W

Sa

b

a

ARRA

KMNRSW

1

rbe1aiopabdspsuB

PT

G

0d

Behavioural Processes 84 (2010) 696–704

Contents lists available at ScienceDirect

Behavioural Processes

journa l homepage: www.e lsev ier .com/ locate /behavproc

ithin-litter variance in rat maternal behaviour

onia A. Cavigelli a,b,∗, Christina M. Ragana,b, Catherine E. Barretta,1, Kerry C. Michaela

Department of Biobehavioral Health, The Pennsylvania State University, University Park, PA 16802, United StatesInterdepartmental Graduate Department Program in Neuroscience, The Pennsylvania State University, University Park, PA 16802, United States

r t i c l e i n f o

rticle history:eceived 24 December 2009eceived in revised form 7 April 2010ccepted 9 April 2010

eywords:other–neonate interactionseonatal experience

a b s t r a c t

As a first step in determining the influence of maternal behaviour on sibling behavioural variance, wetested whether rat mothers differentially interact with neonates within the same litter. We also testedwhether fading of an ink-mark on individual pups could provide an index of within-litter variance inmaternal licking in laboratory rats. In Study 1, during the first postnatal week we distinguished individ-ual Sprague–Dawley rat pups across 4 litters by placing an ink-mark on the skin and quantified variancein maternal licking frequency toward each pup and compared fading of individual pup marks to thefrequency of maternal licks received and to four pup characteristics that could influence mark-fading.

odentprague–Dawley ratsithin-clutch variance

In Study 2, neonate mark-fading (a proxy for maternal licking) was compared to adolescent and adultoffspring behaviour across 8 litters. Results indicated that: (1) there are substantial and consistent dif-ferences in how much rat mothers lick same-sex siblings within a litter, (2) differential licking rates canbe documented with a non-observational method (ink-mark-fading), and (3) within-litter variance inmaternal behaviour may relate to sibling behavioural variance. The findings indicate a viable research

ental

model for future experimwithin families.

. Introduction

Adult siblings show considerable variation in behaviouralesponse biases, and in a variety of mammalian species adultehaviour has been shown to be influenced by early familial socialxperiences, including early mother–infant interactions (Daniels,986; Moore and Power, 1992; Rubin and Burgess, 2002; Caldji etl., 1998; Cameron et al., 2005; Maestripieri et al., 2006). Assum-ng that early interactions between mother and infant differ fromne sibling to the next, variance in adult offspring behaviour mayartially result from early within-family variance in maternal inter-ctions. As a first step toward understanding the role of maternalehaviour in the development of sibling variability, we carefullyocumented the extent to which maternal behaviour varies amongiblings within litters of a common laboratory rat. For this pur-

ose, we chose a rat strain that produces large litters and for whichignificant within-litter variation in sibling behaviour has been doc-mented (Cavigelli and McClintock, 2003; Cavigelli et al., 2006).ecause maternal behaviour can be closely observed and accurately

∗ Corresponding author at: 315 Health & Human Development Building - East,ennsylvania State University, University Park, PA 16802, USA.el.: +1 814 863 0210; fax: +1 814 863 7525.

E-mail address: sac34@psu.edu (S.A. Cavigelli).1 Present address: Graduate Program in Neuroscience, Emory University, Atlanta,A 30322, United States.

376-6357/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.beproc.2010.04.005

studies on causes and consequences of differential maternal investment

© 2010 Elsevier B.V. All rights reserved.

quantified in the laboratory situation, and because environmentalconditions can be controlled and manipulated, this system couldprovide an ideal model to test the role of maternal behaviour in thedevelopment of sibling behavioural variance.

Field studies with non-mammalian species indicate thatmothers differentially provision (e.g. egg/offspring size, feeding)offspring within the same clutch, and that variable offspringprovisioning may be a method for mothers to maximize their repro-ductive fitness by maximizing offspring variability to best cope withunpredictable or temporally variable environments (Crump, 1981;Gottlander, 1987; McGinley et al., 1987; Williams, 1994; Dziminskiand Roberts, 2006; Marshall et al., 2008; Crean and Marshall, 2009).In mammalian species, Moore and colleagues showed variance inmaternal behaviour within rat litters, with laboratory Long-Evansrat dams differentially licking male and female offspring withina litter. Males received twice as much licking in the anogenitalregion as compared to female pups during postnatal weeks 2 and3, and this extra tactile stimulation was important for male off-spring sex-specific behavioural and neuroendocrine development(Moore and Morelli, 1979; Moore, 1984; Moore and Power, 1992).In these findings, it is important to note that this differential lick-ing of male pups, important for sex-specific development, was most

apparent during the second and third weeks of life, whereas recentstudies have implicated differences in maternal stimulation dur-ing the first postnatal week in the development of fear-associatedbehavioural and neurological biases (Francis et al., 1999; Meaney,2001; Champagne et al., 2003). The above results indicate that

ural Pr

moh

eaaledaitmeic12mnadltilomv

sbanptb(mac–sfliotnf2

2

2

atnldmf

S.A. Cavigelli et al. / Behavio

ammalian and non-mammalian mothers differentially ‘provision’ffspring within a clutch and that this differential behaviour mayave long-term impacts on offspring development.

In laboratory rats and other mammalian species, there is clearvidence that significant between-mother variability exists in hownd how much dams touch and interact with their offspring,nd that these early mother–infant interactions can have long-asting influences on offspring behaviour (Meaney, 2001; Herrerat al., 2004; Maestripieri et al., 2006). In laboratory rats, extensiveata from Long-Evans rats indicate that “high-licking/groomingnd arched-back nursing” mothers (high LG-ABN) spend approx-mately twice as much time licking their offspring comparedo “low LG-ABN” mothers and adult offspring of low LG-ABN

others appear more fearful, with less exploration in a novelnvironment, increased startle responses, and elevated circulat-ng ACTH and glucocorticoid levels in response to acute restraintompared to adult offspring of high LG-ABN mothers (Liu et al.,997; Caldji et al., 1998; Francis et al., 1999; Champagne et al.,003). Based on the data from laboratory rats, we know that earlyaternal–offspring interactions can influence pup behavioural and

euronal development, and that same-sex siblings vary in theirdult behaviour; however, we have no evidence that mothersifferentially interact with same-sex neonates within the same

itter. The current study lays the groundwork to later determinehe extent to which within-litter variance in maternal behaviournfluences behavioural variance among same-sex siblings using aaboratory rodent model (Sprague–Dawley rats). Based on resultsf the current study, future studies could use this model to deter-ine the role of mothers in the development of sibling behavioural

ariance.To determine if early rat maternal behaviour varies among

ame-sex, same-age offspring, we carefully observed maternalehaviour toward individual offspring within rat litters and testedpossibly integrative measure of within-litter variance in mater-al behaviour. These observations were conducted during the firstostnatal week, a time when rat maternal behaviour is high andhought to have a significant influence on offspring fear-associatedehavioural development (Rosenblatt, 1969; Francis et al., 1999).In the current study we did not examine the role that neonates

ay play in these early developmental interactions but this is anrea that we are actively studying and acknowledge as another cru-ial factor in early social interactions and behavioural developmente.g. Stern, 1997; Moore and Chadwick-Dias, 1986.) Across two

tudies, we tested the following hypotheses: (a) maternal lickingrequency will consistently differ among same-sex pups within aitter during the first week of life (Study 1), (b) fading of a standard-zed ink-mark placed on individual pups will provide an estimatef differential maternal licking rates but not of neonate characteris-ics like activity, growth, etc. (Study 1), and (c) fading of individualeonate marks during the first postnatal week will predict offspring

ear-related behaviour during adolescence and adulthood (Study).

. Materials and methods

.1. General

Two studies were conducted to: (a) quantify within-litter vari-nce in maternal behaviour during the first postnatal week, (b)est a surrogate non-observational measure of within-litter mater-

al licking variance, and (c) compare the surrogate measure to

ater offspring behaviour. Maternal licking behaviour was observeduring the first postnatal week because this is a time when rataternal licking and nursing can influence the development of pup

ear-related behaviour and precedes the time when sex differences

ocesses 84 (2010) 696–704 697

in maternal anogenital-licking rates are most pronounced (Mooreand Morelli, 1979; Richmond and Sachs, 1984; Francis et al., 1999;Meaney, 2001; Champagne et al., 2003). In addition, our pilot workindicated greater within-litter variance in maternal licking dur-ing postnatal week 1 compared to week 2. Study 1 involved dailymarking of individual pups and eight daily observations of mater-nal licking to examine within-litter variance in maternal behaviour(hypothesis a) and to test if fading of an ink-mark on pups’ skinprovided an estimate of maternal licking frequency during this firstweek (hypothesis b). Study 2 made use of the ink-mark-fading mea-sure to examine the variance and temporal change in fading withinlitters during the first postnatal week and to compare this vari-ance and temporal change to rates of maternal licking observed inStudy 1 to determine the influence of the daily pup-marking proto-col on later offspring behaviour, and to determine the relationshipbetween neonate mark-fading and adolescent/adult fear-relatedoffspring behaviour both between and within litters (hypothesisc).

For these initial studies on natural variance in within-littermaternal pup-licking, we did not cull litters in order to examineun-manipulated variance in maternal licking. None of the littersstudied included runts. Date of birth was identified as postnatal day0 (PND 0). Pups were weaned on PND 22 and housed with same-sex littermates. On PND 35, all rats were re-housed into groupsof 3 (sometimes 2) same-sex littermates. In both studies, mothersand pups were housed in solid-bottom plastic cages with corncobbedding (43.5 cm × 23.5 cm × 20.5 cm), in a 12L:12D light schedulewith lights on at 20:00 h EST. Food and water were available adlibitum and the colony room was maintained at 22 ◦C with ∼50%humidity. Cages were first cleaned after PND 8 and cleaned twice aweek thereafter. All methods adhered to standards specified in theGuide for the Care and Use of Laboratory Animals (National ResearchCouncil, 1996).

2.2. Study 1: within-litter variance in maternal licking and asurrogate measure of maternal licking (i.e. neonateink-mark-fading)

2.2.1. AnimalsFour litters were bred from 4 unrelated pairs of Sprague–Dawley

rats (Charles River, Wilmington, MA). The litters contained 15–18pups and were all close to parity in sex ratio (68 offspring: 35 males,33 females). On a daily basis during the first postnatal week, allpups within each litter were temporarily separated from the damto be weighed and tagged with a unique ink-mark on the ven-trum and dorsum for easy individual identification during maternallicking observations. The degree of fading for individual pup ink-marks was quantified during the daily weighing/marking sessions,and daily maternal licking observations (8/day) were conductedthroughout this first postnatal week. These methods are furtherdescribed below.

2.2.2. Individual pup-markingAll pups were uniquely identified with a black ink-mark on their

dorsal and ventral surface. Marking was conducted daily from PND1–7 at the same time of day immediately following one of the eightdaily maternal observations (13:30 h). (A prior study with Long-Evans rats showed that a comparable ink-mark placed on pupsduring the first postnatal week did not affect rates of maternallicking compared to unmarked pups, Champagne et al., 2003.) Tomark pups, the mother was removed from the home cage for 15 min

while experimenters marked each pup with a large (2.5 cm high)individually-specific letter, number, or symbol on the dorsum andventrum using a non-toxic black marker (Sharpie® Fine Point, OakBrook, IL). Sharpie® pens were used instead of Codman® surgicalpens (Johnson and Johnson, Brunswick, NJ; e.g. Champagne et al.,

6 ural Pr

2mduttotn

2

i1((nseapiBefpcaael

2

tuagdtasfhitce

ietnmfcmibtawSal1

98 S.A. Cavigelli et al. / Behavio

003) because in test trials the Sharpie pens left a more distinctark than the Codman pens. The darkness of the mark was stan-

ardized so that each pup received a maximally-saturated marksing three even passes of the marker with the pups’ skin stretchedaut. Pups were sequentially weighed and marked and returned toheir home cage, carefully placing them in contact with the restf the pups. When all pups were marked, and 15 min had elapsed,he mother was returned to the home cage and observed to verifyormal contact with the pups.

.2.3. Maternal behaviourOn a daily basis, eight 30-min observations were conducted dur-

ng PND 1–7: six observations during the light phase (9:00, 11:00,3:00, 15:00, 17:00, and 19:00 h) and two during the dark phase7:00 and 21:00 h). During dark phase observations, four red bulbs25-W) located near the cage facilitated observations. Cages wereot disturbed during the first postnatal week. For each observationession, two experimenters were present: one to identify and callvents/time and one to scribe and assist with pup identifications necessary. Each time the female licked a pup, we recorded theup identification and the type of licking: anogenital-licking – licked

n anogenital region, or body-licking – licked in any other region.ecause we were interested in frequency of licks directed towardach pup within a litter and because dams quickly switch lickingrom one pup to another, we recorded the frequency of individualup-licking bouts and not the duration. For the purpose of quantifi-ation, termination of an individual pup-licking bout was defineds when the mother directed her attention away from the pup fort least 5 s. In pilot observations, we found that licking bouts withach individual pup were relatively short, with only 7% of boutsasting more than 3 s.

.2.4. Fading of individual pup marksInk-mark-fading was quantified every day in conjunction with

he daily pup weighing and marking procedure. A 5-point scale wassed to quantify mark-fading, with ‘1’ indicating very little fadingnd ‘5’ indicating the greatest fading. A template with a black-to-rey gradient showing the five fading levels was used as a referenceuring scoring. The template was made in Microsoft Excel, usinghe table cell-shading option and five levels of grey (90, 70, 50, 30nd 10%). The coder scored the level of mark-fading for the dor-al and for the ventral marks separately and was unaware of howrequently each pup was licked on any given day. Each pup wasandled only once per day, with each handling session including

nk-mark-fading coding, pup weighing, then re-marking the pup onhe dorsum and ventrum and placing the pup back into the homeage. This daily marking/handling procedure took less than 90 s forach pup.

Neonate characteristics that may influence mark-fading: Becausendividual neonate marks may fade as a result of several differ-nt pup characteristics during the postnatal period, we recordedhe following information for each neonate, in addition to mater-al licking: growth, body contact with other siblings or mother,ovement, and an index of body rubbing. Growth was calculated

rom daily weight measures. Body contact and movement data wereollected at 3-min intervals (point samples) throughout all 30-in focal observations. Body contact was recorded for each pup

f they were located at the center of a huddle where they hadoth dorsal and ventral contact with another pup, the dam, orhe cage bottom. Movement was recorded for each pup that wasctively moving (crawling or wriggling) at a point sample. Finally,

e estimated body rubbing based on movement and contact data.

ee ‘Analyses’ for more detailed information on how each vari-ble was calculated. Because rat eccrine (sweat) glands are onlyocated on the palmar and plantar tubercles (Ring and Randall,947), we were not concerned that individual differences in pup

ocesses 84 (2010) 696–704

sympathetic activity or sweating rates would influence fading of theink-marks.

2.2.5. AnalysesWe compared maternal licking frequencies for the MOST- and

LEAST-licked tertile of pups within litters by using a repeated-measures ANOVA that included maternal identity to control forlitter effects. We tested whether neonate ink-mark-fading relatedto: (1) maternal licking frequency, (2) neonate growth rate, (3)neonate body contact with others – estimated from pup visibility,(4) neonate movement, and (5) neonate body rubbing – estimatedfrom the contact and movement measures. Comparisons were con-ducted using correlations and a linear multiple regression withmeans for the above 5 variables calculated across PND 1–7 as pre-dictor variables and mean ink-mark-fading calculated across PND2–7 as the outcome variable. (This time-lagged analysis was usedto account for the fact that degree of ink-fading on each day likelyresulted from behaviour during the previous 24 h.) The five predic-tor variables were calculated for each pup in the following ways.(1) Maternal licking was the mean frequency of body or anogen-ital licks received per hour of observation across PND 1–7. (2)Neonate growth was estimated as the linear slope of daily weightmeasures (g/day). (3) Body contact was estimated by summing thenumber of point samples when a pup was not visible (i.e. in thecentre of a huddle where body contact was high as opposed toon the periphery or separate from the huddle) across PND 1–7.(4) Neonate movement was estimated by summing the numberof point samples when a pup was moving and dividing by thenumber of point samples when the pup was visible. The denom-inator controlled for the fact that pup movement could only berecorded during point samples when they were visible. (5) Rub-bing was estimated by summing the number of point samplesin which a pup was not visible (i.e. in close body contact withothers) and in which they were visible and moving. This finalmeasure was used to account for the fact that body contact andpup movement both increase skin friction and potentially increasemark-fading. One day of ink-mark-fading measures were lost for5 of the 68 pups in this study, thus the total number of pups inthe above analyses was 63. All variables were normally-distributedand thus regular parametric tests were used and data were nottransformed.

2.3. Study 2: within-litter variance in neonate ink-mark-fadingand relationship to later adolescent and adult offspring behaviour

2.3.1. AnimalsTwelve litters were bred, each including 15–17 pups (with most

litters close to parity in sex ratio, 180 offspring: 87 males, 93females). Pups from 8 of these litters (122 offspring: 63 males, 59females) were individually-marked during the first postnatal weekto determine if the degree of mark-fading for an individual was con-sistent across the week, and then offspring behavioural responsesto two novelty arenas (PND 25 and 50) and the elevated plus maze(PND 70) were measured. Pups from the remaining 4 litters (58 off-spring: 24 males, 34 females) were not marked during the neonatalperiod, and their adolescent and adult behaviour was tested andcompared to the behaviour of the marked/handled litters.

2.3.2. Individual pup-markingPup-marking of all ‘marked/handled’ pups was conducted daily

in Study 2 in the same way that it was conducted in Study 1.

2.3.3. Fading of individual pup marksMark-fading was scored daily for all pups by using the same

method used in Study 1. The only difference in methodology for

ural Pr

Svs

2

awacbMare

woiwbTofca

13wicnrtatatoaiarltnoibsp

taaws(I4baia

S.A. Cavigelli et al. / Behavio

tudy 2 was that two coders simultaneously scored fading of indi-idual pup marks while consulting one another on each mark-fadecore.

.3.4. Offspring adolescent and adult behaviourWe measured adolescent and adult responses to two novelty

renas and to the elevated plus maze (EPM). Response to noveltyas measured because low-locomotor responses to this test are

ssociated with elevated corticosterone production in males, indi-ating a reasonable measure of fear, and because it elicits variableehavioural responses from same-sex rat siblings (Cavigelli andcClintock, 2003; Cavigelli et al., 2006). The EPM was selected asstandard measure of anxiety or fear in rodents, and behavioural

esponses on the arena have been associated with early postnatalxperiences in laboratory rats.

Adolescent responses to physical and social novelty: Adolescentillingness to explore complex novel environments was assessed

n PND 25 and 50 using two novelty tests – the novel phys-cal and novel social arenas (Cavigelli et al., 2007). The arenas

ere designed to mimic the novel room situation used to assessehavioural inhibition in young children (Garcia-Coll et al., 1984).he arenas are similar to the ‘Open Field’ (Hall, 1934), but inrder to maximize the range of behavioural responses to novelty,ear-provoking/threatening stimuli were minimized; the arenasontained rat-sized objects, had opaque walls, a clear plastic cover,nd some urine-soaked bedding from all cages in the colony room.

For both the physical and social novelty tests, the arena was22 cm × 122 cm with 46-cm walls and a Plexiglas cover with a× 3 grid with 9 equal squares for locomotion coding. The test roomas dimly illuminated with a 90-W red light bulb. The novel phys-

cal test included four rat-sized objects placed ∼10 cm from eachorner. The novel social test included two cages (in opposite cor-ers of the arena), one of which contained an unrelated same-sexat. Rats were tested in the novel physical test on PND 25 and inhe novel social test on PND 50. Testing occurred between 13:00nd 16:00 h, during the dark (active) period of the light cycle, andest order was randomized. Rats were individually transported infamiliar metal rat-sized dish from their home cage. The rat and

he dish were gently placed into a clear mouse housing cage turnedn its side and lowered into the corner of the test arena. The dishnd cage provided a familiar and protected start point. The exper-menter left the room and coded behaviour from a TV monitor inn adjacent room. After testing, the rat and the metal bowl wereemoved from the arena, the rat was placed in a new cage, fecal pel-ets were removed from the test arena, and the next rat transportedo the arena in the familiar metal bowl. (With this method, we doot see significant test order effects.) The frequency of lines crossedn the overlaid grid was recorded during the 5-min test. To assessndividual responses to novelty, we summed locomotion scores inoth the physical and social arenas to obtain a mean locomotioncore which we have previously shown to relate to corticosteroneroduction in Sprague–Dawley rats (Cavigelli et al., 2007).

Adult response to elevated plus maze (EPM): Rats were tested onhe EPM at 70 days of age in dim red light (11–13 lux in the openrms, 2–3 lux in the closed). To maximize anxiety, testing occurredt the beginning of the light period (between 20:00 and 22:00 h),hen daily activity normally begins to decrease. The maze con-

isted of two open and two closed black plastic perpendicular arms50.2 cm × 10.8 cm) elevated 75 cm above the ground (Lafayettenstrument, Lafayette, IN). Black walls on the closed arms were

0 cm high. Test order was randomized. Rats were individuallyrought from their colony room to the behavioural testing roomnd placed in the centre of the maze facing a closed arm. The exper-menter left the room and scored behaviour from a TV monitor inn adjacent room.

ocesses 84 (2010) 696–704 699

Over a 5-min period, closed and open arm entries were recorded.Entry into an arm was defined as four limbs crossing the thresh-old. To eliminate familiar con-specific odours, the apparatus wascleaned with 10% ethanol after each test. After testing, the animalwas placed in a clean new cage until all cage mates were tested.Our behavioural measure was a commonly used reverse index offear or anxiety-like behaviour – the proportion of open arm entriesrelative to total arm entries – which controls for overall activity inthe arena.

2.3.5. AnalysesWe determined if individual differences in pups’ ink-mark-

fading were relatively consistent across PND 2–7. In other words,did a neonate with relatively low-fading on PND2 consistently havelow-fading across all days of the first postnatal week, and vice versafor high-fading pups? From each family, we selected one male andone female neonate with the MOST and LEAST fading on PND 2(i.e. total of two males and two females from each family), thenused a repeated-measures ANOVA to determine if the MOST andLEAST faded pups on PND2 continued to have differential fadingscores across PND 3–7. (We repeated this analysis using the threeMOST and LEAST faded males and females from each litter.) In addi-tion, we tested whether there was a sex difference in mark-fadingacross PND 3–7 using a repeated-measures ANOVA. For all ANOVAanalyses, mother’s identity was used as an independent variable toaccount for between-litter variance in licking and mark-fading.

As daily handling of litters has been shown to significantlyalter adult offspring stress reactivity (Caldji et al., 2000; Plotskyand Meaney, 1993), we compared marked and unmarked offspringopen arm entries on the EPM as adults to determine the influ-ence of the pup-marking procedure on later offspring behaviour. AnANOVA was used to compare relative open arm entries by adultsthat were either marked or unmarked as pups, using sex of theoffspring as an additional factor.

To test whether the mark-fading measure replicated previousfindings that high-licking mothers produce offspring with loweranxiety-like behaviour than low-licking mothers (e.g. Meaney,2001), we used a series of multiple regression analyses to compareneonate mark-fading to offsprings’ adolescent and adult behaviourin the novel arenas and EPM. To control for within-family effects,we simultaneously compared mean mark-fading for each litter andmean fading for each individual pup to the offsprings’ behaviour inthe different tests. Mean mark-fading for litters and individual pupswere calculated across PND 2–7. Mean litter mark-fading includedboth sexes because no significant difference in mark-fading wasseen between males and females (t104 = 0.398, ns). Behaviouralanalyses were conducted separately for male and female offspring,since the influence of early maternal interactions on behaviouraldevelopment may differ across sexes.

3. Results

3.1. Study 1

3.1.1. Maternal behaviourWhen a mother body-licked a pup, 95% of the time we were

able to record the identity of that pup. When mothers licked apup’s anogenital region, we were able to identify the pup 86% ofthe time because the pup’s body was often underneath the female,making identification more difficult. On average, each pup in a lit-

ter was licked once every hour (body lick range: 0.10–0.88 licks/h;anogenital lick range: 0.08–0.91 licks/h). The two forms of lick-ing were highly positively correlated such that individual pupsthat received frequent body licks also received frequent anogeni-tal licks (Pearson correlation: r68 = 0.343, p < 0.01). Given the lower

700 S.A. Cavigelli et al. / Behavioural Processes 84 (2010) 696–704

Fig. 1. Frequency of daily body- and anogenital-licking bouts during first week oflife for the MOST and LEAST-licked pup tertiles in Study 1 (n = 4 litters; 12 males and1e(o

ralleFw

tplaAFqp(idndtcr

3

stwpyaowbvmrppp

Table 1Results of multiple linear regression relating neonatal events to mean ventral anddorsal ink-mark-fading during first week of life. Neonatal events include: (1) fre-quency of maternal body licks, (2) neonate growth (slope of body weight change,g/day), (3) body contact with others, (4) pup movement, and (5) rubbing index acrossPND 1–7. Mean ink-mark-fading was calculated as a mean of ventral and dorsalfading across PND 2–7.

Neonatal events (PND 1–7) Coefficient p

Maternal licks/Hr observed 0.601 0.04Neonate growth (slope of body weight change, g/day) −0.430 0.15Body contact (pup not visible during point sample) −0.006 0.99

were still significant when we used the three MOST and three LEASTfaded males and females from each family (repeated-measuresANOVA: F1,4 = 5.18, p < 0.05). As in Study 1, the peak in ink-mark-fading occurred on PND 4–6, which is was also the time of maximalmaternal licking in Study 1.

2 females in each of the two groups, e.g., 3 MOST and 3 LEAST-licked males fromach litter). Lines depict mean daily licking frequency and error bars indicate SEM.When data are limited to pups of one or the other sex, or to either of the two kindsf maternal licking – body or anogenital – the results are similar.)

ates of pup identification for anogenital-licking, we analysed body-nd anogenital-licking data separately. However, given the corre-ation between the two kinds of licking, we present data from bothick-types together where appropriate. Males and females receivedqual amounts of maternal licking across PND 1–7 (body licks:1,60 = 2.24, p = 0.14; anogenital licks: F1,60 = 0.75, p = 0.39); thus,here appropriate, we present results combined across the sexes.

Same-sex siblings received differential amounts of licking fromhe mother and these differences were consistent across the firstostnatal week. The tertile of pups that were MOST-licked within a

itter consistently received more than twice as many licks (body andnogenital) as the LEAST-licked tertile (Fig. 1, repeated-measuresNOVA: body licks – F1,42 = 13.37, p < 0.01; anogenital licks –1,41 = 16.50, p < 0.001). In addition, maternal licking was most fre-uent on PND 4–6. There was a trend for the MOST-licked tertile ofups to weigh approximately 5% less than the LEAST-licked tertilerepeated-measures ANOVA: F1,44 = 3.23, p = 0.079). This differencen weight was consistent across the first postnatal week; i.e. theifference in weight was not more pronounced at either the begin-ing or end of this week. In terms of pup behaviour, there were noifferences in body contact, movement or body rubbing betweenhe MOST- and LEAST-licked tertiles (repeated-measures ANOVAs:ontact – F1,44 = 0.046, p = 0.83; movement – F1,44 = 0.035, p = 0.85;ubbing – F1,44 = 0.105, p = 0.75).

.1.2. Fading of individual pup ink-marksIndividual pup ink-mark-fading scores on the dorsal and ventral

urfaces were highly positively correlated – i.e. if a pup’s mark onhe dorsal surface faded a lot, then the mark on its ventral surfaceas also likely to have faded a lot (Pearson correlation: r63 = 0.523,< 0.0001). Given this relationship, we conducted statistical anal-ses using mean dorsal and ventral fading for each pup, as wells mean fading on each surface separately. Mean ink-mark-fadingn pups’ dorsal and ventral surfaces during the first postnatal weekas closely associated with the frequency with which they received

ody, but not anogenital, licks (Pearson correlation: body lickss. dorsal mark-fading, r63 = 0.272, p < 0.05; body licks vs. ventral

ark-fading, r63 = 0.328, p < 0.01; anogenital licks vs. dorsal fading,

63 = −0.037, p = 0.78; anogenital licks vs. ventral fading, r63 = 0.059,= 0.645; body licks vs. mean dorsal/ventral fading, r63 = 0.343,< 0.01; anogenital licks vs. mean dorsal/ventral fading, r63 = 0.012,= 0.93; Table 1). Mark-fading was not linearly associated with pup

Movement (point samples when pup moving) −1.787 0.76Rubbing index (body contact + movement) −0.020 0.98

Adjusted R2 = 0.164, F5,62 = 3.43, p = 0.009.

characteristics that influence rubbing or stretching of the skin –i.e. neonate growth, body contact (non-visibility), movement whenvisible, or overall rubbing (i.e. body contact + movement when vis-ible; Table 1).

In addition to the association between overall mean body-licking and ink-mark-fading in the first postnatal week, therewas a temporal association between daily body-licking and dailyink-mark-fading during this week. Specifically, on days when body-licking was most frequent (PND 4–6), mean dorsal and ventralfading were also the greatest (Fig. 2; r5 = 0.894, p < 0.05). Thistemporal synchrony was not evident when comparing daily mark-fading to daily levels of other factors associated with stretching ofthe skin (growth, contact, movement and rubbing, all ps > 0.10).

3.2. Study 2

3.2.1. Fading of individual pup ink-marksThe range of mean mark-fading (dorsal and ventral) across PND

2–7 was 2.5–3.8 on a 5-pt scale. There were no sex differencesin ink-mark-fading across PND 2–7 (repeated-measures ANOVA:F1,5 = 0.002, p = 0.98). When using the one MOST and LEAST fadedmale and female from each litter, pups with the MOST mark-fadingon PND 2 had consistently more faded marks across PND 3–7 ascompared to pups with the LEAST faded marks on PND 2 (repeated-measures ANOVA: F1,4 = 16.79, p < 0.001; Fig. 3). These differences

Fig. 2. During the first postnatal week, daily ink-mark-fading on pups’ ventral anddorsal surfaces and maternal body-licking of her pups peaked during PND 4–6 inStudy 1 (n = 4 litters; 8 males and 8 females in each group, evenly selected acrosslitters). Lines depict mean mark-fading and error bars indicate SEM.

S.A. Cavigelli et al. / Behavioural Processes 84 (2010) 696–704 701

Ffwa

3

pato(em

taffftetf

Faufd

Table 2Relation between neonate ink-mark-fading and adolescent behaviour in male (a andb) and female (c and d) offspring. To relate mark-fading to adolescent behaviourat both family- and individual-levels, mean ink-mark-fading was calculated foreach litter (‘Litter fading’) and for each pup (‘Individual fading’). Multiple regres-sion results are reported for locomotion in novel arenas (a and c) and for relativefrequency of open arm entries in elevated plus maze (b and d).

Males

Predictor Std. coefficient t p

a. Locomotion in novel arenasLitter fading 0.560 2.86 0.006Individual fading −0.438 2.23 0.029

Adjusted R2 = 0.089, F2,61 = 4.07, p = 0.022

b. Relative frequency of open arm entries in EPMLitter fading 0.580 3.18 0.002Individual fading −0.127 0.70 0.490

Adjusted R2 = 0.212, F2,61 = 9.45, p = 0.000

Females

Predictor Std. coefficient t p

c. Locomotion in novel arenasLitter fading 0.203 1.08 0.284Individual fading 0.104 0.56 0.581

Adjusted R2 = 0.050, F2,56 = 2.53, p = 0.088

d. Relative frequency of open arm entries in EPM

ig. 3. Neonate ink-mark-fading during first week of life for the MOST and LEASTaded pup for each litter in Study 2 (n = 8 litters; 8 males and 8 females in each group,ith 1 male and 1 female from each litter/group). Lines depict mean mark-fading

nd error bars indicate SEM.

.2.2. Offspring adolescent and adult behaviourPups that were ink-marked on a daily basis during the first

ostnatal week showed less anxiety-like behaviour on the EPM asdults. Adult offspring that had been marked as neonates enteredhe open, unprotected arms of the maze approximately 25% moreften than those that had not been marked/handled as neonatesANOVA main effect of marking: F1,175 = 11.72, p < 0.001). This influ-nce was more pronounced in males than in females (ANOVAarking × sex interaction: F1,175 = 5.36, p < 0.05; Fig. 4).Offspring from litters with high levels of mark-fading (sugges-

ive of more overall maternal licking) displayed less fearful andnxiety-like behaviour as adolescents and adults than offspringrom low mark-fading litters. Specifically, and as expected if mark-ading provided an index of mean maternal licking rates, malesrom litters with a high degree of mark-fading moved more in

he novel arenas (Table 2a, first predictor called ‘Litter fading’) andntered open arms more frequently (Table 2b, first line) comparedo males from low mark-fading litters. Females from high mark-ading litters entered open arms more frequently than those from

ig. 4. Behavioural response to elevated plus maze (specifically, proportion of openrm entries relative to total arm entries) for adult rats that were either marked ornmarked during PND 1–7 in Study 2 (n = 8 marked litters with 63 males and 59emales total and 4 marked litters with 24 males and 34 females total). Bar heightepicts means and error bars indicate SEM.

Litter fading 0.465 2.51 0.015Individual fading −0.410 2.21 0.031

Adjusted R2 = 0.074, F2,56 = 3.30, p = 0.044

low mark-fading litters (Table 2d, first line). There was no differencein female locomotion in novel environments according to meanlitter mark-fading (Table 2c, first line).

Within families, behavioural outcomes relative to individual pupmark-fading were the opposite of those found between families.Specifically, males within a litter with more faded marks movedsignificantly less in the novel arenas than their brothers with lessfading (Table 2a, second predictor called ‘Individual fading’). Therewas no significant effect of within-family mark-fading variance onmale open arm entries in the EPM, although the direction of thisrelationship is opposite to that seen between litters (Table 2b, sec-ond line). Females with more ink-mark-fading than their sistersentered EPM open arms less frequently than their less-faded sis-ters (Table 2d, second line). There were no within-litter differencesin female behaviour in the novel arenas (Table 2c, second line).

4. Discussion

It is well-established that quality and quantity of maternalbehaviour can vary significantly among mothers of the samespecies and that maternal behaviour (e.g. nursing and licking inrat mothers) has a significant influence on the behavioural devel-opment of offspring. For example, high-licking rat mothers tend toproduce more exploratory offspring than low-licking mothers asassessed in a variety of behavioural paradigms (Caldji et al., 1998;Francis et al., 1999; Liu et al., 1997; Meaney, 2001). In the currentstudies, we found that the amount of variance in maternal lickingpreviously documented across rat mothers was also seen within sin-gle mothers licking the different pups in a litter. Specifically, across4 litters, a third of the pups in each litter consistently received twiceas much licking as the LEAST-licked pups in each litter (Fig. 1). Theseresults support our first hypothesis that maternal licking consis-tently differs among same-sex siblings during the first week of life.

In support of our second hypothesis, daily standardized ink-marksplaced on the dorsal and ventral surface of pups faded in relationto the amount of body-licking that they received, with frequently-licked pups having more faded marks than infrequently-licked pups

7 ural Pr

(mmtcmp

lls1wntoawtepwflmstw

4b

atntadiaosmaeh(a

fwfilmsttirbopc

02 S.A. Cavigelli et al. / Behavio

Table 1), and mark-fading was most pronounced on days whenaternal licking was most frequent (PND 4–6, Fig. 2). Also, ink-ark-fading related to maternal body-licking frequency but not

o anogenital-licking frequency or to neonate characteristics thatould have increased stretching or rubbing of the skin and thearks (i.e. more rapid pup growth, increased body contact, frequent

up movement, and/or increased body rubbing; Table 1).Consistent with the above within-litter variance in maternal

icking, we found within-litter variance in ink-mark-fading in 8itters in Study 2, with a peak in fading on PND 4–6 which is con-istent with the peak in maternal licking and mark-fading in Study(Fig. 3). These results further support our second hypothesis thatithin-litter variance in maternal licking can be estimated from aon-observational measure. With more examination, we may findhat the ink-mark-fading may provide an integrative measure ofverall licking rates because daily fading may closely reflect themount of maternal licking completed over a 24-h period. Finally,ith regards to our third hypothesis, pups from highly-faded lit-

ers (indicative of increased maternal licking) were more active andxploratory in novelty and anxiety-related behavioural tests com-ared to pups from less-faded litters (Table 2), which is consistentith prior studies showing that high-licking mothers produce less

earful offspring (e.g. Meaney, 2001). Interestingly, pups within aitter that had high mark-fading compared to their same-sex litter

ates were less active and exploratory in these tests compared toiblings with low mark-fading (Table 2). These results warrant fur-her studies to determine the causes, consequences and function ofithin-litter variance in maternal licking.

.1. Possible function of within-litter variance in maternalehaviour

In terms of function, it is possible that variance in maternalttention within-litters is an adaptive behaviour that mothers useo increase overall litter behavioural variance and survival. If mater-al behaviour shapes offspring adult behaviour, then it may behat mothers are ‘hedging their bets’ by licking some pups within

litter more than others and thereby producing behaviourallyiverse offspring, with some phenotypes better able to reproduce

n one kind of environment and others better able to reproduce innother environment. For example, ‘cautious’ or less exploratoryffspring may have greater success in environments with manykillful predators; whereas in more favourable conditions cautionay be unnecessary or harmful and ‘curious’ or ‘bold’ individu-

ls may be more successful (Dingemanse et al., 2004; Cameront al., 2005). In unpredictable environmental conditions this bet-edging may be one way to maximize the overall success of a litterCrump, 1981; McGinley et al., 1987; Marshall et al., 2008; Creannd Marshall, 2009).

The fact that between litters we found that neonate mark-ading (an index of maternal licking) was positively associatedith increased exploratory behaviour, but that within litters we

ound an opposite association, suggests that within-litter variancen maternal licking may have a different function than between-itter variance in maternal licking. Perhaps, within a litter, mothers

ay be less involved in ‘shaping’ the future diversity of her off-pring (as is seen among litters), and more involved in respondingo early diversity that already exists among neonates within a lit-er. Within-litter variance in licking may simply reflect variancen neonate behaviours or signalled needs, to which the mother

esponds accordingly. For example, pups within a litter that laterecome fearful adolescents and adults may be more solicitousf and therefore receive more maternal licking during the firstostnatal week compared to pups that are less fearful as adoles-ents/adults.

ocesses 84 (2010) 696–704

Examples of pup behaviours that solicit maternal licking in ratsinclude contacting mother’s snout, probing her ventrum, and tak-ing longer to attach to nipples during a nursing bout (Moore andChadwick-Dias, 1986; Stern and Lonstein, 1996; Stern, 1997; Sternand Lonstein, 2001). In the current study we did not collect data onhow these behaviours differed among siblings, however, in Study1 we did see a trend for high-licked siblings to weigh less thanlow-licked siblings. This is comparable to results from a prior studyshowing that Sprague–Dawley mothers spend more time nursingcross-fostered pups that were prenatally malnourished comparedto those that were well-nourished (Galler and Tonkiss, 1991). In thisprior study, there was no evidence that the cue for malnourishmentwas smaller body weight, suggesting that some other pup charac-teristic (potentially behaviour) was involved in soliciting increasedmaternal behaviour. In birds it has been shown that differencesin sibling begging behaviour are positively correlated with mater-nal food provisioning (Gottlander, 1987) and theory and researchwith humans suggests that mothers that are well-provisioned (per-haps comparable to ad libitum fed laboratory rat dams) will biastheir attention toward offspring that require additional assistanceto thrive (Beaulieu and Bugental, 2008).

The results of prior studies and the current study supportthe hypothesis that mammalian within-litter variance in mater-nal behaviour may be ‘driven’ by early within-litter variance inneonate behaviour as opposed to an a priori maternal decision tointeract with certain pups more than others. This ‘pup-driven’, asopposed to a ‘mother-driven’ scenario could explain the seeminglyopposite relationship found between neonate ink-mark-fadingand adolescent/adult offspring behaviour when comparing thesevariables within- vs. between-litters. Sibling behavioural vari-ance may be present near and shortly after birth, which is notsurprising given genetic, epigenetic and prenatal influences onbehavioural development (Darnaudéry and Maccari, 2008; Muellerand Bale, 2008; Seckl and Meaney, 2004), and these early dif-ferences among siblings may stimulate within-litter variance inmaternal behaviour in well-provisioned mothers. Evidence ofwithin-litter variance in laboratory rat maternal behaviour inthe current study provides the necessary background for futureexperimental studies of the function, cause and consequencesof within-family variance in maternal behaviour. For example,manipulation of maternal provisioning in laboratory rats wouldprovide an optimal test of the contingent model of within-familyvariance in maternal investment which theorizes that well-provisioned mothers will bias maternal investment toward moreneedy offspring while poorly-provisioned mothers will bias invest-ment toward the more hardy offspring (Bugental and Beaulieu,2003).

4.2. Non-observational ink-mark-fading index of maternal licking

When comparing rat offspring of different mothers, thoseof highly-attentive mothers (high LG-ABN) have decreased fearresponses compared to offspring of low LG-ABN dams (Caldji etal., 2003). In the current study, we found similar results whenwe compared mean litter ink-mark-fading (as a potential sur-rogate measure of maternal licking; Fig. 2) to mean offspringadolescent/adult behaviour. This replication of findings supportsour surrogate measure of maternal licking rates within rat litters,however, further refinement and validation of this measure arerequired. For example, mark-fading – either on the ventrum or dor-sum – was not related to rates of maternal anogenital-licking, which

is a crucial aspect of maternal behaviour in this species. Becauseanogenital-licking is important for male neonate behavioural andneural development in rats (Moore, 1984; Moore and Power, 1992),a refined marking index, for example that includes alternate markplacement between the anus and genitalia, may provide greater

ural Pr

ittsutmmvdcvviaiaso

owtrna2cablan

5

iwpdmaoomLePevstbmoomiDswtdt

S.A. Cavigelli et al. / Behavio

nformation on anogenital-licking rates. It should be noted thathe process of adding ink-marks and measuring the fading ofhese marks on pups had a significant influence on male off-pring behaviour in the elevated plus maze (Fig. 4), and thus thetility of this measure is limited to studies in which this long-erm behavioural influence of marking does not affect key study

easures/processes or the relative variance of these key outcomeeasures. In addition, because of this long-term impact of the

ery short marking procedure, it is important to use a highly stan-ardized protocol for marking and measuring individual pups. Aombination of ink-mark-fading measures and behavioural obser-ations may provide an ideal complex measure of within-litterariance in maternal behaviour. Either way, the benefits of thenk-mark-fading index are numerous – overall fading may providen integrative measure of maternal licking, the marks are non-nvasive, minimally-intrusive, and temporary, and they may proven inexpensive and relatively simple method to apply to otherpecies. These benefits support further refinement and applicationf the method.

In the current studies, we introduced a daily stressor of 15 minf maternal removal and 90 s of neonate handling and markingith a felt tip pen to identify individual pups within litters. Given

hat prenatal stress can abolish sex differences in anogenital-lickingates and generally decrease pup-licking frequency, and that post-atal handling can increase rates of maternal behaviour (Powernd Moore, 1986; Smith et al., 2004; Champagne and Meaney,006), it is possible that the daily marking protocol used in theurrent study reduced maternal licking variance both between-nd within-litters. If it is true that the variance in maternalehaviour in manipulated litters is less than in un-manipulated

itters, than there is even greater reason to examine the causesnd consequences of this early variance in familial experience foreonates.

. Conclusions

The results of the current research indicate significant variabil-ty in the frequency with which rat mothers lick their offspring

ithin the same litter during the first week of life – with someups receiving more than twice as much licking than others. Thisifference is relatively stable during the first week of life anday be related to later adolescent and adult behavioural vari-

nce among siblings. Because of the known interactive naturef maternal-neonate behaviour during this early stage of devel-pment – i.e. neonate behaviours influence rates and forms ofaternal attention (Moore and Chadwick-Dias, 1986; Stern and

onstein, 1996, 2001; Stern, 1997) and maternal behaviours influ-nce pup behaviour and development (Moore, 1984; Moore andower, 1992; Liu et al., 1997; Caldji et al., 1998, 2003; Francist al., 1999; Meaney, 2001) – further exploration of within-litterariance in neonate and maternal behaviour is warranted. Wehow strong evidence that a non-observational method to quan-ify within-litter variance in maternal licking may complementehavioural observations. The current results indicate that ratothers in controlled and well-provisioned conditions (i.e. the lab-

ratory) show differential ‘provisioning’ of maternal licks to theffspring within a litter, which may be analogous to other forms ofaternal provisioning biases identified in non-mammalian species

n the natural environment (e.g. Gottlander, 1987; Williams, 1994;ziminski and Roberts, 2006). Further work is required to under-

tand the function (causes and long-term consequences) of thisithin-litter variance in mammalian maternal behaviour, and

he laboratory rat provides an interesting model to study thisevelopmental process across a variety of environmental condi-ions.

ocesses 84 (2010) 696–704 703

Acknowledgements

We thank J.M. Mateo, Michelle Scott, and several anonymousreviewers for extensive and constructive comments on a draft ofthis manuscript, and R. Anolik, W. Bahar, A. Bruscke, E. Cheng,L. Hugar, A. Inthavongsa, C.E. Kovacsics, S. Leathers, K. Mehta, R.Moser, J. Quinn, and J. Smith for their expert assistance with datacollection and processing. This research was supported by fundsfrom the College of Health & Human Development and the Child,Youth and Family Consortium at the Pennsylvania State University.

References

Beaulieu, D.A., Bugental, D., 2008. Contingent parental investment: an evolutionaryframework for understanding early interaction between mothers and children.Evol. Hum. Behav. 29, 249–255.

Bugental, D., Beaulieu, D.A., 2003. A bio-social cognitive approach to understandingand promoting the outcomes of children with medical and physical disorders.In: Kail, R. (Ed.), Advances in Child Development and Behavior, vol. 31. AcademicPress, New York, NY, pp. 129–258.

Caldji, C., Tannenbaum, B., Sharma, S., Francis, D., Plotsky, P.M., Meaney, M.J.,1998. Maternal care during infancy regulates the development of neural sys-tems mediating the expression of neural systems mediating the expression ofbehavioural fearfulness in adulthood in the rat. Proc. Natl. Acad. Sci. U.S.A. 95,5335–5340.

Caldji, C., Francis, D., Sharma, S., Plotsky, P.M., Meaney, M.J., 2000. The effects of earlyrearing environment on the development of GABAA and central benzodiazepinereceptor levels and novelty-induced fearfulness in the rat. Neuropsychophar-macology 22, 219–229.

Caldji, C., Diorio, J., Meaney, M.J., 2003. Variations in maternal care alter GABAA

receptor subunit expression in brain regions associated with fear. Neuropsy-chopharmacology 28, 1950–1959.

Cameron, N.M., Champagne, F.A., Parent, C., Fish, E.W., Ozaki-Kuroda, K., Meaney,M.J., 2005. The programming of individual differences in defensive responsesand reproductive strategies in the rat through variation in maternal care. Neu-rosci. Biobehav. Rev. 29, 843–865.

Cavigelli, S.A., McClintock, M.K., 2003. Fear of novelty in infant rats predicts adultcorticosterone dynamics and an early death. Proc. Natl. Acad. Sci. U.S.A. 100,16131–16136.

Cavigelli, S.A., Stine, M.M., Kovacsics, C., Jefferson, A., Diep, M.N., Barrett, C.E., 2007.Behavioral inhibition and glucocorticoid dynamics in a rodent model. Physiol.Behav. 92, 897–905.

Cavigelli, S.A., Yee, J.R., McClintock, M.K., 2006. Infant temperament predicts life spanin female rats that develop spontaneous tumors. Horm. Behav. 50, 454–462.

Champagne, F.A., Francis, D.D., Mar, A., Meaney, M.J., 2003. Variations in mater-nal care in the rat as a mediating influence for the effects of environment ondevelopment. Physiol. Behav. 79, 359–371.

Champagne, F.A., Meaney, M.J., 2006. Stress during gestation alters postpartummaternal care and the development of the offspring in a rodent model. Biol.Psychiatry 59, 1227–1235.

Crean, A.J., Marshall, D.J., 2009. Coping with environmental uncertainty: dynamicbet hedging as a maternal effect. Philos. Trans. R. Soc. B 364, 1087–1096.

Crump, M.L., 1981. Variation in propagule size as a function of environmental uncer-tainty for tree frogs. Am. Nat. 117, 724–737.

Daniels, D., 1986. Differential experiences of siblings in the same family as predictorsof adolescent sibling personality differences. J. Pers. Soc. Psychol. 51, 339–346.

Darnaudéry, M., Maccari, S., 2008. Epigenetic programming of the stress response inmale and female rats by prenatal restraint stress. Brain Res. Rev. 57, 571–585.

Dingemanse, N.J., Both, C., Drent, P.J., Tinbergen, J.M., 2004. Fitness consequences ofavian personalities in a fluctuating environment. Proc. R. Soc. Lond. B: Biol. Sci.271, 847–852.

Dziminski, M.A., Roberts, J.D., 2006. Fitness consequences of variable maternal pro-visioning in quacking frogs (Crinia georgiana). J. Evol. Biol. 19, 144–155.

Francis, D.D., Diorio, J., Liu, D., Meaney, M.J., 1999. Nongenomic transmission acrossgenerations in maternal behaviour and stress responses in the rat. Science 286,1155–1158.

Galler, J.R., Tonkiss, J., 1991. Prenatal protein malnutrition and maternal behaviourin Sprague–Dawley rats. J. Nutr. 121, 762–769.

Garcia-Coll, C., Kagan, J., Reznick, J.S., 1984. Behavioral inhibition in young children.Child Dev. 55, 1005–1019.

Gottlander, K., 1987. Parental feeding behaviour and sibling competition in the piedflycatcher Ficedula hypoleuca. Ornis. Scand. 18, 269–276.

Hall, C.S., 1934. Emotional behavior in the rat: defecation and urination as measuresof individual differences in emotionality. J. Comp. Psychol. 18, 385–403.

Herrera, E., Reissland, N., Shepherd, J., 2004. Maternal touch and maternal child-

directed speech: effects of depressed mood in the postnatal period. J. Affect.Disord. 81, 29–39.

Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S.,Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glu-cocorticoid receptors, and hypothalamic–pituitary–adrenal responses to stress.Science 277, 1659–1662.

7 ural Pr

M

M

M

M

M

M

M

M

M

N

P

Stern, J.M., Lonstein, J.S., 1996. Nursing behaviour in rats is impaired in a smallnestbox and with hyperthermic pups. Dev. Psychobiol. 29, 101–122.

Stern, J.M., Lonstein, J.S., 2001. Neural mediation of nursing and related maternalbehaviors. Prog. Brain Res. 133, 263–278.

04 S.A. Cavigelli et al. / Behavio

aestripieri, D., McCormack, K., Lindell, S.G., Higley, J.D., Sanchez, M.M., 2006.Influence of parenting style on the offspring’s behaviour and CSF monoaminemetabolite levels in crossfostered and noncrossfostered female rhesusmacaques. Behav. Brain Res. 175, 90–95.

arshall, D.J., Bonduriansky, R., Bussière, L.F., 2008. Offspring size variation withinbroods as a bet-hedging strategy in unpredictable environment. Ecology 89,2506–2517.

cGinley, M.A., Temme, D.H., Geber, M.A., 1987. Parental investment in offspring invariable environments: theoretical and empirical considerations. Am. Nat. 130,370–398.

eaney, M.J., 2001. Maternal care, gene expression, and the transmission of indi-vidual differences in stress reactivity across generations. Annu. Rev. Neurosci.24, 1161–1192.

oore, C.L., 1984. Maternal contributions to the development of masculine sexualbehaviour in laboratory rats. Dev. Psychobiol. 17, 347–356.

oore, C.L., Chadwick-Dias, A.-M., 1986. Behavioural responses of infant rats tomaternal licking: variations with age and sex. Dev. Psychobiol. 19, 427–438.

oore, C.L., Morelli, G.A., 1979. Mother rats interact differently with male and femaleoffspring. J. Comp. Physiol. Psychol. 93, 677–684.

oore, C.L., Power, K.L., 1992. Variation in maternal care and individual differencesin play, exploration, and grooming of juvenile Norway rat offspring. Dev. Psy-chobiol. 25, 165–182.

ueller, R., Bale, T., 2008. Sex-specific programming of offspring emotionality after

stress early in pregnancy. J. Neurosci. 28, 9055–9065.

ational Research Council, 1996. Guide for the Care and Use of Laboratory Animals.National Academy Press, Washington, DC, 125 pp.

lotsky, P.M., Meaney, M.J., 1993. Early, postnatal experience alters hypothalamiccorticotrophin-releasing factors (CRF) mRNA, median eminence CRF content andstress-induced release in adult rats. Brain Res. Mol. Brain Res. 18, 195–200.

ocesses 84 (2010) 696–704

Power, K.L., Moore, C.L., 1986. Prenatal stress eliminates differential maternal atten-tion to male offspring in Norway rats. Physiol. Behav. 38, 667–671.

Richmond, G., Sachs, B.D., 1984. Maternal discrimination of pup sex in rats. Dev.Psychobiol. 17, 87–89.

Ring, J.R., Randall, W.C., 1947. The distribution and histological structure of sweatglands in the albino rat and their response to prolonged nervous stimulation.Anat Rec. 99, 7–19.

Rosenblatt, J.S., 1969. The development of maternal responsiveness in the rat. Am.J. Orthopsychiatry 39, 36–56.

Rubin, K.H., Burgess, K.B., 2002. Parents of aggressive and withdrawn children. In:Bornstein, H. (Ed.), Handbook of Parenting, vol. 1, 2nd edition. Children andParenting, Lawrence Erlbaum Associates, Mahwah, NJ, pp. 383–418.

Seckl, J.R., Meaney, M.J., 2004. Glucocorticoid programming. Ann. N. Y. Acad. Sci.1032, 63–84.

Smith, J.W., Seckl, J.R., Evans, A.T., Costall, B., Smythe, J.W., 2004. Gestational stressinduces post-partum depression-like behaviour and alters maternal care in rats.Psychoneuroendocrinology 29, 227–244.

Stern, J.M., 1997. Offspring-induced nurturance: animal–human parallels. Dev. Psy-chobiol. 31, 19–37.

Williams, M.D., 1994. Intraspecific variation in egg size and egg composition in birds:effects on offspring fitness. Biol. Rev. 68, 38–59.