Neurotox Res (2013) 24:55–62 DOI 10.1007/s12640-012-9359-y
ORIGINAL ARTICLE
NMDA Preconditioning Attenuates Cortical and Hippocampal Seizures Induced by Intracerebroventricular Quinolinic Acid Infusion Samuel Vandresen-Filho • Alexandre A. Hoeller • Bruno A. Herculano • Marcelo Duzzioni • Filipe S. Duarte • Tetsadeˆ C. B. Piermartiri • Carina C. Boeck Thereza C. M. de Lima • Jose´ Marino-Neto • Carla I. Tasca
•
Received: 26 August 2011 / Revised: 25 October 2012 / Accepted: 29 October 2012 / Published online: 27 November 2012 Ó Springer Science+Business Media New York 2012
Abstract Searching for new therapeutic strategies through modulation of glutamatergic transmission using effective neuroprotective agents is essential. Glutamatergic excitotoxicity is a common factor to neurodegenerative diseases and acute events such as cerebral ischemia, traumatic brain injury, and epilepsy. This study aimed to evaluate behavioral and electroencephalographic (EEG) responses of mice cerebral cortex and hippocampus to subconvulsant and convulsant application of NMDA and quinolinic acid (QA), respectively. Moreover, it aimed to evaluate if EEG responses may be related to the neuroprotective effects of NMDA. Mice were preconditioned with NMDA (75 mg/kg, i.p.) and EEG recordings were performed for 30 min. One day later, QA was injected (36.8 nmol/site) and EEG recordings were performed during 10 min. EEG analysis demonstrated NMDA preconditioning promotes spike-wave
discharges (SWDs), but it does not display behavioral manifestation of seizures. Animals that were protected by NMDA preconditioning against QA-induced behavioral seizures, presented higher number of SWD after NMDA administration, in comparison to animals preconditioned with NMDA that did display behavioral seizures after QA infusion. No differences were observed in latency for the first seizure or duration of seizures. EEG recordings after QA infusion demonstrated there were no differences in the number of SWD, latency for the first seizure or duration of seizures in animals pretreated with saline or in animals preconditioned by NMDA that received QA. A negative correlation was identified between the number of NMDAinduced SWD and QA-induced seizures severity. These results suggest a higher activation during NMDA preconditioning diminishes mice probability to display behavioral seizures after QA infusion.
S. Vandresen-Filho (&) B. A. Herculano T. C. B. Piermartiri C. I. Tasca Departamento de Bioquı´mica, CCB, Universidade Federal de Santa Catarina, Trindade, Floriano´polis, SC 88040-900, Brazil e-mail:
[email protected]
Keywords NMDA preconditioning Quinolinic acid Seizures Electroencephalography
A. A. Hoeller M. Duzzioni F. S. Duarte T. C. M. de Lima Departamento de Farmacologia, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil C. C. Boeck Programa de Po´s-graduac¸a˜o em Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil
Abbreviations EEG Electroencephalographic i.c.v. Intracerebroventricular NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor QA Quinolinic acid, 2,3-pyridine dicarboxylic acid SWD discharge
J. Marino-Neto Departamento de Cieˆncias Fisiolo´gicas, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil
Introduction
J. Marino-Neto Instituto de Engenharia Biome´dica, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil
Brain preconditioning refers to a state of transient brain tissue tolerance to a lethal insult evoked by a prior mild insult through the induction of the tissue endogenous
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protective mechanisms (Dirnagl et al. 2003). A wide range of preconditioning models has been demonstrated, such as chemical agents, hypoxia, and ischemia. Preconditioning with N-methyl-D-aspartate (NMDA) has shown to protect neurons in vitro (Boeck et al. 2005) and in vivo (Boeck et al. 2004; Vandresen-Filho et al. 2007). In vivo model of NMDA preconditioning can be evoked by a non-convulsant dose of NMDA, and it is known to be neuroprotective against kainate toxicity (Ogita et al. 2003) or ischemia (Miao et al. 2005). NMDA preconditioning also prevents seizures and cell death induced by quinolinic acid (QA) in mice (Boeck et al. 2004). Although neuroprotective effects from NMDA preconditioning have been demonstrated, the neural mechanisms involved such protection remains unclear. QA is a selective NMDA receptor (NMDAR) agonist, with similar NMDA potency, and it induces clonic–tonic seizures after intracerebroventricular (i.c.v.) administration. QA toxicity, after NMDAR activation, has been related to the elevation of cytosolic concentrations of free Ca2?, ATP exhaustion, and free radicals formation (Stone et al. 2000; Vega-Naredo et al. 2005). Besides its agonist action at NMDAR, QA can also overstimulate glutamatergic transmission by means of modulating glutamate transport. Specifically, QA inhibits glutamate uptake in cultured astrocytes (Tavares et al. 2002), it decreases glutamate uptake into synaptic vesicles (Tavares et al. 2000) and in hippocampal slices evaluated after QA i.c.v. infusion (Piermartiri et al. 2009). Furthermore, QA toxicity has been implicated in various pathological conditions, such as Huntington and Parkinson’s diseases (Stone 2001), and dementia associated with HIV infection (Heyes et al. 1989). Concerning brain preconditioning, it induces protection through upregulation of proteins associated to cell survival and downregulation of proteins related to cell death. A recent study from our laboratory has shown the inhibition of signaling pathways, as the cyclic AMP-dependent protein kinase (PKA), phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinases (MAPK/ERK), prevented neuroprotection against QA-induced seizures evoked by NMDA preconditioning (de Araujo Herculano et al. 2011), demonstrating specific signaling pathways are involved in the neuroprotective mechanism of preconditioning. Since NMDA preconditioning promotes epileptic tolerance against QA-induced seizures, which can be detected through electroencephalographic (EEG) analyses, it is plausible NMDA preconditioning modulates ionic channels, such as calcium and potassium channels, which play a role in the electrical activity in the brain. Therefore, we aimed to determine if there are interactions between the EEG responses of the cerebral cortex and hippocampus to a
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subconvulsant dose of NMDA and a convulsant dose of QA, and also, if those EEG responses are related to the neuroprotective effects of NMDA preconditioning.
Materials and Methods Materials NMDA and QA were from Sigma (St. Louis, MO, USA). The anesthetic chloral hydrate was obtained from Crista´lia (Itapira, SP, Brazil). Animals Male adult Swiss albino mice (30–40 g) were maintained on a 12 h light/12 h dark schedule at 25 °C. Mice were housed in plastic cages with food and water ad libitum. The animals were allowed to adapt to the laboratory conditions for, at least, 1 week before behavioral assessment. All experiments were designed to minimize animal suffering, limit the number of animals used and they were approved by the local Ethical Committee for Animal Research (CEUA/UFSC). Surgical Procedures Animals were anesthetized with chloral hydrate (700 mg/ kg; i.p.). Stereotaxic surgery and infusion techniques were carried out as previously described (Schmidt et al. 2000). Briefly, on a stereotaxic apparatus, the skull skin was removed and a 27-gauge/7-mm guide cannula was placed at 1 mm posterior to bregma, 1 mm right from midline, and 1 mm above lateral brain ventricle. The guide cannula was implanted 1.5 mm ventral to the superior surface of the skull and fixed with jeweler acrylic cement. The tip of the 30-gauge infusion cannula protruded 1 mm beyond the guide cannula, aiming for the lateral ventricle. In addition, EEG recordings were carried out through bipolar twisted NiCr wire electrodes (tip diameter 150 lm) implanted on the cortical area surface (AP = ? 0.5; ML = ? 1.5) and CA1 hippocampal area (AP = -1.65; ML = ? 1.5; DV = -2.0), according to Paxinos and Franklin (2001). A stainless steel screw was implanted in the occipital area and it served as a reference electrode. Methylene blue (4 ll) was injected through the cannula to confirm infusions effectiveness. Animals without significant contrast in lateral brain ventricle were discarded. After the experiments, the correct location of implanted electrodes was verified in sections using a cryostat microtome (LeicaÒ CM1850, Germany), followed by slices examination under light microscope (NikonÒ, Japan).
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NMDA Preconditioning and QA Infusion NMDA was dissolved in saline solution (NaCl 0.9 %) and adjusted to pH 7.4 with NaOH. Animals were pretreated with NMDA in a low non-convulsant dose (75 mg/kg; 10 ml/kg; i.p.; n = 16) (Boeck et al. 2004) or with an i.p. injection of vehicle (saline 0.9 %; 10 ml/kg, n = 14) 48 h after the cannula implantation and 24 h before QA or phosphate-buffered saline (PBS) administration. Animals were observed for 30 min immediately after NMDA administration. Seizures were induced by the chemoconvulsant QA in an i.c.v. infusion (4 ll of a 9.2-mM solution) (Schmidt et al. 2000). Mice were observed for 10 min for the occurrence of wild running, clonic, tonic, or tonic– clonic seizures lasting for more than 5 s. Mice that did not display seizures during these 10 min were considered protected. In this way, NMDA-preconditioned mice that displayed behavioral seizures induced by QA are referred as NQc (NMDA plus QA-convulsed mice) and NMDApreconditioned mice that did not display behavioral seizures are referred as NQnc (NMDA plus QA nonconvulsed mice). A quantitative scale was developed, based on previous studies (Cruz et al. 2003; Marganella et al. 2005), to evaluate QA-induced seizure severity: 0 = no response; 1 = immobility and excessive grooming ? paroxysmal scratching; 2 = circling and rearing; 3 = wild running; 4 = jumping and falling; 5 = forepaw clonus and tail hypertonus; 6 = generalized tonic–clonic convulsions; and 7 = generalized tonic convulsion and death. Latency and duration of generalized clonic or tonic–clonic convulsions seizures were also determined. Electroencephalographic Recordings EEG recordings were carried out using a digital polygraph system (BIOPAC System, MP-100/WSW, Inc.). Signals were amplified 20,0009, filtered through a built-in 60-Hz notch filter, digitalized at a sampling rate of 256 Hz and recorded by means of the acquisition software ACQKnowledge (v. 3.2). Experiments were conducted in a glass tank (0.3 9 0.5 9 0.4 m) located inside a Faraday cage (1.0 9 0.6 9 0.7 m). Two days after surgery, mice were individually placed in the glass tank, the acquisition cable was connected to the microconnector on the animal head and biosignals were captured using the BIOPACÒ system. Baseline 1 was carried out before preconditioning (on the first experimental day) during 20 min. After that, animals (n = 4) received an i.p. injection of saline or NMDA (according to the preestablished protocol) and EEG recordings were taken during 30 min. Baseline 2 was recorded on the next day, when mice were submitted to QA or PBS i.c.v. infusion and recorded during 10 min. Time
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period procedures for EEG recordings were obtained from a previous study (Torres et al. 2010). Electrical brain activity and animal behaviors were recorded simultaneously through a webcam (Orbit Logitech QuickcamÒ) located inside the Faraday cage. EEG recordings were analyzed by direct visual inspection since EEG changes included the presence of polyspikes or sharp waves with amplitudes bigger than twice the background tracings. Statistical Analysis Results were analyzed through one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test when it was appropriated. Results are presented as mean ± SEM. Differences were considered significant at [95 % confidence.
Results Behavioral Evaluation of Seizures After saline pretreatment, QA infusion induced tonic and clonic seizures in all animals (Table 1) and a mortality rate of 17 % was observed. As previously demonstrated (Boeck et al. 2004; Vandresen-Filho et al. 2007), NMDA preconditioning (75 mg/kg, i.p.) did not induce tonic and/or clonic seizures, and promoted 50 % reduction in the incidence of QA-induced seizures. NMDA-preconditioned mice that displayed behavioral seizures after QA infusion (NQc) did not show any alteration on latency or duration of seizures when compared to QA-treated mice (Table 1). Also, QA-treated and NQc mice presented behavioral alterations like wild running, jumping, falling and clonic, tonic, or tonic–clonic seizures followed or not by death. Spontaneous behavioral analysis revealed NMDA-preconditioned mice that did not display behavioral-related seizures (NQnc), such as tonic or clonic seizures, presented a very low profile on the severity index analysis, when compared to NQc and QA groups (Fig. 1). Electroencephalographic Results EEG recordings were carried out to investigate a probable long-term effect over electrical brain activity caused by NMDA preconditioning, as well as EEG recording alterations promoted by QA infusion. A representative photomicrograph demonstrating an electrode implantation site in the CA1 region of the hippocampus is shown in Fig. 2. As it is shown in Fig. 3, EEG visual inspection after saline injection did not show electrographic alterations, and it was characterized by low voltage and fast activity, mainly in periods where animals denoted an alert immobility behavior. This
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Table 1 Incidence, latency and duration of seizures after NMDA preconditioning and QA infusion Groups
Incidence of seizures: number of convulsed mice/total number of mice
Latency for seizures onset (s)
Duration of seizures (s)
Control
0/14
–
–
NMDA
0/16
–
–
QA
14/14
30.27 ± 16.25
31.18 ± 18.58
NQnc
8/16
–
–
NQc
8/8
31.60 ± 23.58
26.20 ± 13.12
Mice were treated with vehicle (saline, control group) or NMDA (75 mg/kg, i.p.) 24 h before QA infusion (36.8 nmol, i.c.v.) and were observed for 10 min for the occurrence of convulsions. Data are presented as mean ± SD (ANOVA showed no difference among groups) NQnc NMDA plus QA non-convulsed mice, NQc NMDA plus QA convulsed mice
Fig. 1 Severity scale obtained from quinolinic acid (QA)-induced seizures after preconditioning with N-methyl-D-aspartate (NMDA) in mice. Mice were treated with vehicle (saline 0.9 %, i.p.) or NMDA (75 mg/kg, i.p.) 24 h before QA infusion (36.8 nmol, i.c.v.), and they were observed for 10 min for the occurrence of behavioral changes. NQnc NMDA plus QA non-convulsed mice, NQc NMDA plus QA convulsed mice. Values are expressed as mean ± SEM. p \ 0.01 compared with other groups. Asterisk indicates statistical difference from QA and NQc groups (ANOVA followed by Tukey’s test)
EEG profile was similar to the baseline 1 made before the pretreatment injections of all groups. Interestingly, even though systemic NMDA injection did not promote considerable behavioral alterations (besides increased incidence in the immobility behavior duration with no seizure-related behavior, data not shown), approximately two minutes (140.5 ± 37.5 s) after NMDA injection, low voltage and fast EEG activity took place, mainly, through high voltage spike-wave discharges (SWDs). These SWD lasted for 36.6 ± 5.12 s, approximately, in both cortical and hippocampal areas (Fig. 3), and normally related to immobility behavior. Electrographic NMDA-seizures were also characterized by infrequent presence of high voltage sharp wave discharges in both cortex and hippocampus.
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In all control mice, saline pretreatment did not alter EEG recordings compared to spontaneous EEG activity in the cortex or in the hippocampus (Fig. 3). EEG analysis revealed NMDA preconditioning promoted SWD, predominantly, and sharp wave discharges (Fig. 3). These alterations in EEG recordings induced by NMDA preconditioning were generally accompanied of animal immobility. SWD were detected simultaneously in cerebral cortex and hippocampus, therefore EEG changes from baseline in cerebral cortex were associated with EEG changes in hippocampus both in latency, duration, and number of SWD (Fig. 3). Baseline 2 was performed 24 h after saline or NMDA injection, as it can be seen in Fig. 3. At this time, electrographic seizure-related alterations caused by NMDA are not observed in such a way that baseline 2 of NMDA-treated mice is similar to baseline 1. Further, comparison of baseline 2 from saline or NMDA-treated mice shows no visual differences (Fig. 3). Such EEG profile, with low voltage and fast activity, is also observable in PBS tracings that were preceded by saline injection. On the other hand, mice that received QA and were pretreated with saline showed high voltage sharp wave discharges in the EEG recordings, followed by behavioral alteration as forelimbs myoclonic seizures immediately after i.c.v. infusion of QA. These high voltage poly-spiking waves were intermittent with fast activity and isolated spikes with high voltages, normally culminating in a tonic seizure and resulting in the mouse death. Interestingly, even though animals, which were pretreated with NMDA and did not display convulsive behavior after QA injection (NQnc group), electrographic alterations were clearly evident during the recording period, with high voltages poly-spiking waves associated with low activity in both cortex and hippocampus. These electrographic alterations persisted during the major part of the recordings, and they were associated with increased exploratory-related behaviors. However, animals pretreated with NMDA and followed by QA-induced seizures (NQc group) displayed high voltage poly-spiking waves correlated with fast activity, both in cortical and hippocampal areas. Quantification of SWD number, latency and duration of seizures are depicted in Table 2. EEG recordings revealed a significant increase of SWD incidence in the NQnc group, when compared to the NQc group after NMDA preconditioning. No differences were observed on other evaluated parameters (Table 2). A significant negative correlation was identified between the number of NMDA-induced SWD and seizures severity, as it was evaluated by the severity scale we have developed to measure QA-induced seizures in mice (r = -0.9507 Pearson’s correlation, p = 0.0003) (Fig. 4a). This means the higher the number of NMDA-induced SWD, the lower the probability of displaying QA-induced tonic– clonic seizures. On the other hand, no significant correlation was found between the number of NMDA-induced
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Fig. 2 Photomicrograph (right) and schematic drawing (left), based on Paxinos and Franklin Atlas (2001) of coronal sections from mice brain showing the electrode implantation site in the CA1 region of the hippocampus
Fig. 3 Electrographic recordings of seizures observed in mice after NMDA preconditioning and quinolinic acid (QA) infusion. Representative recordings from simultaneous cortical (Cx) and hippocampal (Hp) electroencephalography (EEG) recordings are shown. Mice
received intraperitoneal injection of saline (0.9 %,) or NMDA (75 mg/kg) and EEG was recorded for 30 min. After 24 h, mice were injected with PBS (4 ll, pH 7.4) or QA infusion (36.8 nmol, i.c.v.) and EEG was recorded for 10 min
SWD and QA-induced SWD (r = -0.057 Pearson’s correlation, p = 0.8927) (Fig. 4b).
been related to neurotoxicity since it leads to calcium influx and generation of free radicals, promoting necrotic or apoptotic cell death. The excitotoxicity caused by NMDAR overactivation has been associated to a wide range of neurologic disorders, such as epilepsy, Alzheimer’s, and Huntington’s diseases (Estrada Sanchez et al. 2008; Hynd et al. 2004). Besides this classical excitotoxicity, mild activation of NMDAR had been shown to exert neuroprotective actions. In vitro, subtoxic concentrations
Discussion NMDAR activation had been shown to exert neurotoxic and neuroprotective effects depending on the degree of activation. For a long time, overactivation of NMDAR had
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Table 2 Number of spike-wave discharges (SWDs), latency and duration of electroencephalographic seizures after NMDA preconditioning and QA infusion Group
Number of SWD after NMDA injection
Latency for seizures onset after NMDA
Duration of seizures after NMDA
QA
–
–
–
Number of SWD after QA 2.0 ± 0.3
Latency for seizures onset after QA (s)
Duration of seizures after QA (s)
15.5 ± 5.6
30.3 ± 9.4
NQnc
8.5 ± 1.04
177.0 ± 71.5
34.1 ± 12.2
2.25 ± 1.3
301.0 ± 172.6
18.3 ± 11.6
NQc
3.5 ± 0.6*
104.0 ± 18.0
30.8 ± 12.0
4.25 ± 0.9
34.25 ± 23.6
35.7 ± 16.4
Mice were treated with NMDA (75 mg/kg, i.p.) and EEG recordings were performed for 30 min. One day later, mice were infused with QA (36.8 nmol, i.c.v.) and were EEG recordings were performed for 10 min. Latency for seizure onset and latency are presented in seconds. Data are presented as mean ± SEM QA saline plus QA-treated mice, NQnc NMDA plus QA non-convulsed mice, NQc NMDA plus QA convulsed mice. * Statistical difference from NQnc group (ANOVA followed by Tukey’s test)
Fig. 4 Analysis of the relationship between the severity scale of convulsions, NMDA-induced spike-wave discharges (SWDs) and QA-induced SWD. Mice (n = 4) were treated with vehicle (saline 0.1 %, i.p.) or a subconvulsant dose of NMDA (75 mg/kg, i.p.). After 24 h, they were submitted to QA infusion (36.8 nmol, i.c.v.) and
cortical and hippocampal EEG changes were recorded for 10 min. a Correlation between NMDA-induced SWD and QA-induced seizures severity (r = -0.9507 Pearson’s correlation, p = 0.0003). b Correlation between NMDA-induced SWD and QA-induced SWD (r = -0.8927 Pearson’s correlation, p = 0.057)
of NMDA and, in vivo, subconvulsant doses of NMDA have been used to promote cell protection against a wide range of brain insults, in the so-called NMDA preconditioning (Ogita et al. 2003). However, the mechanisms involved in such protection were not fully elucidated. The results here reported, showed that EEG alterations following NMDA preconditioning were correlated to the posterior response of mice to QA infusion. So, we have demonstrated a higher number of SWD during NMDA preconditioning diminishes the probability of developing clonic and/or tonic seizures after QA treatment. Cerebroventricular injection of QA overstimulates motor systems, promoting a typical sequence of behavioral alterations, ranging from wild running and jumping to generalized tonic–clonic seizures. Herein, based on studies using NMDA to induce seizures, we have presented a severity scale to better evaluate QA-induced seizures (Cruz et al. 2003; Marganella et al. 2005). NMDA preconditioning promoted cortical and hippocampal epileptiform activity, which was not accompanied by behavioral seizures. However, animals display immobility, staring, and freezing behavior, similar to those findings observed by
previous studies (Sawant et al. 2010). These EEG alterations were characterized by the presence of spike and/or sharp wave discharges, as it was expected after NMDAR activation and easily detected in the cortex and hippocampus (Kabova et al. 1999). The new interesting finding, in this study, was the negative correlation observed between the number of SWD registered after NMDA treatment, which was associated with diminished behavioral seizures susceptibility. We observed that NQnc animals presented a greater number of SWD during NMDA preconditioning in comparison to the NQc group. This is an important finding in the present study, since we could demonstrate that a strong depolarization is necessary to the induction of tolerance. This is in accordance with studies that used other chemical agents to induce preconditioning, and they observed strong stimulation is related to epileptic tolerance (Borges et al. 2007; Najm et al. 1998). However, it was demonstrated that epileptic tolerance may also be induced by low levels of depolarization through preconditioning induced by domoic acid (Sawant et al. 2010). Here, we demonstrated that NMDA preconditioning was not able to alter QA-induced seizure activity in EEG
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recordings, even in those animals which did not display convulsive behaviors. In the same way, it was observed kainic acid-induced preconditioning did not alter EEG activity during status epilepticus induced by a convulsive dose of kainic acid (Hatazaki et al. 2007). Although brain preconditioning had demonstrated to be neuroprotective, it was observed EEG traces between epileptic preconditioned and status epilepticus mice are similar. However, as we have shown in the current study, these observations are derived from short-term EEG analysis (Jimenez-Mateos et al. 2010). It has been demonstrated that NMDA and AMPA receptors are involved in the generation of hippocampal EEG traces (Geocadin et al. 2005; Leung and Shen 2004). Moreover, it has been shown ischemic preconditioning prevents severe ischemia-induced downregulation of GluR2 subunit, thus preventing the increase in calcium influx via AMPA/kainate receptors (Tanaka et al. 2002; Sommer and Kiessling 2002; Deng et al. 2003). In this way, modulation of NMDA or AMPA receptors composition may be involved in seizure tolerance induced by preconditioning. Mechanisms underlying epileptic tolerance induced by preconditioning remain unclear. However, it is believed the tolerance to seizures is acquired through the impairment of excitatory transmission, as well as improvement of inhibitory transmission. It has been shown preconditioning can induce downregulation and suppression of genes related to subunits of voltage-dependent calcium and sodium channels and glutamate receptors. Moreover, preconditioning induces upregulation of the glial glutamate transporter, EAAT1, that could promote greater clearance of glutamate from synaptic cleft (Jimenez-Mateos et al. 2008). It has also been demonstrated brain preconditioning promotes inhibitory transmission through downregulation of aminobutyrate aminotransferase, the enzyme which metabolizes GABA and downregulation of GABA transporter (GAT1) (Borges et al. 2007). Furthermore, it has been observed that an adenosine A1 receptor antagonist prevented NMDAinduced preconditioning, even in vitro or in vivo (Boeck et al. 2004, 2005; Ogita et al. 2003). Activation of adenosine A1 receptor has been related to a decreased release of excitatory transmitters, and it is involved in neuroprotection (Poli et al. 1991; Ralevic and Burnstock 1998). Thus, these alterations could lead to a decrease in neuronal excitability and increase neuronal inhibition, which may be responsible for epileptic tolerance. We have recently demonstrated that the inhibition of PKA, PI3K, and MAPK/ERK signaling pathways activation prevents anticonvulsant effects of NMDA preconditioning (de Araujo Herculano et al. 2011). However, inhibition of the Ca2?/ calmodulin-dependent protein kinase II and protein kinase C signaling pathways did not alter the preconditioning promoted by NMDA. Then, specific survival pathways are
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activated to induce cellular responses which are responsible for brain tolerance to insults. In conclusion, this study has demonstrated epileptic tolerance induced by NMDA against QA-induced seizures was related to the degree of depolarization in the cerebral cortex and hippocampus during preconditioning, as it was confirmed by the correlation between seizure score and EEG data. Therefore, EEG recording can be used as a tool to predict the induction or not of an epileptic tolerance promoted by neuroprotective agents, and also, it can be useful for evaluating electrical mechanisms related to the protective action of preconditioning. Acknowledgments This work was supported by grants from CNPq, CAPES, FAPESC (NENASC Project—PRONEX—CNPq/FAPESC), FINEP-IBN/Net (01.06.0842-00), and INCT for Excitotoxicity and Neuroprotection to C.I. Tasca. C.I.T., J.M.N., and T.C.M.L. are recipients of CNPq productivity fellowship. Conflict of interest interest.
The authors declare that there is no conflict of
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