Modulation of intracellular ATP determines adenosine release and functional outcome in response to metabolic stress in rat hippocampal slices and cerebellar granule cells

*School of Life Sciences, University of Warwick, Coventry, UK

†Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, University of Dundee,

Dundee, UK

AbstractCerebral ischaemia rapidly depletes cellular ATP. Whilst thisdeprives brain tissue of a valuable energy source, theconcomitant production of adenosine mitigates the damagingeffects of energy failure by suppressing neuronal activity.However, the production of adenosine and other metabolites,and their loss across the blood–brain barrier, deprives the brainof substrates for the purine salvage pathway, the primarymeans bywhich the brainmakes ATP. Because of this, cerebralATP levels remain depressed after brain injury. To test whethermanipulating cellular ATP levels in brain tissue could affectfunctional neuronal outcomes in response to oxygen/glucosedeprivation (OGD), we examined the effects of creatine andD-ribose and adenine (RibAde). In hippocampal slices creatine

delayed ATP breakdown, reduced adenosine release, retardedboth the depression of synaptic transmission and the anoxicdepolarization caused by OGD, and improved the recovery oftransmission. In contrast, RibAde increased cellular ATP,caused increased OGD-induced adenosine release and accel-erated the depression of synaptic transmission, but did notimprove functional recovery. However, RibAde improved theviability of cerebellar granule cells when administered afterOGD. Our data indicate that RibAde may be effective inpromoting recovery of brain tissue after injury, potentially viaenhancement of salvage-mediated ATP production.Keywords: adenosine, ATP, cerebral metabolism, creatine,D-ribose, ischaemia.J. Neurochem. (2014) 128, 111–124.

The brain relies on a constant supply of oxygen and glucose tomaintain the high levels of intracellular ATP required for it tofunction. This makes the brain particularly vulnerable tointerruptions in nutrient supply, for example, during cerebralischaemia. As a result, the depletion of ATP during ischaemiaresults in a rapid loss of neuronal function and viability (Lipton1999). Furthermore, the recovery of post-ischaemic ATP maytake longer than 24 h (Kleihues et al. 1974) and is jeopardizedby a delayed secondary decline (Folbergrova et al. 1995;Phillis et al. 1996; Paschen et al. 2000; Kimura et al. 2002).This prolongs the period of energy depletion and leaves thebrain both less able to deploy reparative mechanisms andmorevulnerable to acute secondary insults (Kirino 2002).Whilst ATP depletion renders cells less able to meet

energetic demands, ATP metabolism yields the purinenucleoside adenosine which, via activation of A1 receptors,inhibits synaptic transmission and neuronal excitability andthereby helps cells to preserve energy homeostasis duringischaemia (Rudolphi et al. 1992; Dale and Frenguelli 2009).

In addition, adenosine activates A2 receptors in blood vesselsand thereby increases cerebral blood flow and the supply ofnutrients (Phillis 1989; Pelligrino et al. 2011). Accordingly,adenosine is regarded as an important endogenous neuro-protective agent (Stone et al. 2009).However, the breakdown of ATP during ischaemia results

in losses to the circulation of adenosine and other ATPmetabolites, such as inosine and hypoxanthine (Hillered et al.1989; Kobayashi et al. 1998; Valtysson et al. 1998; Weigand

Received April 26, 2013; revised manuscript received July 30, 2013;accepted August 6, 2013.Address correspondence and reprint requests to Bruno G. Frenguelli,

School of Life Sciences, University of Warwick, Coventry CV4 7AL,UK. E-mail: [email protected] used: 8-CPT, 8-cyclopentyl theophylline; aCSF, artifi-

cial cerebrospinal fluid; CGC, cerebellar granule cells; EC, energycharge; fEPSP, field excitatory post-synaptic potential; OGD, oxygen/glucose deprivation; RibAde, D-ribose and adenine; TAN, total adeninenucleotides.

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 111--124 111

JOURNAL OF NEUROCHEMISTRY | 2014 | 128 | 111–124 doi: 10.1111/jnc.12397

et al. 1999). Since the brain uses these metabolites in thepurine salvage pathway to restore ATP levels (Gerlach et al.1971; Mascia et al. 2000; zur Nedden et al. 2012), this,together with the production of the unsalvageable metabolitesxanthine and uric acid (Kanemitsu et al. 1988; Stover et al.1997), results in a long-lasting depletion of ATP substrates inthe brain and likely underpins the reduction and incompleterecovery of post-ischaemic ATP levels. Accordingly, beingable to manipulate the availability of purine salvage pathwaymetabolites may be a viable means to improve bioenergeticand functional recovery in the injured brain.To test the influence of cellular ATP on adenosine release

and the recovery of function after oxygen/glucose deprivation(OGD), we have used two manipulations known to affectcellular ATP levels. We demonstrate that creatine, whichbuffers ATP breakdown via its creatine kinase-mediatedconversion to phospho-creatine and transfer of its phosphategroup to ADP, delayed ATP loss, reduced the production ofadenosine and slowed the OGD-induced depression oftransmission, but improved recovery of synaptic transmissionafter prolonged OGD. In contrast, RibAde, which improvesATP levels in hippocampal slices (zur Nedden et al. 2011),increased the production of adenosine during OGD and bothaccelerated the OGD-induced depression of excitatory syn-aptic transmission and delayed its recovery after brief OGD,consistent with increased adenosine release. RibAde did notenhance recovery of synaptic transmission after prolongedOGD, but post-OGD treatment with RibAde improved cellviability in cerebellar granule cells.Our results suggest that enhancing purine salvage with

RibAde may be an effective strategy to improve cell survivaland function after brain injury. Given the extent to which Riband Ade are tolerated by humans we propose that RibAdemerits consideration as a potential post-injury neurorestor-ative therapy.

Methods

Materials

Creatine, D-ribose, adenine, 8-cyclopentyl theophylline (8-CPT),Hoechst and propidium iodide (PI) were purchased from SigmaAldrich (Gillingham, Dorset, UK). All the salts for the artificialcerebrospinal fluid (aCSF) for hippocampal slice experiments andKrebs solution for the preparation of cerebellar granule cell cultureswere purchased from Fisher Scientific (Loughborough, Leicester-shire, UK). Neurobasal medium and B27 were from Invitrogen(Paisley, Renfrewshire, UK).

Preparation of hippocampal slices

Male Sprague–Dawley rats (P 19–27) were obtained from anin-house colony and killed by cervical dislocation in accordancewith Schedule 1 of the UK Government Animals (Scientific Proce-dures) Act 1986 and with local Ethical Review procedures. Sagittalbrain slices (400 lm), composed of hippocampus and overlyingneocortex, were then cut in ice-cold, 11 mM Mg2+-containing aCSF

using a Microm HM 650 V microtome, as described previously(zur Nedden et al. 2011). Slices were transferred to an incubationchamber (50–100 mL) (Edwards et al. 1989) and submerged incontinuously circulating, oxygenated standard aCSF kept at 34°C.The composition of the standard aCSF solution was NaCl 124 mM;KCl 3 mM; CaCl2 2 mM; NaHCO3 26 mM; NaH2PO4 1.25 mM;D-glucose 10 mM; MgCl2 1 mM; pH 7.4 with 95% O2/5% CO2.

Preparation of cerebellar granule cells

Cerebellar granule cells (CGCs) were prepared from P 6–8 Sprague–Dawley rats, as described previously (Tomaselli et al. 2008; Wallet al. 2010). For each preparation, one pup was killed by cervicaldislocation in accordance with Schedule 1 of the UK GovernmentAnimals (Scientific Procedures) Act 1986 and with local EthicalReview procedures. CGCs were plated at a density of 1.3 9 105 onpoly-l-ornithine coated borosilicate glass coverslips (VWR, 16 mmdiameter). Neurons were cultured for 7–8 days in Neurobasalmedium, containing KCl 25 mM, B27 2%, glutamine 1.5 mM andPen/Strep 0.01% in a humidified atmosphere with 5% CO2, beforebeing used for experiments.

Induction of oxygen/glucose deprivation

In hippocampal slices, OGD was induced by exchanging standard orsupplemented aCSF with aCSF containing 10 mM sucrose and pre-equilibrated (> 1 h) with 95% N2/5% CO2. To achieve a partialrecovery of synaptic transmission in combination with a partialreduction of the total adenine nucleotide pool against which tocompare the effects of interventions of the purine salvage pathway,we chose an OGD exposure period for HPLC analyses of 8 min, thevalue for the 75th percentile of the mean time to anoxic depolar-ization (AD) in control slices. For electrophysiological recordings,slices were exposed to OGD until AD, as determined by thedisappearance of the presynaptic fibre volley. For biosensormeasurements, slices were exposed to 5 min OGD, To avoid theoccurrence of AD, which would otherwise result in irrevocable lossof synaptic transmission (Frenguelli 1997; Pearson et al. 2006;Frenguelli et al. 2007). Reperfusion was achieved by transferringthe slices or switching the perfusion system back to standard orsupplemented aCSF for periods up to 60 min.

In cerebellar granule cells, OGD was induced by replacingNeurobasal medium (Invitrogen) (supplemented with 0.01% Pen/Strep, 1.5 mM glutamine, 25 mM KCl and 2% B27 supplement)with glucose-free supplemented phosphate-buffered saline (PBS),which had been pre-equilibrated with 95% N2/5% CO2 for 3 h in asealed chamber. Cerebellar granule cells were then transferred toanother chamber, which was purged with 95% N2/5% CO2 for afurther 30 min. Thereafter, the chamber was thoroughly sealed withParafilm and returned to the incubator for 6 h. Control cells weretreated with glucose-containing supplemented PBS for 6 h undernormoxic conditions. After this period of OGD the OGD medium,and for control cells the glucose-containing supplemented PBS wasreplaced with the previous Neurobasal culture medium, with orwithout Rib and/or Ade for a 12–14 h reperfusion period.

Nucleotide extraction and high performance liquid chromatography

measurements

For each condition, the tissue nucleotides of two brain slices wereextracted and subsequently analysed by reverse-phase ion pairing

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 111--124

112 S. zur Nedden et al.

HPLC, as described previously (zur Nedden et al. 2009). Theprotein content was determined by Bradford assay and all values areexpressed as nmol/mg protein.

Electrophysiological recordings

For electrophysiological recordings, slices were transferred to arecording chamber and submerged in 2–3 mL of solution andperfused at a flow rate of 6–8 mL/min at 33.5 � 0.5°C. A smallplatinumharp strungwith nylonfibreswas gently placed on the slice toprevent it frommoving. A twisted bipolar Teflon-coated tungstenwireelectrode (100 lm diameter) was placed in stratum radiatum tostimulate the Schaffer collateral/commissural pathway every 15 s.Field excitatory post-synaptic potentials (fEPSPs) were recorded fromstratum radiatum in area CA1 of the hippocampus with a glassmicroelectrode filled with aCSF (1 MΩ) as described previously(Frenguelli et al. 2007; zur Nedden et al. 2011). The stimulusintensity was adjusted to 50–60% of that required to evoke apopulation spike. Stimulus parameters and acquisition and analysis offEPSPs were under the control of LTP software (Anderson andCollingridge 2001). For input/output curves, the current was increasedstepwise from 10 lA to 300 lA and the slope was measured for eachfEPSP. For paired-pulse ratio measurements the two pulses weregiven at pulse intervals ranging from 50 to 250 ms. The time to 50%inhibition (T50) upon OGD induction was determined for eachindividual experiment by a Boltzmann sigmoidal equation usingGraphpad Prism (GraphPad Software Inc., San Diego, CA, USA).

Adenosine biosensor measurements

Adenosine and null microelectrode biosensors (50 lm diameter/500 lm length) were purchased from Sarissa Biomedical Ltd(Coventry, UK). Biosensor recordings from stratum radiatum of theCA1 region were performed as described previously (zur Neddenet al. 2011). After each experiment, the biosensors were taken out ofthe tissue and calibrated with 10 lM adenosine in the recordingchamber. To monitor the non-specific response to electroactivesubstances, 10 lM serotonin was applied and if the response wassmaller than 50 pA, the adenosine signal during the experiment wasassumed to be specific. Since no non-specific electroactive releasecould be detected on the null biosensor, adenosine release wascalculated without subtraction of the null trace and the values aregiven as lM′ (lM prime) to reflect that the adenosine biosensormeasures adenosine and its metabolites (Frenguelli et al. 2007).Since adenine gives a signal on the adenosine biosensor, because ofthe presence of xanthine oxidase, RibAde (as well as creatine) waswashed out of the slices for at least 30–45 min before the biosensorswere inserted and electrophysiological recordings were started45 min later. By that time (> 1 h) HPLC analysis indicates thattissue adenine levels decline to baseline, whereas tissue ATP levelsare still maintained (zur Nedden et al. 2011).

Hoechst/PI staining of cerebellar granule cells

Cells were incubated in the dark at 37°C for 10 min with Hoechst(Sigma Aldrich) (cell permeable blue stain; 8 lM), followed by afurther 5 min with (PI; cell impermeable red stain; 5 lg/mL). Cellswere washed twice with PBS and fixed with 4% paraformaldehydefor 15 min. After washing cells thoroughly with PBS, coverslipswere mounted onto microscope slides using Vectashield (VectorLaboratories, Peterborough, Cambridgeshire, UK) and sealed with

nail vanish. Imaging was conducted with a 209 objective on anepifluorescence microscope (Nikon) and four to five images werecaptured of each coverslip, at distributed locations that wererepresentative of the cells present.

Hoechst and PI images were merged in Photoshop (Adobe, SanJose, CA, USA) and cells were assessed as being either viable (blue;Hoechst staining alone), or non-viable (pink; co-merged Hoechstand PI staining). Only cells greater than 3 lm in diameter wereincluded in the analysis. The percentage of cell death was calculatedfor each image (% cell death = dead cells 9 100/total cell number),and the five values for each condition averaged to give the totalpercentage of cell death per condition.

Statistical analysis

All values are expressed as mean � SEM. For the electrophysio-logical and adenosine biosensor measurements, n values refer to thenumber of slices per experimental condition, which for most cases isalso equal to the number of animals used. Slices were used induplicate for nucleotide extraction. For statistical analysis of morethan two groups, one-way ANOVA with Bonferroni’s multiplecomparison test was applied, whereas for comparisons betweentwo independent groups unpaired t-tests were used. Calculationswere carried out with Graphpad Prism 6; p-values < 0.05 wereconsidered as statistically significant.

Results

Differential modulation of tissue purine metabolites by

RibAde and creatine before, during and after metabolic

stress

Figure 1 shows representative HPLC chromatograms of theeffects of 5 and 10 min OGD on brain slice adeninenucleotides and metabolites. After 5 min OGD, there was adramatic ~70% decrease in ATP (from 13.5 � 1.1 to4.0 � 0.9 nmol/mg protein): after 10 min OGD, ATP wasalmost completely lost (93% decrease, 0.9 � 0.1 nmol/mgprotein; n = 6–8; p < 0.001, one-way ANOVA), with corre-sponding increases in AMP and adenosine. In parallelelectrophysiological experiments in hippocampal slices weconsistently observed almost full recovery of the fEPSP after5 min OGD, (see below) but none after 10 min exposure toOGD, despite there being good recovery of ATP levels evenafter only 10 min reperfusion [Fig. 1, 89% of control after5 min OGD (12.0 � 0.9 nmol/mg protein) and ~50% after10 min OGD (6.9 � 0.7 nmol/mg protein)]. Accordingly, toexamine the influence of RibAde and creatine on both tissueenergetics and synaptic transmission we performed subse-quent HPLC measurements in slices exposed to 8 min OGD,which allowed some recovery of synaptic transmission inslices incubated in standard aCSF (see below), and againstwhich any influence of RibAde or creatine could be compared.Immediately after slice cutting, brain slices were trans-

ferred to incubation chambers containing standard aCSF orchambers containing either 1 mM D-ribose/50 lM adenine(RibAde), to increase basal ATP levels (zur Nedden et al.2011), or with 1 mM creatine to increase phospho-creatine


Cellular ATP determines OGD adenosine release 113

levels and buffer the initial decline of ATP during metabolicstress (Fig. 2a). The concentration of RibAde (1 mM/50 lMrespectively) was based on previous results (zur Neddenet al. 2011), whilst creatine was used at an equimolarconcentration to Rib (1 mM) and which has previously beenshown to exert near maximal effects in vitro (Perasso et al.2013).After 3 h slices were transferred to an identical incubation

chamber and exposed to 8 min OGD in standard aCSF.Slices were then transferred back to the standard orsupplemented aCSF for 1 h reperfusion, at which point theywere processed for HPLC measurements. The changesobserved in the levels of individual adenine nucleotides,adenosine and inosine monophosphate (IMP) in standardslices or slices pre-treated with RibAde or creatine are shownin Fig. 2. IMP serves as an index of the activity of the purinesalvage pathway (Fig. 2a) since the deamination of AMP toinosine monophosphate (IMP) is inhibited during metabolicstress when ATP levels are reduced (Torrecilla et al. 2001;zur Nedden et al. 2012). It was not possible to measurephospho-creatine and creatine levels with this HPLC method

since the retention time was very close to the void volume ofthe column (~1.5 min) and was contaminated by otherunretained materials. However, it has been shown thattreatment of brain slices with 1 mM creatine increases basalphospho-creatine levels after a 2 h treatment period (Bales-trino et al. 1999).As shown previously (zur Nedden et al. 2011), RibAde

treatment resulted in a significant elevation of basal ATPlevels (Fig. 2b, Table S1) and of the total adenine nucleotidepool (TAN; Fig. 2e; Table S1) compared to standard andcreatine-treated slices but which was not translated intohigher basal adenosine levels (Fig. 2f, Table S1). In contrast,creatine did not change any of the metabolites tested undercontrol conditions.During exposure to 8 min OGD, we observed a significant

decline in ATP levels (Fig. 2b), and significant increases inAMP (Fig. 2d), adenosine (Fig. 2f) and IMP levels (Fig. 2g)in all conditions tested (Table S1). RibAde treatment resultedin significantly higher adenosine levels duringOGD comparedto both standard and creatine-treated slices (Fig. 2f; Table S1),suggesting that the greater ATP pool results in greaterproduction of adenosine during energy crisis. Creatine-treatedslices on the other hand displayed significantly lower levels ofadenosine during OGD than standard slices, when a directcomparison was made using an un-paired t-test between thetwo conditions (p = 0.0218; Fig. 2f). This is likely because ofincreased buffering capacity of the phospho-creatine pool andthe delayed degradation of ATP. Buffering of ATP by creatineis also supported by the significantly elevated ADP levelsduring OGD (Fig. 2c) and the tendency to reduced productionof AMP (Fig. 2d). These observations suggest that whereasadenosine release may be enhanced during OGD by RibAde,creatine may reduce adenosine release.IMP levels after 8 min OGD were significantly higher in

RibAde-treated slices compared to control slices (Fig. 2g;Table S1). Since the conversion of AMP to IMP is inhibitedduring metabolic stress (Torrecilla et al. 2001; zur Neddenet al. 2012), this suggests increased salvage of accumulatedhypoxanthine and/or Ade via the purine salvage pathway(Fig. 2a). In contrast, creatine-treated slices had significantlylower IMP levels (Fig. 2g) compared to RibAde-treatedslices, once again suggesting a lack of metabolites for thepurine salvage pathway.Upon reperfusion, RibAde treatment resulted in signifi-

cantly elevated ATP levels (Fig. 2b; Table S1). Despite theimproved absolute recovery of ATP in RibAde-treated slicesthere was still an overall 10% reduction of the TAN poolupon reperfusion, which was similar (13%) to that observedfor control slices (Fig. 2e; Table S1). In contrast, creatinetreatment prevented the OGD-induced reduction of TANlevels (Fig. 2e; Table S1).IMP levels in control and RibAde-treated slices remained

significantly elevated compared to their preceding baselinevalues, and were elevated above post-OGD IMP levels in

Fig. 1 Changes in tissue adenine nucleotides and metabolites duringand after oxygen/glucose deprivation. Representative HPLC chroma-tograms from brain slices under control conditions (top row) showing

the adenine nucleotides ATP, ADP and AMP (arrows), metabolites andguanine nucleotides (numbered 1–6). The middle row shows theeffects of 5 min oxygen/glucose deprivation (OGD) on these molecules(left panel) and after 10 min reperfusion (right panel). The lower panels

show chromatograms for 10 min OGD (left) and 10 min reperfusion(right). Double arrow represents vertical scale bar of 100 arbitrary units.



creatine-treated slices, suggesting the persistence ofincreased purine salvage activity for some considerable timeafter metabolic stress.

These results show that it is possible, by RibAde orcreatine pre-treatment to modulate the decline and recoveryof intracellular adenine nucleotide levels during OGD, as

(a)

(b) (c)

(d) (e)

(f) (g)

Fig. 2 Differential effects of ribose/adenine and creatine on adenine

nucleotides, adenosine and IMP levels after oxygen/glucose depriva-tion and reperfusion. (a) Simplified scheme of ATP metabolism and thepurine salvage pathway. Indicated are the site of action of creatine (Cr)

and its reversible conversion by creatine kinase to phospho-creatine(PCr), which serves as a phosphate donor to ADP to support ATPlevels during energy crises, the entry of ribose (Rib) into the purine

salvage pathway to form IMP with hypoxanthine, and conversion of D-ribose and adenine (RibAde) to AMP. Brain slices were incubated instandard artificial cerebrospinal fluid (aCSF) (open bars) or pre-treated

with RibAde (light grey bars, 1 mM/50 lM) or creatine (dark grey bars,1 mM) for 3 h at 34°C before being exposed to 8 min oxygen/glucose

deprivation (OGD). For reperfusion, slices were transferred back to

control aCSF or aCSF supplemented with these substances. HPLCanalysis was performed immediately and (b) ATP, (c) ADP, (d) AMP,(e) total adenine nucleotides (TAN) as well as (f) adenosine and (g)

IMP levels were measured. All values are expressed as mean � SEM,n = 4–5, *p < 0.05, **p < 0.01, ***p < 0.001 between indicatedgroups, and ††p < 0.01, †††p < 0.001, ††††p < 0.0001 compared to

corresponding pre-OGD values; one-way ANOVA with Bonferroni’smultiple comparison test. ^A direct comparison between standardand creatine-treated slices during 8 min of OGD revealed significantly

reduced adenosine production in creatine-treated slices compared tothose in standard aCSF (unpaired t-test, p = 0.0218).



well as the production of ATP degradation and precursormetabolites such as adenosine. In addition, there is evidenceof stimulation of the purine salvage pathway by RibAde.Moreover, these data reaffirm our previous conclusion (zurNedden et al. 2011) that RibAde is effective in enhancingATP levels in brain tissue having been previously injured: inthe case of brain slices, by their preparation.

Creatine preserves energetic parameters during OGD

The relative amounts of individual adenine nucleotides in theTAN pool, as calculated by the energy charge (EC;EC = (ATP + 0.5ADP)/TAN) (Atkinson 1968) or theATP/AMP ratio (Hardie and Hawley 2001; zur Neddenet al. 2011), are commonly used parameters to determine theenergetic state of a tissue, since these ratios better reflect therelationship of energy production (ATP) and energy con-sumption (ADP, AMP) than their absolute concentrations.As seen in Fig. 3, none of the treatments tested had any

significant effects on the basal EC values or the ATP/AMPratio, which, at least for EC, approaches values recorded invivo (0.94–0.96). This suggests that brain slices attain abioenergetic equilibrium, regardless of absolute adeninenucleotide levels. However, during OGD, creatine-treatedslices had significantly higher EC values compared tostandard and RibAde-treated slices (Fig. 3a; Table S2),because of a better preservation of ATP and ADP levels asseen in Fig. 2b and c. During reperfusion EC recoverednearly completely and there was no difference in any of theconditions tested, despite the fact that TAN levels decreasedin control and RibAde-treated slices (Fig. 2e), onceagain suggesting the establishment of a new energeticequilibrium.During OGD, the ATP/AMP ratio substantially decreased

in standard, RibAde and creatine-treated slices (Fig. 3b;Table S2), but remained significantly higher in creatine-treated slices. After 1 h reperfusion, the ATP/AMP ratio wassignificantly lower compared to control in standard slices butnot in RibAde- or creatine-treated slices.These data suggest that boosting the cellular ATP pool

with RibAde does not influence the decline and recovery ofenergetic parameters during metabolic stress, but creatine, byvirtue of buffering the breakdown of ATP, preserves theenergetic status of the tissue for longer.

Modulation of intracellular adenine nucleotide levels by

RibAde and creatine influences adenosine release and

synaptic transmission in response to OGDRibAde and creatine significantly increased and decreased,respectively, the levels of tissue adenosine during OGD(Fig. 2f). We therefore hypothesized that this might bereflected in both the amount of adenosine released duringOGD, and the rate at which excitatory synaptic transmissionwas depressed during OGD, as this is known to depend inlarge part upon adenosine release and the activation of

adenosine A1 receptors (Pearson et al. 2006; Dale andFrenguelli 2009).Accordingly, we used simultaneous recordings of fEPSPs

and adenosine biosensors to measure directly and in real time

(a)

(b)

Fig. 3 Creatine delays the decline of energetic parameters after

oxygen/glucose deprivation. Brain slices were incubated in standardartificial cerebrospinal fluid (aCSF) (open bars) or pre-treated withD-ribose and adenine (RibAde) (light grey bars, 1 mM/50 lM) or

creatine (dark grey bars, 1 mM) for 3 h at 34°C before being exposedto 8 min oxygen/glucose deprivation (OGD). For reperfusion, sliceswere transferred back to control aCSF or aCSF supplemented with

these substances. HPLC analysis was performed immediately and (a)the energy charge (EC = (ATP + 0.5ADP)/TAN) as well as (b) the ATP/AMP ratio were determined. All values are expressed as mean �SEM, n = 4–5, **p < 0.01, between indicated groups and ††p < 0.01,

††††p < 0.0001 compared to corresponding pre-OGD values; one-wayANOVA with Bonferroni’s multiple comparison test.



the OGD-induced release of adenosine in RibAde- andcreatine-treated slices and the effect this had on synaptictransmission. Slice were treated for 3–6 h with RibAde orcreatine and these substances were washed out in standardaCSF for at least 30 min before slices were transferred intothe recording chamber (see Methods). Creatine, like RibAde(zur Nedden et al. 2011), had no significant effects on basalsynaptic transmission, such as input–output curves or paired-pulse ratios (data not shown). Forty-five min after insertionof the biosensors, fEPSPs were recorded for 20–30 min andthen slices were exposed to brief (5 min) OGD to permitrecovery of the fEPSP during subsequent reperfusion instandard aCSF (Fig. 4).RibAde-treated slices released significantly more adeno-

sine during OGD and upon reperfusion (Fig. 4a;16 � 1.3 lM’ compared to 11 � 0.4 lM’ in control slices,n = 4, p < 0.05, one-way ANOVA), whereas creatine-treatedslices released significantly less adenosine (5 � 0.5 lM’,n = 3, p < 0.01 compared to control slices and p < 0.001compared to RibAde-treated slices, one-way ANOVA) asmeasured at the peak of the post-OGD purine efflux(Frenguelli et al. 2003). These observations correlated withthe observed effects on the OGD-induced depression ofsynaptic transmission, with RibAde accelerating the depres-sion of the simultaneously recorded fEPSP and creatinedelaying the depression (Figs 4b and 5a and b for additionalquantification). Indeed, complete depression of the fEPSP increatine-treated slices only occurred during the adenosineefflux associated with reperfusion [Fig. 4a and b, arrows andsee Frenguelli et al. (2003)]. Furthermore, the greater releaseof adenosine in RibAde-treated slices (Fig. 4a) resulted in asubstantially delayed recovery of synaptic transmission(Fig. 4b). In contrast, there was little difference in therecovery rates in standard and creatine-treated slices, whichmay reflect a similar rate of clearance of adenosine from thesynaptic cleft.

Creatine, but not RibAde, improves the recovery of synaptictransmission after OGD

The previous electrophysiological and adenosine biosensorstudies allowed the depression and recovery of synaptictransmission to be observed during a brief, sublethal episodeof OGD. To test whether manipulation of intracellularnucleotides by RibAde and creatine influenced recovery ofsynaptic transmission after more prolonged, injurious,episodes of OGD, we extended the duration of OGD untilthe appearance of the AD. The AD indicates the time ofcomplete loss of ion homeostasis and neuronal membranepotential (Somjen 2001) and the point at which under normalcircumstances there is an irreversible loss of synaptictransmission (Frenguelli 1997; Somjen 2001; Balestrinoet al. 2002; Frenguelli et al. 2007).The occurrence of the AD was indicated by the disap-

pearance of the presynaptic fibre volley, at which point we

(a)

(b)

Fig. 4 Modulation of intracellular adenine nucleotides with D-riboseand adenine (RibAde) or creatine influences adenosine release andsynaptic transmission in response to brief oxygen/glucose deprivation:

Brain slices were incubated in standard artificial cerebrospinal fluid(aCSF) or pre-treated with RibAde (1 mM/50 lM) or creatine (1 mM)for 3–6 h at 347°C before being exposed to oxygen/glucose depriva-

tion (OGD) for 5 min. (a) Averaged traces for adenosine biosensorrecordings of control slices (black trace, n = 4), slices pre-treated withRibAde (light grey trace, n = 4) or creatine (dark grey trace, n = 3).Note the accelerated release of adenosine during OGD (black bar) and

the larger post-OGD efflux in RibAde-treated slices, and the reducedlevels of release during both these phases in creatine-treated slices.Scale bar measures 2 min and 5 lM’ Adenosine. (b) Time-course of

field excitatory post-synaptic potentials (fEPSPs) recorded simulta-neously with adenosine biosensor recordings shown in (a) duringexposure and recovery to 5 min OGD (black bar) for control (open

squares) and RibAde- (light grey circles) and creatine- (dark greytriangles) treated slices. Inset are representative fEPSPs normalized topre-OGD baseline transmission and superimposed with respect totiming before (i), during (ii) and after (iii) exposure to OGD, as indicated

on the time-course plot. Note the accelerated decline and delayedrecovery of fEPSPs in RibAde-treated slices (light grey traces andcircles), which reflects the increased extracellular adenosine levels

observed in (a), and the full depression of creatine-treated fEPSPs onlyduring the post-OGD adenosine efflux (arrows). In (a) traces representthe mean of three to four experiments, whilst the values in (b) are

expressed as mean � SEM. fEPSPs for i and iii are averages of threesequential fEPSPs, whereas ii is an individual fEPSP. Scale bar forfEPSPs measures 10 ms.



(a) (b)

(c)

(d)

Fig. 5 Creatine, but not D-ribose and adenine (RibAde), delays theanoxic depolarization and improves functional recovery after prolonged

oxygen/glucose deprivation. Brain slices were incubated in standardartificial cerebrospinal fluid (aCSF) or pre-treated with RibAde orcreatine for 3–6 h at 34°C before being exposed to oxygen/glucosedeprivation (OGD). (a) The decline of excitatory synaptic transmission

during the first 4 min of OGD (black bar) with representative fieldexcitatory post-synaptic potential (fEPSP) traces before (a) and during(b) the inhibition of the fEPSP. Note the acceleration of the depression

in RibAde-treated slices (light grey circles and fEPSPs), and the delayin creatine-treated slices (dark grey triangles and fEPSPs) comparedto standard aCSF (open squares and black fEPSPs). (b) The time to

50% inhibition of the fEPSP (T50), as calculated by fitting a Boltzmannsigmoidal curve to each individual experiment in (a). Symbols in (a andb) refer to: Control slices (open squares, n = 12) and slices treated

with RibAde (light grey circles, 1 mM/50 lM, n = 10) or creatine (darkgrey triangles, 1 mM, n = 8). (c) Brain slices were exposed to OGDuntil the presynaptic fibre volley disappeared, which marks the time ofthe anoxic depolarization (AD; open squares, n = 25), slices pre-

treated with RibAde (light grey circles; 1 mM/50 lM; n = 7) or creatine(dark grey triangles; 1 mM; n = 8). (d) Reperfusion was induced as

soon as the presynaptic fibre volley disappeared. It took ~1 min for thefibre volley to reappear. Slices were reperfused with the treatment theyreceived before OGD, which was either standard aCSF (open squares;n = 7), or aCSF supplemented with RibAde (light grey circles; 1 mM/

50 lM; n = 5) or creatine (dark grey triangles; 1 mM; n = 4). Therecovery of the fEPSP during 1 h of reperfusion from interleaved andcontemporaneous experiments is shown, with inset representative

fEPSPs from the experiments depicted in (a) taken at time c. AllRecordings were performed at 33.4 � 0.2°C at a flow rate of 7–8 mL/min after a ≥ 3 h recovery period from slice cutting. Values in (a and d)

are presented as mean � SEM and values in (b and c) as individualexperiments with the mean shown as a black horizontal line.**p < 0.01, ****p < 0.0001, one-way ANOVA with Bonferroni’s multiple

comparison test. fEPSPs for a and c are averages of three sequentialfEPSPs, whereas b is an individual fEPSP. Scale bar for fEPSPsmeasures 1 mV and 5 ms.



switched the OGD solution to the standard or supplementedaCSF for 1 h. Importantly, neither the age of the animal (19–27 day old rats were used) nor the age of the slice aftercutting (3–8 h) had an effect on the time to AD (data notshown).As per the previous experiments using adenosine biosen-

sors, RibAde and creatine differentially affected the OGD-induced depression of the fEPSP (Fig. 5a), which wequantified in terms of the time to 50% inhibition of theinitial fEPSP response (Fig. 5a and b, 106 � 2.5 s forRibAde-treated slices, 128 � 2.7 s for control slices, and168 � 7 s for creatine-treated slices n = 8–12, p < 0.01 andp < 0.0001, one-way ANOVA).However, after this initial depression of synaptic trans-

mission, the subsequent duration of the OGD episode, asdetermined by the disappearance of the fibre volley, wasaffected by creatine pre-treatment. As seen in Fig. 5c thetime to AD was not different between control(7.4 � 0.2 min; n = 25) and RibAde-treated slices(7.2 � 0.2 min; n = 7; p > 0.05, one-way ANOVA). How-ever, creatine (n = 8) significantly delayed the occurrence ofthe AD (10.9 � 0.6 min; p < 0.0001 compared to standardand RibAde-treated slices, one-way ANOVA). One slice,incubated for 10 h in creatine, displayed unusually delayedAD at 17 min and was not considered further, whilst in sixout of eight slices treated with creatine (1 mM) we observedepileptiform activity during OGD, which occurred approx-imately 1 min before the AD. This epileptiform activity isconsistent with reduced extracellular adenosine and reducedactivation of adenosine A1 receptors (Etherington andFrenguelli 2004).One hour after the end of the period of OGD and despite

the greater adenosine release in RibAde-treated slices(Fig. 4a and b) the recovery of synaptic transmission wasnot different between control (Fig. 5d, 18.7 � 4.9% of theinitial fEPSP baseline; n = 12) and RibAde-treated slices(26.7 � 5.6%; n = 5). The incomplete recovery of synaptictransmission in RibAde-treated slices was not because ofpersistently increased extracellular adenosine levels, sinceapplication of 8-CPT (1 lM), a selective A1 receptorantagonist, only resulted in 28% increase of fEPSP baseline(n = 3, data not shown), which is similar to the effect of 8-CPT on standard slices in control conditions [~21% increase(zur Nedden et al. 2011)]. This suggests that althoughextracellular adenosine levels might be elevated 1 h afterreperfusion in RibAde slices, as they are in standard slicessubjected to OGD (Pearson et al. 2006), they do not aloneaccount for the incomplete recovery of synaptic transmission.In contrast, creatine-treated slices, despite being subjected toa longer period of OGD showed a virtually completerecovery of synaptic transmission (99.9 � 9.3% for crea-tine-treated slices, n = 8; p < 0.001 compared to all othergroups, one-way ANOVA; Fig. 5d), which may reflect betterpreservation of ATP levels during OGD and the delaying of

the AD. However, this protection was not without limit: inthe one slice where the AD was delayed to 17 min,reperfusion resulted in no recovery of synaptic transmission.Pre-incubation with lower concentrations of creatine (0.1

and 0.5 mM, n = 4 and 5, respectively, data not shown)delayed the AD (9.1 � 0.9 and 9.75 � 0.4 min respec-tively), did not provoke epileptiform activity, and resulted ina slight, but insignificant, improved recovery of synaptictransmission compared to controls [40.5 � 20.1% (range 0–95%) and 38.2 � 11% (range 12–70%)]. For the lowerconcentration of creatine (0.1 mM) there was a strong linearcorrelation between recovery and pre-incubation duration(r = 0.998, p = 0.0022). This suggests that given enoughtime the phosphocreatine pool can accumulate to levelsachieved more rapidly with higher concentrations. In contrastto the protective effects of pre-incubation with creatine, nobenefits were observed when standard slices were reperfusedwith 1 mM creatine-containing aCSF after the AD hadoccurred, achieving recovery levels (33.9 � 8.8% recovery;n = 6; data not shown) no different from standard andRibAde-treated slices (one-way ANOVA).These results show that RibAde, by increasing pre-OGD

ATP levels, or increasing adenosine levels during OGD doesnot affect the time to AD or improve functional recovery,whereas, in agreement with previous reports (Balestrinoet al. 1999), delaying the degradation of ATP by creatinesupplementation increases the time to AD by 60% andpromotes functional recovery of synaptic transmission.

RibAde improves post-OGD cell viability in cerebellargranule cells

Prophylactic treatment of brain tissue with substances thatcan delay the degradation of ATP, such as creatine have beenshown to be neuroprotective in the in vivo ischaemic brain(Berger et al. 2004; Perasso et al. 2009, 2013). In agreementwith these reports our results show that creatine pre-treatmentbetter maintains cellular levels of ATP, the energy charge,and the ATP/AMP ratio during OGD. This corresponds withfunctional effects such as delaying both the OGD-induceddepression of excitatory synaptic transmission and the timeto anoxic depolarization, and improving the functionalrecovery after OGD. However, this occurs at the expenseof extracellular adenosine, and a propensity towardsenhanced glutamatergic excitation leading to epileptiformactivity. In addition, the opportunities to pre-treat the at-riskbrain are limited.Accordingly, we sought to specifically address whether the

selective ability of RibAde to stimulate the purine salvagepathway and to both improve ATP levels in controlconditions, and to provide a larger reservoir of adenosine,could be deployed to promote recovery after injury in amodel where protracted post-injury treatment was possible.As an in vitro model for post-injury intervention, we used

cultures of CGC, since they are reported to contain 95%



neurons (Thangnipon et al. 1983) and are therefore a goodmodel system to study modulation of post-OGD cell viabilityin a relatively pure neuronal population.After 6–7 days in vitro CGCs were exposed to 6 h OGD

and thereafter the OGD medium was replaced by theprevious Neurobasal culture medium, which was supple-mented with or without Rib and/or Ade. Rib and Ade had noadverse effects on cell viability, when added for 24 h up to aconcentration of 10 mM and 100 lM, respectively(31 � 1.4% cell death in control cells, n = 8; 31 � 3% celldeath in cells which were treated with 10 mM Rib and50 lM Ade, n = 3; 28 � 6% in cells that were treated with100 lM Ade, n = 3, p > 0.05, one-way ANOVA; data notshown).After 12–14 h reperfusion, there was no significant

difference in the number of dead cells between cells thatwere continuously cultured in Neurobasal medium(30 � 4%, n = 4, data not shown) and cells that wereexposed to 6 h PBS + glucose and subsequent 12–14 hreperfusion in Neurobasal medium (PBS; 25 � 9%, n = 3,P > 0.05, one-way ANOVA, Fig. 6). However, there wasconsiderable cell death in cells exposed to 6 h OGD andsubsequent 12–14 h reperfusion in Neurobasal medium(OGD; 82 � 8%, p < 0.0001 compared to 6 h PBS, one-way ANOVA). Rib alone, at concentrations of 1, 2.5 and 5 mMhad no beneficial effect on cell viability (Rib; 82 � 3% celldeath; n = 3 for each concentration; pooled data (n = 9)shown in Fig. 6). However, the co-application of Ade(50 lM) converted previously inactive concentrations ofRib (1, 2.5 and 5 mM) into concentrations that significantlyreduced cell death in CGCs when administered after OGD(RibAde; 56 � 4% cell death; n = 3 for each RibAdecombination; p < 0.01 and p < 0.001 (n = 9) when com-pared to control cells and cells treated with 1–5 mM Ribalone respectively).These data suggest that RibAde can substantially improve

cell viability even when administered in the post-injuryperiod and offers a potential novel therapeutic strategy in themetabolically compromised brain.

Discussion

Our data demonstrate that it is possible to modulate thedecline and recovery of intracellular ATP associated withmetabolic stress by increasing the pre-OGD tissue ATPcontent with RibAde or buffering ATP metabolism withcreatine. This also has measurable functional consequenceson the release of adenosine during OGD and the decline andrecovery of synaptic transmission. Creatine treatmentdelayed the degradation of ATP, sustained energy chargeduring OGD, maintained post-OGD ATP levels and resultedin a nearly complete recovery of synaptic transmission afterOGD, likely via delaying the anoxic depolarization. Incontrast, RibAde-treatment resulted in increased tissue ATP

levels under basal conditions and after OGD, increasedadenosine release, and improved cell viability even whenadministered in the post-OGD period.

Fig. 6 Oxygen/glucose deprivation (OGD)-induced cell death incerebellar granule cells is reduced by D-ribose and adenine (RibAde).Cerebellar granule cells were exposed to 6 h OGD followed by12–14 h reperfusion in unsupplemented Neurobasal medium or

Neurobasal medium supplemented with either ribose alone (Rib; 1,2.5 or 5 mM; n = 3 for each) or RibAde (50 lM Ade and either 1, 2.5 or5 mM Rib; n = 3 for each combination). Upper images: Cell death was

analysed by Hoechst (blue, all cells) and propidium iodide (PI) (red,dead cells) staining after a 12–14 h reperfusion period. All pink cells(blue + red) are dead cells. Rib concentration in the images was

1 mM. Scale bar measures 20 lm. Lower graph shows the percentageof cell death in the four conditions tested: [(PI positive cells 9 100)/Hoechst positive cells]. All values are expressed as mean � SEM;n = 3–9; **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA

with Bonferroni’s multiple comparison test.



Pre-treatment with creatine maintains ATP levels, delays the

anoxic depolarization and improves the functional recovery

of synaptic transmission

Creatine has previously been shown to delay the time to ADin brain slices (Kass and Lipton 1982; Lipton and Whitting-ham 1982; Balestrino et al. 1999) and improve the anoxia-induced impairment of protein synthesis (Carter et al. 1995).In addition, pre-administration of creatine and Rib wereeffective in preserving cell viability in cardiac myocytesexposed to OGD (Caretti et al. 2010).Although these positive attributes of creatine have been

translated into in vivo models of cerebral ischaemia, thelimited penetration of creatine across the blood–brain barrier(Perasso et al. 2013), likely due to the lack of creatinetransporters on astrocytic endfeet (Andres et al. 2008),necessitates prolonged pre-treatment for beneficial effects tobe observed. Accordingly, addition of creatine after theischaemic insult (Berger et al. 2004), or acute prophylacticcreatine administration for 1 week in adult animals (Zhuet al. 2004) were not protective, whereas prolonged pre-treatment (1 month) was effective in reducing post-ischaemiccaspase-mediated cell death in mice (Zhu et al. 2004).Nonetheless, since creatine is well tolerated in humans,prophylactic treatment with creatine of high-risk strokepatients has been suggested (Zhu et al. 2004; Gualano et al.2011), whilst a novel phospho-creatine-Mg-acetate complexhas been developed which can cross the blood–brain barrierindependently of the creatine transporter and may reduce pre-treatment times (Perasso et al. 2009, 2013). In addition, thewidespread use of creatine amongst amateur and professionalsportspeople (Gualano et al. 2012) has led to the suggestionthat athletes at risk of head injury, for example Americanfootball players, may inadvertently be pre-treating withcreatine and mitigating against the concussive blows theyexperience (Sullivan et al. 2000).In this study, creatine delayed the OGD-induced depres-

sion of synaptic transmission. Whilst this has been explainedpreviously as better maintenance of ATP and prolongedsupport for transmitter release (Perasso et al. 2013), analternative explanation is the reduced release of extracellularadenosine, demonstrated here using direct measurement withadenosine biosensors, and hence reduced activation ofinhibitory adenosine A1 receptors. Indeed, adenosine A1

receptor antagonists or knockout of A1 receptors greatlydelay the hypoxic or OGD-induced depression of synaptictransmission at times when intracellular ATP would beseverely depleted and adenosine levels correspondingly high(Dale and Frenguelli 2009). In fact, as was shown some timeago, A1 antagonists, by preserving synaptic activity duringmetabolic stress, may actually accelerate ATP depletion(Lipton and Robacker 1982). Thus, the actions of creatine onthe depression of synaptic transmission likely revolve aroundthe limiting of adenosine release rather than effects on ATPmaintenance per se. This reduction of adenosine release

during OGD may also explain the propensity towardsepileptiform activity observed in creatine-treated slices. Incontrast, the creatine-induced delay in the appearance of theAD and improved recovery of synaptic transmission areprobably because of increased phospho-creatine levels andbetter maintenance of cellular ATP.

RibAde stimulates the purine salvage pathway to both

increase tissue ATP and increase adenosine release during

oxygen/glucose deprivation

The heart, like the brain, relies upon the purine salvagepathway for the restoration of adenine nucleotides (Zimmer1992, 1998). Indeed, the positive effects of Rib or Rib andAde on the recovery of post-ischaemic myocardial ATP andTAN in in vitro and in vivo preparations are well documented(Zimmer 1982, 1996; Mauser et al. 1985; St Cyr et al. 1986;Zimmer et al. 1989; Muller et al. 1998; Smolenski et al.1998; Perkowski et al. 2007). Furthermore, improved post-ischaemic ATP recovery has been linked to an enhancedfunctional recovery of the heart, as seen in improved cardiaccontractility (Pasque and Wechsler 1984; Lamberts et al.2007; Schneider et al. 2008), and may also provide benefit inrenal ischaemic injury (Nishiyama et al. 2009; Sato et al.2009). In humans, administration of Rib to patients sufferingfrom congestive heart failure (Pliml et al. 1992; Omran et al.2003, 2004; MacCarter et al. 2009) has been shown to havebeneficial effects on cardiac function, likely via improvedcardiac energy metabolism, which has led to suggestions thatRib should be used therapeutically (Pauly and Pepine 2000;Shecterle et al. 2010).We have previously shown that RibAde increases basal

ATP and TAN levels in brain slices into a range comparablewith the levels found in the in vivo brain, once the damagededges of slices are taken into account (zur Nedden et al.2011). Furthermore, this elevated intracellular ATP trans-lated into increased extracellular adenosine release duringhigh frequency stimulation of afferent fibres in hippocampalslices, and resulted in the raising of the threshold for theinduction of long-term potentiation (zur Nedden et al. 2011).In this study, we have demonstrated further metabolic andfunctional effects of RibAde in brain tissue.RibAde resulted in greater intracellular production of IMP

and adenosine during OGD as demonstrated by HPLCanalysis of tissue adenine-based compounds. Given thatAMP deamination to IMP is inhibited by low levels of ATPand the production of adenosine via AMP is favoured(Torrecilla et al. 2001), the increase in IMP productionacross all conditions suggests the stimulation of the purinesalvage pathway (Fig. 2a). RibAde produced a significantincrease of IMP above control and creatine-treated slices,which implies that the activation of this pathway can bepromoted by providing the appropriate substrates, which maybe important when considering strategies to improve themetabolically compromised brain. Promotion of the purine



salvage pathway would have the additional benefit ofreducing the production of non-salvageable xanthine(Fig. 2a) and its metabolites uric acid and the reactiveoxygen species, H2O2 (Addis et al. 2012). This may befurther enhanced by use of clinically relevant xanthineoxidase inhibitors, such as allopurinol, which have beenshown to preserve ATP and improve outcome in animalmodels of cerebral ischaemia (Phillis et al. 1995). Further-more, the elevated levels of IMP in control and RibAde-treated slices 1 h after reperfusion may reflect a prolongedactivation of the salvage pathway, and might offer anextended window of opportunity during which it may bepossible to intervene therapeutically.The increased levels of intracellular adenosine in RibAde-

treated slices translated into greater and more rapid adenosinerelease into the extracellular space during OGD and resultedin a more rapid depression of excitatory synaptic transmis-sion and a slower recovery after brief periods of OGD.However, this did not translate into an improvement ofsynaptic function over control slices in the period afterprolonged OGD. This is perhaps not unexpected given thatRibAde did not preserve tissue ATP during OGD and did notdelay, in contrast to creatine, the AD.There is an apparent dichotomy in the higher levels of ATP

observed in RibAde-treated slices after OGD compared tocontrol slices and the failure of synaptic transmission torecover to any greater extent. It is possible that the recoveryperiod (60 min) was not sufficient to observe a protractedrecovery of synaptic transmission, given the comparablelevels of post-OGD ATP in creatine-treated slices in whichsynaptic transmission rapidly returned. It is also possible thatthe high levels of ATP are found in cells or compartmentsnot directly involved in synaptic transmission, or, as we havepreviously suggested, (Frenguelli 1997), that synapticdysfunction after OGD is highly localized and may not reflect,at least in the early stages after metabolic stress, a generalmetabolically compromised state. A related issue is thecellular locus of the enhanced ATP and adenosine release.Our brain slice studies do not address this directly, but sinceboth neurones and astrocytes possess the purine salvageenzymes hypoxanthine phosphoribosyltransferase (EC2.4.2.8), and adenine phosphoribosyltransferase (EC2.4.2.7) (Zoref-Shani et al. 1995), and are both capable ofreleasing adenosine (Dale and Frenguelli 2009), it is likelythat both neurones and astrocytes will respond to RibAde.What is more significant are our observations that basal ATP

levels in slices can be improved by RibAde, and that exposingcerebellar granule cells to RibAde after OGD reduces celldeath. Whether the latter protective action in CGCs can beattributed to accelerated recovery of ATP, as opposed to someother action of RibAde, such as the antioxidant properties ofribose and the pentose phosphate pathway (Addis et al. 2012;Riganti et al. 2012) remains to be determined, although itshould be noted that ribose alone was ineffective in improving

cell viability in CGCs. Since a common feature of bothpreparations is that they have suffered an insult that hasresulted in reduced ATP levels, RibAde may be most effectivein tissue suffering from reduced ATP. As such RibAde mayhave valuable restorative, as opposed to prophylactic proper-ties, in the traumatized brain.Accordingly, given the prior safe use of both ribose and

adenine in humans, there is great potential for the use ofRibAde as a post-brain injury supplement to assist in therestoration of intracellular ATP levels, ionic and metabolichomeostasis, and in replenishing the pool of the anticonvul-sant purine nucleoside adenosine.

Acknowledgements

SzN was supported by a PhD Studentship from Research intoAgeing. We thank Professor N. Dale for the use of his HPLCapparatus and helpful advice during the course of the study, and DrMark Wall for helpful comments on the manuscript. SzN and ASDhave no conflict of interest to declare. BGF is a Director of SarissaBiomedical, the company that manufactures the adenosine biosen-sors used in this study.

Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher's web-site:

Table S1. Tissue content of ATP and metabolites.Table S2. Values of Energy Charge and the ATP/AMP ratio.

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Modulation of intracellular ATP determines adenosine release and functional outcome in response to metabolic stress in rat hippocampal slices and cerebellar granule cells