Maternal deprivation induces deficits in temporal memory and cognitive flexibility and exaggerates synaptic plasticity in the rat medial prefrontal cortex

Neurobiology of Learning and Memory 98 (2012) 207–214

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Maternal deprivation induces deficits in temporal memory and cognitive flexibility and exaggerates synaptic plasticity in the rat medial prefrontal cortex Aurélie Baudin a,b,c,1, Kévin Blot a,b,c,1, Catherine Verney d,e,f, Lucie Estevez a,b,c, Julie Santamaria a,b,c, Pierre Gressens d,e,f, Bruno Giros a,b,c,g, Satoru Otani a,b,c, Valérie Daugé a,b,c, Laurent Naudon a,b,c,⇑ a

INSERM, UMRs 952, Physiopathologie des Maladies du Système Nerveux Central, 9 quai Saint Bernard, F-75005 Paris, France CNRS, UMR 7224, Physiopathologie des Maladies du Système Nerveux Central, 9 quai Saint Bernard, F-75005 Paris, France UPMC Univ. Paris 06, Physiopathologie des Maladies du Système Nerveux Central, 9 quai Saint Bernard, F-75005 Paris, France d INSERM, U676, Hôpital Robert-Debré, 48, boulevard Sérurier, 75019 Paris, France e Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France f PremUP, 75006 Paris, France g Douglas Hospital Research Center, Department of Psychiatry, McGill University, 6875 Boulevard Lasalle Verdun, Montreal, QC, Canada b c

a r t i c l e

i n f o

Article history: Received 24 May 2012 Revised 27 July 2012 Accepted 12 August 2012 Available online 24 August 2012 Keywords: Maternal deprivation (MD) Prefrontal cortex (PFC) Cognitive tasks Stereology In vivo electrophysiology Rat

a b s t r a c t Early life adverse events can lead to structural and functional impairments in the prefrontal cortex (PFC). Here, we investigated whether maternal deprivation (MD) alters PFC-dependent executive functions, neurons and astrocytes number and synaptic plasticity in adult male Long-Evans rats. The deprivation protocol consisted of a daily separation of newborn Long-Evans pups from their mothers and littermates 3 h/day postnatal day 1–14. Cognitive performances were assessed in adulthood using the temporal order memory task (TMT) and the attentional set-shifting task (ASST) that principally implicates the PFC and the Morris water maze task (WMT) that does not essentially rely on the PFC. The neurons and astrocytes of the prelimbic (PrL) area of the medial PFC (mPFC) were immunolabelled respectively with anti-NeuN and anti-GFAP antibodies and quantified by stereology. The field potentials evoked by electrical stimulation of ventral hippocampus (ventral HPC) were recorded in vivo in the PrL area. In adulthood, MD produced cognitive deficits in two PFC-dependent tasks, the TMT and ASST, but not in the WMT. In parallel, MD induced in the prelimbic area of the medial PFC an upregulation of long-term potentiation (LTP), without any change in the number of neurons and astrocytes. We provide evidence that MD leads in adults to an alteration of the cognitive abilities dependent on the PFC, and to an exaggerated synaptic plasticity in this region. We suggest that this latter phenomenon may contribute to the impairments in the cognitive tasks. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Early life adversity, such as emotional neglect and/or sexual, physical and psychological abuse may predispose individuals to psychiatric conditions (for review see McCrory, De Brito, & Viding,

Abbreviations: AFR, animal facility rearing rats; ASST, attentional set-shifting task; D, maternally deprived rats; HPC-mPFC, hippocampal-medial prefrontal cortex pathway; LTP, long-term potentiation; MD, maternal deprivation; mPFC, medial prefrontal cortex; MS, maternal separation; PFC, prefrontal cortex; PrL, prelimbic area; TMT, temporal order memory task; ventral HPC, ventral hippocampus; WMT, Morris water maze task. ⇑ Corresponding author. Address: INSERM, UMRs 952, CNRS UMR 7224, UPMC Paris 6, Physiopathologie des Maladies du Système Nerveux Central, Université Pierre et Marie Curie, 9 Quai Saint Bernard, 75005 Paris, France. Fax: +33 1 44 27 60 69. E-mail address: [email protected] (L. Naudon). 1 The first two authors contributed equally to this work. 1074-7427/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2012.08.004

2010). For the infant, the interaction with its mother is the most important environmental factor, since a variety of its physiological systems responds to specific elements of this interaction. The animal models of maternal separation (MS) have been useful tools to demonstrate that the disruption of mother–child interaction results in an alteration of normal growth and development of the infant. Particularly, several studies showed that long MS procedures (P3 h per day) induce long-lasting effects on the hypothalamo– pituitary–adrenal axis in male rats, resulting in an increased response to stress (for review see Pryce & Feldon, 2003). We have previously developed a particular model of MS, a model of maternal deprivation (MD) which consisted of a daily separation of newborn Long-Evans pups from their mothers and also from their littermates for 3 h per day from postnatal days 1 to 14. Later, the adult maternally deprived (D) rats are compared with animal facility rearing (AFR) rats, which have experienced human intervention for animal care (Pryce & Feldon, 2003). We have

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shown that MD leads in male rats to an enhanced anxiety and reactivity to stress, increased preference for sucrose, hypersensitivity to the rewarding effect of morphine and morphine dependence (Mourlon et al., 2010; Vazquez, Farley, Giros, & Daugé, 2005; Vazquez, Giros, & Daugé, 2006; Vazquez, Penit-Soria et al., 2005). The prefrontal cortex (PFC), which orchestrates the integration of cognition, emotion and action through its cortical and subcortical networks, has been described as a brain region very sensitive to the detrimental effects of the exposure to chronic stress (for review see Arnsten, 2009). The MS indeed results in various impairments of the PFC in adolescent and adult rats, including modified proteins expression (Brenhouse & Andersen, 2011; Chocyk, Dudys, Przyborowska, Mac´kowiak, & We˛dzony, 2010), changed dendritic morphology (Monroy, Hernandez-Torres, & Flores, 2010; Muhammad & Kolb, 2011; Pascual & Zamora-Leon, 2007) and altered neuronal activity (Benekareddy, Goodfellow, Lambe, & Vaidya, 2010;Stevenson, Halliday, Marsden, & Mason, 2008). In human, functional imaging studies reveal structural and functional impairments in the PFC in patients with psychiatric disorders, particularly those reporting childhood maltreatment or adversity (Tomoda et al., 2009; van Harmelen et al., 2010). However, to date, the details as to how the MS affects cognitive functions in rodent models and how such cognitive changes are supported at the cellular level are largely unknown. In the present study, we aimed to examine the influence of MD on cognitive performance in adult male rats. Following MS protocols, several studies have investigated behavioral tasks such as the novel object recognition task (Aisa, Tordera, Lasheras, Del Rio, & Ramirez, 2007; Benetti et al., 2009; Hulshof et al., 2011), the Morris water maze task (WMT) (Aisa et al., 2007; Grace, Hescham, Kellaway, Bugarith, & Russell, 2009; Hui et al., 2011; Huot, Plotsky, Lenox, & McNamara, 2002; Lai et al., 2006; Mello, Benetti, Cammarota, & Izquierdo, 2009; Pryce, Bettschen, Nanz-Bahr, & Feldon, 2003; Uysal et al., 2005; Zhu et al., 2010) and the radial arm maze task (Sandstrom & Hart, 2005). The majority of these studies described cognitive impairments in MS rats, with the exception of Pryce et al. (2003), who observed an improvement in memory performance, and some studies showing no effects (Grace et al., 2009; Lai et al., 2006). Here, we chose to use two tasks in which the performance relies critically on the PFC and its networks as demonstrated by inactivation or lesion of the PFC. First, the temporal order memory task (TMT) assesses the ability of rats to discriminate objects that have been encountered at different times in the past (Hannesson, Howland, & Phillips, 2004; Mitchell & Laiacona, 1998). Second, the attentional set-shifting task (ASST) is equivalent to the Wisconsin card-sorting test used in human subjects to diagnose frontal lobe damage; in ASST, complex stimuli differ along perceptual dimensions, allowing the assessment of the capacity of rats to acquire, maintain and shift attentional set (Birrell & Brown, 2000; McAlonan & Brown, 2003). We completed these studies by testing the spatial learning of AFR and D rats in the WMT, for which the role of the PFC does not appear to be critical (for review see Wang & Cai, 2008). Long-term potentiation (LTP) and long-term depression are models of activity-dependent facilitation and reduction of synaptic transmission thought to underlie learning and memory (Bliss & Collingridge, 1993). It is still unknown however whether MD modifies synaptic transmission in the PFC and, if so, whether such changes are correlated with changes in executive cognitive functions. The hippocampal-medial PFC (HPC-mPFC) pathway is a monosynaptic glutamatergic projection (Ferino, Thierry, & Glowinski, 1987; Jay & Witter, 1991) that can support plastic changes as it can be potentiated, depotentiated and depressed (for review Laroche, Davis, & Jay, 2000). Importantly, this pathway is known to be involved in executive functions (Devito & Eichenbaum, 2011; Marquis, Goulet, & Dore, 2008; Wang & Cai, 2008). Therefore, we conducted a series of in vivo electrophysiological experiments to

monitor synaptic responses in the HPC-mPFC pathway. We focused on LTP in the prelimbic (PrL) area of the medial PFC (mPFC) using high-frequency stimulation protocol previously described in the studies above-mentioned. In addition, as previous studies have suggested that maternal separation can lead to change in the number of cells in the brain (Leventopoulos et al., 2007; Llorente et al., 2009; Musholt et al., 2009), we tested, by immunohistochemical labeling and stereological quantification, whether MD altered the number of neurons and astrocytes in the PrL area of the mPFC. 2. Methods and materials 2.1. Maternal deprivation procedure Experimental procedure and animal care were performed in accordance with local committee guidelines and the European Communities Council Directive of November 24, 1986 (86/609/ EEC). Four series of 20 pregnant Long-Evans rats were received on day 14 of gestation (Janvier, Le Genest St. Isle, France). The dams gave birth 1 week after inclusion. MD was performed as previously described (Mourlon, Naudon, Giros, Crumeyrolle-Arias, & Daugé, 2011; Mourlon et al., 2010). On the postnatal day 1, litters were cross-fostered culled to 8–12 pups, half females-half males randomly chosen. Neonates of the maternal deprivation group were individually placed in temperature- (30–34 °C) and humidity-controlled cages. D pups were isolated 3 h daily (2 pm–5 pm) from days 1 to 14. AFR pups remained with their mothers during this period and received no specific handling other than changing the bedding in their cages once a week. On day 22, pups were weaned and housed in groups of two or three. Only male rats (AFR, n = 50; D, n = 49) were included in the study, and each individual has been used only for one test or quantification. 2.2. TMT procedures During the four days preceding the task, 11 AFR and 11 D rats (80 days old) were handled daily and habituated to the open-box (100  100  60 cm; lighting, 50 lux) and the test room as previously described (Naudon, Hotte, & Jay, 2007). At the fifth day, the task consisted of two consecutive sample phases and one test phase of 4 min each. In the first sample phase, rats were allowed to explore two identical objects (12–15 cm height) fixed (PatafixÒ) on the floor of the box in position 3 cm from the back and 12 cm from the side walls. One hour later, rats received a second sample phase with two copies of a new object. After an additional delay of 3 h, one ‘‘old object’’ from the 1st sample phase and one ‘‘recent object’’ from the 2nd sample phase were presented during the test phase. The time exploring each object (i.e. directing the nose to the object at a distance 6 2 cm) was scored on videotape. The discrimination ratio was calculated as ((time spent exploring the old object) (time spent exploring the recent object))/(time spent exploring both objects). 2.3. ASST procedures The procedure was adapted from Birrell and Brown (2000). In a white open rectangular box (45  70  60 cm; lighting, 50 lux), two digging ceramic bowls (7 cm diameter, 4 cm depth) were placed at one end of the box, with a central divider between them. The bait was a piece of chocolate-flavored cereal (ChocapicÒ) hidden at the bottom of the bowl that contains the relevant stimulus. Chocolate powder was added to each bowl to ensure that the rat might not recognize the bait to the smell. Each bowl was defined by an odor-medium association. A removable wall separated the bowls compartment from the other half of the box and indicated

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the beginning and end of each trial when raised and lowered. During one week, 12 AFR and 12 D rats (80–100 days old) were restricted to 10 g of food per day. During the task, only one bowl was baited, rats had to find the reward in 10 min. An error was recorded if they dug first in the unbaited bowl. Testing continued until rats reached a criterion level of six consecutive correct trials. Briefly, rats underwent seven consecutive trial sessions in the same order: simple discrimination, compound discrimination, reversal 1, intradimensional shift, reversal 2, extradimensional shift, and reversal 3 (for details, see Table S1 in Supplementary Information).

count if the bottom of the cell was observed within the volume of the dissector (height of the section thickness excluding 2 lm thick guard zone). For each rat, from section thickness, the number of sampling areas and the number of counted cells, the software calculated the total number of neurons and astrocytes in the PrL and the volume of this cortical area. Moreover, a coefficient of Schaeffer (coefficient of error) was calculated; values below 0.05 assessed the reliability of the cell distribution.

2.4. WMT procedures

Height AFR and 7 D rats (90–170 days old) were anesthetized by urethane (1.5 g/kg, i.p.) and placed in a stereotaxic frame (Kopf, CA, USA) with body temperature maintained at 37 ± 0.2 °C. By the use of manual manipulators (Narishige, Japan), a recording electrode (Teflon-coated tungsten wire, external diameter 200 lm, A–M Systems, WA, USA) was placed in the mid layer of the PrL area (3.3 mm anterior to the bregma, 0.8 mm lateral to the midline, 3.5 mm from the brain surface, according to Paxinos and Watson (2005)), and a stimulating electrode (bipolar Teflon-coated stainless steel wire, external diameter 200 lm) in the ventral HPC (5.0–6.0 mm posterior to the bregma, 5.2–5.8 mm lateral to the midline, and 4.0– 7.0 mm from the brain surface). The electrode positions were adjusted to obtain the maximum amplitude of postsynaptic potential, and the intensity of stimulation was set to evoke 60% of the maximum response to start experiments (300–700 lA). The postsynaptic potentials were evoked at 0.033 Hz in the mPFC by delivering the constant current, monophasic square pulses of 250 ls width (A360 stimulus isolator, WPI, FL, USA) to ventral HPC and fed to a differential AC amplifier (model 1700, A–M Systems, WA, USA) with 100 times amplification and the filtration set at 1.0 Hz and 5 kHz. The signals were digitized at 10 kHz through a Digidata 1322A interface (Molecular Devices, CA, USA), and recorded and stored by the use of Elphy data acquisition-analysis program developed by Dr. G. Sadoc (Institut Alfred Fessard, CNRS, France) in a PC computer for later analysis. After a stable baseline response recording for at least 30 min, high-frequency stimulation was delivered at the test pulse intensity (two series in 6 min interval of 10 trains of 50 pulses delivered at 250 Hz). The postsynaptic potentials amplitudes were grouped for each successive 2 min period (i.e. four responses) to reduce variability and expressed for each animal as percentage changes from the 30 min baseline level.

The water maze consisted of a circular pool (150 cm diameter, 60 cm height) filled to a depth of 29 cm with water (24 ± 1 °C) made opaque (Acusol OP 301 opacifier, Rohm Ihaas, France). Distal extra-maze cues were arranged around the pool. The rats, 12 AFR and 12 D, were required to locate a stationary hidden platform (9 cm diameter; 1 cm below water surface) situated in the center of one of the four quadrants that divided the pool. Over 15 trials (3 trials per day for 5 consecutive days; 1 h inter-trial interval), rats were placed in the pool at one of four equidistant locations, and they were tested to assess the escape latency and distance traveled. After a delay of 2 days, the maze was run with the platform removed, and rats were tested for the time spent in the quadrant previously containing the platform (one probe trial). Behaviors were recorded and analyzed using a computerized video-tracking system (View Point, Champagne au Mont d’Or, France). Rats were 75–85 days old at the time of testing. 2.5. Stereological counting of NeuN and GFAP labeled cells Following decapitation 7 AFR and 7 D rats (90 days old), the brains were fixated in 4% formalin for 7 days and embedded in paraffin. Serial coronal sections of the PFC (20 lm) were cut at the level of the mPFC (Fig. 4A). For immunohistochemistry, deparaffinized sections were heated in citrate buffer for antigen retrieval at 92 °C, prior to overnight incubation with the primary antibodies. Antibodies were directed against neuronal nuclei (NeuN) (1/500, mouse monoclonal; Chemicon, Temecula, CA, USA) and glial fibrillary acidic protein (GFAP) (1/1000, mouse monoclonal; Sigma–Aldrich, Saint-Quentin Fallavier, France). The antibodies we chose have been previously used in the rat brain to identify neurons (for review see Sanchez et al., 2009) and astrocytes (for review see Del Carmen Gómez-Roldán et al., 2008); their characterizations are indicated in the Supplementary material. These antibodies were detected using an avidin–biotin–horseradish peroxidase kit (Vector), as instructed by the manufacturer. Diaminobenzidine was used as a chromogen (Gressens et al., 2008). To test the specificity of the secondary antibodies, sections were processed without first antibodies and displayed no immunoreactivity. The evaluation of the number of neurons and astrocytes in the PrL was carried out using the Stereoinvestigator software program (MicroBrightField, Williston, VT, USA) associated to a Leica DMR microscope. The software program involves the combining of optical dissector counting with fractionator sampling (Schmitz & Hof, 2005). Cell counting was performed on 7–9 sections per rat, spaced by 200 lm. Uniform grid areas were placed over the PrL on sections labeled with anti-NeuN antibodies (350  350 lm2) and anti-GFAP antibodies (275  275 lm2). The size of the sampling areas (optical dissector) was 50  50 lm2 for NeuN labeling and 75  75 lm2 for GFAP labeling. The software program automatically and randomly moves from sampling areas that include appropriate ‘‘acceptance’’ and ‘‘forbidden’’ lines. Only cells, which fell within the sampling area or were touching the ‘‘acceptance’’ line, were counted. In the same way, a cell was only taken in ac-

2.6. Electrophysiology procedures

2.7. Statistical analysis Differences among groups were tested for significance by oneway ANOVA with ‘‘MD’’ as variable, and ‘‘stages of discrimination’’ and ‘‘daily trial’’ as repeated measures for the ASST and the WMT. Post hoc analyses were performed when required by Fisher’s least significance difference test. Regarding electrophysiological results, ANOVA repeated measures were used to compare the post-episode postsynaptic potentials changes between groups. Two-tailed Student t-test was used to compare the percent changes occurring at the end of the 3-h recording period post-tetani (i.e. last 15 min period) between groups. 3. Results 3.1. Object temporal order memory performance During the sample phases, AFR and D rats spent the same total time exploring both objects (data not shown). During the test phase, the total amount of time to explore both ‘‘old’’ and ‘‘recent’’ objects was again similar (Fig. 1A). However, the discrimination ra-

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tio was significantly lower in D rats in comparison with AFR rats (P < 0.01) (Fig. 1B). Indeed, the AFR rats spent less time exploring the recent object that the old object (respectively 3.3 ± 0.6 s and 6.2 ± 0.9 s, P < 0.05) but not the D rats (6.8 ± 1.5 and 5.9 ± 1.3). It was noticed no difference in body weight between AFR and D rats the day of the test. It was the same for all the different tests presented in this article (data not shown). 3.2. Attentional set-shifting performance The restricted diet did not result in any difference in weight gain between AFR and D rats (data not shown). The D rats differed significantly from AFR rats in their overall performance in the task (F(1, 22) = 22.7 ; P < 0.001). More specifically, there was a significant interaction between the factors ‘‘MD’’ and ‘‘stages of discrimination’’ (F(6, 132) = 3.6; P < 0.01). In comparison with AFR rats, D rats required a higher number of trials to criterion for three stages of discrimination, compound discrimination (P < 0.01), Reversal 1 (P < 0.001) and extradimentional shift (P < 0.05) (Fig. 2).

Fig. 2. AFR and D rats performance in the attentional set-shifting task. The number of trials to reach the criterion of six consecutive correct trials (trials to criterion) was significantly higher during the CD, Rev1 and ED stages in the D rats in comparison with AFR rats. Results are expressed as mean ± SEM, n = 12.  P < 0.05,  P < 0.01 and  P < 0.001 vs. AFR rats. Discrimination stages: CD, compound discrimination; ED, extradimensional shift; ID, intradimensional shift; Rev1, reversal 1; Rev2, reversal 2; Rev3, reversal 3; SD, simple discrimination.

3.3. Memory performance in the WMT The latency to escape to the platform decreased over the five consecutive days similarly in AFR and D rats (F(4, 88) = 56.5, P < 0.001) (Fig. 3A). The distance traveled decreased in parallel, with no significant changes in the the swimming speed between the two groups (data not shown). During the one probe trial (day 8), the time spent visiting each quadrant was different according to the quadrant (F(3, 66) = 6.8, P < 0.01). AFR and D rats spent a significantly higher amount of time in the quadrant previously containing the platform (>25%) in comparison with the other quadrants (data not shown), without differences between groups (Fig. 3B). 3.4. Stereological counting of neurons and astrocytes in the PrL We established that the parameters related to the counting (i.e. the number of investigated section, the section thickness, the number of sampling areas, the number of counted cells and the Schaeffer coefficient) were accurate and identical in AFR and D rats (Table 1). The estimated volume of the PrL (Fig. 4A) was not different between AFR and D rats (respectively, 3.00 ± 0.09 mm3 and 2.97 ± 0.06 mm3). The D rats showed no difference in the number of NeuN immunoreactive neurons (Fig. 4B) and GFAP immunoreactive astrocytes (Fig. 4C) in the PrL in comparison with AFR rats (Fig. 4D and E).

Fig. 3. AFR and D rats performance in the Morris water maze task. (A) Through the course of the WMT, both the AFR and D rats learned the task as demonstrated by a significant day effect for escape latency. (B) During the 90-s probe trial, AFR and D rats spent a similar percent of time in the quadrant that previously had contained the hidden platform. Results are expressed as mean ± SEM, n = 12.

3.5. Effect of MD on the LTP of the HPC-mPFC pathway Tetanic stimulation to ventral HPC induced clear increases of the amplitude of the HPC-mPFC responses in both groups. Analysis with ANOVA repeated measures revealed significantly larger LTP in the D rats as compared to the AFR rats during the 3 h post-tetani period: (F(1, 13) = 19.3, P < 0.001 (Fig. 5). Two-tailed t-test indicated a significant difference in the response increase at 3 h after tetani between these two groups (P < 0.01). Thus, MD facilitated the induction of LTP in HPC-mPFC pathway. 4. Discussion

Fig. 1. AFR and D rats performance in the object temporal order memory task. (A) During the test phase, the total time of exploration was not different between AFR and D rats. (B) In contrast with AFR rats, D rats failed to discriminate between the two objects as indicated by the discrimination ratio. Results are expressed as mean ± SEM, n = 11.  P < 0.01, vs. AFR rats.

We showed that MD produced cognitive deficits in the TMT and the ASST, two PFC-dependent tasks, but not in HPC-dependent WMT, in adult male Long-Evans rats. In parallel, MD was also found to significantly impact on synaptic plasticity, by upregulating LTP in the HPC-mPFC pathway. In the TMT, in contrast with AFR rats, D rats were unable to discriminate between old and recent objects. Recently, it has been shown that separated and non-separated rats were both able to perform successfully in the TMT (Grace et al., 2009). However, in this latter study, conducted with Sprague–Dawley rats, the pups were not individually isolated, a condition that is known to represent a much less severe manipulation than the present MD (McCormick, Kehoe, & Kovacs, 1998). Previously, we have reported unaffected performance in the object recognition task in D rats (Mourlon et al., 2010). Interestingly, this is consistent with the observation that the specific inactivation of the PrL area of the

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Fig. 4. Number of NeuN and GFAP immunoreactive cells in the prelimbic region of the medial prefrontal cortex of AFR and D rats. (A) Schematic delineation of the prelimbic (PrL) and infralimbic (IL) subregions of the medial prefrontal cortex (mPFC). Serial histological sections were defined from their anteriority from bregma, (anteriorposteriority 4.20 mm to 2.52 mm according to Paxinos and Watson (2005). In the PrL, (B) NeuN immunoreactive cells presented typical morphology of neurons and (C) GFAP immunoreactive cells displayed star-shaped morphology of astrocytes (AFR rat). Immunoreactive cells were indicated with white arrows. Scale bar = 50 lm. (D) NeuN immunoreactive neurons and (E) GFAP immunoreactive astrocytes in the PrL were in equal number in AFR and D rats. Results are expressed as mean ± SEM, n = 7.

Table 1 Parameters related to counting conditions of anti-Neun and anti GFAP-labeled cells in the prelimbic region of the medial prefrontal cortex.

NeuN AFR D GFAP AFR D

Number of investigated sections

Section thickness

Number of sampling areas

Number of counted cells

Schaeffer coefficient of error

8.1 ± 0.1 8.1 ± 0.1

13.4 ± 0.6 13.2 ± 0.5

174.3 ± 6.6 170.7 ± 4.1

555.9 ± 19.1 533.8 ± 13.1

0.039 ± 0.002 0.039 ± 0.001

14.7 ± 0.5 14.6 ± 0.2

235.9 ± 7.8 235.4 ± 5.3

411.6 ± 8.2 408.7 ± 9.5

0.039 ± 0.001 0.039 ± 0.001

8 8

None of the parameters listed in the table above, was different between AFR and D rats. Mean ± SEM, n = 7.

mPFC disrupts the performance in the TMT but not in the object recognition task (Hannesson et al., 2004). To our knowledge, the consequences of a MS on the cognitive flexibility in ASST had never been studied. The learning by D rats of the odor-reward associations in the ASST and escape-platform in the WMT confirmed that MD did not preclude in adulthood the ability to learn a rule (Aisa et al., 2007; Grace et al., 2009; Hui et al., 2011; Lai et al., 2006; Mello et al., 2009; Pryce et al., 2003; Zhu et al., 2010). Although AFR and D rats were capable to perform the seven successive stages of the ASST, D rats showed a weaker overall performance, which results in a significant cognitive deficit during the compound discrimination, reversal 1 and extradimentional shift. These three stages of discrimination are characterized by the appearance of a new level of complexity never encountered before by the rats: i.e., in compound discrimination, a new perceptual dimension disconnected of the reward is introduced; in reversal 1, the rules applied previously become no longer valid; and in extradimentional shift, the previously relevant perceptual dimension must be disregarded to shift to a new dimension (Birrell & Brown, 2000). D rats seemed to have a reduced

ability to adapt to the introduction of each of these new levels of complexity, i.e. to switch attention and to perform strategy changes. In contrast, MD did not appear to prevent the retention of the learning necessary for the resolution of a next stage in the ASST. Indeed, when the D rats were confronted with a difficulty equivalent to that encountered previously (for instance reversal 2 vs. reversal 1), their performance did not significantly differ from those of AFR rats. The role of the PFC in processing events that are novel and in adapting flexibility to a changing environment has been well demonstrated (Floresco, Ghods-Sharifi, Vexelman, & Magyar, 2006; Kehagia, Murray, & Robbins, 2010). Brown and colleagues reported that the lesion of the PrL, a sub-region of the mPFC, impaired the extradimentional set-shifting, while the lesion of the orbital PFC impaired reversal learning. However, the difficulties to achieve the extradimentional shift or reversal stages were only transitory, and rats ended eventually to complete the various stages of the ASST (Birrell & Brown, 2000; McAlonan & Brown, 2003). This may be because, although the PFC plays a key role, the cognitive performances in the ASST are dependent on neural circuits that include several other interconnected brain regions

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Fig. 5. Effect of MD on the LTP of the HPC-mPFC pathway. Each point represents mean ± SEM of averaged responses to four test stimuli given at 30 s intervals. Values are expressed as the percentage changes relative to the baseline (30 min). Tetanic stimulation (two series) is indicated by the arrow and was applied after the baseline period in the D rats (n = 8) and the AFR rats (n = 7). MD procedure induces a significant facilitation of LTP induction compared to the AFR group (p < 0.001, ANOVA). Representative averaged waveforms were taken 3 h after LTP induction in an AFR rat (gray) and in a D rat (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

such as the nucleus accumbens (Floresco et al., 2006), thalamus (Block, Dhanji, Thompson-Tardif, & Floresco, 2007) and ventral HPC (Marquis et al., 2008). MD had no effects on spatial cognition in the WMT in adulthood for both the learning of the platform position and the spatial retention memory. The consequences of MS on the performance in this task have been controversial. For the learning they ranged from negative (Zhu et al., 2010) to positive (Pryce et al., 2003) via neutral effects (Aisa et al., 2007; Grace et al., 2009; Hui et al., 2011; Lai et al., 2006; Mello et al., 2009). As well, the retention memory could be impaired (Aisa et al., 2007; Hui et al., 2011; Mello et al., 2009) or preserved (Grace et al., 2009; Lai et al., 2006). It has been reported that the mPFC could participate in spatial navigation through its interactions with the hippocampus (Churchwell, Morris, Musso, & Kesner, 2010; Wang & Cai, 2008) and that its lesion or inactivation impaired the WMT performance (Compton, Griffith, McDaniel, Foster, & Davis, 1997; Wang & Cai, 2008). However, several other studies contradicted these points (Granon & Poucet, 1995; Lacroix, White, & Feldon, 2002; Sullivan & Gratton, 2002). In particular, de Bruin and colleagues (1994, 2001), using protocols with either invisible or visible platforms, demonstrated that mPFC was required for strategy flexibility but not for spatial learning and memory. Moreover, recently it was shown that the mPFC participates in remote memory retrieval (25 days) in the WMT but not in recent memory retrieval (5 days) (Lopez et al., 2012). Taken together, these behavioral data suggest that the temporal memory and attentional set-shifting deficits observed in adult D rats may reflect dysfunction of the PFC and networks in which it is involved. Recently, memory deficits in a delayed win-shift procedure that is dependent on the PFC has been observed in adolescent MS rats (Brenhouse & Andersen, 2011). The lack of effect of MD on the performance in the WMT may result from the lesser involvement of the PFC in the completion of this task. The number of neurons and astrocytes assessed in the mPFC were similar in D and AFR rats. In a previous study, it has been described that MS induced a reduction of GFAP-immunoreactive astrocytes in brain regions, including the PFC, implicated in stress-related behavior in Fischer male rats (Leventopoulos et al., 2007). It is worthy of note that in contrast to what was observed in this strain, MD did not result in reduced sucrose consumption, a depressive-like behavior, in Long-Evans male rats (Mourlon

et al., 2010; Rüedi-Bettschen et al., 2006). The stress induced by a MS likely operates differently during the development depending on the different strains. Indeed, a recent study highlighted the critical role of genetic predisposition on the effect of MS in rat (Sterley, Howells, & Russell, 2011). In our study, the potential role played by the PFC in cognitive impairments caused by MD, does not appear to be dependent on the number of neurons and astrocytes in this region. Instead, it could be related to change in cell morphology, such as the length and numbers of dendrites and astrocyte branches. Studies of the dendritic morphology in MS models have produced conflicting results, since MS induced either decreases in dendritic length and spine density (Monroy et al., 2010; Pascual & ZamoraLeon, 2007) or increases in spine density (Muhammad & Kolb, 2011). Changes in astroglia-neurone interactions could be also involved since astrocytes are integral functional elements of the synapses, responding to neuronal activity and regulating synaptic transmission and plasticity (for review see Araque & Navarrete, 2010; Sidoryk-Wegrzynowicz, Wegrzynowicz, Lee, Bowman, & Aschner, 2011). Under our experimental conditions, we were not able to examine these hypotheses, but we tested for the first time to our knowledge, the possible change in synaptic plasticity in the HPC-mPFC pathway in MS models. Remarkably, we found that D rats show significantly facilitated LTP in the HPC-mPFC synaptic responses in comparison to AFR rats. Because LTP is a well-established neuronal model underlying learning and memory, the facilitation of LTP induction was more likely expected to be accompanied by an improved performance in the cognitive tasks. It could also be conceivable that, in D rats, the enhancement in LTP underlay an enhanced learning of an initial event which led to the deficits in the following learning. However, to our knowledge, this hypothesis and, more generally, how an increased LTP could be correlated with cognitive impairments have never been investigated. On the other hand, we observed that the performance during the SD, i.e. the initial learning stage in ASST, was similar in D and AFR rats. There are examples in the literature that link neuronal plasticity induction to behavioral deficits. For example, an enhanced plasticity induction in the hippocampus can be correlated with spatial learning impairments (Han, Tian, Zhang, Ren, & Yang, 2011; Kaksonen et al., 2002; Moser, Krobert, Moser, & Morris, 1998). Moreover, it has been shown that prenatal cocaine exposure resulted in enhanced LTP induction in mPFC in vitro (Huang, Liang,

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& Hsu, 2011), and cognitive deficits including impairments in memory (Morrow, Elsworth, & Roth, 2002; Thompson, Levitt, & Stanwood, 2005) and flexibility (Garavan et al., 2000). Regarding MS models, MD also has been shown to result in a modification of synaptic plasticity in different brain regions in adulthood: i.e. it enhanced LTP in the hippocampus (Kehoe & Bronzino, 1999) and both LTP and long-term depression in basolateral amygdaladentate gyrus pathway (Blaise, Koranda, Chow, Haines, & Dorward, 2008). However, how MD induced a facilitation of LTP induction in the HPC-mPFC pathway remains to be elucidated. It has been found that early separation induced an up-regulation of neuronal plasticity-related genes in the PFC (Benekareddy et al., 2010). Interestingly, LTP in HPC-mPFC pathway is NMDA receptor-dependent (Jay, Burette, & Laroche, 1995), and previous reports suggested that LTP deficits or saturation occur under increased level of basal NMDA receptor activity in certain pathological conditions, which are amenable to reversal by the weak NMDA receptor-blocking drug, memantine (Frankiewicz & Parsons, 1999). In this regard, conversely, a reduced basal neuronal activity in the mPFC of MS rats (Stevenson et al., 2008) may have resulted in the facilitation of LTP induction in these rats. Indeed, it is possible that early MS permanently affects the synaptic plasticity since the first 2–3 postnatal weeks are a critical period for NMDA receptor expression and establishment of synaptic connections (Babb et al., 2005). 5. Conclusion Our data showed that early life events, such as MD, might trigger alteration of PFC-dependent cognitive performance in adulthood. Moreover, we show for the first time in an early stress rodent model, an enhanced LTP in the mPFC associated with the impairments in these cognitive tasks. Further studies should be conducted to determine the neurobiological mechanisms in the PFC underlying the association between childhood trauma and impaired cognitive performance in adulthood. Particularly, it would be important to understand how the facilitation of LTP in the MD rats disturbs the cognitive performance in these rats. A better understanding of how neuronal plasticity is modified by early stress events could generate therapeutic targets for mood disorders. Acknowledgement Leslie Schwendimann for her technical advice; Myriam Richer for her participation in immunohistochemistry studies. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nlm.2012.08.004. References Aisa, B., Tordera, R., Lasheras, B., Del Rio, J., & Ramirez, M. J. (2007). Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology, 32, 256–266. Araque, A., & Navarrete, M. (2010). Glial cells in neuronal network function. PhilosophicalTransactions Royal Society London B: Biological Science, 365, 2375–2381. Arnsten, A. F. (2009). Stress signalling pathways that impair prefrontal cortex structure and function. Nature Reviews Neuroscience, 10, 410–422. Babb, T. L., Mikuni, N., Najm, I., Wylie, C., Olive, M., Dollar, C., et al. (2005). Pre- and postnatal expressions of NMDA receptors 1 and 2B subunit proteins in the normal rat cortex. Epilepsy Research, 64, 23–30. Benekareddy, M., Goodfellow, N. M., Lambe, E. K., & Vaidya, V. A. (2010). Enhanced function of prefrontal serotonin 5-HT(2) receptors in a rat model of psychiatric vulnerability. Journal of Neuroscience, 30, 12138–12150. Benetti, F., Mello, P. B., Bonini, J. S., Monteiro, S., Cammarota, M., & Izquierdo, I. (2009). Early postnatal maternal deprivation in rats induces memory deficits in

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