Summary:Purpose: Adenosine is a major negative neuromodulator of synaptic activity in the central nervous system and can exert anticonvulsant and neuroprotective effects in many experimental models of epilepsy. Extracellular adenosine can be formed by a membrane-anchored enzyme ecto-5 -nucleotidase. The purposes of this study were to characterize the role of adenosine receptors in modulating status epilepticus (SE) induced by pilocarpine and evaluate its neuroprotective action. Ecto-5 -nucleotidase activity was studied during the different phases of pilocarpine-induced epilepsy in rats.Methods: Adult rats were pretreated with different adenosinergic agents to evaluate the latency and incidence of SE induced by pilocarpine in rats. The neuroprotective effect also was evaluated.Results: A proconvulsant effect was observed with DPCPX and DMPX that reduced the latency of SE in almost all rats. Pretreatment with the MRS 1220 did not alter the incidence of SE but reduced the latency to develop SE. An anticonvulsant and neuroprotective effect was detected with R-PIA. Rats pretreated with R-PIA had a decreased number of apoptotic cells in the hippocampus, whereas pretreatment with DPCPX did not modify the hippocampal damage. An intensification of neuronal death was observed in the dentate gyrus and CA3 when rats were pretreated with DMPX. MRS-1220 did not modify the number of apoptotic cells in the hippocampus. An increase in the ecto-5´-nucleotidase staining was detected in the hippocampus during silent and chronic phases.Conclusions: The present data show that adenosine released during pilocarpine-induced SE via A1-receptor stimulation can exhibit neuroprotective and anticonvulsant roles. Similar effects could also be inferred with A2a and A3 adenosinergic agents, but further experiments are necessary to confirm their roles. Ecto-5´-nucleotidase activity during silent and chronic phases might have a role in blocking spontaneous seizures by production of inhibitory neuromodulator adenosine, besides taking part in the mechanism that controls sprouting.

Summary:Purpose: This study was performed to study the role of adenosine triphosphate (ATP) in the brain of pilocarpine-induced chronic epileptic rats.Methods: ATP-mediated changes in intracellular calcium were studied by the fura-2 method. Immunohistochemistry and Western blotting methods were used to localize and quantify P2X7 receptors in these animals.Results: The fluorometric study in chronic rats revealed a biphasic response indicating the presence of P2X7 receptors.The Western blotting study showed an increase of 80% of P2X7 expression in chronic rats compared with the control group. P2X7 immunoreactivity resembled mossy fiber sprouting at the dentate gyrus of epileptic animals.Conclusions: These results suggest that purinergic receptors may participate in the pathophysiology of temporal lobe epilepsy. Key Words: Epilepsy-ATP-P2X7-PilocarpineRat.The role of adenosine triphosphate (ATP) as a peripheral and central neurotransmitter is mediated by P2 purinoceptors (1). The response of ATP as a fast neurotransmitter is mediated by ionotropic P2X receptors that have a high permeability to calcium ions. Seven subtypes of these receptors have been cloned (P2X1-7), and some of them (P2X4, P2X6, and P2X7) are abundant in hippocampus, amygdala, thalamus, and cortex (2). In contrast to other subtypes, P2X7 can form a 900-Da pore when activated by ATP, and this pore can dialyze the cytoplasmatic content, producing a large change in the intracellular ion homeostasis (3). Moreover, in vitro study in hippocampal slices showed that ATP released by electrical stimulation of Schaffer collateralcommissural afferents take part in excitatory synaptic transmission (4). ATP released from nerve endings stimulates an increase in intracellular Ca 2+ in rat brain and can activate the microglia that are able to release several cytotoxic and protective substances (5,6). However, there is no evidence that ATP-mediated calcium entry plays a pathophysiologic role in epilepsy. This study was performed to study the ATP role in temporal lobe epilepsy (TLE) using pilocarpine-induced epileptic rats. Thus, we studied hippocampal ATP-mediated calcium influx, and we localized and quantified purinergic P2X7 receptors. METHODSChronic epileptic animals were obtained 60 days after status epilepticus induced by pilocarpine (380 mg/kg, i.p.). For the control group, a saline solution was used instead of pilocarpine. The intracellular calcium response mediated by ATP was measured with fluorometry. P2X7 expression was studied by immunohistochemistry (IHC) and quantified by Western blotting.For the fluorometric study, hippocampi slices were submitted to the fura-2 method described by Grynkiewicz et al. (7). In brief, rats from epileptic (n ‫ס‬ 5) and control (n ‫ס‬ 5) groups were killed, their brains removed and placed in cold artificial cerebrospinal fluid (aCSF) containing (in mM): 130 NaCl, 24 NaHCO 3 , 10 D-glucose, 1.3 NaH 2 PO 4 , 3.5 KCl, 5 MgCl, and 0.2 CaCl 2 . Afterward, the brains were sliced (500 m) in a vibratome. Hippocampal slices were inc...