Differential Effects of Short- and Long-Term Early Maternal Separation on Subsequent Maternal...

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Differential Effects of Short-and Long-Term Early MaternalSeparation on SubsequentMaternal Behavior in RatsKarin J. Bodensteiner a , Loraina L. Ghiraldi b &Stephanie S. Miner ca University of Wisconsin–Stevens Pointb St. Lawrence Universityc Toledo ZooPublished online: 04 Apr 2012.

To cite this article: Karin J. Bodensteiner , Loraina L. Ghiraldi & Stephanie S. Miner(2012) Differential Effects of Short- and Long-Term Early Maternal Separation onSubsequent Maternal Behavior in Rats, The Journal of General Psychology, 139:2,78-99, DOI: 10.1080/00221309.2012.661377

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The Journal of General Psychology, 2012, 139(2), 78–99Copyright C© 2012 Taylor & Francis Group, LLC

Differential Effects of Short- andLong-Term Early Maternal Separation

on Subsequent Maternal Behavior in Rats

KARIN J. BODENSTEINERUniversity of Wisconsin–Stevens Point

LORAINA L. GHIRALDISt. Lawrence University

STEPHANIE S. MINERToledo Zoo

ABSTRACT. Female Sprague-Dawley pups were separated from mothers every other dayfor 8 hr (long-term separation/LTS), 4 hr (short-term separation/STS), or 0 hr (no separa-tion/NS) from postnatal day 2–20. In adulthood, they were mated and tested for maternalbehaviors during two lactations. It was expected that females separated from mothers aspups would show deficits in maternal behavior as adults. Contrary to expectations, LTSshowed better nest building and grouped young faster during both lactations. LTS werefirst to display aggression and displayed more aggression during the second lactation. No-tably, while some measures decreased from first to second lactation in NS and STS, LTSmaintained levels of maternal care. These results suggest that extended periods of maternalseparation may exaggerate some aspects of maternal behavior.

Keywords: maternal behavior, maternal separation, parity, rats, neonatal experience, post-partum aggression, nest building

The authors thank the following colleagues at St. Lawrence University for their assis-tance with this research: Michael Schuckers, Associate Professor of Mathematics, Statistics& Computer Science; Joseph Erlichman, Professor of Biology; Serge Onyper, AssistantProfessor of Psychology; Thomas Greene, Professor of Psychology; and Elizabeth Mac-Dougall, LVT, Animal Facilities Supervisor. The authors are grateful for the financialsupport provided by the Hilary Valentine Fund (Psychology Department, St. LawrenceUniversity) and The William B. Bradbury Junior Faculty Award (awarded to Karin J.Bodensteiner).

Address correspondence to Loraina L. Ghiraldi, Department of Psychology, FlintHall, St. Lawrence University, 23 Romoda Drive, Canton, NY 13617, USA; [email protected] (e-mail).

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Bodensteiner, Ghiraldi, & Miner 79

STRESSFUL EVENTS DURING CHILDHOOD, SUCH AS PARENTAL LOSS,NEGLECT, OR ABUSE, are risk factors for development of psychopathologiesin later life (Harris, Brown, & Bifulco, 1986; Servant & Parquet, 1994; Lupie,King, Meaney, & McEwen, 2001). Furthermore, individuals who experience mal-treatment as children often show impulsive aggression and antisocial personalities(Widom, 1989; Dodge, Bates, & Pettit, 1990). In rodents, the maternal behaviorrepertoire, necessary for nutritional, thermal, and protective support of young, isknown to influence physiological, neuroendocrine, and behavioral outcomes (e.g.,Caldji, Tannenbaum, Sharma, Francis, Plotsky, & Meaney, 1998; Caldji, Fran-cis, Sharma, Plotsky, & Meaney, 2000; Liu et al., 1997), and maternal separationparadigms have often been used as animal models for studying effects of neonatalstress on subsequent behavior (see Lehmann & Feldon, 2000; Kikusui & Mori,2009 for review).

Both natural and artificial variations in maternal-neonatal interactions maylead to functional changes that persist well into adulthood (Caldji et al., 1998;Caldji et al., 2000; Francis, Diorio, Liu, & Meaney, 1999; Liu et al., 1997; Meaney,Aitken, van Berkel, Bhatnagar, & Sapolsky, 1988; Sakhai, Kriegsfeld, & Francis,2011). For example, offspring of mothers with high levels of licking, grooming, andarched-back nursing demonstrated decreased behavioral fearfulness in responseto a novel environment as adults (Caldji et al., 1998). Indeed, it has been suggestedthat exposure to short periods of maternal separation, also known as the handlingparadigm, may be beneficial, resulting in decreased fearfulness, improved responseto stress, and better maternal responsiveness in adulthood (Meaney et al., 1988;Caldji et al., 1998; Rees & Fleming, 2001; Huot, Gonzalez, Ladd, Thrivikraman,& Plotsky, 2004), possibly as a result of increased maternal care upon mother-pup reunion (Pryce, Bettschen, Feldon, 2001; Zimmerberg, Rosenthal, & Stark,2003; Marmendal, Roman, Eriksson, Nylander, & Fahlke, 2004). In contrast,extended periods of repeated maternal separation can lead to a prolonged stressresponse, increased emotionality, poorer learning, and aberrant maternal behaviorin adulthood (Boccia & Pederson, 2001; Hall, 1998 for review; Kuhn & Schanberg,1998; Rees & Fleming, 2001).

In fact, subjecting pups to maternal separation as neonates can instigatechanges in maternal behavior when those pups give birth to their own offspring.In one study, Boccia and Pederson (2001) found that females exposed to 3-hrmaternal separation showed deficits in nest building, nursing, and pup grooming,compared to females that experienced 15-min maternal separation. Females ex-posed to 3-hr maternal separation also demonstrated an increased latency to attacka novel male and decreased total number of attacks toward the male (Boccia &Pederson, 2001). In another study, Lovic, Gonzalez, and Fleming (2001) reportedthat pups separated from their mothers for an average of 5 hr per day licked andcrouched over their own pups less than controls. It is interesting that Rees andFleming (2001) found that while 3 hr of daily maternal separation did not cause

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80 The Journal of General Psychology

deficits in later maternal behavior, brief separation (15 min) enhanced maternalbehavior of previously separated females.

Research has indicated that maternal behavior and responsiveness to youngcan also be influenced by reproductive experience (pregnancy and lactation).Although some studies revealed no differences in maternal behavior betweenprimiparous and multiparous rats (e.g., Moltz & Robbins, 1965), other evidencespeaks to the contrary. For example, parturient rabbits demonstrated better nestbuilding (Ross, Denenberg, Sawin, & Meyer, 1956), and parturient female ratsdemonstrated improved pup retrieval and response times from the first to secondlactation (Carlier & Noirot, 1965). In addition, parous female rats were moreresponsive to pups than nulliparous females, even when not pregnant (Mayer& Rosenblatt, 1984). Rats with prior pregnancy and parturition experience alsodisplayed decreased latency to respond to pups when tested 25 days after lastpup exposure (Bridges, 1975), and maternal experience, either via pup exposureor parity, allowed for retention of maternal behaviors up to 80 days after initialpup experience (Scanlan, Byrnes, & Bridges, 2006). In mice, parity appears toinfluence postpartum aggression, with aggression increasing across the first threelactation periods and then declining (Svare & Gandelman, 1976). These variationsin levels of maternal aggression may be due, in part, to changes in postpartumhormones (Svare & Gandelman, 1976), and it is has been suggested that parity mayalter neural and/or endocrinological responsiveness to pup-related cues (Kinsley,Mann, & Bridges, 1989; Scanlan et al., 2006).

In the current study, effects of duration of maternal separation on subse-quent maternal behavior were examined. It was hypothesized that as duration ofmaternal separation increased, maternal behavior, including lactation, nest build-ing, nest defense, pup grouping, and hovering, would decrease. Given the evidencesupporting the role of maternal care in development of the hypothalamic-pituitary-adrenal (HPA) axis (Stanton, Wallstrom, & Levine, 1987; Stanton & Levine, 1990;Meaney et al., 1996 for review), we were also interested in possible effects of ma-ternal separation on the hypothalamic-pituitary-gonadal (HPG) axis. Therefore,reproductive measures, including latency to first estrus, latency to pregnancy, andnumber of pups were assessed. In addition, effects of parity on maternal behaviorwere assessed by mating the female subjects a second time. It was hypothesizedthat although maternal behavior would likely increase across all experimentalgroups, those that experienced early maternal separation would continue to showdeficits in maternal behavior.

Method

Subjects

Subjects were female young born to pregnant Sprague-Dawley rats (N = 9)obtained from Zivic Suppliers (Zelienople, PA, USA) 1 week prior to parturition.

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Upon arrival, dams were individually housed in translucent polypropylene cages(46 × 25 × 21 cm) lined with bedding (BetaChip; Heat Treated Hardwood Labo-ratory Bedding, NEPCO, Columbia, KY). Food (Prolab RMH 3000, Brentwood,MO) and water were available ad libitum. Throughout the study, animals werehoused in the university vivarium, which was maintained at a temperature of 21± 4◦C and kept on a 12/12 hr light/dark cycle, with lights on at 8:00 a.m.

From the nine litters, three were randomly designated as controls, or no sepa-ration (NS, 0-hr separation), three as short-term separation (STS, 4-hr separation),and three as long-term separation (LTS, 8-hr separation). Postnatal day (PND) 0was defined as parturition beginning by 1:00 p.m.

Separation Procedure

On PND 1, numbers of pups and sex ratios were recorded, and litters wereculled to four females each. Average pup weight for each litter was then recordedby using a digital scale (Mettler Toledo, PG5002-S).

Maternal separation began on PND 2 at approximately 9:00 a.m. and wasconducted every other day through PND 20. The dam was removed from thehome cage before pups were removed. Each litter was weighed and average pupweight was recorded. Control pups were weighed and immediately returned totheir cage. During the separation, each litter was removed from the home cageand placed in a new cage lined with a Chinet Carry Tray (Van Leer, Flare 20951)covered with bedding. For separations conducted on PND 2, 4, and 6, a heatingpad (Soft Heat Moist or Dry Heating Pad, Model No. HP910; Kaz Incorporated,Hudson, NY) was placed beneath each cage with the temperature maintained at35◦C. For separations on PND 8, 10, and 12, the temperature of the heating padwas maintained at 33◦C. For the final four separations, the heating pad was notused and the ambient temperature was approximately 26◦C. At the end of theseparation period, pups were returned to the dam in the home cage.

Following weaning on PND 23, total pup number was decreased to 24, n = 8per separation group. Pups were then housed in groups of four (with others of thesame separation group) until PND 35, when they were moved to individual cages.

Estrus Testing

To determine timing of first estrus, vaginal smears were collected daily fromPND 35 until the presence of squamous, cornified epithelial cells was confirmed(Fox, 1976).

Timed-Mating of Female Subjects

Vaginal smear testing began again on PND 75 in preparation for mating.When a female was determined to be in estrus, she was housed with a novel

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Sprague-Dawley male approximately 2 months of age (Charles River Supplier,Wilmington, MA). Upon confirmation of a vaginal plug, latency to observation ofthe plug was recorded, and the female was individually housed for the duration ofgestation. Mating resulted in N = 22: NS (n = 8), STS (n = 7), and LTS (n = 7).

Day of Parturition

Two days prior to parturition, each subject was provided with two papertowels for nesting material. Following parturition, the time line was designatedaccording to postpartum day (PPD). All subjects remained undisturbed on the dayof parturition (PPD 0). Litter size, sex ratio, and average pup weight were recordedon PPD 1. Litters were then culled to four pups (two males and two females).

Maternal Behavior Testing

Maternal behavior testing was conducted twice per week (specifically, on PPD2, 4, 8, 10, 14, and 16) between 10:00 a.m. and 4:00 p.m. Measures of maternalbehaviors included lactation performance, nest building, nest defense, groupingof pups, and hovering over pups. These variables were operationally defined asfollows:

• Lactation Performance. Lactation performance was measured indirectly byrecording the average weight of the four pups for each subject on each testday over the 3 weeks of lactation.

• Nest Building. Nest building was measured quantitatively and qualitatively.Beginning on PPD 1, eight nestlets (each a 2 × 2 in. square of compressedcotton) weighing approximately 20 g, were placed in the food well undersome food for easy access. Amount of cotton used (in grams) was recordedon each test day. In addition, nest quality was rated on a scale of 0–4, similarto procedures used by Quinones-Jenab, Batel, Schlussman, Ho, and Kreek(1997). Briefly, nests received a score of 0 when no nest was built, a scoreof 1 when nesting material was concentrated in a single corner but withoutdefinite shape, a score of 2 when the nest showed some organization ofcotton, but without complex structure, a score of 3 for a semicircular nestwith raised walls, and a score of 4 for a full nest with tall walls.

• Aggression Testing. Aggression testing occurred during five maternal testdays (PPD 2, 4, 8, 10, and 14). Aggression testing began by removing thelitter from the home cage, at which time the litter was weighed. The cagetop was also removed and the subject was given a 3-min habituation periodto adjust to removal of her pups. An unfamiliar adult male conspecific wasthen placed into the home cage, and latency to first aggressive response,number of attacks (a bout of physical contact with biting), and numberof lunges (a rush toward the intruder that falls short of physical contact)

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exhibited by the subject were recorded for 5 min. At the end of the session,the male was removed and returned to his home cage. A total aggressionscore, lunges plus attacks, was tallied.

• Grouping and Hovering. Following the aggression test, the subject wasallowed to remain undisturbed for 3 min. Pups were then placed into thecenter of the home cage. Latency for the subject to move each pup into agroup and number of pups grouped within a 5-min period was recorded. Ifall pups were grouped in less than 5 min, the time was recorded and thehovering measure was assessed. If all pups were not grouped at the end ofthe 5 min period, a score of 300 s was given, and remaining pup(s) weremanually placed in a group to conduct the hovering assessment. Hoveringmeasurements were recorded by placing the subject at the opposite end ofthe cage from her pups. Latency to hover (assume a nursing position overthe pups) was measured, along with time spent hovering within a 5-minsession. Number and duration of hovering bouts were recorded.

To assess effects of prior maternal experience on subsequent maternal careexhibited by females exposed to maternal separation as young, these subjectswere mated a second time and assessed on the same measures using the sameprocedures.

Data Analyses

Data collected during each pregnancy and lactation were analyzed with one-way or mixed (separation group × test day) ANOVAs and Tukey HSD post hoctests (alpha = .05) using SPSS v.16 (IBM).

Results

Separation Procedure

A one-way ANOVA showed no differences in average pup weights amonglitters prior to creating the treatment groups by initiating maternal separationprocedures on PND 1. No group differences in pup weights were found duringlactation on any of the 10 separation days.

Estrus Testing

A one-way ANOVA did not reveal a significant difference between separationgroups for latency to enter first estrus (F(2, 21) = 3.043, p = .069, η2 = .227).The difference, however, approached significance, suggesting that LTS subjectsentered first estrus somewhat earlier than STS subjects. These data are presentedin Table 1.

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Timed-Mating and Day of Parturition

During the first mating and pregnancy, for those NS (n = 8), STS (n = 7), andLTS (n = 7) subjects that became pregnant, a one-way ANOVA revealed no effectof separation on latency to observe a vaginal plug, number of pups delivered, oraverage pup weight of subjects’ litters on PPD 1.

During the second mating and pregnancy, for those NS (n = 5), STS (n =6), and LTS (n = 5), subjects in which pups were carried to full term, a one-wayANOVA revealed no significant differences in latency to see a plug. There was noeffect of separation group on average pup weights on PPD 1.

Although statistical significance was not achieved, ANOVA revealed a trendtoward a difference between separation groups in number of pups delivered fol-lowing the first (F(2, 19) = 2.947, p = .077, η2 = .237) and second (F(2, 13) =3.441, p = .063, η2 = .346) pregnancies, with LTS subjects delivering the greatestnumber of pups. See Table 1.

Maternal Behavior Testing

Lactation PerformanceFor the first lactation, a 3 × 6 mixed ANOVA on average pup weights revealed

no significant effect of separation on subjects’ lactation performance over the 3weeks of testing.

During the second lactation, a 3 × 6 mixed ANOVA revealed a strong trendtoward an interaction effect on lactation performance (F(10, 65) = 1.964, p = .052,η2 = .016), with LTS pups weighing more than NS pups (M = 14.14, SEM = 0.45;M = 12.06, SEM = 0.25, respectively) on PPD 10.

Nest BuildingA 3 × 6 mixed ANOVA revealed no main effect of separation on quality

ratings of nests built during the first lactation; however, there was a significantinteraction of separation group and test day (F(10, 95) = 3.041, p = .002, η2 =.062). Tukey HSD post hoc test for interaction effects (unconfounded comparisons;p < .05) indicated that LTS subjects built higher quality nests compared with NSon PPD 10 and STS on PPD 14. During the second lactation period, no effect ofmaternal separation on nest quality was observed. These results are illustrated inFigure 1.

During the first lactation, a significant main effect of separation group onamount of cotton (in grams) used in the nest was found (F(2, 19) = 3.681, p =.045). Tukey HSD post hoc test showed that LTS used significantly more cottonthan NS (M = 10.473, SEM = 2.191; M = 2.458; SEM = 2.050, respectively; p =.038). There was also a significant interaction of separation group and test day onthe amount of cotton used (F(10, 95) = 2.415, p = .013, η2 = .048). Tukey HSD

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FIGURE 1. Nest quality rating (M ± SEM) during the first and second lactationas a function of maternal separation group and postpartum test day. Duringthe first lactation, a significant group × day interaction (p = .002) revealed thatLTS subjects built higher quality nests compared with than NS on PPD 10 andSTS on PPD 14. During the second lactation, no differences were found. (Note:Scores for NS subjects during the second lactation fell below scoring criteriaand were too minimal to illustrate.)

post hoc test for interaction effects (unconfounded comparisons; p <.05) indicatedthat LTS subjects used more cotton than NS on PPD 4, 10, 14, and 16 and morethan STS on PPD 14 and 16. These data are illustrated in Figure 2.

There was also a significant main effect of separation on amount of cottonused in nests during the second lactation (F(2, 13) = 4.011, p = .044, η2 = .302).Tukey HSD post hoc test revealed that LTS subjects used significantly more cottonthan NS subjects (M = 11.036, SEM = 2.744; M = .044, SEM = 2.744; p = .035).See Figure 2.

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FIGURE 2. Amount (g) of cotton (M ± SEM) used in the nest during the first(p = .045) and second (p = .044) lactations as a function of maternal separationgroup and postpartum test day. There was a significant effect of separationduring both lactations (p = .045 and p = .044, respectively), with LTS subjectsusing more cotton than NS subjects during both lactations. (Note: Scores for NSsubjects during the second lactation were too minimal to illustrate graphically.)

Aggression TestingNo effect of maternal separation on postpartum aggressive behavior was

observed during the first lactation. During the second lactation, however, a 3 × 5mixed ANOVA revealed a significant main effect of separation group on latencyto display aggression (F(2, 13) = 5.798, p = .016, η2 = .266). Tukey HSD posthoc test indicated that latency to first aggressive response in LTS (M = 207.284,SEM = 20.218) was significantly shorter than NS (M = 298.048, SEM = 20.218;p = .019) and STS (M = 281.444, SEM = 18.456; p = .044) subjects.

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FIGURE 3. Mean total aggression (M ± SEM) during the first and secondlactation as a function of maternal separation group and postpartum test day.There was no effect of maternal separation during the first lactation; how-ever, an effect was observed during the second lactation (p = .043), with LTSdisplaying the most aggression overall.

Furthermore, a statistically significant difference in mean total aggression wasalso observed during the second lactation (F(2, 13) = 4.029, p = .043, η2 = .266).Although means indicate that LTS subjects displayed more overall aggression thanSTS and NS, the Tukey HSD post hoc test did not reach statistical significance(p = .62; p = .072, respectively). These results are shown in Figure 3.

Grouping and HoveringDuring the first and second lactations, no effect of maternal separation on

latency to begin hovering, total time spent hovering, or total number of hovering

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FIGURE 4. Latency (s) to begin grouping pups (M ± SEM) during the first andsecond lactations as a function of maternal separation group and postpartumtest day. A significant effect of separation was found during the second lactationonly (p = .005), with LTS grouped young faster than STS subjects duringthe second lactation. Average latencies were greater at the second lactationcompared to the first (p = .02), but LTS subjects showed the least amount ofchange from the first to the second lactation.

bouts was observed. As expected, as pup age increased, latency to hover increasedand total number of bouts decreased.

Although maternal separation did not affect latency to begin grouping pupsduring the first lactation, during the second lactation, there was a significant maineffect of separation on latency to begin grouping pups (F(2, 13) = 8.033, p = .005,η2 = .118). Tukey HSD post hoc test revealed that LTS subjects began groupingpups faster than STS subjects (M = 109.818, SEM = 18.161; M = 213.643, SEM =17.493, respectively; p = .004). These data are illustrated in Figure 4.

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During the first lactation, a 3 × 6 mixed ANOVA revealed a significant maineffect of separation on time to complete grouping pups (F(2, 19) = 4.804, p =.021, η2 = .033). Tukey HSD post hoc test indicated that LTS subjects took lesstime to group pups than NS subjects (M = 160.785, SEM = 12.487; M = 212.263,SEM = 11.680, respectively; p = .019).

There was also a significant main effect of maternal separation on time tocomplete grouping pups during the second lactation (F(2, 13) = 4.708, p = .029,η2 = .082). Tukey HSD post hoc test indicted that LTS subjects grouped fasterthan STS subjects (M = 198.16, SEM = 16.459; M = 266.49; SEM = 15.025,respectively; p = .023). See Figure 5.

Effects of Parity

For those subjects that sustained two successful pregnancies and lactations,differences in maternal behaviors between first and second lactations were an-alyzed within each maternal separation group using 2 (parity) × 3 (separationgroup) × 6 (postpartum test day) mixed ANOVAs.

Analyses revealed a main effect of parity on latencies to begin grouping pups(F(1, 26) = 6.193, p = .02, η2 = .021), with average latency greater during thesecond lactation M = 161.49, SEM = 10.631) compared with the first (M =124.08, SEM = 10.631). There was also a main effect of postpartum test day onlatency to begin grouping pups (F(5, 130) = 51.382, p < .0001, η2 = .512); asseen in Figure 4, latency increased with advancing lactation.

Similarly, there was a main effect of parity on latency to complete groupingpups (F(1, 26) = 15.239, p = .001, η2 = .046), with increased latencies duringthe second lactation, as illustrated in Figure 5.

To focus solely on effects of parity, maternal behaviors during first and secondlactations were collapsed over test days and analyzed separately within each ma-ternal separation group. It was found that in NS subjects, number of hovering boutsdecreased from first to second lactation (F(1, 8) = 13.333, p = .006, η2 = .161).STS subjects showed a longer latency to begin grouping young (F(1, 10) = 5.174,p = .046, η2 = .064) and a longer latency to group all four pups (F(1, 10) =18.226, p = .002, η2 = .140) during the second lactation. LTS subjects, however,showed no differences in maternal behavior as a function of parity. These resultscan be seen in Figures 4 and 5, respectively.

Discussion

Maternal care in rats follows a typical behavioral progression from birth toweaning (Grota & Ader, 1969) and exposure to maternal separation, combinedwith changes in maternal behavior upon mother and pup reunion (e.g., Kosten &Kehoe, 2010; Pryce et al., 2001; Zimmerberg et al., 2003), may lead to long-term

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FIGURE 5. Latency (s) to complete grouping all four pups (M ± SEM) duringthe first and second lactations as a function of maternal separation group andpostpartum test day. A significant effect of separation was found at the firstlactation (p = .021), with LTS showing shorter latencies than NS, and duringthe second lactation, with LTS grouping faster than STS subjects. Latencies tocomplete grouping during the second lactation were significantly longer thanthose of the first lactation (p = .001).

alterations in neural, endocrinological, and/or behavioral outcomes in pups. Dur-ing the first lactation, although there were no differences in aggression or hovering,maternal separation affected nest building and pup grouping, with LTS subjectsusing more cotton, building higher quality nests, and taking less time to completegrouping young than NS and/or STS subjects. During the second lactation, LTSsubjects continued to use more nesting material and take less time to group pups.Maternal separation did not affect hovering during the second lactation, but it didaffect aggression; LTS subjects demonstrated decreased latencies to first aggres-sion and increased total aggression. It is interesting that although some measures

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of maternal behavior decreased from first to second lactation in NS and STSsubjects, LTS subjects maintained increased levels of maternal care.

Contrary to expectations, these results suggest that extended maternal separa-tion may function to enhance some aspects of subsequent adult maternal behaviorand may increase responsiveness to parity. This idea, in general, disagrees withmuch of the previous literature, which has typically indicated a deficit in maternalbehavior following maternal separation during early development. For example,exposing rat pups to 3 hr of daily maternal separation lowered subsequent lev-els of nest building, nursing, and pup grooming; increased latency to attack; anddecreased number of aggressive responses (Boccia & Pederson, 2001). Similarly,pups that experienced 5 hr of maternal separation licked and crouched over theirown pups less than controls (Lovic et al., 2001). In a more recent study, althoughlactating females displayed increased pup-directed behavior (e.g., pup-licking) anddecreased non-pup-directed behavior (e.g., burrowing) immediately following re-union of isolated (1-hr/day) and handled (5–10 min/day) litters, those femalesexposed to isolation as neonates did not show this pattern toward their own iso-lated litters (Kosten & Kehoe, 2010). On the other hand, 3 hr of daily maternalseparation had few negative effects on later maternal behavior, and 15 min ofdaily separation enhanced later maternal behavior in adult rats (Rees & Fleming,2001). Such differences in maternal responsiveness between studies may be ex-plained, at least in part, by variations in duration of maternal separation, timingof the separation during development, number of separation episodes, isolationconditions, and/or choice of control group (Lehmann & Feldon, 2000). Therefore,the increased length of time and alternate day exposure to maternal separationin the present study may have been involved in eliciting the differential maternalresponsiveness observed in LTS subjects.

Presence and/or number of littermates, in addition to other aspects of the earlyrearing environment, including colony room sensory cues, can also influence latermaternal responsiveness (e.g., Moore & Morelli, 1979; Moretto, Paclik, & Flem-ing, 1986; Stern & Johnson, 1990). In Rees and Fleming (2001), experimentallitters contained six pups (male and female), whereas Boccia and Pederson (2001)used litters of eight female pups and placed dams in a room containing no otherrats. In addition, subjects that had undergone maternal separation as neonateswere compared to control rats (no maternal separation) obtained from a commer-cial supplier. In the study by Lovic et al. (2001), litters of 12 pups were housedindividually during the maternal separation procedure but were placed in the nestand covered with nesting material upon return to the home cage. Here, the controlgroup was removed and returned immediately to the home cage, a procedure thattook less than 15 s. And in yet another twist on the maternal separation paradigm,Kosten and Kehoe (2010) subjected litters of 12 pups to isolation from the damand littermates, but handled pups remained with littermates, and the control groupwas age-matched non-handled pups. Thus, observed discrepancies in maternalbehaviors between studies is likely the result of procedural differences, including

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potential differences associated with litter size and contact (e.g., Bautista, Garcia-Torres, Prager, Hudson, & Rodel, 2010). Nevertheless, the present study revealedinteresting and consistent differences in maternal behaviors in LTS subjects. Fur-thermore, because procedural factors remained consistent across separation groupswithin the current study, they cannot sufficiently account for the increased ma-ternal behavior exhibited by LTS subjects. However, to compare results betweenstudies and develop a truly robust model of early life stress, standardization ofmaternal separation protocols will be necessary (Lehmann & Feldon, 2000).

Despite the lack of procedural consistency across studies, maternal separationparadigms have been used as a model of stress induction for many years, withlonger-term separation protocols often leading to increased activity of the HPAaxis and the adult stress response (e.g., Meaney et al., 1988; Caldji et al., 2000).Naturally occurring variations in maternal responsiveness to pups (licking andgrooming) have been associated with differences in the stress response in adult-hood (e.g., Liu et al., 1997; Caldji et al., 1998). Several hormones, particularlycorticosterone, have been shown to increase during maternal separation, possiblyprecipitating long-term effects on subjects through an alteration of hippocampalneurons (Meaney et al., 1988; Vallee, Mayo, Maccari, Le Moal, & Simon, 1996;McCormick, Kehoe, & Kovacs, 1998). Therefore, in the present study, effects ofextended separation of young from the mother during early life could arise fromtwo potential sources: (1) direct stress experienced by pups in lost or insufficientmaternal care (e.g., nursing and sensory stimuli) during separation, and/or (2)quality or type of maternal care provided to pups upon reunion. We did not mea-sure hormone levels or the mothers’ behaviors following separations in the presentstudy; however, assuming different (possibly more intense?) care upon mother-litter reunion (e.g., Pryce et al., 2001; Marmendal et al., 2004; Zimmerberg et al.,2003), altered neurobiological development in those young (particularly LTS sub-jects in the present study), would not be surprising. Moreover, these modificationsmay precipitate altered maternal behavior in mothers during subsequent lactations(e.g., Kinsley & Lambert, 2008), as well as in offspring toward their own youngin adulthood (e.g., Fleming, O’Day, & Kraemer, 1999).

Although some studies have found little to no effects of maternal separationon reproductive parameters (Kosten & Kehoe, 2010; Rhees, Lephart, & Eliason,2001), mounting evidence suggests that female reproductive function is modulatedby maternal care. In the present study, nonsignificant trends in the data suggestedLTS subjects entered first estrus earlier, delivered more pups during the secondpregnancy, and demonstrated increased lactation performance during the secondpostpartum period. These data suggest an effect of extended maternal separationon reproductive parameters, possibly through altered HPG axis activity. This sug-gestion is in agreement with studies that have shown an influence of neonatalhandling on sexual behavior and reproduction. For example, a reduction in adultsexual behavior was observed in male and female rats that were handled for 1min or subjected to aversive stimuli (e.g., bright light, low temperature, or noise)

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for 10 min daily (Padoin, Cadore, Gomes, Barros, & Lucion, 2001), and neonatalhandling for 1 min daily induced anovulatory estrous cycles in adulthood (Gomes,Frantz, Sanvitto, Anselmo-Franci, & Lucion, 1999). Indeed, natural variations inmaternal care impact sexual behavior and reproductive outcome. High levels ofmaternal licking and grooming over the first week postpartum were associatedwith decreased sexual receptivity, lower lordosis ratings, and decreased pregnan-cies upon reaching adulthood (Cameron, Fish, & Meaney, 2008), whereas femaleoffspring of mothers demonstrating low levels of licking and grooming demon-strated increased sexual receptivity, increased plasma LH and progesterone duringproestrus, an increased positive feedback effect of estradiol on both LH and GnRHexpression in the medial preoptic area (MPOA), and increased estrogen receptor α

(ERα) expression in the anteroventral paraventricular nucleus (AVPVn) (Cameron,Del Corpo, Diorio, McAllister, Sharma, & Meaney, 2008). It is interesting thatfemale offspring of low licking and grooming mothers also entered puberty earlierthan those of high licking and grooming mothers (Cameron, Del Corpo et al.,2008), and were more often chosen by male rats in a mate preference task, indi-cating increased attractivity (Sakhai et al., 2011). Again, maternal responsivenessof subjects’ mothers was not measured in the present study, so we are unable todetermine if differences in maternal behavior of our subjects’ mothers existed,or if they influenced the observed differences in reproductive outcome betweentreatment groups. However, given recent data supporting a role for maternal carein development of neuroendocrine systems that regulate reproduction and sexualbehavior (see Cameron, Shahrokh et al., 2008 for review), a similar influence ofmaternal separation on LTS subjects in the present study seems likely.

Research indicates that expression of maternal care and maternal aggressionare related, and developmentally, share a similar hormonal foundation (e.g., Rosen-blatt, Factor, & Mayer, 1994). For maternal aggression in the present study, LTSsubjects demonstrated shorter latencies to first aggressive response and an increasein total aggressive behaviors during the second lactation. These findings agree withSvare and Gandelman (1976), who reported an increase in maternal aggressionacross the first three lactation periods in mice. Although maternal aggression andnest defense would obviously be important for protective support of young, levelsof maternal aggression may also affect fecundity, as animals with higher over-all levels of aggression produced more offspring than animals exhibiting lowerlevels (Lloyd & Christian, 1969; Svare & Gandelman, 1976). Indeed, this trendwas observed in the present study. The possibility of a relationship between ag-gression and fecundity is intriguing and suggests a biologically relevant adaptivemechanism behind these behavioral changes (Cameron, Champagne, Parent, Fish,Ozaki-Kuroda, & Meaney, 2005; Svare & Gandelman, 1976). Aggression resultsfrom the present study should be interpreted with caution, however, as there waslittle postpartum aggression displayed in any of the groups. In fact, the NS (con-trol) subjects showed little maternal aggression, a behavior typically observedin postpartum females (e.g. Erskine, Barfield, & Goldman, 1980; Flannelly &

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Flannelly, 1987; Mayer, Reisbick, Siegel, & Rosenblatt 1987). This caution shouldalso be extended to the interpretation of nest-building behavior, which was alsominimal in NS subjects. An explanation for the relatively low level of maternalaggression and nest building in NS subjects is not immediately apparent, but maybe related to unintended effects of handling inherent in our protocol.

Maternal behavior and responsiveness to pups is also influenced by parity,and improvements in maternal behavior following maternal experience have beendocumented in rats (Bridges, 1975; Carlier & Noirot, 1965; Mayer & Rosen-blatt, 1984; Scanlan et al., 2006), rabbits (Ross et al., 1956), and mice (Svare &Gandelman, 1976). Thus, we hypothesized that levels of maternal responsivenesswould increase across all groups from the first to second lactation. Contrary toour expectations, however, some measures of maternal behavior decreased fromthe first to second lactation in NS and STS subjects. LTS subjects, on the otherhand, maintained or increased levels of maternal behavior, suggesting that fe-males subjected to longer periods of maternal separation as pups may be affecteddifferently by the physiological and/or experiential factors that influence mater-nal responsiveness in multiparous mothers. An abundance of research indicatesthat reproductive and maternal experience alters the female brain (for review, seeKinsley & Lambert, 2008), possibly via neural and/or endocrinological alterationsassociated with gestational and postpartum hormones, pup-related stimuli, parity(Kinsley et al., 1989; Kinsley et al., 2008; Scanlan et al., 2006), and subtle strain-specific strategies (Champagne, Curley, Keverne, & Bateson, 2007). Mountingevidence also suggests that this neural plasticity may be long-lasting and precipi-tate enhanced cognitive (i.e., spatial), motivational (i.e., foraging), and behavioral(i.e., anxiety, fearfulness, and aggression) ramifications for the female (Byrnes &Bridges, 2006; Kinsley & Lambert, 2008; Kinsley et al., 2008). Such alterationscan initiate a trickle-down effect on quality and duration of more proximal behav-iors associated with care provided by the mother to her young. In the present study,evidence of maternal enhancements was noted in LTS subjects with prior mater-nal experience (at the second lactation); an explanation for the lack of maternalenhancements in STS and NS subjects is not immediately apparent.

In summary, in contrast to previous literature reporting deficits in maternal be-havior following maternal separation, we found that chronic, extended periods ofmaternal separation prior to weaning enhanced some aspects of maternal behaviorand heightened responsiveness to parity. Extended periods of maternal separationalso had an impact on reproductive parameters. Although the ecological benefitsof these changes are not yet clear, these results paint a more complex picture of thematernal separation experience. Clearly, additional research is required, particu-larly for the potential development of a cross-species model, and an understandingof individual differences and identification of risk factors relevant to humans (forreviews, see Beery & Francis, 2011; Meaney, 2010; Zhang & Meaney, 2010). Pro-cedural differences between studies complicate comparison of results, however,and protocol standardization would greatly assist efforts to further unravel the

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neural, hormonal, and behavioral changes associated with the maternal separationexperience.

AUTHOR NOTES

Karin J. Bodensteiner is an Associate Professor of Biology at the University of Wisconsin–Stevens Point. Her research interests broadly relate to reproductive health and includehormones and behavior, ovarian folliculogenesis, and reproductive toxicology. Loraina L.Ghiraldi is an Associate Professor of Psychology at St. Lawrence University. Her researchinterests relate to hormonal and environmental influences on cognitive processes and sexual,aggressive, and maternal behaviors in animal models. Stephanie S. Miner earned her MAin Psychology, with an Advanced Certificate in Animal Behavior and Conservation, atCUNY Hunter College, New York, NY. She is currently employed at the Toledo Zoo inOhio as the enrichment coordinator, focusing on environmental enrichment and behavioralresearch.

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Original manuscript received November 25, 2011Final version accepted January 22, 2012

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