Neuroscience 298 (2015) 455–466

TRACING THE TRAJECTORY OF BEHAVIORAL IMPAIRMENTSAND OXIDATIVE STRESS IN AN ANIMAL MODEL OF NEONATALINFLAMMATION

M. MACRAE, a T. MACRINA, a A. KHOURY, b

M. M. MIGLIORE b AND A. C. KENTNER a*

aSchool of Arts & Sciences, Health Psychology Program,

MCPHS University (formerly Massachusetts College of Pharmacy

& Health Sciences), Boston, MA 02115, United States

bSchool of Pharmacy, MCPHS University, Boston, MA 02115, United

States

Abstract—Exposure to early-life inflammation results in

time-of-challenge-dependent changes in both brain and

behavior. The consequences of this neural and behavioral

reprogramming are most often reported in adulthood.

However, the trajectory for the expression of these various

changes is not well delineated, particularly between the

juvenile and adult phases of development. Moreover, inter-

ventions to protect against these neurodevelopmental dis-

ruptions are rarely evaluated. Here, female Sprague–

Dawley rats were housed in either environmental enrich-

ment (EE) or standard care (SC) and their male and female

offspring were administered 50 lg/kg i.p. of lipopolysaccha-

ride (LPS) or pyrogen-free saline in a dual-administration

neonatal protocol. All animals maintained their respective

housing assignments from breeding until the end of the

study. LPS exposure on postnatal days (P) 3 and 5 of life

resulted in differential expression of emotional and cogni-

tive disruptions and evidence of oxidative stress across

development. Specifically, social behavior was reduced in

neonatal-treated (n)LPS animals at adolescence (P40), but

not adulthood (P70). In contrast, male nLPS rats exhibited

intact spatial memory as adolescents which was impaired

in later life. Moreover, these males had decreased prefrontal

cortex levels of glutathione at P40, which was normalized in

adult animals. Notably, EE appeared to offer some protec-

tion against the consequences of inflammation on juvenile

social behavior and fully prevented reduced glutathione

levels in the juvenile prefrontal cortex. Combined, these

time-dependent effects provide evidence that early-life

inflammation interacts with other developmental variables,

specifically puberty and EE, in the expression (and preven-

tion) of select behavioral and molecular programs.

� 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.neuroscience.2015.04.0480306-4522/� 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

*Corresponding author. Tel: +1-617-274-3360 (O); fax: +1-617-732-2959.

E-mail address: amanda.kentner@mcphs.edu (A. C. Kentner).Abbreviations: ANOVA, analysis of variance; EDTA,ethylenediaminetetraacetic acid; EE, environmental enrichment; LPS,lipopolysaccharide; MIA, maternal immune activation; P, postnatal day;SC, standard care.

455

Key words: inflammation, maternal care, enrichment, devel-

opment, oxidative stress, corticosterone.

INTRODUCTION

Exposure to inflammatory mediators at distinct critical time

periods (i.e., fetal or neonatal development) results in long-

term brain changes and corresponding behavioral

disruptions in rats and mice. The occurrence of such

disruptions is dependent on the timing of early-life

challenge and has been reported most often in mature

animals (Meyer et al., 2006a, 2008b).Moreover, the conse-

quences of reprogramming the brain following exposure to

inflammatory mediators such as lipopolysaccharide (LPS)

and polyriboinosinic–polyribocytidilic acid (Poly:IC) appear

to have a regulated time course (Harre et al., 2008; Forrest

et al., 2012; Garay et al., 2013; Khalil et al., 2013; Liu et al.,

2013). For example, neonatal (n)LPS led to hippocampal

NMDA receptor subtype expression that was juxtaposed

between acute (i.e., increased expression) and chronic

(i.e., decreased expression) time points (Harre et al.,

2008). Time-dependent emergence of behavioral impair-

ments is also apparent between adolescence and adult-

hood (Fan et al., 2011; Stolp et al., 2011; Dinel et al.,

2014). Mice challenged with nLPS had increased anxiety-

like behavior at postnatal day (P)30 that was resolved by

P90. The opposite expression pattern was observed with

respect to depressive-like behavior in the forced swim test

(Dinel et al., 2014) while repeated nLPS i.p. injections in

rats were associated with increased duration and number

of entries into the light chamber of the light/dark test in

adulthood, but not at P21. The reverse was seen with

respect to disrupted prepulse inhibition response at P20,

which was not sustained following maturity (Stolp et al.,

2011). In general, the time course of behavioral and neural

manifestations following early inflammatory stressors is

not well understood. Therefore, in the present paper, we

incorporate both juvenile and adult developmental time

points into the evaluation of reprogramming following

nLPS.

The developmental disruptions that follow some

inflammatory challenges are interesting given their

similarities to the timing and phenotypical expression of

autism and schizophrenia, particularly with respect to

cognitive functioning (Zuckerman et al., 2003; Meyer

et al., 2006b; Boksa, 2010). The parallels in the

456 M. MacRae et al. / Neuroscience 298 (2015) 455–466

pathogenesis between these neurodevelopmental disor-

ders and early-life inflammation also extend to social

impairments, imbalances in dopamine, reductions in

reelin and NMDA receptor expression, in addition to dys-

regulation in immune system mediators such as cytoki-

nes, chemokines and other chronic modifications

precipitated by immune activation (Coyle et al., 2003;

Meyer et al., 2008a; see Shi et al., 2003; Patterson,

2009). Notably, oxidative stress has been proposed as a

mechanism underlying disruptions in animal models of

immune activation, as well as schizophrenia and autism

(Do et al., 2000; Boksa, 2010; Gu et al., 2015).

Maternal immune activation (MIA) induces oxidative

stress in fetal brain and the depletion of glutathione, which

protects cells from oxygen-free radicals. Pretreatment

with the antioxidant N-acetylcysteine increases

L-cysteine levels, prevents LPS-induced decreases in

fetal glutathione (Lante et al., 2007; Paintlia et al.,

2008), and protects against associated deficits in juvenile

spatial memory (Lante et al., 2007).

Environmental enrichment (EE) protocols have

demonstrated success as an intervention for autism and

schizotypal personality in human rehabilitation settings

(Raine et al., 2003; Woo and Leon, 2013), and a clinical

trial is currently in progress evaluating EE for infants at

risk for cerebral palsy (Morgan et al., 2014). Recently,

EE prevented social interaction impairments and hypotha

lamic–pituitary–adrenal activation following MIA (Connors

et al., 2014). Moreover, there is evidence for enriched

environments preventing/reducing oxidative stress in ani-

mal models of cerebral hypoperfusion (Cechetti et al.,

2012), Alzheimer’s disease (Herring et al., 2011), and

aging (Kempermann et al., 2002), each of which are asso-

ciated with inflammatory activation. Therefore, we

employed EE in order to evaluate its ability to counteract

oxidative stress and behavioral disruptions in a dual-

administration nLPS model. Overall the purpose of this

paper was to (1) map out the developmental timing of

the emerging brain and behavioral impairments following

neonatal inflammation, and (2) evaluate the protective

effect of the environment against these behavioral disrup-

tions and reductions in glutathione, an indicator of oxida-

tive stress.

EXPERIMENTAL PROCEDURES

Animals and housing

Virgin female and male Sprague–Dawley rats were

obtained from Charles River (Wilmington, MA, USA) and

housed at 20 �C on a 12-h light/dark cycle (0700–1900

light) with free access to food and water. Female rats

were pair-housed in one of two conditions: EE (large

multi-level cage with toys, tubes, chew bone, Nestlets�and ramps; Critter Nation, Muncie IN, USA), or

Standard Care (SC; standard cage with tube, chew

bone, and Nestlets�). Toys and tubes were changed

twice weekly in the EE group to ensure novelty of the

condition. Male rats were maintained in standard

housing. Experimental procedures were approved by

the MCPHS University Institutional Animal Care and

Use Committee and were carried out in compliance with

the Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC). A flowchart of the

study procedures is located in Fig. 1.

Breeding, parturition and litter phenotype

Following acclimatization to laboratory conditions female

rats were bred and pregnancy was confirmed by

continued weight gain and visible teats during the later

phase of gestation. Approximately two days prior to

parturition, SC dams (n= 12) were placed into

individual cages and EE females (n= 8; one female

was not pregnant for a final n= 7) were separated from

their partner, in a cleaned cage, by a physical divider

that allowed for olfactory, auditory, and some tactile and

visual contact. This separation allowed for the correct

identification of dams and their litters after birth

[postnatal day (P)1] and was maintained until weaning

(P22).

On P3 all litters were adjusted to 12 pups, creating an

equal distribution of males and females wherever

possible. A dual lipopolysaccharide (LPS; Escherichiacoli, serotype 026:B6; L-3755, Sigma, St. Louis, MO,

USA) administration protocol was used on P3 and P5.

Pups were injected i.p. with 50 lg/kg of LPS or an

equivolume of pyrogen-free saline, in a balanced

manner within litters. Their skin was then colored with

non-scented non-toxic markers in order to identify the

neonatal drug treatment received (described previously

Connors et al., 2014). Weaning involved the removal of

each dam and placing her offspring into a clean cage,

maintaining their respective neonatal housing conditions.

EE offspring were housed in same-sex groups of four–six

while SC animals were housed in pairs; all animals were

weighed once per week following weaning.

Maternal behavior

In order to ensure that the nLPS protocol did not influence

maternal behavior we monitored passive maternal care

between P3 and P6, three times daily (7:30, 12:30,

17:30 h); each session consisted of ten observations and

a total composite score was calculated for each postnatal

day. Dams (n= 12 SC; 7 EE) were evaluated for 1-min

intervals per observation (with at least 5 min between

each interval). Behaviors scored included the frequency

of licking/grooming, pup retrieval, and nest building

(digging, retrieving Nestlets� to the nest), in addition to

high arched-back, low arched-back (blanket) and passive

nursing (dam lying on either her back or side). Finally, the

total duration of time on the nest was recorded.

On P7 we employed a maternal preference test. Four

rat pups from each neonatal treatment/sex were grouped

together in different areas of a large arena (72 cm �72 cm � 36 cm). Large amounts of home cage nesting

material were placed in one section of the arena, equal

distance from the location of each pup group. Dams were

then placed into this arena and latency to retrieve

(seconds) was recorded. Surprisingly, few dams from

either housing condition demonstrated retrieval behaviors

so we only evaluated a subset of the original set of dams

(n= 6 SC; 7 EE). Post hoc, we simply measured the

Fig. 1. Flow chart of study procedures.

M. MacRae et al. / Neuroscience 298 (2015) 455–466 457

percentage of time dams spent in direct contact (sniffing,

standing over, nursing, licking) with each neonatal

treatment group/sex (total seconds spent with group/600

second test) * 100. Given the failure of dams to retrieve in

the maternal preference test, we evaluated maternal

retrieval times in the home cage (n= 12 SC; 7 EE).

From each litter, two pups from every neonatal

treatment/sex were scattered across the home cage

floor. Latency to begin retrieval and total time to collect all

pups from the litter (and each group) back to the nest

were logged. Maternal anxiety level was also evaluated in

an open field test for 10 min (n= 12 SC; 6 EE). One EE

rat was not tested in the maternal anxiety test due to

experimental error. The black plexiglas open field arena

(40 cm � 40 cm � 28 cm) was divided into a 25-square

grid. The percentage of entries into the center of the open

field [(central entries/total entries) � 100] was calculated

as an index of anxiety level (Paris et al., 2011). For the

remainder of the study, only one pup per sex and neonatal

treatment groupwas used per litter for each behavioral and

molecular time point described below.

Offspring behavior

Social behavior and spatial memory were evaluated in

male and female offspring, in a counter-balanced

manner on P40 (n= 7–10) and P70 (a separate group

of rats; n= 6–10). Beforehand, rats were habituated to

a black test arena (40 cm � 40 cm � 28 cm), over three

separate days, for 10 min daily. The discrepancy

between the total number of litters and offspring

evaluated in the behavioral tests is due to a file transfer

failure between the video camera and computer, which

resulted in data loss.

Social interaction: To evaluate social behavior, rats

were introduced to a novel SC-housed conspecific

(n= 8) of the same sex and age, for 10 min in an

identical manner as used previously by this laboratory

(Connors et al., 2014). In brief, we evaluated animals on

the frequency and duration of each of the following behav-

iors: allogrooming, mounting/crawling, approaching/fol-

lowing. We categorized these observations into

composite scores to detail the total duration of time spent

initiating vs. receiving social contact.

Object-in-place: A novel object was positioned in each

corner of the arena and rats were given 5 min to explore.

After a 1-h delay objects were replaced with identical

copies of the four items, except that the placement of

two of the objects switched location. Animals were

returned to the arena for an additional 5 min of

exploration (as described in Connors et al., 2014). A dis-

crimination ratio was calculated [(total time exploring

moved objects � total time exploring permanent

objects)/(total time exploring both objects)].

Blood sampling and plasma corticosterone

One week following the P70 behavioral tests, a random

sample of adult rats (n= 5–6) were restrained in a

plastic cone for 10 min, following a baseline tail blood

collection. At baseline, the rat’s tail was lanced close to

the tip and a microvette (CB 300 K2E; Sarstedt,

Germany) was used to collect tail blood. The microvette

was coated with EDTA to prevent coagulation of blood.

Tail blood samples were collected at 15, 30, 60, and

120 min following restraint. Samples were kept on ice,

centrifuged and plasma aliquots were stored at �75 �Cuntil processing. Blood sampling took place between

10:00 and 1:00 pm to limit potential circadian rhythm

effects. Plasma corticosterone levels were measured in

duplicate by ELISA according to the small sample assay

protocol of the standard testing kit (ADI-900-097, Enzo

Life Sciences, Farmingdale, NY, USA). The minimum

detectable concentration was 26.99 pg/ml, and the intra-

and inter-assay coefficients of variation were 6.6% and

7.8%, respectively.

Brain collection and analysis

On P40 and P90, 2 weeks following adult blood sampling,

rats were deeply anesthetized with a mixture of

Ketamine/Xylazine (40–80 mg/kg, i.p./5–10 mg/kg, i.p.)

and perfused intracardially with a phosphate-buffered

solution. Brains were quickly removed and placed over

ice. The prefrontal cortex and hippocampus were

immediately dissected, frozen on dry ice and stored at

�75 �C until processing.

Glutathione

Tissue concentrations of prefrontal cortex and

hippocampal glutathione were determined via a

fluorometric assay detection kit according to the

manufacturer’s instructions (ab65322, Abcam,

Cambridge MA, USA). Briefly, tissue was homogenized

458 M. MacRae et al. / Neuroscience 298 (2015) 455–466

and diluted 1:5. Samples were mixed with GST reagent

and monochlorobimane (supplied in kit) and incubated

at 37 �C for 1 h. The lower limit of detection was 1 nmol

and the assay to assay variability was within 5%.

Fluorescence was measured with excitation at 380 nm

and emission at 460 nm with a gain of 35.

Statistical analysis

All analyses were conducted using SPSS version 21.0

software. Passive maternal care behaviors (i.e.,

licking/grooming, nursing postures, total nursing

frequency, duration on nest) were evaluated using a

repeated measures design with ‘housing condition’ (SC,

EE) as the independent variable and observation day

(P3,P4,P5,P6) as the repeated variable. For the

remaining maternal behavior evaluations (i.e., overall

duration on nest, latency to start retrieval and open field)

one-way analysis of variances (ANOVAs) were employed

with housing condition as the independent factor. With

respect to neonatal treatment group comparisons (i.e.,

social behavior, object recognition test) we used three-

way ANOVAs with housing condition, ‘neonatal drug’

(nLPS, nsaline), and ‘sex’ (male, female) as the

independent factors. Plasma corticosterone, body weight,

and differences between young and adult glutathione

levels were similarly evaluated using a three-way

repeated measures ANOVA with time (corticosterone:

baseline, 15, 30, 60, and 120 min; body weight: P22–

P90; glutathione: P40 vs P90) as repeated factors. Area

under the curve for the corticosterone response was

evaluated using a three-way ANOVA, as above.

Greenhouse–Geisser corrections were applied to

violations to the assumption of sphericity. Significant

main effects were further assessed via pairwise t-tests

and Levene’s was applied in the occurrence of unequal

variances on the post hoc assessments. Bonferroni alpha

adjustments were applied as appropriate.

RESULTS

Maternal behavior

There were no significant housing, time, or housing by time

effects observed for the total number of pup retrievals,

licking/grooming, or nest building behaviors recorded

(p> 0.05; Fig. 2A shows total observations). Dams

reared in SC demonstrated more instances of low crouch

nursing (F(1,17) = 6.299, p= 0.023; Fig. 2B shows total

observations) however no other effects of nursing were

observed. There was a significant housing by time

interaction for the total duration of time spent on the nest

(F(3,51) = 3.069, p = 0.036; Fig. 2C). SC dams spent

more time on the nest on P5 (F(1,17) = 11.443,

p= 0.004) and P6 (1, 17) = 5.047, p= 0.038)

compared to EE dams. Overall there was no evidence of

passive maternal care variation as a function of neonatal

inflammation. With respect to the maternal preference

test, there was a significant three-way interaction

between housing condition, neonatal drug treatment, and

sex (F(1,44) = 4.618, p= 0.037; Fig. 2D). Follow-up

analyses with Bonferroni alpha corrections did not reveal

preferences for either sex or neonatal nLPS vs. nsaline

pups (p> 0.016). In the home cage retrieval task EE

dams had shorter latencies to begin (F(1,17) = 5.201,

p= 0.036; Fig. 2E) and fully (F(1,17) = 5.713,

p= 0.029; Fig. 2F) retrieve their litters compared to

standard housed rats. There were no neonatal treatment

differences in the latency of retrieval in the home cage

(p> 0.05; Fig. 2F). Based on the significantly decreased

latency for EE dams to retrieve their pups, we evaluated

maternal anxiety in an open field. There were no

differences in maternal housing conditions on percent of

entries into the center (p> 0.05; Fig. 2G) suggesting that

these animals were not anxious. Moreover, there were no

differences in body weights between nLPS and nsaline-

treated pups (p> 0.05) indicating that potential subtle

differences in maternal care did not affect this measure.

However, following application of the Greenhouse–

Geisser correction there was a significant interaction

between time and sex (F(2.736,150.453) = 114.753,

p= 0.0001) with male rats registering heavier than

females across time.

Social interaction

Juvenile social behavior was associated with a significant

neonatal treatment by housing interaction

(F(1,55) = 4.216, p= 0.045) in that nLPS-treated

animals spent a lower percentage of time in social

contact compared to their nsaline-treated standard

housed counterparts (t(29) = �2.084, p= 0.046;

Fig. 3A). There were no significant differences in either

the duration or frequency of initiating social engagement

(p> 0.05; Fig. 3B, C). However, there was significant

neonatal treatment by housing interactions with respect

to the duration (F(1,55) = 14.999, p= 0.0001) and

frequency (F(1,55) = 19.862, p= 0.0001) of social

behavior directed toward the rats by their conspecifics.

Specifically, SC nLPS-treated animals received lower

durations (t(29) = �2.600, p= 0.015; Fig. 3D) and

frequencies (t(29) = �3.063, p= 0.005; Fig. 3E) of

contacts directed toward them compared to SC-housed

nsaline rats. There were no differences between nLPS

and nsaline-treated rats housed in EE in terms of either

initiating or receiving contact (p> 0.05). Notably,

following Bonferroni alpha corrections, EE-housed

animals treated with nsaline had lower frequencies

(t(27) = �3.075, p= 0.005; Fig. 3E) but not durations

of contact initiated toward them compared to standard

housed controls.

A three-way ANOVA of adult social behavior showed

a main effect of drug on the frequency of contacts

initiated (F(1,56) = 4.735; p= 0.034). Follow-up tests

did not reveal any specific effects of drug on this

measure (p> 0.05, Fig. 3H). Total frequency receiving

contact revealed a significant interaction between sex

and housing condition (F(1,56) = 7.013, p= 0.010)

where male EE rats had fewer contacts initiated toward

them than standard housed rats (t(29) = �2.526,p= 0.017; Fig. 3J). There were no differences observed

on any other adult social measure (p> 0.05; Fig. 3F,

G, I). Therefore, nLPS rats show improvements in social

interactions across development.

Fig. 2. Maternal behavior. (A) Total frequency of pup retrieval, licking/grooming, and nest building across housing groups, (B) total frequency of

each nursing behavior (low, high, passive) and a combined score of all nursing behaviors, (C) total time (seconds) spent on the nest across all four

passive maternal observation days (P3, P4, P5, P6) and a combined total duration spent on the nest, (D)% pup preference in a novel context, (E)

latency to start pup retrieval and, (F) latency to retrieve the full litter in the home cage, (G) % of entries into the center of an open field (maternal

anxiety index). Rats were reared in either standard (SC) or environmentally enriched (EE) housing and offspring treated with neonatal (n)LPS or

nsaline; data are expressed as mean ± SEM; *p< 0.05, **p< 0.01.

M. MacRae et al. / Neuroscience 298 (2015) 455–466 459

Object-in-place

The three-way ANOVA for the juvenile discrimination ratio

revealed an effect of sex (F(1,58) = 4.786, p= 0.033)

but no effect of either neonatal treatment or housing

condition (p> 0.05; Fig. 4A). Post hoc follow up with

Levene’s confirmed a sex difference in that males had

higher discrimination ratios than females (t(56.373) =2.181, p= 0.033; Fig. 4A).

With respect to the adult discrimination ratio, a three-

way ANOVA uncovered an interaction between neonatal

treatment and sex (F(1,53) = 5.222, p= 0.026). Post

hoc follow up confirmed that adult male nLPS rats had

lower discrimination ratios compared to nsaline animals

(t(27) = �4.129, p= 0.0001; Fig. 4B). Overall, male

juvenile nLPS rats had an intact object-in-place memory

while their adult counterparts did not.

Plasma corticosterone response to a stressor

Evaluations of plasma corticosterone showed a significant

interaction between sex and time (F(1,39) = 16.594,

p= 0.0001) with females having significantly higher

levels of baseline (t(35.002) = �2.707, p= 0.010) and

Fig. 3. Social interaction data for male (left) and female (right) rats treated with neonatal (n)LPS or nsaline and reared in either standard (SC) or

environmentally enriched (EE) housing. Graphs illustrate (A, F) % of time in social contact, total (B, G) duration (seconds), and (C, H) frequency

initiating contact toward a novel conspecific, and total (D, I) duration (seconds) and (E, J) frequency receiving contact from a novel conspecific on

postnatal days 40 (top panel) and 70 (bottom panel), respectively. Mean ± SEM; ⁄p< 0.05, ⁄⁄p> 0.01.

460 M. MacRae et al. / Neuroscience 298 (2015) 455–466

Fig. 4. Object-in-place discrimination ratios for male (left) and female

(right) juvenile (top) and adult (bottom) rats following neonatal (n)LPS

or nsaline challenge. Animals were raised in standard (SC) or

environmentally enriched (EE) conditions. Mean ± SEM; *p< 0.05,**p> 0.01.

M. MacRae et al. / Neuroscience 298 (2015) 455–466 461

stress-induced corticosterone at 60 (t(30.921) = �4.607,p= 0.001) and 120 (t(39.689) = �2.547, p= 0.014)

minutes following restraint. There were no

consequences of neonatal treatment or housing

condition on either basal or stress-induced plasma

corticosterone. There was also a main effect of sex for

the area under the curve analysis with females having

higher levels than males (F(1,39) = 22.428, p= 0.0001).

Glutathione

ANOVA analysis for juvenile prefrontal cortex glutathione

level revealed a three-way interaction between neonatal

treatment, housing condition and sex (F(1,46) = 6.682,

p= 0.013). Male nLPS rats housed in standard

conditions had lower prefrontal glutathione levels than

nsaline-treated juveniles (t(12) = �2.406, p= 0.033,

Fig. 5A). There were no neonatal treatment differences

between juvenile males housed in EE (p> 0.05,

Fig. 5A) suggesting a protective effect of EE against

nLPS. Prefrontal glutathione level was higher in female

standard housed nLPS rats (t(12) = 4.010, p= 0.002,

Fig. 5A) and nsaline EE rats compared to nsaline SC

controls (t(8.965) = 2.863, p= 0.019, Fig. 5A), perhaps

indicative of a compensatory mechanism against

oxidative stress in females. There were no significant

neonatal treatment, housing or sex effects on adult

glutathione levels in the prefrontal cortex (p> 0.05,

Fig. 5B). With respect to juvenile hippocampal

glutathione levels, there was a two-way interaction

between housing condition and sex (F(1,46) = 11.759,

p= 0.001). Specifically, hippocampal glutathione levels

were augmented in female EE rats compared to their

SC counterparts (t(26) = 2.994, p= 0.006; Fig. 5C).

Adult hippocampal levels were significantly higher in

female rats compared to males (F(1,58) = 4.423,

p= 0.040); there was no additional neonatal treatment

or housing differences. Interestingly, a repeated

measures ANOVA (P40, P90) revealed that

hippocampal glutathione significantly increased across

all groups as animals matured (F(1,29) = 28.381;

p= 0.001; Fig. 5C, D), offering a potential explanation

for susceptibility to behavioral disruptions (i.e., social)

during the neonatal period following an inflammatory

triggering event.

DISCUSSION

The present work suggests that nLPS-induced

reprogramming is expressed in a time, sex, and

environmental context dependent manner. We

demonstrate that SC, but not EE, male and female rats

treated with a dual LPS protocol as neonates had

disrupted juvenile social interactions which were

remitted by maturity. In contrast, nLPS male animals

exhibited intact spatial memory as adolescents which

was impaired in later life. Moreover, SC nLPS males

had decreased prefrontal cortex levels of glutathione at

P40, which was normalized in adult animals. Overall,

these time-dependent effects provide additional

evidence that early-life inflammation interacts with other

developmental variables, specifically puberty

(Zuckerman et al., 2003; Meyer et al., 2006a) and EE,

in the expression (and prevention) of select behavioral

and molecular anomalies.

Considerations of environment

Since disruption of the nest and administration of LPS to

neonates may impact maternal care we evaluated several

behaviors to determine if dams treated their offspring

differently as a function of neonatal treatment and

housing. Although maternal licking and grooming (L/G)

is well recognized to have robust effects on offspring

development (Caldji et al., 1998; Kaffman and Meaney,

2007) we did not see changes on this measure in

response to our housing manipulation protocol. Instead,

we consistently show that EE dams spend less time on

the nest, which appears to differntially program later off-

spring anxiety-like behavior for novel vs. familiar environ-

ments (Connors et al., 2015). We also assessed whether

dams had preferences for particular offspring treatment

groups using maternal retrieval and preference tests. In

general, neonatal inflammation did not significantly depre-

ciate the value of pups by their mother. This suggests that

maternal care did not contribute to inflammatory-mediated

changes in the physiological and behavioral measures

evaluated here. However, we were surprised that there

was not a maternal preference for male vs. female pups,

as reported previously (Moore, 1985). Despite evidence

that MIA disrupts maternal care (Meyer et al., 2006b;

Penteado et al., 2014), in line with other observations

Fig. 5. Markers of oxidative stress in juvenile (left) and adult (right) offspring raised in standard (SC) or environmentally enriched (EE) housing

following neonatal (n)LPS or nsaline. Graphs depict glutathione levels expressed as nmol/mg in (A, B) prefrontal cortex and (C, D) hippocampus.*p< 0.05, **p> 0.01.

462 M. MacRae et al. / Neuroscience 298 (2015) 455–466

(Spencer et al., 2006; Bilbo et al., 2007), neonatal inflam-

mation in the present study did not alter dam behavior.

Interestingly, EE dams began retrieving their pups

sooner and gathered together the full litter more quickly

than standard housed rats. Based on the apparent

urgency of this responding we tested if they had

heightened levels of anxiety. Evaluations of the open

field test suggested that this was not the case. Previous

studies have shown that placement into EE in later life

reduces anxiety-like behavior in male rats (Pena et al.,

2009; Ravenelle et al., 2013), but we report no difference

compared to standard housed dams. Given the impact of

EE housing on maternal care, it is difficult to discern

between the contribution of these two variables, which

ought to be considered for all other measures of the study

when assessing the benefits of environmental

experience.

Our enriched environment appeared to offer some

protection against the early social deficits that followed

nLPS. Previously, EE has mitigated various animal

models of disease and trauma (Dahlqvist et al., 2004;

Spires et al., 2004; Johnson et al., 2013; Connors et al.,

2014), and increased social behavior (Morley-Fletcher

et al., 2003; Schneider et al., 2006) and preference direc-

ted toward animals reared in this condition (Mitra and

Sapolsky, 2012). Although EE seemed to prevent further

nLPS-induced reductions in social behavior, overall socia-

bility levels were lower in EE animals than we anticipated.

One major difference from past EE research is that our

animals received neonatal injections which may have

been interpreted as a stressor by the EE groups, affecting

behavior. Indeed, injection stress can impact experimen-

tal outcomes (Lapin, 1995; O’Callaghan et al., 2002)

and early-life stressors (i.e., maternal separation;

Morley-Fletcher et al., 2003; Holland et al., 2014) disrupt

social behavior.

Despite reported benefits of EE, particularly with

respect to cognition (van Praag et al., 2000), we did not

demonstrate protection against later-life spatial impair-

ments in nLPS males. Moreover, EE did not improve

the sustained inability of female rats to perform the

object-in-place discrimination task. Many EE studies are

confounded by the use of running wheels so the role of

physical activity vs. other elements of enrichment (i.e.,

social or novelty) cannot be clearly established.

Therefore we did not include running wheels in our

enriched housing. Given the benefits of hippocampal neu-

rogenesis as a consequence of wheel running (van Praag

et al., 1999), it is likely that the former environmental

manipulation would be more beneficial in the prevention

and rehabilitation of neuroinflammatory cognitive chal-

lenges, more so than enrichment alone. That said, EE

appeared to offer benefits against oxidative stress follow-

ing nLPS by preventing reduced glutathione levels in the

juvenile prefrontal cortex. An alternative interpretation is

that EE itself led to a reduction of glutathione in control

animals which may account for the lack of differences

between SC-nLPS and EE-nsaline juvenile males.

Given that our EE paradigm includes twice weekly toy

changes and varied social encounters it may be that our

animals interpreted these component(s) of their housing

as stressful. Indeed, chronic unpredictable mild stress is

known to reduce brain glutathione (Kumar et al., 2011).

However, there were no differences between SC-nsaline

and EE-nsaline/EE-nLPS glutathione levels in the juvenile

prefrontal cortex. Moreover, there were no indications of

M. MacRae et al. / Neuroscience 298 (2015) 455–466 463

dysregulation in resting plasma corticosterone or stress

recovery as a consequence of EE. Notably, others have

reported elevations in basal corticosterone and more effi-

cient stress recovery following 6 weeks of EE housing in

adulthood (Konkle et al., 2010). This is relevant because

our data suggest that enrichment protocols established

and maintained in early-life may not interfere with impor-

tant stress research endpoints. Indeed, evidence sug-

gests that environmental complexity may enhance

rather that diminish reproducibility in research, compared

to attempts at standardization across laboratories

(Richter et al., 2009).

Interestingly, juvenile EE females had elevated levels

of hippocampal glutathione compared to controls. This

effect was not sustained into adulthood, which may

confirm evidence that some EE benefits are more

enduring in males compared to females (Pena et al.,

2009). However, the observation that hippocampal glu-

tathione increased in both sexes across development

indicates that the effect was not lost in females but rather

protection from inflammatory-mediated oxidative stress

was conferred to both sexes across maturity.

Considerations of sex

Notably, lower social engagement following MIA in the

early-to-mid gestational period has been reported to

occur in a sex-specific manner with males but not

females being affected (Taylor et al., 2012; Connors

et al., 2014). However, in SC-housed rats, we demon-

strate reduced social contact in juveniles of both sexes

using the dual administration nLPS model. Autism, a neu-

rodevelopmental disorder associated with disrupted

social interactions, is reported four times more frequently

in males than females (CDC, 2014). Current hypotheses

posit that inflammation during early development may

be a factor involved in the etiology of this disorder

(Patterson, 2009). Given that in animal models the inutero effects are observed (or at least reported) most

often in males, it is possible that the underlying inflamma-

tory mechanism has a limited critical time period with

respect to its influence on females, accounting for the

lower incidence in this sex. Indeed, later MIA at P17

resulted in disrupted social interaction in both male and

female mice (Bitanihirewe et al., 2010) which we show

here using nLPs in rats. However, when relating the rele-

vance of these animal models to neurodevelopmental dis-

orders such as autism, further examination of sex

differences is warranted. This is evident given that

research and diagnostic practices often demonstrate a

sex bias toward males, potentially accounting for the

increased number of males reportedly affected (Lai

et al., 2015).

Prefrontal glutathione levels were higher in juvenile

female SC-nLPS rats compared to SC-nsaline controls

which may indicate a compensatory mechanism against

oxidative stress in females. It should be noted that

estrous cycles were not evaluated in these animals and

cyclic hormones could underlie some reported

differences. Additional work is necessary to determine

the role of reproductive hormones on glutathione-related

markers throughout the central and peripheral nervous

systems as findings have been inconsistent/difficult to

compare between humans and animal models (Browne

et al., 2008; Ozacmak and Sayan, 2009; Lee et al.,

2012; Priyanka et al., 2013; Mitra et al., 2015).

Considerations of time

Early-life inflammation is commonly associated with

spatial memory disruptions in a variety of behavioral

tasks (Dinel et al., 2014; Vorhees et al., 2015; Zhang

and van Praag, 2015). Moreover, studies demonstrate

the existence of ‘critical windows’ during which a chal-

lenge must take place in order for specific behavioral out-

comes to occur (Harre et al., 2008). Here, we confirm and

provide further evidence that these disruptions are also

mediated in a time-dependent manner with respect to

the trajectory of when they are expressed (i.e., adoles-

cence vs. adulthood). For example we show that juvenile

spatial memory is intact in male nLPS rats, but discrimina-

tion ability is compromised after maturity. Delays in cogni-

tive processes following early-life inflammation have been

reported previously. Specifically, MIA results in disrupted

latent inhibition in post-pubescent, but not juvenile, ani-

mals (Zuckerman et al., 2003; Meyer et al., 2006b).

Relatedly, Dinel et al. (2014) observed that P14 nLPS ele-

vated anxiety-like behavior in juvenile but not mature

mice, while the opposite time course was seen for

depressive-like symptoms in the forced swim test. We

extend upon these observations by reporting differential

expression of social disruptions between the juvenile

and adult periods of development. Furthermore, the pat-

tern of these social impairments was consistent with indi-

cators of oxidative stress in the prefrontal cortex of male

nLPS rats.

Our previous work suggests that overall decreased

social interaction following early-life inflammation is due

to reduced contact directed toward gestational and

neonatally treated LPS male rats (Connors et al., 2014;

MacRae et al., 2015). We show this here, but in both male

and female neonatally challenged animals. Notably, gen-

eral willingness to engage with a novel conspecific was

not disrupted, corresponding to previous reports (Ibi

et al., 2009). Specifically social behaviors initiated by

adult mice were not decreased following neonatal inflam-

mation – unless animals were repeatedly paired with the

same intruder, in which case they habituated more quickly

than controls (Ibi et al., 2009). However, the investigators

did not report the amount of contact directed toward the

neonatally treated animals. Still, these findings are in par-

allel with our data suggesting that (a) early-life inflamma-

tion may not necessarily decrease social interest directly

and/or (b) social impairments following neonatal exposure

are not as explicit in adulthood vs. the juvenile period.

Overall, this is interesting given that autistic individuals

are more likely to be rejected or overlooked by peers

and many express a desire for social interaction (Dean

et al., 2014). Moreover, some individuals reportedly show

improved emotional responsiveness, social reciprocity,

and socialization with age (McGovern and Sigman,

2005; Shattuck et al., 2007).

Together, these data consistently suggest a

programed timing in the emergence of impairments

464 M. MacRae et al. / Neuroscience 298 (2015) 455–466

following early-life inflammation in that (1) some

emotional disruptions are apparent by adolescence and

disappear post puberty while (2) maturity results in the

gradual unfolding of cognitive detriments. Since our

animals were housed under the same conditions (EE or

SC) between each juvenile and adult test point, it is

possible that puberty may be the contributing factor to

these outcomes. Future work must delineate the specific

hormones and other regulators underlying the trajectory

of affective, cognitive and associated molecular changes

in each specific inflammatory model (pre vs. postnatal).

CONCLUSIONS

With respect to early-life inflammation rarely do studies

simultaneously evaluate both juvenile and adult phases

of development, and very few investigate adolescent

development at all. Here, we demonstrate that nLPS-

induced reprogramming effects follow a sex and time-

dependent trajectory in terms of their expression.

Overall, this work extends upon the literature confirming

that early-life stress interacts with developmental

milestones (i.e., puberty) and the environment in the

expression (and prevention) of select behavioral and

molecular programs.

AUTHOR CONTRIBUTIONS

M.M., T.M., A.K., M.M.M., ran the experiments, M.M.M. &

A.C.K. analyzed data, M.M.M. & A.C.K. designed the

study and A.C.K. wrote the manuscript.

DISCLOSURES AND POTENTIAL CONFLICT OFINTERESTS

None.

Acknowledgments—We are grateful for the departmental support

provided by MCPHS University, the MCPHS Summer

Undergraduate Research Fellowship (SURF) awarded to M.M

and the Summer Healthcare Internship Program Grant awarded

to T.M.

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(Accepted 21 April 2015)(Available online 29 April 2015)