Antagonism of P2X7 receptors enhances lorazepam action in delaying seizure onset in an in vitro model of status epilepticus.

Approximately 30% of patients with status epilepticus (SE) become refractory to two or more antiseizure medications (ASMs). There is thus a real need to identify novel targets against which to develop new ASMs for treating this clinical emergency. Among purinergic receptors, the ionotropic ATP-gated P2X7 receptor (P2X7R) has received attention as a potential ASM target. This study evaluated the effect of the selective P2X7R antagonist A740003 on acute seizures in the dentate gyrus (DG) of hippocampal brain slices, where P2X7Rs are highly expressed, with a view to establishing the potential of P2X7R antagonists as a therapy or adjunct with lorazepam (LZP) in refractory SE. Extracellular electrophysiological recordings were made from the DG of male mouse hippocampal slices. Spontaneous seizure-like events (SLEs) were induced by removing extracellular Mg2+ and sequentially adding the K+ channel blocker 4-aminopyridine and the adenosine A1 receptor antagonist 8-cyclopentyltheophylline, during which the early and late application of A740003 and/or lorazepam was evaluated. Our study revealed that, in the absence of changes in mRNA for P2X7Rs or inflammatory markers, P2X7R antagonism did not reduce the frequency of SLEs. However, A740003 in conjunction with LZP delayed the onset of seizures. Furthermore, our results support the need for employing LZP before seizures become refractory during SE as delayed application of LZP increased seizure frequency. These studies reveal possible sites of intervention that could have a positive impact in patients with high risk of suffering SE.

The mammalian purine salvage pathway as an exploitable route for cerebral bioenergetic support after brain injury.

Purine-based molecules play ancient, fundamental, and evolutionarily-conserved roles across life on Earth, ranging from DNA and RNA, to the universal energy currency, ATP. In mammals, the two primary routes for the synthesis of the adenine nucleotides ATP, ADP and AMP, and, as a consequence, the major bioactive metabolite adenosine, are the de novo purine biosynthesis (DNPB) pathway, and the purine salvage pathway (PSP). Of the two, the PSP dominates in both the mammalian brain and heart. This is because the PSP utilizes the breakdown products of ATP, occasioned by the high energy demands of these organs, to rapidly regenerate adenine nucleotides. This resynthesis route, while efficient and energetically favourable, leaves these organs vulnerable to loss of salvageable metabolites, with the potential for protracted depletion of the means to synthesize ATP, and the ability to deploy neuro- and cardioprotective adenosine. Having previously shown that hippocampal cellular ATP and adenosine release can be increased by supplying substrates for the PSP (d-ribose and adenine), we now explore the expression of DNPB and PSP enzymes in hippocampal neurons and astrocytes based on available transcriptomic data. We find that key enzymes of the PSP are expressed at higher levels than those in the DNPB pathway, and that PSP enzymes are expressed at higher levels in neurons than in astrocytes. These data reflect the importance of the PSP in the mammalian brain and imply that pharmacological targeting of the PSP may be particularly beneficial to neurons at times of metabolic stress. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression.

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

Synedrella nodiflora Extract Depresses Excitatory Synaptic Transmission and Chemically-Induced In Vitro Seizures in the Rat Hippocampus.

Extracts of the tropical Cinderella plant Synedrella nodiflora are used traditionally to manage convulsive conditions in the West African sub-region. This study sought to determine the neuronal basis of the effectiveness of these plant extracts to suppress seizure activity. Using the hippocampal slice preparation from rats, the ability of the extract to depress excitatory synaptic transmission and in vitro seizure activity were investigated. Bath perfusion of the hydro-ethanolic extract of Synedrella nodiflora (SNE) caused a concentration-dependent depression of evoked field excitatory postsynaptic potentials (fEPSPs) recorded extracellularly in the CA1 region of the hippocampus with maximal depression of about 80% and an estimated IC50 of 0.06 mg/ml. The SNE-induced fEPSP depression was accompanied by an increase in paired pulse facilitation. The fEPSP depression only recovered partially after 20 min washing out. The effect of SNE was not stimulus dependent as it was present even in the absence of synaptic stimulation. Furthermore, it did not show desensitization as repeat application after 10 min washout produced the same level of fEPSP depression as the first application. The SNE effect on fEPSPs was not via adenosine release as it was neither blocked nor reversed by 8-CPT, an adenosine A1 receptor antagonist. In addition, SNE depressed in vitro seizures induced by zero Mg2+ and high K+ -containing artificial cerebrospinal fluid (aCSF) in a concentration-dependent manner. The results show that SNE depresses fEPSPs and spontaneous bursting activity in hippocampal neurons that may underlie its ability to abort convulsive activity in persons with epilepsy.

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor.

Despite being among the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, they suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and they highlight the dramatic effect that chemical modifications can have on the overall binding mechanism, paving the way for the rational development of a structure-kinetics relationship of A1R agonists.

Purines: From Diagnostic Biomarkers to Therapeutic Agents in Brain Injury.

The purines constitute a family of inter-related compounds that serve a broad range of important intracellular and extracellular biological functions. In particular, adenosine triphosphate (ATP) and its metabolite and precursor, adenosine, regulate a wide variety of cellular and systems-level physiological processes extending from ATP acting as the cellular energy currency, to the adenosine arising from the depletion of cellular ATP and responding to reduce energy demand and hence to preserve ATP during times of metabolic stress. This inter-relationship provides opportunities for both the diagnosis of energy depletion during conditions such as stroke, and the replenishment of ATP after such events. In this review we address these opportunities and the broad potential of purines as diagnostics and restorative agents.

The Purine Salvage Pathway and the Restoration of Cerebral ATP: Implications for Brain Slice Physiology and Brain Injury.

Brain slices have been the workhorse for many neuroscience labs since the pioneering work of Henry McIlwain in the 1950s. Their utility is undisputed and their acceptance as appropriate models for the central nervous system is widespread, if not universal. However, the skeleton in the closet is that ATP levels in brain slices are lower than those found in vivo, which may have important implications for cellular physiology and plasticity. Far from this being a disadvantage, the ATP-impoverished slice can serve as a useful and experimentally-tractable surrogate for the injured brain, which experiences similar depletion of cellular ATP. We have shown that the restoration of cellular ATP in brain slices to in vivo values is possible with a simple combination of D-ribose and adenine (RibAde), two substrates for ATP synthesis. Restoration of ATP in slices to physiological levels has implications for synaptic transmission and plasticity, whilst in the injured brain in vivo RibAde shows encouraging positive results. Given that ribose, adenine, and a third compound, allopurinol, are all separately in use in man, their combined application after acute brain injury, in accelerating ATP synthesis and increasing the reservoir of the neuroprotective metabolite, adenosine, may help reduce the morbidity associated with stroke and traumatic brain injury.

The combination of ribose and adenine promotes adenosine release and attenuates the intensity and frequency of epileptiform activity in hippocampal slices: Evidence for the rapid depletion of cellular ATP during electrographic seizures.

In addition to being the universal cellular energy source, ATP is the primary reservoir for the neuromodulator adenosine. Consequently, adenosine is produced during ATP-depleting conditions, such as epileptic seizures, during which adenosine acts as an anticonvulsant to terminate seizure activity and raise the threshold for subsequent seizures. These actions protect neurones from excessive ionic fluxes and hence preserve the remaining cellular content of ATP. We have investigated the consequences of manipulation of intracellular ATP levels on adenosine release and epileptiform activity in hippocampal slices by pre-incubating slices (3 h) with creatine (1 mM) and the combination of ribose (1 mM) and adenine (50 µM; RibAde). Creatine buffers and protects the concentration of cellular ATP, whereas RibAde restores the reduced cellular ATP in brain slices to near physiological levels. Using electrophysiological recordings and microelectrode biosensors for adenosine, we find that, while having no effect on basal synaptic transmission or paired-pulse facilitation, pre-incubation with creatine reduced adenosine release during Mg2+- free/4-aminopyridine-induced electrographic seizure activity, whereas RibAde increased adenosine release. This increased release of adenosine was associated with an attenuation of both the intensity and frequency of seizure activity. Given the depletion of ATP after injury to the brain, the propensity for seizures after trauma and the risk of epileptogenesis, therapeutic strategies elevating the cellular reservoir of adenosine may have value in the traumatized brain. Ribose and adenine are both in use in man and thus their combination merits consideration as a potential therapeutic for the acutely injured central nervous system.

Proof of concept and feasibility studies examining the influence of combination ribose, adenine and allopurinol treatment on stroke outcome in the rat.

BACKGROUND: Cerebral ischaemia results in a rapid and profound depletion of adenosine triphosphate (ATP), the energy currency of the cell. This depletion leads to disruption of cellular homeostasis and cell death. Early replenishment of ATP levels might therefore have a neuroprotective effect in the injured brain. We have previously shown that the ATP precursors, D-ribose and adenine (RibAde), restored the reduced ATP levels in rat brain slices to values similar to those measured in the intact rodent brain. The aim of this study was to assess whether RibAde, either alone or in combination with the xanthine oxidase inhibitor allopurinol (RibAdeAll; to further increase the availability of ATP precursors), could improve outcome in an in vivo rodent model of transient cerebral ischaemia. METHODS: After 60 min occlusion of the middle cerebral artery, and upon reperfusion, rats were administered saline, RibAde, or RibAdeAll for 6 h. Baseline lesion volume was determined by diffusion-weighted MRI prior to reperfusion and final infarct volume determined by T2-weighted MRI at Day 7. Neurological function was assessed at Days 1, 3 and 7. RESULTS: Ischaemic lesion volume decreased between Days 1 and 7: a 50% reduction was observed for the RibAdeAll group, 38% for the RibAde group and 18% in the animals that received saline. Reductions in lesion size in treatment groups were accompanied by a trend for faster functional recovery. CONCLUSION: These data support the potential use of ribose, adenine and allopurinol in the treatment of cerebral ischaemic injury, especially since all compounds have been used in man.

Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity.

A series of N(6)-bicyclic and N(6)-(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N(6)-adamantyl substitution in combination with 5'-N-ethylcarboxamido or 5'-hydroxymethyl groups. In addition, we determined that 5'-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compounds might have therapeutic potential.

Combined electrophysiological and biosensor approaches to study purinergic regulation of epileptiform activity in cortical tissue.

BACKGROUND: Cortical brain slices offer a readily accessible experimental model of a region of the brain commonly affected by epilepsy. The diversity of recording techniques, seizure-promoting protocols and mutant mouse models provides a rich diversity of avenues of investigation, which is facilitated by the regular arrangement of distinct neuronal populations and afferent fibre pathways, particularly in the hippocampus. NEW METHOD AND RESULTS: We have been interested in the regulation of seizure activity in hippocampal and neocortical slices by the purines, adenosine and ATP. Via the use of microelectrode biosensors we have been able to measure the release of these important neuroactive compounds simultaneously with on-going epileptiform activity, even of brief durations. In addition, detailed numerical analysis and computational modelling has produced new insights into the kinetics and spatial distribution of elevations in purine concentration that occur during seizure activity. COMPARISON AND CONCLUSIONS: Such an approach allows the spatio-temporal characteristics of neurotransmitter/neuromodulator release to be directly correlated with electrophysiological measures of synaptic and seizure activity, and can provide greater insight into the role of purines in epilepsy.

Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations.

The activation of Group I metabotropic glutamate receptors (GI mGluRs) in the hippocampus results in the appearance of persistent bursts of synchronised neuronal activity. In response to other stimuli, such activity is known to cause the release of the purines ATP and its neuroactive metabolite, adenosine. We have thus investigated the potential release and role of the purines during GI mGluR-induced oscillations in rat hippocampal areas CA3 and CA1 using pharmacological techniques and microelectrode biosensors for ATP and adenosine. The GI mGluR agonist DHPG induced both persistent oscillations in neuronal activity and the release of adenosine in areas CA1 and CA3. In contrast, the DHPG-induced release of ATP was only observed in area CA3. Whilst adenosine acting at adenosine A1 receptors suppressed DHPG-induced burst activity, the activation of mGlu5 and P2Y1 ATP receptors were necessary for the induction of DHPG-induced oscillations. Selective inhibition of pannexin-1 hemichannels with a low concentration of carbenoxolone (10 µM) or probenecid (1 mM) did not affect adenosine release in area CA3, but prevented both ATP release in area CA3 and DHPG-induced bursting. These data reveal key aspects of GI mGluR-dependent neuronal activity that are subject to bidirectional regulation by ATP and adenosine in the initiation and pacing of burst firing, respectively, and which have implications for the role of GI mGluRs in seizure activity and neurodevelopmental disorders.

Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase.

Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

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

Cerebral ischaemia rapidly depletes cellular ATP. Whilst this deprives brain tissue of a valuable energy source, the concomitant production of adenosine mitigates the damaging effects 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 brain of substrates for the purine salvage pathway, the primary means by which the brain makes ATP. Because of this, cerebral ATP levels remain depressed after brain injury. To test whether manipulating cellular ATP levels in brain tissue could affect functional neuronal outcomes in response to oxygen/glucose deprivation (OGD), we examined the effects of creatine and d-ribose and adenine (RibAde). In hippocampal slices creatine delayed ATP breakdown, reduced adenosine release, retarded both the depression of synaptic transmission and the anoxic depolarization caused by OGD, and improved the recovery of transmission. In contrast, RibAde increased cellular ATP, caused increased OGD-induced adenosine release and accelerated the depression of synaptic transmission, but did not improve functional recovery. However, RibAde improved the viability of cerebellar granule cells when administered after OGD. Our data indicate that RibAde may be effective in promoting recovery of brain tissue after injury, potentially via enhancement of salvage-mediated ATP production.

Measurement of purine release with microelectrode biosensors.

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.

Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors.

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists.

While the position of adenosine as an endogenous anticonvulsant is well established, it is unclear to what extent its precursor, ATP, contributes to seizure activity via P2 receptors. In this study we have addressed this issue through the use of ATP biosensors and agonists and antagonists of ATP P2 receptors to detect the release and role of ATP, respectively, during electrically-evoked electrographic seizure-like events (eSLEs) in rat hippocampal slices. The broad-spectrum P2 receptor antagonists RB-2 and PPADS (10µM) caused a small ∼30% inhibition of eSLE duration, and a reduction in intensity. This inhibition of eSLEs was partially reproduced with the P2X(1,2/3,3) antagonist NF023 (10µM), but not the P2X(7) antagonist BBG (10µM). However, the P2X receptor agonist α,ß-meATP did not enhance eSLEs, but instead reduced their duration. Furthermore, we could discern no role for P2Y(1) receptors in electrically-evoked eSLEs: both the P2Y(1) antagonist MRS2179 (10µM) and the P2Y(1) receptor agonist 2-methylthioADP (10µM) were without effect on eSLEs. Consistent with a minor role for ATP P2 receptors on eSLEs we could detect no ATP release during eSLEs, although appreciable quantities of adenosine were detected, which had a pronounced inhibitory action on eSLEs via A(1) receptors. We conclude that the role of ATP P2 receptors in modulating electrographic seizure activity is limited, at least in models such as this one requiring electrical stimulation of afferent fibres. We further conclude that the presence and action of adenosine under these conditions may primarily reflect direct release of this purine.

Intracellular acidification causes adenosine release during states of hyperexcitability in the hippocampus.

Decreased pH increases extracellular adenosine in CNS regions as diverse as hippocampus and ventral medulla. However, thus far there is no clear consensus whether the critical pH change is a decrease in intracellular and/or extracellular pH. Previously we showed that a decrease in extracellular pH is necessary and a decrease in intracellular pH alone is not sufficient, to increase extracellular adenosine in an acute hippocampal slice preparation. Here we explored further the role of intracellular pH under different synaptic conditions in the hippocampal slice. When synaptic excitability was increased, either during gamma-aminobutyric acid type A receptor blockade in CA1 or after the induction of persistent bursting in CA3, a decrease in intracellular pH alone was now sufficient to: 1) elevate extracellular adenosine concentration, 2) activate adenosine A1 receptors, 3) decrease excitatory synaptic transmission (CA1), and 4) attenuate burst frequency in an in vitro seizure model (CA3). Hippocampal slices obtained from adenosine A1 receptor knockout mice did not exhibit these pH-mediated effects on synaptic transmission, further confirming the role of adenosine acting at the adenosine A1 receptor. Taken together, these data strengthen and add significantly to the evidence outlining a change in pH as an important stimulus influencing extracellular adenosine. In addition, we identify conditions under which intracellular pH plays a dominant role in regulating extracellular adenosine concentrations.

An ion-pair reversed-phase HPLC method for determination of fresh tissue adenine nucleotides avoiding freeze-thaw degradation of ATP.

Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe an ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze-thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results in a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen.