洛伐他汀减轻NMDA诱导的兴奋性毒性损害

目的 探讨洛伐他汀(Lovastatin,LOV)对NMDA诱导的大鼠皮质神经元兴奋性毒性损害的神经保护作用.方法 选用胚胎17d的SD大鼠,取皮质神经元接种培养.通过台盼蓝排除实验评估细胞活力、TUNEL染色检测凋亡细胞及免疫荧光细胞化学技术测定神经元形态.结果 台盼蓝排除实验显示500nmol/L洛伐他汀预处理3d显著减轻NMDA诱导的大鼠皮质神经元兴奋性毒性损害,而不能减少蛋白激酶C抑制剂星形孢菌素诱导的细胞凋亡.LOV和L-甲羟戊酸(MVA)共同预处理不能减轻NMDA诱导的兴奋性毒性损害,提示其保护作用依赖于降低胆固醇水平.洛伐他汀的神经保护作用呈剂量和时程依赖性.TUNEL染色显示洛伐他汀预处理能减少NMDA诱导的细胞凋亡.免疫荧光细胞化学结果显示洛伐他汀能改善NMDA诱导的MAP-2神经元形态损害.结论 洛伐他汀选择性地减轻NMDA诱导的大鼠皮质神经元兴奋性毒性损害及形态损害,提示洛伐他汀对兴奋性毒性损害相关神经病理有潜在的神经保护作用.     

17β-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors

Several lines of evidence suggest that 17β-estradiol (βE₂) has neuroprotective properties. The risk and severity of dementia are decreased in women who have received estrogen therapy, and βE₂ protects neurons in vitro against death from a variety of stressors. Neuroprotection by βE₂ has been suggested to be due to free radical scavenging. We demonstrate an additional neuroprotective mechanism whereby βE₂ protects against NMDA-induced neuronal death by directly inhibiting the NMDA receptor.     

Triptolide protects astrocytes from hypoxia/ reoxygenation injury

Astrocytes in an in vitro murine astrocyte model of oxygen and glucose deprivation/hypoxia and reoxygenation were treated with different concentrations of triptolide (250, 500, 1 000 ng/mL) in a broader attempt to elucidate the protection and mechanism underlying triptolide treatment on astrocytes exposed to hypoxia/reoxygenation injury. The results showed that the matrix metalloproteinase-9, interleukin-1β, tumor necrosis factor α and interleukin-6 expressions were significantly decreased after triptolide ...     

Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons

Immunoblot analysis, using antibodies against distinct N-methyl-d -aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.     

Sulforaphane protects primary cultures of cortical neurons against injury induced by oxygen-glucose deprivation/reoxygenation via antiapoptosis

Objective To determine whether sulforaphane (SFN) protects neurons against injury caused by oxygen-glucose deprivation/reoxygenation (OGD/R) and, if so, to investigate the possible mechanisms. Methods Primary cultures of neurons were prepared from the cerebral cortex of 1-day-old Sprague-Dawley rats. On days 5-6 in vitro, the neurons were exposed to OGD for 1 h, followed by reoxygenation for 24 h. Cells were treated with 0, 0.1, 0.2, 0.5, 1, 2.5, or 5 μmol/L SFN, with or without 10 μmol/L LY294002, a PI3K-specific inhibitor, during OGD/R (a total of 25 h). After 24-h reoxygenation, MTT was used to assess viability and injury was assessed by Hoechst 33258/propidium iodide (PI) staining; immunofluorescence staining and Western blot were performed to detect molecular events associated with apoptosis. Results The MTT assay showed that 1 μmol/L SFN significantly increased viability, and Hoechst 33258/PI staining showed that the numbers of injured neurons were reduced significantly in the SFN group. Furthermore, immunofluorescence staining and Western blot showed that SFN increased Bcl-2 and decreased cleaved caspase-3 levels. Moreover, LY294002 inhibited the phosphorylated-Akt expression evoked by SFN, decreased Bcl-2 expression and increased cleaved caspase-3 expression. Conclusion SFN protects neurons against injury from OGD/R and this effect may be partly associated with an anti-apoptosis pathway.     

Gypenosides protect primary cultures of rat cortical cells against oxidative neurotoxicity

Gypenosides (GPs) were tested for their ability to protect primary cultures of immature cortical cells against oxidative glutamate toxicity. In immature neural cells, glutamate cytotoxicity is known to be mediated by the inhibition of cystine uptake, leading to depletion of intracellular glutathione (GSH). The depletion of GSH impairs cellular antioxidant defenses resulting in oxidative stress and cell death. We found that pretreatment with GPs (100-400 microg/ml) significantly protected cells from glutamate-induced cell death. It was therefore of interest to investigate whether GPs protect cortical cells against glutamate-induced oxidative injury through preventing GSH depletion. Results show that GPs significantly up-regulated mRNAs encoding gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione reductase (GR) and enhanced their activities for GSH synthesis as well as recycle. Furthermore, GPs lowered the consumption of GSH through decreased accumulation of intracellular peroxides, leading to an increase in the intracellular GSH content. GPs were also found to prevent lipid peroxidation and reduce the influx of Ca(2+) which routinely follows glutamate oxidative challenge. GPs treatment significantly blocked glutamate-induced decrease in levels of Bcl-2 and increase in Bax, leading to a decrease in glutamate-induced apoptosis. Thus, we conclude that GPs protect cortical cells by multiple antioxidative actions via enhancing intracellular GSH, suppressing glutamate-induced cytosolic Ca(2+) elevation and blocking glutamate-induced apoptosis. The novel role of GPs implies their remarkable preventative and therapeutic potential in treatment of neurological diseases involving glutamate and oxidative stress.     

N-acetylaspartylglutamate and beta-NAAG protect against injury induced by NMDA and hypoxia in primary spinal cord cultures

The acidic dipeptide N-acetylaspartylglutamate (NAAG) is the most prevalent peptide in the central nervous system. NAAG is a low potency agonist at the NMDA receptor, and hydrolysis of NAAG yields the more potent excitatory amino acid neurotransmitter glutamate. beta-NAAG is a competitive inhibitor of the NAAG hydrolyzing enzyme N-acetylated alpha-linked acidic dipeptidase (NAAG peptidase activity) or glutamate carboxypeptidase II, and may also act as a NAAG-mimetic at some of the sites of NAAG pharmacological activity. Since NAAG has been shown to have neuroprotective characteristics in a number of experimental preparations, it is the purpose of the present study to specifically evaluate the possible efficacy of NAAG and beta-NAAG against NMDA- and hypoxia-induced injury to spinal cord mixed neuronal and glial cell cultures. NAAG (500-1000 microM) protected against NMDA- or hypoxia-induced injuries to spinal cord cultures, and the nonhydrolyzable analog beta-NAAG (250-1000 microM) completely eliminated the loss of viability caused by either insult. Both peptides also attenuated NMDA-induced increases in intraneuronal Ca(2+). Nonspecific mGluR antagonists, pertussis toxin, a stable cAMP analog, and manipulation of NAAG peptidase activity did not by themselves alter cell damage and did not influence the neuroprotective effects of NAAG. NAAG was not protective against kainate- or AMPA-induced cellular injury, while beta-NAAG was partially neuroprotective against both insults. At 2 mM, NAAG and beta-NAAG reduced neuronal survival and increased intraneuronal Ca(2+); these effects were only marginally attenuated by dizocilpine and APV. The results indicate that NAAG and beta-NAAG protect against excitotoxic and hypoxic injury to spinal cord neurons, and do so predominantly by interactions with NMDA and not mGluR receptors.     

Concentration dependence of adenosine and the protection of rat cortical neurones during anoxia

Aglycaemic / anoxic slices of rat olfactory cortex lose all electrical activity. On reoxygenation, 10 μM adenosine enhanced recovery from 23 ± 7% to 53 ± 12%; an increased tissue endurance of 5–7 min. 100 μM adenosine slightly depressed recovery to 11.5 ± 2.1%. Dipyridamole increasedwhereas adenosine deaminase reduced recovery. These observations question the therapeutic effectiveness of high adenosine concentrations.     

NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice

PURPOSE: N-methyl D-aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)-induced seizures are used to average NMDA receptors-evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA-induced seizures and hippocampal damage in vivo. METHODS: Mice were pretreated with nonconvulsant doses of NMDA for different times before i.c.v. QA infusion and observed for the occurrence of seizures. Hippocampal slices from mice were assayed to measure cellular viability. RESULTS: NMDA preconditioning presented 53% protection against QA-induced seizures, as well as QA-induced cellular death in the hippocampus. The NMDA receptor antagonist, MK-801, prevented the protection evoked by NMDA preconditioning. The adenosine A1 receptor antagonist, CPT, prevented the protection evoked by NMDA preconditioning against QA-induced seizures, but not against QA-induced hippocampal cellular damage. The adenosine A1 receptor agonist, CPA, did not mimic the NMDA preconditioning-evoked protective effects. CONCLUSIONS: These results suggest that in vivo preconditioning with subtoxic doses of NMDA protected mice against seizures and cellular hippocampal death elicited by QA, probably through mechanisms involving NMDA receptors operating with adenosine A1 receptors.     

Topically applied clonidine protects the rat retina from ischaemia/reperfusion by stimulating α2-adrenoceptors and not by an action on imidazoline receptors

Ischaemia was induced to the rat retina by raising the intraocular pressure above the systolic blood pressure for 45 min. After a reperfusion period of 5 days, alterations in the localisation of choline acetyltransferase (ChAT) and calretinin immunoreactivities, a reduction in the thickness of the inner retinal layers and a decline in the b-wave amplitude of the electroretinogram were recorded. These changes were blunted when clonidine was injected intraperitoneally before or after ischaemia or when applied topically by a specific regime. Other α₂-adrenoceptor agonists, brimonidine and apraclonidine, acted in a similar way to clonidine when applied topically but because of the number of experiments carried out a comparison between the effectiveness of the different α₂-adrenoceptor agonists was not possible. The protective effect of clonidine was attenuated when the α₂-adrenoceptor antagonists yohimbine or rauwolscine were co-administered, suggesting that the mechanism of action of the drug is to stimulate α₂-adrenoceptors. In addition, the imidazoline receptor ligands, BU-226 and AGN-192403 did not blunt the effect of ischaemia/reperfusion, supporting the notion that the protective action of the α₂-adrenoceptor agonists does not involve imidazoline sites but rather the activation of α₂-adrenoceptors. The protective effect of 0.5% clonidine appeared to be greater when topically applied to the eye that received ischaemia than when applied by the same regime to the contralateral eye. These studies suggest that while most of topically applied clonidine reaches the retina by a systemic route one cannot rule out additional pathways.     

Effect of NMDA Receptor Blockade on Melatonin and Activity Rhythm Responses to a Light Pulse in Rats

The possible role of the excitatory amino acids as mediators of the acute suppression and subsequent delay by light of pineal melatonin production was studied in rats using the NMDA receptor antagonist MK-801. Saline or MK-801 in doses up to 3 mg/kg (IP), was administered 15 min before a 15-min light pulse (200 lx), 4 h after dark onset, and the excretion of 6-sulphatoxymelatonin (aMT.6S) determined. Under these conditions saline injected/light exposed animals exhibited an acute, total but transient suppression of urinary aMT.6S excretion and a delay in the onset of aMT.6S the following night of 1.5 ± 0.2 h. MK-801 failed to block either the acute or phase delaying effect of light (onset delayed by 2.2 ± 0.4 h). Pretreatment with MK-801 (3 mg/kg) failed to block the effects of shorter, less intense light pulses 15 min before the pulse (e.g., 1 min/2 lx; onset delayed by 2.0 ± 0.4 h following saline, 1.5 ± 0.1 h following MK-801) or 60 min before a short duration low intensity pulse. In other experiments MK-801 (1 and 3 mg/kg) failed to affect aMT.6S excretion when injected in the dark at the time of lights out or 4 h after dark onset. NMDA (10 and 30 mg/kg) injection at the time of lights out or 4 h after darkness did not mimic the effects of a light pulse by decreasing aMT.6S excretion or causing a delay in the onset of excretion the following night. Finally MK-801 (3 mg/kg) injected 4 h after dark failed to block the phase delaying effects of a 15 min light pulse (200 lx) on running activity in rats. These results do not support the hypothesis that excitatory amino acids in the retino-hypothalamic tract acting on the NMDA receptor subtype and terminating in the suprachiasmatic nucleus mediate the photic influences upon rat pineal melatonin and activity rhythms. Copyright © 1996 Elsevier Science Inc.     

Selectively increased sensitivity of cerebellar granule cells to AMPA receptor-mediated excitotoxicity in a mouse model of Batten disease

Batten disease, a lysosomal storage disorder, is caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3-/-) mouse model of the disease exhibits many characteristic pathological features of the human disorder. Here, we show that Cln3-/- mice, similarly to Batten disease patients, have a deficit in cerebellar motor coordination. To explore the possible cellular cause of this functional impairment, we compared the vulnerability of wild type (WT) and Cln3-/- cerebellar granule cell cultures to different toxic insults. We have found that cultured Cln3-/- cerebellar granule cells are selectively more vulnerable to AMPA-type glutamate receptor-mediated toxicity than their WT counterparts. This selective sensitivity was also observed in organotypic cerebellar slice cultures. Our results suggest that lack of the CLN3 protein has a significant influence on the function of AMPA receptors in cerebellar granule neurons, and that AMPA receptor dysregulation may be a major contributor to the cerebellar dysfunction in Batten disease.     

Ruthenium red neurotoxicity and interaction with gangliosides in primary cortical cultures

Ruthenium red (RR) is an inorganic polycationic dye able to exert several effects on the nervous system, including neurodegeneration, both in vivo and in cell cultures. Gangliosides have been shown to protect cultured neurons against several damaging conditions, and it has been postulated that RR can interact with the negative charges of the sialic acid residues of these molecules. In the present work we have tested the effect of the trisialoganglioside GT1b and the monosialoganglioside GM1 on the RR-induced neuronal damage in primary cortical cultures, as well as on the binding of RR to synaptosomes. GT1b at 100–200 μM concentrations partially protected against RR-induced neurodegeneration, as judged by light microscopy and by measurement of the reduction of a tetrazolium salt, while GM1 was ineffective. GT1b, but not GM1, also partly blocked both RR binding and its diminution in the culture medium occurring during incubation. These results suggest that the three negative charges of GT1b enable it to interact with RR and as a consequence the entrance of the dye into the cells is blocked and neurotoxicity is diminished, although other mechanisms of protection cannot be excluded. Endogenous polysialic acid–containing molecules do not seem to be involved in RR effects, since the removal of sialic acid residues by treatment with neuraminidase did not prevent the cell damage. J. Neurosci. Res. 49:72–79, 1997. © 1997 Wiley-Liss Inc.     

Post-injury treatment with lipopolysaccharide or lipooligosaccharide protects rat neuronal and glial cell cultures

Traumatic brain injury (TBI) is a major cause of disability in civilians and military personnel worldwide that is caused by the acceleration force of a primary shockwave, blast wind or the force of a direct contact. Following the primary injury, secondary injury is caused by activation of the immune response due to an influx of neuro-inflammatory cells, increased production of inflammatory cytokines, and edema. In ischemia models pre-conditioning with lipopolysaccharide (LPS) has been shown to be neuroprotective, and post-injury conditioning with LPS was found to be protective in a spinal cord and an acute brain injury model. In this study, we utilized an in vitro scratch model of TBI to assess the effect of post-injury treatment with Escherichia coli LPS and Neisseria meningitidis lipooligosaccharide (LOS) on cell death and cytokine induction by assessing the level of lactate dehydrodgenase released from cells and rat multiplex cytokine assays. Our results showed that post-injury treatment of C6 glioma cells with either the LPS or the LOS reduced cell death when compared to scratched controls treated with media only. Post-injury treatment of the primary mixed neuronal cultures with LPS reduced cell death and resulted in a significant up-regulation in IL-10 when compared to controls. With LOS post-scratch treatment of the primary cell cultures, we found that IL-1α, IL-1β, IL-6, and TNF-α were significantly upregulated in addition to IL-10 compared to the media-only controls. The results strongly support additional testing of the neuroprotective ability of post-injury treatment with LPS or LOS in models of TBI.     

Iridoids from Scrophularia buergeriana attenuate glutamate-induced neurotoxicity in rat cortical cultures

In previous work, we isolated 7 neuroprotective iridoid glycosides from the 90% MeOH fraction of Scrophularia buergeriana (Scrophulariaceae). We therefore investigated the mode of action of 8-O-E-p-methoxycinnamoyl-harpagide (8-MCA-Harp), the most potent neuroprotective iridoid, and its aglycone, harpagide (Harp) using primary cultures of rat cortical cells in vitro. 8-MCA-Harp only revealed its neuroprotective activity in a pretreatment paradigm; this iridoid had more selectivity in protecting neurons against N-methyl-D-aspartate (NMDA)-induced neurotoxicity as opposed to that induced by kainic acid (KA). On the other hand, Harp exerted significant neuroprotective activity when it was administered either before or after glutamate insult and protected cultured neuronal cells from neurotoxicity induced by NMDA or KA. Furthermore, Harp significantly prevented the decrease of glutathione, an antioxidative compound in the brain, in our cultures. Finally, 8-MCA-Harp and Harp could successfully reduce the overproduction of nitric oxide and the level of cellular peroxide in cultured neurons. Collectively, these results suggested that Harp and 8-MCA-Harp protected primary cultured neurons against glutamate-induced oxidative stress primarily by acting on the antioxidative defense system and on glutamatergic receptors, respectively.     

Adenovirus-mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSVβgal), expressing β-galactosidase (β-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSVβgal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of β-gal expression following Adv/RSVβgal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSVβgal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated. © 1996 Wiley-Liss, Inc.     

Contribution of NMDA and nonNMDA glutamate receptors to synchronized excitation and cortical output in the primary motor cortex of the rat

Application of a GABA (gamma-aminobutyric acid) type A receptor antagonist through a microdialysis probe into the forelimb primary motor cortex (MI) of ketamine anesthetized rats induced the appearance of paroxysmal field potentials recorded in the supragranular layers of the MI and concomitant electromyographic (EMG) activity in the contralateral forelimb. Application of a nonNMDA (N-methyl-d-aspartate) glutamate receptor antagonist in conjunction with the GABA type A receptor antagonist completely blocked the paroxysmal field potentials and the EMG activity of the contralateral forelimb, while a NMDA receptor antagonist had no effect. The results indicate that the spread of activity within the primary motor cortex and the motor cortex output are mediated by nonNMDA receptors.     

Network activity and spike discharge oscillations in cortical slice cultures from neonatal rat

Network bursts and oscillations are forms of spontaneous activity in cortical circuits that have been described in vivo and in vitro. Searching for mechanisms involved in their generation, we investigated the collective network activity and spike discharge oscillations in cortical slice cultures of neonatal rats, combining multielectrode arrays with patch clamp recordings from individual neurons. The majority of these cultures showed spontaneous collective network activity [population bursts (PBs)] that could be described as neuronal avalanches. The largest of these PBs were followed by fast spike discharge oscillations in the beta to theta range, and sometimes additional repetitive PBs, together forming seizure-like episodes. During such episodes, all neurons showed sustained depolarization with increased spike rates. However, whereas regular-spiking (RS) and fast-spiking (FS) neurons fired during the PBs, only the FS neurons fired during the fast oscillations. Blockade of N-methyl-d-aspartate receptors reduced the depolarization and suppressed both the increased FS neuron firing and the oscillations. To investigate the generation of PBs, we studied the network responses to electrical stimulation. For most of the stimulation sites, the relationship between the stimulated inputs and the evoked PBs was linear. From a few stimulation sites, however, large PBs could be evoked with small inputs, indicating the activation of hub circuits. Taken together, our findings suggests that the oscillations originate from recurrent inhibition in local networks of depolarized inhibitory FS interneurons, whereas the PBs originate from recurrent excitation in networks of RS and FS neurons that is initiated in hub circuits.     

Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures

There is an increasing demand for in vitro test systems to detect neurotoxicity for use in chemical risk assessment. In this study, we evaluated the applicability of rat primary cortical cultures grown on multi-well micro-electrode arrays (mwMEAs) to detect effects of chronic 14-day exposure to structurally different insecticides or methylmercury on neuronal activity (mean spike rate; MSR).Effects of chronic exposure to α-cypermethrin, endosulfan, carbaryl, chlorpyrifos(-oxon), methylmercury or solvent control [14 days exposure, initiated after baseline recording at day in vitro (DIV)7] were studied in five successive recordings between DIV10 and DIV21. The results were compared to effects of acute exposure to these same compounds (activity recorded immediately after the start of exposure after baseline recording at DIV10-11).Chronic 14-day exposure to methylmercury, chlorpyrifos and α-cypermethrin inhibited MSR, all with a lowest-observed effect concentration (LOEC) of 0.1 μM, while exposure to endosulfan increased MSR [LOEC: 1 μM]. No significant effects were observed for chlorpyrifos-oxon and carbaryl. Similar to the observations in the chronic 14-day exposure studies, MSR was inhibited by acute 30-min exposure to methylmercury, chlorpyrifos, and α-cypermethrin [LOECs: 1 μM, 10 μM, and 1 μM, respectively], whereas endosulfan increased MSR [LOEC: 0.3 μM]. While not observed in the chronic 14-day exposure study, acute exposure to chlorpyrifos-oxon and carbaryl resulted in inhibition of MSR [LOECs: 10 μM, and 100 μM, respectively]. Effects on median interspike intervals (mISI; a measure for neuronal firing pattern) were not detected following chronic 14-day or acute 30-min exposure, except for increased mISI at acute chlorpyrifos and α-cypermethrin exposures at concentrations that also inhibited MSR.These data indicate that the effects of chronic 14-day exposures to methylmercury and insecticides at low concentrations on spontaneous neuronal activity in vitro can be predicted in rapid acute screening studies using mwMEAs.     

An activity-dependent switch from facilitation to inhibition in the control of excitotoxicity by group I metabotropic glutamate receptors

Activation of group I metabotropic glutamate receptors (mGlu1 or -5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5-dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a "neuroprotective mode". The characteristics of the activity-dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1-45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype-selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity-dependent switch in regulating excitotoxic neuronal death and, therefore, the recent "history" of these receptors is critical for the response to agonists or antagonists.