Dexamethasone-induced acute excitotoxic cell death in the developing brain

    Dexamethasone-induced acute excitotoxic cell death in the developing brain Dmitriy A. Lanshakov, Ekaterina V. Sukhareva, Tatjana S. Kalinina, Nikolay N. Dygalo PII: DOI: Reference:

S0969-9961(16)30030-4 doi: 10.1016/j.nbd.2016.02.009 YNBDI 3698

To appear in:

Neurobiology of Disease

Received date: Revised date: Accepted date:

19 May 2015 12 January 2016 7 February 2016

Please cite this article as: Lanshakov, Dmitriy A., Sukhareva, Ekaterina V., Kalinina, Tatjana S., Dygalo, Nikolay N., Dexamethasone-induced acute excitotoxic cell death in the developing brain, Neurobiology of Disease (2016), doi: 10.1016/j.nbd.2016.02.009

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ACCEPTED MANUSCRIPT Dexamethasone-induced acute excitotoxic cell death in the developing brain Dmitriy A. Lanshakova, Ekaterina V. Sukharevaa, Tatjana S. Kalininaa,b, Nikolay N. Dygaloa,b*

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Functional Neurogenomics Laboratory, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russian Federation. b Novosibirsk State University, Novosibirsk, Russian Federation. *

Corresponding author at: Functional Neurogenomics Laboratory, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russian Federation. E-mail address: [email protected] (N. Dygalo)

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E-mail address: [email protected] (D. Lanshakov) E-mail address: [email protected] (E. Sukhareva)

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E-mail address: [email protected] (T. Kalinina)

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The authors declare no conflicts of interest.

Abstract.

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There is substantial evidence that the use of glucocorticoids in neonates is associated with an increased risk of neurodevelopmental disorders. However, it remains unclear how treatment with low doses of dexamethasone (DEX) may result in behavioral abnormalities without evident signs of immediate neurotoxicity in the neonatal brain. It is possible that cells vulnerable to the pro-apoptotic effects of low doses of DEX escaped detection due to their small number in the developing brain. In agreement with this suggestion, low-dose DEX treatment (0.2 mg/kg) failed to induce apoptosis in the cortex or hippocampus proper of neonatal rats. However, this treatment was capable of inducing apoptosis specifically in the dorsal subiculum via a two-step mechanism that involves glutamate excitotoxicity. Application of DEX leads to increased activity of CA1/CA3 hippocampal MAP2-positive neurons, as determined by c-fos expression at 0.5-1 hour after DEX injection. Five hours later, the apoptotic markers (fragmented nuclei, active caspase-3 and TUNEL labeling) increased in the dorsal subiculum, which receives massive glutamatergic input from CA1neurons. Pretreatment with memantine, an antagonist of glutamate NMDA receptors, dose dependently blocked the DEX-induced expression of apoptotic markers in the subicular neurons and astrocytes. These findings provide new insights into the mechanisms of DEXinduced neurotoxicity as well as on the mechanism of therapeutic action of antagonists of NMDA receptors against neurobehavioral disorders caused by neonatal exposure to glucocorticoids. Keywords: dexamethasone, c-fos, excitotoxicity, apoptosis, caspase-3, DNA fragmentation, memantine, hippocampus, subiculum, neonatal

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ACCEPTED MANUSCRIPT Introduction. Developing brain, especially during pre- and early postnatal periods, is significantly influenced by glucocorticoids. Physiological concentrations of these hormones are essential

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for normal neuronal development (Abrahám et al., 2006; Baud et al., 2001; Feng et al., 2009),

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whereas high glucocorticoid levels induce cell loss, reduce neurogenesis and glial

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proliferation, attenuate dendrite formation, inhibit brain growth and cause abnormalities of brain structure and function (Barrington, 2001; de Kloet et al., 2014; Lupien et al., 2009; Reynolds, 2013). Glucocorticoid treatment is widely used to prevent chronic lung disease in

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premature infants. However, perinatal use of glucocorticoids is associated with an increased risk of neurodevelopmental disorders (Cheong et al., 2013; Damsted et al., 2011; Hirvikoski et al., 2007; Yeh et al., 2004). Therefore, the mechanisms underlying adverse effects of

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therapeutic doses of glucocorticoids should be investigated in more detail. Since the discovery of glucocorticoid-induced increase of extracellular glutamate levels in the brain (Stein-Behrens et al., 1992), excitotoxicity caused by this neurotransmitter

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was suggested to play an important role in the hormone-related loss of neurons (Jacobs et al.,

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2006; Popoli et al., 2011). Numerous studies have shown that prolonged (for days or weeks) exposure of animals (Bhatt et al., 2013; Chang et al., 2013; Zuloaga et al., 2012) or cell

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cultures (Flavin, 1996; Lu et al., 2003; Yu et al., 2010, 2011; Zhu et al., 2006) to high doses of glucocorticoids promoted excitotoxic cell death. At the same time, treatment with glucocorticoids for shorter periods usually did not cause signs of cell death as, for example,

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when neonatal rat pups were injected with a single dose (0.031 - 0.5 mg/kg) of dexamethasone (DEX) (Feng et al., 2009). Moreover, DEX (0.1 to 100 µM) added to neuronal or astrocyte cultures for 12 hours not only failed to induce an increase in the rate of spontaneous cell death but instead protected cells from the toxic effects of N-methyl-Daspartate or hypoxia (Baud et al., 2001). An intriguing observation is that low doses of glucocorticoids can induce behavioral abnormalities without evident signs of immediate neurotoxicity in the neonatal brain. Thus, even a single neonatal injection of DEX resulted in abnormal behavior of rat pups in novel environment (Menshanov et al., 2014) and impaired learning abilities of adult rats in behavioral tests (DeKosky et al., 1982). It can be proposed that a single injection of low-dose DEX also can lead to a selective cell death of most vulnerable cells. However, the loss of these cells had evidently escaped detection, possibly due to small numbers of such cells in the developing brain.

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ACCEPTED MANUSCRIPT To explore this possibility, we injected neonatal rat pups with low-dose DEX and searched for cells that were activated and/or committed apoptosis in the brain after the treatment. c-Fos expression was used as a marker of neuronal activation (Kovács, 1998).

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Hippocampal neurons may be primary targets of DEX because they express high levels of

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glucocorticoid receptors during perinatal development (van Eekelen et al., 1991). Regions of axonal projections of these neurons were screened for apoptosis at 6 hours after the treatment.

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Active caspase-3 staining, fragmented nuclei staining and staining for DNA damage were used to monitor apoptotic cell death in the hippocampus, subiculum and cortex. The excitotoxic nature of DEX-induced cell death was confirmed by the blockade of glutamate

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NMDA receptors with the antagonist memantine.

Animals and Experimental Design.

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Materials and methods.

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All animal procedures were performed in compliance with the EU Directive 2010/63/EU, and

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all protocols were approved by the Institutional Animal Care and Use Committee. Pregnant Wistar rats were individually housed (22-24°C, natural illumination) with free access to food

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and water. The day of birth was considered the first postnatal day - P1. At P2, litters were culled to 8 pups. Pups of both sexes were used in experiments and randomly divided into the untreated, saline- or Dex -treated groups. At P3, the animals were injected subcutaneously with dexamethasone-21-phosphate (DEX, KRKA, 0.2 mg/kg in 20 µl of saline). Memantine

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(M, Sigma) was administered (5 or 20 mg/kg in 20 µl of saline) singly as well as 2 h before the DEX injection at P3 by subcutaneous route to the animals of additional control and DEXtreated groups. Control animals were injected with saline (20 µl) or left untreated. The pups were either taken for immunohistochemical analysis (n = 4 per treatment group) or determination of mRNA levels (n = 8 per treatment group).

mRNA analysis Animals were decapitated at 0.5, 1 and 2 h after DEX administration. Each timetreatment group was represented by 8 animals from 3-4 litters. Hippocampi were rapidly dissected and frozen in liquid nitrogen. Total cellular RNA was isolated using a single-step acidic phenol extraction as was described previously (Shishkina et al., 2012). Only RNA samples with OD 260/280 ratios close to 2.0 were selected for reverse transcription. TaqMan® Gene Expression Assays were used to measure c-fos and beta-actin mRNA levels 3

ACCEPTED MANUSCRIPT (c-fos: Rn02396759_m1, beta-actin: Rn00667869_m1) on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). All reactions were carried out in triplicate on cDNA samples in 96-well optical plates according to the manufacturer’s protocol

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in 25 µl of 1 × TaqMan® Universal PCR Master Mix (Applied Biosystems). The real-time

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PCR consisted of one cycle of 50 oC for 2 min and 95 oC for 10 min, followed by 40 cycles each of 95 oC for 15 s and 60 oC for 1 min. The comparative ddCT method was used to manufacturer’s manual (Applied Biosystems).

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Immunohistochemistry

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calculate mRNA expression relative to the beta-actin as an endogenous control according to

Rat pups treated with DEX, memantine (M) and DEX+M as well as animals of control

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groups were anesthetized with avertin 1 and 6 h after DEX and/or 8 h M administration. Then, animals were transcardially perfused with 0.1 M phosphate-buffer (PB), followed by fixative 4% paraformaldehyde in 0.1 M PB. Brains were removed from the skull, postfixed overnight

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in the same fixative at 4°C and cryoprotected with 30% sucrose solution overnight. The brains

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were frozen at -80 oC in OCT (Tissue-Tek® O.C.T™, Sakura Finetek). 18-μm-thick coronal cryostat sections were taken and mounted on superfrost slides. Further steps were as described

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previously (Baranek et al. 2012).

Sections were incubated with primary antibodies in 1.5% BSA overnight at 4°C. CFos immunoreactivity was detected with a rabbit anti-c-Fos antibody (dilution 1:200; Cell Signaling). Hippocampal neurons and astrocytes were labeled with chicken anti-Map2

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(Millipore, 1:300) and mouse anti-GFAP (Millipore, 1:200) respectively. Active caspase-3 was labeled with rabbit anti-cleaved caspase-3 antibody (Cell Signaling; 1:200), the signal was visualized with indirect method using biotinylated goat anti-rabbit secondary antibody (Santa Cruz) and HRP conjugated streptavidin (Abcam). For double immunolabeling of cleaved caspase-3 with cell type markers, rabbit anti-cleaved caspase-3 antibody (Cell Signalig; 1:200) in cocktail either with mouse-anti SATB2 (Santa Cruz; 1:200) or mouse antiGFAP (Millipore; 1:200) were incubated in 1.5% BSA at 4°C overnight for labeling SATB2positive neurons and GFAP-positive astrocytes. Satb2 that expressed in the hippocampal CA1 neurons in the developing rodent brain (Balamotis et al., 2012) was used as a neuronal marker. For all double labeling experiments secondary antibodies were purchased from Invitrogen. Donkey anti-rabbit Alexa-488-conjugated, donkey anti mouse Alexa-555conjugated and donkey anti chicken Alexa-594-conjugated antibodies (all in 1:350 dilution), were used. TUNEL assay was performed using the Roche In situ Cell Death Kit POD 4

ACCEPTED MANUSCRIPT according to the manufacturer’s instructions. The FITC- TUNEL staining was visualized by fluorescence microscopy. Fragmented nuclei were visualized with DAPI nuclear counterstain. Hippocampal and subiculum regions were identified according to the rat brain atlas

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(Paxinos and Watsoin, 2007). Sections (4 sections per animal, 4 animals from 3-4 different

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litters in each group) were analyzed by confocal laser-scanning microscopy (Zeiss LSM 780) at the Microscopic Centre of the Institute of Cytology and Genetics, Novosibirsk, Russia. The

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following channel settings were used for CLSM imaging: DAPI - 405 nm, FITC and Alexa 488 – 488 nm, Alexa 555 and Alexa 594 with 561 nm laser lines. 63x objective, 39 μm, 46 μm and 46 μm pinholes with 405 nm (5% power), 561 nm (10% power) and 488 nm (12%

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power) lasers respectively were used. Z-stacks were collected with 0,18 μm optical slices. Panoramic images of the hippocampus were created using tile scan function (10 x objective and 200μm pinhole with lasers power 16%). Results were expressed as the mean number of

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labeled cells per mm2 of the region of interest.

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Statistics

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Statistical analyses were performed using STATISTICA software. The numbers of cfos-, cleaved caspase-3-, and TUNEL-positive cells and of cells with fragmented nuclei were

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analyzed by one-way ANOVA. The time profiles of mRNA expression for c-fos were analyzed using two-way ANOVA with treatment and time as factors. ANOVAs were followed by Tukey's post hoc test. The results were considered significant at probability level

Results

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less than 0.05.

Activation of hippocampal neurons by DEX C-fos mRNA level. Injection of DEX resulted in more than a two-fold increase in c-fos mRNA levels in the neonatal rat hippocampus (F (3, 41) = 14.14, P < 0.001; Fig. 1). This marker of cell activation was significantly elevated already at 0.5 h, reached maximum at 1 h and decreased 2 h after DEX treatment. Saline injection (a stressor for pups) also caused a slight increase in the hippocampal c-fos mRNA levels that reached statistical significance at 1 h (F (3, 36) = 3.92, P < 0.05) in comparison with intact control (zero point of the X-axis). C-Fos immunohistochemistry. Immunohistochemical analysis confirmed activation of hippocampal cells by DEX. As shown in the Figure 2, c-Fos-positive immunofluorescence was strong in CA1, moderate in 5

ACCEPTED MANUSCRIPT CA3, low in other hippocampal subfields and practically absent in the cortical regions adjacent to hippocampus or in subiculum at 1 h after the hormonal treatment. Double labeling of c-Fos-positive cells with neuronal marker MAP2 or astrocyte marker GFAP revealed that

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DEX specifically activated CA1 and CA3 neurons (Fig. 3). The numbers of c-fos-MAP2

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positive neurons were significantly elevated in CA1 and CA3 1 h after DEX injection compared to control groups (Figs. 3A and 3C; F(2, 9)=17.359, p=0.001 for CA1 and F(2,

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21)= F(2, 9)=8.426, p<0.01 for CA3)). Also, the number of neurons activated by DEX in CA1 was significantly higher (p<0.05) than in CA3. DEX didn’t change the numbers of c-fos positive astrocytes in both investigated hippocampal regions at 1 hour after injection (Figs. 3B

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and 3D; F(2, 9)=0.037, NS for CA1 and F(2, 9)=0.108, p=0.899, NS for CA3).

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Acute DEX-induced apoptosis in the neonatal rat brain

To evaluate the consequences of DEX-induced activation of hippocampal neurons on brain cell viability, apoptotic markers were analyzed 6 h after hormonal treatment. This time

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interval was chosen because apoptosis needs several hours to occur after exposure to cell

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death inducing stimuli. In these experiments, the application of DEX, which increased c-fos expression in CA1 and CA3 neurons, had no effect on the numbers of TUNEL-positive cells

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in the proper hippocampus 6 h after treatment (Fig. 4A-B). However, unexpectedly, we found a significant 5-fold increase in the number of TUNEL-positive cells in the dorsal subiculum (Fig. 4E-F; F(2,9)=30.09, p<0.001). DEX-induced activation of apoptosis in the subiculum was also confirmed by the increase in numbers of fragmented nuclei (Figs. 4E and 4G; F(2,

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9)=28.80, p<0.001) as well as by the increased number of cleaved caspase-3-positive cells (Figs. 5A-F; F(2, 9)=12.01, P < 0.01). The acute DEX-induced cell death was limited to the subiculum. There were no signs of apoptosis not only in the proper hippocampus but also in the cortex at 6 h after DEX treatment (Figs. 4 and 5).

Excitotoxic cell death in the neonatal rat subiculum after DEX treatment The specific apoptosis of subicular cells which, as evidenced by c-Fos expression, were not directly activated by DEX suggests an indirect action of DEX on viability of these cells. Most of the axon terminals of the glutamatergic CA1 neurons that were activated by DEX innervate neurons in the dorsal subiculum (O'Mara et al., 2001). If DEX-induced cell death in the subiculum was mediated by glutamate excitotoxicity, then it should be prevented by the blockade of glutamate receptors. With the aim to test this hypothesis, we have administered glutamate NMDA receptor antagonist memantine two hours before DEX 6

ACCEPTED MANUSCRIPT injection. Memantine had no appreciable effect on the expression of apoptotic markers in the subiculum (Fig. 6). However, this antagonist of NMDA receptors significantly attenuated glucocorticoid-induced increases in the numbers of cleaved caspase-3-positive (Fig. 6A; F (6,

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21) =14.122, p<0.05) as well as TUNEL-positive (Fig. 6B; F (5, 42) =9, 1527, p<0.05) cells

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in the dorsal subiculum in a dose dependent manner.

To determine types of the subicular cells in which DEX treatment induced apoptosis,

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these cells were colabeled for active caspase-3 and neuronal marker SATB2 (Fig. 7A) as well as for active caspase-3 and marker of astrocytes GFAP (Fig. 7B). DEX significantly increased the numbers of caspase-3-SATB2 colabeled neurons (Fig. 7C) as well as caspase-3-GFAP

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colabeled astrocytes (Fig. 7D) in the subiculum at 6 h after the treatment. Morphology of active caspase-3-positive subicular cells (Figs. 5E and 7A) showed that most of these cells

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were neurons. Though both cell types were affected by DEX, hormone-induced increase in the number of caspase-3-SATB2 positive neurons was 2.5 times higher than that of caspase-3GFAP astrocytes (Fig. 7). Administration of memantine before DEX significantly decreased

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the numbers of caspase-3-SATB2 neurons (Fig. 7C; F(6, 21)=4.1985 p<0.05) as well as of

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caspase-3-GFAP astrocytes (Fig. 7D; F(6, 21)=3.2549 p<0.05). The effects of both doses of memantine on the relatively low level of DEX-induced apoptosis of subicular astrocytes were

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similar. Higher level of DEX-induced apoptosis of subicular neurons was decreased by pretreatment with the antagonist of NMDA receptors in a dose dependent manner.

Discussion

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There is substantial evidence that the use of glucocorticoids in neonates is associated with neurodevelopmental side effects. These effects include a decrease in brain volume, attention deficits, impaired memory, poorer intellectual performance such as, for example, cognitive disabilities at school age (Barrington, 2001; Cheong et al., 2013; Damsted et al., 2011; Hirvikoski et al., 2007; Yeh et al., 2004). In animal studies, perinatal administration of DEX, even at low doses, also caused neurological dysfunctions in rats (Ichinohashi et al., 2013), mice (Li et al., 2014) and monkeys (Pryse et al., 2011). Neurobehavioral consequences of perinatal exposure to high levels of glucocorticoids are thought to result from damage of the brain cells. In the present study, a single low dose of DEX (0.2 mg/kg) caused cell death in the subiculum of neonatal rats. This dose is close to the dose that stimulated maturation of cholinergic synapses (0.15 mg/kg), while higher doses of DEX (0.6-2.4 mg/kg) inhibited synaptic development (Kreider et al., 2005). The signs of cell death were not found previously 7

ACCEPTED MANUSCRIPT following acute exposure to moderate doses of glucocorticoids for less than 24 h (Baud et al., 2001; Feng et al., 2011; Menshanov et al., 2013). Unlike low-acute doses, high doses of DEX induce evident apoptosis in the brain. Hippocampal neural progenitor cells of newborn rats

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show an increase in apoptotic markers on the next day after a single injection of DEX (0.5

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mg/kg) (Sze et al., 2013). A single exposure of neonatal mice to DEX (3 mg/kg) was shown to produce selective and rapid cerebellar neural progenitor cell apoptotic death (Noguchi et

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al., 2008). DEX in cumulative doses more than 0.5 mg/kg applied for several days inhibited cell proliferation and induced apoptosis in the brain and in neuronal cell cultures (Chang et al., 2013; Flavin, 1996; Lu et al., 2003; Yu et al., 2010, 2011; Zhu et al., 2006). The

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differences between the results of prolonged high-dose and short time low-dose experiments could not be explained by insufficient time for generation of glucocorticoid-induce surge of

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extracellular glutamate or the time needed for the later to cause cell death. Indeed, a single dose of corticosterone or DEX caused a 2-3-fold increase in extracellular glutamate concentration in the brain for 30-60 min within an hour after injection (Abrahám et al., 1996;

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Venero, Borrell, 1999), and bold application of glutamate for only 5-15 min in culture

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medium resulted in induction of neuronal apoptosis, evident at 2 h (Gleichmann et al., 2009; Gut et al., 2013). A possible explanation for the differences between high- and low-dose

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experiments may be that acute moderate doses of glucocorticoids induce death of only most vulnerable cells, the loss of which might have escaped detection due to their small number in the developing brain.

In the neonatal rat brain, glucocorticoid receptors are highly expressed in the

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hippocampal and especially in CA1 cells (van Eekelen et al., 1991) and therefore, these cells should be most sensitive to glucocorticoids. Indeed, an increase in c-fos mRNA and protein expression, a marker of neuronal activation (Kovács, 1998), was found in CA1/CA3 hippocampal subfields in a short time after DEX administration. Existing information on the effects of glucocorticoids on c-fos expression in the hippocampus is inconsistent and related to adult rats. It was reported that corticosterone replacement to adrenalectomized rats had no effect on c-fos mRNA levels in CA1 and CA3 (Hansson, Fuxe, 2008). In agreement with these data, DEX did not change the expression of c-Fos protein in the hippocampus of intact rats (Fazekas et al., 2006). However, DEX given 1h before restrain and 2 h before immunohistochemistry reduced stress-induced activation of c-Fos expression (de Medeiros et al., 2005). Contrary to these data, adrenalectomy attenuated hippocampal c-Fos response to 1 h of restraint and corticosterone replacement in drinking water normalized this response (Fevurly, Spencer, 2004). Moreover, a single-dose of corticosterone resulted in a significant, 8

ACCEPTED MANUSCRIPT time-dependent increase in c-Fos immunoreactivity in the granule cell layer of the dentate gyrus, as well as in regions CA1 and CA3 of the hippocampus (Szakacs et al., 2010). Although the mechanism of DEX-induced activation of hippocampal neurons of neonatal rats

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remains to be determined, the rapid increase in c-fos mRNA expression with the peak at 1 h,

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suggests that, besides activation of hormone-dependent genes, rapid nongenomic steroid signaling also could not be excluded (Groeneweg et al., 2012). Experiments with antagonist

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of glucocorticoid receptors RU486 give clear evidence for the involvement of these receptors in rapid activation of c-Fos expression by stress (Gutièrrez-Mecinas et al., 2011). Correlation of in vivo firing rates of individual neurons with their expression levels of the immediate

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early gene c-fos was demonstrated (Peter et al., 2013).

Activation of CA1 hippocampal cells, most of which are glutamatergic neurons

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(Klausberger, Somogyi, 2008), was not accompanied by an increase of apoptotic markers in this brain region of 3-day-old rats 6 h after DEX treatment. Apoptosis occurs at high frequency in the neonatal hippocampus (Menshanov et al., 2006). However, a single DEX

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injection was not able to induce an increase of cell death in this brain region of neonatal rats

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within 1-5 days after treatment (Feng et al., 2009, 2015). Nevertheless, a single injection of DEX to 3-4-day-old rats can lead to behavioral abnormalities in neonatal pups (Menshanov et

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al., 2014) as well as in adult animals (DeKosky et al., 1982). Cumulatively, these data suggest that even a single dose of DEX may have adverse effects on neurodevelopment that, evidently, originated outside the hippocampus proper. The subiculum is the major output structure of the hippocampal formation. It receives

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massive glutamatergic input from the hippocampal CA1 region (O'Mara, 2005; O'Mara et al., 2001). Glucocorticoids affect glutamatergic pathways by increasing extracellular glutamate (Abraham et al., 1996; Moghaddam et al., 1994; Virgin et al., 1991; Wang and Wang, 2009). Inhibition of glutamate uptake (Benitez-Diaz et al., 2003; Chang et al., 2013; Moghaddam et al., 1994) as well as an increase of the glutamate release (Wang and Wang, 2009) may be involved in the increasing effect of glucocorticoids on extracellular glutamate concentrations. Thus, DEX-induced activation of CA1 neurons, found in the present study, is capable of increasing extracellular glutamate levels in the subiculum that can induce excitotoxic death of the subicular cells. Unlike DEX, which activated only CA1 neurons and had no effect on the c-fos expression in the hippocampal astrocytes, glutamate is able to affect both glial (Matute et al., 2006) and neuronal (Lai et al., 2014) cell types. Appearance of apoptotic markers in cells of both types 6 h after DEX treatment is in line with the idea of a glutamate-mediated glucocorticoid effect on the viability of neurons and astrocytes in the subiculum. Support for 9

ACCEPTED MANUSCRIPT this idea came from the observation that pretreatment with memantine, an antagonist of NMDA receptors, dose dependently blocked the DEX-induced expression of apoptotic markers in the subicular cells. In general, these results suggest a two-step mechanism of

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cytotoxic effect of DEX on the developing brain (Fig. 8). The process is initiated by

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activation of glucocorticoid-sensitive hippocampal glutamatergic neurons. The axon terminals of these neurons release glutamate in the subiculum. Excessive concentrations of this

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neurotransmitter induce excitotoxic death of the second components of the process – glutamate-sensitive subicular astrocytes and neurons.

Subiculum is thought to be important for memory and its lesion can disrupt the

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memory process and operant learning (O'Mara, 2005). An impairment of working memory in children after prenatal treatment with DEX (Hirvikoski et al., 2007) as well as an impairment

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of acquisition of memory in adult rats (DeKosky et al., 1982), juvenile and adult mice (Li et al., 2014) after neonatal DEX treatment have been reported. Thus, at least some of the longterm neurobehavioral effects of developmental exposure to DEX may be attributed to damage

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of subicular cells. Importantly, the cognitive deficits produced by neonatal exposure to DEX

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could be prevented by pretreatment with antagonist of glutamate receptors (Li et al., 2014). In conclusion, the results of this study demonstrate that neonatal treatment with low

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dose of DEX caused apoptosis specifically in the dorsal subiculum via a two-step mechanism that involves glutamate excitotoxicity. It was also shown that pretreatment with memantine, an antagonist of NMDA receptors, dose dependently blocked the DEX-induced apoptosis of the cells in the subiculum. These results provide new insights into the mechanisms of DEX-

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induced neurotoxicity as well as on the mechanism of therapeutic action of antagonists of NMDA receptors against neurobehavioral disorders caused by neonatal exposure to glucocorticoids.

Acknowledgments The work was funded by RFBR No. 13-04-31314; RSF No. 14-15-00115.

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Figure legends

Fig. 1. Time-course of c-fos mRNA expression in the hippocampus of 3-day-old rats after injection of DEX or saline. Data represent mean ± SEM of 8 individual animals. * p<0.05

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compared with zero time point and corresponding saline treated groups as analyzed by oneway ANOVA followed by Tukey's post hoc test.

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Fig. 2. Representative images of c-fos immunostaining in the hippocampus of DEX-treated 3-

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day-old rats 1 h after treatment. A – intact, B – saline, C – dexamethasone treated groups.

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Arrowheads indicate c-Fos positive cells. Scale bar - 200 µm.

Fig. 3. Representative images of double immunostaining: (A) for c-Fos and neuronal marker MAP2, (B) for c-Fos and astrocyte marker GFAP in hippocampal CA1 region of DEX-treated

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3-day-old rats 1 h after treatment. Arrowheads indicate c-Fos positive nuclei. Scale bar - 20 µm. Numbers of c-Fos positive neurons (C) and astrocytes (D) in CA1 and CA3 hippocampal regions of DEX-treated 3-day-old rats as was determined by double immunostaining for cFos and neuronal marker MAP2 or astrocyte marker GFAP. INT – intact, SAL – saline, DEX – dexamethasone. Data represent mean ± SEM of 4 individual animals. * p<0.05 and ** p<0.01 – the difference in total numbers of c-Fos-positive cells in CA1 and CA3 hippocampal v

regions of DEX-treated rats compared with corresponding control groups; p<0.05 and w

p<0.01– the difference in numbers of double labeled c-Fos–MAP2–positive cells in CA1

and CA3 hippocampal regions of DEX-treated rats compared with corresponding control groups as analyzed by one-way ANOVA followed by Tukey's post hoc test. Also, vp<0.05 as compared with numbers of double labeled c-Fos–MAP2–positive cells in CA1.

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ACCEPTED MANUSCRIPT Fig. 4. Representative images of TUNEL and DAPI staining in the brain of DEX-treated 3day-old rats 6 h after treatment. (A-D) - TUNEL staining in the forebrain. (A) – intact. (B) – DEX-treated. Subicular regions marked by rectangles on A and B are shown at higher

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magnification at (C) and (D) respectively. Arrowheads indicate TUNEL- positive cells. Scale

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bar - 100 µm; (E) - TUNEL staining and fragmented nuclei (DAPI stain) in the subiculum of DEX-treated animals. Arrowheads indicate TUNEL- positive cells with fragmented nuclei.

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Scale bar - 20 µm. Numbers of cells with DNA damage (F) and fragmented nuclei (G) in the subiculum of DEX-treated 3-day-old rats as was determined by TUNEL (F) and DAPI (G) staining. INT – intact, SAL – saline, DEX – dexamethasone. Data represent mean ± SEM of 4

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ANOVA followed by Tukey's post hoc test.

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individual animals. ** p<0.01 compared with both control groups as analyzed by one-way

Fig. 5. Expression of active caspase-3 in the brain of DEX-treated 3-day-old rats 6 h after treatment. (A-E) - Representative images of active caspase-3 immunostaining (A-B) - in the

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forebrain of (A) – intact and (B) – DEX-treated pups. Subicular regions marked by rectangles

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on A and B are shown at higher magnification at (C) and (D) respectively. (A-D) - scale bars - 100 µm. (E) Subicular region of DEX-treated animal at higher magnification. Scale bar -

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10µm. Black arrowheads indicate active caspase-3-positive cells, white arrowheads – cell debris. (F) Numbers of active caspase-3-positive cells in the subiculum. INT – intact, SAL – saline, DEX – 0.2 mg/kg of DEX. Data represent mean ± SEM of 4 individual animals. ** p<0.01 compared with both control groups as analyzed by one-way ANOVA followed by

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Tukey's post hoc test.

Fig. 6. Effects of 2-hour pretreatment with antagonist of NMDA receptors memantine on the numbers of active caspase-3-immunoreactive cells (A) and TUNEL-positive cells (B) in the subiculum of DEX-treated 3-day-old rats 6 h after DEX treatment. INT – intact, SAL – saline, DEX – 0.2 mg/kg of DEX, M5 – 5 mg/kg of memantine, M20 – 20 mg/kg of memantine, M5D – 5 mg/kg of memantine + 0.2 mg/kg of DEX, M20D – 20 mg/kg of memantine + 0.2 mg/kg of DEX. Data represent mean ± SEM of 4 individual animals. ** p<0.01 compared with all other groups except the M5D group, * p<0.05 compared with INT, SAL and DEX groups, & p<0.01 compared with DEX and M5D groups as analyzed by one-way ANOVA followed by Tukey's post hoc test.

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ACCEPTED MANUSCRIPT Fig. 7. Effects of 2-hour pretreatment with antagonist of NMDA receptors memantine on the numbers of active caspase-3-immunoreactive neurons and astrocytes in the subiculum of DEX-treated 3-day-old rats as was determined by double immunostaining for active caspase-3

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and neuronal marker SATB2 (A) or astrocyte marker GFAP (B) 6 h after DEX treatment.

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Representative images of double immunostaining: (A) for active caspase-3 and neuronal marker SATB2; (B) for active caspase-3 and astrocyte marker GFAP. Arrowheads indicate

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double labeled cells. Scale bar - 20 µm. Numbers of active caspase-3-immunoreactive neurons (C) and astrocytes (D). INT – intact, SAL – saline, DEX – 0.2 mg/kg of DEX, M5 – 5 mg/kg of memantine, M20 – 20 mg/kg of memantine, M5D – 5 mg/kg of memantine + 0.2

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mg/kg of DEX, M20D – 20 mg/kg of memantine + 0.2 mg/kg of DEX. Data represent mean ± SEM of 4 individual animals. ** p<0.01 compared with INT, SAL, M5 and M20 groups

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except the M5D group, * p<0.05 compared with INT, SAL and DEX groups, & p<0.05 compared with DEX group as analyzed by one-way ANOVA followed by Tukey's post hoc

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Fig. 8. The scheme of the proposed mechanism of glutamate-mediated cytotoxic effect of DEX in the developing brain. The first component of the mechanism (1) is glucocorticoid-

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sensitive hippocampal glutamatergic neurons activated by the hormone. The axon terminals of these neurons release glutamate in the subiculum. Excessive concentrations of glutamate

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induce excitotoxic death of the second component – (2) glutamate-sensitive subicular cells.

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ACCEPTED MANUSCRIPT Graphical abstract

A two-step mechanism of glutamate-mediated cytotoxic effect of dexamethasone in the

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ACCEPTED MANUSCRIPT Highlights Low-dose DEX treatment induces c-fos expression in CA1 neurons of neonatal rats.

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DEX increases apoptotic markers expression specifically in subiculum of rat pups.

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Antagonist of NMDA receptors prevents DEX-induced apoptosis of the subicular

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Toxic effect of DEX on developing brain is mediated by glutamate excitotoxicity.

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cells.

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