Postnatal development in vasopressin deficient Brattleboro rats with special attention to the hypothalamo-pituitary–adrenal axis function: the role of maternal genotype

Int. J. Devl Neuroscience 27 (2009) 175–183

Contents lists available at ScienceDirect

International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu

Postnatal development in vasopressin deficient Brattleboro rats with special attention to the hypothalamo-pituitary–adrenal axis function: the role of maternal genotype Do´ra Zelena *, Zsuzsa Mergl, Ga´bor B. Makara Hungarian Academy of Sciences, Institute of Experimental Medicine, Szigony u. 43, 1083 Budapest, Hungary

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 July 2008 Received in revised form 31 October 2008 Accepted 12 November 2008

Anomalies in hormonal and neurotransmitter status during perinatal period can lead to lifespan alterations in the central nervous system. Vasopressin is present early in the brain and has various mitogenic, metabolic and physiological actions, e.g. in water homeostasis or in the regulation of the hypothalamo-pituitary–adrenal (HPA) axis. Therefore we examine the possible role of vasopressin in perinatal development with special attention to the influence of maternal genotype and to the HPA axis regulation. We compared homozygous vasopressin deficient (di/di) Brattleboro rats to their heterozygous (di/+) littermates both from di/+ and di/di mother. Higher locomotion due to reduced adaptation was present at preweaning. During the first 10 days of life the di/di pups from di/di mother were the smallest, while in the later perinatal period the genotype of the pups became the more important determinant of the somatic development, namely the di/di pups from both mothers had reduced weight gain. Generally the lack of vasopressin in the pups fastened the somatic development (pinna detachment, eye and ear opening, incisor eruption) however the neurobehavioral development (palmar grasp reflex, righting reflex, negative geotaxis, etc.) was not influenced profoundly by either the mother’s or the pup’s genotype. The lack of vasopressin in pups abolished the 24 h maternal separation induced adrenocorticotrop hormone (ACTH) elevation while the accompanying corticosterone rises were even higher. The vasopressin deficiency of the mother reduced the resting ACTH and all corticosterone levels in all pups. So we can conclude that the lack of vasopressin speeds up the development, probably there is a greater drive for self-sufficiency in these animals. The mother’s vasopressin deficiency reduced the HPA axis reactivity of the pups. The role of vasopressin in the HPA axis regulation is important during the perinatal period independently from the mother’s genotype. The large discrepancy between ACTH and corticosterone regulation requires further studies. ß 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Keywords: Vasopressin Neonatal period ACTH Corticosterone Maternal separation

1. Introduction Anomalies in hormonal and neurotransmitter status during early stages of brain development can lead to lifespan alterations in functioning of the central nervous system (Anisman et al., 1998). The neuropeptide vasopressin is considered to play an important role in the water homeostasis (Decaux et al., 2008) and the immaturity of the posterior pituitary with inadequate vasopressin was postulated to contribute to the decreased ability of newborn animals to concentrate urine. However, later studies demonstrated that the hypothalamo-neurohypophyseal system is already pre-

* Corresponding author. Tel.: +36 1 210 9400x290; fax: +36 1 210 9951. E-mail address: [email protected] (D. Zelena). Abbreviations: ACTH, adrenocorticotrop hormone; CRH, corticotropin-releasing hormone; di/+, heterozygous vasopressin deficient Brattleboro rat; di/di, homozygous vasopressin deficient Brattleboro rat with diabetes insipidus; HPA, hypothalamo-pituitary–adrenal axis; RIA, radioimmunoassay. 0736-5748/$34.00 ß 2008 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2008.11.003

sent prenatally and immunoreactive vasopressin was detected in the plasma shortly after birth (Sinding et al., 1980a,b). Besides its role in water homeostasis vasopressin is also involved in the regulation of the hypothalamo-pituitary–adrenal (HPA) axis (Antoni, 1993). The HPA axis, a fundamental effector of stress reaction, plays a crucial role in coping with challenges. The main hypothalamic component of this axis is the corticotrophinreleasing hormone (CRH), while vasopressin, co-localized in the parvocellular neurons of the paraventricular nucleus of the hypothalamus, may potentiate its effect (Evans et al., 1993). To find out the role of vasopressin in physiological functions the genetically vasopressin deficient Brattleboro strain offers a good tool (Bohus and de Wied, 1998). However, the literature is inconsistent, because there are reports on normal as well as on reduced adrenocorticotrop hormone (ACTH) rise after an acute challenge in adult vasopressin deficient animals (Buckingham and Leach, 1980; Kjaer et al., 1993; Makara et al., 2004; Zelena et al., 2004). Moreover, the breeding conditions (e.g. the genotype of the

176

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

mother) may have profound influence not only on prenatal development (Snijdewint et al., 1988), but also on the stress reactivity of the pups in later life (Zelena et al., 2003b). Namely, reduced ACTH secretion given to 60 min restraint was measured in vasopressin deficient offspring of vasopressin deficient mothers only. Even less is known about the regulation of the HPA axis during development. In rats the HPA axis is already functional in late gestation, however, data are controversial as to when the axis becomes fully functional (Palkovits and Mitro, 1968). It was demonstrated that the regulation of hypothalamic CRH gene expression was not mature perinatally (Grino et al., 1989), whereas the regulation of hypothalamic vasopressin gene expression matured very early (Grino and Oliver, 1992). So during the rat perinatal period vasopressin may be the main regulator of the HPA (Avishai-Eliner et al., 1995; Levine, 2002; Zelena et al., 2008) in contrast to adulthood, where CRH seems to be the more important regulator (Aguilera et al., 2004). The role of vasopressin was extended also to other brain function such as circadian rhythmicity, memory consolidation, and body temperature regulation. Vasopressin seems to act centrally as a neurotransmitter rather than a hormone. From the sites of synthesis in the cell bodies of the supraoptic, paraventricular and suprachiasmatic nuclei of the hypothalamus, an extensive network of vasopressinergic nerve fibers emerges, which synaptically innervates extrahypothalamic brain areas such as the olfactory bulb, spinal cord and septum (Koolhaas et al., 1998). Since vasopressin is present early in the brain and has various mitogenic, metabolic and physiological actions, one might expect vasopressin to be of importance for normal brain and behavioral development, as well (Boer, 1983). Indeed, the absence of vasopressin in the Brattleboro rat coincides with impaired body growth and brain development (Boer et al., 1982b, 1984; van Norde et al., 1983). Prenatal situation of the mother, i.e. vasopressin-deficiency, significantly contributes to the developmental disturbance of the vasopressin deficient pups (Snijdewint et al., 1988). The above-mentioned circumstances led us to examine the possible role of vasopressin in perinatal development with special attention to the influence of maternal genotype and the HPA axis regulation. Three questions arose: (I) is the functional lack of vasopressin measurable during the perinatal period? Dlouha et al. (1982) concluded that the syndrome of hereditary diabetes insipidus appears at the onset of weaning period, but we wanted to re-examine it from other points of view. The most characteristic defect of the vasopressin deficient Brattleboro pups was the reduced size of cerebellum (Boer et al., 1982a), nevertheless their locomotion was not yet studied. (II) Does the lack of vasopressin in pups as well as the genotype of the mother influence the somatic and psychomotor development? (III) Has the genotype of the mother an influence on HPA axis reactivity of the pups already during the perinatal period? One of the widely studied natural stimuli of the pup’s HPA axis is maternal separation (Desbonnet et al., 2008; Suchecki et al., 1993), which has a profound effect for later life (Barna et al., 2003). This is a good model for child abuse/ neglect, and the long-term alterations reflect depression-like changes (Desbonnet et al., 2008). The importance of vasopressin in depression was supported by our previous findings in adult Brattleboro rats (Mlynarik et al., 2007) and we already demonstrated an important role of maternal vasopressin in 24 h maternal separation induced ACTH elevation, too (Zelena et al., 2008). 2. Materials and methods 2.1. Animals Brattleboro rats were maintained at the Institute of Experimental Medicine in a colony started from breeder rats from Harlan, Indianapolis, IN, USA. We compared the vasopressin deficient homozygous (di/di) rats with congenital diabetes insipidus to heterozygous (di/+) control rats from the same litters (Bohus and de

Wied, 1998; Zelena et al., 2003b). Data from male and female pups were pooled. The 0 day was determined when the offspring was found with the mother on the usual observation at around 9 h. Only those families were involved where six or more pups were born and the litters were culled to eight based upon a random selection right after birth (removed or added among mothers with same genotype who gave birth on the same day). Under usual breeding conditions homozygous vasopressin deficient (di/di) fathers are mated with heterozygous (di/+) mother rats, and only di/+ and di/di rats are born. Pups from this kind of mating were used for examining the appearance of vasopressin deficiency during the perinatal period (question I: pituitary vasopressin content, polyuria, osmolarity, locomotion). For studying the somatic and psychomotor development (question II) and HPA axis changes (question III), di/di mothers were also mated with di/+ male rats and the family history of each rat was recorded. After 4-day mating the females were separated from the males and pups were kept together with their mother only. The genotype of the pups was inferred from the vasopressin content of the pituitary at the end of examination period (see later). Rats were kept in controlled environment (23  1 8C, 50–70% humidity, 12 h light starting at 07.00 h) and given commercial rat chow (Charles River, Hungary) and tap water ad libitum. The number of animals is given in legends between parentheses. All studies were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and approved by the Animal Welfare Committee of the Institute of Experimental Medicine, Budapest, Hungary. 2.2. Signs of vasopressin deficiency 2.2.1. Vasopressin measurement The genotype of the parents was inferred from their water consumption after weaning, which was increased in vasopressin deficient animals (di/+: 31.4  0.7 ml/ day (n = 39), di/di: 166.1  13.2 ml/day (n = 36)) [F(1, 72) = 114.0, p < 0.01]. The genotype of the pups was inferred from the vasopressin content (described in Section 2.5) of the whole pituitary. The vasopressin levels were arranged in ascending order (Fig. 1A) and animals below the limit of detection of the assay were designated as di/di, while animals above the limit were designated as di/+. To follow the development we determined the pituitary vasopressin content on postnatal day 5–10–20 and in adulthood (approximately 10-week old). 2.2.2. Polyuria Half of the 9-day-old pups were separated from their mother for 24 h. At the end of separation period the urinary bladder was emptied by gentle lower abdominal pressure and the amount of urine was calculated from body weight changes. Plasma osmolarity was measured by vapor pressure osmometry. 2.2.3. Preweaning locomotor activity Behavior of the 20-day-old pups was tested in a plastic cage with rectangle floor (36 cm  40 cm) and walls (40 cm). Floor was divided into nine equal squares of 12 cm  13.3 cm. The rat was placed in the middle square and its activity was counted by number of line-crosses. Each measurement lasted for 5 min. Floor of cage was washed with water and dried before the next animal. 2.3. Physical measures and neurobehavioral development Separate set of pups was observed daily, between 9:00 and 11:00 a.m. Mother was removed from home cage for less than 10 min. Each pup of a litter was observed individually for approximately 2 min, then immediately returned to their home cage. Care was taken to minimize the duration of separation from the dam. The mean day of appearance of each of the below parameters was calculated. On 21 postnatal day, the offsprings were decapitated for genotype determination (see Section 2.5). Each rat was weighed daily between 1 and 14 postnatal days. Pinna detachment: When the curved ear straightening beginning at 1 postnatal day. Incisor eruption: Observation of superior and inferior teeth was tested beginning on 7 postnatal day. Ear channel opening was tested beginning on 9 postnatal day. Eye opening: Opening of both eyes was tested beginning on 12 postnatal day (Lazarini et al., 2004; Moraes et al., 2004; Ohta et al., 1998). Palmar grasp reflex: Grasping of a paper clip with forepaws if stroked was studied between 1 and 9 postnatal days. Righting reflex: Offsprings were placed on its back on a smooth, flat surface and allowed to assume the normal upright position with all four feets on the table. The criterion of maturation was a latency smaller than 15 s to return to prone position recorded between 0 and 4 postnatal days. Cliff aversion: Offsprings were placed with their heads and front paws over the edge of a table. The latency to retract their body from the edge was recorded between 2 and 7 postnatal days. An aversive response was considered as positive when it was performed in less than 10 s. Pivoting locomotion: Offsprings were placed on a table. The time to rotate through 908 on hind legs was recorded between 2 and 6 postnatal days. The criterion of maturation was a 908 rotation occurring in less than 10 s.

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

177

Negative geotaxis: Offsprings were placed on a 458 inclined foam-rubber board with their nose pointing down. The animals had 15 s to rotate the body through 1808. This reflex was measured between 4 and 9 postnatal days (Lazarini et al., 2004; Moraes et al., 2004; Ohta et al., 1998). 2.4. HPA axis studies—maternal separation On a separate set of 9-day-old di/di and di/+ pups we investigated the effects of 24 h long maternal deprivation on plasma ACTH and corticosterone levels. In each litter half of the pups were separated from their mothers, whereas the other half remained undisturbed. During maternal deprivation littermates were kept together in a novel cage with normal bedding without providing additional treatment. All pups were killed by decapitation on day 10. ACTH and corticosterone levels were determined in trunk blood. 2.5. Hormone measurements Blood was collected on ice-cold Eppendorf tubes containing 20 ml of 20% sodium–EDTA and centrifuged at 3000 rpm for 20 min at 4 8C. Plasma was stored at 20 8C till the hormone assay. Plasma ACTH was measured by radioimmunoassay (RIA) in 50 ml unextracted plasma as described earlier (Zelena et al., 1999). The ACTH antibody was raised in rabbit in the Institute of Experimental Medicine, Hungarian Academy of Sciences (Budapest, Hungary) and was directed against the middle part of the h-ACTH1–39 molecule. The sample no. 8514 was used. The antibody is highly specific, showing 0.2% cross-reaction with a-MSH and no significant cross-reaction with g-MSH, CLIP, ACTH11–24, ACTH25–39, ACTH1–14, and ACTH1–19. The intra-assay coefficient of variation was 7.23%. All samples from a particular experiment were assessed in the same RIA. Plasma corticosterone was measured in 10 ml unextracted plasma by RIA as described earlier (Zelena et al., 2003a). The corticosterone antibody was developed against corticosterone-carboximethyloxime bovine serum albumin in our Institute. 125 I-labelled corticosterone-carboxymethyloxime-tyrosinemethyl ester was used as tracer. The interference from plasma transcortin was eliminated by inactivating transcortin at low pH. Assay sensitivity was 1 pmol. The intra-assay coefficient of variation was 7.5%. All samples from a particular experiment were measured in one RIA. Vasopressin content was also measured by RIA. Pituitaries were collected in 100 ml 0.1N HCl and frozen at 20 8C. After thawing, pituitaries were boiled for 5 min then were ultrasonically homogenized and refrozen. On the next day, samples were thawed and centrifuged for 20 min at 3000 rpm. The supernatants were stored at 20 8C till hormone measurement. The rabbit anti-vasopressin antiserum was obtained from Dr. M. Vecsernye´s (Szent-Gyo¨rgyi Medical University, Szeged, Hungary). The vasopressin content was measured in 50 ml, 100-fold diluted, unextracted homogenisate. The assay sensitivity was 4 pg. The intra- and inter-assay coefficients of variation were 10.7% and 17.7%, respectively. 2.6. Statistical analysis Data were analyzed by analysis of variance using ANOVA/MANOVA module of the STATISTICA software package (Tulsa, OK, USA). Multiple pairwise comparisons where appropriate were made by the Newman–Keuls method. When the effect of pup’s genotype was significant an additional regression analysis was performed to find out if there was a correlation between pituitary vasopressin content and the examined parameter, or once vasopressin reached some minimum threshold value, then the full effect was present. Values are presented as mean  S.E.M. The level of statistical significance was taken as p < 0.05 in all statistical analyses.

3. Results 3.1. Signs of vasopressin deficiency in pups The vasopressin content of the whole pituitary was under the detection limit in di/di animals throughout their life (Fig. 1A and B). In contrast in di/+ pups the vasopressin content increased between postnatal day 5 and 10 to approximately 2.5-fold, while this level was constant during the later postnatal phase, then increased further in adulthood (approximately 2-fold). Statistically the effect of age [F(3, 173) = 35.1, p < 0.01], genotype [F(3, 173) = 417.0, p < 0.01] and their interaction [F(3, 173) = 33.4, p < 0.01] were significant with significant pairwise comparison in the Newman– Keuls test indicated in the figures. In control, unseparated animals, the 24 h weight gain was lower in 10-day-old di/di pups than in di/+ animals. Using 24 h maternal separation as a stimulus, all separated pups lost some weight, which was significantly lower in di/di offspring (Fig. 2A). The effect

Fig. 1. (A) Vasopressin content of the pituitary (ng). For inferring to the offspring’s genotype the vasopressin content of pituitaries was arranged in ascending order (number of samples) and animals below the limit of detection of the assay were considered to be vasopressin deficient. (B) Age dependence of pituitary vasopressin content (ng/pituitary). Vasopressin deficient (di/di) animals had undetectable level, while in control animals (di/+) the vasopressin content of the pituitary increased gradually with age. Offsprings of di/+ mothers were used (n = 8–31). **p < 0.01 vs. day 5; ##p < 0.01 vs. di/+; ++p < 0.01 vs. day 10 and 20.

of separation [F(1, 143) = 953.1, p < 0.01], genotype [F(1, 143) = 40.4, p < 0.01] and also their interaction [F(1, 143) = 13.8, p < 0.01] was highly significant. There was a significant urine retention in separated pups, which was not affected by the genotype, however the undisturbed di/di pups have a tendency for higher urine retention than their di/+ littermates (Fig. 2B). Two-way ANOVA revealed a significant separation effect [F(1, 40) = 14.4, p < 0.01], while neither the effect of genotype nor the interaction between genotype  separation was significant on body weight changes because of emptying the bladder. The plasma osmolarity was already increased in 10-day-old di/ di animals without any effect of separation (Fig. 2C) [genotype: F(1, 144) = 426.02; p < 0.01]. There were no further changes in adulthood among basal conditions [genotype: F(1, 10) = 47.1; p < 0.01]. The locomotor consequence of the vasopressin deficiency was the enhanced line-crossing in an open field measurable around weaning (Fig. 3A) [genotype: F(1, 14) = 8.39; p < 0.01]. Presumably this is the consequence of reduced adaptation of di/di offspring to a new environment (Fig. 3B). 3.2. Signs of vasopressin deficiency in mothers The vasopressin deficiency of the mother did not influence the pregnancy rate of the females using 4-day mating (Table 1). On the other hand, the vasopressin deficient mothers have significantly less offspring than their di/+ littermates [F(1, 8) = 18.96; p < 0.01; from seven series of mating]. The number, as well as the body weight ratio of male/female pups were not different between the two mother groups, however we could confirm that the number

178

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

Fig. 2. (A) Changes during 24 h maternal separation in 10-day-old pups of di/+ mothers. The body weight of di/di animals was lower, but separation induced body weight decrease in both genotype (n = 30–43). (B) At the end of 24 h separation urine retention was measured by weight changes after emptying the bladder without significant effect of the genotype (n = 7–13). (C) Plasma osmolarity (measured by freezing point depression) was higher in di/di animals already in 10-day-old pups without further increase in adulthood (n = 31–43). **p < 0.01 vs. control, unseparated rats; ##p < 0.01 vs. di/+.

but not the weight of males was higher, and this was independent from the genotype of the mother (number of male pups: 4.67  0.52; number of female pups: 3.30  0.28) [F(1, 14) = 5.33; p = 0.037].

3.3. Somatic development When we followed the early body weight changes up to 14 day, we concluded that the growth of the di/+ pups was not influenced

Fig. 3. Locomotion at perinatal period: openfield in 20-day-old pups. (A) During the 5 min observation period the number of line-crossing was significantly higher in di/di rats. (B) Number of line-crossing measured in every 1 min represents a habituation in di/+ but not in di/di animals (n = 5–11). *p < 0.05 vs. first 1 min; #p < 0.05; ##p < 0.01 vs. di/+.

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

179

Table 1 Comparison of the reproductive capacity of di/+ and di/di Brattleboro mothers. Mother

Pregnancy rate (%) Number of pups Ratio of male/female pups Weight of male/female pups (10 days)

p

di/+

di/di

38.79  5.52 (n = 105) 9.34  0.29 (no. of delivery = 56) 1.76  0.3 1.03  0.02

35.85  9.48 (n = 96) 6.92  0.35 (no. of delivery = 44) 1.23  0.12 1.015  0.015

0.83 0.002 0.25 0.78

by the genotype of the mother (Fig. 4). In contrast, the di/di offspring of the di/di mothers showed reduced body weight gain compared either to their di/+ littermates or to the di/di offspring of di/+ mothers. Approximately in the 10-day-old pups, the influence of maternal genotype diminished and the genotype of the pups became the more important determinant of the somatic development. That meant that the di/di offspring of di/+ mother began to reduce their weight. There was a highly significant influence of the pup-genotype [F(1, 40) = 15.85; p < 0.01], time [F(13, 520 = 684.2; p < 0.01], time  pup-genotype [F(13, 520) = 7.1; p < 0.01] and time  pup-  mother-genotype interactions [F(13, 520) = 7.4; p < 0.01], while the effect of mother-genotype was also significant although to a lower level [F(1, 40) = 4.4; p = 0.043]. There was a significant positive correlation between the pituitary vasopressin content and body weight of pups during the whole examination period. We should mention, that the vasopressin was measured just at one time-point (at the end), therefore only the last day should be taken into consideration (r = 0.52; p < 0.01). First sign of the somatic development was the ear straightening (pinna detachment) in the 3-day-old pups, which was not influenced by either genotype (mothers’ and pups’) (di/+ mother di/+ pup: 2.89  0.12, di/di pup: 2.63  0.11, di/di mother di/+ pup: 2.93  0.22, di/di pup: 3.05  0.11 (day)). The differences could be covered by the uncertain determination of the delivery (max. 23 h

difference can be between the same day old litters because we check the litters once a day). The upper incisors break through earlier (0.5–1 day) than the lower ones, both at about 10-day-old age [F(1, 106) = 20.2; p < 0.01]. Both teeth of the vasopressin deficient pups break through earlier than their littermates, but the genotype of the mother had no profound effect on this (Fig. 5A). In the case of upper incisor two-way ANOVA revealed significant pup-genotype effect [F(1, 103) = 31.2; p < 0.01] with marginally significant mother pup-genotype interaction [F(1, 103) = 3.14; p = 0.079]. There

Fig. 4. Body weight changes in the offspring. During the first 10 days of life the di/di offsprings of di/di mothers have reduced weight gain compared both to di/+ offspring and di/di offspring of di/+ mothers. In later life-period the di/di offspring of di/+ mother had reduced weight gain, too (n = 14–33). *p < 0.05; ** p < 0.01 compare mothers (respective circles to triangles); #p < 0.05; ##p < 0.01 compare pups (respective open to filled symbols).

Fig. 5. Neurobehavioral development. (A) Upper incisor eruption occurred earlier in di/di offspring of both (di/+ and di/di) mothers (n = 14–33). (B) Ear opening was accelerated in di/di pups of di/+ mother but there was a delay in di/di offspring of di/ di mother (n = 14–33). (C) Eye opening occurred earlier in di/di offspring (n = 6–29). The values are represented as mean  S.E.M. days of age when each parameter first occurred. *p < 0.05; **p < 0.01 compare offsprings; ##p < 0.01 compares mothers.

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

180

was a significant positive correlation between pituitary vasopressin content and incisor eruption (r = 0.47; p < 0.01). The ear channels were completely opened at around 12-day-old age (Fig. 5B). The vasopressin deficient animals of di/+ mother were ‘‘faster’’, their ear opened earlier. In contrast the vasopressin deficient di/di pups of the di/di mothers developed ‘‘slower’’ in this respect. Two-way ANOVA showed a significant mother-  pupgenotype interaction [F(1, 103) = 6.83; p = 0.01]. Both the mothers’ and pups’ vasopressin deficiency led to earlier eye opening at 14 day compared to either the effect of di/+ mother or to di/+ pups (Fig. 5C). There was a significant effect of mother-genotype [F(1, 54) = 5.16; p = 0.027], as well as the genotype of the pups [F(1, 54) = 13.09; p < 0.01], while their interaction showed just marginally significance [F(1, 54) = 3.63; p = 0.06]. There was a significant positive correlation between pituitary vasopressin content and eye opening (r = 0.28; p = 0.034). 3.4. Behavioral development The first, ancient reflex appeared was the grasp palmar reflex detectable already around birth. In the offspring of vasopressin deficient mothers this reflex developed earlier but its disappearance occured later, so it persisted longer (Table 2). With two-way ANOVA the mother-genotype significantly influenced both the appearance [F(1, 102) = 5.55; p = 0.02] and the disappearance [F(1, 98) = 24.3; p < 0.01] of the reflex, while the disappearance of the reflex showed also a significant mother-  pup-genotype interaction [F(1, 98) = 4.71; p = 0.03]. In the case of the second reflex (righting) at around day 2, there was only a tendency for the mothers’ vasopressin deficiency to accelerate the appearance [F(1, 103) = 3.23; p = 0.075]. Both the pivoting locomotion at around day 3 and the cliff aversion at around day 5 were not influenced by the genotype of the mother or pup. The latest studied reflex was the negative geotaxis in the 5–6day-old animal without any significant influence of mother- or pup-genotype. However, some tendencies were present [effect of pup-genotype: F(1, 103) = 2.82; p = 0.09; mother-  pup-genotype interaction: F(1, 103) = 3.61; p = 0.06]. 3.5. HPA axis changes The 24 h maternal separation induced a significant rise in the ACTH levels of the 10-day-old di/+ pups of control, di/+ mothers (Fig. 6A, approximately 4-fold increase). The di/di pups of the same mothers had increased resting levels, but failed to react to maternal separation at all. The lack of vasopressin in the mothers led to reduced resting ACTH levels in di/+ pups (23% reduction) with a similar tendency in di/di animals (p = 0.07, 30% reduction). Table 2 Neurobehavioral development in Brattleboro pups: effect of maternal genotype. Mother

di/+

di/di

Offspring

di/+

di/di

di/+

di/di

Palmar grasp reflex Disappearance of palmar reflex Righting reflex Pivoting locomotion Cliff aversion Negative geotaxis

1.45  0.2 5.83  0.3

1.38  0.2 5.33  0.2

0.86  0.1# 6.54  0.3#

1.22  0.1 7.15  0.2##

2.23  0.2 3.48  0.3 5.59  0.2 5.94  0.2

2.48  0.2 2.70  0.2 5.47  0.2 5.90  0.2

1.93  0.3 3.62  0.2 5.43  0.1 5.39  0.2

2.00  0.2 3.68  0.2 5.98  0.1 6.13  0.2

The values are represented as mean  S.E.M. days of age that the parameter first occurred (except other indicated) (n = 13–33). The appearance as well as the disappearance of palmar grasp reflex was influenced by the mother’s genotype, while other reflexes revealed just marginally significance. # p < 0.05, ##p < 0.01 vs. di/+ mother same genotype.

Fig. 6. Influence of maternal genotype on the HPA axis reactivity. (A) ACTH (fmol/ ml) levels were significantly lower in separated di/di offspring compared to their littermates from both types of mother. (B) Corticosterone (pmol/ml) levels were significantly higher in di/di offspring. The vasopressin deficiency of the mother significantly reduced the overall corticosterone levels (n = 10–20). *p < 0.05; **p < 0.01 vs. control, unseparated rats; #p < 0.05; ##p < 0.01 vs. di/+ pups; +p < 0.05 vs. di/+ mother.

In contrast, the ACTH rise of di/+ pups to maternal separation was even more pronounced (approximately 6-fold increase). The di/di offsprings of the di/di mothers have also increased resting ACTH levels compared to their di/+ littermates, and significantly smaller reaction to maternal separation. Statistical analysis by three-way ANOVA (mother-genotype, pup-genotype, separation) revealed a highly significant effect of pup-genotype [F(1, 116) = 50.4; p < 0.01], separation [F(1, 116) = 115.2; p < 0.01] and pupgenotype  separation interaction [F(1, 116) = 85.9; p < 0.01], while the mother-genotype  separation interaction was also significant, but only to a lower level [F(1, 116) = 5.02; p = 0.027]. The correlation between pituitary vasopressin content and plasma ACTH levels was not significant just in the case, when we examined the basal and stressed levels separately (basal: r = 0.37; p < 0.01; stressed: r = 0.6; p < 0.01). The 24 h maternal separation induced a significant corticosterone rise in di/+ offspring of di/+ mothers (Fig. 6B, approximately 13-fold increase), which was even higher than the ACTH increase in the same animals at the same time-point. The lack of vasopressin in the pups led to higher resting and stress-induced corticosterone levels, however in di/di pups the increase to 24 h maternal separation was just 3.4-fold due to the already elevated resting levels. The vasopressin deficiency of the mothers was unable to influence the pattern of the changes, however the overall corticosterone levels in their pups were smaller than in di/+ mothers (e.g. corticosterone rise in di/+ pups of di/di mother was just approximately 9-fold). The three-way ANOVA showed highly significant effect of pup-genotype [F(1, 115) = 31.1; p < 0.01] and

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

separation [F(1, 115) = 329.8; p < 0.01] and a lesser significant effect of mother-genotype [F(1, 115) = 5.34; p = 0.022] without any interaction. There was a significant negative correlation between pituitary vasopressin content and plasma corticosterone levels (r = 0.32; p < 0.01). 4. Discussion The following facts were found: (I) reduced weight gain, higher plasma osmolarity, resting ACTH and corticosterone levels were detected already in 10-day-old vasopressin deficient animals. Higher locomotion due to the reduced adaptation was also present in these rats around weaning. (II) During the first 10 days of life the di/di pups from di/di mother were the smallest, while in the later perinatal period the genotype of the pups became the more important determinant of the somatic development. Generally the lack of vasopressin in the pups hastened the somatic development, however the neurobehavioral development was not profoundly influenced by either the mothers’ or the pups’ genotype. (III) The lack of vasopressin in the pups abolished the 24 h maternal separation induced ACTH elevation, while the accompanying corticosterone rises were even higher. The vasopressin deficiency of the mother reduced the resting ACTH levels but stimulated the stressed levels of di/+ offspring and caused an overall reduction in corticosterone secretion. Number of environmental, nutritional, hormonal and neurotransmitter factors is known to influence the development of brain and behavior (Boer et al., 1982a). To exclude the first two factors we compared homozygous diabetes insipidus pups with their heterozygous littermates (Bohus and de Wied, 1998; Zelena et al., 2003b), therefore all observed differences are due to endogenous factors related to vasopressin deficiency. The reproductive capacity of the Brattleboro rats was already summarized (Boer et al., 1981; Sokol and Zimmerman, 1982). We could demonstrate that our colony has the same potency with no difference in pregnancy rate (our results show lower pregnancy rate while we examined the conclusion of 4-day mating only) but significantly smaller litter size of di/di mothers (Table 1). According to the literature the plasma oxytocin level of vasopressin deficient animals is increased throughout the life (Boer et al., 1988; Bundzikova et al., 2008). Perhaps, as a consequence, the gestation length was reported to be shorter with 3.5 h in di/di mothers (Boer et al., 1982a). On the other hand, the sensitivity of the uterus to oxytocin was found to be reduced by Haldar et al. (1982) and normal by Goren et al. (1980). We did not detect any problem with delivery. It is also present in human being that more males than females were born and the male offsprings are heavier (Nielsen et al., 1997), but the sex ratio was never studied before in Brattleboro rats. The hypothalamo-neurohypophyseal system is already present prenatally (Sinding et al., 1980b), however Dlouha et al. (1982) reported that the role of vasopressin in the water homeostasis regulation became important just at weaning. Here we demonstrated that among resting conditions the di/di pups show some signs of polyuria already at 10 postnatal day (Fig. 2). Because the licking by mother is the main stimulus of urination during the perinatal period (Wu and de Groat, 2006), therefore the separated pups show urine retention. In this case the role of vasopressin disappears. The other sign of diabetes insipidus in 10-day-old pup is the already elevated plasma osmolarity, which is very similar to that detectable in adult di/di animals (Ideno et al., 2003). The reduced weight gain as well as the elevated resting plasma ACTH and corticosterone levels can also be the consequence of perinatal vasopressin deficiency (Domokos et al., 2008). The reduced size of the cerebellum throughout the life is the most characteristic and obvious defect of the di/di Brattleboro rats (Boer et al., 1982a), and we demonstrated one possible behavioral

181

consequence of this deficit in 20-day-old pup showing an impaired adaptation to the locomotor activity in a novel surrounding (Fig. 3). The adaptational disability of Brattleboro rats was already shown in adulthood (Brito, 1983), probably due to disturbed memory processes (Colombo et al., 1992). In contrast, the overall enhanced locomotion suggests an upregulation rather than a deficit in the motor-coordination, however this phenomenon disappears in adulthood (Mlynarik et al., 2007). The loss of V1a receptors (due to N-methyl-D-aspartate administration) was already described to induce hyperlocomotion in rats (Matsuoka et al., 2005), so it might be that perinatally the reduced activation of V1a receptors can influence the locomotor behavior, while in adults the compensatory mechanisms may overcome this effect. All studied somatic and psychomotor parameters developed slightly earlier in Brattleboro rats than is usual in the widely studied Wistar rat (for comparison see Lazarini et al., 2004). The effect of maternal genotype was the strongest one on somatic growth during the first 10 days of life, when the effect of vasopressin deficiency in pups was additive with the mother’s vasopressin deficiency (Fig. 4). In later postnatal period the genotype of the pups became more important, as it was true for other studied factors, as well. Taken into consideration other somatic factors (incisor eruption, eye opening, and ear opening) the overall impression was that the lack of vasopressin in the pups leads to enhanced somatic development, which is in contrast to that what we expected (Fig. 5). The strong correlation between pituitary vasopressin content and different developmental parameters supports further the developmental role of vasopressin. An earlier opening of the eyes in di/di Brattleboro rats was already described (Boer, 1985) and we could confirm it. The vasopressin deficiency of the mother had no uniform effect, but could accelerate the eye opening. Examining the neurobehavioral development we did not find big differences (Table 2). The problem could be that the uncertain determination of delivery could cover any effect. The only significant effect was the earlier appearance and later disappearance of the palmar reflex, one of the most ancient and widespread reflexes in mammals. So we can conclude that the lack of vasopressin speed up the development, probably there is a greater drive for self-sufficiency in these animals. In contrast to the general view, more vulnerable animals do not have definitely delayed development. There is no doubt that vasopressin supports the ACTH secreting role of CRH in adulthood (Antoni, 1993; Buckingham, 1982). On the other hand, in the neonate rat vasopressin seems to be the dominant secretagogue (Avishai-Eliner et al., 1995; Levine, 2002; Zelena et al., 2008) (Fig. 6). Its role seems to be limited to the stressrelated rises and not to the maintenance of the resting levels. The increased resting hormone levels observed in the 10-day-old di/di rat pups suggest that during an important developmental period di/di rat pups are exposed to high endogenous ACTH and corticosterone levels, which may have considerable acute and delayed effects. It was surprising that in vasopressin deficient pups the 24 h maternal separation induced a pronounced corticosterone rise without ACTH elevation. Our previous work confirmed that this phenomenon is not due to the different time-courses of the two hormones (Zelena et al., 2008), not restricted to the 10 postnatal day and to the maternal deprivation, but also true with other stimuli like hypnorm injection (fentanyl-fluanison combination, anesthetic). It cannot be detected in adulthood (Zelena et al., 2007), possibly due to the development of compensatory mechanisms. Based upon accumulating observations it is clear that ACTH and corticosterone plasma levels do not go parallel in each situation. We think, that a certain amount of ACTH is required to maintain the corticosterone synthesis. In case ACTH elevation is occured, it is the strongest stimulus to corticosterone secretion. In its absence other secretagogues come in the front. These

182

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183

mechanisms are present in ‘‘normal’’ animals (humans?), but their importance is to assist ACTH. The main question of the present work was the role of maternal genotype. It was likely, that the aspects of maternal phenotype might be the main factor regulating the offspring’s HPA axis development, since maternal behavior was reported to influence the reactivity of the HPA axis (Champagne and Meaney, 2001; Zelena et al., 2003b). However, we could not demonstrate a profound effect of the mother’s vasopressin deficiency on the pup’s HPA axis reactivity. The only finding, which did not fit into our previous work (Zelena et al., 2008), as well as into our present data, was that the 24 h maternal separation induced a significant (although quite small) ACTH rise in di/di offspring of di/di mother. However, the elevation is much lower than in the di/+ offspring. Indeed, all of these changes might suggest some disturbances in the HPA axis regulation in the offspring of di/di mother. The following conclusions could be made: the functional signs of vasopressin deficiency appear already perinatally. The lack of vasopressin leads to accelerated development with reduced body weight. In the regulation of somatic growth the role of maternal genotype was important just in the first 10 days of life. The role of vasopressin in the HPA axis regulation seems to be important during the perinatal period independently from the mother’s genotype. However, there was a big discrepancy between ACTH and corticosterone regulations (Zelena et al., 2008), which requires further studies. Acknowledgements Supported by T 043161, NN71629 OTKA and 059/2006 ETT grants. References Aguilera, G., Nikodemova, M., Wynn, P.C., Catt, K.J., 2004. Corticotropin releasing hormone receptors: two decades later. Peptides 25, 319–329. Anisman, H., Zaharia, M.D., Meaney, M.J., Merali, Z., 1998. Do early-life events permanently alter behavioral and hormonal responses to stressors? Int. J. Dev. Neurosci. 16, 149–164. Antoni, F.A., 1993. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14, 76–122. Avishai-Eliner, S., Yi, S.J., Newth, C.J., Baram, T.Z., 1995. Effects of maternal and sibling deprivation on basal and stress induced hypothalamic-pituitary–adrenal components in the infant rat. Neurosci. Lett. 192, 49–52. Barna, I., Ba´lint, E., Baranyi, J., Bakos, N., Makara, G.B., Haller, J., 2003. Genderspecific effect of maternal deprivation on anxiety and corticotropin-releasing hormone mRNA expression in rats. Brain Res. Bull. 62, 85–91. Boer, G.J., Boer, K., Swaab, D.F., 1982a. On the reproductive and developmental differences within the Brattleboro strain. Ann. NY Acad. Sci. 394, 37–45. Boer, G.J., Van Rheenen-Verberg, C.M., Uylings, H.B., 1982b. Impaired brain development of the diabetes insipidus Brattleboro rat. Brain Res. 255, 557–575. Boer, G.J., 1983. Vasopressin: a necessary factor for normal brain development? Int. J. Dev. Neurosci. 1, 227. Boer, G.J., Dozy, M.H., Uylings, H.B.M., 1984. Cerebellar DNA and tissue water changes in the brain of diabetes insipidus Brattleboro rats are already present at birth. Int. J. Dev. Neurosci. 2, 301–304. Boer, G.J., 1985. Vasopressin and brain development: studies using the Brattleboro rat. Peptides 6 (Suppl. 1), 49–62. Boer, G.J., van Heerikhuize, J., van der Woude, T.P., 1988. Elevated serum oxytocin of the vasopressin-deficient Brattleboro rat is present throughout life and is not sensitive to treatment with vasopressin. Acta Endocrinol. (Copenhagen) 117, 442–450. Boer, K., Boer, G.J., Swaab, D.F., 1981. Reproduction in Brattleboro rats with diabetes insipidus. J. Reprod. Fertil. 61, 273–280. Bohus, B., de Wied, D., 1998. The vasopressin deficient Brattleboro rats: a natural knockout model used in the search for CNS effects of vasopressin. Prog. Brain Res. 119, 555–573. Brito, G.N., 1983. The behavior of vasopressin-deficient rats (Brattleboro strain). Physiol. Behav. 30, 29–34. Buckingham, J.C., Leach, J.H., 1980. Hypothalamo-pituitary–adrenocortical function in rats with inherited diabetes insipidus. J. Physiol. 305, 397–404. Buckingham, J.C., 1982. Potentiation of hypothalamic corticotropin releasing activity by vasopressin: studies in the Brattleboro rat. Ann. NY Acad. Sci. 394, 580–586. Bundzikova, J., Pirnik, Z., Zelena, D., Mikkelsen, J.D., Kiss, A., 2008. Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats. Cell. Mol. Neurobiol..

Champagne, F., Meaney, M.J., 2001. Like mother, like daughter: evidence for nongenomic transmission of parental behavior and stress responsivity. Prog. Brain Res. 133, 287–302. Colombo, G., Hansen, C., Hoffman, P.L., Grant, K.A., 1992. Decreased performance in a delayed alternation task by rats genetically deficient in vasopressin. Physiol. Behav. 52, 827–830. Decaux, G., Soupart, A., Vassart, G., 2008. Non-peptide arginine–vasopressin antagonists: the vaptans. Lancet 371, 1624–1632. Desbonnet, L., Garrett, L., Daly, E., McDermott, K.W., Dinan, T.G., 2008. Sexually dimorphic effects of maternal separation stress on corticotrophin-releasing factor and vasopressin systems in the adult rat brain. Int. J. Dev. Neurosci. 26, 259–268. Dlouha, H., Krecek, J., Zicha, J., 1982. Postnatal development and diabetes insipidus in Brattleboro rats. Ann. NY Acad. Sci. 394, 10–20. Domokos, A., Mergl, Z., Barna, I., Makara, G.B., Zelena, D., 2008. Congenital vasopressin deficiency and acute and chronic opiate effects on hypothalamo-pituitary–adrenal axis activity in Brattleboro rats. J. Endocrinol. 196, 113–121. Evans, M.J., Marshall, A.G., Kitson, N.E., Summers, K., Donald, R.A., 1993. Factors affecting ACTH release from perfused equine anterior pituitary cells. J. Endocrinol. 137, 391–401. Goren, H.J., Geonzon, R.M., Hollenberg, M.D., Lederis, K., Morgan, D.O., 1980. Oxytocin action: lack of correlation between receptor number and tissue responsiveness. J. Supramol. Struct. 14, 129–138. Grino, M., Young 3rd, W.S., Burgunder, J.M., 1989. Ontogeny of expression of the corticotropin-releasing factor gene in the hypothalamic paraventricular nucleus and of the proopiomelanocortin gene in rat pituitary. Endocrinology 124, 60–68. Grino, M., Oliver, C., 1992. Ontogeny of insulin-induced hypoglycemia stimulation of adrenocorticotropin secretion in the rat: role of catecholamines. Endocrinology 131, 2763–2768. Haldar, J., Kupfer, L., Sokol, H.W., 1982. Decreased sensitivity to oxytocin of uteri from homozygous Brattleboro rats. Ann. NY Acad. Sci. 394, 46–49. Ideno, J., Mizukami, H., Honda, K., Okada, T., Hanazono, Y., Kume, A., Saito, T., Ishibashi, S., Ozawa, K., 2003. Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector expressing arginine– vasopressin in Brattleboro rats. Mol. Ther. 8, 895–902. Kjaer, A., Knigge, U., Bach, F.W., Warberg, J., 1993. Impaired histamine- and stressinduced secretion of ACTH and beta-endorphin in vasopressin-deficient Brattleboro rats. Neuroendocrinology 57, 1035–1041. Koolhaas, J.M., Everts, H., de Ruiter, A.J., de Boer, S.F., Bohus, B., 1998. Coping with stress in rats and mice: differential peptidergic modulation of the amygdala– lateral septum complex. Prog. Brain Res. 119, 437–448. Lazarini, C.A., Lima, R.Y., Guedes, A.P., Bernardi, M.M., 2004. Prenatal exposure to dichlorvos: physical and behavioral effects on rat offspring. Neurotoxicol. Teratol. 26, 607–614. Levine, S., 2002. Regulation of the hypothalamic-pituitary–adrenal axis in the neonatal rat: the role of maternal behavior. Neurotox. Res. 4, 557–564. Makara, G.B., Mergl, Z., Zelena, D., 2004. The role of vasopressin in hypothalamopituitary–adrenal axis activation during stress: an assessment of the evidence. Ann. NY Acad. Sci. 1018, 151–161. Matsuoka, T., Sumiyoshi, T., Tanaka, K., Tsunoda, M., Uehara, T., Itoh, H., Kurachi, M., 2005. NC-1900, an arginine–vasopressin analogue, ameliorates social behavior deficits and hyperlocomotion in MK-801-treated rats: therapeutic implications for schizophrenia. Brain Res. 1053, 131–136. Mlynarik, M., Zelena, D., Bagdy, G., Makara, G.B., Jezova, D., 2007. Signs of attenuated depression-like behavior in vasopressin deficient Brattleboro rats. Horm. Behav. 51, 395–405. Moraes, A.P., Schwarz, A., Spinosa, H.S., Florio, J.C., Bernardi, M.M., 2004. Maternal exposure to diphenhydramine during the fetal period in rats: effects on physical and neurobehavioral development and on neurochemical parameters. Neurotoxicol. Teratol. 26, 681–692. Nielsen, B.B., Liljestrand, J., Hedegaard, M., Thilsted, S.H., Joseph, A., 1997. Reproductive pattern, perinatal mortality, and sex preference in rural Tamil Nadu, south India: community based, cross sectional study. BMJ 314, 1521–1524. Ohta, R., Matsumoto, A., Nagao, T., Mizutani, M., 1998. Comparative study of behavioral development between high and low shuttlebox avoidance rats. Physiol. Behav. 63, 545–551. Palkovits, M., Mitro, A., 1968. Morphological changes in the hypothalamo-pituitary–adrenal system during early postnatal period in rats. Gen. Comp. Endocrinol. 10, 253–262. Sinding, C., Robinson, A.G., Seif, S.M., Schmid, P.G., 1980a. Neurohypophyseal peptides in the developing rat fetus. Brain Res. 195, 177–186. Sinding, C., Seif, S.M., Robinson, A.G., 1980b. Levels of neurohypophyseal peptides in the rat during the first month of life. I. Basal levels in plasma, pituitary, and hypothalamus. Endocrinology 107, 749–754. Snijdewint, F.G., Boer, G.J., Swaab, D.F., 1988. Prenatal development of the Brattleboro rat is influenced by genotype and lysine vasopressin treatment of the mother. Biol. Neonate 53, 295–304. Sokol, H.W., Zimmerman, E.A., 1982. The hormonal status of the Brattleboro rat. Ann. NY Acad. Sci. 394, 535–548. Suchecki, D., Mozaffarian, D., Gross, G., Rosenfeld, P., Levine, S., 1993. Effects of maternal deprivation on the ACTH stress response in the infant rat. Neuroendocrinology 57, 204–212. van Norde, W., Uylings, H.B., Boer, G.J., 1983. Impaired cerebellar development and underlying structural deficits in the vasopressin deficient Brattleboro rat—a morphometrical study. Int. J. Dev. Neurosci. 1, 244.

D. Zelena et al. / Int. J. Devl Neuroscience 27 (2009) 175–183 Wu, H.Y., de Groat, W.C., 2006. Maternal separation uncouples reflex from spontaneous voiding in rat pups. J. Urol. 175, 1148–1151. Zelena, D., Kiem, D.T., Barna, I., Makara, G.B., 1999. Alpha 2-adrenoreceptor subtypes regulate ACTH and beta-endorphin secretions during stress in the rat. Psychoneuroendocrinology 24, 333–343. Zelena, D., Mergl, Z., Foldes, A., Kovacs, K.J., Toth, Z., Makara, G.B., 2003a. Role of hypothalamic inputs in maintaining pituitary–adrenal responsiveness in repeated restraint. Am. J. Physiol. Endocrinol. Metab. 285, E1110–E1117. Zelena, D., Mergl, Z., Makara, G.B., 2003b. Maternal genotype influences stress reactivity of vasopressin-deficient brattleboro rats. J. Neuroendocrinol. 15, 1105–1110.

183

Zelena, D., Fo¨ldes, A., Mergl, Z., Barna, I., Kova´cs, K.J., Makara, G.B., 2004. Effects of repeated restraint stress on hypothalamo-pituitary–adrenocortical function in vasopressin deficient Brattleboro rats. Brain Res. Bull. 63, 521–530. Zelena, D., Domokos, A., Mergl, Z., Makara, G.B., 2007. The role of vasopressin in chronic stress-induced hypothalamo-pituitary–adrenal axis hyperactivity: studies on brattleboro rats with repeated restraint. In: Levine, B.A. (Ed.), Neuropeptide Research Trends. Nova Publishers, Hauppauge, NY, pp. 189–212. Zelena, D., Domokos, A., Barna, I., Mergl, Z., Haller, J., Makara, G.B., 2008. Control of the hypothalamo-pituitary–adrenal axis in the neonatal period: adrenocorticotropin and corticosterone stress responses dissociate in vasopressin-deficient Brattleboro rats. Endocrinology 149, 2576–2583.