intensification of maternal care by double-mothering boosts cognitive function and hippocampal...

Intensification of Maternal Care by Double-Mothering Boosts CognitiveFunction and Hippocampal Morphology in the Adult Offspring

Francesca R. D’Amato,1* Claudio Zanettini,1 Carmelo Sgobio,2 Celeste Sarli,1

Valentina Carone,1 Anna Moles,1,2 and Martine Ammassari-Teule1,2

ABSTRACT: Mice born from high care-giving females show, as adults,low anxiety levels, decreased responsiveness to stress, and substantialimprovements in cognitive function and hippocampal plasticity. Giventhe relevance of this issue for preventing emotional and cognitive abnor-malities in high-risk subjects, this study examines the possibility to fur-ther enhance the beneficial effects observed in the progeny by augment-ing maternal care beyond the highest levels females can display instandard laboratory conditions. This was produced by placing a secondfemale with the dam and its litter in the rearing cage from the partumuntil pups weaning. Maternal behavior of all females was scored duringthe first week postpartum, and behavioral indices of emotionality, pre-stress and poststress corticosterone levels, cognitive performance, andhippocampal morphology were assessed in the adult offspring. Wefound that pups reared by female dyads received more maternal carethan pups reared by dams alone, but as adults, they did not exhibitalterations in emotionality or corticosterone response estimated in basalcondition or following restraint stress. Conversely, they showedenhanced performance in hippocampal-dependent tasks including long-term object discrimination, reactivity to spatial change, and fear condi-tioning together with an increase in dendritic length and spine densityin the CA1 region of the hippocampus. In general, the beneficial effectsof dyadic maternal care were stronger when both the females were lac-tating. This study demonstrates that double-mothering exerts a long-term positive control on cognitive function and hippocampal neuronalconnectivity. This experimental manipulation, especially if associatedwith increased feeding, might offer a concrete possibility to limit orreverse the consequences of negative predisposing conditions fornormal cognitive development. VVC 2010 Wiley-Liss, Inc.

KEY WORDS: mice; maternal behavior; emotionality; cognition;hippocampus; dendrite arbor; dendritic spines

INTRODUCTION

Natural occurring variations in maternal care modulate developmentof genetically programmed emotional responses in the progeny (Weaveret al., 2004). For example, adult rats born from dams classified asHIGH with respect to the frequency of their arched-back nursing(ABN) and licking/grooming (LG) behaviors show reduced plasma adre-nocorticotropic hormone (ACTH) and corticosterone response to acute

stress, enhanced glucocorticoid feedback sensitivity,and decreased anxiety in comparison with rats bornfrom LOW ABN-LG mothers (Liu et al., 1997,2000a; Champagne et al., 2003). Following crossfos-tering, however, the behavioral and hormonal profileof rats appears to be determined by the pattern ofmaternal care displayed by their adoptive mothers(Anisman et al., 1998; Francis et al., 2003).

Effects of maternal care have also been detected inrelation to cognition. Specifically, in comparison withoffspring of HIGH ABN-LG mothers, offspring ofLOW ABN-LG mothers shows spatial memory impair-ments associated with a variety of hippocampal neuro-nal alterations including decreased synaptic density andsynaptogenesis, low levels of NMDA receptors, andreduced BDNF expression (Liu et al., 2000b; Bredyet al., 2003a,b). Recently, alterations in the morphologyand the plasticity of CA1 hippocampal cells have alsobeen reported in adult LOW LG offspring (Champagneet al., 2008). Consistent with these observations, pro-longed mother–pup separation, which drastically reducecare-giving during the first weeks of life, increased apo-ptosis and decreased both neurotrophic factor expres-sion and mossy fiber density in the hippocampus of theadult progeny (Lee et al., 2001; Huot et al., 2002;Roceri et al., 2002). Conversely, a short period ofmother–pup separation (15 min of daily handling),which tends to elicit an increase in maternal care, hadpositive consequences on neural systems and cognition(McIntosh et al., 1999; Lehmann et al., 2002).

These studies therefore indicate that pups benefitingfrom intense maternal stimulation, especially LG(Meaney and Szyf, 2005), will be those showing,when adults, the lowest levels of emotionality and themost proficient hippocampal function. Surprisingly,the consequences of increasing maternal care beyondstandard laboratory levels have poorly been investi-gated, despite the relevance of this issue to individualswith abnormal copying to stress or cognitive deficits.In the present study, we experimentally intensifiedmaternal care from birth to weaning by placing a lac-tating or a nonlactating female together with the damin the rearing cage. We then assessed the impact ofdyadic rearing, with or without additional milk sup-ply, on emotional behaviors, corticosterone levels, cog-nitive abilities, and hippocampal morphology in adultmale offspring. Here, we report the paradoxical find-

1CNR Institute of Neuroscience, Rome, Italy; 2Department of Experi-mental Neurology, Santa Lucia Foundation, Rome, ItalyGrant sponsor: Telethon Italy; Grant number: GGP05220; Grant sponsor:Regione Lazio Funds for ‘‘Sviluppo della Ricerca sul Cervello.’’*Correspondence to: Francesca R. D’Amato, CNR Institute of Neuro-science, Via del Fosso di Fiorano 64, 00143 Rome, Italy.E-mail: [email protected] for publication 23 October 2009DOI 10.1002/hipo.20750Published online 19 January 2010 in Wiley Online Library(wileyonlinelibrary.com).

HIPPOCAMPUS 21:298–308 (2011)

VVC 2010 WILEY-LISS, INC.

ing that double-mothering does not affect the emotional profileof the progeny, but boosts hippocampal function andmorphology.

MATERIALS AND METHODS

Subjects

Male and female NMRI outbred mice (Harlan) were housedseparately in groups of four to five in transparent high-tempera-ture polysulfone cages (27 3 21 3 14 cm3) with water andfood available ad libitum. Room temperature (21 1 18C) anda 12:12 h light–dark cycle (lights on at 07.00 p.m.) were keptconstant. Mice were mated when they were 12 weeks old. Mat-ing protocol consisted of housing two females with one maleduring 15 days. After males were removed, females wereassigned to one of the three following experimental conditionsaccording to their reproductive status inferred from their bodyweight increase: pregnant female housed alone (P); pregnantfemale housed with one nonpregnant female (P/NP); two preg-nant females housed together (P/P).

Around the expected day of partum, the cages were inspectedtwice a day. After delivery, the number of pups and the identity ofthe mother in the P/P cages were recorded. In the P/P condition,after at least 1 day elapsed between the two partum, the youngerlitter was left in the cage while the older one was removed. P/Pcages with females delivering on the same day or at a time intervalsuperior to 5 days were discarded. In all conditions, cages withfewer than eight pups or an unbalanced sex-ratio were also dis-carded. The day of delivery was considered as PND0 and experi-mental conditions (P, P/NP, and P/P) were referred to hereafter asL, L/NL, and L/L since former pregnant (P) females were now lac-tating (L) females. Experimental litters were culled to eight pups(four males and four females) at PND1. Pups from the same litterwere weaned, separated by sex, and housed in standard cages offour at PND28. Experiments were run using four batches of malemice dedicated to offspring physiology (Batch 1), behavior(Batches 2 and 3), and brain morphology (Batch 4) measure-ments. All experiments were conducted under license from theItalian Department of Health and in accordance with Italian regu-lations on the use of animals for research (legislation DL 116/92)and NIH guidelines on animal care.

Maternal Care

Maternal care given by dams and, when present, by the sec-ond female was checked from PND1 to PND7 in a total of 31litters (Batches 2 and 3: L, N 5 11; L/NL, N 5 7; L/L, N 513). Two maternal behaviors were recorded: nursing postures(NP, Champagne et al., 2007) and LG pups. Two sessions ofdata recordings were set every day (10.00–11.00 h and 16.00–17.00 h). The occurrence of NP and LG was monitored usingan instantaneous sampling method (one sampling every 4 min,for a total of 15 sampling points per session). The number of

sampling points during which females (single or dyads) werefound to display maternal care was plotted in each group.

Offspring Physiology

Mice from Batch 1 (L, N 5 35; L/NL, N 5 33; L/L, N 527) with no more than two males from the same litter wereused for assessing body weights, brain weights, corticosteronelevels, and white adipose tissue weights.

Body weights

Pups were weighted at five age-points: PND1, PND7,PND28, PND60, and PND120. Body weight before weaning(PND1 and PND7) referred to the average litter weight.

Corticosterone levels and brain weights

Assessment of hypothalamo–pituitary–adrenal (HPA) axisfunction and brain weights was performed at PND120. Micewere transferred from the animal house to the experimentalroom adjacent to the surgery room at 11.00. Serum corticoster-one levels were measured in basal condition (L 5 8, L/NL 5 8;L/L 5 10) or following a 30-min period of restraint stress (L 511, L/NL 5 7; L/L 5 12) in an opaque black Plexiglas tube(diameter 5 3 cm). Immediately after mice were removed fromtheir home cage (baseline) or from the tubes (poststress), theywere sacrificed by decapitation and their brains were weighted.Blood samples were collected and centrifuged at 48C, 4,000rpm, for 20 min, and the serum was stored at 2208C for laterradioimmunoassay. All samples were collected between 12.30and 14.30 h. Serum levels of corticosterone were measured usinga radioimmunoassay kit (ICN). The assay sensitivity was 0.017ng/ml. All measures were carried out in duplicates. The inter-and intra-assay coefficients of variation were 9.3 and 6.1%,respectively. Cross reactivity with desoxycorticosterone was0.34%. The analysis was performed by BIOS lab service, Italy,under the Good Laboratory Practice and certified for quality lab-oratory standards under the UNI European Norm ISO 9001.

White adipose tissue weight

In the same mice, the white adipose tissue (WAT) was col-lected. WAT samples were weighted and estimated in percent-age of total body weights.

Offspring Behavior

Mice from Batch 2 (L, N 5 14; L/NL, N 5 12; L/L, N 514, with no more than two males taken from the same litter)underwent anxiety, long-term object novelty discrimination,and active avoidance learning tests. The tests were administeredaccording to the same sequence in the three mice groups, start-ing at PND 60. The intertask interval was 7 days.

Anxiety

Anxiety was estimated in the open field and in the elevatedplus-maze test. The open field consisted of a square arena

DOUBLE-MOTHERING IMPROVES COGNITIVE PERFORMANCE IN MICE 299

Hippocampus

(42 3 42 cm2) surrounded by Plexiglas walls (25-cm-high). Inthis test, the 12 squares adjacent to the walls represent a pro-tected field, referred to as ‘‘arena periphery’’ and the other 4squares represent an exposed field or ‘‘arena center.’’ The ani-mals were transferred to the experimental room and left in for1 h. The test started by placing the mouse at the center of thearena and letting it move freely for 5 min. Mouse behavior wascontinuously recorded by a video camera placed over the fieldand then analyzed using a video-tracking software (Smart 1.1).The arena was carefully cleaned with a 10% alcohol solutionafter every session. The dependent variables were the time spentin the arena center and the total distance traveled. The elevatedplus-maze consisted of two open arms (27 3 5 cm2) divergingperpendicularly from two enclosed arms (27 3 5 3 15 cm3).The maze was made of gray plastic material and was elevatedto 38.5 cm above the floor. The subjects were individuallytested during one single 5-min session under low illuminationconditions. All mice were placed at the intersection of the twoarms (central platform: perimeter 5 cm 3 4), but facing anopen arm. Each test was video-recorded and analyzed later onusing a keyboard connected to a computer. The dependent var-iables were the number of entries (four paws inside) and thetime spent in each arm.

Object novelty long-term discrimination

Object novelty long-term discrimination (Ennaceur andDelacour, 1988) was assessed in the same square open field.On Day 1, mice were given two 3-min sessions of explorationin the empty arena separated by a 2-h interval. The samplingsession was run 24 h later. During this session (Day 2), micewere exposed for 5 min to two identical objects (a black plastictube of 10 cm in height and 4 cm in diameter). The noveltysession was run 24 h after the sampling session. During thissession (Day 3), mice were exposed for 5 min to two differentobjects. One object was a black plastic tube identical to thetubes presented during the sampling session. The other objectwas a disk made of white plastic material of (8 cm in diameterand 2 cm in thickness) inserted vertically on rectangular blackplastic base (6 3 4 3 2 cm3). The behavior of mice on Days2 and 3 was video-recorded and analyzed later on using a key-board connected to a computer. The time spent exploring eachobject during the sampling and the novelty sessions wasrecorded.

Active avoidance

Active avoidance testing was carried out in a battery of 8two-way shuttle-boxes (40 3 10 3 15 cm3). Each shuttle-boxwas divided into two compartments by a partition with anopening at the floor level connecting the two compartments.The shuttle boxes had a transparent cover with a light bulb (10W) attached above each compartment. The floor was a stainlessgrid. Mice ran one active avoidance session (duration: 40 min,80 avoidance trials) for five consecutive days. Each trial con-sisted of a 30 s light signal [conditioned stimulus (CS)] pre-sented in one compartment 5 s before the onset of an electric

foot-shock (0.7 mA, 25 s) in the other compartment [uncondi-tioned stimulus (US)]. Three dependent variables wererecorded: escape responses (crossings during US presentation),avoidance responses (crossings within the 5 s the CS was pre-sented alone), and intertrial responses (crossings independent ofUS or CS presentation).

Mice from Batch 3 (N 5 6 per rearing condition, with eachindividual taken from a different litter) were first examined formotor activity, habituation of object exploration, reactivity tospatial change, and reactivity to object change in one singleexperiment involving six sessions with a 3-min intrasessioninterval (Ammassari-Teule et al., 1995). Seven days later, thesame mice were subjected to contextual fear conditioning.Shock sensitivity was then measured in the same mice 7 daysafter the contextual fear test. The tasks were administered inthe same order in the three mice groups. Data collection wasperformed using video recordings that were analyzed on a com-puter equipped with the Ethovision software (Noldus, TheNetherlands).

Motor activity

Mice were placed in a circular open field of 60 cm in diame-ter made of gray plastic material with the floor divided intosectors by black lines. The open field was surrounded by a 30-cm-high circular wall on which a conspicuous stripped pattern,20 cm wide and 10 cm high (alternating 1.5 cm wide verticalwhite and black bars), was attached. Motor activity was esti-mated by recording the distance covered and the number of pe-ripheral and central sectors traversed in the empty open fieldduring a 5-min session (Session 1).

Habituation of object exploration

From Session 2, five objects were introduced in the open field:(a) a gray plastic cube (side 5 4 cm) inserted on a same coloredhexagonal base (side 5 4 cm, height 5 2 cm); (b) a black plexi-glas cylinder (diameter 5 8 cm; height 5 18 cm); (c) a transpar-ent plastic tube (height 5 16 cm; diameter 5 3 cm) inserted ona black plastic circular base (diameter 5 6 cm; height 5 2 cm);(d) a white plexiglas cylinder (height 5 2 cm; diameter 5 5 cm)with a small red plastic sphere on the top (3 cm in diameter); (e)a red and white spool (height 5 12 cm; diameter of the top andthe base 5 5 cm) with a small electric bulb on the top. The ini-tial arrangement was a square with a central object. A sixthobject (f ) was used to examine the reactivity to object change. Itconsisted of a red ceramic coffee cup (diameter of the top 5 5cm). From Sessions 2–4, mice were allowed to explore the fieldand the objects for 5 min during which the duration of contactswith each object was recorded. A contact was defined as the sub-ject’s snout actually touching the object.

Reactivity to spatial change

In Session 5, the initial configuration was modified bychanging the position of two objects. Mice were allowed again

300 D’AMATO ET AL.

Hippocampus

to explore the objects, and the duration of contacts with thedisplaced objects and the nondisplaced ones was recorded.

Reactivity to object change

Session 6 was run to habituate mice to the new arrangementof objects. In Session 7, one familiar nondisplaced object (thespool) was substituted by a novel object (red ceramic coffeecup). Mice were still allowed to explore the objects, and theduration of contacts with the substituted object and the famil-iar ones was recorded.

Contextual Fear conditioning

Mice were first handled for 3 days in the animal house, thenfor two additional days in the experimental room to minimizetheir emotional reaction to the novel environment. On the fifthday, each mouse was placed in a transparent Plexiglas cage (283 28 3 10 cm3) with a removable grid floor made of stainlesssteel rods. After a 120-s free exploration period, the mouse wasexposed to a series of five nonsignaled foot-shocks (duration 52 s; intensity 5 0.7 mA, separated by a 1-min interval) deliv-ered through the grid floor. Contextual fear memory wasassessed 24 h later by placing back the mice in the condition-ing chamber for 5 min. Motor activity and freezing behaviorswere recorded during conditioning and testing by means of avideo camera mounted 60 cm above the ceiling of the cage andconnected to a computer equipped with the Ethovision soft-ware (Noldus, The Netherlands). During the conditioning ses-sion, the time spent freezing (absence of all but respiratorymovements associated with a crouching posture) was recordedfor 1 min following the last foot-shock. During the testing ses-sion, fear memory was estimated using activity suppressionratios [activitytesting/(activityconditioning 1 activitytesting)] (Ana-gnostaras et al., 2000).

Shock sensitivity

Mice were individually placed in the conditioning chamber,and the intensity of the shock was progressively increased fromzero until the value eliciting paw licking and jumping. Foreach mouse, the minimal intensity eliciting jumping wasretained as the score.

Offspring Morphology of Hippocampal andPrimary Visual Cortex Neurons

Golgi-Cox impregnation of brain tissue

At PND60, mice from Batch 4 (N 5 4 per condition) wereanesthetized with chloral hydrate (400 mg/kg) and perfusedintracardially with 0.9% saline. The brains were dissected andimpregnated using a standard Golgi-Cox solution (1% potas-sium dichromate/1% mercuric chloride/0.8% potassium chro-mate) according to the method described by Glaser and Vander Loos (1981). The brains immersed in the Golgi-Cox solu-tion were stored at room temperature for 6 days, transferred toa sucrose solution (30%) for 5 days, then sectioned coronally

(150 lm) using a vibratome. Sections were mounted on gelati-nized slides, stained according to the Gibb and Kolb (1998)method, and covered with Permount.

Measurement of dendritic length anddendrite nodes

Fully impregnated pyramidal neurons laying in the CA1region of the hippocampus and in the primary visual cortexarea were initially identified under low magnification (203/0.5NA). Within each hemisphere, three neurons displaying dendri-tic tree without obvious truncations were then analyzed underhigher magnification (633/0.75 NA). Because no interhemi-spheric difference was detected, the data were pooled so thatsix neurons per brain area were considered in each analysis.Measurements were carried out using a microscope (DMLB,Leica) equipped with a camera (resolution 2,600 3 2,600, Axi-ocam, Carl Zeiss AG, Germany) and the KS300 3.0 system(Carl Zeiss AG, Germany). Morphological measurements weremade by an experimenter blind to the mice group. The lengthand the number of nodes of the dendritic trees were quantifiedtracing the entire basal and apical dendrites and then perform-ing Sholl analyses. Briefly, using the center of the soma as refer-ence point, dendrite length and branch points (nodes) weremeasured as a function of their radial distance from the somaby adding up all values in each successive concentric segment(segment radius 5 25 lm). The number of radius segmentswas 9 (from 25 to 225 lm) for the basal dendrites and 25(from 25 to 625 lm) for the apical dendrites.

Spine density

Measurements were performed under the same magnificationin four CA1 and primary visual cortex neurons laying in eachhemisphere. For each neuron, five 20-lm dendrite segmentswere sampled 50 lm away from soma to exclude the spine-depleted zone arising from the cell body. All protrusions werecounted as spines if they were in direct contact with the den-dritic shaft. The average spine density (number of spines per20 lm of dendrite length) was estimated by focusing in andout with the fine adjustment. Because this method has provento produce reliable result (Horner and Arbuthnott, 1991), noattempt was made to introduce a correction factor for hiddenspines. A total 160 segments (five segments on eight neuronsin four mice) was examined in each group. Values were aver-aged for each neuron and means compared among groups.

Statistical analysis

One-way ANOVAs with the factor ‘‘group’’ were used foranalyzing maternal behaviors (NP and LG), offspring physiol-ogy (brain and body weights, corticosterone levels, WAT), anxi-ety (open field exploration and plus-maze), motor activity, reac-tivity to spatial and object change, preshock and postshockfreezing to the context, foot-shock sensitivity, and spine densitydata. Tukey’s HSD tests were used for post-hoc paired-compari-sons. For the reactivity to spatial and to object change, intra-


Hippocampus

group comparisons of exploration directed toward the displacedversus the nondisplaced objects, and the novel versus the famil-iar objects were performed by means of post-hoc planned com-parison tests. Two-way ANOVAs were used for analyzing long-term object novelty discrimination (main factors group andobject) active avoidance (main factor ‘‘group,’’ repeated factor‘‘days’’), object exploration in the circular open field (main fac-tor ‘‘group,’’ repeated factor ‘‘sessions’’), dendritic length andnodes (main factor ‘‘group,’’ repeated factor ‘‘segment radius’’).

RESULTS

Maternal Care

Results are shown in Figure 1. The amount of NP (Fig. 1A)differed across rearing conditions [effect of ‘‘group,’’ F(2,28) 59.53, P < 0.001]. Subsequent pair comparisons revealed thatpups reared by dams with second lactating females wereexposed to more NP than pups reared by dams with a cyclingfemale (L/L vs. L/NL: t 5 3.58, P < 0.01) or by dams alone(L/L vs. L: t 5 3.82, P < 0.01). Lactating partners in the L/Lgroup showed more NP than cycling females (t 5 3.32, P <0.01). Surprisingly, the amount of nursing displayed by biologi-cal mothers varied across conditions [F(2,28) 5 6.34, P <0.01], since dams rearing their pups with a cycling female pro-duced significantly less NP than dams alone (L/NL vs. L: t 523.55, P < 0.01) or with a lactating female (L/NL vs. L/L: t5 23.20, P < 0.01). This reduction was likely due to the factthat, within female dyads, the cycling female competed moreefficiently with the mother for NP than did the second lactat-ing female (percentage of conursing, L/NL condition 5

12.5%; LL condition 5 35%). The amount of LG (Fig. 2B)also differed among conditions [F(2,28) 5 16.07, P < 0.001].Specifically, pups reared by two females were those receivingmore LG (L/NL vs. L: t 5 5.78, P < 0.001; L/L vs. L: t 54.17, P < 0.001). However, differently from what wasobserved for NP, the three biological mothers were now foundto display the very same amount of LG [no effect of ‘‘group,’’F(2,28) 5 0.60, ns]. Thus, the global difference in maternalstimulation across dyadic rearing conditions was due to the factthat the cycling females were more engaged in LG than the sec-ond lactating females (L/NL vs. L/L: t 5 3.07, P < 0.01).

Offspring Physiology

The data are shown in Table 1. Body weights averaged perlitter were similar in all groups at PND1 [F(2,42) 5 1.87, ns],

FIGURE 1. Maternal care. (A) Dams (white bars) exhibitedless nursing postures (NP) when the second females (gray bars)were nonlactating (NL condition). (B) Pups experiencing dyadicrearing received more LG compared to pups reared by dams alone.Dams (white bars) produced the same amount of LG in all condi-tions, but among the second females (gray bars), those that werenot lactating produced more LG than those that were lactating. L,dams alone; L/NL, dams with nonlactating females; L/L, damswith lactating females. *P < 0.01, differences in total maternalcare; §P < 0.01, differences between dams maternal care; ◦P <0.01, differences between second females maternal care.

FIGURE 2. Behavioral measurements. (A) During the test ses-sion of the long-term recognition memory test, mice reared by twolactating females explored the novel object significantly more thanthe familiar. (B) In Session 1, all groups covered an equivalent dis-tance (in cm) in the empty circular open field. (C) The time (insec) spent exploring the five objects decreased similarly in allgroups across sessions 2–4. (D) In session 5, only mice experienc-ing dyadic rearing showed a significant reactivity to spatial change.(E) In session 7, all mice significantly reacted to object change. (F)In the contextual fear test, mice reared by one dam showed signifi-cantly higher residual activity (activity suppression ratios) thanmice reared by female dyads among which no difference wasfound. L, dams alone; L/NL, dams with cycling females; L/L, damswith lactating females. *P < 0.05, **P < 0.01.


Hippocampus

but differed at PND7 [F(2,42) 5 15.89, P < 0.001] withpups reared by two lactating females showing significantlyhigher weights (P < 0.01 for both comparisons). This differ-ence was still evident after weaning [PND28: F(2,92) 5 36.99,P < 0.001] and in the adulthood [PND60: F(2,92) 5 9.83, P< 0.001; PND120: F(2,49) 5 8.98, P < 0.001]. In agreementwith this, white adipose tissue estimated at PND120 washigher in L/L mice [F(2,49) 5 8.64, P < 0.001]. Brainweights examined at PND60 did not differ significantlybetween groups [F(2,15) 5 1.72, ns, values not shown]. Corti-costerone levels measured at PND120 did not differ signifi-cantly between groups, either in baseline condition [F(2,23) 50.03, ns] or following restraint stress [F(2,27) 5 0.52, ns].

Offspring Behavior

Anxiety

The data are shown in Table 2. The percentage of time spentin the central sector of a square open field and the number ofentries in this sector did not vary between groups. The threemice groups also showed an equivalent percentage of entries,

and of time spent in the open arms of a plus-maze, as well as asimilar total number of entries in both arms.

Object novelty long-term discrimination

During the sample session of the long-term recognitionmemory test, all mice spent the same total time exploring thetwo identical objects [no effect of ‘‘group,’’ F(2,37) 5 0.70, ns]and each object was explored for an equivalent duration [noeffect of ‘‘object category,’’ F(1,37) 5 2.48, ns]. During thetest session that was run after a 24-h delay (Fig. 2A), the novelobject (new) was explored globally more than the familiar one(old) in all groups [effect of ‘‘object category,’’ F(1,37) 5 7.50,P < 0.01], but only LL mice showed a significant long-termreaction to object novelty [significant effect of the ‘‘group 3object category’’ interaction, F(2,37) 5 3.83, P < 0.05 signifi-cant effect of ‘‘object category’’ in L/L, t 5 23.26, P < 0.01].

Active avoidance learning

The number of conditioned responses increased similarly inall groups during the course of training [effect of ‘‘days,’’

TABLE 1.

Mean Body Weights (6SEM) of Litters at PND1 and PND7 and of Males From PND28 Onwards, and Percentage of White Adipose Tissue at

PND120 and Corticosterone Levels in Adult Animals in Baseline and After 30 min of Restraint Stress

Groups

Body weighta (g)

% WATb

at PND120

Corticosterone level (ng/ml)

PND1 PND7 PND28 PND60 PND120

PND120

(baseline)

PND120

(30-min restraint)

L 14.07 (0.33) 43.08 (0.84) 25.42 (0.26) 39.13 (0.54) 51.90 (1.23) 2.85 (0.15) 39.62 (12.30) 266.54 (14.15)

L/NL 13.68 (0.22) 42.55 (0.76) 25.62 (0.28) 38.10 (0.61) 48.29 (0.96) 2.32 (0.23) 37.80 (6.01) 302.67 (39.96)

L/L 14.70 (0.45) 52.85* (1.76) 28.65* (0.32) 41.62* (0.46) 55.87** (1.09) 3.42** (0.15) 40.81 (9.70) 265.14 (21.97)

L: dam alone; L/NL: dam plus a cycling female; L/L: dam plus a lactating female.Tuckey–Kramer post-hoc test: *P < 0.01, L/L versus L/NL and L at the same age; **P < 0.01, L/L versus L/NL at the same age.aBody weights averaged per litter at PND1 and PND7, per individual from PND28 onwards.b% WAT: Percentage of total white adipose tissue on body weight.

TABLE 2.

Behavioral Performance of Male Mice in Anxiety Tests

La L/NLa L/La F (2/37) P

Open field

% Time in C.S. 13.26 (1.62) 15.07 (1.26) 13.87 (1.39) 0.39 ns

% Entries in C.S. 20.50 (1.38) 22.54 (1.80) 20.33 (1.57) 0.58 ns

Plus maze

% Entries in O.A. 43.06 (3.79) 46.63 (3.28) 45.78 (2.91) 0.31 ns

% Time in O.A. 45.74 (5.14) 48.47 (4.25) 49.58 (4.16) 0.19 ns

Total entries 21.00 (1.16) 23.33 (1.95) 19.64 (1.39) 1.52 ns

Independent mice groups were tested during one 5-min session in the open field or in the plus maze. No effect of the rearing condition was found for anymeasurement.L: dam alone; L/NL: dam plus a cycling female; L/L: dam plus a lactating female; C.S.: central sectors of the squared open field; O.A.: open arms of the plus maze.aValues represent mean (SEM).


Hippocampus

F(4,108) 5 71.69, P < 0.001; no effect of ‘‘group,’’ F(2,108)5 1.11, or of the ‘‘group’’ 3 ‘‘days’’ interaction: F(8,108) 51.15]. The mean correct responses recorded in each group bythe end of training were in the same range (L 5 56.6 6 4.68;L/NL 5 57.12 6 11.45; L/L 5 67.75 6 4.47, data notshown).

Motor activity and habituation to objects

As shown in Figure 2B, all mice traveled an equivalent dis-tance in the empty open field during Session 1 [no effect of‘‘group,’’ F(2,15) 5 0.23, ns]. From Sessions 2–4, all miceshowed the same reduction of the time spent exploring of thefive objects present in the field across sessions [effect of ‘‘ses-sions,’’ F(2,31) 5 77.43, P < 0.001].

Reactivity to spatial and object change

As shown in Figure 2D, the displaced object (DO) was glob-ally more explored than the nondisplaced ones (NDO) [signifi-cant effect of ‘‘object category,’’ F(1,15) 5 9.64, P < 0.001],but intragroup comparisons revealed that only mice experienc-ing dyadic rearing significantly reacted to spatial change (L/NL:t 5 2.58, P < 0.05; L/L: t 5 2.98, P < 0.05; L: t 5 0.89,ns). In Session 7 (Fig. 2E), the novel object (SO) was globallymore explored than the familiar ones (NSO) [significant effectof ‘‘object category,’’ F(1.15) 5 57.02, P < 0.001], and allmice significantly reacted to object change (t-tests, P < 0.05for all comparisons).

Contextual fear conditioning

In the training session, no between-group difference wasfound for the time spent freezing after the last shock [F(2,15)5 0.13, ns, data not shown]. In the test session (Fig. 2E),group differences were observed for activity suppression ratio[F(2,15) 5 6.39, P < 0.01]. Pair comparisons revealed thatmice reared by one dam (L) showed higher residual activity(ratio of activity suppression) than mice reared by femaledyads (P < 0.05 for each comparison). Activity suppressionratios did not differ between L/NL and L/L mice.

Shock sensitivity

The minimal shock intensity eliciting jumping did not varybetween groups [F(2,15) 5 0.84, P > 0.5, data not shown].

Offspring Neuronal Morphology

Hippocampus

Curves depicting sholl analyses for dendrite length andbranch nodes, and histograms showing spine density values inthe apical and basal dendrite compartment of CA1 neurons areshown in Figure 3 (top). In the basal compartment (right partof the curve), the analysis of variance (ANOVA) performed ondendritic length data did not reveal a main effect of ‘‘group’’[F(2,9) 5 3.57, ns], but a significant effect of ‘‘segment radius’’[F(8,72) 5 393.21, P < 0.001] and of the ‘‘group 3 segment

radius’’ interaction [F(16,72) 5 3.81, P < 0.01]. Post-hoccomparisons then showed that dendrites lying 75–100 lmfrom the soma were significantly longer in mice reared byfemale dyads (P < 0.05 for L vs. L/NL and L/L). No between-group difference was found for the number of nodes [F(2,9) 52.81, ns]. For this comparison, there was only an effect of ‘‘ra-dius segment’’ [F(8,72) 5 160.04, P < 0.001] indicating thatbranch nodes decreased similarly in all groups as a function oftheir distance from the soma. In the apical compartment (leftpart of the curve), there was only an effect of ‘‘radius segment’’[dendritic length: F(24,216) 5 80.04, P < 0.001; nodes:F(24,216) 5 30.08, P < 0.001]. For spine density, differenceswere found in the basal compartment [F(2,105) 5 16.84, P <0.01] with post-hoc comparisons showing fewer spines in Lmice compared to L/NL (P < 0.01) and L/L (P < 0.001). Inthe apical compartment, although the ANOVA showed only amarginal effect of ‘‘group’’ [F(2,105) 5 3.01, P 5 0.08], post-hoc tests revealed a difference between L and LL mice (P <0.05), with more spines counted in the latter group.

Visual cortex

The sholl analysis curves and histograms showing spine den-sity values for visual cortex neurons are shown in Figure 3(bottom). No ‘‘group’’ effect for dendritic length or branchnodes was found in any compartment. ANOVAs revealed onlya significant effect of ‘‘segment radius’’ [basal dendritic length:F(8,72) 5 50.21, P < 0.001; apical dendrite length: F(24,216)5 26.70, P < 0.001; basal nodes: F(24,216) 5 42.57, P <0.001; apical nodes: F(2,24) 5 20.70, P < 0.001]. In agree-ment with previous observations (Restivo et al., 2009), visualcortex neurons exhibited higher spine density values than CA1neurons. These values, however, did not differ significantlybetween rearing conditions [apical dendrites: F(2,105) 5 2.76,ns; basal dendrites: F(2,105) 5 3.01, ns].

DISCUSSION

Our findings show that, as expected, pups reared by femaledyads received more maternal care than pups reared by damsalone. As adults, however, male mice experiencing double-mothering did not differ in their emotional or hormonal profilein baseline condition or in response to stress from mice rearedby their mothers alone, but exhibited enhanced cognitive per-formance, longer dendritic branch length, and increased spinedensity on CA1 pyramidal cells. In general, the beneficialeffects of dyadic maternal care on cognition and hippocampalmorphology were stronger when both the females werelactating.

Pups reared by two lactating females had access to morenutrients and accordingly showed higher body weights fromPND7 than pups receiving milk from their dams only. AtPND120, body weights of L/L mice were no longer differentfrom those of L mice and those of L/NL mice were still lower


Hippocampus

(Table 1). The slower growth observed in the latter group ispresumably due to the fact that dams caged with cyclingfemales displayed less nursing thereby reducing the time pupshad access to milk. Interestingly, the higher body weights of L/L offspring were accompanied by an increased percentage ofwhite adipose tissue in the adulthood confirming that postnatalovernutrition is a risk factor for obesity (Bassett and Craig,

1988). Nevertheless, the same body weights of adult micereared in the L/L and L conditions exclude that the behavioraland morphological differences these two mice groups show canbe ascribed to metabolic factors.

As shown in Figure 1, the amount of care directed towardpups was indeed globally superior in double-mothering condi-tions, but the care given by dams in each dyad varied according

FIGURE 3. Morphological measurements. (A) Hippocampalmorphology. Top: Neurolucida drawing of a typical pyramidalneuron in the CA1 region of the hippocampus and photograph ofa 20-lm Golgi-stained basal dendrite segment indicative of spinedensity in each experimental condition. Bottom: Sholl analysiscurves for dendritic length in the apical (left) and basal (right)dendrite compartment, and histograms showing spine density val-ues in each experimental condition. In the basal compartment,dendrites lying 75–100 lm from the soma were significantly

longer in mice reared by female dyads than by the dams alone; nogroup difference was observed in the apical dendrite compartment.In the basal compartment, spine density was significantly higher inmice reared by female dyads. (B) Primary visual cortex morphol-ogy. Top and Bottom: The same as for CA1 hippocampal neurons.No difference imputable to the rearing condition was observed fordendritic length and spine density in primary visual cortex neu-rons. L, dams alone; L/NL, dams with cycling females; L/L, damswith lactating females. *P < 0.01, **P < 0.001.


Hippocampus

to the hormonal characteristics of the second female. For exam-ple, although dams displayed the very same amount of LGwhen they were alone or in dyads, their NP scores were signifi-cantly reduced in the presence of cycling females. This observa-tion suggests a competition/cooperation between females in thedisplay of nursing postures in the L/NL condition that did notoccur in the L/L condition. In agreement with the strong inter-est of cycling females toward pups, these females were alsothose displaying the higher LG scores. It is therefore apparentthat not only the amount but the quality of the maternal carevaries according to the maternal status of the second femaleand that this aspect should be systematically controlled in situa-tions of maternal care sharing.

Based on multiple reports showing that pups exposed tohigh maternal solicitation show lower stress responsiveness inadulthood (Liu et al., 1997; Champagne et al., 2003, 2008),our hypothesis was that augmenting maternal care experimen-tally would have further enhanced coping capabilities to stressin the adult progeny. Unexpectedly, we found that adult malemice reared in standard or dyadic conditions showed similaranxiety behaviors and neuroendocrine responses (Tables). Spe-cifically, no significant difference was detected between groupswhen behavioral indices of anxiety/emotionality were examinedand no variation imputable to the rearing condition was foundin either prestress or poststress corticosterone measurements.These findings lend therefore further support to the hypothesisof a neonatal bond between mother and pups (Moles et al.,2004), which would hinder any other dam to play a criticalrole in offspring emotional development. Indeed, the presentobservations apparently disagree with data showing that moth-ers rearing their litters in communal nests are also more fre-quently engaged in LG behaviors and that pups experiencingcommunal nesting show decreased emotionality and earlieremergence of their social status but no enhancement of cogni-tion (Branchi, 2009; Curley et al., 2009). However, an obviousproperty of communal nesting is that increases interactionswith multiple dams and peers, making it difficult to estimatethe maternal component in the reduction of pups’ emotionality.Differently, the present protocol fixes a constant number ofpups per litter in order the only factor varying across rearingconditions is the number of females and consequently theamount of care (and of milk) pups receive. Considering thatdams LG was similar in all conditions while total LG differedacross groups, two hypotheses can be proposed to explain theabsence of dyadic rearing effect on emotionality. First, as previ-ously suggested, only maternal LG is effective in modulatingoffspring emotional responses so that additional LG from thesecond female becomes irrelevant. Second, above a certainthreshold, LG, indifferently given by the dam or the secondfemale, is no longer effective.

Conversely, we found that mice reared by females dyadsshowed marked improvements in hippocampal-dependentbehaviors (Fig. 2). For example, both L/L and L/NL mice weremore reactive to spatial change and displayed lower activitysuppression ratios with stronger freezing in the contextual fearconditioning test than L mice. Only L/L mice, however,

showed increased investigation/preference for the novel objectduring the discrimination session of the long-term recognitionmemory task. Consistent with a selective enhancement of hip-pocampal function in mice experiencing double-mothering, noeffect of the rearing condition emerged in active avoidancelearning. This task requires the formation of explicit stimulus-response associations and is therefore largely hippocampal-inde-pendent (Pittenger et al., 2006).

Quantification of dendrite length and spine density inGolgi-Cox-impregnated tissue has been used to examine theeffects of a variety of early life manipulations on neuronal mor-phology of brain regions critical for learning (Kolb et al.,1998). The hippocampus maturation, which requires about 3weeks in rodents (Pokorny and Yamamoto, 1981), makes thisbrain region particularly sensitive to any form of perinatal envi-ronmental stimulation including maternal care. The reportedbeneficial effects of high LG on hippocampal function includeenhanced expression of NR2A and NR2B subunits of theNMDA receptor, increased release of neurotrophic factors (Liuet al., 2000b; Bredy et al., 2003a,b), stronger long-term poten-tiation (LTP), and longer dendrite branches with a higher spinedensity in CA1 cells from the adult offspring (Champagneet al., 2008). Our findings that double-mothering increasesdendrite length and spine density in the basal dendrite com-partment of CA1 adult neurons (Fig. 3) is consistent with thesefindings and overall demonstrate that intensification of mater-nal behaviors above standard laboratory levels promotes a highdegree of innervation in the CA1 region of L/L and L/NLmice, which likely contributes to their behavioral phenotype.The selectivity of hippocampal remodeling is confirmed by theobservation that no morphological change occurred in the pri-mary visual cortex. Indeed, this latter finding also indicates thatthe high object discrimination performance of L/L mice cannotbe ascribed to an enhancement of visual informationprocessing.

The observation that L/L mice outmatched L/NL mice inlong-term recognition memory and CA1 spine density clearlyindicates that combining more maternal care with a surplus ofnutrients represents the most favorable condition for boostinghippocampal function. Beyond superior milk intake, L/L pupsare also those absorbing the larger quantity of colostrum, i.e.,the pre-milk fluid produced postpartum by mother’s mammil-lary glands. Colostrum contains immune and growth factors,and there is evidence that adult rats chronically exposed tocolostrinin, a complex of polypeptides derived from the colos-trum of sheep, show learning improvements (Popik et al.,1999). Colostrum, however, decreases rapidly after partum and,given that adoptive lactating mothers were the first to deliverwithin the dyad, it is unlikely that L/L pups absorbed signifi-cantly more colostrum than the other pups. Thus, additionalmilk supply might enlarge double-mothering effects on cogni-tion and hippocampal morphology. Indeed, maternal care rep-resents the earliest source of environmental stimulation and,given that pups are deaf and blind at birth, the relevance ofincreased tactile and olfactory perinatal maternal stimulation isobviously superior to any other form of enrichment they could


Hippocampus

be exposed to in the perinatal phase. Protocols mixing physical(large cages with toys) and social enrichment (other dams withtheir litters, juveniles, and sire) from birth to weaning have alsobeen shown to be beneficial for neural plasticity and cognition(Sale et al., 2004; Meaney and Szyf, 2005; Nithianantharajahand Hannan, 2006), but make it difficult identifying whichstimuli are actually crucial. For example, the presence of thesire in the cage still needs to be clarified (Orefice and Hein-richs, 2008). Differently, our data demonstrate that the pres-ence of a second adult female during the first stages of develop-ment exerts a positive control on hippocampal morphology andcognitive function. This simple experimental manipulation,especially if associated with increased milk intake, might offer aconcrete possibility to limit or reverse the consequences of neg-ative predisposing conditions for normal cognitive develop-ment. Therapeutic implications of double-mothering wouldtherefore be examined in rodent models of brain disorder withgenetic cognitive disturbances including fragile-X, Down syn-drome, or various forms of early brain injury.

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intensification of maternal care by double-mothering boosts cognitive function and hippocampal...

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