Hormones and Behavior 57 (2010) 284–290

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Hormones and Behavior

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Potentiated effect of systemic administration of oxytocin on hypertonic NaCl intakein food-deprived male rats

Antonio Bernal ⁎, Javier Mahía, Amadeo PuertoPsychobiology, University of Granada, Campus of Cartuja, Granada 18071, Spain

⁎ Corresponding author. Psicobiología, UniversidadCartuja s/n, Granada, Spain. Fax: +34 958 24 62 39.

E-mail address: antoniobernal@ugr.es (A. Bernal).

0018-506X/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.yhbeh.2009.12.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 July 2009Revised 15 December 2009Accepted 18 December 2009Available online 4 January 2010

Keywords:OxytocinSubcutaneous administrationFood-deprivationWater intakeNaCl intakeFood intakeUrine volumeNatriuresisMale rat

Subcutaneous administration of oxytocin (OT) increases water intake and sodium/urine excretion in food-deprived male rats. This study analyzes the effect of OT administration (at 0830 and 1430h) on theconsumption of water and hypertonic NaCl (1.5%). In the first experiment, injections of OT increased theintake of hypertonic NaCl (but not of water) in food-deprived rats but not in ad lib-fed animals during thesecond 12 h (2030 to 0830) of the treatment day. The net concentration of the fluid consumed by OT/deprived animals was close to isotonic. In the second experiment, the initial effect of OT administration wasan increase in urine volume and urinary sodium excretion and concentration by food-deprived animalsduring the first 12 h (0830 to 2030). These findings suggest that in food-deprived animals, systemicadministration of OT induces NaCl intake as a consequence of previous urine loss and urinary sodiumexcretion.

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Introduction

Maintenanceof appropriate levels of sodium in theorganismrequiresthe participation of various physiological and behavioral regulatorymechanisms. Sodium appetite was described over 70 years ago by CurtRichter, who found that adrenalectomy induced the consumption ofhypertonic saline solutions (Richter, 1936). Body sodium balance is aneuroendocrine-dependent process involving hormones such as angio-tensin II and aldosterone, which protect against the loss of sodium in thebody and generate an appetite for salt (Epstein, 1982; Fitzsimons, 1998;Fregly and Rowland, 1985; Johnson and Thunhorst, 1997). Conversely,sodium intake might be inhibited by hormones such as atrial natriureticpeptide, which is involved in the excretion of extracellular body sodium(Antunes-Rodrigues et al., 1986; McCann et al., 1996).

Oxytocin (OT) is a neurohormone secreted from the neurohypoph-ysis through nerve fibers from the supraoptic nucleus and magnocel-lular component of the paraventricular nucleus (Binkley, 1995; Leng etal., 2005; Morris, 2006). In rats, results of the i.c.v. administration of OTor OT antagonists and peripheral administration of high doses of OTsuggest that this neuropeptide inhibits sodium appetite (Blackburn etal., 1992a,b) and water intake (Arletti et al., 1990; Fitts et al., 2003).However, systemic administration of lower doses ofOT increasedwaterintake (Bernal et al., 2007) and polyethylene glycol (PEG)-inducedwater and NaCl intakes (Stricker and Verbalis, 1987).

Systemic administration of OT has also been implicated in theexcretion of urine and body sodium (Conrad et al., 1986; Gimpl andFahrenholz, 2001; Haanwinckel et al., 1995; Huang et al., 1995;Schmidt et al., 1990; Stoeckel and Freund-Mercier, 1989; Verbalis etal., 1991; Walter et al., 2000; Windle et al., 1995, 1997), physiologicalprocesses closely linked to hypovolemic thirst and sodium hunger(Fitzsimons, 1961; Johnson and Thunhorst, 1997; Phillips andSumners, 1998; Stricker, 1966, 1981; Stricker and Sved, 2000).Systemic OT administration increases water intake, a behavioral effectthat appears to be dependent on the sodium intake, since it is onlyobserved in animals that are food deprived or fed with low sodiumdiets but not in those fed with standard food (Bernal et al., 2007).

With this background, the main objectives of this study were todetermine whether the peripheral administration of OT enhances theintake of hypertonic NaCl (Experiment 1) and whether it inducessodium excretion prior to these ingestive effects (Experiment 2). Itwas specifically examined whether the food intake influences thebehavioral effects of OT by using ad lib-fed and food-deprived animalsin both experiments.

Materials and methods

Experiment 1

Subjects and groupsTwenty-eight adult male Wistar rats from the breeding colony at

the University of Granada (290–340 g) were used in this study.

Fig. 1. Timeline of experimental procedure and measurements in Experiment 1. During the first 48 h (days 1 and 2), animals had ad lib access to water and food. At 0830 on day 3,food was withdrawn from half of the animals. Water was always available for all animals. At 2030 on day 3, a bottle with 1.5% NaCl was placed in the metabolic cage. Ad lib-fed andfood-deprived Experimental and Control groups received s.c. administration of OT or Vehicle, respectively, at 0830 and 1430 on day 3 (see text for further details).

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Animals were housed in individual cages in a roommaintained at 21–23 °C on a 12/12-h light–dark cycle (lights on at 0830). Use andhandling of the animals followed animal research guidelines estab-lished by European Community Council Directive (86/609/EEC) andSpanish Law (1201/2005).

After several days of habituation to the laboratory, the animalswere randomly divided into four groups of 7 animals as follows: OTwith food available (OT/ad lib), Control with food available (C/adlib), OT without food available (OT/deprived), and Control withoutfood available (C/deprived). The animals were then given ad libitumaccess to water and food (Sandermus dry chow with 0.25% NaCl,Unión Alimentaria Sanders, Sanders S.A, Madrid) for a 48-h baselineperiod.

Experimental procedureThe experiment lasted 72 h. Body weight and water and food

intakes were recorded daily during the 48-h pre-injection period(measures obtained during the second 24 h were consideredbaseline values). During the final 24 h of the experiment (day 3),animals were placed in metabolic cages with water available ad lib(Tecniplast 3701MO-000). Food was withdrawn from the OT/deprived and C/deprived groups. The OT/ad lib and C/ad lib groupshad ad libitum access to food and water throughout the experiment(Fig. 1).

At 0830 on day 3, OT/ad lib and OT/deprived animals received asubcutaneous (s.c.) (Stricker and Verbalis, 1987; Uvnäs-Moberg et al.,1996) injection of 0.5 ml with 11 μg of OT diluted in distilled water(Oxytocin, Laboratorios Iven, Madrid) at the midline of the lumbarregion. As previously shown, this dose generates a significant

Table 1Water and food intakes and body weight on day 2 (baseline), and water intake, urine volumefor first 12 h) in Experiment 1.

Experiment 1

24-h baseline (day 2) First 12 h o

Groups n Water intake(ml)

Food intake(g)

Body weight(g)

Water intak(ml)

C/ad lib 7 37±2 25±1 331±19 7.9±1.6OT/ad lib 7 34±2 25±1 318±11 7.2±0.6C/deprived 7 33±3 25±1 300±28 4.5±0.9OT/deprived 7 31±6 24±1 314±29 4.9±1.3

OT administration and food-deprivation produced a fall in water “balance” and body weighincreased urine excretion (vs. C/deprived and OT/ad lib animals). Water “balance”was comgroups; OT: Oxytocin groups).

a pb0.01 ad lib-fed vs. food-deprived groups.b pb0.01 OT vs. Control groups.c pb0.01 OT/deprived vs. C/deprived.d pb0.01 OT/deprived vs. OT/ad lib.

natriuretic response when measured after 6 h (Bernal et al., 2007).At 1430, a second dose was administered in an identical manner. C/adlib and C/deprived groups were injected with distilled waterfollowing the same procedure.

This administration route appears to allow the hormone to enterthe bloodstream progressively through the initial formation of asubcutaneous bolus that gradually decreases in volume over time. Weused a double s.c. administration in order to maximize the effect ofthis neurohormone, which has a plasmatic half-life of only a fewminutes (Binkley, 1995).

At 2030 (6 h after the second injection), the water intake, urinevolume, and body weight of animals were recorded (using a redlight to avoid interference with the light–dark cycle), and asolution of 1.5% NaCl was introduced into the metabolic cage (Falkand Young, 1956; Kutscher and Steilen, 1973; Stellar, 1954; Weinerand Stellar, 1951). After another 12 h (at 0830 on day 4), thewater intake, NaCl intake, urine volume, and body weight wererecorded.

Statistical analysisModule ANOVA/MANOVA of the Statistica software was used to

analyze data during baseline and the first and second 12-h periodand entire 24-h period of the treatment day. MANOVA (Raostatistic) was automatically performed when multiple dependentvariables were specified. Water and food intakes and body weightwere analyzed for baseline. For the first 12 h of the treatment day,water intake and urine volume were also analyzed. The water“balance” (water intake minus urine volume) was calculated fromthese data, and a MANOVA was performed to jointly analyze this

, water “balance”, body weight change, and food and NaCl intakes at 2030 on day 3 (data

f treatment day (day 3)

e Urine volume(ml)

Water “balance”(ml)

Body weight change(g)

Food intake(g)

6.3±0.8 1.7±1.7a −7.1±2.1a 7.4±0.98.0±0.4 −0.8±0.5a,b −11.6±1.7a,b 5.5±0.76.5±0.7 −2.0±0.3 −15.3±1.1 –

13.1±1.3c,d −8.1±1.9b −20.7±1.7b –

t. The combination of food-deprivation and OT administration (OT/deprived animals)puted as the water intake minus urine volume (C: Control groups; depr.: food-deprived

286 A. Bernal et al. / Hormones and Behavior 57 (2010) 284–290

balance with body weight change for the first 12 h. For the second12 h of the treatment day, water and NaCl intakes and urine volumewere analyzed. Fluid “balance” (water plus NaCl intakes minus urinevolume for the second 12 h) were calculated from these data, and aMANOVA was performed to jointly analyze these balances withbody weight change for the second 12 h. A further MANOVA wasperformed to analyze fluid “balance” and body weight change after24 h (data for first plus second 12-h period, see Fig. 1).

ANOVAs (F values) were used to analyze: the body weight ofanimals before each s.c. administration; the amount of food consumedby ad lib-fed animals during days 2 and 3 of the experiment; theamount of water and food consumed from 0830 to 2030 of thetreatment day; and thewater, water plusNaCl, net fluid concentration,and food consumed by groups during the second 12 h (0830 to 2030).

Planned comparisons (F values) between the pairs of groups thatdiffered in only one aspect (C/ad lib vs. C/deprived or OT/ad lib andOT/deprived vs. C/deprived or OT/ad lib) were used to analyzesignificant effects. The Bonferroni correction for multiple comparisonswas applied. Data are presented as means±SEM.

Experiment 2

Subjects, groups and experimental procedureForty-four adult male Wistar rats (n=11 animals per group) from

the breeding colony at the University of Granada were kept under thesame conditions as in Experiment 1. They received the injection of OTor distilled water at 0830 and 1430 (as in Experiment 1), and waterintake, urine volume, and body weight were recorded at 2030, whenthe experiment ended.

Urinary sodium concentrations (mEq/l) were measured by using anautomatic analyzer (Ilyte analyzer, Instrumentation Laboratory, USA).Plasma sodium concentration that resulted from fluid intake and urineloss from 0830 to 2030 were estimated by using the following formula(Stricker, 1981): 1000⁎[(0.150) (0.69) (BW)+sodium “balance”]/[(0.69) (BW)+water “balance”], where BW was the body weight (ingrams) of the animal at the time of testing (2030). The sodium “balance”(in mEq) was computed as the difference between ingested sodium infood and sodium loss in urine (from 0830 to 2030).

Fig. 2. Water and 1.5% NaCl intakes (A) and urine volume (B) of the four groups (n=7) durifluid consumption and urine volume were significantly higher in OT/deprived animals thandeprived vs. C/deprived).

Statistical analysisMANOVAs were used to analyze water and food intake and body

weight during the baseline period; water “balance” and body weightchange from 0830 to 2030; and urinary sodium excretion andconcentration of the urine excreted from 0830 to 2030.

ANOVAs were used to analyze the body weight of animals beforeeach s.c. administration; water and food intakes and sodium “balance”from 0830 to 2030; and estimated plasma sodium concentration at2030.

Planned comparisons (C/ad lib vs. C/deprived and OT/ad lib andOT/deprived vs. C/deprived and OT/ad lib) were used to analyzesignificant effects and urine excretion from 0830 to 2030 (with theBonferroni correction for multiple comparisons). Data are presentedas means±SEM.

Results

Experiment 1

Baseline data and body weights of groups before s.c. administrationof substances

Water and food intakes during baseline did not significantly differamong the four animal groups (Table 1). There were no differences inbody weight among groups before the first (Table 1) or second s.c.administration of substances (326±20 g for C/ad lib, 308±11 g forOT/ad lib, 287±27 g for C/deprived and 299±29 g for OT/deprivedat 1430). The amount of food consumed by C/ad lib and OT/ad libgroups during day 3 did not significantly differ from that consumedduring baseline (25±1 vs. 25±1 for C/ad lib and 22±1 vs. 25±1 forOT/ad lib respectively).

Data for first 12 h (0830 to 2030) of the treatment day (Table 1)

Water intake and urine volume. Twelve hours after the first injection(at 2030 on day 3), the water intake and urine volume of groups weresignificantly dependent on the interaction between food availabilityand substance injected (Rao (2,23)=3.8; pb0.04). Urine volume wasgreater in OT/deprived than in OT/ad lib (F(1,24)=17.2; pb0.01) or

ng the second 12 h (2030 to 0830) of the treatment day (Experiment 1). NaCl and totalin OT/ad lib and C/deprived groups (#pb0.01 OT/deprived vs. OT/ad lib; ⁎pb0.01 OT/

Fig. 3. Fluid “balance” (A) and body weight change (B) of the four groups (n=7) during the second 12 h (2030 to 0830) of the treatment day (Experiment 1). Fluid “balance” wascomputed as water plus NaCl intakes minus urine volume (●pb0.01 ad lib-fed vs. food-deprived groups; ■pb0.01 OT vs. Control groups).

287A. Bernal et al. / Hormones and Behavior 57 (2010) 284–290

C/deprived animals (F(1,24)=28.7; pb0.01). However, no significantdifferences in water consumption were observed among the fourgroups.

Water “balance” and body weight change. Water “balance” and bodyweight were reduced by food-deprivation (Rao (2,23)=14.7;pb0.01) and by OT administration (Rao (2,23)=6.5; pb0.01).

Food intake in ad lib-fed groups. No significant differences in foodintake were observed between the ad lib-fed OT and Control groups.

Data for second 12 h (2030 to 0830) of the treatment day

Water and NaCl intakes, urine volume, and fluid intake concentration.During the 12 h of darkness, NaCl intake and urine volume weresignificantly related to the interaction between food availability andsubstance injected (Rao (3,22)=6.3; pb0.01). NaCl intake wasgreater in OT/deprived than in OT/ad lib (F(1,24)=17.1; pb0.01)and C/deprived animals (F(1,24)=43.7; pb0.01) (Fig. 2A). The totalfluid intake (NaCl pluswater) was also greater in OT/deprived animals(F(1,24)=12.1; pb0.01 vs. OT/ad lib group and F(1,24)=16.1;pb0.01 vs. C/deprived group). The volume of urine excreted wasgreater in the OT/deprived group than in the OT/ad lib (F(1,24)=25.0;pb0.01) and C/deprived (F(1,24)=9.0; pb0.01) groups (Fig. 2B).

Table 2Fluid “balance” and body weight change of animals after 24 h of day 3 in Experiment 1.

Groups 24 h of treatment day

n Fluid “balance” (ml) Body weight change (g)

C/ad lib 7 16.8±2.0a 4.7±1.6a

OT/ad lib 7 19.3±1.4a 2.8±1.6a

C/deprived 7 2.4±1.4 −26.5±1.4OT/deprived 7 3.6±2.1 −24.7±2.4

Fluid “balance” was computed as the net water plus NaCl intakes minus urine volume.After 24 h, no significant differences were observed between C/ad lib and OT/ad libgroups or between C/deprived and OT/deprived groups (C: Control groups; depr.:food-deprived groups; OT: Oxytocin groups).

a pb0.01 ad lib-fed vs. food-deprived groups.

However, the water intake did not differ among the four groups ofanimals (Fig. 2A).

The net concentration of the fluid consumed was significantlyrelated to the interaction between food availability and substanceinjected (F(1,24)=6.1; pb0.03). The concentration of the fluid con-sumedby theOT/deprivedgroupwasvirtually isotonic (0.7±0.08%) andsignificantly greater than that consumed by the C/deprived group(0.26±0.10%) (F(1,24)=14.9; pb0.01); the net concentration of fluidconsumed in the OT/ad lib group and C/ad lib group was 0.55±0.05%and 0.50±0.10%, respectively.

Fluid “balance” and body weight change. Fluid “balance” and bodyweight were reduced by food-deprivation (Rao (2,23)=94.8;pb0.01) and by OT administration (Rao (2,23)=9.0; pb0.01) (Fig. 3).

Food intake in ad lib-fed groups. ANOVA results showed nosignificant differences in food intake between ad lib-fed groups(17.7±1.5 for C/ad lib and 16.4±0.6 for OT/ad lib).

Behavioral and physiological data after 24 h (Table 2)After 24 h (from 0830 on day 3 to 0830 on day 4), body fluid

“balance” and body weight change were greater in ad lib-fed (vs.food-deprived) groups (Rao (2,23)=129.6; pb0.01) and did notdiffer between OT and Control groups Table 2.

Experiment 2

Baseline data and body weights of groups before s.c. administration ofsubstances

Water and food intake during baseline did not significantly differamong the four animal groups (Table 3). Results obtained show nodifferences in body weight among groups before the first (Table 3)or second s.c. administration of substances (415±17 g for C/ad lib,420±17 g for OT/ad lib, 391±8 g for C/deprived and 406±11 g forOT/deprived at 1430).

Data for first 12 h (0830 to 2030) of the treatment day

Water intake, urine volume, water “balance”, and body weight change(Table 3). As in the first experiment, urine volume was greater in

Table 3Water and food intakes and body weight on day 2 (baseline), and water intake, urine volume, water “balance”, body weight change, and food and NaCl intakes at 2030 on day 3 (datafor first 12 h) in Experiment 2.

Experiment 2

24-h baseline (day 2) First 12 h of treatment day (day 3)

Groups n Water intake(ml)

Food intake(g)

Body weight(g)

Water intake(ml)

Urine volume(ml)

Water “balance”(ml)

Body weight change(g)

Food intake(g)

C/ad lib 11 35±3 20±2 423±21 6.0±0.6 3.8±0.5 2.6±0.7a −9.6±0.7a 4.8±0.8OT/ad lib 11 34±3 21±2 431±17 5.8±0.9 5.6±0.9 −0.2±1.3a,b −12.8±2.4a,b 6.4±0.8C/deprived 11 33±2 20±2 402±09 6.6±0.6 4.1±0.7 2.1±0.7 −11.7±1.5 –

OT/deprived 11 30±1 20±1 422±14 4.8±0.2 9.2±0.6c,d −5.0±0.5b −19.1±2.2b –

As in Experiment 1, OT administration and food-deprivation produced a fall in water “balance” and body weight. The combination of food-deprivation and OT administration (OT/deprived animals) increased urine excretion (vs. C/deprived and OT/ad lib animals). Water “balance” was computed as the water intake minus urine volume (C: Control groups;depr.: food-deprived groups; OT: Oxytocin groups).

a pb0.01 ad lib-fed vs. food-deprived groups.b pb0.01 OT vs. Control groups.c pb0.01 OT/deprived vs. C/deprived.d pb0.01 OT/deprived vs. OT/ad lib.

288 A. Bernal et al. / Hormones and Behavior 57 (2010) 284–290

OT/deprived than in OT/ad lib (F(1,31)=14.2; pb0.01) or C/deprived(F(1,31)=28.7; pb0.01) animals at 12 h after the first injection (2030on day 3).Water “balance” and bodyweight were increased in Controlversus OT groups (Rao (2,28)=15.2; pb0.01) and in ad lib-fed versusfood-deprived groups (Rao (2,28)=3.9; pb0.01).

Natriuresis and urinary sodium concentration. Urinary sodiumexcretion (Fig. 4A) and urinary sodium concentration (Fig. 4B) weresignificantly related to the interaction between food availability andsubstance injected (Rao (2,38)=3.4, pb0.05). OT increased urinarysodium excretion and concentration in ad lib-fed (OT/ad lib vs. C/adlib) (F(1,39)=52.2; pb0.01 and F(1,39)=52.0; pb0.01) and food-deprived (OT/deprived vs. C/deprived) (F(1,39)=123.5; pb0.01and F(1,39)=48.0; pb0.01) animals. A larger amount of sodium (F(1,39)=8.2; pb0.01) was excreted by the OT/deprived animalsthan by the OT/ad lib group. Food-deprivation reduced the urinarysodium concentration in OT animals (F(1,39)=7.0; pb0.012) (Fig. 4B).

Food intake in ad lib-fed groups. ANOVA results showed no signi-ficant differences in food intake between ad lib-fed groups (Table 3).

Sodium “balance”. Sodium “balance” was related to the interactionbetween food availability and substance injected (F(1,39)=7.3;pb0.01). Sodium balance of OT/deprived animals (−3.03 mEq) wassignificantly lower than that of C/deprived (−0.29±0.06 mEq; F(1,39)=118.1; pb0.01) or OT/ad lib animals (−2.04±0.29 mEq; F(1,39)=14.7; pb0.01). The sodium balance of OT/ad lib animals wassignificantly lower than that of C/ad lib animals (−0.27±0.08 mEq; F(1,39)=46.5; pb0.01).

Estimated plasma sodium concentration. Plasma sodium concen-tration was greater in Control versus OT groups (F(1,29)=87.15;pb0.001) (148.79±0.57 mEq/l for C/ad lib, 144.27±1.38 mEq/l forOT/ad lib, 147.78±0.34 mEq/l for C/deprived and 140.94±0.49 forOT/deprived animals).

Discussion

This study reports the effects of systemic OT administration onurine excretion and water and NaCl intakes in ad lib-fed and food-deprived rats.

First twelve hours

OT administration initially increased urine excretion in food-deprived animals during the first 12 h (0830 to 2030) of the treatmentday. The polyuric effect of OT was accompanied by an increase in

urinary sodium excretion and concentration but not water intake(Tables 1 and 3 and Fig. 4). These renal effects may be related to thefood-deprivation effect as well as to the hormonal action, since theresponses in OT/deprived animals significantly differed from those inOT/ad lib and C/deprived animals. Dose-dependent polyuric andnatriuretic effects were previously observed in food-deprived animalsat 6 h after the s.c. injection of 2.7–11 μg of OT (Bernal et al., 2007).These results are in agreement with a potential action on OT receptorsin kidney (Gimpl and Fahrenholz, 2001; Stoeckel and Freund-Mercier,1989).

The polyuric effect of OT and its lack of effect on water intake gaverise to a reduction in the water “balance” and body weight of food-deprived animals during the first 12 h (0830 to 2030) of the treatmentday (Tables 1 and 3). The natriuretic effect of OT in conjunction withthe unavailability of sodium also reduced the sodium “balance” of theOT/deprived animals.

In ad lib-fed animals, OT administration did not increase urineexcretion or water intake and produced a lesser natriuretic response.Consequently, the deficits in water and sodium “balances” and in bodyweight shown by OT/ad lib animals were less marked than those inOT/deprived animals (Tables 1 and 3 and Fig. 4).

Second twelve hours

During the second 12 h (2030 to 0830), animals in the OT/deprived group consumedmore 1.5% NaCl (Fig. 2) than did animals inthe OT/ad lib and C/deprived groups, despite the aversive taste of thissolution (Falk and Young, 1956; Kutscher and Steilen, 1973; Stellar,1954; Weiner and Stellar, 1951). This higher consumption ofhypertonic NaCl was accompanied by increases in urine excretion,water “balance”, and body weight, with no increase in water intake(Figs. 2 and 3). In a mouse study, Kutscher and Steilen (1973) alsoobserved that food-deprivation alone induced higher consumption ofthe same hypertonic solution (1.5% NaCl) without modifying waterconsumption, which the authors ascribed to the excretion of bodysodium. In ad lib-fed animals, however, the water, NaCl and foodintake and urine volume were not significantly affected by OT,although a net increase in body fluid “balance” and body weight wasproduced. As a result, the changes in body fluid “balance” and bodyweight of both OT-treated groups matched those observed in Controlanimals (Table 2). Recovery of normal fluid “balance” and bodyweight was also observed in OT-treated animals with access to wateralone (Bernal et al., 2007). Compensation over time for the effects ofOT has also been observed in the study of other behaviors. Thus,Lokrantz et al. (1997) found that the intraoral intake of glucose wasinitially inhibited after OT administration but subsequently increased,with no difference in total intake between treated and control

Fig. 4. Urinary sodium excretion (A) and concentration (B) of the four groups (n=11) during the first 12 h (0830 to 2030) of the treatment day (Experiment 2). Urinary sodiumexcretion and concentration were greater in OT-treated animals (■pb0.01 OT/ad lib vs. C/ad lib; #pb0.012 OT/deprived vs. OT/ad lib; ⁎pb0.01 OT/deprived vs. C/deprived).

289A. Bernal et al. / Hormones and Behavior 57 (2010) 284–290

animals. Likewise, Björkstrand and Uvnäs-Moberg (1996) reported aninitial reduction and a subsequent increase in food intake and bodyweight after OT administration, and Petersson et al. (1999) observedan initial increase and subsequent reduction in arterial pressure.

The combined intake of 1.5% NaCl and water by OT/deprivedanimals led to their consumption of a virtually isotonic solution at asignificantly higher concentration than was consumed by C/deprivedanimals. This behavior is characteristic of animals in hypovolemicstate, which show preference for isotonic solutions, and of animalswith simultaneous access to hypertonic NaCl and water. This isbecause isotonic solutions are the most appropriate for restoring thevolume and composition of extracellular body fluid (Kutscher andSteilen, 1973; Stricker and Jalowiec, 1970) and are probably theoptimal method for alleviating volemic thirst (Smith and Stricker,1969). Hence, consistent with the sodium excretion observed in thepresent study, the estimated plasma sodium concentrations werelower in OT-treated animals than in control animals. When OT-treated animals have access to water and a more hypertonic solution(3% NaCl), the intake of a net isotonic solution implies an increase inboth NaCl and water intakes (Stricker and Verbalis, 1987).

The loss of body fluids and salts generates an increase in bothsodium appetite and water intake (Fitzsimons, 1961; Johnson andThunhorst, 1997; Phillips and Sumners, 1998; Stricker, 1966, 1981;Stricker and Sved, 2000). An increase in water intake was alsoobserved after OT administration to food-deprived animals withaccess to water alone (Bernal et al., 2007). Conversely, the i.p.injection of hypertonic doses of NaCl (1.5, 3.0 and 5.0%) progressivelyreduces the polydipsic effect of OT (submitted).

Our behavioral findings (NaCl intake) are compatible with theNaCl intake observed using sodium deficient diets and lower OT doses(≈2.6 to 5.3 μg/rat) released over 24 h by minipumps (Stricker andVerbalis, 1987). The physiological results (natriuresis) obtained areconsistent with the dose-dependent natriuretic effect of s.c. OT infood-deprived animals (Bernal et al., 2007) and evenwith the effect ofs.c. OT at physiological plasma concentrations in rats fed ad lib withlow sodium diets (Verbalis et al., 1991).

In contrast, peripheral administration of higher doses of OT(≥150 μg/rat) to ad lib-fed rats (with standard diet) exertscontroversial effects (Olson et al., 1991), inhibiting food and fluidintake in some studies (Arletti et al., 1989, 1990, but see Arletti et al.,

1993) but not in others (Björkstrand and Uvnäs-Moberg, 1996;Uvnäs-Moberg et al., 1996). It should be taken into account that 2‰ ofperipherally administered OT can cross the blood–brain barrier andreach the brain (Jones and Robinson, 1982), where it acts on OTreceptors related to ingestive behaviors (Gimpl and Fahrenholz, 2001;Verbalis, 1999). It has been demonstrated that i.c.v. administration ofOT inhibits food and water intakes (Arletti et al., 1990), whereas i.c.v.administration of OT receptor antagonist enhances angiotensin- andnaloxone-induced sodium appetite and induces per se the intake ofwater (Blackburn et al., 1992a,b; Fitts et al., 2003). It should be bornein mind that Arletti et al. (1990) examined the ingestive behavior oftheir animals for only 15–60 min after OT administration, a muchshorter time period than studied in the present and other previousstudies (Bernal et al., 2007; Stricker and Verbalis, 1987; Uvnäs-Moberg et al., 1996). Hence, it is also possible that the effects of OTadministration may differ over the course of time, with an initialinhibitory effect that is subsequently compensated for (Björkstrandand Uvnäs-Moberg, 1996).

In summary, this study demonstrates that systemic OT adminis-tration to food-deprived male rats increases their urine volume andurinary sodium excretion and concentration and induces hypertonicNaCl intake. The reduction in dietary sodium (vs. OT/ad lib animals)may facilitate the NaCl intake observed in OT/deprived animals.Consistent with this possibility, OT administration potentiates thevolemic NaCl intake induced by PEG administration in animals fed adlibwith sodium deficient diets (Stricker and Verbalis, 1987). Likewise,OT-induced increases in water intake (as only available fluid) wereobserved in food-deprived animals and in rats fed ad lib with lowsodium diet (0.04% NaCl) but not in rats with free access to a standarddiet (Bernal et al., 2007). Differences in the effects of OT betweenfood-deprived and ad lib-fed rats include not only NaCl and waterintakes but also intraoral glucose intake and body weight (Lokrantz etal., 1997; Sohlström et al., 1999).

The negative sodium and water “balances” induced by OTadministration in food-deprived rats would presumably enhanceangiotensin system activity, triggering salt intake (Fitzsimons, 1998;Stricker and Verbalis, 1987), although other factors cannot be ruledout. Further studies are required to specifically confirm that the lossof urine and/or mineral salts are determining factors for thepolydipsia (Bernal et al., 2007) and increased intake of hypertonic

290 A. Bernal et al. / Hormones and Behavior 57 (2010) 284–290

NaCl (present data) produced when OT is administered to food-deprived animals.

Acknowledgments

This study was submitted by the first author in partial fulfillmentof requirements for his PhD in Psychology (Psychobiology) at theUniversity of Granada, Granada (Spain). We are grateful to RichardDavies for his assistance with the English version of the text and to D.Vilchez (Iven/Rodivet Laboratory, Guadix) for the generous gift ofoxytocin. This research was supported in part by the University ofGranada, Andalusian Regional Government (CTS-430), and SpanishMinistry of Education and Culture/Feder (National R+D Plan:BSO2003-06627, SEJ2006-06710 and SEJ2007-61839).

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