Rat Cerebrocortical Slices Research Articles

Akt phosphorylation and cell survival after hypoxia-induced cytochrome c release in superfused respiring neonatal rat cerebrocortical slices.

Phosphorylation of Akt before hypoxia (30 min) and during reoxygenation (4 h) was evaluated in superperfused neonatal rat cerebrocortical slices (350 microm, P7, Sprague-Dawley). Cytosolic cytochrome c intensities in Western blots, which were increased at the end of hypoxia. were decreased during reoxygenation. Western blot intensities of phosphorylated Akt (phospho-Akt), nearly undetectable at the end of hypoxia, recovered quickly during reoxygenation, in a trend opposite that for cytochrome c. At 1.5 h and 4 h after hypoxia they became larger or the same as before hypoxia. Total Akt was unchanged by hypoxia and reoxygenation. Phosphocreatine (PCr) and nucleotide triphosphates (NTP) were measured in parallel 14.1 Tesla ex vivo 31P NMR superfused brain slice studies. PCr and alpha-NTP were nearly undetectable at the end of hypoxia. Although they recovered quickly after hypoxia, they were lower than before hypoxia. Reductions in phospho-Akt during hypoxia were consistent with the general unavailability of basic high energy phosphates. Preferential phosphorylation of Akt after hypoxia suggested that substantial reductions in intracellular energy, as indicated by PCr and NTP, might be tolerated by processes important for generating phospho-Akt. Additionally, the post-hypoxia increase in phospho-Akt might have contributed to concomitant reductions in cytosolic cytochrome c.

NTP and PCr responses to hypoxia by hypothermic and normothermic respiring, superfused, neonatal rat cerebrocortical slices: an NMR spectroscopy study at 14.1 Tesla.

Although mechanisms of hypothermic neuroprotection during oxygen deprivation have long been investigated, further characterizations of various molecular mechanisms are appropriate. Anticipating future studies of hypothermia and hypoxia/ischemia, we investigated the extent to which our ex vivo, NMR-based, superfused brain slice model might be helpful. (Slices are approximately 350 microm thick, with 18 slices per 8 mm NMR tube.) 31P NMR spectroscopic measurements were made of hypothermia-induced changes in high energy phosphates, while simultaneously monitoring and controlling tissue temperature, using 1H NMR, the high spectroscopic resolution available at 14.1 Tesla (600 MHz for protons), and a recently published protocol. NTP and PCr concentrations in healthy, well-oxygenated slices decreased to (55 +/- 15)% and (66 +/- 30)% of their respective values at 28.0 degrees C when warmed to 38.0 degrees C, in approximate agreement with earlier in vivo studies by others. During 30 min hypoxia NTP and PCr decreased to non-observable values, regardless of temperature. After reoxygenation, NTP and PCr recoveries as percentages of respective prehypoxia values were (63% +/- 16%; 70%) +/- 5%) for hypothermic slices (28.0 degrees C), and (46% +/- 13%; 41% +/- hypothermic neuroprotection during oxygen deprivation in this model, which appears suitable for use in further studies.

Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices.

Cellular injury was evaluated in superfused cerebrocortical slices (350 micro m) from 7-day-old Sprague-Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.

Halothane attenuates the cerebroprotective action of several Na + and Ca 2+ channel blockers via reversal of their ion channel blockade

We have previously shown the involvement of Na + channel as well as N-type and P/Q-type Ca 2+ channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na + and Ca 2+ channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na + channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca 2+ channel blockers, such as verapamil, ω-agatoxin IVA and ω-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, ω-agatoxin IVA and ω-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na + and Ca 2+ channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na + and Ca 2+ channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na + and Ca 2+ channel blockers.

Inhibitory effects of intravenous anaesthetic agents on K(+)-evoked glutamate release from rat cerebrocortical slices. Involvement of voltage-sensitive Ca(2+) channels and GABA(A) receptors.

It is widely accepted that most general anaesthetic agents depress the central nervous system (CNS) by potentiation or activation of the GABA(A) receptor-mediated Cl(-) conductance. These agents also reportedly inhibit voltage-sensitive Ca(2+) channels (VSCCs), thus depressing excitatory transmission in the CNS. However, in this regard there are few functional data at the level of neurotransmitter release. In this study we examined the effects of VSCC antagonists and a range of intravenous anaesthetic agents on K(+)(40 mM)-evoked glutamate release from rat cerebrocortical slices in the absence and presence of the GABA(A) receptor antagonist bicuculline (100 microM). We employed both selective and non-selective VSCC antagonists, the anaesthetic barbiturates thiopental, pentobarbital and phenobarbital, the non-anaesthetic barbiturate barbituric acid, the non-barbiturate anaesthetics alphaxalone, propofol and ketamine and the GABA(A) receptor agonist, muscimol. Glutamate released into the incubation medium was determined by a glutamate dehydrogenase-coupled assay. Omega-agatoxin IV(A) (P-type VSCC), omega-conotoxin MVII(C) (P/Q-type VSCC) and Cd(2+) (non-selective) essentially abolished glutamate release whilst nifedipine (L-type VSCC) and omega-conotoxin GVI(A) (N-type VSCC) reduced release by less than 30%. The concentrations producing 50% of the maximum inhibition (IC(50)) for thiopental, pentobarbital, phenobarbital, alphaxalone, propofol and ketamine were (in microM) 8.3, 22, 112, 6.3, 83 and 120, respectively. Barbituric acid produced a small (about 20%) inhibition. With the exception of ketamine, the IC(50) values for these anaesthetic agents were increased threefold by bicuculline (100 microM). In addition, muscimol significantly inhibited release by 26% with an IC(50) of 1.1 microM. In summary, a range of anaesthetic agents at clinically achievable concentrations inhibit glutamate release and this inhibition of release appears to be due mainly to direct inhibition of P/Q-type VSCCs, although activation of the GABA(A) receptor plays a role in this response.

Nociceptin/orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices

We have previously demonstrated that nociceptin/orphanin FQ (N/OFQ), inhibits K + depolarisation-evoked glutamate release from rat cerebrocortical slices. In this study we have examined the effects of N/OFQ on glutamate release from rat cerebellar and brain stem slices as there are regional differences in nociceptin/orphanin FQ receptor (NOP) expression. Slices were depolarised with two pulses of 46 mM K + (S 1 and S 2) with N/OFQ added after S 1. Glutamate (non-radioactive) was measured using a fluorescence-based assay. N/OFQ effects were assessed by measuring area under S 1 and S 2 release curves and calculation of S 2/S 1 ratios. In cerebellar slices K + evoked S 2/S 1 ratio was 1.17±0.10 ( n=28). This was reduced in a concentration dependent (EC 50 22 nM; E max 46%) and naloxone (10 μM) insensitive manner by N/OFQ. In the brain stem K + evoked glutamate release was considerably reduced compared to cerebellum. In several preparations K + failed to evoke a significant release. In those that did K + evoked S 2/S 1 ratio was 1.03±0.07 ( n=13). A total of 100 nM N/OFQ reduced this by 38±12% and this response was naloxone insensitive. Due to this small response and its variability we could not construct a full concentration response curve. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate.

Effects of ammonia and hepatic failure on the net efflux of endogenous glutamate, aspartate and taurine from rat cerebrocortical slices: modulation by elevated K + concentrations

Cerebrocortical minislices derived from control rats (“control slices”) and from rats with thioacetamide (TAA)-induced hepatic failure showing moderate hyperammonemia and symptoms of hepatic encephalopathy (HE) (“HE slices”), were incubated with physiological saline in the absence or presence of 5 mM ammonium acetate (“ammonia”), at potassium ion (K +) concentrations ranging from 5 to 15 mM. The efflux of endogenous aspartate (Asp), glutamate (Glu) and taurine (Tau) to the incubation medium was assayed by HPLC. At 5 mM K +, perfusion of control slices with ammonia did not affect Glu and slightly depressed Asp efflux. Raising K + concentrations in the incubation medium to 7.5 led to inhibition of Glu and Asp efflux by ammonia and the inhibitory effect was further potentiated at10 mM K +. The inhibition was also significant at 15 mM K +. This suggests that, depression of excitatory neurotransmission associated with acute hyperammonemia is more pronounced under conditions of intense neuronal activity than in the resting state. HE moderately increased the efflux of Glu and Asp, and the stimulatory effect of HE on Glu and Asp efflux showed virtually no variation upon changing K + concentration up to 15 mM. Ammonia strongly, and HE moderately, increased Tau efflux at 5 mM K +. However, both the ammonia- and HE-dependent Tau efflux decreased with increasing K + concentration in the medium and was no longer significant at 10 mM concentration, indicating that intense neuronal activity obliterates the neuroprotective functions of this amino acid triggered by hyperammonemia.

Orexin A and B evoke noradrenaline release from rat cerebrocortical slices.

1. Orexin A and B, recently identified in the rat hypothalamus are endogenous neuropeptide agonists for the G-protein coupled orexin-1 (OX1) and orexin-2 (OX2) receptors. 2. In the present study, we have examined the effects of orexin A, B and raised extracellular K(+) on noradrenaline release from the rat cerebrocortical slice. We have compared this with other sleep-wake-related (excitatory) neurotransmitters; dopamine, glutamate, serotonin and histamine. 3. Neurotransmitter release studies were performed in rat cerebrocortical slices incubated in modified Krebs buffer (with and without Ca(2+)+EGTA 1 mM) with various concentrations of orexin A, B and K(+) for various times. 4. Orexin A and B-evoked (10(-7) M) noradrenaline release was time-dependent reaching a maximum some 10 min after stimulation. K(+) (40 mM) evoked release was also time dependent but reached a maximum after 6 min. Orexin A, B and K(+) stimulation of release was concentration dependent with pEC(50) and E(max) (% of basal) values of 8.74+/-0.32 (1.8 nM) and 263+/-14% and 8.61+/-0.38 (2.4 nM) and 173+/-7% and 1.43+/-0.02 (37 mM) and 1430+/-70%, respectively. Orexin-evoked release was partially extracellular Ca(2+) dependent. 5. Of the other transmitters studied there was a weak orexin A and B stimulation of glutamate release. In contrast K(+) evoked dopamine, glutamate, histamine and serotonin release with pEC(50) and E(max) (% of basal) values of 1.47+/-0.05 (34 mM) and 3430+/-410%, 1.38+/-0.04 (42 mM) and 1240+/-50%, 1.47+/-0.02 (34 mM) and 480+/-10% and 1.40+/-0.05 (40 mM) and 560+/-60% respectively. 6. We conclude that the neuropeptides orexin A and B evoke noradrenaline release from rat cerebrocortical slices.

Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices.

Nociceptin/orphanin FQ (NC), the endogenous ligand for the G-protein coupled nociceptin receptor (NCR), has a modulatory role in various physiological processes including neurotransmitter release. We have examined the effects of NC, the analogues NC(1-13)NH2 and [F/G]NC(1-13)NH2 and the competitive antagonist [Nphe1]NC(1-13)NH2 (Nphe1) on glutamate efflux during an acute simulated ischaemic challenge in rat cerebrocortical slices. The increase in glutamate efflux seen with ischaemia was inhibited by NC (EC50 250 nM). At micromolar concentrations, the analogues were found to have a similar effect on glutamate efflux compared to NC. In all cases, inhibition of glutamate efflux was abolished by Nphe1 (30 microM). These results suggest a neuroprotective action for NC.

Involvement of Na + and Ca 2+ channel activation and resultant nitric oxide synthesis in glutamate-mediated hypoxic injury in rat cerebrocortical slices

The role of Na + and Ca 2+ channels in glutamate-mediated hypoxic injury was investigated in slices of the rat cerebral cortex. Hypoxic injury was determined by mitochondrial reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide after exposure of brain slices to 30-min of hypoxia/glucose deprivation followed by 3-h of reoxygenation. Endogenous glutamate release was markedly elevated during hypoxia/glucose deprivation, but it returned almost to basal level during reoxygenation. Hypoxic injury was prevented by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Combined treatment with ω-conotoxin GVIA, ω-agatoxin IVA, and tetrodotoxin reversed the hypoxic injury, although none of these agents alone or nifedipine was effective. Moreover, a novel Na +/Ca 2+ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] significantly inhibited the hypoxic injury. Several inhibitors of nitric oxide synthase also blocked the hypoxic injury. Consistently, nitric oxide synthesis, as estimated from cyclic GMP formation in the extracellular fluids, was enhanced during hypoxia/glucose deprivation. NS-7 and other Na + and Ca 2+ channel blockers suppressed the enhancement of nitric oxide synthesis, although these compounds alone, or in combination, did not reduce hypoxic glutamate release. These findings suggest that hypoxic injury in rat cerebrocortical slices is triggered by glutamate and subsequent enhancement of nitric oxide synthesis through activation of both Na + and Ca 2+ channels. Thus, the simultaneous blockade of both Na + channel as well as N-type and P/Q-type Ca 2+ channels is required to sufficiently reverse the hypoxic injury.

Involvement of peroxynitrite and hydroxyradical generated from nitric oxide in hypoxia/reoxygenation injury in rat cerebrocortical slices

The changes in nitric oxide (NO) formation during hypoxia and reoxygenation were measured in slices of rat cerebral cortex, and the possible involvement of NO and its decomposition products, including peroxynitrite and hydroxyradical, in the hypoxia/reoxygenation injury was subsequently investigated. NO formation estimated from cGMP accumulation in the extracellular fluids was enhanced during hypoxia and to a lesser extent in the reoxygenation period. The mRNA for inducible NO synthase (NOS) was detected 3–5 h after reoxygenation, although neuronal NOS mRNA decreased after reoxygenation. Several NOS inhibitors such as N G-monomethyl- l-arginine and N G-nitro- l-arginine blocked not only the NO formation but also the hypoxia/reoxygenation injury as determined by lactate dehydrogenase (LDH) leakage. The hypoxia/reoxygenation injury was prevented by peroxynitrite scavengers including deferoxamine and uric acid, or several hydroxyradical scavengers such as dimethylthiourea, 2-mercaptopropionylglycine and d(-) mannitol. In addition, the hypoxia/reoxygenation injury was attenuated by poly(ADP–ribose)synthetase inhibitors such as banzamide, 3-aminobenzamide and 1,5-isoquinolinediol. On the other hand, both N-morpholinosidnonimine, a peroxynitrite generator, and hydroxyradical-liberating solution containing FeCl 3–ADP and dihydroxyfumarate caused a marked LDH leakage in normoxic slices. These findings suggest that the enhanced formation of NO causes hypoxia/reoxygenation injury after degradation to peroxynitrite and hydroxyradical and the resultant activation of poly(ADP–ribose)synthetase.

Effects of intravenous anesthetic agents on glutamate release: a role for GABAA receptor-mediated inhibition.

Many anesthetic agents are known to enhance the alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation. Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37 degrees C, samples for glutamate assay were obtained at 2-nmin intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1-100 microM) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1-300 micro/M), propofol (10 microM), or ketamine (30 microM) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1). Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 +/- 0.33 (mean +/- SD, n = 96). Ketamine and thiopental produced a concentration-dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 /microM, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 microM. Bicuculline also reversed the effects of propofol but not those of ketamine. The authors' data indicate that thiopental, propofol, and ketamine inhibit K+-evoked glutamate release from rat cerebrocortical slices. The inhibition produced by thiopental and propofol is mediated by activation of GABAA receptors, revealing a subtle interplay between GABA-releasing (GABAergic) and glutamatergic transmission in anesthetic action.

Immunohistochemical nitrotyrosine distribution in neonatal rat cerebrocortical slices during and after hypoxia

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl- d-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia ( pO 2<5 mmHg) with or without 10 μM MK-801 treatment; with exposure to 10, 100 and 1000 μM NMDA; and no treatment (control). In each experiment, twenty 350-μm thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague–Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-μm frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% ( p<0.001) and NT-stained neurons by 54.1% ( p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic–ischemic brain injury and NMDA-induced brain injury.

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices

We have examined the effects of the recently described heptadecapeptide nocistatin on K +-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n=7) did not inhibit K +-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7±8.3% ( P<0.05, n=6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

Reversal of Thiopentone-Induced Inhibition of K+-evoked Glutamate Release From Rat Cerebrocortical Slices by the GABAA antagonist, Bicuculline

Reversal of Thiopentone-Induced Inhibition of K+-evoked Glutamate Release From Rat Cerebrocortical Slices by the GABAA antagonist, Bicuculline

A novel Na+/Ca2+ channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices.

The substance 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7) has been developed recently as a cerebroprotective compound with Na+ and Ca2+ channel blocking action. In the present study, the effect of NS-7 in an in vitro model of hypoxic injury was examined and the possible involvement of Na+ and Ca2+ channels in the hypoxic injury subsequently determined. When slices of rat cerebral cortex were exposed to hypoxia/glucose deprivation followed by reoxygenation and restoration of the glucose supply, marked leakage of lactate dehydrogenase (LDH) occurred 3-6 h after reoxygenation. This hypoxia/reoxygenation-induced injury was blocked almost completely by the removal of extracellular Ca2+ or by chelating intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM). In addition, combined treatment with the N-type Ca2+ channel blocker omega-conotoxin GVIA and the P/Q-type Ca2+ channel blocker omega-agatoxin IVA significantly reduced LDH leakage, although neither of these Ca2+ channel blockers alone, nor nimodipine, an L-type Ca2+ channel blocker, was effective. On the other hand, several Na+ channel blockers, including tetrodotoxin, local anaesthetics and antiepileptics, significantly reduced the hypoxic injury. NS-7 (3-30 microM) concentration-dependently inhibited LDH leakage caused by hypoxia/reoxygenation, but had no influence on the reduction of tissue ATP content and energy charge during hypoxia and glucose deprivation. It is suggested that blockade of Na+ and Ca2+ channels is implicated in the cerebroprotective action of NS-7.

Expression of c- fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis : Brain Research 785 (1998) 262–278

Expression of c- fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis : Brain Research 785 (1998) 262–278

31P]/[1H] nuclear magnetic resonance study of mitigating effects of GYKI 52466 on kainate-induced metabolic impairment in perfused rat cerebrocortical slices.

Kainic acid (KA) has long been used in experimental animals to induce status epilepticus (SE). A mechanistic implication of this is the association between excitotoxicity and brain damage during or after SE. We evaluated KA-induced metabolic impairment and the potential mitigating effects of GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine] in superfused rat cerebral cortical slices. Interleaved [31P]/[1H] magnetic resonance spectroscopy (MRS) was used to assess energy metabolism, intracellular pH (pHi), N-acetyl-L-aspartate (NAA) level, and lactate (Lac) formation before, during, and after a 56-min exposure to 4 mM KA in freshly oxygenated artificial cerebrospinal fluid (oxy-ACSF). In the absence of GYKI 52466 and during the KA exposure, NAA, PCr, and ATP levels were decreased to 91.1 +/- 0.8, 62.4 +/- 3.9, and 59.1 +/- 4.3% of the control, respectively; Lac was increased to 118.2 +/- 2.1 %, and pH, was reduced from 7.27 +/- 0.02 to 7.13 +/- 0.02. During 4-h recovery with KA-free ACSF, pHi rapidly and Lac gradually recovered, NAA decreased further to 85.5 +/- 0.3%, and PCr and ATP showed little recovery. Removal of Mg2+ from ACSF during KA exposure caused a more profound Lac increase (to 147.1 +/- 4.0%) during KA exposure and a further NAA decrease (to 80.4 +/- 0.5%) during reperfusion, but did not exacerbate PCr, ATP, and pHi changes. Inclusion of 100 microM GYKI 52466 during KA exposure significantly improved energy metabolism: the PCr and ATP levels were above 76.6 +/- 2.1 and 82.0 +/- 2.9% of the control, respectively, during KA exposure and recovered to 101.4 +/- 2.4 and 95.0 +/- 2.4%, respectively, during reperfusion. NAA level remained at 99.8 +/- 0.6% during exposure and decreased only slightly at a later stage of reperfusion. Our finding supports the notion that KA-induced SE causes metabolic disturbance and neuronal injury mainly by overexcitation through non-N-methyl-D-aspartate (NMDA) receptor functions.

Modulation of extracellular GABA levels in the retina by activation of glial P2X-purinoceptors.

1. In the rat retina, gamma-aminobutyric acid (GABA) released as a transmitter is inactivated by uptake mainly into glial cells (Müller cells). Activation of P2-purinoceptors in Müller cells increases [Ca2+]i and the present study was undertaken to see whether this action affected the glial release of [3H]-GABA from the superfused rat isolated retina. 2. Adenosine 5'-triphosphate (ATP) and the P2X-purinoceptor agonists, alpha,beta-methylene-ATP (alpha,beta-meATP) and beta,gamma-methyleneATP (beta,gamma-meATP) significantly increased the KCl-evoked release of [3H]-GABA from the retina. 3. Adenosine and the P2Y-purinoceptor agonist, 2-chloroATP, had no effect on the KCl-evoked release of [3H]-GABA from the retina. However, 2-methylthioATP (2-Me-S-ATP) significantly enhanced the evoked release of [3H]-GABA. 4. The effect of ATP on the glial release of [3H]-GABA was abolished by the P2-antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. When the superfused retina was exposed to the GABA uptake inhibitor, SKF89976A, the enhancing effect of alpha,beta-meATP on the KCl-evoked release of GABA was abolished. 6. The KCl-evoked release of [3H]-GABA from the frog retina and rat cerebrocortical slices, which take up GABA mainly into neurones, was not affected by ATP or alpha,beta-meATP. 7. We concluded that the glial Müller cells in the rat retina possess P2-receptors, activation of which increases the 'release' of preloaded [3H]-GABA apparently by reducing uptake. On balance, the results suggest the involvement of P2X-purinoceptors, although we cannot exclude the possibility that P2Y-purinoceptors may be involved. Our results suggest that ATP, as well as being a conventional transmitter in the retina, may be involved in neuronal-glial signalling and modulate the extracellular concentration of GABA.

Expression of c- fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis

Respiring neonatal rat cerebrocortical slices were exposed for 30 min to toxic concentrations of N-methyl- d-aspartate (NMDA; 100 μM, 500 μM and 1000 μM). In situ hybridization was used to study c- fos and hsp70 mRNA before, during, and for 8 h after NMDA exposure. Cell swelling and nuclear morphology were assessed using Cresyl violet (Nissl) staining. Possible evidence for apoptosis was examined using in situ terminal transferase d-UTP nick-end labeling (TUNEL) staining and agarose–gel electrophoresis of extracted slice DNA. After NMDA administration c- fos and hsp70 mRNA expression increased, with maxima occurring, respectively, at 1 h and 4 h after NMDA exposure. When treatment with dizocilpine (MK-801; 10 μM), a non-competitive NMDA antagonist, was started before NMDA exposures, expression of both c- fos and hsp70 mRNA was decreased to values near control, indicating that activation of NMDA receptors induces both genes. Only a minority of induced cells expressed FOS protein and no HSP70 protein expression was seen. These apparent failures of translation might be related to the stress response. Histologically, 1000 μM NMDA produced substantial necrosis, with no evidence of apoptosis. Evidence for apoptosis was found at the two lower NMDA concentrations, which produced TUNEL-positive fragmented nuclei and faint ladder patterns in DNA electrophoresis. Dizocilpine pre-treatment blocked NMDA-induced necrosis and attenuated TUNEL-positive staining in slice parenchyma. TUNEL-positive staining with a different morphology was found in the injury layer, a region 50- μm thick where mechanical trauma was inflicted when slices were cut from brain. When slices received dizocilpine immediately after decapitation, TUNEL-positive staining no longer occurred in the injury layer, in agreement with previous cell culture studies that implicated NMDA receptor activation after mechanical trauma to neurons. We conclude that at the toxic doses studied, NMDA receptor activation results primarily in necrosis. However, data at low NMDA concentrations are consistent with a small amount of apoptosis.