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Neuropharmacology 67 (2013) 511e520

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Effects of oxygen and glucose deprivation on synaptic transmission inrat dentate gyrus: Role of A2A adenosine receptors

Giovanna Maraula a, Chiara Traini a, Tommaso Mello b, Elisabetta Coppi a, Andrea Galli b, Felicita Pedata a,Anna Maria Pugliese a,*

aDepartment of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, ItalybGastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Florence, Italy

a r t i c l e i n f o

Article history:Received 27 June 2012Received in revised form29 November 2012Accepted 3 December 2012

Keywords:Adenosine A2A receptorSynaptic potentialsAnoxic depolarizationCerebral ischemiaBrdUDCX

Abbreviations: aCSF, artificial cerebrospinal fluidDMSO, dimethyl sulphoxide; OGD, oxygenegluco4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triBrdU, 5-Bromo-20-deoxyuridine; DCX, doublecortin.* Corresponding author. Tel.: þ39 055 4271 325; fa

E-mail address: annamaria.pugliese@unifi.it (A.M.

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.12.002

a b s t r a c t

The hippocampus is comprised of two distinct subfields that show different responses to hypoxic-ischemic brain injury: the CA1 region is particularly susceptible whereas the dentate gyrus (DG) isquite resistant. Our aim was to determine the synaptic and proliferative response of the DG to severeoxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contributionof A2A adenosine receptor antagonism to recovery of synaptic activity after OGD.

Extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) in granule cells of theDG in brain slices prepared from male Wistar rats were used. A 9-min OGD is needed in the DG to alwaysinduce the appearance of anoxic depolarization (AD) and the irreversible block of synaptic activity, asrecorded up to 24 h from the end of the insult, whereas only 7-min OGD is required in the CA1 region.Selective antagonism of A2A adenosine receptors by ZM241385 significantly prevents or delays theappearance of AD and protects from the irreversible block of neurotransmission induced by 9-min OGDin the DG.

The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zoneof DG in slices prepared from 5-bromo-20-deoxyuridine (BrdU) treated rats was investigated. Slices werefurther incubated with an immature neuronal marker, doublecortin (DCX). The number of BrdUþ cellswas significantly decreased 6 h after 9-min OGD and this effect was antagonized by ZM241385. After 24 hfrom the end of 9-min OGD, the number of BrdUþ cells returned to that found before OGD and increasedarborization of tertiary dendrites of DCXþ cells was observed.

The adenosine A2A antagonist ZM241385 protects from synaptic failure and from decreasedproliferation of immature neuronal cells at a precocious time after OGD.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The hippocampus is comprised of two distinct subfields thatshow different responses to hypoxic-ischemic brain injury. The CA1region is particularly susceptible whereas the dentate gyrus (DG),which serves as a gateway to the hippocampus, is quite resistant(Wang et al., 2005), probably because of its regenerative capacity,even in adulthood (Altman, 1962; Altman and Das, 1965; Bartleyet al., 2005). Recent studies have demonstrated the presence of

; AD, anoxic depolarization;se deprivation; ZM241385,azin-5-ylamino]ethyl)phenol;

x: þ39 055 4271 280.Pugliese).

All rights reserved.

multipotent neural stem cells in the subgranular zone (SGZ) of theDG (Yamashima et al., 2007), able to proliferate and differentiateinto neurons, astrocytes and oligodendrocytes (Sharp et al., 2002)as a response tomultiple factors, including hypoxic-ischemic injury(Liu et al., 1998; Takagi et al., 1999). An increase in DG cell prolif-eration has been demonstrated in different animal models of brainischemia in vivo (Liu et al., 1998; Wang et al., 2005) or in oxygeneglucose deprived hippocampal slice cultures (Chechneva et al.,2006). Synaptic activity in the perforant pathway appears to beimportant for maintenance of DG cell proliferation (Cameron et al.,1995; Wang et al., 2005). The recovery of neurotransmission foundin DG at least 30 days after the end of global ischemia in the ratin vivo is attributable to the newly generated neurons elicited byischemia (Wang et al., 2005). Between 26 and 35 days afterischemic injury in vivo up to 60% of the newborn cells exhibitneuronal characteristics (Liu et al., 1998; Wang et al., 2005).

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520512

Failure of synaptic activity is the earliest consequence of cere-bral ischemia (Hofmeijer and van Putten, 2012; Pugliese et al., 2006,2007; Traini et al., 2011) and accounts for electric silence in thepenumbra (Astrup et al., 1981; Eccles et al., 1966; Symon, 1980;Symon et al., 1977). In brain slices subjected to oxygeneglucosedeprivation (OGD), ATP concentrations decline first in synaptic ascompared to other slice regions (Lipton and Whittingham, 1982),suggesting that synaptic failure is a result of energy exhaustion.

One of the early events occurring during ischemia, either in vivoor in vitro, is the increase in extracellular concentrations of aden-osine (Latini and Pedata, 2001). Adenosine acts as a modulator inthe brain, activating four receptor subtypes, A1, A2A, A2B and A3(Fredholm et al., 2001), all of which are expressed in the brain(Dixon et al., 1996). Evidence suggests that A1, A2A and A3 receptorsplay a role in this pathological event (Burnstock et al., 2011; Pedataet al., 2007). The protective role of adenosine on synaptic trans-mission is mainly attributed to well-established inhibitory tonethrough activation of the A1 receptor subtype (Latini and Pedata,2001). Depression of synaptic activity during ischemic insult isconsidered a compensatory mechanism to balance the oxygensupply and consumption in order to preserve neuronal integrity(Guillemin and Krasnow, 1997). Unfortunately, the development ofA1 selective agonists as possible anti-ischemic drugs has beenhindered by cardiovascular side effects, including sedation, brady-cardia. Therefore, attention has focused on the contribution of theA2A adenosine receptors in order to identify putative targets fortherapeutic intervention. Reverse transcriptase polymerase chainreaction and in situ hybridization have shown that adenosine A2Areceptors are expressed in the CA1, CA3 and DG of the hippocampus(Cunha et al., 1994). A2A mRNA expression is mainly localized in thepyramidal and granular cells, the same hippocampal regions thatshow adenosine A1 receptor mRNA expression (Cunha et al., 1994).

It is well established that the selective block of adenosine A2Areceptors is protective in different in vivo models of cerebralischemia (Bona et al., 1997; Gao and Phillis, 1994; Melani et al.,2003, 2006, 2009; Monopoli et al., 1998; von Lubitz et al., 1995).In agreement, the brain damage induced by transient focalischemia is attenuated in A2A knockout mice (Chen and He, 1999).We have recently demonstrated in the model of oxygeneglucosedeprived hippocampal slices that two selective adenosine A2Areceptor antagonists, ZM241385 and SCH58261 (with less efficacy),are protective during OGD in the CA1 region by delaying theappearance of anoxic depolarization (AD), astrocyte activation, andby improving neuronal survival and recovering synaptic activityunder slice reperfusion with oxygenated-glucose-containing arti-ficial cerebrospinal fluid (aCSF) (Pugliese et al., 2009). The majorprotective effect of A2A receptor antagonists has been attributed toreduced glutamate outflow from neurons (Marcoli et al., 2003;Pedata et al., 2003, 2005) and therefore, to reduced excitotoxicdamage.

Our research focuses on the synaptic and proliferative responseof the DG to severe OGD and on the contribution of A2A adenosinereceptor antagonism to the recovery of synaptic activity of DG inacutely isolated rat hippocampal slices. A preliminary account ofthis work has been communicated (Maraula et al., 2012).

2. Materials and methods

2.1. Slice preparation

All animal procedures were conducted according to the Italian Guidelines forAnimal Care, DL 116/92, application of the European Communities Council Directive(86/609/EEC). Experiments were carried out on acute hippocampal slices (Puglieseet al., 2006), prepared from Male Wistar rats (Harlan; Udine, Italy, 150e200 gbody weight). Animals were killed with a guillotine under anesthesia with etherand their hippocampi were rapidly removed and placed in ice-cold oxygenated (95%O2e5% CO2) aCSF of the following composition (mM): NaCl 125, KCl 3, NaH2PO4 1.25,

MgSO4 1, CaCl2 2, NaHCO3 25 and D-glucose 10. Slices of hippocampi (400 mm thick)were cut using a McIlwain tissue chopper (The Mickle Lab. Engineering, Co. Ltd.,Gomshall, U.K.) and kept in oxygenated aCSF for at least 1 h at room temperature(RT). A single slice was then placed on a nylon mesh, completely submerged ina small chamber (0.8 ml) and superfused with oxygenated aCSF (31e32 �C) ata constant flow rate of 1.5e1.8 ml min�1. Changes in superfusing solutions (OGD ordrugs) reached the preparation in 60 s and this delay was taken into account in ourcalculations.

2.2. Extracellular recording

The field potential recordings were performed in the CA1 region of the stratumradiatum following stimulation of the Schaffer collaterals and in the mid-molecularlayer of DG following stimulation of the perforant pathway. Test pulses (80 ms,0.066 Hz) were delivered through a bipolar nichrome electrode positioned into theSchaffer collaterals for CA1 region and into the perforant pathway for DG recordings.Evoked extracellular potentials were recorded with glass microelectrodes (2e10 MU, Harvard Apparatus LTD, Edenbridge, UK) filled with 150 mM NaCl andplaced into the CA1 region or into the DG. Test stimuli were delivered to the Schaffercollaterals or perforant pathway every 15 s. Responses were amplified (200�, BM622, Mangoni, Pisa, Italy), digitized (sample rate, 33.33 kHz), and stored for lateranalysis with LTP (version 2.30D) program (Anderson and Collingridge, 2001).

Stimulus-response curves were obtained by gradual increases in stimulusstrength at the beginning of each experiment, when a stable baseline of evokedresponse was reached. The test stimulus pulse was then adjusted to produce a fieldexcitatory postsynaptic potential (fEPSP) whose slope and amplitude was 40%e50%of the maximum and was kept constant throughout the experiment. The fEPSPamplitude was routinely measured and expressed as the percentage of theaverage amplitude of the potentials measured during the 5 min precedingexposure of the hippocampal slices to OGD. In some experiments, both theamplitude and initial fEPSP slope were quantified, but because no appreciabledifferences between these two parameters were observed in drug effect and OGD,only the amplitude measurement is expressed in the Figures. Simultaneously withfEPSP amplitude, AD, induced by 7, 9 or 30-min OGD, was recorded as negativeextracellular direct current (d.c.) shifts. The d.c. potential is an extracellularrecording considered to provide an index of the polarization of cells surroundingthe tip of the glass electrode (Farkas et al., 2008). AD latency, expressed in min,was calculated from the beginning of OGD; AD amplitude, expressed in mV, wascalculated at the maximal negativity peak. In the text and bar graphs, AD amplitudevalues were expressed as positive values.

Conditions of OGD were obtained by superfusing the slice with aCSF withoutglucose and gassed with nitrogen (95% N2e5% CO2) (Pedata et al., 1993). This causeda drop in pO2 in the recording chamber fromw500 mm Hg (normoxia) to a range of35e75 mmHg (after 7-min OGD) (Pugliese et al., 2003). At the end of the ischemicperiod, the slice was again superfused with normal, glucose-containing, oxygenatedaCSF. Throughout this paper, the terms ‘untreated OGD slices’ or ‘treated OGD slices’refer to hippocampal slices in which OGD episodes of different duration wereapplied in the absence or in the presence of drugs, respectively. Control slices werenot subjected to OGD or drug treatment, but were incubated in oxygenated aCSF foridentical time intervals. The selective adenosine A2A receptors antagonist,ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol), was applied 15min before, during and 5min after 9 or 30 min OGD. Aconcentration of 100 nM ZM241385 was used and was chosen according to pub-lished data (Latini et al., 1999; Lopes et al., 2002; Pugliese et al., 2003). At thisconcentration, ZM241385 was still selective for rat A2A adenosine receptors, but ata concentration which exceeded its Ki value, as determined by binding experiments(Fredholm et al., 1994).

2.3. Immunohistochemical assay

2.3.1. Newly generated neuronsProliferating cells were detected by using the DNA replication marker 5-Bromo-

20-deoxyuridine (BrdU), a thymidine analog which incorporates into the DNA of allcells during the S-phase. To determine the phenotype of the newly born cells,doublecortin (DCX), an immature neuronal marker, was used. DCX is expressed innewborn hippocampal granule cells during the first 3 weeks after mitosis (Brownet al., 2003).

BrdU treatments. Two intraperitoneal injections (i.p.) of BrdU (50 mg/kg) weregivenwith the interval of 6 h for three consecutive days. BrdUwas dissolved in salinesolution (sterile 0.9% NaCl) and prepared freshly every day of injection. After 24 hfrom last injection, the animal was sacrificed and hippocampal slices prepared asdescribed before.

2.3.2. BrdU and DCX stainingAfter extracellular recordings, slices were maintained in separate chambers in

oxygenated aCSF at RT before fixing which was performed at different time points:after 3, 6 or 24 h from the end of OGD. Control slices were monitored for electro-physiological activity and fixed at corresponding times from cutting in the sameexperimental day. Hippocampal slices (400 mm) were then fixed overnight using

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520 513

1 ml of 4% ice-cold paraformaldehyde and then cryoprotected in a sucrose solution(18%) for at least 48 h. Then, slices were glued on frozen cubes of agar (4%) preparedin 18% sucrose solution, and then re-sliced into 50-mm thick slices with a cryostat.Superficial slices were excluded, while those obtained from the inner part wereplaced in antifreeze solution (30% ethylene glycol, 30% glycerol in phosphate buffer)at �20 �C, until immunohistochemical assay.

Day 1. Slices were stained using the free-floating method described byGiovannini et al. (2001, 2003). Hippocampal slices were placed in 24-well plates andrinsed for 10 min in phosphate-buffered salinee0.3% Triton X-100 (PBS-TX). Briefly,DNA denaturation was achieved by treatment with 2 M HCl at 36 �C for 15 min andthen rinsed in borate buffer for 10 min (0.1 M, pH 8.5 at RT). They were extensivelywashed with PBS containing 0.3% Triton X-100 and then incubated on a shaker for1 h at RT with PBS containing 0.3% Triton X-100, 0.05% NaN3, 10% normal goat serumand 10% normal horse serum (blocking solution). For double-labeling experiments,after a PBS solution containing 0.3% Triton X-100 rinse, sections were incubatedwithat least 250 ml in mouse monoclonal anti-BrdU antibody (1:300; Abcam, Cambridge,UK) and rabbit monoclonal anti-DCX antibody (1:500; Abcam, Cambridge, UK)diluted in blocking solution, overnight at 4 �C on a shaker.

Day 2. Primary antibodies were removed and slices were washed several timeswith PBS solution containing 0.3% Triton X-100. From this step, all procedures werecarried out in the dark. Sections were then incubated under agitation for 2 h at RTwith a horse anti-mouse fluorescent secondary antibody (Alexa Fluor 488, 1:500,Invitrogen, Ltd, UK) dissolved in blocking buffer. The secondary antibody was thenremoved and the slices were washed several times with PBS solution containing0.3% Triton X-100 and incubated at RT with a goat anti-rabbit fluorescent secondaryantibody (Alexa Fluor 635, 1:500, Invitrogen Ltd, UK) dissolved in blocking buffer.After 2 h of incubation at RT, the secondary antibody was removed and slices werewashedwith PBS solution 0.3% Triton X-100. Finally, after an extensivewashout withdistillate water, slices were mounted onto gelatin-coated slides for microscopicexamination using Pro-Long mounting medium (Invitrogen Ltd, UK).

Image analysis of double immunofluorescent labeling was performed by a SP2-AOBS confocal laser-scanning microscope (Leica Microsystems, Mannheim,Germany) through a 20� 0.5 NA air objective and 40� 1.2 NA oil-immersionobjective, using laser excitation at 488 and 635 nm. Images were acquired as Z-stacks of the entire slice thickness (50 mm), with sampling at 1.5-mm intervals for theentire slice depth (50 mm) and assembled intomontageswith Image J software (NIH;http://rsb.info.nih.gov/ij/) and Adobe Photoshop 7.0 (Adobe Systems, MountainView, CA, USA). 3D volume rendering and animations of confocal images was per-formed with Volocity software (Perkin Elmer, Milan, Italy).

Double-labeled neurons were quantified in a confocal plane throughout the area(750 � 750), captured at 20� magnification, and the total number of BrdUþ cellswere counted by eye. In particular, all quantification analysis were performed blindby two different experimenter and results were averaged. The number of slices usedfor immunohistochemical analysis in each experimental conditions corresponds tothe number of rats used. Only those cells that were found within the granule celllayer, in the SGZ, defined as the 20-mm band under the granule cell layer, and in thehilus were included in the analysis. We used the dorsal portion of the hippocampussince it is known to be most severely and consistently affected by ischemia (Aueret al., 1989).

2.4. Drugs

ZM241385 was purchased from Tocris (Bristol, UK). The A2A antagonist wasdissolved in dimethyl sulphoxide (DMSO). Stock solutions of 1000e10,000 times thedesired final concentration were stored at �20 �C. The final concentration of DMSO(0.05% and 0.1% in aCSF) used in our experiments did not affect either fEPSPamplitude or the depression of synaptic potentials induced by OGD (not shown).BrdU was purchased from Sigma (Sigma-Aldrich, Italy).

2.5. Statistics

Statistical significance was evaluated by Student’s paired or unpaired t tests.Analysis of variance (one-way ANOVA), followed by NewmaneKeuls multiplecomparison post hoc test was also used, as appropriate. P-values from both Student’spaired and unpaired t tests are two-tailed. Data were analyzed using softwarepackage GRAPHPAD PRISM (version 4.0; GraphPad Software, San Diego, CA, USA). Allnumerical data are expressed as the mean � standard error of the mean (SEM).

3. Results

3.1. Severe, 9-min long, OGD always induces the appearance of ADand the irreversible depression of fEPSP in the DG

Seven-min OGD induces an irreversible depression of synapticactivity in the CA1 area (Pugliese et al., 2003, 2007; Traini et al.,2011) and is considered severe in this hippocampal subfield.Therefore, in an early set of experiments we evaluated the effects of

7-min OGD in the DG. Fig. 1A shows that in the CA1 region, 7-minOGD induced the appearance of AD (evaluated as d.c. shift) and anirreversible loss of synaptic activity in all slices tested (4.0 � 3.1% ofrespective pre-ischemic values, Fig. 1C). AD peaked at 6.1 � 0.2 min(n ¼ 11) (calculated from the beginning of ischemic insult), with anamplitude of 7.4 � 0.6 mV (n ¼ 11). At variance with the resultsobtained in the CA1 area, 7-min OGD in DG induced the appearanceof AD only in 5 out of 11 slices examined (Fig. 1B). In these 5 slices,AD peaked at 6.1�0.2 minwith an amplitude of 8.4� 0.4 mV. In all5 slices, 7-min OGD was followed by a partial recovery of fEPSP(18.6 � 4.1% of respective pre-ischemic values, Fig. 1C). In theremaining 6 slices, no AD was recorded and consistent fEPSPrecovery was observed (81.4 � 4.7% of respective pre-ischemicvalues, Fig. 1C). During 7-min OGD the depression of fEPSP ampli-tude is significantly slower in DG than CA1 (Fig. 1D). These resultsindicate varying susceptibility of DG and CA1 to 7-min OGD andsuggest that a longer time should be chosen to study the effect ofsevere OGD in the DG of acutely isolated hippocampal slices. Thistime was identified by prolonging the time of OGD exposure.

It is known that 30-min OGD in the CA1 area of the hippo-campus is invariably associated with irreversible failure of synaptictransmission and tissue damage (Pearson et al., 2006; Puglieseet al., 2006). Fig. 2 illustrates the time of the appearance and theamplitude of depolarizing d.c. shift in the CA1 (Fig. 2A) and DG(Fig. 2B). It shows that, while AD appeared with a mean latency of6.2 � 0.16 min (n ¼ 18, Fig. 2C) in the CA1 region, it was alwaysdelayed to 7.6 � 0.3 min (n¼ 14, P< 0.001, Fig. 2C) in the DG. In theDG, the mean peak amplitude of the d.c shift (5.6 � 0.9 mV, n ¼ 14)(Fig. 2D) was reduced, but not significantly, in comparison with theCA1 area (7.3 � 0.48 mV) (n ¼ 18, Fig. 2D).

Therefore, in following experiments, the duration of the OGD inthe DGwas prolonged to 9 min. With application of 9-min OGD, ADappeared in all recorded slices (Fig. 3A). In this set of experimentsAD peaked at 7.7 � 0.2 min (n ¼ 51) with a mean amplitude of5.4 � 0.3 mV (n ¼ 51) (Fig. 3A). No fEPSP recovery (4.2 � 1.2% ofrespective pre-ischemic values, n ¼ 51) was observed up to 60 min(Fig. 3D) nor 24 h (see also Fig. 4) after the end of 9-min OGD in allthe slices tested.

3.2. Selective antagonism of A2A adenosine receptors by ZM241385significantly prevents or delays the appearance of AD and protectsfrom the irreversible fEPSP depression induced by 9-min OGD

The effect of adenosine A2A receptor antagonist was studied on9-min OGD experimental protocol. The selective adenosine A2Areceptor antagonist ZM241385 was applied 15 min before, duringand 5min after the end of OGD. ZM241385 (100 nM, n¼ 32) did notchange fEPSP amplitude under basal, normoxic, conditions (from1.2 � 0.07 mV before to 1.2 � 0.05 mV, calculated after 15 minZM241385 before OGD application).

The A2A antagonist prevented the appearance of AD in 20 out of32 slices tested (Fig. 3B). In these 20 slices, in which AD was absent,an almost complete fEPSP recovery was observed (95.2 � 5.0% ofrespective pre-ischemic conditions, calculated 60 min after the endof OGD, Fig. 3D). In 4 of 32 slices, ZM241385 significantly delayedthe appearance of AD (AD latency: 9.44 � 0.16 min, calculated fromthe beginning of OGD, P < 0.05, Fig. 3C). In these 4 slices a modest,but significant fEPSP amplitude recovery occurred (24.8 � 10.6% ofrespective pre-ischemic values, P < 0.05, not shown). In theremaining 8 out of 32 slices, ZM241385 was not effective, since nochange on AD appearance or fEPSP recovery (not shown) wasdetected. In these 8 slices AD peaked at 8.1 � 0.2 min, with a meanamplitude of 4.4� 0.6 mV (Fig. 3C). On the basis of these results, wecan conclude that in DG the A2A antagonist was effective in 75% ofthe slices tested.

Fig. 1. Effects of 7-min OGD in the CA1 region and in the DG of rat hippocampal slices. AD was recorded as a negative d.c. shift in response to 7-min OGD. The d.c. shift was alwaysrecorded in CA1 (A, n ¼ 11), while in the DG (B, n ¼ 11) AD was recorded in 5 out of 11 slices tested. C. Graph shows the time-course of 7-min OGD effect on fEPSP amplitude,expressed as percent of pre-ischemic baseline, both in the CA1 (mean � SEM, n ¼ 11) or in the DG (mean � SEM, n ¼ 11). Note that in the CA1 region the ischemic insult leads to thegradual reduction until disappearance of fEPSPs amplitude, that does not recover after washing in oxygenated aCSF. In the DG, after reperfusion in normal oxygenated aCSF,a consistent fEPSP recovery was evident in 6 out of 11 slices. In these 6 slices, 7-min OGD application does not induce AD appearance. In the remaining 5 out of 11 slices, in which ADappears during 7-min OGD, however, a recovery of fEPSPs is also recorded. The arrow indicates a portion of the graph that is shown in D on a different x-scale. D. Time-course offEPSP amplitude (expressed as percent of pre-ischemic baseline) during 7-min OGD in the CA1 (mean � SEM, n ¼ 11) or in the DG (mean � SEM, n ¼ 5, hippocampal slices in whichAD was present during OGD; n ¼ 6, OGD treated slices in which AD was absent during OGD). At least **P < 0.001, unpaired two-tailed Student’s t test between CA1 and corre-sponding time-points of 7-min OGD slices recorded in DG in which AD was present (n ¼ 5). Gray bar: OGD time duration.

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520514

We also compared the time of AD appearance, in the absence orin the presence of ZM241385, during 30-min OGD. As illustrated inFig. 3E, during 30-min OGD, AD appeared in untreated OGD sliceswith a mean latency of 7.6 � 0.3 min and a mean peak amplitude of5.6 � 0.7 mV (n ¼ 14). When 30-min OGD was applied in thepresence of 100 nM ZM241385, the d.c. shifts were always delayed(AD latency of 9.9 � 0.6 min, n ¼ 8, P < 0.01, Fig. 3E, left), while ADamplitude was not significantly modified (3.55 � 0.4 mV, n ¼ 8,Fig. 3E, right).

In Fig. 4, trace examples of fEPSPs recorded from a typicalexperiment at different times are shown. From 1 to 24 h after slicecutting, fEPSP was still recordable (Fig. 4 A,D). Similar results wereobtained from 5 other control slices. Conversely, in a slice to which9-min OGD was applied, no recovery of fEPSP (but only of theafferent fiber volley) was detected at least up to 24 h after the endof the insult (Fig. 4B,E). Similar results were obtained from 7 otherslices. When 9-min OGD was applied in the presence of ZM241385(100 nM), a stable fEPSP was recorded either 1 h (Fig. 4C) or 24 h(Fig. 4F) after the end of the insult. Similar results were obtainedfrom 4 other slices. This demonstrates that the recovery of neuro-transmission produced by ZM241385 lasts up to 24 h after the endof OGD.

3.3. Effects of OGD and adenosine A2A receptors on cell proliferationand maturation in the DG of rat hippocampal slices

In a parallel series of experiments, we investigated whether 9-min OGD modified cell proliferation and maturation in the SGZ,

by performing immunohistochemical analysis for BrdU and DCX,respectively. BrdU-immunoreactive (BrdUþ) cells were detectedboth in the SGZ or in the hilus in control slices at all times examined(Fig. 5A,D and G). The number of BrdUþ proliferating cells in theSGZ was not modified after 3 h from the ischemic insult carried outeither in the absence or in the presence of ZM241385 (Fig. 5G).Conversely, 9-min OGD significantly reduced the number of BrdUþ

cells in the SGZ, compared to control, after 6 h from the end of theinsult (Fig. 5B,G); from 24.4� 3.2 in control, n¼ 9 to 15.5�1.7 afterOGD, n ¼ 8, P < 0.05 (Fig. 5G). This effect was completely coun-teracted in the presence of ZM241385 (Fig. 5C,G). After 24 h fromOGD the number of BrdUþ cells in the SGZ was restored, reachingcontrol level (Fig. 5E,G). At this time after OGD, ZM241385 did notmodify the number of BrdUþ cells (Fig. 5F,G).

The number of BrdUþ cells in the hilus (control: 9.25 � 1.4 after6 h and 23 � 2.55 after 24 h) was not affected either by OGD alone(8.9� 3.1 after 6 h fromOGD and 21.6� 2.4 after 24 h fromOGD) orin combination with 100 nM ZM241385 (7.4 � 1.6 after 6 h fromOGD and 23.1 � 3.6 after 24 h from OGD).

The microtubule-associated protein DCX is a cytoskeleton-associated protein that is transiently expressed during adult neu-rogenesis. In control slices, DCX-immunoreactive neuroblasts(DCXþ) were clearly observed in the SGZ (Figs. 5 and 6). Theirprocesses appeared very short and without tertiary dendrites,either 6 (Fig. 5A) or 24 h after cutting (Fig. 6A, see also short moviein supplementary data). After 3 (not shown) or 6 h (Fig. 5B,C) fromthe end of OGD, the morphology of DCXþ cells was not modifiedeither in the absence or in the presence of the A2A antagonist. In

Fig. 2. The appearance of AD recorded in the DG during 30-min OGD is delayed if compared to CA1. AD was recorded as a negative d.c. shift during 30-min OGD in the CA1 region (A,n ¼ 18) and in the DG (B, n ¼ 14). Gray bar: OGD time duration. C. Each column represents the mean � SEM of AD latency recorded either in the CA1 or in the DG during 30-minOGD. AD latency was measured from the beginning of OGD insult. Note that in the DG the appearance of AD is significantly delayed. **P < 0.001, unpaired two-tailed Student’s t testcompared to CA1. D. Each column represents the mean � SEM of AD amplitude recorded in the CA1 and in the DG hippocampal subfields during 30-min OGD. The number (n) ofslices tested is reported inside columns.

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520 515

contrast, after 24 h from OGD carried out either in the absence(Fig. 6B) or in the presence of ZM241385 (Fig. 6C), the DCXþ neu-roblasts showed a well-developed arborization of tertiarydendrites. Finally, the vast majority of BrdUþ cells in the SGZ lay inthe proximity of DCXþ cells in all experimental groups (Fig. 6, seealso short movie in supplementary data), showing that BrdUþ nucleibelong to DCXþ cells.

Supplementary videos related to this article can be found onlineat http://dx.doi.org/10.1016/j.neuropharm.2012.12.002.

4. Discussion

The present results demonstrate that an episode of OGD longer(9 min) than that needed (7 min) in the CA1 region is necessary toinduce the appearance of AD and the irreversible depression ofsynaptic activity in the adult rat DG.

The duration of an ischemic insult affects the severity ofsynaptic damage. In rat CA1, short ischemic (2e5 min) episodesdefinitely depress fEPSP amplitude but never elicit the appearanceof AD, a clear sign of cell damage (Somjen, 2001) and synaptictransmission fully recovers when slices are reperfused with glucosecontaining oxygenated aCSF (Fowler, 1992; Frenguelli et al., 2007;Latini et al., 1999; Pearson et al., 2006; Pugliese et al., 2003). Inagreement with previous works (Frenguelli et al., 2007; Puglieseet al., 2007, 2009; Traini et al., 2011), we report here that inacutely isolated CA1 hippocampal slices, the application of 7-minOGD always induces the appearance of AD and an irreversibleloss of neurotransmission. Application of 7-min OGD in DG induces

AD and the block of synaptic activity only in 45% of the slicesexamined. In the DG, in order to constantly induce the appearanceof AD and synaptic block, it is necessary to prolong OGD duration to9 min, in agreement with previous observations (Aitken and Schiff,1986; Balestrino et al., 1989; Kass and Lipton, 1986). The CA1susceptibility to prolonged and severe AD depolarization mayemphasize the selective vulnerability of CA1 neurons to hypoxicand ischemic damage. The generation of AD is complex andmultifactorial (see: Somjen, 2001). After OGD initiation, the largeefflux of potassium into the extracellular space combined withactivation of sodium and calcium channels, triggers sustaineddepolarization of hippocampal cells that coincides with theappearance of AD. Increased intracellular calcium and/or massiveglutamate receptor activation are additional mechanisms thatoccur to produce AD (Tanaka et al., 1997; Yamamoto et al., 1997) andthat contribute to cell damage during ischemia (Somjen, 2001). Adelay in the appearance of AD can be obtained by treating the sliceswith glutamate receptor antagonists (Tanaka et al., 1997;Yamamoto et al., 1997; see also in Somjen, 2001), demonstratingthe importance of glutamate in triggering AD. Therefore, whenconsidering the possible reasons for the different sensitivities ofCA1 and DG to ischemia, differences in the capability of cell(especially neuron) depolarization and in the concomitant ionicredistribution and glutamate excitotoxicity might be importantfactors. It has been demonstrated that in rat hippocampal slices,CA1 accumulates twice as much Ca2þ during anoxia as DG (Kassand Lipton, 1986). A study conducted on gerbil hippocampus byin situ hybridization has demonstrated that mRNA levels of GLAST

Fig. 4. ZM241385 allowed recovery of synaptic potentials up to 24 h after 9-min OGD. In control conditions a stable fEPSP can be recorded both at 1 h (A) and at 24 h after cutting(D). At different times from the end of OGD only the afferent fiber volley can be recorded in untreated OGD slice, both after 1 (B) or 24 h (E). In 100 nM ZM241385 OGD-treatedslice a fEPSP was present up to 24 h (C: after 1 h; F: after 24 h). Each trace, taken from a typical experiment, represents the average of 3 consecutive evoked fEPSPs. Calibration:0.5 mV, 10 ms.

Fig. 3. The selective adenosine A2A receptor antagonist, ZM241385, prevents or delays the appearance of AD and counteracts the irreversible loss of neurotransmission induced by9-min OGD in the DG. AD was recorded as a negative d.c. shift in response to 9-min OGD in control (A, n ¼ 51) or in a group of hippocampal slices in which OGD was applied in thepresence of 100 nM ZM241385 (C, n ¼ 12). The A2A adenosine receptor antagonist (100 nM) completely prevented the appearance of AD in 20 slices (B) during 9-min OGD. D. Graphshows the time course of 9-min OGD effects on fEPSP amplitude in untreated OGD slices (mean � SEM, n ¼ 51) and in 100 nM ZM241385 treated OGD slices (mean � SEM, n ¼ 20) inwhich AD was absent during OGD. fEPSPs amplitude is expressed as percent of respective pre-ischemic baseline. Gray bar: OGD time duration. E. Each column represents themean � SEM of AD latency (left) and AD amplitude (right) recorded in the DG during 30-min OGD, both in the absence (n ¼ 14) or in the presence of ZM241385 (100 nM, n ¼ 8).Note that ZM241385 significantly delayed AD development (*P < 0.01, unpaired two-tailed Student’s t test compared to untreated OGD slices). AD latency was measured from thebeginning of OGD insult. The number (n) of slices tested is reported inside columns.

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Fig. 5. Temporal profile of cell proliferation in the SGZ of DG. A-F. Double labeling immunohistochemical analysis for BrdU (green) and DCX (red) staining (20�). Note that the vastmajority of BrdUþ cells lay in proximity of DCXþ cells. Scale bar ¼ 100 mm. SGZ: subgranular zone. BrdU, 5-Bromo-20-deoxyuridine; DCX, doublecortin. G. Quantification of BrdUþ

cells in the SGZ of the DG at different time points after the end of OGD. In control conditions the quantification was evaluated at corresponding times after cutting. Each columnshows the total number of BrdUþ cells in the SGZ. Bars represent the mean � SEM. In parentheses is the number of slices investigated. *P < 0.05 vs control, One-way ANOVAfollowed by post hoc NewmaneKeuls test. The regions in the box are shown at a higher magnification in Fig. 6.

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520 517

(glutamate/aspartate transporter) and GLT1 (glutamate trans-porter1) were intensely expressed in the DG if compared to CA1.After forebrain ischemia, the mRNA levels of glutamate trans-porters in DG are unchanged while they are markedly decreased bythe seventh day after ischemia in the CA1 (Fujita et al., 1999). Theseobservations indicate that a greater glutamate excitotoxicityaccounts for the higher vulnerability of CA1 neurons in comparisonto DG to ischemia, indicative of cell death (Fujita et al., 1999).Moreover, reoxygenation after OGD increased reactive oxygenspecies (ROS) levels in the hippocampal CA1 layers but did not havesignificant effects in the stratum granulosum of the DG (Feketeet al., 2008). All together, these observations support that DG isless excitable during OGD and is less damaged under reperfusion incomparison to CA1. This might account for the major resistance ofDG to develop AD and the irreversible depression of synaptictransmission. A further difference between DG and CA1 was thatafter 7-min OGD in the DG, a partial recovery of neurotransmissionis observed, even in the presence of AD. This effect was neverrecorded in the CA1, where the appearance of AD and synapticdepression were concomitant “all or nothing” phenomena. It islikely that the different characteristics of excitatory glutamatergictransmission in DG during OGD account for a lower spreading of ADand for the consequent recovery of neurotransmission by a group ofneurons, undamaged during OGD.

The decrease in fEPSP amplitude during OGD is slower in DGthan CA1. The predominant, although not exclusive, mechanismthat accounts for the reduction in fEPSPs during the first 4e5min ofischemia is a decrease in glutamatergic neurotransmission causedby activation of adenosine A1 presynaptic receptors, as welldescribed in CA1 hippocampal slices (Fowler, 1992; Gribkoff et al.,1990; Latini et al., 1999; Pedata et al., 1993). A delayed failure of

synaptic transmission in the DG in comparison to CA1 may be dueto reduced adenosine release during severe OGD or to a lower A1receptors density. An in vitro receptor autoradiography, used tomeasure the regional binding site densities of the adenosinereceptors in rat brain, demonstrate a reduced A1 receptor expres-sion in DG in comparison to CA1 (Cremer et al., 2009). Furthermore,unlike CA1, stimulation of A1 receptors in DG does not suppressexcitatory synaptic input to dentate interneurons during anoxia, asdemonstrated in rat hippocampal slices (Doherty and Dingledine,1997). We might mention that adenosine agonists are more effec-tive in inhibiting acetylcholine release in the CA1 than the DG, andthat DPCPX, a selective A1 adenosine antagonist, increases basalacetylcholine release in the CA1, but not in the DG (Cunha et al.,1994). Therefore, differences in adenosine receptor abundance oractivity between DG and CA1 likely account for the differentsusceptibility to ischemic insult.

Twenty four hours after cutting, control acutely-isolatedhippocampal slices were always viable although fEPSPs werereduced in amplitude in comparison to those recorded at earliertimes. Differently from CA1 (Pugliese et al., 2009), 24 h aftercutting, the recorded potentials in the DG never showed seizure-like activity. The reason for this might be a higher resistance ofDG to the formation of tissue-destructive oxygen radicals (Feketeet al., 2008), which are unavoidably produced after prolongedexposition of the slices to a medium containing 95% O2 (D’Agostinoet al., 2007; Pomper et al., 2001).

Here we report the presence of BrdUþ cells in the DG and in thehilus of hippocampal slices and of DCX-labeled cells located in theSGZ, at the border between the hilus and the granule cell layer. Themicrotubule associated protein DCX is a cytoskeleton-associatedprotein that is transiently expressed during adult neurogenesis.

Fig. 6. In the SGZ the vast majority of BrdUþ cells co-localized with DCXþ cells. AeC.Confocal images of the DG double labeling for BrdU and DCX staining showing that thevast majority of BrdUþ nuclei (green) belong to DCXþ cells (red) (40�). Double-stainedcells are clearly visible in the SGZ of DG in control (A) and 24 h from the end of OGDcarried out either in the absence (B) or in the presence of 100 nM ZM241385 (C). Insetsin AeC: higher magnifications better indicate the double BrdU/DCX staining in a samecell. Note that after OGD, applied in the absence or in the presence of the A2A antag-onist, DCXþ cells have well-developed tertiary dendrites. SGZ: subgranular zone. BrdU,5-Bromo-20-deoxyuridine; DCX, doublecortin. Scale bars: 20 mm; Inset: 7.5 mm.

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520518

Antibodies against DCX are among the most widely used markersfor detection of neuronal progenitors and identifies type-2b andtype-3 cells, which belong to blast-like late progenitors and exhibitthe first morphological signs of neuronal maturation (Brandt et al.,2003; Brown et al., 2003, Klempin et al., 2011). Staining for DCX isproximal to BrdUþ nuclei, indicating that most often the same typeof cells are stained by the two markers and that neuron committedcells are proliferating.

The number of BrdUþ cells in the SGZ is not decreased 3 h afterOGD, but is decreased 6 h after OGD in comparison to control slices.This reduction is likely due to acute damage after the insult andindicates that a part of BrdUþ proliferating cells encountered ondeath. In the adult rat activation of NMDA receptors in the DGrapidly decreases the number of proliferating cells identified with3H-thymidine incorporation, whereas blockade of NMDA receptorsrapidly increases the number of cells in the S phase, as indicated byan autoradiography and immunohistochemistry study (Cameronet al., 1995).

Although 9-min OGD rapidly induces a block of synaptictransmission that persists up to 24 h, the number of BrdUþ cells inthe SGZ is not yet affected 3 h after OGD in comparison to controlslices. We cannot exclude that part of BrdUþ cells are alreadydamaged or died following the strong depolarization induced bysevere OGD, but that they are still retaining BrdU. The reduction incell proliferation in the SGZ observed after 6 h is restored 24 h afterthe end of OGD, when a comparable number of BrdUþ cellsbetween control and OGD slices was found. Since BrdU wasadministered i.p. prior to isolation of hippocampal slices, the re-

establishment of the number of proliferating cells can beexplained by the increased proliferation of residual BrdUþ cells,with new daughter cells now exhibiting BrdU labeling.

A major resistance of the DG to glutamate excitotoxicity and toROS production might account for the persistence of proliferativecapabilities of the BrdUþ cells in the SGZ of the DG after the severeOGD period.

Severe OGD modifies neuronal progenitor cell morphology inthe SGZ. For the first time we demonstrate, in this in vitro model,that 24 h after OGD the number of dendrites of DCX-labeled cells isincreased, as indicated by qualitative immunohistochemical anal-ysis. DCX is a microtubule-associated protein necessary forneuronal migration; its expression is required for the developmentof dendritic arborisation, as demonstrated in primary hippocampalneurons (Cohen et al., 2008). Reduction in the arborisation of thedendritic tree is connected with dendrite instability since reductionof the expression levels of DCX in migrating neuroblasts alters theirmigration (Cohen et al., 2008). In our experiments the increase inDCX expression after OGD may be considered an input for cellmigration and maturation toward neuronal phenotype.

In the present work we demonstrate that the selective adeno-sine A2A receptor antagonist, ZM241385, delays the occurrence ofAD, improves neuronal survival following 9-min OGD in the DG, asdemonstrated by the significant recovery of otherwise disruptedneurotransmission, and antagonizes reduction of BrdUþ cellnumber 6 h after OGD. Similar results were obtained in the CA1,where several A2A receptor antagonists delayed the appearance ofAD and prevented from the irreversible failure of synaptic activityinduced by 7-min OGD (Pugliese et al., 2009).

During OGD, endogenous adenosine is released in largeamounts from hippocampal slices reaching extracellular concen-trations between 5 (3 min after OGD) and 23e30 mM (5 min afterOGD) (Latini et al., 1999; Pearson et al., 2006). These concentrationscan maximally stimulate A2A adenosine receptors which have anaffinity for adenosine in nanomolar range (Fredholm et al., 1994,2011). Endogenous adenosine released by prolonged OGD, actingthrough A2A receptors, facilitates glutamate outflow from hippo-campal slices, as demonstrated by the decrease in glutamateoutflow induced by the A2A adenosine receptor antagonistZM241385 (Sperlagh et al., 2007). Pharmacological activation ofadenosine A2A receptors increases glutamate outflow and increasessynaptic transmission in the CA1 area of hippocampal slices(Diógenes et al., 2004; Lopes et al., 2002; Tebano et al., 2005).Furthermore, A2A receptor stimulation potentiates NMDAresponses in the CA1 hippocampus (Tebano et al., 2005). NMDAreceptor activation is essential to AD initiation and propagation(Somjen, 2001). In the rat hippocampus, adenosine A2A receptorshave been localized on glutamatergic terminals (Rebola et al.,2005b) and at postsynaptic sites (Rebola et al., 2005a). They areexpressed not only in the CA1 and CA3 but also in the DG, wherethey are mainly localized in the pyramidal and granular cells,respectively (Cunha et al., 1994). Selective adenosine A2A receptorantagonism, by removing A2A receptor potentiation of glutamatetransmission in the DG, may reduce the participation of glutamatein triggering AD. Severe insults, such as ischemia, can lead toincreased neurogenesis as an intrinsic compensatory response toneuronal cell death in the hippocampus (Gould and Tanapat, 1997;Kee et al., 2001; Liu et al., 1998). Synaptic recovery as well asneurogenetic processes in the DG are prevented by gamma irradi-ation applied focally to the rat head (Wang et al., 2005). Thisobservation supports that the better recovery of neurotransmissionin DG, in comparison to the CA1 region, observed after several daysfrom the insult is attributable to DG regenerative capabilities.

Observation that the A2A ligand antagonizes reduction of BrdUþ

cell number 6 h after OGD indicates that it protects proliferating

G. Maraula et al. / Neuropharmacology 67 (2013) 511e520 519

cells from ischemic damage. Besides participating to excitotoxicdamage, adenosine A2A receptors are known to increase brain-derived neurotrophic factor which supports cell proliferation andsurvival (Assaife-Lopes et al., 2010; Tebano et al., 2008). However,no data are to date in the literature concerning the role of adeno-sine A2A receptors on adult neurogenesis. Our recent resultssupport the involvement of adenosine A2A receptors in this processby demonstrating that the selective A2A antagonist SCH58261reverses the inhibitory effect exerted by the selective A2A agonistCGS 21680 onmaturation of oligodendrocyte progenitor cells (OPC)in the firsts 10 days of in vitro differentiation (Cellai et al., 2012).

In summary, in this paper we describe the higher resistance ofthe DG to severe OGD in comparison to the CA1 area of thehippocampus. We also demonstrate the presence of proliferatingneuronal progenitor cells in the DG whose maturation is promotedby the severe OGD. The adenosine A2A antagonist ZM241385protects from synaptic failure and from loss of immature neuronalcells at a precocious time after OGD.

Disclosure

We confirm that we have read the Journal’s position on issuesinvolved in ethical publication and affirm that this report isconsistent with these guidelines. All the authors have no conflictsof interest.

Acknowledgments

This investigation was supported by grants from IRCCS CentroNeurolesi ‘Bonino-Pulejo’, Messina, Italy; Italian Ministry of Health;Fondazione Ente Cassa di Risparmio di Firenze, Italy (FP, TM andAG). We would like to thank Dr. Daniele Lana for technical support.Animals were kept in the ‘Centro Stabulazione Animali da Labo-ratorio’ (Cesal) of the University of Florence.

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