFO (Kisley et al. 1999). Thus, our studyexamining Fos IR elicited by endogenously generatedAng II may have revealed the effect that the authors ofthis previous study proposed.

It is interesting that OeB and weight loss selectivelydecreased Iso-elicited Fos IR and AT1 receptor mRNAin the SFO. Earlier studies demonstrated that oestrogendecreases neural activity and AT1 receptor binding in theSFO (Kisley et al. 1999; Tanaka et al. 2001); however, toour knowledge, ours is the first study to demonstrate thatweight loss also produces similar effects. This reduction

in AT1 receptor mRNA probably underlies the bluntedIso-elicitedFosIRthatweobservedinbothOeBandweightloss rats.

Consistent withthis idea, Iso produced similar increasesin PRA, an accurate indicatorof circulating Ang II (Stockeret al. 2000), in weight loss and oil-treated rats. Moreover,comparisons with our previous study (Krause et al. 2003)indicate percentage increases from basal PRA after Isoin OeB (421%) and oil- (288%) treated rats similar tothose in the present study (oil, 260%; weight loss, 543%).PRA commonly is used as an indicator of plasma Ang IIlevels; however, it is a measurement of the amount ofangiotensin I generated during a set period of time, andnot the quantity of Ang II produced. Therefore, OeBor weight loss may interfere with other components ofthe biosynthetic pathway, such as angiotensin convertingenzyme, and result in different levels of circulating Ang II.In this regard, increased basal PRAs have been reported inrats (e.g. Katayama & Lee, 1985) and humans (e.g. Krakoff,1973; Pallas et al. 1977) with oestrogen replacement;however, these studies employed long-term oestrogenreplacement or used very high oestrogen doses. Thus,methodological differences may explain the discrepancybetween the results of these studies and our previous studyin which we did not observe an effect of oestrogen on basal

PRA in rats (Krause et al. 2003). In any case, it is clearthat the attenuation of Iso-elicited Fos IR we observed inthe SFO is not due to decreased PRA after OeB or weightloss. Thus, we suggest that OeB and weight loss inhibitSFO responses to circulating Ang II by down-regulatingthe expression of the AT1 receptor.

Site-specific injection of AT1 receptor antagonists intothe SFO decreases Iso-elicited water intake (Fitts, 1994);therefore, it would be expected that reductions in AT1receptor mRNA in the SFO would be accompaniedby an attenuation of Iso-elicited water intake as was



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observed in the present study. Weight loss rats, whichhad decreased AT1 mRNA in the SFO, drank significantlyless water after Iso (3.6 1.1 ml) than did oil-treated rats(7.2 1.2 ml) and these intakes were very similar to thoseobserved in our previous study (Krause et al. 2003) thatexamined the effects of OeB on water intake after Iso(oil, 6.0 0.5 ml; OeB, 2.8 0.8 ml). Thus, central and

behavioural responses to RAS activation are very similarin OeB and weight loss rats. We cannot rule out thepossibilitythattheacutenatureoftheweightlossafterfoodrestriction may affect responses to circulating Ang II bydifferent mechanisms than does the more gradual weightloss that accompanies OeB replacement. Nonetheless,taken together, the obtained data suggest that weight losscontributes to OeB attenuation of Ang II-stimulated waterintake and central activation.

ItispossiblethatOeBandweightlosshavesimilareffectson the central and behavioural responses to circulatingAng II, albeit through different mechanisms. Although

we did not phenotype Fos-positive cells in this study,it is possible that Iso activated different populations ofcells in the SFO of OeB and weight loss rats. The SFOcontains cholinergic receptors and activation of thesereceptors by direct injection of carbachol also stimulatesdrinking (Mangiapane & Simpson, 1983). Moreover, theSFO is responsive to other circulating factors that initiatewater consumption such as amylin (Riediger et al. 1999).However, the nature of our stimulus and the fact that bothOeB and weight loss rats had attenuated AT1 mRNA in theSFO make these possibilities unlikely.

Although we propose that weight loss is an importantcontributing factor for the observed effects, an intriguingalternative explanation focuses on weight gain. In the rat,ovariectomy is associated with hyperphagia that results in1216% increase in body weight over 5 weeks and muchof this weight gain is due to increased adipose tissue(McElroy & Wade, 1987). Obese post-menopausal womenhave elevated blood pressure that is greatly attenuated bya 5% weight loss (Engeli et al. 2005). Additionally, thehypertension that is observed in obese rats is reversed byadministrationofAT1receptorantagonists(Boustanyetal.2005). These studies suggest that weight gain enhancescardiovascular responses to circulating Ang II. In thepresentstudy,oil-treatedrats,whichincreasedbodyweight

by 2.5%, had greater Fos IR in the SFO and augmentedwater intakes relative to ovariectomized rats that ate lessandlost weight.Moreover, theweightgain that occurs afterovariectomy did not affect basal PRA, which is consistentwithstudiesexaminingtheeffectofobesityonPRA(Faloiaet al. 2002; Becker et al. 2003). Thus, weight gain alsomay enhance the central and behavioural responses tocirculating Ang II.

Interestingly, Becker et al. (2003) reported that obeserats had a 100% increase in AT1 receptors in the proximal

tubule of the kidney and suggested that up-regulation ofthese receptors may contribute to the observed hyper-tension by increasing water and sodium reabsorption. Inthis regard, the onset of hypertension is accelerated inobese rats given access to NaCl solutions, implicating abehavioural component to the high blood pressure thatis observed in obese rats (Dobrian et al. 2003). In our

study, oil-treated rats had greater AT1 mRNA in the SFOrelative to ovariectomized rats that lost weight, suggestingthat increased body weight also may up-regulate AT1receptors in the brain. Given the role of AT1 receptors inthe SFO to promote the ingestion of water and sodium(see Fitzsimons, 1998, for review), it seems likely thatincreased AT1 receptors in the SFO may further exacerbatehypertension in obese subjects by augmenting water andsodium intake. In other words, weight gain may result inreceptor-mediated enhancement of renal and behaviouralresponses to Ang II with deleterious consequences forcardiovascular health.

The mechanism responsible for modulation of AT1receptorsby changesin body weight is unknown. However,severalcomponentsoftheRASarepresentinadiposetissue(Karlsson et al. 1998), suggesting a role for adipose RASin obesity-related hypertension. In fact, angiotensinogenin circulation and in adipose tissue is elevated in obesehypertensive rats (Boustany et al. 2004). Additionally,obesehypertensive post-menopausal women have elevatedcirculating angiotensinogen (Engeli et al. 2005). Recentwork by Kurdi et al. (2005) showed that components ofthe RAS are mediators of gene expression, an observationthat may provide insights into the mechanism underlyingbody weight effects on the regulation of AT1 receptors.

In summary, the current study examined the effectof OeB or weight loss on central responses to increasedcirculating Ang II. Both oestrogen and weight lossdecreasedtheresponsivenessofSFOneuronstocirculatingAng II, as indicated by decreased Fos IR, and selectivelyreduced the expression of AT1 mRNA in the SFO.Similar to our previous studies of oestrogen effects onRAS-stimulated water intake (Krause et al. 2003), weightloss also reduced water intake elicited by Iso but did notaffect stimulated PRAs. Thus, we propose that the weightloss that accompanies OeB replacement in ovariectomizedrats contributes to OeB effects on RAS-stimulated water

intake via down-regulation of AT1 receptors in the SFO.

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Acknowledgements

We thank Lisa Eckel, Frank Johnson and Zuoxin Wang for their

valuable advice and assistance. This work was supported by NIH

grants DK063754 (E.G.K.), DC006360 (K.S.C.), T32 NS07437(T.L.S.) and DC04785 (R.J.C.).


) at CAPES Usage on October 8 2010jp physoc orgDownloaded from J Physiol (

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