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In utero extracellular dehydration modifies thirst in neonatal rats
Appetite 51 (2008) 599–603
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Appetite journal homepage: www.elsevier.com/locate/appet
Research report
In utero extracellular dehydration modifies thirst in neonatal rats C. Perillan, M. Costales, M. Vijande, J. Arguelles * Area de Fisiologia, Departamento de Biologia Funcional, Facultad de Medicina, C/Julian Claveria s/n 33006 Oviedo, Universidad de Oviedo, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2008 Received in revised form 7 March 2008 Accepted 21 April 2008
The main aim of this study was to investigate the effect of maternal extracellular dehydration during pregnancy in rats on the development of thirst mechanisms in the offspring. Pregnant rats underwent three episodes of extracellular dehydration induced by injecting s.c. 15 ml/kg b.w. of a 20% wt/vol solution of polyethylene glycol (PEG) in saline. The treatment given on days 14, 17 and 20 postconception is thought to induce endocrine and natriophilic responses similar to those elicited by vomiting. The offspring were tested for their responses to three different thirst stimuli at 2, 4 and 6 days of age. Like the controls, the offspring from PEG-treated mothers responded to beta stimulation by isoproterenol at 6 days of age. However, they failed to respond to cellular dehydration (NaCl hypertonic injection) at 2 days of age or to extracellular dehydration by PEG on day 4. In conclusion, offspring exposed to in utero extracellular dehydration do not respond to cellular dehydration at 2 days of age or to extracellular dehydration at 4 days of age, whereas control pups had already developed an appropriate response to these stimuli. According to these results, it therefore seems that in utero conditions determine the development of adaptive thirst responses in offspring. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Thirst Ontogeny Gestation Extracellular dehydration Development NaCl
Introduction Several adaptations in maternal haemodynamics and hormonal and biochemical variables occur during pregnancy that enable normal foetal growth and development. Changes in in utero environmental conditions have a potential impact and, to a certain extent, ‘‘program’’ postnatal development of offspring, even in adult life (Barker, 1995; Godfrey & Barker, 2000). For instance, an association between the systolic blood pressure of progeny and maternal vomiting during gestation in humans has been reported (Malaga et al., 2005), adding a new element of evidence to the ‘‘Barker hypothesis’’. Episodes of extracellular dehydration by polyethylene glycol administration in pregnant rats led to an increased salt appetite in adult offspring (Nicolaı¨dis, Galaverna, & Metzler, 1990). An increased salt appetite has also been found in offspring when acute and repeated mineralofluid losses had occurred during pregnancy after diuretic-natriuretic treatment (Galaverna, Nicolaı¨dis, Yao, Sakai, & Epstein, 1995). Arguelles, LopezSela, Brime, Costales, and Vijande (1996) have suggested that a high salt environment in utero conditions the mechanisms underlying cardiovascular responsiveness to pressor substances like angiotensin II. Pups from mother rats with diet-induced hypertension appear to be insensitive to the hypertensive effects of sodium chloride
* Corresponding author. E-mail address: [email protected] (J. Arguelles). 0195-6663/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.appet.2008.04.015
(Langley-Evans & Jackson, 1996). Handelmann, Russell, Gainer, Zerbe, and Bayorh (1983) and Handelmann and Sayson (1984) showed that neonatal exposure to vasopressin induced long-term changes in renal responses to this hormone. Arguelles, Brime, LopezSela, Perillan, and Vijande (2000) and Perillan, Costales, Diaz, Vijande, and Arguelles (2004) have described modifications in the ingestive behaviour of offspring from hypereninemic, hypertensive and natriophilic mothers in rats. In addition to immediate homeostatic responses, the developing organism may make predictive adaptive responses of no immediate advantage, though with long-term consequences (Gluckman & Hanson, 2004). This ‘‘developmental origins of health and disease’’ concept may have important biological, medical and socioeconomic implications. Taken as a whole, these findings suggest that a hydrosaline and hormonally-altered environment during pregnancy may modify, among others, certain thirst responses later in life. Water drinking responsiveness to thirst stimuli has a well known sequential activation pattern. Newborn rats respond to cellular dehydration at 2 days of age, to hypovolemia at 4 days and to beta-adrenergic activation not before 6 days (Wirth & Epstein, 1976). Changes in the ontogeny of drinking behaviour after the manipulation of the RAS system during pregnancy has already been reported (Perillan et al., 2004; Perillan, Costales, Vijande, & Arguelles, 2007). The purpose of the present study was to elucidate the potential influence of acute changes in maternal homeostasis induced by extracellular dehydration on the development pattern
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of drinking behaviour in offspring after standardized dipsogenic challenges. Methods Animals All experiments were performed on Wistar rats (from the University of Oviedo vivarium), housed individually in a light and temperature controlled room (12 h light/dark; 21 1 8C). Animal care was in accordance with guidelines from EEC Directive 86/609 and the study had the approval of the Institutional Animal Ethical Committee. The rats had free access to a standard laboratory diet, tap water and 2.7% NaCl (liquids available from graduated glass tubes fitted with glass spouts), where appropriate. A group of 30 female rats (250–300 g) were mated and pregnancy-assessed by daily vaginal smears, revealing the presence of spermatozoids on day 1 of pregnancy. They were then divided in two groups as follows: (a) Polyethylene glycol rats (D-PEG). On day 14, 17 and 20 postconception, the pregnant rats received a 15 ml/Kg s.c. injection of PEG (MERCK) 20% wt/vol in isotonic saline (Fitzsimons, 1961; Stricker, 1981). (b) Control (D-sPEG). Female rats were injected with saline vehicle on days 14, 17 and 20 postconception and were used as the control for the PEG group.
All injections were made under light ether anaesthesia. To prevent the rats from compensating for their treatment-induced hypovolemia, water was not available during 12 h after injection (Nicolaı¨dis et al., 1990). Volume intakes of the two fluids (water and 2.7% saline) were recorded after 5, 10, 15, 30, 60, 120 min and 24 h. Daily water intake and 2.7% saline was recorded in all groups. Immediately after completion of the intake test period in the pups, these were sacrificed by decapitation and 5 ml of blood were collected. Hematocrit was determined immediately and the remaining blood was centrifuged at 4 8C. Plasma osmolality (vapour pressure osmometer – WESCOR) and [Na+] plasmatic (Flame Photometer – CORNNING G410) were measured. The remaining plasma was stored at 20 8C for renin activity determinations (RIA: DRG DIAGNOSTIC, Sensitivity 0.2 ng, intraassay variance: 12.5%).
(SIGMA): 500 mg/kg, in 6-day-old pups. The other three pups from each litter were subcutaneously injected with 0.15 M NaCl (control). All subcutaneous injections were made over the scapulae at a volume of 1.25 ml/100 g b.w. just before testing, except when PEG was the challenge, in which case the litter was injected 4 h prior to testing. (D) Testing: The pups were weighed and replaced in the box after the challenge for a 2-h test period. Skin temperature was still maintained at 33 1 8C under the 25-W lamp. The apparatus for thirst testing the pups consisted of an infusion pump delivering room temperature distilled water through a PE-50 plastic spout extending 2 mm beyond the blunted end of a needle. The infusion rate was 0.7 ml/min. Each rat was offered a 15 s bout every 15 min, over a period of 2 h (9 bouts for each rat). It could either lick, struggle or remain active. (E) End of testing. At the end of the 2-h test period, each rat was weighed. Finally, percent weight gain over the test period was calculated. Weight gain was used as a measure of water ingested, since there was no other source of weight gain (no mother available). Evaporative loss is assumed to be the same for all challenge and control rats, and there is no spontaneous excretion in the suckling rat according to Wirth and Epstein (1976). At the end of the test, each pup was decapitated using a razor blade, its blood was collected in heparinized capillary tubes and hematocrit and osmolality were determined. Statistical analysis The results are presented as means S.E. The unpaired ‘‘t’’ Student test was used where appropriate. Values of p < 0.05 were deemed statistically significant. Results Mothers The number of D-PEG impregnated rats giving delivery was smaller than in the D-sPEG group (70% vs. 87%, N.S.). The number of pups per litter and the duration of pregnancy were not significantly affected by treatment. Biological data obtained during pregnancy are shown in Table 1. Salt and water intake significantly increased (p < 0.01) during 1, 2 and 24 h after extracellular dehydration in mother dams by PEG treatment on days 14, 17 and 20 of pregnancy (Fig. 1A and B). However, water and salt intakes returned to normal values (vs. control) on the other days of pregnancy (data not shown). Maternal blood parameters are shown in Table 2. No significant differences in hematocrit, plasma osmolality, PRA levels and Na+ plasma were found in D-sPEG vs. D-PEG dams.
Intake testing in offspring Offspring The day of birth was designated as ‘‘day 0’’. The final inspection of litters was at 17:00 h P.M. Litters, usually 8–12 pups, were kept intact until the day of testing. The protocol first described by Wirth and Epstein (1976) consisted of five steps: (A) Deprivation: four hours prior to testing, the litter size was adjusted to 8 pups (4 females and 4 males). The pups were separated from the dam during the last 2 h and placed under a 25-W lamp; skin temperature was monitored using a thermistor probe and was maintained at 33 8C. Pups were weighed to the nearest 0.01 g before and after deprivation. (B) Nursing: pups were then returned to the dam for 1 h and 45 min and re-weighed. In order to minimize the effect of hunger on subsequent thirst testing, one pup of each sex that had gained the least weight during the nursing period was excluded. (C) Challenge: Three pups from each litter were subcutaneously injected with one of the following thirst stimuli: (i) hypertonic saline (1 M): 2.5 mmol/ 100 g b.w., in 2-day-old pups; (ii) polyethylene glycol (PEG) (MERCK,): 30% wt/vol, in 4-day-old pups; and (iii) isoproterenol
D-PEG offspring (O-PEG) showed a significantly lower (p < 0.05–0.01) body weight at 2 and 6 days of age vs. descendants of control dams (O-sPEG). No differences were found at 4 days of age (Fig. 2). Hypertonic challenge at 2 days. At 2 days of age, O-sPEG significantly responded to subcutaneous hypertonic saline, drinking more than vehicle-injected pups (p < 0.01); whereas O-PEG Table 1 Influence of treatment on pregnancy Group
Duration of pregnancy (days)
Pups per litter
D-sPEG D-PEG
21.3 0.4 (9) 21.7 0.4 (14)
12.4 0.8 (9) 12.0 0.8 (14)
Values are presented as means S.E.M. (n); D-sPEG = pseudo polyethylene glycol treated dams; D-PEG = polyethylene glycol treated dams.
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Fig. 1. Water (A) and saline (NaCl 2.7%) intake (B) by PEG-treated dams (D-PEG; n = 14) and by pseudo-treated dams (D-sPEG; n = 9) over a 24-h test period. (**p < 0.01).
Table 2 Blood parameters (dams) Group
Hematocrit (%) Osmolality (mOsm) PRA (ng/ml/h AngI) Na+ (mEq/l)
D-sPEG 39.3 1.4 (9) 302.6 3.5 (9) D-PEG 38.8 1.4 (14) 302.9 2.4 (14)
0.8 0.5 (5) 1.0 1.3 (5)
165.5 23.4 (4) 162.5 38.5 (4)
Values are presented as means S.E.M. (n); PRA = plasma renin activity; DsPEG = pseudo polyethylenglycol treated dams; D-PEG = polyethylenglycol treated dams.
Fig. 2. Body weight of offspring from PEG-treated dams (O-PEG) and from pseudotreated dams (O-sPEG) on the day of intake tests (2, 4 and 6 days old). (n in brackets at the bottom of bars; *p < 0.05; **p < 0.01).
Fig. 3. Water intake by offspring from PEG-treated dams (O-PEG) and by offspring from pseudo-treated dams (O-sPEG) over a 2-h test period. Thirst challenges: (A) NaCl (1 M) at 2 days of age; (B) PEG at 4 days of age; (C) Isoproterenol at 6 days of age. (n in brackets at the bottom of bars; *p < 0.05; **p < 0.01).
failed to respond (Fig. 3A). No differences were found between OPEG and O-sPEG after vehicle injection or after hypertonic saline. Hypovolemic challenge at 4 days. At 4 days of age, PEG induced a significant response in O-sPEG (p < 0.01). However, it was totally ineffective in stimulating water intake in O-PEG. No differences were found between O-PEG and O-sPEG after vehicle injection (Fig. 3B). Isoproterenol challenge at 6 days. At 6 days of age (Fig. 3C), isoproterenol caused significant water intake both in O-sPEG and in O-PEG (p < 0.01). Blood parameters. Blood parameters of pups measured at the end of ingestive testing are shown in Table 3. At 2 days of age, no alterations in blood parameters were found. At 4 days of age, hematocrit was lower in O-PEG vs. O-sPEG (p < 0.05), both in vehicle- and PEG-treated pups; however, plasma osmolality in PEG-treated O-PEG was significantly higher than in PEG-treated O-
C. Perillan et al. / Appetite 51 (2008) 599–603
602 Table 3 Offspring blood parameters Treatment
Hematocrit (%) O-sPEG
O-PEG
2-Days 2-Days 4-Days 4-Days 6-Days 6-Days
31.1 0.8 (10) 29.3 1.2 (11) 30.6 0.9*,a (13) 31.1 1.2*,b (12) 30.2 0.8*,a (12) 29.9 1.2*,c (11)
29.5 0.8 28 1.5 27.6 0.9 27.8 0.9 27.2 0.7 26.9 0.7
(vehicle) (NaCl 1 M) (vehicle) (PEG) (vehicle) (isoproterenol)
Osmolality (mOsm)
(9) (8) (16) (15) (12) (14)
O-sPEG
O-PEG
308.5 10.2 (10) 314 5.0 (12) 295.4 4.6 (10) 293.2 3.9*,b (10) 288.3 11.9 (6) 282.6 14.0 (5)
308 7.6 (9) 322.3 16.4 (9) 306.1 8.0 (16) 307.4 4.1 (15) 287.9 5.9 (12) 287.7 16.12 (13)
Values are presented as means S.E.M. (n); O-sPEG = offspring from pseudo polyethylene glycol treated dams; O-PEG = offspring from polyethylene glycol treated dams. a Vehicle (O-sPEG vs. O-PEG). b PEG (O-sPEG vs. O-PEG). c Isoproterenol (O-sPEG vs. O-PEG). * p < 0.05.
sPEG (p < 0.05). At 6 days of age, hematocrit was lower both in vehicle- or isoproterenol-treated O-PEG vs. O-sPEG (p < 0.05); whereas no changes were found in osmolality regardless of treatment. Discussion The main aim of this study was to investigate the influence of maternal extracellular dehydration on the maturation of the dipsic mechanisms in offspring. Several alterations of the ontogeny of salt and water intake mechanisms after materno-foetal hydromineral challenges have been reported (Fitzsimons, 1998). Different studies (Contreras & Kosten, 1983; Mouw, Vander, & Wagner, 1978) have demonstrated that salt deprivation in pregnant rats determines increased water intake in adult offspring. Conversely, high salt intakes during pregnancy and lactation have been shown to induce high blood pressure in offspring (Contreras, 1989). Arguelles et al. (2000) reported water and salt intake modifications along with changes in cardiovascular responses to angiotensin II in offspring from hypertensive and natriophilic mother rats. The early programming of hydromineral homeostasis has also been clearly demonstrated by Katovich, Aerni, Cespedes, and Rowland (2001), who found differences in pressor responses to angiotensin in adult rats which had been subjected to different maternal salt consumptions during pregnancy. Nicolaı¨dis et al. (1990) showed that a prenatal history of maternal extracellular dehydration by PEG treatment in rats, which induces high water and salt intakes, is sufficient to increase offspring preference for salty solutions. Increased salt appetite has also been found in offspring after acute diuretic-natriuretic treatment during pregnancy (Galaverna et al., 1995). These experimental paradigms imply the activation of the renin– angiotensin–system (RAS). The origin of such early imprinting has also been investigated. Maternal prenatal salt loading was shown to induce changes in angiotensinogen mRNA in the foetal liver and brain (Mao et al., 2007), providing unequivocal proof that hydromineral challenges in mothers affect the expression of genes in foetuses. Therefore, new light is now shed on the long-term influence of acute changes in maternal salt and water balance on offspring and adult thirst behaviour leading to a better understanding of these processes. Drinking in response to thirst stimuli has a known maturation chronology in neonatal rats (Wirth & Epstein, 1976). These authors were the first to report that pups already drink after cellular dehydration at 2 days of age, after hypovolemia at 4 days and after beta-adrenergic stimulation at 6 days. In our experiments, PEG treatment of pregnant dams did not affect litter size (number of pups per litter). Bird and Contreras
(1986) also failed to find changes in litter size using experimental protocols associated with forced salt intakes during pregnancy. However, we did find a reduction in body weight in pups from PEGtreated dams (O-PEG) at 2 and 6 days of age. This may reflect some impairment of pregnancy conditions produced by PEG treatment that affects foetal and perinatal development. Our results support this hypothesis, since no significant response to hypertonic injection (1 M NaCl) was found in 2-day-old pups from treated mothers (O-PEG). It is worth noting that the overall blood changes in suckling rats (at 2, 4 or 6 days of age) that were subjected to treatments that could affect drinking behaviour are difficult to interpret. Discrepancies from the expected results were found in different cases in our experiments. This also occurred and was duly acknowledged in the original paper by Wirth and Epstein (1976), who described the pattern of consummatory maturation. The deficit in the maturation of thirst mechanisms lasts at least 4 days, as the extracellular stimulus for thirst (PEG injection) in OPEG still fails to evoke significant drinking responses at that time; whereas O-sPEG rats manage to regulate their hydrosaline homeostasis after volume depletion, as expected. Many drugs, particularly chemicals for anaesthesia, can affect foetuses in utero. These drugs can influence foetal development and may also have an impact on imprinting. As both experimental and control groups received ether anaesthesia, the final results observed in this study may therefore not only be caused by PEG treatments during pregnancy, but may also be caused by PEG injections when anaesthesia was induced. Although ether anaesthesia itself may not be sufficient to cause the changes determined in the present study, since the control group was similarly anaesthetized, ether anaesthesia together with PEG during pregnancy may however have produced what was observed in the experiments. From the point of view of drinking behaviour, the O-PEG group normalizes its maturation level at 6 days of age, when these pups responded normally to isoproterenol, as did control pups (O-sPEG). The above information demonstrates that maternal PEG treatment which induces high water and salt intake and volume depletion is associated with the induction of more or less severe changes in the maturation of the dipsogenic responses of progeny. We can only speculate as to the mechanisms that are responsible for the observed alterations in the maturation of thirst mechanisms and drinking. We hypothesize at least three non-mutually exclusive possible causes: (1) The PEG treatment in mothers induces a generalized retardation in maturation; (2) the alteration of the RAS in the mothers and/or foetuses modifies the development of the neural substrate necessary for appropriate thirst responses; and (3) maternal behaviour is altered by the experimental procedure, which in turn affects the development pattern of foetuses and pups.
C. Perillan et al. / Appetite 51 (2008) 599–603
Evidence in favour of the first hypothesis is the lower weight observed in our pups. However, the weight of 6-day-old pups which behave normally is significantly lower than that of control pups. As regards the possible implication of RAS modification in the observed phenomenon, it has been demonstrated that offspring from mother rats treated with DOCA, which depresses RAS, respond normally to beta-adrenergic activation (at 6 days of age) and fail to respond to cellular (2 days of age) and extracellular (4 days of age) dipsogenic stimuli (Perillan et al., 2007). However, enhanced responses were found in 6-day-old pups from hyperreninemic dams (Perillan et al., 2004). From this point of view, offspring from PEG-treated mothers showed a similar pattern of maturation to that observed in offspring from DOCA-treated dams.As to the third hypothesis, regarding the possibility of behavioural changes in mothers as a result of the alterations observed in the offspring in our experimental paradigm, several studies on the ingestive behaviour of offspring from mothers on a high salt diet have suggested that maternal diet could produce both physiological changes during gestation and lactation in mothers and behavioural maternal changes which could eventually condition that of their offspring (Contreras, 1993; Contreras, Wong, Henderson, Curtis, & Smith, 2000) A combination of factors related with all three hypotheses is also plausible, since some experiments showed the existence of significant changes in maternal behaviour in spontaneous hypertensive rats, which at the same time have RAS alterations (Gouldsborough, Black, Johnson, & Ashton, 1998; Rose & McCarty, 1994). Our results provide one more indication that gestational hydrosaline and endocrine status could be determinant in the development of dipsogenic mechanisms, assessed by means of independent ingestive tests in neonatal rats. The plausible alteration in gene expression after early exposure to hydrosaline imbalances (which in turn could determine the organization of neural foetal development) opens up the possibility of delayed alterations. More research is needed in this field to assess a possible physiological or physiopathological role of these findings in late consummatory performance and hydrosaline homeostasis of adult or old individuals. References Arguelles, J., Brime, J. I., Lopez-Sela, P., Perillan, C., & Vijande, M. (2000). Adult offspring long-term effects of high salt and water intake during pregnancy. Hormones and Behaviour, 37, 156–162. Arguelles, J., Lopez-Sela, P., Brime, J. I., Costales, M., & Vijande, M. (1996). Changes of blood pressure responsiveness in rats exposed in utero and perinatally to a highsalt environment. Regulatory Peptides, 66, 113–115.
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Barker, D. J. P. (1995). Fetal origins of coronary heart disease. British Medicine Journal, 311, 171–174. Bird, E., & Contreras, R. J. (1986). Dietary salt affects fluid intake and output patterns of pregnant rats. Physiology and Behaviour, 37, 365–369. Contreras, R. J. (1989). Differences in perinatal NaCl exposure alters blood pressure levels of adult rats. American Journal of Physiology, 256, R70–R77. Contreras, R. J. (1993). High NaCl intake of rat dams alters maternal behavior and elevates blood pressure of adult offspring. American Journal of Physiology, 264, R296–R304. Contreras, R. J., & Kosten, T. (1983). Prenatal and early postnatal sodium chloride intake modifies the solution preferences on adult rats. Journal of Nutrition, 113, 1051– 1062. Contreras, R. J., Wong, D. L., Henderson, R., Curtis, K. S., & Smith, J. C. (2000). High dietary NaCl early in development enhances mean arterial pressure of adult rats. Physiology and Behaviour, 71, 173–181. Fitzsimons, J. T. (1961). Drinking by rats depleted of body fluid without increase in osmotic pressure. Journal of Physiology London, 159, 297–309. Fitzsimons, J. T. (1998). Angiotensin, thirst, and sodium appetite. Physiological Reviews, 78, 583–686. Galaverna, O., Nicolaı¨dis, S., Yao, S. Z., Sakai, R. R., & Epstein, A. N. (1995). Endocrine consequences of prenatal sodium depletion prepare rats for high need-free NaCl intake in adulthood. American Journal of Physiology, 269, R578–R583. Gluckman, P. D., & Hanson, M. A. (2004). Living with the past: Evolution, development, and patterns of disease. Science, 17, 1733–1736. Godfrey, K. M., & Barker, D. J. (2000). Fetal nutrition and adult disease. American Journal of Clinical Nutrition, 71, 1344S–1352S. Gouldsborough, I., Black, V., Johnson, I. T., & Ashton, N. (1998). Maternal nursing behaviour and the delivery of milk to the neonatal spontaneously hypertensive rat. Acta Physiologica Scandinava, 162, 107–114. Handelmann, G. E., Russell, J. T., Gainer, H., Zerbe, R., & Bayorh, M. (1983). Vasopressin administration to neonatal rats reduces antidiuretic response in adult kidneys. Peptides, 4, 827–832. Handelmann, G. E., & Sayson, S. C. (1984). Neonatal exposure to vasopressin decreases vasopressin binding sites in the adult kidney. Peptides, 5, 1217–1219. Katovich, M. J., Aerni, J. D., Cespedes, A. T., & Rowland, N. E. (2001). Perinatal dietary NaCl level: Effect on angiotensin-induced thermal and dipsogenic responses in adult rats. Physiology and Behaviour, 72, 621–627. Langley-Evans, S. C., & Jackson, A. A. (1996). Rats with hypertension induced by in utero exposure to maternal low-protein diets fail to increase blood pressure in response to a high salt intake. Annals of Nutrition & Metabolism, 40, 1–9. Malaga, I., Arguelles, J., Diaz, J. J., Perillan, C., Vijande, M., & Malaga, S. (2005). Maternal pregnancy vomiting and offspring salt taste sensitivity and blood pressure. Pediatric Nephrology, 20, 956–960. Mao, C., Lv, J., Zhu, H., Zhou, Y., Chen, R., Feng, X., et al. (2007). Fetal functional capabilities in response to maternal hypertonicity associated with altered central and peripheral angiotensinogen mRNA in rats. Peptides, 28(6), 1178–1184. Mouw, D. R., Vander, A. J., & Wagner, V. (1978). Effects of prenatal and early postnatal sodium deprivation of subsequent adult thirst and salt preference in rats. American Journal of Physiology, 234, F59–F63. Nicolaı¨dis, S., Galaverna, O., & Metzler, C. H. (1990). Extracellular dehydration during pregnancy increases salt appetite of offspring. American Journal of Physiology, 258, R281–R283. Perillan, C., Costales, M., Diaz, F., Vijande, M., & Arguelles, J. (2004). Thirst changes in offspring of hyperreninemic rat dams. Pharmacology Biochemistry and Behaviour, 79, 709–713. Perillan, C., Costales, M., Vijande, M., & Arguelles, J. (2007). Maternal RAS influence on the ontogeny of thirst in the rat. Physiology and Behaviour, 92, 554–559. Rose, J. L., & McCarty, R. (1994). Maternal influences on milk intake in SHR and WKY pups. Physiology and Behaviour, 56, 901–906. Stricker, E. M. (1981). Thirst and sodium appetite after colloid treatment in rats. Journal of Comparative and Physiological Psychology, 95, 1–9. Wirth, J. B., & Epstein, A. N. (1976). Ontogeny of thirst in the infant rat. American Journal of Physiology, 230, 188–198.
Contents lists available at ScienceDirect
Appetite journal homepage: www.elsevier.com/locate/appet
Research report
In utero extracellular dehydration modifies thirst in neonatal rats C. Perillan, M. Costales, M. Vijande, J. Arguelles * Area de Fisiologia, Departamento de Biologia Funcional, Facultad de Medicina, C/Julian Claveria s/n 33006 Oviedo, Universidad de Oviedo, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 January 2008 Received in revised form 7 March 2008 Accepted 21 April 2008
The main aim of this study was to investigate the effect of maternal extracellular dehydration during pregnancy in rats on the development of thirst mechanisms in the offspring. Pregnant rats underwent three episodes of extracellular dehydration induced by injecting s.c. 15 ml/kg b.w. of a 20% wt/vol solution of polyethylene glycol (PEG) in saline. The treatment given on days 14, 17 and 20 postconception is thought to induce endocrine and natriophilic responses similar to those elicited by vomiting. The offspring were tested for their responses to three different thirst stimuli at 2, 4 and 6 days of age. Like the controls, the offspring from PEG-treated mothers responded to beta stimulation by isoproterenol at 6 days of age. However, they failed to respond to cellular dehydration (NaCl hypertonic injection) at 2 days of age or to extracellular dehydration by PEG on day 4. In conclusion, offspring exposed to in utero extracellular dehydration do not respond to cellular dehydration at 2 days of age or to extracellular dehydration at 4 days of age, whereas control pups had already developed an appropriate response to these stimuli. According to these results, it therefore seems that in utero conditions determine the development of adaptive thirst responses in offspring. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Thirst Ontogeny Gestation Extracellular dehydration Development NaCl
Introduction Several adaptations in maternal haemodynamics and hormonal and biochemical variables occur during pregnancy that enable normal foetal growth and development. Changes in in utero environmental conditions have a potential impact and, to a certain extent, ‘‘program’’ postnatal development of offspring, even in adult life (Barker, 1995; Godfrey & Barker, 2000). For instance, an association between the systolic blood pressure of progeny and maternal vomiting during gestation in humans has been reported (Malaga et al., 2005), adding a new element of evidence to the ‘‘Barker hypothesis’’. Episodes of extracellular dehydration by polyethylene glycol administration in pregnant rats led to an increased salt appetite in adult offspring (Nicolaı¨dis, Galaverna, & Metzler, 1990). An increased salt appetite has also been found in offspring when acute and repeated mineralofluid losses had occurred during pregnancy after diuretic-natriuretic treatment (Galaverna, Nicolaı¨dis, Yao, Sakai, & Epstein, 1995). Arguelles, LopezSela, Brime, Costales, and Vijande (1996) have suggested that a high salt environment in utero conditions the mechanisms underlying cardiovascular responsiveness to pressor substances like angiotensin II. Pups from mother rats with diet-induced hypertension appear to be insensitive to the hypertensive effects of sodium chloride
* Corresponding author. E-mail address: [email protected] (J. Arguelles). 0195-6663/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.appet.2008.04.015
(Langley-Evans & Jackson, 1996). Handelmann, Russell, Gainer, Zerbe, and Bayorh (1983) and Handelmann and Sayson (1984) showed that neonatal exposure to vasopressin induced long-term changes in renal responses to this hormone. Arguelles, Brime, LopezSela, Perillan, and Vijande (2000) and Perillan, Costales, Diaz, Vijande, and Arguelles (2004) have described modifications in the ingestive behaviour of offspring from hypereninemic, hypertensive and natriophilic mothers in rats. In addition to immediate homeostatic responses, the developing organism may make predictive adaptive responses of no immediate advantage, though with long-term consequences (Gluckman & Hanson, 2004). This ‘‘developmental origins of health and disease’’ concept may have important biological, medical and socioeconomic implications. Taken as a whole, these findings suggest that a hydrosaline and hormonally-altered environment during pregnancy may modify, among others, certain thirst responses later in life. Water drinking responsiveness to thirst stimuli has a well known sequential activation pattern. Newborn rats respond to cellular dehydration at 2 days of age, to hypovolemia at 4 days and to beta-adrenergic activation not before 6 days (Wirth & Epstein, 1976). Changes in the ontogeny of drinking behaviour after the manipulation of the RAS system during pregnancy has already been reported (Perillan et al., 2004; Perillan, Costales, Vijande, & Arguelles, 2007). The purpose of the present study was to elucidate the potential influence of acute changes in maternal homeostasis induced by extracellular dehydration on the development pattern
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of drinking behaviour in offspring after standardized dipsogenic challenges. Methods Animals All experiments were performed on Wistar rats (from the University of Oviedo vivarium), housed individually in a light and temperature controlled room (12 h light/dark; 21 1 8C). Animal care was in accordance with guidelines from EEC Directive 86/609 and the study had the approval of the Institutional Animal Ethical Committee. The rats had free access to a standard laboratory diet, tap water and 2.7% NaCl (liquids available from graduated glass tubes fitted with glass spouts), where appropriate. A group of 30 female rats (250–300 g) were mated and pregnancy-assessed by daily vaginal smears, revealing the presence of spermatozoids on day 1 of pregnancy. They were then divided in two groups as follows: (a) Polyethylene glycol rats (D-PEG). On day 14, 17 and 20 postconception, the pregnant rats received a 15 ml/Kg s.c. injection of PEG (MERCK) 20% wt/vol in isotonic saline (Fitzsimons, 1961; Stricker, 1981). (b) Control (D-sPEG). Female rats were injected with saline vehicle on days 14, 17 and 20 postconception and were used as the control for the PEG group.
All injections were made under light ether anaesthesia. To prevent the rats from compensating for their treatment-induced hypovolemia, water was not available during 12 h after injection (Nicolaı¨dis et al., 1990). Volume intakes of the two fluids (water and 2.7% saline) were recorded after 5, 10, 15, 30, 60, 120 min and 24 h. Daily water intake and 2.7% saline was recorded in all groups. Immediately after completion of the intake test period in the pups, these were sacrificed by decapitation and 5 ml of blood were collected. Hematocrit was determined immediately and the remaining blood was centrifuged at 4 8C. Plasma osmolality (vapour pressure osmometer – WESCOR) and [Na+] plasmatic (Flame Photometer – CORNNING G410) were measured. The remaining plasma was stored at 20 8C for renin activity determinations (RIA: DRG DIAGNOSTIC, Sensitivity 0.2 ng, intraassay variance: 12.5%).
(SIGMA): 500 mg/kg, in 6-day-old pups. The other three pups from each litter were subcutaneously injected with 0.15 M NaCl (control). All subcutaneous injections were made over the scapulae at a volume of 1.25 ml/100 g b.w. just before testing, except when PEG was the challenge, in which case the litter was injected 4 h prior to testing. (D) Testing: The pups were weighed and replaced in the box after the challenge for a 2-h test period. Skin temperature was still maintained at 33 1 8C under the 25-W lamp. The apparatus for thirst testing the pups consisted of an infusion pump delivering room temperature distilled water through a PE-50 plastic spout extending 2 mm beyond the blunted end of a needle. The infusion rate was 0.7 ml/min. Each rat was offered a 15 s bout every 15 min, over a period of 2 h (9 bouts for each rat). It could either lick, struggle or remain active. (E) End of testing. At the end of the 2-h test period, each rat was weighed. Finally, percent weight gain over the test period was calculated. Weight gain was used as a measure of water ingested, since there was no other source of weight gain (no mother available). Evaporative loss is assumed to be the same for all challenge and control rats, and there is no spontaneous excretion in the suckling rat according to Wirth and Epstein (1976). At the end of the test, each pup was decapitated using a razor blade, its blood was collected in heparinized capillary tubes and hematocrit and osmolality were determined. Statistical analysis The results are presented as means S.E. The unpaired ‘‘t’’ Student test was used where appropriate. Values of p < 0.05 were deemed statistically significant. Results Mothers The number of D-PEG impregnated rats giving delivery was smaller than in the D-sPEG group (70% vs. 87%, N.S.). The number of pups per litter and the duration of pregnancy were not significantly affected by treatment. Biological data obtained during pregnancy are shown in Table 1. Salt and water intake significantly increased (p < 0.01) during 1, 2 and 24 h after extracellular dehydration in mother dams by PEG treatment on days 14, 17 and 20 of pregnancy (Fig. 1A and B). However, water and salt intakes returned to normal values (vs. control) on the other days of pregnancy (data not shown). Maternal blood parameters are shown in Table 2. No significant differences in hematocrit, plasma osmolality, PRA levels and Na+ plasma were found in D-sPEG vs. D-PEG dams.
Intake testing in offspring Offspring The day of birth was designated as ‘‘day 0’’. The final inspection of litters was at 17:00 h P.M. Litters, usually 8–12 pups, were kept intact until the day of testing. The protocol first described by Wirth and Epstein (1976) consisted of five steps: (A) Deprivation: four hours prior to testing, the litter size was adjusted to 8 pups (4 females and 4 males). The pups were separated from the dam during the last 2 h and placed under a 25-W lamp; skin temperature was monitored using a thermistor probe and was maintained at 33 8C. Pups were weighed to the nearest 0.01 g before and after deprivation. (B) Nursing: pups were then returned to the dam for 1 h and 45 min and re-weighed. In order to minimize the effect of hunger on subsequent thirst testing, one pup of each sex that had gained the least weight during the nursing period was excluded. (C) Challenge: Three pups from each litter were subcutaneously injected with one of the following thirst stimuli: (i) hypertonic saline (1 M): 2.5 mmol/ 100 g b.w., in 2-day-old pups; (ii) polyethylene glycol (PEG) (MERCK,): 30% wt/vol, in 4-day-old pups; and (iii) isoproterenol
D-PEG offspring (O-PEG) showed a significantly lower (p < 0.05–0.01) body weight at 2 and 6 days of age vs. descendants of control dams (O-sPEG). No differences were found at 4 days of age (Fig. 2). Hypertonic challenge at 2 days. At 2 days of age, O-sPEG significantly responded to subcutaneous hypertonic saline, drinking more than vehicle-injected pups (p < 0.01); whereas O-PEG Table 1 Influence of treatment on pregnancy Group
Duration of pregnancy (days)
Pups per litter
D-sPEG D-PEG
21.3 0.4 (9) 21.7 0.4 (14)
12.4 0.8 (9) 12.0 0.8 (14)
Values are presented as means S.E.M. (n); D-sPEG = pseudo polyethylene glycol treated dams; D-PEG = polyethylene glycol treated dams.
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Fig. 1. Water (A) and saline (NaCl 2.7%) intake (B) by PEG-treated dams (D-PEG; n = 14) and by pseudo-treated dams (D-sPEG; n = 9) over a 24-h test period. (**p < 0.01).
Table 2 Blood parameters (dams) Group
Hematocrit (%) Osmolality (mOsm) PRA (ng/ml/h AngI) Na+ (mEq/l)
D-sPEG 39.3 1.4 (9) 302.6 3.5 (9) D-PEG 38.8 1.4 (14) 302.9 2.4 (14)
0.8 0.5 (5) 1.0 1.3 (5)
165.5 23.4 (4) 162.5 38.5 (4)
Values are presented as means S.E.M. (n); PRA = plasma renin activity; DsPEG = pseudo polyethylenglycol treated dams; D-PEG = polyethylenglycol treated dams.
Fig. 2. Body weight of offspring from PEG-treated dams (O-PEG) and from pseudotreated dams (O-sPEG) on the day of intake tests (2, 4 and 6 days old). (n in brackets at the bottom of bars; *p < 0.05; **p < 0.01).
Fig. 3. Water intake by offspring from PEG-treated dams (O-PEG) and by offspring from pseudo-treated dams (O-sPEG) over a 2-h test period. Thirst challenges: (A) NaCl (1 M) at 2 days of age; (B) PEG at 4 days of age; (C) Isoproterenol at 6 days of age. (n in brackets at the bottom of bars; *p < 0.05; **p < 0.01).
failed to respond (Fig. 3A). No differences were found between OPEG and O-sPEG after vehicle injection or after hypertonic saline. Hypovolemic challenge at 4 days. At 4 days of age, PEG induced a significant response in O-sPEG (p < 0.01). However, it was totally ineffective in stimulating water intake in O-PEG. No differences were found between O-PEG and O-sPEG after vehicle injection (Fig. 3B). Isoproterenol challenge at 6 days. At 6 days of age (Fig. 3C), isoproterenol caused significant water intake both in O-sPEG and in O-PEG (p < 0.01). Blood parameters. Blood parameters of pups measured at the end of ingestive testing are shown in Table 3. At 2 days of age, no alterations in blood parameters were found. At 4 days of age, hematocrit was lower in O-PEG vs. O-sPEG (p < 0.05), both in vehicle- and PEG-treated pups; however, plasma osmolality in PEG-treated O-PEG was significantly higher than in PEG-treated O-
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602 Table 3 Offspring blood parameters Treatment
Hematocrit (%) O-sPEG
O-PEG
2-Days 2-Days 4-Days 4-Days 6-Days 6-Days
31.1 0.8 (10) 29.3 1.2 (11) 30.6 0.9*,a (13) 31.1 1.2*,b (12) 30.2 0.8*,a (12) 29.9 1.2*,c (11)
29.5 0.8 28 1.5 27.6 0.9 27.8 0.9 27.2 0.7 26.9 0.7
(vehicle) (NaCl 1 M) (vehicle) (PEG) (vehicle) (isoproterenol)
Osmolality (mOsm)
(9) (8) (16) (15) (12) (14)
O-sPEG
O-PEG
308.5 10.2 (10) 314 5.0 (12) 295.4 4.6 (10) 293.2 3.9*,b (10) 288.3 11.9 (6) 282.6 14.0 (5)
308 7.6 (9) 322.3 16.4 (9) 306.1 8.0 (16) 307.4 4.1 (15) 287.9 5.9 (12) 287.7 16.12 (13)
Values are presented as means S.E.M. (n); O-sPEG = offspring from pseudo polyethylene glycol treated dams; O-PEG = offspring from polyethylene glycol treated dams. a Vehicle (O-sPEG vs. O-PEG). b PEG (O-sPEG vs. O-PEG). c Isoproterenol (O-sPEG vs. O-PEG). * p < 0.05.
sPEG (p < 0.05). At 6 days of age, hematocrit was lower both in vehicle- or isoproterenol-treated O-PEG vs. O-sPEG (p < 0.05); whereas no changes were found in osmolality regardless of treatment. Discussion The main aim of this study was to investigate the influence of maternal extracellular dehydration on the maturation of the dipsic mechanisms in offspring. Several alterations of the ontogeny of salt and water intake mechanisms after materno-foetal hydromineral challenges have been reported (Fitzsimons, 1998). Different studies (Contreras & Kosten, 1983; Mouw, Vander, & Wagner, 1978) have demonstrated that salt deprivation in pregnant rats determines increased water intake in adult offspring. Conversely, high salt intakes during pregnancy and lactation have been shown to induce high blood pressure in offspring (Contreras, 1989). Arguelles et al. (2000) reported water and salt intake modifications along with changes in cardiovascular responses to angiotensin II in offspring from hypertensive and natriophilic mother rats. The early programming of hydromineral homeostasis has also been clearly demonstrated by Katovich, Aerni, Cespedes, and Rowland (2001), who found differences in pressor responses to angiotensin in adult rats which had been subjected to different maternal salt consumptions during pregnancy. Nicolaı¨dis et al. (1990) showed that a prenatal history of maternal extracellular dehydration by PEG treatment in rats, which induces high water and salt intakes, is sufficient to increase offspring preference for salty solutions. Increased salt appetite has also been found in offspring after acute diuretic-natriuretic treatment during pregnancy (Galaverna et al., 1995). These experimental paradigms imply the activation of the renin– angiotensin–system (RAS). The origin of such early imprinting has also been investigated. Maternal prenatal salt loading was shown to induce changes in angiotensinogen mRNA in the foetal liver and brain (Mao et al., 2007), providing unequivocal proof that hydromineral challenges in mothers affect the expression of genes in foetuses. Therefore, new light is now shed on the long-term influence of acute changes in maternal salt and water balance on offspring and adult thirst behaviour leading to a better understanding of these processes. Drinking in response to thirst stimuli has a known maturation chronology in neonatal rats (Wirth & Epstein, 1976). These authors were the first to report that pups already drink after cellular dehydration at 2 days of age, after hypovolemia at 4 days and after beta-adrenergic stimulation at 6 days. In our experiments, PEG treatment of pregnant dams did not affect litter size (number of pups per litter). Bird and Contreras
(1986) also failed to find changes in litter size using experimental protocols associated with forced salt intakes during pregnancy. However, we did find a reduction in body weight in pups from PEGtreated dams (O-PEG) at 2 and 6 days of age. This may reflect some impairment of pregnancy conditions produced by PEG treatment that affects foetal and perinatal development. Our results support this hypothesis, since no significant response to hypertonic injection (1 M NaCl) was found in 2-day-old pups from treated mothers (O-PEG). It is worth noting that the overall blood changes in suckling rats (at 2, 4 or 6 days of age) that were subjected to treatments that could affect drinking behaviour are difficult to interpret. Discrepancies from the expected results were found in different cases in our experiments. This also occurred and was duly acknowledged in the original paper by Wirth and Epstein (1976), who described the pattern of consummatory maturation. The deficit in the maturation of thirst mechanisms lasts at least 4 days, as the extracellular stimulus for thirst (PEG injection) in OPEG still fails to evoke significant drinking responses at that time; whereas O-sPEG rats manage to regulate their hydrosaline homeostasis after volume depletion, as expected. Many drugs, particularly chemicals for anaesthesia, can affect foetuses in utero. These drugs can influence foetal development and may also have an impact on imprinting. As both experimental and control groups received ether anaesthesia, the final results observed in this study may therefore not only be caused by PEG treatments during pregnancy, but may also be caused by PEG injections when anaesthesia was induced. Although ether anaesthesia itself may not be sufficient to cause the changes determined in the present study, since the control group was similarly anaesthetized, ether anaesthesia together with PEG during pregnancy may however have produced what was observed in the experiments. From the point of view of drinking behaviour, the O-PEG group normalizes its maturation level at 6 days of age, when these pups responded normally to isoproterenol, as did control pups (O-sPEG). The above information demonstrates that maternal PEG treatment which induces high water and salt intake and volume depletion is associated with the induction of more or less severe changes in the maturation of the dipsogenic responses of progeny. We can only speculate as to the mechanisms that are responsible for the observed alterations in the maturation of thirst mechanisms and drinking. We hypothesize at least three non-mutually exclusive possible causes: (1) The PEG treatment in mothers induces a generalized retardation in maturation; (2) the alteration of the RAS in the mothers and/or foetuses modifies the development of the neural substrate necessary for appropriate thirst responses; and (3) maternal behaviour is altered by the experimental procedure, which in turn affects the development pattern of foetuses and pups.
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Evidence in favour of the first hypothesis is the lower weight observed in our pups. However, the weight of 6-day-old pups which behave normally is significantly lower than that of control pups. As regards the possible implication of RAS modification in the observed phenomenon, it has been demonstrated that offspring from mother rats treated with DOCA, which depresses RAS, respond normally to beta-adrenergic activation (at 6 days of age) and fail to respond to cellular (2 days of age) and extracellular (4 days of age) dipsogenic stimuli (Perillan et al., 2007). However, enhanced responses were found in 6-day-old pups from hyperreninemic dams (Perillan et al., 2004). From this point of view, offspring from PEG-treated mothers showed a similar pattern of maturation to that observed in offspring from DOCA-treated dams.As to the third hypothesis, regarding the possibility of behavioural changes in mothers as a result of the alterations observed in the offspring in our experimental paradigm, several studies on the ingestive behaviour of offspring from mothers on a high salt diet have suggested that maternal diet could produce both physiological changes during gestation and lactation in mothers and behavioural maternal changes which could eventually condition that of their offspring (Contreras, 1993; Contreras, Wong, Henderson, Curtis, & Smith, 2000) A combination of factors related with all three hypotheses is also plausible, since some experiments showed the existence of significant changes in maternal behaviour in spontaneous hypertensive rats, which at the same time have RAS alterations (Gouldsborough, Black, Johnson, & Ashton, 1998; Rose & McCarty, 1994). Our results provide one more indication that gestational hydrosaline and endocrine status could be determinant in the development of dipsogenic mechanisms, assessed by means of independent ingestive tests in neonatal rats. The plausible alteration in gene expression after early exposure to hydrosaline imbalances (which in turn could determine the organization of neural foetal development) opens up the possibility of delayed alterations. More research is needed in this field to assess a possible physiological or physiopathological role of these findings in late consummatory performance and hydrosaline homeostasis of adult or old individuals. References Arguelles, J., Brime, J. I., Lopez-Sela, P., Perillan, C., & Vijande, M. (2000). Adult offspring long-term effects of high salt and water intake during pregnancy. Hormones and Behaviour, 37, 156–162. Arguelles, J., Lopez-Sela, P., Brime, J. I., Costales, M., & Vijande, M. (1996). Changes of blood pressure responsiveness in rats exposed in utero and perinatally to a highsalt environment. Regulatory Peptides, 66, 113–115.
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