Manuela Marcoli

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices.

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.

Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing.

In vitro neuronal cultures exhibit spontaneous electrophysiological activity that can be modulated by chemical stimulation and can be monitored over time by using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. Dissociated networks respond to transmitters, their blockers and many other pharmacological substances, including neurotoxic compounds. In this paper we present results related to the effects, both acute (i.e. 1 hour after the treatment) and chronic (3 days after the treatment), of increasing glutamatergic transmission induced by the application of rising concentrations of glutamate and its agonists (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid - AMPA, N-methyl-D-aspartate - NMDA and AMPA together with cyclothiazide - CTZ). Increase of available glutamate was obtained in two ways: 1) by direct application of exogenous glutamate and 2) by inhibiting the clearance of the endogenously released glutamate through DL-threo-β-benzyloxyaspartate (TBOA). Our findings show that fine modulations (i.e. low concentrations of drug) of the excitatory synaptic transmission are reflected in the electrophysiological activation of the network, while intervention leading to excessive direct stimulation of glutamatergic pathways (i.e. medium and high concentrations of drug) results in the abolishment of the electrophysiological activity and eventually cell death. The results obtained by means of the MEA recordings have been compared to the analysis of cell viability to confirm the excitotoxic effect of the applied drug. In conclusion, our study demonstrates that MEA-coupled cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission and might provide sensitive endpoints to detect acute and chronic neurotoxic effects of chemicals and drugs for predictive toxicity testing.

Glutamate release in human cerebral cortex and its modulation by 5-hydroxtryptamine acting at h 5-HT1D receptors

The release of glutamic acid and its modulation by 5‐hydroxytryptamine (5‐HT) in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply sited tumours. The Ca2+‐dependent K+ (15 mM)‐evoked overflow of glutamate was inhibited by 5‐HT in a concentration‐dependent manner (EC50=2.9 nM; maximal effect ≃50%). The inhibition caused by 5‐HT was antagonized by the 5‐HT1/5‐HT2 receptor antagonist methiothepin. The 5‐HT1B/5‐HT1D receptor agonist sumatriptan mimicked 5‐HT (EC50=6.4 nM; maximal effect ≃50%); the effect of sumatriptan was also methiothepin‐sensitive. Selective 5‐HT1A receptor antagonists could not prevent the inhibition of glutamate release by 5‐HT. The 5‐HT1B/5‐HT1D receptor ligand GR 127935 and the 5‐HT2C/5‐HT1B/5‐HT1D receptor ligand metergoline were unable to prevent the 5‐HT effect; instead they inhibited glutamate release, their effects being abolished by methiothepin. Some 5‐HT1A receptor antagonists also displayed intrinsic agonist activity. The effect of sumatriptan was prevented by ketanserin, a drug known to display much higher affinity for recombinant h 5‐HT1D than for h 5‐HT1B receptors. We propose that neocortical glutamatergic nerve terminals in human brain cortex possess release‐inhibiting presynaptic heteroreceptors that appear to belong to the h 5‐HT1D subtype.

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release.

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X7 receptors, it is still unclear whether neuronal functions can be attributed to P2X7 receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X7 receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non‐neuronal cells and allows exposure of ‘nude’ release‐regulating pre‐synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X7 receptors. Together, our findings suggest that (i) P2X7 receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X7 receptors are localized on glutamatergic nerve terminals; (iii) P2X7 receptors play a significant role in ATP‐evoked glutamate efflux, which involves Ca2+‐dependent vesicular release; and (iv) the P2X7 receptor itself constitutes a significant Ca2+‐independent mode of exit for glutamate.

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+‐dependent vesicular glutamate release and significant Ca2+‐independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3‐(5‐(2,3‐dichlorophenyl)‐1H‐tetrazol‐1‐yl)methyl pyridine (A‐438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A‐438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non‐glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R‐mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.

Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT2C and 5-HT1A receptors

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5‐hydroxytryptamine (5‐HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery. The cyclic GMP elevation produced by 100 μM NMDA was blocked by 100 μM of the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG) or by 10 μM of the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐α] quinoxaline‐1‐one (ODQ). The NMDA effect was prevented by 5‐HT or by the 5‐HT2 agonist (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane ((±)‐DOI; EC50=22 nM). The (±)‐DOI inhibition was insensitive to the 5‐HT2A receptor antagonist MDL 100907 or the 5‐HT2B antagonist rauwolscine; it was largely prevented by 1 μM of the non‐selective 5‐HT2C antagonists mesulergine (5‐HT2A,B,C), ketanserin (5‐HT2A,C) or SB 200646A (5‐HT2B,C); it was completely abolished by 0.1 μM of the selective 5‐HT2C receptor antagonist SB 242084. The NMDA‐induced cyclic GMP elevation also was potently inhibited by the selective 5‐HT2C agonist RO 60‐0175 and by the antidepressant trazodone, both added at 1 μM, in an SB 242084‐sensitive manner. Finally, the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT; 1 μM) inhibited the NMDA‐evoked cyclic GMP response, an effect blocked by the selective 5‐HT1A receptor antagonist WAY 100635. In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5‐HT2C or 5‐HT1A receptors.

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5‐hydroxytryptamine (5‐HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5‐HT receptors. The aim of this study was to subclassify the 5‐HT2 receptor mediating inhibition of the cyclic GMP response elicited by N‐methyl‐d‐aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5‐HT2A, 5‐HT2B, and 5‐HT2C subtypes, differentially affected the inhibition by (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5‐HT2C subtype.

Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein

Background Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. Principal Findings Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1(130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. Conclusion We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

Motor neuron dysfunction in a mouse model of ALS: gender-dependent effect of P2X7 antagonism.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative progressive currently untreatable disease, characterized by selective motor neuron degeneration; the incidence and prevalence of ALS are greater in men than in women. Although some important mechanisms that might contribute to the death of motor neurons have been identified, the mechanisms underlying disease pathophysiology are still uncertain. In particular, the mechanisms underlying the role of gender in ALS and whether treatments should take into account sexual dimorphism remain only partially understood. Recently, the P2X7 receptor for ATP was reported to display neurotoxic potential in motor neuron disorders, and antagonism of the receptor has been suggested to be helpful in these disorders. Studying transgenic mice with superoxide dismutase 1 gene mutations, widely used as model for ALS, may provide a better understanding of pathogenic mechanisms and of toxicity towards motor neurons, also possibly helping to understand whether treatments for ALS should take into account sexual dimorphism. The aim of the work was (1) investigating on gender-dependence of disease progression in the standard model for ALS - the transgenic mouse bearing superoxide dismutase 1 gene mutations - and (2) assessing if a P2X7 receptor antagonist treatment should take into account sexual dimorphism. We evaluated if gender affect the disease course, the motor performance, the weight loss and the lifespan in mice overexpressing mutant superoxide dismutase 1. We measured motor impairment, motor strength and coordination by rotarod and grip strength testing. Further, we assessed if a treatment with the P2X7 receptor antagonist Brilliant Blue G - a dye that can cross the blood-brain barrier, has low toxicity, and has exhibited therapeutic effects in animal models of neurodegenerative diseases - impact on the disease progression, in male and female ALS mice. We found that (1) the onset and the disease progression, and the survival were dependent on gender: male performed worst than female, lost body weight and died before; (2) treatment with the P2X7 receptor antagonist Brilliant Blue G ameliorated the disease progression. The treatment effect was gender-dependent: amelioration was greater in male than in female. In conclusions, we suggest that not only pathogenetic mechanism of motor neuron toxicity but also the drug treatment effectiveness may depend on gender; sexual dimorphism should be considered when investigating on ALS treatment efficacy in the ALS animal model. Our findings also point on the potential relevance of P2X7 receptor antagonism for ALS treatment, and highlight the importance of adopting a sex-specific approach to searching for treatment of ALS.

Glutamate efflux from human cerebrocortical slices during ischemia: vesicular-like mode of glutamate release and sensitivity to A2A adenosine receptor blockade

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 microM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A(2A) receptor antagonist SCH 58261 (1 microM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A(2A) receptors that enhance vesicular-like glutamate release during ischemia; A(2A) receptor antagonists would deserve consideration for their neuroprotective potential.