Brain Struct Funct DOI 10.1007/s00429-015-1154-0

ORIGINAL ARTICLE

Exposure to a mildly aversive early life experience leads to prefrontal cortex deficits in the rat Antonios Stamatakis1 • Vasileios Manatos1 • Theodora Kalpachidou1 Fotini Stylianopoulou1

•

Received: 10 July 2015 / Accepted: 19 November 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Aversive early life experiences in humans have been shown to result in deficits in the function of the prefrontal cortex (PFC). In an effort to elucidate possible neurobiological mechanisms involved, we investigated in rats, the effects of a mildly aversive early experience on PFC structure and function. The early experience involved exposure of rat pups during postnatal days (PND) 10–13 to a T-maze in which they search for their mother, but upon finding her are prohibited contact with her, thus being denied the expected reward (DER). We found that the DER experience resulted in adulthood in impaired PFC function, as assessed by two PFC-dependent behavioral tests [attention set-shifting task (ASST) and fear extinction]. In the ASST, DER animals showed deficits specifically in the intra-dimensional reversal shifts and a lower activation—as determined by c-Fos immunohistochemistry—of the medial orbital cortex (MO), a PFC subregion involved in this aspect of the task. Furthermore, the DER experience resulted in decreased glutamatergic neuron and dendritic spine density in the MO and infralimbic cortex (IL) in the adult brain. The decreased neuronal density was detected as early as PND12 and was accompanied by increased microand astroglia-density in the MO/IL.

Abbreviations ANOVA Analysis of variance ASST Attention set-shifting task CTR Control animals DAB 3,30 -Diaminobenzidine DER Animals denied the expected reward GAD67 Glutamate decarboxylase, MW 67 kDa GFAP Glial fibrillary acidic protein Iba-1 Ionized calcium-binding adapter molecule 1 IL Infralimbic cortex MO Medial orbital cortex NDS Normal donkey serum NGS Normal goat serum PBS Phosphate-buffered saline PFC Prefrontal cortex PND Postnatal day PrL Prelimbic cortex RER Animals receiving the expected reward roCg1 Rostral part of area 1 of cingulate cortex roVLO Rostral part of ventral lateral orbital cortex SEM Standard error of the mean

Introduction Keywords Behavioral flexibility  Neonatal experience  NeuN  Microglia  PFC  Dendritic spines

& Antonios Stamatakis [email protected] 1

Biology-Biochemistry Lab, School of Health Sciences, National and Kapodistrian University of Athens, Papadiamantopoulou 123, 11527 Athens, Greece

Aversive early life experiences in humans have been shown to result in deficits in the function of the prefrontal cortex (PFC). More specifically, neglect, abuse/maltreatment, deprivation of primary caregiver-institutionalization, or growing up in a low socioeconomic class have been associated with defective attentional processes, and reduced behavioral control leading to increased aggression and inability to regulate negative emotions (Noble et al. 2005; Hackman and Farah 2009; Kishiyama et al. 2009; Tomalski and Johnson 2010; Pechtel and Pizzagalli 2011;

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van Harmelen et al. 2014). In addition, neuronal correlates of these behavioral deficits have been described showing structural abnormalities, such as, decreased gray and white matter volume, particularly in the medial PFC, and reduced interhemispheric connections (Tomalski and Johnson 2010; van Harmelen et al. 2010). In our laboratory, we have developed a model of two early experiences in rats in which pups from postnatal day PND10 till PND13 learn to seek for their mother placed at the end of one arm of a T-maze; upon finding her, one group of pups is allowed to be retrieved by her (receiving the expected reward: RER), while the other is denied the maternal contact (denied the expected reward: DER) (Panagiotaropoulos et al. 2009). The DER experience is of a mild adversity (Diamantopoulou et al. 2013), since pups are prohibited from entering the mother-containing cage in spite of having found and approached it. During the time the DER pups remain in front of the entrance of the maternal cage, they receive olfactory cues from the mother, but are deprived of her tactile stimulation: The mother is present but unavailable to the pup. We have thus proposed that, the DER experience could serve as a model of neglect (Stamatakis et al. 2013). Interestingly, the pups exposed to the DER experience show higher activation of their PFC compared to the controls or those exposed to the RER experience (Panagiotaropoulos et al. 2009). As adults, the male DER animals have reduced levels of serotonin (Diamantopoulou et al. 2012) and dopamine (Raftogianni et al. 2014) in their PFC, indicative of hypofrontality. In addition, these animals show increased aggression (Diamantopoulou et al. 2012), which is known to be associated with low PFC serotonin levels both in rodents (Veenema 2009) and in humans (Siever 2008). Similarly, low PFC dopaminergic activity has been linked to behavioral inflexibility (Floresco et al. 2006; Winter et al. 2009; Logue and Gould 2014). In contrast to the DER, the RER animals did not differ from the controls in any of the above mentioned parameters. Based on the above, we proceeded to study the effect of the DER experience on the PFC at both the cellular and behavioral level. Our experimental hypothesis was that the DER animals would exhibit deficits in PFC structure and function. We thus determined the behavior of the adult male DER animals in a PFC-dependent test, the attention set-shifting task (ASST), which is equivalent to the Wisconsin card sorting test used in humans. (Birrell and Brown 2000), to document impairments in PFC function resulting as a consequence of the DER early experience. Furthermore, we investigated whether this early experience also affected PFC structure i.e., neuronal and spine density, since it has been shown that aversive early life experiences i.e., maternal deprivation and

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prenatal stress, negatively affect neuronal and spine density (Bock et al. 2005; Aksic´ et al. 2013; Chocyk et al. 2013), as well as absolute number of neurons (Mychasiuk et al. 2012). We also determined immunohistochemically the number of GABAergic (GAD67?) and glutamatergic (vGluT?) cells, based on the prevalent idea that an imbalance in inhibitory and excitatory neurotransmission often underlies deficits in PFC function (Bielau et al. 2007; Kehrer et al. 2008; Garey 2010; Laruelle 2014, Tebartz van Elst et al. 2014). Since the cellular analyses revealed that the DER experience affected in addition to the medial orbital cortex, also the infralimbic cortex, we tested the DER animals in the fear extinction test which is dependent on this latter PFC area (Barad 2005). In order to investigate the temporal relation between the DER early experience and the cellular deficits in the PFC detected in adulthood, we determined the neuronal and glutamatergic cell density in the PFC on postnatal days 11–13, i.e., during the period of T-maze training. We also determined the density of microglia (Iba-1?) and astroglia (GFAP?), since it has been reported that aversive early life experiences result in alterations of these markers of neuroinflammation (Barros et al. 2006; Llorente et al. 2009; Diz-Chaves et al. 2012; Marco et al. 2013; S´lusarczyk et al. 2015; Zhao et al. 2015), which in turn has been linked to psychopathology (for a review see Re´us et al. 2015).

Materials and methods Animals Wistar rats bred and grown in our colony were used. Animals lived under standard conditions (23 ± 1 °C, 12:12 h light/dark cycle) with food (Kounker-Keramari Bros. & Co., Athens, Greece) and water ad libitum. Prior to birth (postnatal day 0, PND0), each litter was randomly assigned to either of the two experimental groups [pups denied (DER) or receiving (RER) the expected reward, see ‘‘Training in the T-maze’’ for T-maze training], or to the control group (CTR). Throughout the lactation period, fresh wood chip was added to the cage every 4–5 days, without disturbing the litter. On PND22, animals were weaned and housed in same sex, same group (DER, RER, CTR) of 3–4 animals per cage. Following weaning, cages were cleaned weekly by the experimenters with minimal disturbance of the animals. Overall, four different cohorts of animals were used for different experiments: One cohort (#1) of animals was used for the attention set-shifting task (ASST), as well as c-Fos immunohistochemistry, following ASST (seven litters for each group). In cohort #1, from each litter two adult males

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were used for the ASST and one (randomly selected) of these two animals per litter was also used for c-Fos immunolabeling. Adult males from cohorts #1 (not exposed to the ASST) and from a second cohort (#2) were used for NeuN, vGluT, GAD67, Iba-1 or GFAP immunolabeling (one animal from each of 6 different litters per group was used for all neurochemical determinations) or for Golgi-Cox staining for the determination of spine density (one animal from each of six different litters per group). Different animals from the same litter were used for the neurochemical or the morphological experiments. A third cohort (#3) of animals was used for the behavioral experiments of fear extinction (six litters per group). From each litter two adult males were used for fear extinction. A fourth cohort (#4) of animals was used for NeuN, vGluT, GAD67, Iba-1 or GFAP immunolabeling on PND11, PND12, and PND13. For each developmental stage, the same animals were used for all immunolabelings (for each developmental stage, n = 6 for each group; six litters employed per group). All animal experiments were carried out in agreement with ethical recommendation of the European Communities Council Directive of 22 September 2010 (2010/63/ EU). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Training in the T-maze All animals of a litter were exposed either to the RER or DER experience or were used as controls. Animals of the control group were left undisturbed with their mother throughout the lactation period. Training in the T-maze was performed as previously described (Panagiotaropoulos et al. 2009) during PND10–13; briefly, a custom-made T-maze was used, with one arm leading through a small sliding mesh-wire door (9 cm 9 11 cm) to the mothercontaining cage. For control reasons, at the end of the other arm of the T-maze a cage with a virgin female rat was present, albeit with no access from the T-maze. When pups were trained under receipt of expected reward (RER) the door of the mother-containing cage opened and permitted access to the mother; alternatively, it remained always closed, blocking access to the mother, when pups were trained under the condition of denial of expected reward (DER). Each pup of either group (RER, DER) was trained for ten trials, each trial of maximum 60 s duration, per training day. If a pup did not succeed to reach the entrance of the mother-containing cage before the end of the 60 s, it was gently guided to the entrance and either was allowed contact with its mother (RER group) or had to remain in front of the closed door for 20 s (DER group), before the next trial started. At the end of the ten training trials the

pups were returned to their home cage, containing the mother and the whole litter. Behavioral testing in adulthood Attention set-shifting task (ASST) (Table 1) The protocol employed was based on those described previously by Birrell and Brown (2000), Black et al. (2006) and Gregg et al. (2009). Briefly, the testing arena was a non-transparent Plexiglas box (60 cm width, 60 cm length, 60 cm height), divided by an opaque wooden board into two equal compartments: One ‘‘waiting’’ compartment and one testing compartment. In the testing compartment two white porcelain pots (10 cm diameter, 5 cm height) were placed; the position of the pots relative to extra-arena landmarks, as well as the position of the two pots relative to each other was randomly changed between successive trials (see below). Each pot was filled with solid paraffin bits up to a height of *12 mm covered by a thin layer of Frosted Cheerios and on top of these a mesh screen was placed, thus making Cheerios inaccessible. Over the mesh screen, a different digging medium was placed and the ‘‘rewarding’’ pot contained a buried, yet accessible, Frosted Cheerio. For odor trials, both the pots and the digging material were scented using perfume oil. During each phase of the ASST, unique exemplars have been used. On PND49 (after 3 days of food restriction) rats habituated to the testing arena and started their training to dig in the pots for food reward. Digging training lasted for 5 days with a max duration of 30 min/day. Training was terminated either when 30 min had passed or if the rat made ten correct digs, i.e., if the animal displaced the digging material with either its paw or its nose. About 10–15 % of animals in all three groups (CTR, DER, RER) did not manage to learn to dig the covering material in the pots and were excluded from further analysis. If an animal failed to learn the digging task, all the animals of the litter were excluded both from the behavioral test and the c-Fos determination. On PND54, animals were trained to dig for the food chips under Eppendorf tube cups and to ignore Eppendorf tube bottoms [for a list of the stimuli (exemplars) used in all days and phases of the ASST please refer to Table 1]. On PND55, a simple discrimination test was performed, as on PND54, with the type of digging medium being the food predictor. Once an animal had reached criterion (six consecutive correct trials), it was exposed to a compound discrimination task by applying odor as the second sensory dimension. For the compound discrimination, both pots (rewarded and non-rewarded) had the same odor and animals had to learn to ignore odor (not being a predictor of reward). Once the animal had learned which types of

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Brain Struct Funct Table 1 Order of discriminations and stimuli combinations used in the ASST Day

Discriminations

Dimensions

Stimuli (exemplar) combinations

Relevant

Irrelevant

Positive predictor

Negative predictor

Irrelevant

PND54

Simple rule acquisition

Digging medium

none

Eppendorf tube cups

Eppendorf tube bottoms

None

PND55

Simple rule acquisition

Digging medium

none

Small size white glass tubes

Large size white glass bits

None

Compound rule acquisition

Digging medium

Odor

Small size white glass tubes

Large size white glass bits

Apple-cinnamon

Intra-dimensional shift

Digging medium

Odor

Small size white glass tubes

Large size white glass bits

Papaya

Intra-dimensional reversal shift

Digging medium

Odor

Large size white glass bits

Small size white glass tubes

Papaya

Compound Rule acquisition

Digging medium

Odor

Small size black plastic marbles

Large size black plastic marbles

Rose

Intra-dimensional shift

Digging medium

Odor

Small size black plastic marbles

Large size black plastic marbles

Mint

Intra-dimensional reversal shift

Digging medium

Odor

Large size black plastic marbles

Small size black plastic marbles

Mint

Extra-dimensional shift

Odor

Digging medium

Almond

Orange

Aluminum foil rapt into large balls with irregular surface

Second intradimensional shift

Odor

Digging medium

Almond

Orange

Aluminum foil rapt into small balls with irregular surface

Second intradimensional reversal shift

Odor

Digging medium

Orange

Almond

Aluminum foil rapt into small balls with irregular surface

PND56

ASST attention set-shifting task, PND postnatal day

stimuli were relevant as food predictors and which were not, an intra-dimensional set-shifting took place using a different odor (non-predictor). After completion of this phase, a reversal session was performed (intra-dimensional reversal shift), in which the previously non-reinforced digging medium now covered the food reward. Still, odor was irrelevant as a predictor of food presence. On PND56, again animals were exposed to a compound discrimination, an intra-dimensional set shift and an intradimensional reversal shift, using novel digging materials and odors, with the type of digging material being again the predictor of reward. After an intra-dimensional reversal shift trial, each animal was exposed to an extra-dimensional shift, with a novel set of odors and digging materials, but this time odor predicted reward while digging material was irrelevant, i.e., both pots contained the same digging material but each one had a different odor. Once the animals had reached criterion, an intra-dimensional set-shifting (second intra-dimensional rule shifting) took place using a different type of digging material (non-predictor). Then, a second intra-dimensional rule reversal session was performed during which the previously non-reinforced odor was now associated with the food reward. It should be noted that for an animal to reach criterion it had to make

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six consecutive correct choices. All animals were killed 60 min after the end of ASST and one animal per litter was used for c-Fos immunohistochemistry [marker of neuronal activity (see ‘‘Immunohistochemistry for c-Fos’’)]. Fear extinction We used the protocol utilized previously by Toth et al. (2012) with minor modifications. Briefly, fear conditioning was performed as follows: Each animal was placed in the conditioning box (black box, dimensions 25 9 25 9 27 cm, Pansystems) for a habituation period of 3 min. Following habituation, each animal was exposed to five conditioned stimulus-unconditioned stimulus pairings with a 70-s inter-trial interval. The conditioned stimulus (a tone, 5 kHz, 76 dB) was presented for 10 s and co-terminated with a footshock- unconditioned stimulus (0.5 mA, 1 s). Animals remained in the conditioning box for an additional 2 min and then they were returned to their home cages. One day after fear conditioning, each animal was placed in a modified conditioning box: a white cardboard covered the grid metal bars on the floor in the box. In addition, the box walls were also covered with white cardboard cut-outs of various geometrical shapes. Moreover, in order to

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modify the olfactory cues of the testing room, below the floor of the test box, a sponge with rose fragrance was placed. After 3 min habituation, each animal was exposed to 30 conditioned stimulus presentations without the delivery of unconditioned stimulus (each time, tone alone for 10 s), with an inter-trial interval of 70 s. The percent of time each animal spent freezing during the presentation of each of the 30 tone presentations was measured. The freezing behavior of each rat during the 30 conditioned stimulus presentations was collapsed into ten blocks, with the average % freezing time of each block being the average value of three consecutive conditioned stimulus presentations. Tissue preparation Animals used for immunohistochemistry (on PND11, 12, and 13, 2 h after the end of the last training trial or in adulthood) were deeply anesthetized, decapitated and brains were isolated, and flash-frozen in 40 °C isopentane. Sections of 20 lm in thickness were cut on a freezing cryostat (Leica CM1900, Nussloch, Germany) at 15 °C, collected on silane-coated slides and stored at 80 °C. Immunolabelings Immunofluorescence for NeuN, Iba-1, and GFAP Sections were thawed, post-fixed in ice-cold 4 % paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 1 h, and washed in phosphate-buffered saline (PBS) (3 9 5 min) and in PBS containing 0.4 % Triton X-100 (for NeuN or GFAP) or 0.1 % Triton X-100 (for Iba-1) (3 9 5 min). Non-specific staining was blocked by incubating for 1 h at room temperature in PBS containing either 0.4 % Triton X-100 and 10 % normal donkey serum (NDS) for NeuN and GFAP, or 0.01 % Triton X-100, 10 % NDS and 1 % bovine serum albumin for Iba-1. Afterwards, sections were incubated at 4 °C with a mouse monoclonal anti-NeuN antibody (72 h, 1:200, MAB377, Millipore, USA; diluted in PBS 0.4 % Triton X-100 and 4 % NDS), or a rabbit anti-Iba-1 antibody (72 h, 1:500, WEK6254, Wako, Japan; diluted in PBS 0.01 % Triton, 1 % NDS, and 1 % bovine serum albumin) or a rabbit anti-GFAP antibody (24 h, 1:500, Z0334, DakoCytomation, Glostrup, Denmak; diluted in PBS 0.4 % Triton X-100 and 4 % NDS). Following incubation with the primary antibody, slides were washed in PBS (3 9 5 min) and incubated for 2 h at room temperature with either an Alexafluor555-conjugated donkey anti-mouse IgG (A31570, Invitrogen, Oregon, USA), or an Alexafluor488-conjugated donkey anti-rabbit IgG (A21206) secondary antibody (diluted 1:200 in PBS containing 2 % NDS). Following incubation with the

secondary antibody, sections were washed (3 9 5 min) in PBS and coverslipped using Mowiol mounting medium. In each assay brain sections from all groups of animals were processed concurrently. No staining was observed on sections incubated without the primary antibody. Observation and analysis of the stained sections was performed under an epifluorescent microscope (Eclipse E400, Nikon, Japan). Immunohistochemistry for NeuN, vGluT and GAD67 Sections were thawed, post-fixed in ice-cold 4 % paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 1 h, and washed in phosphate-buffered saline (PBS) (3 9 5 min) and in PBS containing 0.4 % Triton X-100 (3 9 5 min). Non-specific staining was blocked by incubation in PBS containing either 0.4 or 0.3 % Triton X-100 (for vGluT or GAD67 immunohistochemistry, respectively) and 10 % normal donkey serum (NDS) for 1 h at room temperature. Afterward, sections were incubated for 72 h at 4 °C with either the goat polyclonal anti-vGluT (1:1000, AB1520, Millipore, USA) or the mouse monoclonal anti-GAD67 (1:750, clone 1G10.2, Millipore, USA). Antibodies were diluted in PBS containing either 0.4 or 0.3 % Triton X-100 (for vGluT and GAD67 immunohistochemistry, respectively) and 4 % NDS. Following incubation with the primary antibody, slides were washed in PBS (3 9 5 min) and incubated for 2 h at room temperature with a biotinylated donkey anti-goat (AP180B, Millipore) or anti-mouse (AP192B, Millipore) secondary antibody (dilution 1:200 in PBS with 2 % NDS). After five rinses in PBS, slides were incubated with the ABC reagent (Vectastain Elite, PK6100) for 60 min at room temperature. Slides were then washed in PBS (3 9 5 min) and stained with 3,30 -diaminobenzidine [DAB (1.7 mM, Sigma-Aldrich, USA)] diluted in Tris-HCl buffer (10 mM, pH7.6) and 0.03 % H2O2 for 2–5 min at room temperature. Finally, they were washed, dehydrated, and coverslipped with DePex (SERVA, Germany) and analyzed under a brightfield microscope (Eclipse E400, Nikon, Japan). In each assay brain sections from all groups of animals were processed concurrently. No staining was observed on sections incubated without the primary antibody. Immunohistochemistry for c-Fos Sections were thawed, post-fixed with ice-cold 4 % paraformaldehyde in 0.1 M phosphate buffer for 1 h and incubated in 1 % H2O2 for 4–5 min. Then, sections were washed in PBS (3 9 5 min) and incubated in blocking solution, containing 0.2 % Triton X-100, 2 % bovine serum albumin, and 0.5 % normal goat serum (NGS) in PBS for 1 h at room temperature. Sections were then

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incubated overnight at 4 °C with the rabbit polyclonal antic-Fos antibody (sc:52, Santa Cruz, USA) diluted 1:2000 in blocking solution. Following primary antibody incubation, slides were processed as described above for vGluT and GAD67 immunohistochemistry using the appropriate secondary biotinylated donkey anti-rabbit antibody (AP132B, Millipore, dilution 1:200 in blocking solution). In each assay, brain sections from all groups of animals were processed concurrently. No staining was observed on sections incubated either without the primary antibody, or with primary antibody preincubated with tenfold excess of the blocking peptide (sc-52 P, Santa Cruz, USA). c-Fos quantification has been performed as described below (‘‘Cell counting’’). Image analysis Cell counting For all immunostainings, images were recorded in digital format using the Infinite Capture v.6.0 (Lumenera Co., Ottawa, Canada) software. For adult animals, cell counting was performed for NeuN, vGluT, GAD67, and Iba-1 in five prefrontal cortex areas: rostral part of area 1 of the cingulate cortex (roCg1), prelimbic cortex (PrL), rostral part of ventral lateral orbital cortex (roVLO), medial orbital cortex (MO), and infralimbic cortex (IL), while for c-Fos in roCg1, PrL, roVLO, and MO. For PND11, 12 and 13 animals cell counting was performed for NeuN, vGluT, and Iba-1 in an area corresponding to the adult MO/IL areas. Cell counting was performed ‘‘blindly’’ by two independent investigators. A ‘‘threshold’’ was set in the image analysis software (ImageJ v.1.46R, NIH, USA) based on the background, non-specific staining, and only cells above this limit were included in the quantification. For each cortical area analyzed in adulthood, systematic randomly selected sections (seven for MO and roVLO, eight for IL and, ten for PrL and roCg1) were evaluated within the following anatomical borders, according to the anatomical atlas of Paxinos and Wattson (2007): for roVLO and MO from bregma 5.64 to 4.20; for PrL from bregma 5.16 to 2.52; for roCg1from bregma 4.20 to 2.52; IL from bregma 3.72 to 2.52. It should be mentioned that the anatomical borders of the VLO have been determined according to Corwin et al. (1994) and Van De Werd and Uylings (2008). For the MO/IL area analyzed in the rat pups, five systematic randomly selected sections were evaluated from plate 97 until plate 100, according to the anatomical atlas of Ramachandra and Subramanian (2011). In each section, immunolabeled cells were counted in two randomly selected, non-overlapping optical fields of 20,660 lm2 area for NeuN, vGluT, and GAD67 immunohistochemistry and of 41,320 lm2 for c-Fos immunolabeling. For each animal,

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the density of positive cells was determined dividing the number of immunopositive profiles by the area of the measuring frame and the average value (number of immunopositive cells/lm2) was calculated from the data from all optical fields in all brain sections analyzed. Composite photomicrographs were produced with the Adobe Photoshop CS2 (Adobe Systems, USA). GFAP immunoreactivity levels The determination of GFAP? astrocytic processes density was performed in the same cortical areas of pups and adult animals as described above (‘‘Cell counting’’). For each area analyzed, five systematic randomly selected sections were evaluated. In each section, two randomly selected non-overlapping optical fields of 20,660 lm2 area were evaluated. For each section, we determined the percent of total area covered by GFAP? processes according to the following procedure: Using the ImageJ image analysis software, the background staining was removed (‘‘rolling ball’’ algorithm with a radius of 1 pixel), then an automatic threshold was applied, so that only processes above the background level were included, and the area covered by these processes was calculated as the percent of the total area of the optical field analyzed. For each brain area of an animal, the density of GFAP? processes was calculated from the data from all optical fields in all brain sections analyzed. Composite photomicrographs were produced with the Adobe Photoshop CS2 (Adobe Systems). Volume of brain areas The total volume of the adult PFC, MO, and IL as well as of the PND13 PFC and MO/IL have been determined using the ImageJ image analysis software. Areas of interest have been outlined in systematically randomly selected sections: for adult animals from bregma 5.64 to 2.52 for the PFC and from bregma 5.64 to 4.20 for MO; for PND13 animals from plate 97 until plate 100 for PFC and MO/IL. The values obtained were summed and multiplied by the section thickness (20 lm) and the spacing of the sections used. The final volumes were expressed as % of the mean volumes of controls. Morphology of NeuN? and vGluT? cells The following morphological characteristics of NeuN? and vGluT? cells have been determined in adult MO and IL, as well as inPND13 MO/IL using the ImageJ software: cross sectional area of cellular profiles, Circularity [4p 9 (Area/ Perimeter2)], and Aspect Ratio (Major Axis/Minor Axis). Area is expressed as % of control value.

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Golgi-Cox staining Adult male animals (PND55-60) were used for Golgi staining with the FD Rapid GolgiStain kit (FD NeuroTechnologies, Ellicott City, MD, USA), according to the manufacturers’ instructions. Briefly, brains were isolated from animals under deep anesthesia, rinsed in ddH2O, incubated in the impregnation solution for 3 weeks, cryoprotected, frozen, and cut into coronal 40 lm sections on a cryostat (Leica CM1900, Nussloch, Germany) at -28 °C. Brain sections were collected on gelatin-coated slides, stained according to the instructions of the kit, dehydrated in vacuum, and mounted with Depex. For dendritic spine density quantification, sections were randomly chosen according to the anatomical atlas of Paxinos and Wattson (2007): for roCg1 from bregma 4.20 to 2.52; for MO from bregma 5.64 to 4.20; IL from bregma 3.72 to 2.52. Three to four images covering the whole thickness of each individual dendrite were obtained using a 1009 oil immersion objective on a bright-field microscope (Eclipse E400), a digital CCD color video-camera and the Infinite Capture v.6.0 (Lumenera Co.) software. Images from the same dendrite were superimposed using Adobe Photoshop CS2 (Adobe Systems) and spine density was estimated by quantifying at least 1700 lm fragments of individual randomly selected dendrites. In order to avoid any confounds of decreased spine density occurring in proximal dendrites, a region at least 30 lm from the soma was used. At least five well-defined apical dendrites in the deep layers of the cortex were used per section and for each animal a total of at least 25 dendrites were analyzed. Also, the length of the apical dendrites has been measured using the ImageJ software and has been expressed as % of the values of controls. Quantification was performed blindly by two independent experimenters and the average spine density values (number of spines/lm) per animal were calculated. Composite photomicrographs were produced with the Adobe Photoshop CS2 (Adobe Systems). Statistical analysis For all immunolabeling experiments and ASST, values for the DER and RER groups were expressed as percent of the respective values of the control group. For the behavioral tests in adulthood, two animals from each litter employed were used. For the two littermates, the average value was calculated for each behavioral parameter determined and these means served as single data points in the subsequent statistical analyses. Data from the fear extinction test were analyzed by repeated measures one-way ANOVA with successive trials as the repeated measures and group of animals (CTR, DER, RER) as the independent factor. Data from immunolabeling experiments in rat pups have been

analyzed by two-way ANOVAs with the group of animals (CTR, DER, RER) and the developmental stage (PND11, 12 or 13) as the independent factors. Data from all other experiments were analyzed by one-way ANOVAs with the group of animals (CTR, DER, RER) as the independent factor. The LSD test for post hoc analysis was used when appropriate. The level of statistical significance was set at 0.05. All tests were performed with the SPSS software (Release 22, SPSS, USA).

Results Adult animals Behavioral deficits in the prefrontal cortex-dependent task of attention set-shifting (ASST) (Fig. 1) In an effort to test the function of the prefrontal cortex (PFC) of the DER animals, we employed the attention set-shifting task, in which animals have to associate the presence of hidden food with visual, tactile or olfactory characteristics of hiding material. Moreover, animals have to distinguish relevant food-predicting cues (e.g., different types of hiding material texture) from irrelevant, distracting ones (e.g., smell of hiding material) and once they acquire the rules for food-presence prediction (one type of texture over another), the rule is shifted ‘‘within the dimension’’, to a different combination of relevant and irrelevant cues (intra-dimensional shift). The intra-dimensional shift is followed by a rule reversal ‘‘within the dimension’’, i.e.,within texture (intra-dimensional reversal shift). Then, a different type of rule shifting takes place, in which the formerly irrelevant cue (i.e., smell of hiding material) becomes the food predictor and the formerly relevant cue (i.e., texture) is now the distractor (extra-dimensional shift). Once the new rule is acquired, a second intra-dimensional shift and then a second intra-dimensional reversal shift takes place (reversal of the type of food-predicting odor). In this, rather complex, behavioral task, all three groups of animals acquired the initial rule, as well as the intra- and the extra-dimensionally shifted rule with equal efficacy (Fig. 1a). Nevertheless, during intra-dimensional reversal shifts DER animals made more mistakes, either by keeping on following the preceding rule, or reverting to it after making some correct choices based on the reversed rule (one-way ANOVA with the group of animals as the independent factor for the first intra-dimensional reversal shift (texture): F2,20 = 5.577, p = 0.013; post hoc test, *DER vs. CTR p = 0.004, #DER vs. RER p = 0.050; for the second intra-dimensional reversal shift (odor): F2,20 = 6.131, p = 0.009; post hoc test,

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*DER vs. CTR p = 0.008, #DER vs. RER p = 0.006; Fig. 1a). The behavior of DER animals in this test, suggested a specific deficit in intra-dimensional reversal

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shifts, where the dimension of the predicting cues remains the same but the specific type of cue is modified. On the contrary, the ability of DER animals to

Brain Struct Funct b Fig. 1 Effect of the DER experience on PFC function as determined

by the Attention Set-Shifting Task (ASST). Behavioral performance in the task (a). Adult DER males were less efficient in making intradimensional rule reversals (*p \ 0.05 DER vs. CTR, #p \ 0.05 DER vs. RER), while they showed no deficits in acquisition of the rule, the intra-dimensional or the extra-dimensional rule shifting. Bars represent means and SEM, n = 7 per group (the average value of two animals from each of seven litters was used in the statistical analysis). Activation of PFC subregions as determined by c-Fos immunohistochemistry. Representative photomicrographs of c-Fos immunostaining (b) and quantification of the results (means ± SEM), n = 7 per group (c). DER animals showed lower activation of the MO, the subregion of PFC involved in the intra-dimensional rule reversal, compared to the CTR and the RER (b, c) (*p \ 0.05 DER vs. CTR, # p \ 0.05 DER vs. RER). Scale bar in b corresponds to 50 lm. roCg1 rostral part of area 1 of cingulate cortex, CTR control, DER animals denied the expected reward, MO medial orbital cortex, PrL prelimbic cortex, RER animals receiving the expected reward, roVLO rostral part of ventral lateral orbital cortex

decipher new rules based on different dimensions of environmental cues was normal. Since the different aspects of performance in the ASST are regulated by specific prefrontal cortex areas, we determined levels of neuronal activity in these PFC areas following ASST by means of c-Fos immunohistochemistry: The density of c-Fos positive cells was lower in the medial orbital cortex (MO) of adult male DER animals compared to that of controls and RER (F2,20 = 5.034, p = 0.018; post hoc test, *DER vs. CTR p = 0.024, #DER vs. RER p = 0.008; Fig. 1b, c), while no differences in c-Fos levels were detected in the rostral part of area 1 of the cingulate (roCg1), the prelimbic (PrL), or the rostral part of ventral lateral orbital cortex (roVLO) (Fig. 1c). Interestingly, MO has been linked to the capacity of rats to perform intra-dimensional reversal shifts (de Bruin et al. 1994; Chudasama and Robbins 2003; Passetti et al. 2002; McAlonan and Brown 2003). Neurochemical and cellular changes in the prefrontal cortex in adulthood (Fig. 2a–d) Motivated by the behavioral deficit exhibited by the adult male DER animals in a task controlled by the PFC, we examined the possibility of concomitant cellular alterations present in the PFC of DER animals. Indeed, the density of NeuN positive cells (neurons) in the medial orbital (MO) and infralimbic cortex (IL) was lower in the adult male DER animals compared to both the controls and the RER (One-way ANOVA with the group of animals as the independent factor, for MO, F2,17 = 21.500, p \ 0.001; post hoc test, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; for IL, F2,17 = 26.725, p \ 0.001; post hoc test, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; Fig. 2a, b), while no differences were found in the roCg1, PrL or roVLO (Fig. 2b).

It should be mentioned that the total volume of the PFC, MO or IL did not differ between the three groups of adult animals (CTR, DER, RER) (Table 2). Moreover, the morphological characteristics of NeuN? or vGluT? cells were similar in the three groups of animals (Table 3). The reduced levels of NeuN cells in the MO and IL of adult male DER animals posed the question of identifying the phenotype of the missing cells. Thus, we performed immunohistochemistry for the glutamatergic marker vGluT and the GABAergic marker GAD67: The density of vGluT positive cells was lower in the MO and IL of DER animals (for MO, F2,17 = 61.806, p \ 0.001; post hoc test, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; for IL, F2,17 = 18.126, p \ 0.001; post hoc test, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; Fig. 2c, d), while no differences were found in roCg1, PrL or roVLO (Fig. 2d). On the other hand, the density of GAD67 positive cells was similar between the three groups in all prefrontal cortex areas examined (Table 4). These results indicate that *30 % of vGluT positive cells (glutamatergic neurons) is missing from the MO and IL of adult male DER animals. It should also be noted that no difference was detected between control and RER animals in the neurochemical parameters evaluated. Interestingly, the levels of Iba-1? (microglial marker) and GFAP? (astroglial marker) in the adult brain did not differ between the three groups (data not shown). Changes in spine density in the prefrontal cortex (Fig. 2e, f) Besides the neurochemical changes identified in the DER group, a decrease in the density of spines of apical dendrites was also detected in the MO and IL: Spine density was lower in both areas in adult male DER animals compared to that in control or RER animals (for MO, F2,17 = 13.290, p \ 0.001; post hoc test, *DER vs. CTR p = 0.002, #DER vs. RER p \ 0.001; for IL, F2,17 = 10.604, p = 0.001; post hoc test, *DER vs. CTR p = 0.002, #DER vs. RER p = 0.001; Fig. 2e, f). No differences were detected in the roCg1 area between the three groups, or in any of the three areas analyzed between the control and the RER animals (Fig. 2f). It should be noted that the total length of apical dendrites was similar in the MO and IL of CTR, DER or RER animals (length relative to controls: For MO, DER 102 ± 17 % and RER 103 ± 12 %; for IL, DER 99 ± 14 % and RER 104 ± 13 %). Fear extinction (Fig. 3) In order to test if the neurochemical and cellular alterations detected in the infralimbic cortex of adult DER

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Brain Struct Funct

Fig. 2 Effects of the DER experience on neuronal and spine density in the PFC of adult rats. Representative photomicrographs of NeuN immunofluorescence (a), vGluT immunohistochemistry (c), and Golgi-Cox staining (e). b, d, f Bargraphs representing quantification of the results as means and SEM, n = 6 per group. Adult male DER animals had lower neuronal (NeuN? cell) density in MO and IL (a, b). This DER-induced decrease was found only in the glutamatergic cells of the MO and IL (c, d). In addition, adult male DER animals

had lower dendritic spine density in MO and IL (e, f). *p \ 0.05 DER vs. CTR, #p \ 0.05 DER vs. RER. Scale bar in a and c corresponds to 25 lm and in e it corresponds to 5 lm. roCg1 rostral part of area 1 of cingulate cortex, CTR control, DER animals denied the expected reward, IL infralimbic cortex, MO medial orbital cortex, PFC prefrontal cortex, PrL prelimbic cortex, RER animals receiving the expected reward, roVLO rostral part of ventral lateral orbital cortex

animals are reflected on the behavioral level, we exposed adult male animals to fear extinction, a behavioral task regulated by the infralimbic cortex (Barad 2005). It should be noted that all three groups of animals (CTR, DER and RER) froze for the same amount of time at the beginning of the fear extinction procedure, documenting that all three groups had a similar level of memory for the conditioned stimulus–unconditioned stimulus association. Yet, DER males showed reduced ability for fear extinction, freezing longer following repeated exposure to the

conditioned stimulus (tone predicting shock) in the absence of the unconditioned stimulus (shock), compared to the other two groups of animals, verifying the hypothesis of a behavioral correlate of the IL cellular alterations in the DER animals (repeated measures oneway ANOVA with the group of animals as the independent factor and extinction trials as the repeated measures: main effect of group, F2,15 = 7.203, p = 0.006; post hoc test, *DER vs. CTR p = 0.032, #DER vs. RER p = 0.002; Fig. 3).

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Brain Struct Funct

Rat pups Neurochemical and cellular changes in the prefrontal cortex during T-maze training (Fig. 4) Most interestingly, the cellular alterations detected in adult DER animals were already present on PND12 and PND13, on the last 2 days of the exposure to the early life DER experience; it should be noted that no cellular alterations Table 2 Volume of PND13 and adult PFC, medial orbital and infralimbic cortex relative to controls Control Mean

SEM

DER (% of control)

RER (% of control)

Mean

Mean

SEM

SEM

PND13 PFC

100

2

98

5

98

2

MO/IL

100

3

100

6

98

5

Adult PFC

100

6

100

2

103

7

MO

100

7

99

6

99

8

IL

100

4

98

5

100

6

Mean values and SEM are given DER animals denied the expected reward, IL infralimbic cortex, MO medial orbital cortex, PFC prefrontal cortex, PND postnatal day, RER animals receiving the expected reward, SEM standard error of mean

Table 3 Morphological characteristics of NeuN and vGluT immunopositive cells in PND13 and adult medial orbital and infralimbic cortex

were detected on PND11, the second day of training. More specifically, on postnatal days 12 and 13, the neuronal density (NeuN? cells) in the MO/IL area was lower in DER pups compared to both the control and RER, while no difference was observed between control and RER animals [two-way ANOVA with the group of animals (CTR, DER, RER) and the developmental stage (PND11, PND12, and PND13) as the independent factors: group 9 developmental stage interaction F4,53 = 3.885, p = 0.009; further post hoc analyses: on PND12, *DER vs. CTR p = 0.001, # DER vs. RER p = 0.002; on PND13, *DER vs. CTR p = 0.002, #DER vs. RER p = 0.002; Fig. 4a, b]. Similarly, on the same postnatal days (PND12 and 13) the density of vGluT positive cells was reduced in the MO/IL area of DER pups (group 9 developmental stage interaction F6,53 = 4.925, p = 0.002; further post hoc analysis for PND12, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; for PND13, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; Fig. 4c, d). On the other hand, the density of GAD67 positive cells was similar between the three groups in MO/IL on all three postnatal days examined (Table 5). Notably, neither the total volume of the PFC and MO/IL (Table 2) nor the morphological characteristics of NeuN? or vGluT? cells differed between the three groups of postnatal animals (CTR, DER, RER) (Table 3).

Control

DER

Mean

SEM

100

5

Mean

RER SEM

Mean

SEM

NeuN? cells PND13 Area (relative to controls)

98

6

97

8

Circularity

0.88

0.01

0.86

0.01

0.86

0.01

Aspect ratio Adult

1.40

0.04

1.42

0.04

1.44

0.02

Area (relative to controls)

100

5

102

2

98

4

Circularity

0.89

0.004

0.90

0.01

0.89

0.004

Aspect ratio

1.34

0.03

1.33

0.02

1.36

0.01

vGluT? cells PND13 Area (relative to controls)

100

4

100

8

103

8

Circularity

0.88

0.004

0.88

0.005

0.87

0.01

Aspect ratio

1.38

0.01

1.40

0.03

1.41

0.04

Adult Area (relative to controls)

100

5

99

3

100

6

Circularity

0.85

0.004

0.86

0.01

0.85

0.01

Aspect ratio

1.43

0.03

1.42

0.01

1.46

0.03

Mean values and SEM are given DER animals denied the expected reward, RER animals receiving the expected reward, PND postnatal day, SEM standard error of mean

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Brain Struct Funct

Moreover, in the MO/IL area of DER pups, we detected on postnatal days 12 and 13, increased density of both microglia and astrocytic processes, indicative of neurodegenerative processes taking place during this period of DER training. Two-way ANOVA revealed for Iba-1? cells, a group 9 developmental stage interaction: F6,53 = 7.955, p \ 0.001; further post hoc analysis showed that on PND12, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001; for PND13, *DER vs. CTR p \ 0.001, #DER vs. RER p \ 0.001 (Fig. 4e, f). Similar analysis for GFAP? processes also yielded a group 9 developmental stage interaction: F6,53 = 3.370, p = 0.017 and further post hoc analysis for PND12, *DER vs. CTR p = 0.007, #DER vs. RER p = 0.004 and for PND13, *DER vs. CTR p = 0.009, #DER vs. RER p = 0.009 (Fig. 4g, h).

Table 4 Density of GABAergic (GAD67 immunopositive) neurons in adult PFC sub-regions relative to controls Control Mean

SEM

DER (% of control)

RER (% of control)

Mean

SEM

Mean

SEM 6

roCg1

100

3

103

5

102

PrL

100

9

101

6

99

5

roVLO MO

100 100

6 3

104 103

7 7

101 102

6 4

IL

100

5

97

6

102

6

Mean values and SEM are given DER animals denied the expected reward, GAD67 glutamate decarboxylase, MW 67 kDa, PFC prefrontal cortex, IL infralimbic cortex, MO medial orbital cortex, PrL prelimbic cortex, RER animals receiving the expected reward, roCg1 rostral part of area 1 of cingulate cortex, roVLO ventral lateral orbital cortex, SEM standard error of mean

Fig. 3 Effect of the DER experience on the performance of adult male rats in fear extinction. DER animals displayed reduced fear extinction compared to both the CTR and RER (*p \ 0.05 DER vs. CTR, #p \ 0.05 DER vs. RER). Points in graphs represent means and SEM, n = 6 per group (the average value of two animals from each of six litters was used in the statistical analysis). CTR control, DER animals denied the expected reward, RER animals receiving the expected reward

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Discussion The results presented herein show that the DER early life experience, in spite of its fairly mild adversity (Diamantopoulou et al. 2013), had negative consequences on PFC structure and function, while the RER animals did not differ from controls in any of the parameters analyzed in the present work. More specifically, adult DER males showed functional deficits indicating behavioral inflexibility, as documented by their impaired performance in two PFC-dependent behavioral tests: the ASST and fear extinction. In the ASST, DER animals showed deficits specifically in the intra-dimensional reversal shifts and a lower activation—as determined by c-Fos immunohistochemistry—of the medial orbital cortex (MO), a PFC subregion involved in reversed stimulus-reward contingency learning (de Bruin et al. 1994; Chudasama and Robbins 2003; Passetti et al. 2002; McAlonan and Brown 2003). At the cellular level, DER animals had lower neuronal and dendritic spine density in the MO—as mentioned above involved in the ASST—and the IL, which plays a key role in fear extinction (Barad 2005). Similar findings showing reduced neuronal density or absolute number of neurons in the PFC have been reported following other early experiences, namely maternal deprivation (Aksic´ et al. 2013) and prenatal stress (Mychasiuk et al. 2012), respectively. The DER-induced decrease in neurons was observed only in the population of glutamatergic cells, while the GABAergic were not affected. The decrease in glutamatergic cells was detected as early as PND12, was present on PND13—at the end of the exposure to the DER procedure—and was clearly observable in the adult brain. These results indicate that it was the process of the DER experience that induced the neuronal loss which persisted

Brain Struct Funct

Fig. 4 Effects of the DER experience on neuronal and glial density in the PFC of rat pups during the postnatal period of training. Representative photomicrographs of NeuN (a) and vGluT immunohistochemistry (c), as well as Iba-1 (e) and GFAP immunofluorescence (g) on PND12. b, d, f, h Bargraphs representing quantification of the results as means and SEM, for each developmental stage n = 6 per group. On PND12 and 13 DER pups had lower neuronal (NeuN? cell) density in the MO/IL (a, b). This DER-induced decrease was

found only in the glutamatergic cells of the MO/IL (c, d). On the contrary, on the same postnatal days (PND12 and 13) DER pups had increased density of micro- (e, f) and astroglia (g, h) in the MO/IL. *p \ 0.05 DER vs. CTR, #p \ 0.05 DER vs. RER. Scale bar in a and c corresponds to 25 lm and in e and f to 10 lm. CTR control, DER animals denied the expected reward, MO/IL medial orbital/infralimbic cortex, PFC prefrontal cortex, PND postnatal day, RER animals receiving the expected reward

into adulthood. Interestingly, the neuronal decrease during the postnatal period (PND12 and 13), was accompanied by an increase in micro- and astroglia, markers of neuroinflammation. No such effect, however, was detectable in the

brain of the adult DER males, indicating that this effect of the early experience was compensated for. The PFC is the brain region which matures last. During the postnatal period, it is still developing with processes

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Brain Struct Funct Table 5 Density of GABAergic (GAD67 immunopositive) neurons, relative to controls, in MO/IL on PND11–13 Control

DER (% of control)

RER (% of control)

Mean

Mean

Mean

SEM

SEM

SEM

PND11

100

3

98

7

99

6

PND12

100

9

102

6

102

7

PND13

100

5

98

4

101

8

Mean values and SEM are given DER animals denied the expected reward, GAD67 glutamate decarboxylase, MW 67 kDa, MO/IL medial orbital/infralimbic cortex, PND postnatal day, RER animals receiving the expected reward, SEM standard error of mean

such as neuronal maturation, in-growth of axonal projections, synaptogenesis, formation of functional networks, and myelination still going on (Petit et al. 1988; Benes et al. 2000; Kolb et al. 2012). It is thus vulnerable to the effects of early life experiences as documented by a number of studies employing different experimental models such as maternal deprivation (Bock et al. 2005; Helmeke et al. 2008; Baudin et al. 2012; Aksic´ et al. 2013; Chocyk et al. 2013; Li et al. 2013), caregiver maltreatment (Blaze and Roth 2013; Ventura et al. 2013), isolation rearing (Pascual et al. 2006; Bock et al. 2008; Krolow et al. 2012; Wall et al. 2012; Baarendse et al. 2013; Swerdlow et al. 2013), neonatal handling (Noschang et al. 2012), and prenatal stress (Green et al. 2011; Mychasiuk et al. 2012; Luoni et al. 2014). Our results add further support to, and expand the literature in this field. Our finding showing that the DER manipulation affected the glutamatergic but not the GABAergic cells in the PFC, indicates that this early experience could alter the balance between excitatory and inhibitory neurotransmission. Another early experience, maternal deprivation, also results in such an imbalance, but has the opposite effect from the DER experience, resulting in altered numbers of GABAergic cells, while the glutamatergic are not affected (Helmeke et al. 2008; Aksic´ et al. 2013). It could be postulated that the components of neuronal circuits are differentially affected depending on the demands posed on the pups by the various types of early life aversive experiences. Nevertheless, the common denominator is that early life adversity can result in an imbalance between excitatory and inhibitory neurotransmission, which has been proposed to underlie a variety of psychopathologies: Such an imbalance can develop as a result either of decreased glutamatergic neurotransmission, as is the case of schizophrenia and autism (Kehrer et al. 2008; Garey 2010; Laruelle 2014; Tebartz van Elst et al. 2014), or increased GABAergic function, as in mood disorders (Bielau et al. 2007). Dendritic spines constitute the primary site of synaptic neurotransmission. Their spatial distribution within brain

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tissue represents functional neuronal networks and their redistribution a morphological correlation of activity-dependent synaptic plasticity. Aberrant dendritic spine density is associated with a variety of brain pathologies (Hinton et al. 1991; Hutsler and Zhang 2010; Duman and Li 2012; Glausier and Lewis 2013). Aversive early life experiences in rodents, such as maternal deprivation, have been shown to result in reduced density of spines in the PFC (Bock et al. 2005; Chocyk et al. 2013). Our present results corroborate these findings and show that an early experience of even mild adversity leads to decreased dendritic spines. Notably, the reduction in dendritic spines is observed in the same subregions of the PFC (MO and IL) where the number of glutamatergic neurons was decreased. These findings illustrate that the DER experience had topographically specific detrimental effects on the cellular organization of the PFC. The process of training under the DER condition induced significant changes in the cellular organization of the PFC, reducing the number of glutamatergic neurons and increasing that of microglia and astrocytes. It should be noted that these changes were first detected on PND12, when three training sessions had already taken place, suggesting that they were not simply DER-experience-induced losses in neurons and increases in glia, but rather a reorganization of local networks, which would need more time to develop. The increase in the density of glia could reflect their involvement in this reorganization, since it has been shown that glia play a key role in synaptic pruning and remodeling (Harry 2013; Katsumoto et al. 2014). Certainly, the increase in glia could also represent a response to the loss of the neurons (Harry 2013; Katsumoto et al. 2014). It appears, on the basis of existing evidence that aversive early experiences, such as prenatal stress (Barros et al. 2006; Diz-Chaves et al. 2012; S´lusarczyk et al. 2015; Zhao et al. 2015) and maternal deprivation (Llorente et al. 2009; Marco et al. 2013) often lead to increases in micro- and astroglia. Notably, increased number of microglia has been associated with different psychopathologies in humans such as schizophrenia, depression, and autism (for a review see Re´us et al. 2015). In our study, the increase in microglia observed in the PND12 and 13 PFC, was no longer detectable in the adult. However, the alterations in the PFC network organization induced during the postnatal period along with the neuronal loss, which persisted into adulthood, could underlie the deficits in PFC function. One of the factors contributing to the DER-experienceinduced cellular abnormalities—loss of neurons, increase in glia—could be corticosterone, whose levels in DER animals fluctuate aberrantly during this critical developmental period: in the DER pups corticosterone is increased on PND10 and then—on PND11 and 12—falls below

Brain Struct Funct

control levels (Diamantopoulou et al. 2013). A tight control of corticosterone levels is necessary for normal brain structure and function. It has been shown that either higher or lower than normal corticosterone levels can lead to neuronal loss (Yu et al. 2008) and to an activation of microglia (Re´us et al. 2015). The DER-experience-induced cellular defects were accompanied by functional deficits of the PFC, as assessed by the performance of the DER adult males in two PFC-dependent tasks, namely the attention set-shifting task (ASST) and fear extinction. These tasks assess different aspects of behavioral flexibility, a prominent feature of PFC function. In both of these two tasks, the DER animals have low performance: Once they have developed, through learning, a specific behavior/strategy they ‘‘stick to’’ it and have difficulties to switch it, in spite of the altered environmental requirements. This behavior in adulthood could be related to the experience they were exposed to as neonates; DER pups were denied contact with the mother within the T-maze, but after the end of the task they were returned to the home cage and received increased maternal care (Diamantopoulou et al. 2013). Thus, they received the expected reward but with a delay (Panagiotaropoulos et al. 2009). This experience could have programmed their behavior in such a way as to persist in an acquired mode of action and reluctance to make switches. ASST is a PFC-dependent test employed in rodents (Birrell and Brown 2000) which is equivalent to the Wisconsin card sorting test, in which children—whose PFC is not fully functional yet—and human adults with PFC pathology have significant deficits (Crone et al. 2004; Goldberg and Bougakov 2005). Relevantly, in rats, an aversive early life experience, i.e., maternal deprivation, has been shown to lead to reduced performance in the ASST (Baudin et al. 2012). In our study, the adult male DER animals could not efficiently make the intra-dimensional reversal shifts, i.e., within a certain modality could not efficiently learn a new association between a stimulus and the reward, ignoring a previously learned one. On the other hand, they performed normally the extra-dimensional shifts, i.e., they could efficiently replace a previously learned association between a modality and the reward, with a new one. Relevantly, the extra-dimensional shifts are controlled by the medial PFC—corresponding to the rostral Cg1and PrL, in our study—(Birrell and Brown 2000) in which no effect of the DER experience was detected. In contrast, the ability for intra-dimensional reversal shifts involves the MO subregion of PFC (de Bruin et al. 1994; Chudasama and Robbins 2003; Passetti et al. 2002; McAlonan and Brown 2003), in which, as mentioned above, the DER experience resulted in decreased neuronal and spine density. Furthermore, our results showed that this particular subregion of the PFC exhibited lower ASST-induced activation (as assessed by expression of the neuronal activity marker, c-Fos) in the

DER animals, adding further support to the role of the MO in the process of intra-dimensional reversal shift. In the fear extinction task, animals are expected to learn that the conditioned stimulus (a tone) does not have the previously learned predictive value of the unconditioned stimulus (shock). The adult male DER animals were less efficient in doing this, than the controls or the RER; they kept on freezing in spite of the fact that presentation of the conditioned stimulus (tone) was not accompanied by the shock anymore. Fear extinction is controlled by a PFC-amygdala circuit, in which the IL is the particular subregion of the PFC involved (Barad 2005). In DER animals the amygdala has been shown to be more activated during fear memory (Diamantopoulou et al. 2013). It thus appears that the IL, which in the DER animals shows decreased neuronal and spine density, is incapable of effectively containing the amygdalar behavioral output (freezing). In agreement with our results, Green et al. (2011) using another aversive early experience, namely prenatal stress, have demonstrated reduced fear extinction. Interestingly, relevant findings have also been obtained for humans, in which aversive early life experiences such as maltreatment have been associated with abnormal persistence of fear memories (Bremner et al. 2005; Teicher and Samson 2013). We have proposed that the DER experience could be a good animal model for human neglect based on previous results from our laboratory showing decreased PFC dopamine (Raftogianni et al. 2014) and serotonin (Diamantopoulou et al. 2012). The present work documents that the DER experience had profound effects on the PFC resulting in structural abnormalities and functional deficits which can be interpreted as lack of behavioral flexibility. Interestingly, most of the symptoms observed in adult humans following early life neglect, such as increased aggression, enhanced fear memory, inability to shift attention, can be ascribed to decreased behavioral flexibility. The results presented herein thus add further support to the validity of the DER experience as a model for neglect. Acknowledgments The authors would like to thank Ms. Konstantina Nikolakaki for her technical assistance in the immunohistochemical and immunofluorescence experiments. This work has been supported by the John S. Latsis Public Benefit Foundation (#12020). The sole responsibility for the content lies with its authors. Compliance with ethical standards Conflict of interest

No potential conflict of interest.

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