Hormones and Behavior 65 (2014) 32–39

Contents lists available at ScienceDirect

Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh

The effects of pregnancy, lactation, and primiparity on object-in-place memory of female rats Katherine Tombeau Cost a,⁎, Thomas D. Lobell a, Zari N. Williams-Yee a,b, Sherryl Henderson c, Gary Dohanich a,b a b c

Program in Neuroscience, Tulane University, 2004 Percival Stern Hall, 6400 Freret Street, New Orleans, LA 70115 USA Department of Psychology, Tulane University, 2007 Percival Stern Hall, 6400 Freret Street, New Orleans, LA 70115 USA Department of Biology, Xavier University, 1 Drexel Drive New Orleans, LA 70125 USA

a r t i c l e

i n f o

Article history: Received 5 June 2013 Revised 4 September 2013 Accepted 26 October 2013 Available online 8 December 2013 Keywords: Pregnancy Lactation Object-in-place Spatial Memory

a b s t r a c t Maternal physiology and behavior change dramatically over the course of pregnancy to nurture the fetus and prepare for motherhood. Further, the experience of motherhood itself continues to influence brain functioning well after birth, shaping behavior to promote the survival of offspring. To meet these goals, cognitive abilities, such as spatial memory and navigation, may be enhanced to facilitate foraging behavior. Existing studies on pregnant and maternal rats demonstrate enhanced cognitive function in specific spatial domains. We adopted a novel object-in-place task to assess the ability of female rats to integrate information about specific objects in specific locations, a critical element of foraging behavior. Using a longitudinal design to study changes in spatial memory across pregnancy and motherhood, an advantage in the object-in-place memory of primiparous female rats compared to nulliparous females emerged during lactation not during pregnancy, and was maintained after weaning at 42 days postpartum. This enhancement was not dependent on the non-mnemonic variables of anxiety or neophobia. Parity did not affect the type of learning strategy used by females to locate a cued escape platform on a dual-solution water maze task. Results indicate that the enhancement of object-in-place memory, a cognitive function that facilitates foraging, emerged after pregnancy during the postpartum period of lactation and persisted for several weeks after weaning of offspring. © 2013 Elsevier Inc. All rights reserved.

Introduction Pregnancy exacts extreme changes in hormone synthesis, release, and action, affecting every bodily system studied to date. These comprehensive shifts in the hormonal milieu maintain an optimal environment for fetal growth and eventual delivery of the offspring, and profoundly remodel the maternal body and brain to prepare for motherhood (Brunton and Russell, 2008; Kinsley and Lambert, 2008; Nelson, 2005). While the changes to the body are obvious, less obvious and less understood, are the changes to the brain and the consequences for behavior throughout pregnancy and motherhood. In rats, most literature indicates that pregnancy and motherhood are associated with enhanced cognition, particularly on tasks that assess spatial learning and memory (Macbeth and Luine, 2010). Because effective and efficient foraging is highly dependent on spatial ability, these cognitive enhancements logically would improve the chances of offspring survival (Pawluski et al., 2006). Although a number of studies have tested the cognitive functions of primiparous rats (single litter) and multiparous rats (multiple litters) after weaning, only a few studies have actually tested cognitive performance during pregnancy and during lactation. For example, on an object ⁎ Corresponding author. Tel: +504-862-3339 E-mail address: [email protected] (K.T. Cost). 0018-506X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yhbeh.2013.10.012

placement task, pregnant rats displayed better spatial working memory in the first and third weeks of pregnancy than nulliparous females, which had never been pregnant (Macbeth et al., 2008). Similarly, on water maze tasks modified to assess spatial working memory, pregnant rats had shorter latencies and traveled shorter distances when learning to reach a submerged escape platform compared to nulliparous females (Bodensteiner et al., 2006; Galea et al., 2000). Together these studies indicate a modest, but significant, improvement in spatial working memory during pregnancy in rats. Although there have been no reports on the cognitive effects of lactation on spatial working memory, primiparous rats displayed impaired spatial reference memory when learning a conventional water maze task during the first week of lactation compared to nulliparous females (Darnaudery et al., 2007). However, on a subsequent retention trial administered 10 days later, these primiparous females explored the probe quadrant significantly more than nulliparous females, displaying better long-term spatial reference memory. Interestingly, cognitive effects persist beyond pregnancy and lactation. Two weeks after weaning, primiparous female rats displayed better spatial memory on a land maze task compared to nulliparous females, sensitized nulliparous females, or primiparous females whose pups had been removed within 24 h of birth (Lambert et al., 2005). Further, primiparous females spent more time by the baited well on a probe trial than all other groups. On a cued version of this same task, both primiparous females and sensitized nulliparous females found the reward

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

faster than nulliparous and primiparous females whose pups had been removed (Lambert et al., 2005). Similarly, on a radial arm maze task administered one month after weaning, primiparous females made fewer working memory errors than nulliparous females, sensitized nulliparous females, and multiparous females (Pawluski et al., 2006). In the same study, females that had experienced pregnancy, but not motherhood, failed to complete the task significantly more often than all other groups and displayed longer latencies to traverse the arms of the maze implicating non-mnemonic factors. Even post-weaning, the combined effects of pregnancy and mothering improved spatial performance more than pregnancy or mothering independently (Pawluski et al., 2006). In contrast, non-spatial working memory does not seem to be improved after pregnancy and lactation. On an object recognition task administered two weeks after weaning, primiparous rats were no different from nulliparous rats in distinguishing a novel object after either a 4-hour or 24-hour delay interval (Lemaire et al., 2006). Therefore, cognitive changes associated with pregnancy and motherhood are selective, with overall improvements in spatial ability but no reported effects on non-spatial ability. Motherhood also confers life-long benefits in place navigation. Two weeks after weaning, primiparous rats traveled shorter distances to reach the submerged escape platform on a water maze task (Lemaire et al., 2006). This improvement was maintained throughout life. When tested at 22 months of age, these primiparous rats again performed better than nulliparous controls. Moreover, the performance of primiparous rats at 6 and 22 months of age was comparable, whereas nulliparous controls had significantly worse performance at 22 months of age compared to 6 months of age (Lemaire et al., 2006). Similarly, 4 months after weaning, primiparous rats continued to display better working memory on the water maze task than nulliparous controls (Kinsley and Lambert, 2008). Consequently, the combination of pregnancy and motherhood exert effects on the spatial ability of female rats that can persist throughout the lifespan (for comprehensive reviews, see Kinsley and Lambert, 2008; Macbeth and Luine, 2010). We recently reported that biological sex and hormonal status influenced the ability of adult rats to remember the locations of objects on the novel object-in-place task that combines elements of traditional measures of object recognition and object location (Cost et al., 2012). Regardless of biological sex or hormonal condition, rats were able to recognize those objects that had been repositioned in new locations after a brief delay interval of 5 min between the sample phase and the test phase. When the delay interval was increased further to 60 min, only ovariectomized females treated with estradiol and progesterone demonstrated intact object-in-place memory, while ovariectomized females and gonadally-intact males treated with vehicle were unable to distinguish moved from unmoved objects. These results indicated a female advantage in the performance of the object-in-place task that was dependent on elevated levels of ovarian hormones (Cost et al., 2012). The current study was intended to extend the literature on the effects of pregnancy, lactation, and motherhood on spatial ability of rats using a longitudinal design across pregnancy, lactation, and post-weaning stages. Because we found previously that ovarian hormones enhanced object-inplace memory in female rats, we predicted that the high levels of hormones associated with pregnancy and lactation would lead to enhancements in performance on the novel object-in-place task. Additionally, as previous studies have found improved spatial ability in primiparous rats, we investigated whether there are effects of pregnancy and maternity on the type of navigational strategy used to learn a dual-solution water maze task because the preference for a place strategy over a stimulus–response strategy may mediate improved spatial ability. Methods Experimental design Cognitive performance was evaluated using the object-in-place task, which assesses memory for the identity and location of objects in space.

33

In a repeated measures design, primiparous female rats (n = 13) were tested at five time points, including prior to mating, during pregnancy, during lactation on post-delivery day (PDD) 14, and twice following weaning of their litters at PDD 28 and PDD 42. For comparison, nulliparous female rats in diestrus (n = 19) were tested at the same five corresponding time points. In addition, following completion of the five tests of object-in-place memory, all rats completed the cued platform water maze task on PDD 44, to assess the type of strategy used to locate a cued escape platform in a standard water maze.(Fig. 1). Subjects Subjects were 32 female adult Long-Evans rats procured from Harlan Laboratories, Inc. (Indianapolis, IN) at 55–60 days of age and pairhoused. An additional 6 males served as breeders. Rats were maintained at an ambient room temperature of 21 °C ±1° on a 12:12 h light–dark cycle with lights on at 1100 h. Harlan 2016 Teklad Global 16% Protein Rodent Diet and water were available ad libitum. Thirteen females were time-mated at 75 days of age by the male breeders with each female receiving three ejaculations on the first day of gestation. All animals were nulliparous at the beginning of the study. All rats in the primiparous group (n = 13) experienced their first pregnancy and lactation over the course of the experiment. Upon delivery of pups, litters were not culled to allow for the possibility that the total number of pups or the number of male pups may influence cognitive performance in pregnancy or the postpartum period. All animal usage was approved by the Tulane University Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996). Estrous cycle monitoring The estrous cycles of all females were monitored by vaginal swabbing daily beginning two weeks prior to behavioral testing and throughout behavioral testing. Vaginal cells were stained with toluidine blue and viewed under a microscope to monitor changes in vaginal cytology across the estrous cycle. Females were tested only during the metestrus or diestrous stage of their cycles, when ovarian steroid levels are low. Pregnant and lactating females were handled similarly to cycling females to control for the effects of daily handling. Cognitive measures The object-in-place task was administered at five separate time points spanning eight days prior to pregnancy to three weeks after weaning of litters. The cued platform water maze task was administered once one day after the final session of the object-in-place task. Object-in-place task The object-in-place task is a procedure developed to test rodent memory that combines the elements of traditional measures of object recognition and object placement (Barker and Warburton, 2009; Barker et al., 2007; Dix and Aggleton, 1999). To perform successfully, rodents must remember the features and locations of four distinct objects in an open field during a delay interval. Rodents prefer novelty, therefore memory for the original arrangement of the objects is indicated by increased time spent investigating the two objects that were relocated after the delay interval. Rats were tested in an open field constructed of matte black Plexiglas (90 cm × 90 cm × 45 cm) elevated 65 cm above the floor and surrounded by a rich assortment of visual cues. Prior to testing of object memory, rats were habituated to the open field for 10 min each day for three or four consecutive days. Habituation began in the late diestrous or early proestrous stage of the cycle so that the test day, when the rat would be in the metestrus or diestrus stage, would immediately follow the habituation days. Our initial subject numbers were purposefully high to allow for attrition due to the

34

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

Fig. 1. Timeline of cognitive testing.

variability in the duration of each phase of the estrous cycle (Mandl, 1951). Each behavioral trial session included a sample phase, a delay interval, and a test phase. During the sample phase, a rat was placed in the center of the open field and allowed 5 min to investigate four distinct objects, each located 15 cm from the corner walls (See Fig. 2A). Following the sample phase, the rat was returned to her home cage for a delay interval. Following the delay interval of 5 or 30 min, the rat again was placed in the center of the open field for the test phase and allowed to investigate the four objects in a new arrangement for 3 min. For the test phase, two of the original four objects, either those on the right or those on the left of the open field, exchanged positions while the two remaining objects remained in their original positions (See Fig. 2B). In total, 12 objects were used over the 6 behavioral sessions, divided into 3 groups of 4 objects, Object Sets A, B, and C. Each set of objects was chosen and balanced for intrinsic interest as based on preliminary studies conducted within our laboratory. Object Set A was presented at the 5min delay interval, and at Test Session 3. Object Set B was presented at Test Sessions 1 and 4. Object Set C was presented at Test Sessions 2 and 5. The first and second object set presentations were each separated by 45 to 54 days. To our knowledge, the longest duration for successful object recognition testing is 21 days (Ozawa et al., 2011), therefore, memory for the objects and their locations should have been negligible after 45 to 54 days. The testing room and open field remained constant, but the placement of objects during the sample and test phases was counterbalanced across all subjects and trials. This procedure allowed for the effects of anxiety and neophobia to be minimized, while assessing object-in-place memory. All surfaces of the open field and all objects were wiped thoroughly with 10% alcohol solution after sample and test phases to eliminate odor cues. In the first behavioral session administered prior to pregnancy, all rats were tested after a 5-min delay interval between the sample and test phases to validate the task. In all subsequent sessions at the five different time points, rats were tested after a 30-min delay interval between the sample and test phases (Cost et al., 2012). The first minute of the test phase is the most sensitive to rats' preference for novelty, therefore behavioral measures rely on this interval (Cost et al., 2012; Dix and Aggleton, 1999). All rats were required to explore all 4 objects during the sample phase for at least 10

combined seconds, and to spend at least 10 combined seconds exploring during the first minute of the test phase to be included in the analysis (Barker et al., 2007; Cost et al., 2012). Rats also were excluded from analysis if objects were incorrectly placed in the arena by the experimenter, or if rats chewed or moved objects or had entered the proestrous or estrous stages of the cycle on the day of testing. The primary measure of object memory on this task was the Discrimination Ratio (DR), defined as the time with moved objects (Mt) minus the time with unmoved objects (Ut) divided by the sum of the time with moved and unmoved objects [DR = (Mt − Ut) / (Mt + Ut)]. Cued platform water maze task Learning strategy was assessed in a cued platform water maze consisting of a circular pool painted white (180 cm diameter × 60 cm height), filled to a level of 28 cm with water made opaque by white non-toxic paint and maintained at a temperature of 23–25 °C (Daniel and Lee, 2004; Pearce et al., 1998; Spritzer et al., 2013). The platform (15 cm length × 15 width cm × 30 cm height) was submerged 2 cm below the surface of the water and marked by a salient visual cue. The cue consisted of a black plastic ball (3 cm diameter) affixed to a piece of threaded rod bolted to the center of the platform that was constructed of galvanized steel (1 cm diameter × 11 cm height) and covered horizontally with pieces of black and pink electrical tape (~ 2 cm each stripe). Prior to training trials, rats were habituated to the water maze for 1 min for 1 day. Training trials. During training, the escape platform was located in the southwest quadrant of the pool. Eight training trials were conducted with the rat entering from each of four locations in randomized order. If a rat failed to find the platform within 60 s an experimenter guided the rat to the escape platform where she remained for 10 s. All training trials were run consecutively with an inter-trial interval of 5 min (Fig. 3A). Probe trials. The cued platform was moved to the opposite side of the pool and the rat entered from the point halfway between the original

Fig. 2. Object-in-place task. (A) Rats investigate 4 objects for 3 min during the sample phase. (B) After a delay of 5 or 30 min, rats investigate the rearrangement of objects during the test phase for 3 min. Increased time spent investigating the relocated objects during the test phase indicates intact object-in-place memory.

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

35

Fig. 3. Cued platform water maze task. (A) Rats learn to swim to a cued platform to escape the water on 8 training trials. (B) Strategy is assessed on two probe trials after the cued platform is relocated to a new quadrant. Swimming directly to the new location of the cued platform indicates the use of a stimulus–response strategy. Returning to the training location of the platform indicates use of a place strategy.

platform location and the relocated platform (Fig. 3B). Navigation directly to the cued platform to within 5 cm during the probe trial indicated use of a stimulus–response strategy while navigation to the quadrant where the platform had been located during training indicated use of a place strategy. Rats were excluded from analysis if they were in the proestrous or estrous stage of the cycle on the day of testing. Statistical analyses A generalized estimating equation analysis, which is an analysis of variance that accounts for missing data points, was used to identify interactions between trial (pre-pregnancy, pregnancy, lactation, postweaning 1, post-weaning 2) and group (nulliparous females, primiparous females) for the object-in-place task. Subsequent analysis with a one-tailed t-test determined if the Discrimination Ratio (DR) for each group was significantly different from chance (DR = 0). The cued platform water maze navigation data were analyzed with χ2 to determine if there were differences in strategy choice (stimulus–response, place) given maternal status. Group differences in escape path lengths were analyzed using mixed model analysis of variance. All statistics were computed using IBM SPSS Statistics for Apple, Version 20.0 (IBM Corporation, Armonk, NY). Significance level was set to .05. Results Object-in-place memory Pretest (pre-pregnancy), 5-min delay Discrimination Ratios for the first minute of the test phase were significantly different from 0 for both diestrous nulliparous females, t(13) = 2.004, p = .033, and diestrous primiparous females, t(9) =

3.069, p = .007, indicating that both groups displayed intact objectin-place memory after a 5-min delay interval (Fig. 3A). When Discrimination Ratios for the first minute of the test phase were compared between nulliparous females and primiparous females, there was no significant group difference, t(22) = .834, p = .413. There were group differences in object investigation during the first minute of the sample phase, t(22) = 2.464, p = .022, in which primiparous females spent more time investigating all objects. However, differences in object exploration in the first minute of the sample did not affect the ability to discriminate moved from unmoved objects, as confirmed in a metaanalysis of previous studies of object recognition (Akkerman et al., 2012). Five nulliparous females and three primiparous females were excluded from test phase analysis (see Table 1). Repeated measures analysis with generalized estimating equation for 30min delays There was a significant effect of group on object-in-place task, such that, averaged across all trials, the groups performed differently on the task, χ2(1) = 4.421, p = .035. There was also a marginally significant effect of trial, such that, averaged across both groups, rats improved performance on the task, χ2(4) = 9.497, p = .050. However, there was no group by trial interaction, χ2(4) = 1.319, p = .858. Test 1 (pre-pregnancy), 30-min delay All subsequent analysis assessed the performance of each group independently against the test value of DR = 0, which indicates chance discrimination. Discrimination Ratios for the first minute of the test phase were not significantly different from 0 for either nulliparous females, t(15) = .631, p = .269, or primiparous females, t(9) = .821, p = .217, indicating that neither group displayed intact object-inplace memory after a 30-min delay interval (Fig. 4B). There was no

Table 1 Rats excluded from analysis at each of the object-in-place test sessions (5-min delay, 30-min delays during pre-pregnancy, pregnancy, lactation, post-weaning 1, and post-weaning 2). P = primiparous females, N = nulliparous females. Reason for exclusion

5-Min delay

30-Min delay Test 1

Irregular cycling Failure to meet exploratory criteria Moving or chewing on objects Recording equipment failure Disruption to the test environment Object misplacement

Test 2

Test 3

Test 4

Test 5

P

N

P

N

P

N

P

N

P

N

P

N

3 – – – – –

2 1 1 – – 1

3 – – – – –

3 – – – – –

– – – – 2 1

4 – – – – 1

– – – – – –

3 – 1 1 – 1

2 – 1 – – –

1 2 1 – – 2

4 1 – – – –

5 4 1 – 1 –

36

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

Fig. 4. (A) Discrimination Ratios for primiparous females and nulliparous females on the object-in-place task after 5-min delay interval between the sample phase and the test phase. (B) Discrimination Ratios for primiparous females and nulliparous females on the object-in-place task at 5 test sessions (pre-pregnancy, pregnancy, lactation, post-weaning 1, and post-weaning 2) after 30-min delay intervals between the sample phase and the test phase. PDD = Post Delivery Day. The Discrimination Ratios for each group were analyzed with a one-tailed t-test against the expected value of 0. Discrimination Ratios significantly different from 0 indicate intact object-in-place memory after the delay interval, *p b .05.

difference between groups in total time spent investigating all objects in the first minute of the sample phase, t(24) = −.652, p = .520. Three nulliparous females and three primiparous females were excluded from test phase analysis (see Table 1). Test 2 (pregnancy, 14 days post-conception), 30-min delay Discrimination Ratios for the first minute of the test phase were not significantly different from 0 for either nulliparous females, t(13) = −.666, p = .259, or primiparous females, t(9) = −.594, p = .284, indicating that neither group displayed intact object-inplace memory after a 30-min delay interval (Fig. 4B). There was no difference between groups in total time spent investigating all objects in the first minute of the sample phase, t(22) = −.107, p = .916. Five nulliparous females and three primiparous females were excluded from test phase analysis (see Table 1). Test 3 (lactation, 14 days postpartum), 30-min delay Discrimination Ratios for the first minute of the test phase were not significantly different from 0 for nulliparous females, t(12) = .631, p = .269, but were significantly different from 0 for primiparous females, t(12) = 2.567, p = .013, indicating that only maternal females displayed intact object-in-place memory after a 30-min delay interval (Fig. 4B). There was no difference between groups in total time spent investigating all objects in the first minute of the sample phase, t(22) = −.146, p = .886. Six nulliparous females were excluded from test phase analysis (see Table 1) and two primiparous females were excluded from sample phase analysis for recording equipment failure. Test 4 (post-weaning 1, 28 days postpartum), 30-min delay Discrimination Ratios for the first minute of the test phase were not significantly different from 0 for nulliparous females, t(12) = .976, p = .174, but were significantly different from 0 for primiparous females, t(9) = 2.868, p = .010, indicating that only maternal females displayed intact object-in-place memory after a 30-min delay interval (Fig. 4B). There was no difference between groups in total time spent

investigating all objects in the first minute of the sample phase, t(21) = .306, p = .763. Seven nulliparous females and three primiparous females were excluded from test phase analysis (see Table 1). Test 5 (post-weaning 2, 42 days postpartum), 30-min delay Discrimination Ratios for the first minute of the test phase were not significantly different from 0 for nulliparous females, t(7) = .113, p = .457, but were significantly different from 0 for primiparous females, t(7) = 2.637, p = .017, indicating that only maternal females displayed intact object-in-place memory after a 30-min delay interval (Fig. 4B). There was no difference between groups in total time spent investigating all objects in the first minute of the sample phase, t(14) = .093, p = .927. Eleven nulliparous females and five primiparous females were excluded from test phase analysis (see Table 1). Litter size and number of males in the litter There was no relationship between the number of pups in a litter and object-in-place memory during Test 2 (pregnancy), b = .041, t(7) = .946, p = .372; Test 3 (lactation), b = −.033, t(10) = −.783, p = .450; Test 4 (post-weaning 1), b = −.040, t(7) = −1.095, p = .306; or Test 5 (post-weaning 2), b = .029, t(5) = .661, p = .533. Additionally, there was no relationship between the number of male pups in a litter and object-in-place memory during Test 2 (pregnancy), b = −.005, t(7) = −.058, p = .955; Test 3 (lactation), b = −.066, t(10) = −.977, p = .350; Test 4 (post-weaning 1), b = −.072, t(7) = −1.115, p = .297; or Test 5 (post-weaning 2), b = .065, t(5) = .935, p = .386. Anxiety Pretest (pre-pregnancy) 5-min delay Anxiety was measured by the percent of time spent in the center of the testing arena during the first minute of the test phase. There was no

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

37

group difference in percent time in the center of the arena in the first minute of the test phase with a 5-min delay, t(22) = 1.227, p = .233. Repeated measures analysis with generalized estimating equation, 30-min delays There were no group differences, χ2(1) = .115, p = .735, no effect of time, χ2(4) = 3.128, p = .537, and no group by time interaction, χ2(4) = 4.377, p = .357, in the percent time in the center of the arena in the first minute of the test phase with a 30-min delay at Tests 1, 2, 3, 4, or 5. Neophobia Pretest (pre-pregnancy) 5-min delay Neophobia was measured as the latency to approach any object during the test phase. There was no group difference in latency to approach an object in the first minute of the test phase with a 5-min delay, t(22) = −.699, p = .492. Repeated measures analysis with generalized estimating equation, 30-min delays There were no group differences, χ2(1) = .370, p = .543, no effect of time, χ2(4) = 2.618, p = .624, and no group by time interaction, χ2(4) = 7.947, p = .094, in the latency to approach an object in the first minute of the test phase with a 30-min delay at Tests 1, 2, 3, 4, or 5. Test 6 (post-weaning 3, 44 days postpartum), cued platform water maze task Path length to reach the platform decreased across trial blocks for both groups, F(1,29) = 10.342, p = .003. There was no effect of group, F(1,29) = 1.508, p = .229, nor a group by trial block interaction, F(1,29) = 1.232, p = .276. These results indicate that both groups, regardless of parity, learned the task equally well as training progressed (Fig. 5A). There were no group differences in strategy choice on the first probe trial, χ2 = .474, p = .491 (see Fig. 5B), nor on the second, χ2 = 1.316, p = .251 (see Fig. 5C), indicating that both groups were evenly split between place and response strategies on both probe trials. One primiparous female was excluded from analysis for irregular cycling. Discussion Parity affects the ability of female rats to remember the locations of specific objects as assessed by the object-in-place task. Both primiparous and nulliparous female rats could remember object-location associations with a short delay interval of 5 min. When the delay interval was extended to 30 min to increase demand on memory, neither primiparous nor nulliparous female rats could remember the location of objects in the object-in-place task prior to parturition. However, 2 weeks after parturition during lactation, primiparous females could remember object-location associations, while nulliparous females were still unable to do so. This improvement persisted after weaning and even at 42 days post-partum, primiparous females still had intact object-in-place memory while nulliparous females did not. Results indicate a persistent advantage for primiparous females on the object-in-place task after long delay intervals that was not related to the non-mnemonic factors of anxiety or neophobia. The object-in-place task is distinct from yet combines elements of object location and object recognition, requiring the integration of information about individual object features and contextual relationships between objects and the environment (Barker and Warburton, 2009, 2011; Barker et al., 2007; Eichenbaum et al., 2007). This unique integration requires the participation of hippocampal and cortical regions than can be individually or collectively influenced by the distinctive hormonal milieu of pregnancy and/or lactation and the environmental enrichment of motherhood. Previous studies have reported an improvement

Fig. 5. Performance on the cued platform water maze task. (A) Both groups learned the task similarly over 8 training trials. (B,C) Neither group showed a preference for a stimulus–response strategy or place strategy when the opportunity to use either strategy was presented on the first (B) and second (C) probe trials.

in spatial working memory during pregnancy (Bodensteiner et al., 2006; Galea et al., 2000; Macbeth et al., 2008), which may be linked to morphological changes in the hippocampus (Macbeth and Luine, 2010). However, we did not find that female rats in their second week of pregnancy were able to maintain associations between objects and their locations in space after delays of 30 min. The lack of improvement in performance by pregnant females in our study compared to previous studies that have reported modest improvements in spatial ability during pregnancy may be due to differences in experimental procedures related to strain or time of testing in the circadian rhythm. Despite these differences in experimental procedures, the paradigm is essentially different because the complex nature of the object-in-place task requires the integration of information related to object placement and object recognition. Encoding the features and locations of objects in a spatial environment for later recognition is a complex operation requiring the contributions of multiple brain regions, including the hippocampus, perirhinal cortex, and the medial prefrontal cortex (Barker and Warburton, 2011; Barker et al., 2007). If the brain is actually being ‘re-modeled’ during pregnancy as some have suggested (Kinsley and Lambert, 2008), then connectivity between regions such as the hippocampus and the perirhinal and

38

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

entorhinal cortices may take longer to manifest or may even be disrupted while these individual areas experience growth and enrichment. Our results indicate that while the changes necessary for object-in-place memory may be facilitated by the hormonal changes of pregnancy, it is not until the postpartum period when cognitive gains are actually realized. Future tract-tracing studies may be able to identify the specific changes in connectivity between brain regions involved in object-inplace memory associated with late pregnancy and early motherhood. In addition to the current results, several reports have revealed persistent spatial memory improvement in primiparous females postweaning relative to nulliparous females (Kinsley and Lambert, 2008; Lambert et al., 2005; Lemaire et al., 2006; Pawluski et al., 2006). The current study attempted to delineate the time frame during which cognitive improvement develops. We identified the three-week period between the second week of pregnancy and the second week of lactation as the key window leading to long-lasting gains in object-in-place memory. Multiple cognitive tests during this critical span will be necessary to further pinpoint the timing and environmental and hormonal factors involved in cognitive improvements in the spatial domain. The motivation for a mother to return to her nest is unknown. In the rat, the amount of time spent foraging may be motivated by caloric intake or by time away from vulnerable young. Mothers may only return to a nest once a sufficient number of calories have been consumed. Alternatively, mothers may be motivated to return to the nest after a specified period of time away, with anxiety increasing as time away from the nest increases. Much research has been done on the rewarding qualities of pups to new mothers (Febo, 2011; Mattson et al., 2003; Numan and Insel, 2003; Numan et al., 2006; Pereira and Morrell, 2010; Seip et al., 2008), which may be relevant to time foraging. In either case, improved foraging would be paramount to pup survival, which is dependent upon both the mother's vitality and presence and also upon her ability to provision her pups with milk. Previous research reports that pup exposure increases foraging ability in primiparous rats compared to primiparous rats that did not have pup exposure, which indicates that environmental variables, such as pup exposure or food availability may have an effect on foraging ability (Lambert et al., 2005). Conversely, Leuner and Gould (2010), using food reduction, found that maternal rats did display enhancement in attentional set shifting compared to nulliparous females, but not in discrimination or reversal learning. Although the object-in-place task does not involve food deprivation, future studies could investigate the role of caloric deprivation on object-in-place memory in lactating dams. On a cued platform water maze task, neither primiparous nor nulliparous females demonstrated a preference for either a place or a stimulus–response strategy on either of two probe trials. Improvements in the spatial ability of primiparous rats demonstrated above and in previous studies (Kinsley and Lambert, 2008; Kinsley et al., 1999; Lambert et al., 2005; Lemaire et al., 2006; Pawluski et al., 2006) seem to be unrelated to strategy preference. Although a previous study did find improvement in place navigation in primiparous rats relative to nulliparous rats (Lemaire et al., 2006), cycle stage during testing was not reported, despite the documented effect of elevated estradiol titers on place navigation (Korol, 2004; Korol and Kolo, 2002). Further, the effects of hormones on spatial working memory and strategy preference appear to be activational rather than organizational in the peripartum, as past research has found that pregnant females have better spatial working memory performance in the water maze than non-pregnant females (Bodensteiner et al., 2006; Galea et al., 2000). However, 44 days postpartum, when all females were in diestrus with low levels of ovarian hormones, primiparous females learned the task at a similar rate to nulliparous females and did not display any preference for place or stimulus–response strategy. Our data do not show a change in strategy preference in primiparous rats. Therefore, improvement on spatial tasks is not the result of the use of a different spatial strategy, that is to say, how the primiparous rats solve the task is not different from nulliparous rats.

Spatial ability, important for acquiring food and water, increasing mating opportunities, and locating good nesting grounds, is intricately linked with survival. As such, evolution has selected for spatial abilities across species, resulting in differences between the polar-to-tropical navigation of humpback whales (Horton et al., 2011), the 140-km migrations of African elephants (Thouless, 1995), and the 1.2-km2 range of cotton rats (Slade and Swihart, 1983). More phenomenally, evolutionary pressures likely resulted in differences in spatial ability and memory within species, based on hormonal influences, indicating that spatial ability is not a static endowment, but subject to change across the lifespan. In many species, from parti-colored bats (Safi et al., 2007) to house cats (Baratt, 1997) to cotton rats (Slade and Swihart, 1983) and even modern humans (Ecuyer-Dab and Robert, 2004) reproductively-aged males have a larger range size, reliant on better spatial abilities. In some species, such as the matrilineal African elephant, maternal females lead the annual migrations following the everchanging availability of water (Thouless, 1995). Between and within each species, spatial ability has been shaped by the need to survive and to produce offspring that survive, resulting in differential aptitude based on the most salient selective forces. The behavioral changes associated with maternal adaptation are legion and species-specific in placental mammals. Changes to behavior are synchronized with pregnancy and parturition (Brunton and Russell, 2008; Rosenblatt et al., 1988). In rats, nulliparous adult females will avoid or even become aggressive toward pups (Fleming and Rosenblatt, 1974). However, a primigravid female will begin to build a nest and will even become interested in pups as her pregnancy progresses (Numan et al., 2006). Once the pups are born, new mothers will engage in a full repertoire of maternal behaviors not previously displayed, including kyphotic nursing, pup retrieval, and licking and grooming of pups (Numan et al., 2006). For the most robust maternal behavior, primiparous females must have both exposure to pregnancy hormones and exposure to pups (Numan et al., 2006). In contrast to nulliparous females, multiparous females find pup-associated stimuli highly rewarding (Afonso et al., 2008, 2009). All of these behaviors increase the likelihood of survival for the pups and the pups' ability to successfully reproduce themselves (Brunton and Russell, 2008). These behaviors are unquestioningly maternal adaptations that follow naturally from pregnancy and parturition. However, in a laboratory environment, this set of maternal behaviors is divorced from the need of the mother to provision herself, as food is often available ad libitum (Bodensteiner et al., 2006; Galea et al., 2000; Lambert et al., 2005; Lemaire et al., 2006; Macbeth et al., 2008; Pawluski et al., 2006). In a more ecological setting, a mother rat would not have access to a daily-filled food hopper just above her nest, necessitating the enhancement of more than just her direct mothering skills for her pups to survive. Selection is brutal but adaptation is subtle. A slight advantage in foraging ability can win a few extra calories a day and, perhaps more importantly, minimize time away from a vulnerable nest that could make a difference between success and failure for a mother and her offspring. Although the literature is inconclusive about the timing and nature of cognitive changes in pregnancy in rats, there may not be an improvement in cognitive function during pregnancy because it is not necessary, and therefore has not been selected for by evolutionary pressures. Although extra calories are needed to maintain a pregnancy, these can be gathered without additional time constraints and a pregnant rat can return to her nest even after long forays without threatening the survival of herself or her fetuses (Wolff, 2007). If spatial ability in pregnancy does not directly increase reproductive success, evolutionary pressures will not select for improved spatial ability during pregnancy. However, once her altricial offspring are born, a maternal rat must nurse her pups, crouch over them to keep them warm, groom them, retrieve them when they crawl away, and protect them from both predators and conspecifics (Lonstein and Fleming, 2001; Numan et al., 2006; Wolff, 2007). Therefore, the demands are increased after delivery. The maternal rat must now find sufficient sustenance to maintain herself

K.T. Cost et al. / Hormones and Behavior 65 (2014) 32–39

and milk production and to return to her defenseless pups as quickly as possible to preserve her reproductive investment. It is during this critical period of early motherhood when selection will act to increase the fitness of the mother by increasing her spatial abilities to allow her to navigate her environment quickly in order to facilitate her sustenance and to protect and nurture her pups in the nest. If the mother does not adequately recall the location of sufficient food stores, she and her pups will perish. If the mother does not return to her nest quickly enough from foraging, she may find her pups have been eaten by a predator or a conspecific. Although the improvements in cognitive function associated with motherhood are modest, they are one element of a larger set of cognitive and behavioral changes subject to selection to facilitate reproductive success (Numan et al., 2006; Wolff, 2007). Of course, it is also possible that the cognitive changes we see are not a product of selection but may have arisen as a byproduct of other factors selected for in the maternal experience. Acknowledgments This research was supported by the Program in Neuroscience at Tulane University, a Phase II Research Enhancement Award, the Weiss Presidential Scholarship, the Louisiana Board of Regents PFUND, and Newcomb Fellowship. The authors gratefully acknowledge the expert supervision of animal care provided by Kimberly Scamardo and Dr. Meg Bakewell for helpful advice and discussions. References Afonso, V.M., Grella, S.L., Chatterjee, D., Fleming, A.S., 2008. Previous maternal experience affects accumbal dopaminergic responses to pup-stimuli. Brain Res. 1198, 115–123. Afonso, V.M., King, S., Chatterjee, D., Fleming, A.S., 2009. Hormones that increase maternal responsiveness affect accumbal dopaminergic responses to pup- and food-stimuli in the female rat. Horm. Behav. 56 (1), 11–23. Akkerman, S., Blokland, A., Reneerkens, O., van Goethem, N.P., Bollen, E., Gijselaers, H.J., et al., 2012. Object recognition testing: methodological considerations on exploration and discrimination measures. Behav. Brain Res. 232 (2), 335–347. Barker, G.R., Bird, F., Alexander, V., Warburton, E.C., 2007. Recognition memory for objects, place, and temporal order: a disconnection analysis of the role of the medial prefrontal cortex and perirhinal cortex. J. Neurosci. 27 (11), 2948–2957. Barker, G.R., Warburton, E.C., 2009. Critical role of the cholinergic system for object-inplace associative recognition memory. Learn. Mem. 16 (1), 8–11. Barker, G.R., Warburton, E.C., 2011. When is the hippocampus involved in recognition memory? J. Neurosci. 31 (29), 10721–10731. Baratt, D.G., 1997. Home range size, habitat utilisation and movement patterns of suburban and farm cats Felis catus. Ecography 20 (3), 271–280. Bodensteiner, K.J., Cain, P., Ray, A.S., Hamula, L.A., 2006. Effects of pregnancy on spatial cognition in female Hooded Long-Evans rats. Horm. Behav. 49 (3), 303–314. Brunton, P.J., Russell, J.A., 2008. The expectant brain: adapting for motherhood. Nat. Rev. Neurosci. 9 (1), 11–25. Cost, K.T., Williams-Yee, Z.N., Fustok, J.N., Dohanich, G.P., 2012. Sex differences in objectin-place memory of adult rats. Behav. Neurosci. 126 (3), 457–464. Daniel, J.M., Lee, C.D., 2004. Estrogen replacement in ovariectomized rats affects strategy selection in the Morris water maze. Neurobiol. Learn. Mem. 82 (2), 142–149. Darnaudery, M., Perez-Martin, M., Del Favero, F., Gomez-Roldan, C., Garcia-Segura, L.M., Maccari, S., 2007. Early motherhood in rats is associated with a modification of hippocampal function. Psychoneuroendocrinology 32 (7), 803–812. Dix, S.L., Aggleton, J.P., 1999. Extending the spontaneous preference test of recognition: evidence of object-location and object-context recognition. Behav. Brain Res. 99 (2), 191–200. Ecuyer-Dab, I., Robert, M., 2004. Spatial ability and home-range size: examining the relationship in Western men and women (Homo sapiens). J. Comp. Psychol. 118 (2), 217–231. Eichenbaum, H., Yonelinas, A.P., Ranganath, C., 2007. The medial temporal lobe and recognition memory. Annu. Rev. Neurosci. 30, 123–152. Febo, M., 2011. A bold view of the lactating brain: functional magnetic resonance imaging studies of suckling in awake dams. J. Neuroendocrinol. 23 (11), 1009–1019.

39

Fleming, A.S., Rosenblatt, J.S., 1974. Maternal behavior in the virgin and lactating rat. J. Comp. Physiol. Psychol. 86, 957–972. Galea, L.A., Ormerod, B.K., Sampath, S., Kostaras, X., Wilkie, D.M., Phelps, M.T., 2000. Spatial working memory and hippocampal size across pregnancy in rats. Horm. Behav. 37 (1), 86–95. Guide for the Care and Use of Laboratory Animals, 1996. Retrieved from http://www.nap. edu/openbook.php?record_id=5140. Horton, T.W., Holdaway, R.N., Zerbini, A.N., Hauser, N., Garrigue, C., Andriolo, A., Clapham, P.J., 2011. Straight as an arrow: humpback whales swim constant course tracks during long-distance migration. Biol. Lett. 7 (5), 674–679. Kinsley, C.H., Lambert, K.G., 2008. Reproduction-induced neuroplasticity: natural behavioural and neuronal alterations associated with the production and care of offspring. J. Neuroendocrinol. 20 (4), 515–525. Kinsley, C.H., Madonia, L., Gifford, G.W., Tureski, K., Griffin, G.R., Lowry, C., et al., 1999. Motherhood improves learning and memory. Nature 402 (6758), 137–138. Korol, D.L., 2004. Role of estrogen in balancing contributions from multiple memory systems. Neurobiol. Learn. Mem. 82 (3), 309–323. Korol, D.L., Kolo, L.L., 2002. Estrogen-induced changes in place and response learning in young adult female rats. Behav. Neurosci. 116 (3), 411–420. Lambert, K.G., Berry, A.E., Griffins, G., Amory-Meyers, E., Madonia-Lomas, L., Love, G., et al., 2005. Pup exposure differentially enhances foraging ability in primiparous and nulliparous rats. Physiol. Behav. 84 (5), 799–806. Lemaire, V., Billard, J.M., Dutar, P., George, O., Piazza, P.V., Epelbaum, J., et al., 2006. Motherhood-induced memory improvement persists across lifespan in rats but is abolished by a gestational stress. Eur. J. Neurosci. 23 (12), 3368–3374. Leuner, B., Gould, E., 2010. Dendritic growth in medial prefrontal cortex and cognitive flexibility are enhanced during the postpartum period. J. Neurosci. 30 (40), 13499–13503. Lonstein, J.S., Fleming, A.S., 2001. Parental behaviors in rats and mice. Curr. Protoc. Neurosci. Suppl. 17 (8), 15 (11-18.15.26). Macbeth, A.H., Gautreaux, C., Luine, V.N., 2008. Pregnant rats show enhanced spatial memory, decreased anxiety, and altered levels of monoaminergic neurotransmitters. Brain Res. 1241, 136–147. Macbeth, A.H., Luine, V.N., 2010. Changes in anxiety and cognition due to reproductive experience: a review of data from rodent and human mothers. Neurosci. Biobehav. Rev. 34 (3), 452–467. Mandl, A.M., 1951. The phases of the oestrous cycle in the adult white rat. J. Exp. Biol. 28, 576–584. Mattson, B.J., Williams, S.E., Rosenblatt, J.S., Morrell, J.I., 2003. Preferences for cocaine- or pup-associated chambers differentiates otherwise behaviorally identical postpartum maternal rats. Psychopharmacology (Berl.) 167 (1), 1–8. Nelson, R.J., 2005. An Introduction to Behavioral Endocrinology, 3rd ed. Sinauer Associates, Inc., Sunderland, MA. Numan, M., Fleming, A.S., Levy, F., 2006. Maternal behavior, In: Neill, J.D. (Ed.), 3rd ed. Knobil & Neill's Physiology of Reproduction, vol. 2. Elsevier, San Diego, pp. 1922–1993. Numan, M., Insel, T.R., 2003. The Neurobiology of Parental Behavior. Springer, New York. Ozawa, T., Yamada, K., Ichitani, Y., 2011. Long-term object location memory in rats: effects of sample phase and delay length in spontaneous place recognition test. Neurosci. Lett. 497 (1), 37–41. Pawluski, J.L., Vanderbyl, B.L., Ragan, K., Galea, L.A., 2006. First reproductive experience persistently affects spatial reference and working memory in the mother and these effects are not due to pregnancy or ‘mothering’ alone. Behav. Brain Res. 175 (1), 157–165. Pearce, J.M., Roberts, A.D., Good, M., 1998. Hippocampal lesions disrupt navigation based on cognitive maps but not heading vectors. Nature 396 (6706), 75–77. Pereira, M., Morrell, J.I., 2010. The medial preoptic area is necessary for motivated choice of pup- over cocaine-associated environments by early postpartum rats. Neuroscience 167 (2), 216–231. Rosenblatt, J.S., Mayer, A.D., Giordano, A.L., 1988. Hormonal basis during pregnancy for the onset of maternal behavior in the rat. Psychoneuroendocrinology 13 (1–2), 29–46. Safi, K., Koenig, B., Kerth, G., 2007. Sex differences in population genetics, home range size and habitat use of the parti-coloured bat (Vespertilio murinus, Linnaeus 1758) in Switzerland and their consequences for conservation. Biological Conservation 137, 28–36. Seip, K.M., Pereira, M., Wansaw, M.P., Reiss, J.I., Dziopa, E.I., Morrell, J.I., 2008. Incentive salience of cocaine across the postpartum period of the female rat. Psychopharmacology (Berl.) 199 (1), 119–130. Slade, N.A., Swihart, Robert K., 1983. Home range indices for the hispid cotton rat (Sigmodon hispidus) in Northeastern Kansas. J. Mammal. 64 (4), 580–590. Spritzer, M.D., Fox, E.C., Larsen, G.D., Batson, C.G., Wagner, B.A., Maher, J., 2013. Testosterone influences spatial strategy preferences among adult male rats. Horm. Behav. 63 (5), 800–812. Thouless, C.R., 1995. Long distance movements of elephants in northern Kenya. Afr. J. Ecol. 33 (4), 321–334. Wolff, J.O., 2007. Social biology of rodents. Integr. Zool. 2 (4), 193–204.