Maternal sleep deprivation inhibits hippocampal neurogenesis associated with inflammatory response in young offspring rats

Neurobiology of Disease 68 (2014) 57–65

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Maternal sleep deprivation inhibits hippocampal neurogenesis associated with inflammatory response in young offspring rats Qiuying Zhao a, Cheng Peng b, Xiaohui Wu a, Yubo Chen a, Cheng Wang a, Zili You a,⁎ a

School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China

b

a r t i c l e

i n f o

Article history: Received 31 October 2013 Revised 8 April 2014 Accepted 14 April 2014 Available online 24 April 2014 Keywords: Sleep deprivation Pregnancy Offspring Neurogenesis Microglial activation Inflammatory response

a b s t r a c t Although sleep complaints are very common among pregnant women, the potential adverse effects of sleep disturbance on the offspring are not well studied. Growing evidence suggests that maternal stress can induce an inflammatory environment on the fetal development. But people are not sure about the consequences of prenatal stress such as the inflammatory responses induced by maternal sleep deprivation (MSD). In the present study, we investigated the effects of MSD on long-term behavioral and cognitive consequences in offspring and its underlying inflammatory response pathway. The pregnant Wistar rats received prolonged sleep deprivation (72 h) on gestational day (GD) 4, 9, and 18, respectively. The post-natal day (PND) 21 offspring showed impaired hippocampus-dependent spatial learning and memory in the Morris Water Maze task and anhedonia in sucrose preference experiment. Quantification of BrdU+ and DCX+ cells revealed a significant decrease in hippocampus neurogenesis in prepuberty offspring, especially for the late MSD (GD 18) group. Real-time RT-PCR showed that after MSD, the expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNFα) increased in the hippocampus of offspring on PND 1, 7, 14 and 21, whereas anti-inflammatory cytokine IL-10 reduced at the same time. Immunofluorescence found that the cells of activated microglia were higher in the brains of MSD offspring. Taken together, these results suggested that the MSD-induced inflammatory response is an important factor for neurogenesis impairment and neurobehavioral outcomes in prepuberty offspring. © 2014 Elsevier Inc. All rights reserved.

Introduction Probably due to pregnancy-associated anatomic, physiological and hormonal changes, women in pregnancy are at particular risk of sleep deprivation or restriction (Pien and Schwab, 2004). The quality of sleep usually deteriorates with the increasing gestational week (Kizilirmak et al., 2012). Recently there has been a growing research interest in the relationship between maternal sleep deprivation (MSD) and development of short- and long-term health disorders of their offspring. Research suggested that MSD can lead to several harmful consequences to their children, and can damage the mother-infant relationship (Pires et al., 2010). It is found to cause a reduction in adrenal weight and ambulatory behavior in pups (Suchecki and Palermo Neto, 1991). Rapid eye movement sleep deprivation in pregnant rats can cause major impairment of masculine behavior in male offspring during adulthood (VelazquezMoctezuma et al., 1993). Sleep restriction during pregnancy may lead to renal morphologic and functional alterations in young offspring (Thomal et al., 2010). MSD is also found to cause anxiety behaviors

⁎ Corresponding author. Fax: +86 28 83208838. E-mail address: [email protected] (Z. You).Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2014.04.008 0969-9961/© 2014 Elsevier Inc. All rights reserved.

(Calegare et al., 2010) and alter sexual behavior of the F1 offspring (Alvarenga et al., 2013). Though sleep deprivation has been shown to cause biochemical and behavioral changes in offspring (Calegare et al., 2010), mechanisms underlying such effects remain largely unknown. Previous studies have demonstrated that gestational stress, including MSD, has long-lasting effects on the HPA axis in the offspring (O'Keane et al., 2012). Maternal stress also increases the inflammatory response in their offspring, which may increase the risk of schizophrenia, autism and bipolar disorder (Andersen and Teicher, 2009; Bale, 2009). More specifically, it is suggested that secretion of inflammatory cytokines, such as IL-1β and IL-6, mediates the neurodevelopmental effects on the offspring (Ashdown et al., 2006; Chang et al., 2010; Meyer et al., 2008). Neurogenesis from neural progenitor cells in the brain is clearly established in two regions including the dentate gyrus (DG) of the hippocampus and the subventricular zone of the lateral ventricle (Gould, 2007). Here, the environment of neural stem cells (NSCs) composed of neighboring cells and soluble factors regulates their proliferation and differentiation (Gage, 2002). Among the factors, microglia, the resident macrophages of the brain, is one of the most important elements (Sierra et al., 2010). Microglial activation and secretion of inflammatory cytokines play a central role as modulators of the NSC microenvironments (niches) in different processes, such as proliferation,

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differentiation, survival and migration (Perry et al., 2007). Once overactivated in embryonic period, microglia continues to be primed and influences neuron survival into adulthood (Block et al., 2007; Carvey et al., 2003). Prenatal stress leads to long-lasting microglial activation during postnatal development and has a long term impact on the adult neurobehavioral performance (Harry and Kraft, 2012). Among stages of the life, prepuberty is highly sensitive to stress (Perlman et al., 2007) and exhibits many neuropsychiatric disorders (Fisher et al., 2010). In light of this, the present study aimed to determine whether MSD could programmingly affect neurodevelopment and behavior of prepuberty offspring, and whether this stress disorder is related to microglial activation and inflammatory response in the affected offspring.

Materials and methods Animals Three-month-old Wistar rats were obtained from Chengdu Dossy Biological Technology Limited Company (Chengdu, China), weighing 250–270 g (female) and 300–330 g (male), respectively. The animals were housed individually in the room with temperature 23–25 °C and humidity 50–60%, on a 12 h light/dark cycle (lights on 08:00–20:00), with water and food available ad libitum. The animals were allowed to habituate to the environment for one week. For breeding, 2 females were brought together with one male. Successful mating was verified the next morning by the presence of a vaginal plug, and the day was determined as gestational day (GD) 0. After mating, the pregnant females were housed individually and used in the sleep deprivation experiment. When weaned, the male offspring (21 days old) were used in the behavioral and molecular tests. Sleep deprivation Sleep deprivation was induced using modified small-platform method (Mirescu et al., 2006). Pregnant Wistar rats (n = 47) for sleep deprivation were individually housed in the experiment device with 8 small platforms (20 cm in height and 6 cm in diameter) surrounded by roomtemperature water in a tank. The water level was about 1 cm below the edge of the platform. The rats fell into the water if they lost muscle tone, forcing them to climb back on the platform and remain awake. All platform exposure began at 10:00 a.m. and ended after 72 h. The control pregnant rats were placed in the experiment device with 4 big platforms (20 cm high and 15 cm diameter). All rats had free access to food and water. After sleep deprivation, the pregnant females were put back to their room individually. The animals were divided into four groups: Early MSD at GD 4 (Early MSD) (n = 15), Middle MSD at GD 9 (Middle MSD) (n = 15), Late MSD at GD 18 (Late MSD) (n = 17), and control group (n = 12). Male offspring were examined when weaning on the 21 d (1–3 male pups from each mother per group). The offspring were divided into four groups: offspring from Early MSD (n = 18), from Middle MSD (n = 15), from Late MSD (n = 30) and the control group (n = 30). All experimental procedures were approved by the Institutional Animal Care and Use Committee, University of Electronic Science and Technology of China.

Behavioral tests All behavior experiments were performed by an experimenter blind to the identity of experimental groups. Body weight was measured for the young offspring rats of the control (n = 10) and the late MSD group (n = 10), then the sucrose preference test was done for these two groups of young offspring rats. The MWM experiment was done with male offspring rats of the control (n = 10), the early MSD (n = 12), the middle MSD (n = 9) and the late MSD group (n = 10).

Body weight and sex ratio Body weight of the control and MSD offspring was measured once a week until weaning (21 days old). It was measured at 10:00 a.m. on Monday every week. The ratio between male and female pups (sex ratio) was determined at weaning (21 days old). Sucrose preference test Before testing, the offspring (21 days old) were deprived of food and water for 20 h. Intake of water or sucrose solution (1%) was measured by weighing the respective container before testing and 2 h after. In each trial, the containers of water and sucrose solution were randomly placed at either the left or the right side of the cage. The sucrose preference was calculated according to the following ratio: SP = sucrose intake (g)/[sucrose intake (g) + water intake (g)]. Morris Water Maze (MWM) The male offspring rats (21 days old) were given four trials per day, for consecutively four days, to find a hidden 9 cm diameter platform located 2 cm below the water surface in a pool 1 m in diameter (Derecki et al., 2010). The water temperature was kept at 20–23 °C. Within the testing room, only distal visual shape and object cues were available to the rats to aid in locating of the submerged platform. The escape latency, i.e., the time required by the rat to find and climb onto the platform, was recorded for up to 60 s. If the rat did not find the platform within 60 s, it was placed on the platform and returned to its home cage after 10 s. The inter-trial interval for each rat was 5 min. On day 5, the platform was removed from the pool, and each rat was tested by a spatial probe trial for 60 s. On days 6 and 7, the platform was placed in the quadrant opposite the original training quadrant, and the rat was retrained for four sessions each day. Data were recorded using the EthoVision automated tracking system (Noldus Information Technology). All MWM testing was performed between 9 a.m. and 2 p.m. during the lights-on phase. Immunohistochemistry The offspring rats (21 days old) from the control (n = 6) and the MSD group (n = 6 for each MSD group) used in the neurogenesis test were also studied in the microglia experiment. To label proliferating cells in the brain, 21 d male rats were given two injections (8:00 and 16:00) of BrdU (50 mg/kg, i.p., Sigma), for consecutively two days. Two hours after the last BrdU administration, the rats were deeply anesthetized with pentobarbital sodium. The rats were then perfused transcardially with pH 7.2 phosphate-buffered saline (PBS) and 4% paraformaldehyde. Brains were collected and postfixed with 4.0% paraformaldehyde for 48 h, then in 30% sucrose for 24 h. The brain samples were cut into 35 μm coronal sections on a sliding vibratome (CM1900; Leica Microsystems, Wetzlar, Germany). Six sequential slices were placed into each well of a 12-well plate containing 0.03% sodium azide in PBS and stored at 4 °C. The sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min, and then treated with 2 N HCl for 30 min at 37 °C followed by wash with 0.1 M borate buffer (pH 8.5). The sections were placed in 10% donkey serum in PBS for 2 h, incubated with primary antibodies overnight at 4 °C, then incubated with fluorescent-dye-conjugated secondary antibodies. For DCX, Iba1 and IL-6 labeling, brain slices were permeabilized with 0.5% Triton X-100 in PBS for 10 min, and then wash with 0.01 M PBS (pH 7.2). The sections were placed in 10% donkey serum in PBS for 2 h, incubated with primary antibodies overnight at 4 °C, then incubated with fluorescent-dye-conjugated secondary antibodies. The primary antibodies were mouse anti-BrdU (1:500; Cell Signaling Technology), goat anti-DCX (1:400; Santa Cruz), goat anti-Iba1 (1:400; Abcam) and mouse anti-IL-6 (1:300; Abcam). The second antibodies were DyLight 488-conjugate donkey anti-mouse (1:300; Jackson

Q. Zhao et al. / Neurobiology of Disease 68 (2014) 57–65

ImmunoResearch) and DyLight 549-conjugate donkey anti-goat (1:300; Jackson ImmunoResearch). The volumes of the neurogenesis subregions in the hippocampus, the DG and granular cell layer (GCL), were estimated on the basis of the Cavalieri principle with every sixth section stained with DAPI antibody. The volume measurements were performed using Image J software (version 1.45 J; National Institutes of Health, Bethesda, MD, USA). The areas of DG and GCL were determined by summing up the computed areas and multiplied with the cutting thickness of the sections and the number of series (Czeh et al., 2010; Novati et al., 2011)

59

immunohistochemistry. Differences were considered significant when p b 0.05. Analyses were conducted using SPSS for Windows® v.17 (SPSS Inc., Chicago, USA).

Results MSD altered spatial learning and memory of prepuberty offspring

To quantify proliferating cells, every sixth section of the brain containing hippocampus was selected and immunolabelled with BrdU and DCX antibodies. Total numbers of positive cells in all slices per animal were multiplied by six to estimate the number of cells per DG (Lu et al., 2011). Ionized calcium-binding adaptor protein-1 (Iba1) was used to assess microglia state based on Iba1 positive stain and cell morphology. Three cell types of microglial activation were defined as follows (Kreutzberg, 1996): cell type I, defined as resting microglial cells, have rod-shaped cell bodies with fine, ramified processes; cell type II have small cell bodies with long thin processes; cell type III, defined as activated microglial cells, have large somas, short thick processes and a rounded amoeboid morphology. Photomicrographs were saved as TIF files without any manipulation, and quantitatively analyzed with the cell counter of Image J software (Version 1.45 J; National Institutes of Health, Bethesda, MD, USA). Positive cells were manually counted using a 40× objective (Olympus BX51).

Spatial learning and memory was assessed using the MWM in prepuberty offspring on post-natal day (PND) 21 through the hidden platform version of this task. There were no significant differences among the four groups in latency to locate the platform on day 1. However, late MSD offspring took significantly longer to find the hidden platform than the control on days 2–4 (Fig. 1A; day 2: F (3, 28) = 6.14, p = 0.001; day 3: F (3, 28) = 3.08, p = 0.032; day 4: F (3, 28) = 6.04, p = 0.001). Early MSD offspring had longer latency than the control on day 4. When the platform was removed for the probe trial on day 5, the middle and late MSD offspring rats spent significantly less time in the training quadrant than the control rats (Fig. 1B; F (3, 28) = 3.54, p = 0.018). To further characterize memory deficits, we also examined the times that the offspring rats crossing the platform in a circular area circumscribing the original platform location, finding that the number of platform crossing was significantly fewer in the late MSD offspring than other groups (Fig. 1C; F (3, 28) = 6.45, p = 0.008). On the reverse trial, when the platform was placed in the quadrant opposite to the original, the increased latency to locate the platform was recorded for early and late MSD groups on day 6 (Fig. 1D; F (3, 28) = 3.44, p = 0.047), but only late MSD rats on day 7 (Fig. 1E; F (3, 28) = 3.32, p = 0.048).

Real time PCR

Effect of MSD on neurogenesis in offspring rats

Male offspring rats (21 days old, n = 8 for each experimental group) were sacrificed by decapitation and the brain was removed using aseptic techniques. The hippocampus was quickly dissected out, placed in sterile tubes, and frozen on dry ice. Total RNA extraction was performed using Trizol reagent (Invitrogen Life Technologies, USA) according to the manufacturer’s protocol. The RNA samples were suspended in 30 μl nucleasefree water. The first strand cDNA was synthesized with the First Strand cDNA Synthesis Kit (Invitrogen Life Technologies, USA). For each reaction, 5 μg RNA was used for reverse transcription, in a mixture of 1 μl oligo (dT) 12-18, 1 μl random primer, and 1 μl dNTP mixture with a final volume of 12 μl, incubated at 65 °C for 5 min, and rapidly cooled on ice. Then the tube was added the following components: 4 μl 5× First strand synthesis buffer, 1 μl Ribonuclease inhibitor and 2 μl DTT. The samples were incubated at 37 °C for 2 min. Then the 1 μl (200 U) of M-MLV reverse transcriptase was added to the final volume of 20 μl at room temperature. The mixture was incubated at 37 °C for 50 min, and heated at 70 °C for 15 min to terminate reaction. The cDNA was stored at −20 °C. All PCR reactions were under the following condition: initial 98 °C for 2 min followed by 38 cycles at 98 °C for 2 s and 60 °C for 10 s (Bio-Rad CFX 96). Each sample was tested in triplicate. The values were normalized against the housekeeping genes GAPDH. Primer sequences were as follows: TNFα, 5 -CATCTTCTCAAAATTCGAGTGACAA-3 , 5 -GGGAGTAG ACAAGGTACAACCC-3 ; IL-1β, 5 -CCCTGCAGCTGGAGAGTGTGG-3 , 5 TGTGCTCTGCTTGAGAGGTGCT-3 ; IL-6, 5 -TCTTGGGAC-TGATGCTGGTG3 , 5 -CAGAATTGCCATTGCACAACTC-3 ; IL-10, 5 -AATTCCCTGGGTGAGA AGCTG-3 , 5 -TCATGGCCTTGTAGACACCTTG-3 ; GAPDH, 5 -ATGACCCCTT CATTGACCTCA-3 , 5 -GAGATGATGACCCTTTTGGCT-3 .

To determine whether 72 h MSD affects neurogenesis of offspring rats, their brain sections were stained for BrdU and DCX (Fig. 2A). Total number of BrdU-positive cells in DG did not differ between the control and the three MSD groups (Fig. 2B). However, quantification of BrdU+ and DCX+ cells were significantly less in the middle and late MSD offspring compare to the control rats (Fig. 2C; F(3, 10) = 38.03, p b 0.001), and a lower ratio of BrdU+ DCX+/BrdU+ in the same two groups (Fig. 2D; F(3, 10) = 69.57, p b 0.001). Since the results demonstrated that the late perinatal period is more sensitive compared to the other two stages, we decided that later experiments involved only the late MSD group and the control. The volumes of hippocampus subregions were measured in DAPI stained sections. The outlines of DG and GCL were showed in Fig. 2E and F, respectively. MSD did not change the volumes of DG and GCL in the hippocampus of prepuberty offspring (Fig. 2G; DG: F(1, 10) = 1.632, p = 0. 230; GCL: F(1, 10) = 2.079, p = 0. 658).

Quantitation of neurogenesis and microglia

Statistical Analysis All data were presented as mean ± SEM. The results of the MWM and real time PCR were analyzed with two-way ANOVA. One-way ANOVA followed by Tukey post hoc test was used to compare the results of the weight, litter size, sex ratio, preference of sucrose and

MSD aroused inflammation response in prepuberty offspring On PND 1, IL-1β, TNFα and IL-6 were all higher in the late MSD group (IL-1β: F(1, 15) = 3.47, p = 0.074; TNFα: F(1, 15) = 44.69, p b 0.001; IL-6: F(1, 15) = 6.46, p = 0.029). On PND 7, only IL-6 was higher (IL1β: F(1, 15) = 2.57, p = 0.130, TNFα: F(1, 15) = 2.21, p = 0.165, IL-6: F(1, 15) = 4.94, p = 0.045). On PND 14, IL-6 was higher in the late MSD group (IL-1β: p = 0.854, TNFα: p = 0.897, IL-6: p = 0.130). On PND 21, IL-6 was higher in the late MSD group than the control, however, IL-1β was lower in the late MSD group (IL-1β: F(1, 15) = 13.34, p = 0.002, TNFα: F(1, 15) = 0.33, p = 0.574, IL-6: F(1, 15) = 5.76, p = 0.032). The result showed that IL-6 was higher in the late MSD group than the control at different weeks. The expression of IL-10 was lower in the late MSD group during the same time (PND 1: F(1, 15) = 5.72, p = 0.029; PND 7: F(1, 15) = 10.48, p = 0.006, PND 14: F(1, 15) = 0.48, p = 0.500, PND 21: F(1, 15) = 6.24, p = 0.027).

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Q. Zhao et al. / Neurobiology of Disease 68 (2014) 57–65

A

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Fig. 1. MSD impaired spatial learning and memory in the MWM task. During the task acquisition (A) the late MSD offspring spent significantly longer time than the control to locate the hidden platform on day 2, 3, and 4. Early MSD offspring rats also required more travel distance to reach the hidden platform than the control animals. A probe trial was performed on day 5, middle and late MSD rats spent longer to successfully locate the original platform location (B), the number of platform crossing was reduced only in the late MSD group (C). Early MSD on day 6 (D), late MSD on day 6 and 7 (D, E) showed significantly increased time in reversal phases. n (Control) = 10, n (Early MSD) = 12, n (Middle MSD) = 9, n (Late MSD) = 10. *p b 0.05, **p b 0.01 compared with the control. Error bars represent SEM.

MSD caused microglial activation in the hippocampus of prepuberty offspring The percentage of Iba1 immunoreactive cells was assessed according to three morphologies of microglia. Representative images of cell type I, II and III microglia were indicated in Fig. 2B. The percentage of type I cell was lower in the late MSD group than in the control. However, the number of cell types II and III were higher in the MSD offspring (Fig. 4B). Significant difference existed between the late MSD group and the control (cell type I: F(1, 10) = 17.98, p b 0.001; cell type II: F(1, 10) = 46.53, p = 0.006; cell type III: F(1, 10) = 82.59, p b 0.001). The total number of Iba1+ cells was more in late MSD than in the control group (Fig. 4C, p = 0.205). The number of resting cells (type I) was lower in DG. The activated cells (type III) were higher after MSD. Fig. 4D showed that IL-6 was generated from activated microglia.

Changes of body weight, litter size and preference of sucrose after late MSD in offspring rats On both PND 1 (Fig. 5A) and PND 21 (Fig. 5B), the body weight of the MSD offspring was significantly lower than that of the control (Fig. 5A: F (1, 15) = 52.25, p b 0.001; Fig. 5B: F(1, 15) = 4.48, p = 0.041). Litter size and sex ratio were evaluated at PND 21 (Fig. 5C). The litter size was smaller in the late MSD group (F(1, 15) = 10.47, p = 0.012), but there was no statistical differences in the sex ratio, (F (1, 15) = 0.29, p = 0.605). In terms of sucrose preference (Fig. 5D), MSD offspring rats (72%) were significantly lower than the control (82%) (F(1, 15) = 10.42, p = 0.008).

Discussion This report showed that MSD resulted in cognitive deficits and behavioral abnormalities in prepuberty male offspring rats. The lowered level of neurogenesis was associated with microglial activation and inflammatory cytokines response events. More than two-thirds of pregnant women report poor sleep quality and lowered sleep duration, especially in the third trimester (Hollenbach et al., 2013). Although the maternal sleep loss in human is usually chronic and partial, due to such sleep deprivation or restriction is difficult or even impossible to perform in human, multiple platform protocols for sleep deprivation in animal models are widely accepted in research (Calegare et al., 2010; Thomal et al., 2010). These models are free of immobility stress and forced activity, decreasing other interference factors. With pregnant Wistar rats exposed to prolonged sleep deprivation (72 h), the body weight of prepuberty offspring was lower than the control on PND 1 and PND 21. Since birth weight can influence brain development of child and adolescent (Walhovd et al., 2012), children with abnormal birth weight have higher risk of psychiatric disorder and neurologic disabilities. Evidence shows that low birth weight is related to schizophrenia (Abel et al., 2010; Holloway et al., 2013) and bipolar affective disorder (Zucchi et al., 2013). The smaller litter size of the MSD group than the control indicated that the litter size was affected by prenatal sleep deprivation, though the mechanism remains to be explored (Baker et al., 2008). The sucrose preference experiment showed anhedonia in the MSD offspring, which has long been considered an experiential deficit and a core clinical feature in schizophrenia (Strauss, 2013). In the MWM task, the rats were learning to locate the submerged platform on day 1, so there was no significant difference in MWM at first day. On day 2–4, the latency differences to locate the platform

Q. Zhao et al. / Neurobiology of Disease 68 (2014) 57–65

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Fig. 2. Effects of MSD on offspring hippocampal neurogenesis. (A) Representative images of BrdU and DCX labeled cells in the dentate gyrus (DG); (B) Total numbers of BrdU+ cells; (C) Total numbers of all BrdU+DCX+ cells. Middle and late MSD reduced the number of BrdU+DCX+ cells in the DG. (D) Percentages of BrdU+DCX+ out of all BrdU+ cells per DG. The percentage for the middle MSD and the late MSD offspring was lower than that for the control. Representative example of DAPI in the DG (E) and GCL (F) subregions; (G) Cell layer volumes of DG and GCL in hippocampus. The volume of DG and GCL showed no difference between offspring rats of the MSD and control groups. n = 6 for each experimental group. *p b 0.05; all values are means ± SEM. Scale bars: 20 μm.

reflected spatial learning and memory of rats, which is a sensitive indicator of hippocampal function. In this study, late MSD offspring (not early or middle MSD group) spent significantly longer time than the control to locate the hidden platform, indicating deficits in spatial learning and memory in the late MSD offspring rats. Various studies have revealed that prenatal stress impairs hippocampus-dependent spatial learning and memory (Abdul Aziz et al., 2012; Wilcoxon et al., 2005), and causes schizophrenia-like behaviors in rodent animals (Zhang et al., 2012). These data indicated that MSD has strong long-lasting effects associated with increased risk for neuropsychiatric disorders in offspring. In general, there was a strong impact on adult offspring when their mother experienced stress during early fetal development (Lucassen et al., 2009, 2010). Our results observed in the offspring of MSD mothers were in line with the literatures, showing sleep restriction as a stressful stimulus to pregnant rats. It is important to note that sleep deprivation involves, to variable degrees, imposition of nonspecific stress, which may interact with the effects attributable to sleep loss per se (Meerlo et al., 2009; Thomal et al., 2010). The sleep restriction treatment resulted in lower hippocampal volume even without major activation of the HPA axis, a classic sign of stress (Novati et al., 2011). As with human fetus, the development of fetal rats is usually divided into three trimester equivalents. The timing of the prenatal stress (early, middle or late gestation) appears to be critical (Mueller and Bale, 2007; Van Waes et al., 2009).

Since a brain growth spurt occurs in the third trimester of gestation (Gil-Mohapel et al., 2010), it is a critical time when marked growth and differentiation takes place. The third trimester appears to be a particularly sensitive period to the deleterious effects of stress exposure such as MSD. In agreement with other experiments (Ashdown et al., 2006), this research proved that late pregnancy is more sensitive to sleep loss. Prenatal stress subjected during the late stage of pregnancy suppresses cell proliferation and maturation (Grigoryan and Segal, 2013), alters neuronal morphology and impairs neurogenesis (Rayen et al., 2011). The results in our MWM test demonstrated that the late perinatal period is more sensitive compared to the other two stages. The quantification of BrdU+ and DCX+ cells revealed a significantly lower neurogenesis in the DG in middle and late MSD offspring, especially for the late MSD (GD 18) group. These results supported the concept that prenatal stress can induce lasting and profound changes in the offspring, potently inhibit neurogenesis (Lucassen et al., 2009). Adult neurogenesis is now thought to contribute to the structural integrity of the hippocampus, a region particularly sensitive to stress (McCormick et al., 2012). The hippocampal integrity is involved in cognition and emotional regulation (Meerlo et al., 2009; Novati et al., 2011). Long-lasting reduction in neurogenesis and impairment of hippocampal functions have been reported after prenatal stress (Lemaire et al., 2006). Late MSD in this study inhibited hippocampal neurogenesis and impaired hippocampus-

Q. Zhao et al. / Neurobiology of Disease 68 (2014) 57–65

IL-10

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Fig. 3. MSD aroused inflammatory cytokines expression in prepuberty offspring. (A) TNFα and IL-6 were increased in hippocampus on PND 1. On PND 7 (B) and PND 14 (C), only IL-6 enhanced obviously. IL-6 was significantly higher than in the control, while IL-1β was lower for the late MSD offspring on PND 21 (D). For the same time, IL-10 was always lower in the late MSD group. n = 8 for each experimental group. *p b 0.05, **p b 0.01; all values are means ± SEM.

dependent spatial learning and memory in the offspring. Stress-induced down-regulation of neurogenesis leads to deconstruction of neural circuits, which is probably the main culprit for behavioral deficits and neuropsychiatric disorders (Oomen et al., 2010; Sahay et al., 2011; Sompol et al., 2011). The results in the neurogenesis test also proved that the late pregnant period is more sensitive compared to the other two stages. Thus, in the later experiments, only the late MSD group was chosen to evaluate other effects of prenatal stress. However, the volumes of DG and GCL areas, the primary neurogenesis subregions in the hippocampus, showed no difference in the late MSD offspring rats. Although a decrease in the production of new neurons contributes to hippocampal volume reductions, the alteration of hippocampal neurogenesis may be too limited to explain the hippocampal shrinkage (Novati et al., 2011; Oomen et al., 2011). In this study, MSD caused morphological changes of microglia in the hippocampus. The MSD offspring had less resting microglial cells (type I), but more activated microglia (type III). The type II, probably the state before activation, was up-regulated in the MSD offspring. Furthermore, the total number of microglia was enhanced after late MSD. These results indicated that microglia is activated in the prepuberty offspring whose mother experienced sleep deprivation. Upon environmental stimulus, ramified microglial cells transform into amoeboidform microglia, and this transformation is indicative of the gradual shift in the microglial functions (Walker et al., 2014). The increase of type III cells reflected amoeboid was predominant to ramified microglia in late MSD prepuberty offspring rats. Amoeboid microglia are thought to be reactive states to initiate an inflammatory cytokines secretion (Kettenmann et al., 2011). The morphological transformation of microglia in MSD offspring was accompanied by releasing of pro-inflammatory cytokines,

such as IL-6, TNFα and IL-1β. The microglia are induced to a primed state by prenatal stress (Giovanoli et al., 2013), and along with the elevation of pro-inflammatory cytokines (Harry and Kraft, 2012), which lasts long period and leads to adult vulnerability to learning and memory deficits, susceptibility to affective disorders and other neurobehavioral consequences (Howerton and Bale, 2012). Microglia comprise approximately 12% of cells in the brain, with the highest concentrations in the hippocampus, olfactory telencephalon, basal ganglia and substantia nigra (Block et al., 2007). The microglial activation is often divided into two phenotypic profiles: the classical and the alternative activation/deactivation state (Michelucci et al., 2009). The characterization of microglial phenotype is mainly based on the transcription profile or their secreted molecules of activated microglia (Colton, 2009; Michelucci et al., 2009). The alternative phenotype, sometimes called neuroprotective microglial phenotype, is important when switching from a classical inflammatory response to a reduction of pro-inflammatory mediators, an increase of antiinflammatory cytokines and neurotrophic factors. The classical or alternative activation state of the microglia can be reflected by the expression profiles of pro- vs anti-inflammatory cytokines. Classical activated microglia may contribute to reduction of neurogenesis and dysfunction of neurotrophic system by release of inflammatory mediators, including cytokines such as TNF-α, IL-1β and IL-6 (Cerciat et al., 2010), which will ultimately cause local inflammation and neurodegeneration (Griffin, 2006; Perry et al., 2007). Recent evidences suggest that glia consist the major niches and play a key role in controlling multiple steps of neurogenesis (Wake et al., 2013). These cytokines can change the niches of neurons and impact neurogenesis. As shown in Fig. 3, the pro-inflammatory cytokines IL-1β and IL-6 were increased and anti-

Q. Zhao et al. / Neurobiology of Disease 68 (2014) 57–65

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Fig. 4. MSD caused microglial activation in offspring hippocampus. (A) Representative example of morphology of microglia. White arrows indicate representative microglia. (B) Morphology of Cell type , П and Ш and the ratio of each type. Compared with the control, the offspring rats with MSD mothers had less cell type but more type П and type Ш. (C) Total number of microglia in the control and the late MSD group. (D) Representative fluorescent images of the late MSD brain slices examined for Iba1 and IL-6 immunoreactivity in the DG. The color of microglia is red, IL-6 is green. Arrows represent activated microglia, which released IL-6. n = 6 for each experimental group. *p b 0.05, **p b 0.01; all values are means ± SEM. Scale bars: 20 μm.

inflammatory cytokine IL-10 was reduced after MSD. It has been shown that the activation of microglia is associated with a reduction of new neurons in the rodent hippocampus, mainly due to decreased survival of the new neurons (Kohman and Rhodes, 2013; Monje et al., 2003). However, decreasing of IL-10 can aggravate neurogenesis impairment (Luo and Chen, 2012; Meyer et al., 2008). This indicates that shift from the alternative activation to the classical activation state promotes neurogenesis deficit. Enhanced levels of the anti-inflammatory cytokine IL-10 during prenatal development can prevent the emergence of multiple physiological and behavioral abnormalities in the adult offspring after mother exposure to prenatal stress (Meyer et al., 2008; Pan et al., 2013). The dysregulation between pro- and anti-inflammatory during embryogenesis can bring about a variety of behavioral abnormalities and neuropathologies in mental illness (Deverman and Patterson,

2009; Meyer et al., 2005), partly due to the inhibition of neurogenesis (You et al., 2011; Zhang et al., 2013). Chronic prenatal stress has long-term effects on the HPA axis and hypothalamic structure (Frodl and O'Keane, 2013). The effects of prenatal stress in second generation are also identified to be epigenetically modified in the germline (Suter et al., 2011). Besides HPA axis and epigenetic programming, neurogenesis reduction in hippocampus from prenatal stress was shown in the present research to be related to microglial activation and its neuroinflammatory processes. MSD and prenatal stress can induce activation of the maternal immune system, notably with elevated inflammatory cytokines in the fetal environment together with fetal microglia activation (Chang et al., 2010). The primed microglia markedly increases the vulnerability of the offspring to brain immune challenges (Diz-Chaves et al., 2013; Giovanoli et al., 2013). The

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Fig. 5. Effect of MSD on offspring body weight, litter size, sex ratio and preference of sucrose. (A) Birth weight of offspring in the control and late MSD groups (n = 10/group). (B) Body weight of 21 days old rats (n = 10/group). (C) Difference in litter size and sex ratio (n = 8-15/group). (D) The preference of sucrose in prepuberty offspring (n = 10/group). *p b 0.05, **p b 0.01; all values are means ± SEM.

neuroimmunological change can bring about devastating impact on prepuberty offspring, associated with microglial changes (Giovanoli et al., 2013). The present study, to the best of our knowledge, investigated for the first time the effects of MSD on microglial activation in their prepuberty offspring. It showed that MSD inhibits neurogenesis through inflammatory cytokines released from activated microglia in young offspring rats, and results in behavior changes. The MSD-induced microglial activation is an important factor impairing neurogenesis and cognitive function in prepuberty offspring. However, the pathway and mechanism of activated microglia after MSD need to be further explored. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81371327), Key Technologies R & D Program of Sichuan Province (2013SZ0011), the Achievement Transfer Program of Institutions in Chengdu (12DXYB345JH-002) and the Open Research Fund of State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources (No. 2012003). We are grateful to Prof. Keith M. Kendrick for his valuable suggestions, to Prof. Zujun Yang for his assistance and facilities on immunohistochemistry, to Ms. Xiong Wan for her help in copyediting. References Abdul Aziz, N.H., et al., 2012. Prenatal exposure to chronic mild stress increases corticosterone levels in the amniotic fluid and induces cognitive deficits in female offspring, improved by treatment with the antidepressant drug amitriptyline. Behav. Brain Res. 231, 29–39. Abel, K.M., et al., 2010. Birth weight, schizophrenia, and adult mental disorder: is risk confined to the smallest babies? Arch. Gen. Psychiatry 67, 923–930. Alvarenga, T.A., et al., 2013. Effects of sleep deprivation during pregnancy on the reproductive capability of the offspring. Fertil. Steril. 100, 1752–1757.

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