Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin

Hormones and Behavior 62 (2012) 539–551

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Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin Anna Fodor a, Barbara Klausz a, Ottó Pintér a, Nuria Daviu b, Cristina Rabasa b, David Rotllant b, Diana Balazsfi a, Krisztina B. Kovacs a, Roser Nadal b, Dóra Zelena a,⁎ a b

HAS Institute of Experimental Medicine, Budapest, Hungary Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 6 April 2012 Revised 6 September 2012 Accepted 16 September 2012 Available online 21 September 2012 Keywords: Undisturbed maternal behavior Pup retrieval Elevated plus maze Forced swim Sweet preference Stress Prefrontal cortex Bed nucleus of stria terminalis Medial preoptic nucleus Paraventricular nucleus of the hypothalamus

a b s t r a c t Early mother–infant relationships exert important long-term effects in offspring and are disturbed by factors such as postpartum depression. We aimed to clarify if lack of vasopressin influences maternal behavior paralleled by the development of a depressive-like phenotype. We compared vasopressin-deficient Brattleboro mothers with heterozygous and homozygous normal ones. The following parameters were measured: maternal behavior (undisturbed and separation-induced); anxiety by the elevated plus maze; sucrose and saccharin preference and forced swim behavior. Underlying brain areas were examined by c-fos immunocytochemistry among rest and after swimstress. In another group of rats, vasopressin 2 receptor agonist was used peripherally to exclude secondary changes due to diabetes insipidus. Results showed that vasopressin-deficient rats spend less time licking–grooming their pups through a centrally driven mechanism. There was no difference between genotypes during the pup retrieval test. Vasopressin-deficient mothers tended to explore more the open arms of the plus maze, showed more preference for sucrose and saccharin and struggled more in the forced swim test, suggesting that they act as less depressive. Under basal conditions, vasopressin-deficient mothers had more c-fos expression in the medial preoptic area, shell of nucleus accumbens, paraventricular nucleus of the hypothalamus and amygdala, but not in other structures. In these areas the swim-stress-induced activation was smaller. In conclusion, vasopressin-deficiency resulted in maternal neglect due to a central effect and was protective against depressive-like behavior probably as a consequence of reduced activation of some stress-related brain structures. The conflicting behavioral data underscores the need for more sex specific studies. © 2012 Elsevier Inc. All rights reserved.

Introduction In the emergent field of Social Neuroscience it has been proposed that vasopressin (AVP) exerts an important role in affiliative behaviors in all vertebrates, especially in social recognition/memory and pair bonding (Young and Flanagan-Cato, 2012). However, most of the data about the role of AVP in social behavior was obtained in males and not too much information was available in females (Bosch, 2011; Bosch and Neumann, 2012). AVP is mainly synthesized in the magnocellular cells of the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) whose axons project to the posterior pituitary (Rhodes et al., 1981; Sokol et al., 1976). AVP is then released into the blood-stream upon appropriate stimulation (e.g., hemorrhage or dehydration) to act at the kidneys and blood vessels. The brain also contains several populations of smaller, AVP synthesizing parvocellular neurons, whose projections remain within the brain. These populations are located within the PVN, medial amygdala

⁎ Corresponding author at: 1083 Budapest, Szigony 43, Hungary. Fax: +36 1 210 9951. E-mail address: [email protected] (D. Zelena). 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yhbeh.2012.09.003

(MeA) and suprachiasmatic nucleus (SCN) (Buijs et al., 1978). The medial part of the parvocellular PVN (mpPVN) contains corticotropin-releasing hormone (CRH) producing neurons in colocalization with AVP projecting to the median eminence as part of the so-called hypothalamic–pituitary– adrenocortical (HPA) axis (Antoni, 1993). The female brain AVP system becomes activated around parturition and during lactation. AVP levels peak on the day before parturition in PVN and SON (Caldwell et al., 1987). In the septal area of pregnant rats the release of AVP is greater than in virgin rats (Landgraf et al., 1991). Therefore an involvement of AVP in the behavior of lactating mothers can be supposed. The Brattleboro homozygous rat (di/di) has a spontaneous mutation in the AVP precursor and – as a consequence – AVP is not synthesized, leading to a diabetes insipidus phenotype (Richter and Schmale, 1983; Valtin and Schroeder, 1964). This strain is a good model for studying the role of AVP in physiological and psychological processes without the need of further manipulations (i.e. injections and operations) (Bohus and de Wied, 1998). The mother–infant relationship is an important factor in the development of offspring and there are broad individual differences in mother styles that could affect emotionality and stress reactivity of offspring at

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adulthood (Korosi et al., 2012; Meaney and Szyf, 2005; Veenema, 2012). Maternal behaviors in rodents include a number of subcomponents, such as nest building, pup retrieval, nursing, licking and grooming of pups, and maternal defense of the nest against potential intruders (maternal aggression). Although there are no previous detailed evaluations of undisturbed (natural) and separation-induced maternal care in Brattleboro rats, some data already suggest that the quality of the early life environment and development is affected in those animals. Regarding Brattleboro di/di rats, previous studies indicated that litter size and the pup body weight were lower than for +/+ and di/+ dams (Boer et al., 1982; Zelena et al., 2009b). Further work also indicated that – in comparison to Long-Evans rats – Brattleboro di/di dams made nests of less quality, had a higher incidence of cannibalism and had a lower percent of pups that survived until weaning, although in all of these cases the genotype of the pups was also different (Wideman and Murphy, 1990). Mother's mood could also affect normal mother–infant interaction. Postpartum depression (PPD) is a serious medical condition that affects approximately 10% to 20% of mothers during the first 4 weeks after delivery. Symptoms of PPD can include labile mood with prominent anxiety and irritability and depression (Miller, 2002). In humans, children of depressed mothers tend to show abnormal cognitive, motor and social development and are more likely to experience depression or anxiety later in life (Heim and Nemeroff, 2001). Despite its clinical importance this disease is underdiagnosed and undertreated. Gold et al. were the first to propose a role for AVP in mood disorders (Gold et al., 1978); and several studies since then supported this idea (Dinan and Scott, 2005; Frank and Landgraf, 2008; Ryckmans, 2010). Patients with major depression have significantly elevated plasma AVP compared to healthy controls (van Londen et al., 1997). Depressed patients with the melancholic subtype have significantly greater AVP mRNA both in the SON and PVN (Meynen et al., 2006). A single nucleotide polymorphism of the V1b receptor was associated to major depression (van West et al., 2004). However, despite the more common prevalence of depression in females (Weissman and Klerman, 1977), most of the studies have been done in males. Given all the above, the aims of the present work were to study the effects of the lack of AVP in Brattleboro mothers on: (i) undisturbed maternal and separation-induced pup retrieval behavior and the influence of peripheral administration of a V2 receptor agonist on this behavior, (ii) anxiety and depressive-like behavior, and (iii) basal and stressinduced c-fos activation in several brain areas. Our results may give further details to the role of AVP in the behavior of the mothers (both maternal care and development of PPD) and its underlying brain areas without the need of further manipulations which could be very important in the case of undisturbed maternal care. Methods Animals and experimental conditions Primiparous female Brattleboro rats were maintained at the Institute of Experimental Medicine in a colony started from Harlan, Indianapolis, USA. We compared AVP-deficient homozygous recessive (di/di) rats with congenital diabetes insipidus to heterozygous (di/+) littermates and homozygous dominant (+/+) rats. In the first experiment, twelve females were paired in every group with di/di (+/+ mothers) or +/+ (di/+ and di/di mothers) sexually experienced males to homogenize the genotype of the offspring. Finally the number of dams was 7, 6 and 9 in the +/+, di/+ and di/di groups, respectively. Rats were kept in controlled environment (23±1 °C, 50–70% humidity, 12 h light starting at 07:00 h) and given commercial rat chow (Charles River, Hungary) and tap water ad libitum. They were housed in Makrolon cages (40×25×25 cm) containing sawdust bedding (Charles River, Hungary). The deliveries were after 21 days at average. After parturition, litter size was culled to six (3 females and 3 males) to avoid possible differences

of maternal behavior due to litter size or sex (Dimitsantos et al., 2007; Moore and Morelli, 1979). Dams were observed for maternal behavior (see below) in their home-cages (Ferplast, Maxi, 41.3× 26 ×29.8 cm, placed there right after delivery) during the first postnatal week starting the next day after delivery. Due to diabetes insipidus, cages have to be changed every day, which was done daily after the last observation (at around 22:00 h, 12 h before the next observation, all genotypes). In this first experiment, after maternal behavior measurements, mothers' behavior was evaluated in the elevated plus-maze test (EPM), in the sucrose/ethanol preference test and in the forced swim test (FST) (see below). C-fos expression in response to the FST was also measured by immunocytochemistry and separate dams for each genotype were used as controls, which were undisturbed until perfusion. All behavioral experiments (except maternal behavior) were done during the morning. To exclude the possible influence of diabetes insipidus on maternal behavior in a second experiment 10 di/di dams were implanted subcutaneously with an osmotic minipump (Alzet osmotic minipump, 2002, 0.5 μl/h, 14 days) containing desmopressin (DDAVP, 10 ng/h, V2 receptor agonist, Ferring Ltd.) on the day of delivery under ether anesthesia (approximately 3 min intervention). Further 10 di/di and 10 +/+ mothers underwent sham operation. All other procedures (mating, adjusting litter-size, daily cage change etc.) were the same as previously described. Those rats were only used to measure maternal behavior in a more comprehensive way (both undisturbed behavior and pup retrieval test). Maternal behavior and lactation status change dramatically during the postnatal period. As the maternal care is more important right after birth we have choosen this period for testing undisturbed maternal behavior. The shortest, less disturbing experiments were conducted next (pup retrieval, EPM) as they might have only similar effect as cage cleaning and do not really mean mother–infant separation (approximately 5 min.). Sucrose and/or ethanol consumption for 24 h is also unlikely to influence the behavior during the FST several days later. Time of the lactation period (early or late) was reported earlier not to influence the FST behavior (Walker et al., 1995). To have comprehensive results the anxiety- and depressive-like changes were measured in close vicinity.

Maternal behavior Undisturbed maternal behavior The behavior of each dam was recorded following a procedure adapted from Dimitsantos et al. (2007). The undisturbed maternal behavior was observed for three 60 min daily observation periods, for the first 7 postnatal days. Observations were performed at two periods during the light phase (8:30 and 14:30 h, lights ON at 7:00 h) and at one period during the dark phase (20:30 h, lights OFF at 19:00 h). Within each observation period, the behavior of each mother was scored 20 times spaced 3 min each one (20 observations× 3 periods per day × 7 days = 420 observations/mother) as present or absent. During the first experiment the following behaviors were scored as present or absent: (1) LG: mother licking–grooming any pup (body+ anogenital region), (2) mother nursing pups, and (3) mother out of the nest (no maternal contact). During the second experiment a more detailed analysis was conducted and we analyzed: (1) LG, (2) mother nursing pups in an arched-back posture with rigid limbs (“high kyphosis”), (3) mother nursing in a “blanket” posture in which the mother just lies over the pups (“prone nursing”), but did not have her back arched and there was no obvious extension of her legs, (4) mother nursing in a “passive” posture (“supine nursing”) in which the mother lies on her back or side while the pups are nursed, (5) mother out of the nest and (6) mother drinking (as di/di rats supposed to drink more and the administration of DDAVP should normalize this behavior, we used this parameter for validation of our method).

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Pup retrieval test During the second experimental series, on postnatal day 8, at around 10:00 h, all pups were separated from their mothers into a new cage (Makrolon cages containing sawdust bedding) for 5 min. After this brief separation, the entire litter was returned to their mothers into the maternity cage and the pups were spread all around the cage. The mother was then observed for 10 min to measure: (1) latency to carry the first pup, (2) latency to return the first pup into the nest, and (3) latency to return all pups into the nest (Slamberova et al., 2001). Anxiety- and depression-like behavior Elevated plus-maze (EPM) To measure anxiety, dams were exposed to the EPM between postnatal days 10–15 during the morning hours. The EPM was made of metal, painted dark gray and elevated 80 cm above the floor (arm length 50 cm; arm width 15 cm; central platform 15× 15 cm; closed arm walls height 70 cm). Surface of maze was washed with water and dried prior next animal was introduced. The duration of the test was 5 min. Rats were introduced in the center of the maze facing a closed arm. Animal behavior was videotaped by a camera positioned above the maze and analyzed later on by a computer based event recorder program (h77) by an observer unaware of treatments (Mikics et al., 2005). Percentage of time spent in open arms and percentage of open arm entries (number of open arm entries /number of open plus closed arm entries) were calculated and used as measures of anxiety (entry: at least three paws in an arm). Closed arm entries were considered as indicators of general locomotor activity (File, 2001). Sweet preference Between postnatal days 12–17 dam's sucrose preference versus a solution containing ethanol was measured using a two-bottle, free-choice test (24 h/day). The first bottle contained 2.5 w/v sucrose diluted in tap water and the second bottle contained 8% ethanol v/v in 2.5 w/v sucrose (Huot et al., 2001). Sucrose was added to the alcohol to make the solution more palatable to the rat and increase consumption (Samson, 2000). Fluid consumption was measured by subtracting the final weight of the bottle from the initial weight, and converted it to milliliters (assuming 1 g=1 ml water). The percentage of sucrose solution from the total liquid ingested (sucrose preference) was measured. To confirm the outcome in a separate set of animals a saccharin consumption test was conducted (Mazarati et al., 2008; Plaznik et al., 1989). We have chosen this palatable solution because it has no caloric value. For two days the animals were allowed free access to two bottles of tap water, to acclimatize the animals to the system. Then we measured 0.1 w/v saccharin (Sigma, Budapest, Hungary) versus water intake for 24 h and the saccharin preference was calculated. Forced swimming test (FST) The FST, as originally described by Porsolt et al. (1977), is a widely used pharmacological model for assessing antidepressant activity and active/passive behavior in front of stressful situations (Marti and Armario, 1993). Once between postnatal days 15–20 dams were individually placed in a glass cylindrical tank 40 cm tall and 14 cm in diameter filled with tap water (24±1 °C) at a height of 30 cm. The animals were forced to swim for a 15-min period. The swimming session was videotaped by a camera positioned in front of the water tanks and subjected to analyze later on (see earlier). The following measures were taken: floating (immobility), struggling (climbing) and mild swim. Rats were considered floating when their general activity was minimized to occasional and small movements of legs or tail necessary to keep their heads above the water. Swimming was measured when animals were making mild swimming movements, more than those necessary to merely keep the head above water. Struggling is a vertical intense movement of paws, when animal permanently breaks the water surface. After the

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swimming sessions, the rats were removed from the tank, carefully dried by paper towels and returned to their home cages. Water in tank was changed after each animal.

Immunocytochemistry C-fos activation in response to FST was used as a marker of brain activation (Ons et al., 2004; Rotllant et al., 2002). Two hours after the beginning of FST or at rest (control animals without any previous test) dams were anesthetized by a cocktail of ketamine–xylazine–pipolphen (50–10–5 mg/kg in 2 ml/kg volume intraperitoneal) and perfused transcardially with saline solution (0.9% NaCl) for 2 min, then with 300 ml 4 °C 4% paraformaldehyde. The brains were removed, post-fixed in the same solution overnight at 4 °C. In the next day the brains were flushed and cryoprotected by 20% sucrose in phosphate buffer saline (PBS) and waited until it is completely submerged (usually 2 days). After that we removed the cryoprotective solution and blotted the excess cryoprotective solution with a drying paper; wrapped the samples with aluminum foil and labeled them; froze the brains on dry ice and stored at −80 °C until sectioning. 30 μm sections were cut in the coronal plane on a sliding microtome and were stored at −20 °C. The sections were washed first in PBS for 3×10 min and for blocking the endogen peroxidase in 2% Triton-X-100 and H2O2 solution and again 3×5 min in PBS. The next incubation was in 2% normal horse serum (NHS) for 20 min and 3×10 min in PBS. The c-fos protein was labeled with a rabbit polyclonal antibody raised against the amino terminus of c‐fos p62 (Santa Cruz Biotechnology, USA; sc-52; 1:5000, 4 °C) as described earlier (Halasz et al., 2002). This antibody is highly selective and shows no cross-reactions with other members of the fos protein family. The primary antibodies were detected by biotinylated antirabbit serum (1:500; Southern Biotechnology, Birmingham, AL, USA). After that they were incubated in Avidin-Biotin-Complex (ABC, Vestastain; 1:1000 TRIS) for 1 h and developed using 0.05% diaminobenzidine (DAB, Sigma) and 0.01% H2O2 in PBS. After visual inspection and qualitative analysis of fos-like immunoreactivity all over the brain, quantification was done in those nuclei (or sub-regions) considering relevant to the present experiment. Section planes were standardized according to the atlas of Paxinos and Watson (1998) by an experimenter blind to the treatment groups at the same coordinates for each animal. The background staining allowed us to identify brain areas without counter-staining. For the telencephalon (prefrontal cortex (PFC), nucleus accumbens (Acb), lateral septum, bed nucleus of stria terminalis (BNST)) and medial preoptic area (mPOA) gray scale images were taken with a digital camera (NIKON, DMX 1200) coupled to a bright-field microscope (NIKON, Eclipse E400), using a 40× objective, with no further modifications. For other brain regions (amygdala, SON, SCN, PVN) microscopic images were digitized by an OLYMPUS BX51 camera on magnification 10×. The average of four sections taken at 80 μm intervals was used for the analysis of each structure, including both hemispheres; that is, eight measures per area and animal. To select c-fos immunopositive nuclei as targets for quantification, a PC-based software (Scion Image, Scion Corporation, Frederick, MD, USA) was used and targets were subsequently identified in the captured images by gray level thresholding. For each brain area, the threshold for the labeled signal was defined according to the background staining. Size criteria were applied to exclude structures other than c-fos immunopositive nuclei from measurement therefore, only particles with a minimum area of 100 pixels (magnification 40×) or 20 pixels (magnification 10×) were considered as a cell nucleus. The number of c-fos positive nuclei per mm2 was calculated as the average number obtained of all analyzed images per rat. The following brain areas were investigated: PFC (cingulated cortexCg1, prelimbic area-PrL, infralimbic area-IL), nucleus accumbens (shell— AcbSh and core—AcbC), lateral septum, BNST, amygdala (medial (MeA) and central (CeA) part), mPOA, SON, SCN and PVN (magnocellular, medial and dorsal parvocellular parts) (Fig. 1).

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Fig. 1. Brain areas analyzed for c-fos-like immunoreactivity. Cg1: cingulate cortex, PrL: prelimbic area, IL: infralimbic area, AcbSh: nucleus accumbes shell; AcbC: nuleus accumbens core, LSv: lateral septum, ventral part, BNST: bed nucleus of strai terminalis, mPOA: medial preoptic area, SCN: nucleus suprachiasmaticus, SON: nucleus supraoticus, PVN: nucleus paraventricularis hypothalami, mPVN: magnocellular part of the PVN, mpPVN: medial parvocellular part of the PVN, dpPVN: dorsal parvocellular part of the PVN, CeA: central part of the amygdala, MeA: medial part of the amygdala.

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Statistical analysis Data were analyzed by analysis of variance (ANOVA). For undisturbed maternal behavior a general linear model was used (day and time of day as within-subject factors and genotype as the between-subjects factor). For the simple clarity the data are plotted in different groupings (all observations during the 7 days (420 observations/column), or during a time-period of the 7 days (140 observations/point)). Pup retrieval, EPM, sucrose preference and FST were analyzed by one way ANOVA (between‐subjects factor group or genotype). The run-time of the FST was analyzed by repeated measures ANOVA (time as within-subject factor and genotype as between-subjects factor). C-fos was analyzed by two-way ANOVA (between-subjects factor genotype and stress) or one-way ANOVA (genotype). For post-hoc analysis Fisher test was used and these results are indicated on the figures. Pearson correlations were calculated by multiple linear regression analysis. All statistical analyses were performed by Statistica 9.0 statistical software (StatSoft, Tulsa, USA). All data are expressed as mean± S.E.M. Results Maternal behavior Undisturbed maternal behavior Licking–grooming (LG). In the first experiment, there was a significant difference among genotypes (genotype effect: F(2,19) = 5.2; p = 0.01; Fig. 2A: for increased visibility a summary of all the 420 observation is presented). The di/di rats spent less time licking–grooming their pups, in comparison to the other two genotypes. When a detailed analysis across time was done the highest difference was detectable in the morning, in the middle of the day it disappeared and in the evening it became more explicit (effect of time of day: F(2,38) = 24.4; p b 0.01; Fig. 2B). During the second experiment, a main group effect was found (group effect: F(2,26) = 6.62; pb 0.01; Fig. 2C). We could reproduce the difference in LG between +/+ and di/di mothers and DDAVP treatment did not influence the effect of genotype. Similarly to the first experiment we could detect the highest difference between +/+ and di/di animals during the morning and evening hours without DDAVP effect in di/di dams, while the difference between groups was the smallest during noon (effect of time of day: F(2,52) =7.18; pb 0.01; Fig. 2D). We could not detect any changes in this behavior in the course of the 7 day observation period neither during the first nor during the second experiment. Other maternal behaviors During the first experiment there were no significant differences among genotypes in nursing behavior (Table 1). Therefore during the second experiment we conducted a more detailed analysis of this behavior. Although the total nursing behavior was also not different among the groups (Table 1), di/di mothers spend significantly less time in arched back posture than the +/+ ones and the DDAVP treatment had no effect on it (group effect: F(2,26) = 6.37; p b 0.01; Fig. 3A). Results also showed that both di/di and DDAVP treated di/di dams presented less arched-back than +/+ dams at 14:30 and 20:30 h, but not at 8:30 h (effect of time of day: F(2,52) = 5.04; p b 0.01; Fig. 3B). Dams spent around 50% of all the observations in a blanket posture nursing their pups (Table 1). Although there were no significant differences between +/+ and di/di dams in blanket nursing, DDAVP treated mothers spent significantly less time in this posture (group effect: F(2,26) = 16.4; p b 0.01). The passive nursing behavior showed more fluctuation between 5 and 20% of all observations (Table 1). Both di/di and DDAVP treated di/di dams spend more time in this posture in comparison to +/+ mothers (group effect: F(2,26) = 37.4; p b 0.01). There was no change in these behaviors during the 7 day observation period.

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There was no obvious difference between the total time spent out of the nest both during the first and second experiments (Table 1). However, in the first experiment during the 7 day observation period the +/+ mothers spent every day more and more time out of the nest (day effect: F(6,156) =2.21; pb 0.05), while di/di dams spend more time out of nest already during the first day of observation (genotype×time effect: F(12,114) =1.85; pb 0.05; data not shown). We assumed, that enhanced drinking due to peripheral AVP-deficiency could be the reason for this changed maternal behavior of di/di dams, thus we conducted a second experimental series with DDAVP replacement (which restores AVPdeficiency at the renal V2 receptors). Results showed that di/di mothers spend more time drinking and the DDAVP treatment normalized this behavior independently from the day of observation (group effect: F(2,26) = 35.32; pb 0.01; Fig. 3C). All groups drank less in the evening than during the morning measurements (effect of time of day: F(2,52) =7.89; pb 0.01; Fig. 3D). At the end of the 7 day observation period the weight of the litters was measured and the genotype of the mothers had no influence on it (+/+ mother: 110.6 ± 3.4 g; di/+ mother: 107.3± 4.3 g; di/di mother: 105.0 ±3.7 g for 6 pups). Pup retrieval test In the second experiment (DDAVP replacement), there were no differences between groups in the latency to carry and put the first and last pups to the nest (Table 1). Anxiety- and depression-like behavior Elevated plus-maze (EPM) There was no significant difference between the genotypes in the open arm time (Fig. 4A). In the case of the percent of open arm entries the effect of genotypes was on the border of significance (genotype effect: F(2,19) =3.2; p=0.06; Fig. 4B). The locomotor activity, characterized by the number of closed arm entries, was not different between groups (Fig. 4C). Sweet preference test Regarding sucrose preference as a function of total fluid intake, it was significantly higher in di/di mothers than in the other groups (genotype effect: F(2,19) =8.84; pb 0.01; Fig. 4D). On the other hand, saccharin preference was significantly lower in +/+ animals than in the two other groups (genotype effect: F(2,8) = 5.77; p b 0.05; Fig. 4E). Forced swimming test (FST) In our experiment the di/di rats floated less than the others, but these differences were not under the 0.05 significance level (Fig. 5A). If we analyze the results in 5 min periods the only significant difference was the time effect (time effect: F(2,36) = 5.1; p = 0.01; Fig. 5B), which means that all animals spent fewer time with floating during the last 5 min of the session than during the first 5 min. The struggling behavior showed significant differences among the genotypes (genotype effect: F(2,19) = 5.46; p = 0.01; Fig. 5C). The di/di mothers struggled more than the di/+ and +/+ ones. The occurrence of struggling decreased evenly across time in the +/+ and di/+ groups, but the di/di genotype struggled a lot during the first 10 min and just in the last 5 min this parameter decreased (time effect: F(2,36) = 8.35; p b 0.01; Fig. 5D). The effect of genotype was more evident during the middle 5 min (genotype effect: F(2,18) = 5.06; p = 0.01). The di/di mothers showed less swimming behavior in comparison to the other two groups during the entire session (genotype effect: F(2,19) = 23.6; p b 0.01; Fig. 5E). In the first 5 min there were no statistically significant differences among genotypes, but in the other two time-intervals di/di dams showed less swimming behavior (time effect: F(2,36) = 5.46; p = 0.01; Fig. 5F).

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Fig. 2. Licking and grooming behavior in the three genotypes (A,B) and after peripheral V2 agonist treatment (DDAVP) (C,D). A and C represent the summary of 420 observations, while on B and D the same results are shown at different timepoints of the day (140 observations/point). *pb0.05, **p b 0.01 vs. +/+; +p b 0.05, ++p b 0.01 vs. 8.30 h.

Diving behavior occurred less than 0.5% of the time and there was no any difference among the groups (data not shown). Immunocytochemistry We examined different brain areas, which could have a role in maternal behavior (mPOA, BNST (Numan et al., 1998)), maternal memory Table 1 Maternal behavior in the three genotypes and after DDAVP replacement.

Nursing behavior Out of the nest

Nursing behavior Blanket posture Passive nursing Out of the nest Carrying the first pup First pup in the nest Last pup in the nest

+/+

di/+

di/di

294.6 ± 19.5 107.6 ± 18.6

320.5 ± 7.5 88.2 ± 5.9

289.7 ± 11.9 110.3 ± 11.9

+/+

di/di DDAVP

di/di

305.5 ± 13.8 267.5 ± 9.5 18.9 ± 3.8 106.2 ± 14.7 44.2 ± 14.3 61.0 ± 13.4 206.2 ± 47.5

264.1 ± 21.3 174.5 ± 15.6⁎⁎ 83.9 ± 7.2⁎⁎

291.3 ± 11.2 245.0 ± 10.3 ,# 38.1 ± 5.2⁎⁎ 124.6 ± 10.4 41.3 ± 17.2 44.3 ± 12.9 233.4 ± 104.3

147.8 ± 20.5 37.8 ± 13.4 88.0 ± 25.8 371.6 ± 110.5

Nursing behavior is the sum of observations in arched back + blanket posture + passive nursing. DDAVP- subcutan desmopressin treatment via osmotic minipump. ⁎⁎ p b 0.01 vs. +/+. # p b 0.05 vs. di/di DDAVP.

(Acb (D'Cunha et al., 2011)), in stress (PVN, amygdala, PFC) or in general homeostasis (mPVN and dpPVN, SON, SCN) (Fig. 6 and Table 2). Test naïve dams were used as controls. When we conducted a two way ANOVA analysis, stress had a significant effect on all studied brain areas (p b 0.01; mPOA: F(1,27) = 101.5; AcbSh: F(1,27) = 188.0; dorsal parvocellular PVN (dpPVN): F(1,27) = 172.9; mpPVN: F(1,27) = 159.9; CeA: F(1,28) = 23.54; MeA: F(1,26) = 77.8; see Fig. 6 and for other areas see Table 2), however the main effect of genotype was significant only in case of SON (F(2,24) = 8.74; p b 0.01), and there was a significant genotype × stress interaction only in the case of AcbSh (F(2,27) = 7.73; p b 0.01). As the stress induced effects had different magnitudes (mostly 50–100× increase) than genotype induced effects (1.5–3× increase), the former might mask any effects of the latter. Therefore we also conducted a one way ANOVA analysis on basal and stressed levels, separately. In case of resting levels the effect of genotype was significant on mPOA (F(2,10) = 5.06; p b 0.05), AcbSh (F(2,10) = 36.4; p b 0.01), mpPVN (F(2,10) = 5.22; p b 0.05), CeA (F(2,11) = 9.41; p b 0.01) as well as in the SON (F(2,10) = 10.4; p b 0.01) and mPVN (F(2,10) = 12.1; p b 0.01) and there was a tendency in dpPVN (F(2,10) = 3.04; p = 0.09) and MeA (F(2,9) = 3.38; p =0.08) (see Fig. 6 and Table 2). The basal c-fos activity in the di/di rats was significantly higher than in the +/+ ones. Regarding the stress levels only the AcbSh (F(2,17) = 7.75; p b 0.01) and SON (F(2,14) = 4.15; p b 0.05) showed an effect of genotype and there was a tendency for genotype effect in the case of mPOA

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Fig. 3. Frequencies of the arched back posture (A, B) and drinking behavior (C, D) among 420 observations (A, C) and on different periods of the day (B, D; 140 observations/point) in AVP-deficient dams after DDAVP replacement. **p b 0.01 vs. +/+; ##p b 0.01 vs. di/+; ++p b 0.01 vs. 8.30 h.

(F(2,17) = 2.69; p = 0.09). Surprisingly, stress leads to a reduced activity of SON neurons in all groups. When we examined the increment of changes (stressed level minus average basal level; the upper part of the double columns on Fig. 6) the effect of genotype became significant on mPOA (F(2,17) = 4.04; pb 0.05), AcbSh (F(2,17) = 15.9; p b 0.01), dpPVN (F(2,17) = 3.64; p b 0.05), mpPVN (F(2,17) = 5.49; p = 0.01) and MeA (F(2,17) = 3.75; p b 0.05) and there was a tendency for genotype effect in CeA (F(2,17) = 2.92; p = 0.08) and mPVN (F(2,17) = 2.97; p = 0.08). The elevation induced by stress was smaller in di/di than in the +/+ dams in all areas analyzed. Regarding differences between di/+ and di/di animals, in AcbSh, dpPVN and mpPVN the c-fos activation induced by stress was smaller in di/di rats (see Fig. 6). Correlations There was no correlation between FST-induced c-fos labeling and depressive-like behavior on any studied brain areas. The only significant positive correlation was detectable between total LG behavior and FST-induced c-fos labeling on the AcbSh (r = 0.87, p b 0.01). Discussion Our results demonstrated that the congenital deficit of AVP in Brattleboro rats induces changes in undisturbed maternal behavior

without affecting the separation-induced motivational maternal response, the pup retrieval behavior. Moreover, di/di dams presented reduced depressive-like behavior demonstrated by enhanced sucrose and saccharin preference and struggling time in FST. In general, among rest di/di animals have higher c-fos activity on many brain areas (mPOA, AcbSh, CeA, SON, PVN), but their stress induced elevations were smaller (mPOA, AcbSh, MeA, PVN). Maternal behavior Brattleboro di/di dams spent less time licking–grooming their pups and in arched back posture, an effect not modified by peripheral DDAVP treatment. Thus, the excessive need for water intake did not influence maternal behavior of the AVP-deficient dams. Although no physical signs of maternal maltreatment were detected (body weight of the pups was not different), di/di dams seem to show a pattern of maternal neglect, not doing arched-back or licking–grooming behaviors towards their pups and instead performing just passive, supine nursing. In contrast to undisturbed (naturally occurring) maternal behavior, AVP had no effect on pup retrieval that is a measure of motivational maternal behavior (Hansen, 1994). Because retrieval behavior is initiated by the mother, Terkel et al. (1979) classified it as an active maternal response; and they referred to nursing behavior as a passive maternal response since it is primarily initiated by nuzzling and suckling

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Fig. 4. Behavior on the elevated plus maze (A–C) and during the sucrose (D) and saccharin (E) preference test. *p b 0.05,**p b 0.01 vs. +/+; ##p b 0.01 vs. di/+.

stimulation of the mother's ventral surface by the young. Brain circuitries related to pup retrieval and nursing behavior seems to be different although partially overlapping (see (Gammie, 2005) for a review). For instance, rat dams that were given ventral tegmental area microinfusions of 6-hydroxydopamine during lactation showed a persistent deficit in pup retrieval but were not impaired with respect to nursing (Hansen et al., 1991). Studies using injections lead to conflicting results. Acute injection of both AVP and V1a antagonist was found to diminish, while chronic AVP treatment had no effect on the establishment of maternal behavior (Nephew and Bridges, 2008a, 2008b). On the other hand, elegant studies by Bosch and Neumann on genetically changed animals support the involvement of AVP in maternal behavior (Bosch and Neumann, 2008, 2012). Using Wistar rats selected for low and high anxiety behaviors (LAB and HAB respectively) they demonstrated that HAB rats display more maternal care than LAB in parallel with higher brain AVP levels. As mPOA and BNST are the most studied parts of the neuronal circuitry regulating maternal behavior (Lonstein and De Vries, 2000; Numan, 2006; Numan et al., 1998; Numan and Stolzenberg, 2009; Stack et al., 2002), the role of AVP was specially studied on these areas. Upregulation of V1a receptors in the mPOA of normal Wistar rats, a key structure in the regulation of maternal behavior, induced an increase in maternal care (Kinsley and Lambert, 2006; Numan et al., 1998). On the contrary, administration of an AVP antagonist (V1) inside the mPOA blocked pup retrieval and nursing behavior in parturient dams (Pedersen et al., 1994). In normal Wistar rats, the injection of a V1a antagonist inside the BNST did not alter either undisturbed maternal care or pup retrieval, but reduced maternal aggression (Bosch et al., 2010). These results are in agreement with results found in the present experiment with higher resting c-fos activity in the mPOA, but not in the BNST region of di/di dams.

Another area, the AcbSh exhibited a similar pattern to mPOA. This region is a known brain center of reward (Gardner, 2011) and social interactions, like maternal behavior, are rewarding (Trezza et al., 2011). Indeed, mPOA regulated activation of mesolimbic dopamine system, especially the AcbSh, has a crucial role in the immediate onset and maintenance of maternal behavior after parturition (Afonso et al., 2009; Numan and Stolzenberg, 2009; Stolzenberg et al., 2010). AcbSh dopamine release is directly associated with onset of licking bouts (Champagne et al., 2004). Moreover, AcbSh has a role in maternal memory, as well (D'Cunha et al., 2011; Kinsley and Lambert, 2006). We might assume that enhanced resting activity of this nucleus in di/di dams represents a disturbed rewarding potential of maternal care leading to maternal neglect. In this context, c-fos expression induced by a stimulus (FST) was lower in di/di dams in correlation with diminished LG behavior. Previous studies from our laboratory (Zelena et al., 2003) and others (Casolini et al., 1993; Snijdewint et al., 1988) have shown that maternal genotype has an important impact on the Brattleboro offspring. Our studies showed that di/di adult males born and raised from di/di mothers presented a reduced HPA response to restraint stress, while the same effect was not detected when rats came from di/+ mothers (Zelena et al., 2003). However, the HPA reactivity to stressors seems to be independent from maternal behavior because cross-fostering did not modify the phenotype (Zelena et al., 2003). Moreover, in the HAB/ LAB animal model, where the less anxious, less depressive phenotype in LAB mice is accompanied by lower AVP levels (among many other affected genes), the phenotype of the offspring is not affected either by cross-fostering (Kessler et al., 2010). These results together indicate that genetic differences in AVP in these models could not be strongly modulated by maternal care, but likely influenced by intrauterine events.

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Fig. 5. Behavior during the forced swim test. A, C, and E represent the behavior during the whole 15 min observation, while B, D, and F show the same results divided into three 5 min section. **p b 0.01 vs. +/+; #p b 0.05, ##pb 0.01 vs. di/+; +p b 0.05, ++p b 0.01 vs. first 5 min.

Anxiety- and depression-like changes During the postpartum period the AVP-deficient rats showed less depressive-like behavior, than the control animals. It was demonstrated by the significantly higher struggling and higher sucrose and saccharin preference. In our study we could reproduce some of the changes found in male Brattleboro rats (Mlynarik et al., 2007). As in males these changes were not reversed by peripheral DDAVP treatment (unpublished data), it could be suggested that AVP effects are brain mediated in this case. In the present study di/di dams presented a more active coping behavior in front of stressful situations as measured by an increase in escape behavior (struggling) in the FST (Armario et al., 1988; Marti and Armario, 1993; Pinter et al., 2011). As in its traditional form FST was unreliable in the detection of selective serotonin reuptake inhibitors (Cryan et al., 2005), some modification was introduced (like increased water depth), and in this case struggling and swimming and not floating were considered as the most important parameters (Dimitsantos et al., 2007; Wigger and Neumann, 1999). Those antidepressants that increase serotonergic neurotransmission predominantly increase swimming behavior, and those that increase cathecholaminergic neurotransmission increase

struggling behavior (Cryan et al., 2005). The effect of AVP-deficiency in Brattleboro dams on swimming and struggling behavior was similar to the effect of Reboxetine (selective norepinephrine reuptake inhibitor). Anhedonia, the decreased motivation for a reward mediated behavior, is an important component of clinical depression. In animal models of depression-like behavior such as chronic mild stress (Willner, 1997), and in genetically selected rats based on learned helplessness (Sanchis-Segura et al., 2005) or avoidance (Gomez et al., 2009; Piras et al., 2010), a reduced preference for sweet solutions has been often found. To measure sucrose preference we used a paradigm described by Huot et al. (Huot et al., 2001), which was sensitive to an antidepressant treatment. As the phenotype of one group is diabetic the interpretation of our data was difficult. Therefore, to further support our results, a saccharin preference test was also conducted (Mazarati et al., 2008; Plaznik et al., 1989). The present work showed that di/di dams drank more sucrose than the other two groups, and drank more saccharin than the +/+ mothers, suggesting that they are more resistant to the development of depressive-like behavior. The heterozygous dams in one test were similar to +/+, while in the other to di/di dams, which could be due to changes in sensitivity to taste, caloric value etc. Human data

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Fig. 6. Number of c-fos positive cell nuclei among rest (smaller columns) and 2 h after a 15 min forced swim section (higher, open columns above the smaller ones). mPOA: medial preoptic area, AcbSh: nucleus accumbens shell; mpPVN: medial parvocellular part of the nucleus paraventricularis hypothalami, dpPVN: dorsal parvocellular part of the nucleus paraventricularis hypothalami; CeA: central part of the amygdala, MeA: medial part of the amygdala; *p b 0.05, **p b 0.01 vs. +/+; #p b 0.05, ##p b 0.01 vs. di/+; ++p b 0.01 vs. non-stressed.

also suggests that higher plasma AVP levels in depressed patients have been associated with lower reward-dependence (Goekoop et al., 2009). The role of AVP in plus maze behavior is supported by the fact that male rats treated with a V1R- or with a more specific V1bR-antagonist made more entries into and spent more time on the open arms, indicating reduced anxiety (Griebel et al., 2002; Liebsch et al., 1996). In LAB rats the low anxiety in the EPM is accompanied by low AVP levels (Bosch and Neumann, 2008). Moreover, intracerebroventricular AVP injection enhanced anxiety, while V1a antagonism reduced anxiety in lactating females (Bosch, 2011). However, in the present experimental conditions the increase in open arm time in di/di dams did not reach the statistical significance. Stress- and mood-related brain areas It was already described, that male di/di rats had elevated resting c-fos activity in SON, magnocellular PVN, subfornical organ, mPOA and organum vasculosum laminae terminalis compared to +/+ or Long Evans rats (Bundzikova et al., 2010; Guldenaar et al., 1992; Pirnik et al., 2004). In di/di dams we found higher basal activity in mPOA and AcbSh, as well as in dpPVN, mpPVN, CeA, and MeA and the magnocellular cells of the SON and PVN than in +/+ or di/+ animals. However, there was no obvious difference among the basal levels of different genotypes in PFC regions, AcbC, LSv, BNST and SCN.

The higher basal activity of magnocellular cells of di/di rats could be a possible consequence of the constant osmotic pressure. The homeostatic imbalance of the AVP-deficient rats might lead also to an activation of the dpPVN, as the autonomic center, (Hosoya and Matsushita, 1979; Swanson and Kuypers, 1980). The enhanced neuronal activity of the mpPVN cells suggests an exaggerated resting HPA axis; however, it was not the case (male: (Zelena et al., 2009a), female: our unpublished data) and it would be also in contrast to the less depressive phenotype. Thus, the importance of the resting c-fos activation in mpPVN of AVP-deficient dams remains unclear. The CeA is important in adaptation to chronic somatic stressors (Keen-Rhinehart et al., 2009; Thrivikraman et al., 1997), while MeA is related to the regulation of the response to emotional stress (Cullinan et al., 1995; Jankord and Herman, 2008). The resting c-fos levels of the AVP-deficient rats could be more enhanced in the CeA than in MeA due to the somatic stress of the diabetes insipidus. FST induced an activation of c-fos labeling in almost all studied brain regions in all genotypes. The exception of the SON and the relative mild stimulation of the magnocellular part of the PVN is not a surprise, because the function of these nuclei is to reserve the body osmotic balance, not the regulation of behavior or stress reactivity (Herman et al., 1995; Sawchenko et al., 1993). More interesting was the fact that the stress-induced elevation was smaller on mPOA, AcbSh, dpPVN, mpPVN and MeA of di/di dams compared to +/+ and di/+ rats.

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Table 2 c-fos data on different brain areas (cells/mm2). Brain area

Stress

+/+

di/+

di/di

mPFC-Cg1

Control FST Control FST Control FST Control FST Control FST Control FST Control FST Control FST Control FST

2.2 ± 0.9 204.4 ± 41.7 7.2 ± 2.5 258.7 ± 28.1 25.6 ± 6.6 181.1 ± 24.3 21.3 ± 3.3 88.5 ± 8.4 17.1 ± 4.3 817.7 ± 54.9 62.9 ± 14.0 194.5 ± 21.5 1552.3 ± 195.7 2169.6 ± 265.6 417.9 ± 14.4 240.7 ± 95.9 28.7 ± 10.3 179.3 ± 19.8

2.6 ± 1.1 223.7 ± 26.9 9.3 ± 3.3 207.0 ± 26.9 28.1 ± 3.8 114.5 ± 28.3 25.0 ± 4.1 83.7 ± 14.1 11.2 ± 4.6 828.1 ± 47.3 77.4 ± 24.9 147.7 ± 21.6 1654.9 ± 199.8 2390.7 ± 253.1 436.5 ± 47.4 345.1 ± 68.1 110.3 ± 16.8⁎⁎ 178.6 ± 33.6

1.5 ± 1.5 180.4 ± 39.7 2.2 ± 2.2 209.6 ± 31.8 20.8 ± 3.2 173.0 ± 21.9 15.3 ± 2.2 69.4 ± 8.7 7.8 ± 2.9 759.0 ± 39.8 88.2 ± 38.1 135.8 ± 25.0 1560.4 ± 472.2 2302.0 ± 251.9 ,## 625.1 ± 19.8⁎⁎ 494.8 ± 35.1⁎ 157.5 ± 33.0⁎⁎

mPFC-PL mPFC-IL AcbC LSv BNST SCN SON mPVN

Effect of stress (F(1,27)) 42.2 79.8 52.6 55.7 454.5 13.4 8.36 8.06 8.11

186.1 ± 40.8

FST: forced swim test, animals were perfused 2 h after a 15 min swimming section. mPFC: medial prefrontal cortex, Cg1: cingulate cortex, PrL: prelimbic area, IL: infralimbic area, AcbC: nuleus accumbens core, LSv: lateral septum, ventral part, BNST: bed nucleus of stria terminalis, SCN: nucleus suprachiasmaticus, SON: nucleus supraoticus, mPVN: magnocellular part of the nucleus paraventricularis hypothalami. The effect of stress: p b 0.01 for all brain areas. ⁎ p b 0.05. ⁎⁎ p b 0.01 vs. +/+. ## p b 0.01 vs. di/+.

PVN serves as the origin of the final common pathway in the secretion of glucocorticoid hormones in response to stress (Cullinan et al., 1996). The dpPVN and mpPVN responses are in accordance with the lower hormone response of male Brattleboro rats to physical (McCann et al., 1966; Yates et al., 1971) and emotional (Wiley et al., 1974; Zelena et al., 2009a) stressors. The enhanced basal c-fos activity of di/di dams might lead to habituation to homeostatic challenges (Aguilera, 1994), therefore a new stimulus was unable to induce similar changes in di/di than +/+ or di/+ rats. Nevertheless, the reduced stress-reactivity can contribute to the positive mood influencing effect of the AVP deficiency (Sterner and Kalynchuk, 2011). During swim stress one of the main inputs to PVN comes from the mPOA (Cullinan et al., 1996). Moreover, mPOA is strongly involved in stress-induced suppression of pulsatile LH secretion (Li et al., 2009). Although Acb is not the primary center of mood, its deep brain stimulation can contribute to reduction of the symptoms of mood disorders in humans (Bewernick et al., 2010). As this stimulation leads to inhibition rather than stimulation of the cells, we might assume that the reduced activation of the AcbSh region also contributes to the less depressive phenotype of our di/di dams. In previous studies FST caused a significant increase in the extracellular AVP concentration in the amygdala (Ebner et al., 2002). Moreover, bilateral V1 receptor antagonist injection into the amygdala reduced the floating time of the animals, so it had antidepressant effects (Salome et al., 2006). Conclusions We confirmed the important role of AVP in the development of undisturbed maternal response via central mechanism, which clearly dissociated from the separation-induced maternal retrieving, where AVP had no effect. Although the present data on mood suggest that AVP antagonists might have positive impact on PPD, the negative side effects on maternal behavior may limit their usage. Potential translational significance of the data is the ongoing development of AVP antagonists to treat depression. Our results underscore the need for more sex specific studies. The researchers should place greater emphasis on maternal behavior than standard testing paradigms (like EPM, sucrose preference, and FST) for depression and anxiety in PPD research, as the negative effects of PPD on offspring health are mediated through maternal behavior (Nephew and Bridges, 2011). Besides the most-studied brain areas (mPOA, BNST or mpPVN, CeA) the AcbSh could deeply contribute both to maternal behavior as well as depressive-like changes related to AVP.

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