Mixed Neuronal Cultures Research Articles

Synaptic connections in a dissociated mixed culture of rat dorsal root ganglion and spinal cord neurons

Postsynaptic currents and action potentials recorded from neurons in a mixed culture of rat dorsal root ganglion and spinal cord cells are described. The existence of mutual synaptic connections between the above two types of neurons is demonstrated.

Identity of SMCT1 (SLC5A8) as a neuron‐specific Na+‐coupled transporter for active uptake ofl‐lactate and ketone bodies in the brain

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Matrices with Compliance Comparable to that of Brain Tissue Select Neuronal over Glial Growth in Mixed Cortical Cultures

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.

158. Real-Time Visualization of Neuronal Differentiation and Neurite Projection Using an Indicator HSV Amplicon Vector

Top of pageAbstract Stem cell-based replacement therapy has great potential in treating neurological disorders, including brain and spinal cord injuries. Although several studies have demonstrated neuronal and glial differentiation of transplanted neural stem cells (NSCs) in vivo, the efficacy of the transplanted NSCs to generate neurons and of the newly generated neurons to form new synaptic connections with the remaining host neurons still needs clarification. To address these issues, we have constructed an HSV amplicon-based indicator vector that allows visualization of neuronal differentiation and neurite projection. Synapsin I (SYN) is a well characterized gene that expresses selectively in neurons. Thiel and colleagues found that a 400-bp 5' untranslated promoter region of SYN directed prominent expression of a reporter gene in various neuronal cell lines, but that no expression was detected in cell lines derived from non-neuronal tissue (Thiel G et al., PNAS 1991). On the other hand, microtubule- associated protein tau-reporter fusion protein has been used as a genetic reporter to visualize neuronal processes in vivo (Callahan CA et al., PNAS 1994). We, therefore, combined the two genetic elements in the context of HSV amplicon vectors, producing an indicator herpes simplex virus (HSV) amplicon vector, HSV-SYN-tauR, that carries tau fused with a monomeric red fluorescent protein (mRFP, Campbell et al., PNAS 2002) under the control of the 400- bp SYN promoter. When a mixed culture of mouse cortical neurons and glia was transduced with the HSV-SYN-tauR amplicon vector, RFP reporter was expressed only in neurons along their neurites, allowing visualization of every axon, dendrite, and spine. Time-lapse imaging of mixed cortical cultures transduced with the HSV-SYN-tauR amplicon vector allowed noninvasive real-time monitoring of extension and retraction of the neurites of live neurons. This indicator HSV amplicon vector could prove useful in studying neuronal differentiation, neurite projections, and regeneration of neuronal networks in vitro and in vivo.

Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l-arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [3H]-thymidine incorporation after drug treatment. Incubation for 24h with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [3H]-thymidine incorporation in a dose-dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8-bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S-nitrosylation mechanism. SNAP blocked the increase of [3H]-thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [3H]-Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [3H]-thymidine incorporation in retinas from 9-day-old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or “in vivo” through cyclic GMP and ERK-independent pathways.

Neuroprotective activity of glucocorticoids against NMDA toxicity in mouse cortical neuron-astrocyte cultures: Potential role of the tPA-PAI-1 axis

Given the potent anti-inflammatory effects exerted by glucocorticoids under various pathological situations, their use has been proposed to protect the brain against cerebral ischemia, neuro-inflammatory diseases and trauma. However, the efficiency of these treatments remains a matter of debate, since both neuroprotective and neurotoxic effects have been observed depending on the experimental models used (Abraham et al., J Neuroendocrinol., 13: 749, 2001). First we observed that purified neuron and astrocyte populations both express the mRNA encoding for the glucocorticoid receptor (as determined by RT PCR), suggesting that both cell type might respond to glucocorticoid. In order to further understand the role of glucocorticoids in brain tissues, we have tested the effect of dexamethasone (a potent glucocorticoid) in a model of mixed murine cultures of neurons and glia subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity. We observed that pre-treatment of the cultures with dexamethasone significantly reduced NMDA-induced neuronal necrosis. We also described that dexamethasone (dose and time dependently) induced within 12–24 hours of treatment a significant increase in both the synthesis and secretion of type 1 plasminogen activator inhibitor (PAI-1) in astrocyte cultures. Previous experiments in the laboratory have shown in the same paradigm of NMDA-induced toxicity that tPA markedly increases the deleterious action of NMDA and that this potentiating action of tPA is abolished by PAI-1 (Buisson et al., FASEB J, 12: 1683, 1998). Since dexamethasone led to a significant decrease in t-PA activity (as assessed by zymography assay), our results suggest that glucocorticoids might exert a neuroprotective activity against NMDAR-mediated toxicity by interacting with the t-PA/PAI-1 axis.

Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical‐scavenging activity

Minocycline is neuroprotective in animal models of a number of acute CNS injuries and neurodegenerative diseases. While anti-inflammatory and anti-apoptotic effects of minocycline have been characterized, the molecular basis for the neuroprotective effects of minocycline remains unclear. We report here that minocycline and a number of antioxidant compounds protect mixed neuronal cultures in an oxidative stress assay. To evaluate the role of minocycline's direct antioxidant properties in neuroprotection, we determined potencies for minocycline, other tetracycline antibiotics, and reference antioxidant compounds using a panel of in vitro radical scavenging assays. Data from in vitro rat brain homogenate lipid peroxidation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays show that minocycline, in contrast to tetracycline, is an effective antioxidant with radical scavenging potency similar to vitamin E. Our findings suggest that the direct antioxidant activity of minocycline may contribute to its neuroprotective effects in some cell-based assays and animal models of neuronal injury.

Adeno‐associated virus‐mediated gene transfer of human aromatic l‐amino acid decarboxylase protects mixed striatal primary cultures from l‐DOPA toxicity

Although L-DOPA is the drug of choice for Parkinson's disease, prolonged L-DOPA therapy results in decreased drug effectiveness and the appearance of motor complications. This may be due in part to the progressive loss of the enzyme, aromatic L-amino acid decarboxylase (AADC). We have developed an adeno-associated virus vector (AAV-hAADC) that contains human AADC cDNA under the control of the cytomegalovirus promoter. Infusion of this vector into the striatum of parkinsonian rats and monkeys improves L-DOPA responsiveness by improving AADC-mediated conversion of L-DOPA to dopamine. This is now the basis of a proposed therapy for advanced Parkinson's disease. A key concern has been that over-production of dopamine in striatal neurons could cause dopamine toxicity. To investigate this possibility in a controlled system, mixed striatal primary rat neuronal cultures were prepared. Exposure of cultures to high concentrations of L-DOPA induced the following changes: cell death in nigral and striatal neurons, aggregation of neurofilaments and focal axonal swellings, abnormal expression of DARPP-32, and activation of astroglia and microglial cells. Transduction of cultures with AAV-hAADC resulted in efficient and sustained neuronal expression of the AADC protein and prevented all the L-DOPA-induced toxicities. The protective effects were due primarily to AADC-dependent conversion of L-DOPA to dopamine and an increase in induction of vesicular monoamine transporter resulting in dopamine storage in cultured cells. These results suggest a neuroprotective role for AADC gene transfer against L-DOPA toxicity.

Astrocytes exert a pro-apoptotic effect on neurons in postnatal hippocampal cultures

We investigated conditions that promote basal and activity-dependent neuronal apoptosis in postnatal rat hippocampal cultures. Low-density mixed cultures of astrocytes and neurons exhibited lower sensitivity than high-density cultures to basal neuronal death and activity-sensitive neuronal death, induced with glutamate receptor blockers, sodium channel blockers, or calcium channel blockers. Although elevations of [Ca 2+] i protect neurons from apoptosis, low-density microcultures and mass cultures exhibited only minor differences in resting [Ca 2+] i and Ca 2+ current density, suggesting that these variables are unlikely to explain differences in susceptibility. Astrocytes, rather than neurons, were implicated in the neuronal loss. Several candidate molecules implicated in other astrocyte-dependent neurotoxicity models were excluded, but heat inactivation experiments suggested that a heat-labile factor is critically involved. In sum, our results suggest the surprising result that astrocytes can be negative modulators of neuronal survival during development and when the immature nervous system is challenged with drugs that dampen electrical excitability.

The impact of excitotoxic blockade on the evolution of injury following combined mechanical and hypoxic insults in primary rat neuronal culture

Traumatic brain injury (TBI) involves alterations in neuronal physiology, often complicated by secondary hypoxic or hypotensive events. Excitotoxicity is an important process induced in both TBI and hypoxic or ischemic insults to the brain. We investigated two hypotheses: (1) excitotoxicity is more prominent following combined mechanical and hypoxic injury than either alone; (2) both AMPA and NMDA receptor activation mediate combined mechanical and hypoxic injury. Media in primary mixed neuronal cultures were replaced with conditioned media containing MK801 (NMDA antagonist) and/or NBQX (AMPA/kainate antagonist). Cultures were then subjected to mechanical injury. Afterward, media were exchanged for hypoxic media containing the antagonist, and plates were placed in hypoxia chambers for 7 h. At 24 h following hypoxia, LDH release, trypan blue uptake, and morphologic changes were assessed. Blockade had no effect after mechanical injury. After hypoxia, MK801 and combined MK801/NBQX decreased LDH and trypan blue to control levels. NBQX alone after hypoxia had less impact. After combined mechanical injury and hypoxia, both MK801 and NBQX partially reduced LDH and trypan blue. Combining the antagonists led to reduction to control values for both endpoints. We conclude that excitotoxic processes are more prominent after combined than isolated injuries in neurons and that increased cell death is mediated by both NMDA and AMPA receptor activation following combined injuries.

Prenatal infection and risk for schizophrenia: IL-1beta, IL-6, and TNFalpha inhibit cortical neuron dendrite development.

Prenatal exposure to infection increases risk for schizophrenia, and we have hypothesized that inflammatory cytokines, generated in response to maternal infection, alter neuron development and increase risk for schizophrenia. We sought to study the effect of cytokines generated in response to infection-interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha), and interleukin-6 (IL-6)-on the dendritic development of cortical neurons. Primary mixed neuronal cultures were obtained from E18 rats and exposed to 0, 100, or 1000 units (U)/ml of IL-1beta, TNFalpha, IL-6, or IL-1beta+TNFalpha for 44 h. MAP-2-positive neurons were randomly identified for each condition and the number of primary dendrites, nodes, and total dendrite length was determined. We found that 100 U of TNFalpha significantly reduced the number of nodes (27%, p=0.02) and total dendritic length (14%, p=0.04), but did not affect overall neuron survival. A measure of 100 U IL-1beta+TNFalpha significantly reduced the number of primary dendrites (17%, p=0.006), nodes (32%, p=0.001), and total dendritic length (30%, p<0.0001), although it did not affect overall neuron survival. At 1000 U, each cytokine significantly reduced the number of primary dendrites (14-24%), nodes (28-37%), as well as total dendritic length (25-30%); neuron survival was reduced by 14-21%. These results indicate that inflammatory cytokines can significantly reduce dendrite development and complexity of developing cortical neurons, consistent with the neuropathology of schizophrenia. These findings also support the hypothesis that cytokines play a key mechanistic role in the link between prenatal exposure to infection and risk for schizophrenia.

(−)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection

Group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) modulate neurotoxicity of excitatory amino acids and beta-amyloid-peptide ( βAP), as well as epileptic convulsions, most likely via presynaptic inhibition of glutamatergic neurotransmission. Due to the lack of subtype-selective ligands for group-III receptors, we previously utilized knock-out mice to identify mGluR4 as the primary receptor mediating neuroprotection of unselective group-III agonists such as L-AP 4 or (+)-PPG, whereas mGluR7 is critical for anticonvulsive effects. In a recent effort to find group-III subtype-selective drugs we identified (+/−)-PHCCC as a positive allosteric modulator for mGluR4. This compound increases agonist potency and markedly enhances maximum efficacy and, at higher concentrations, directly activates mGluR4 with low efficacy. All the activity of (+/−)-PHCCC resides in the (−)-enantiomer, which is inactive at mGluR2, -3, -5a, -6, -7b and -8a, but shows partial antagonist activity at mGluR1b (30% maximum antagonist efficacy). Chimeric receptor studies showed that the binding site of (−)-PHCCC is localized in the transmembrane region. Finally, (−)-PHCCC showed neuroprotection against βAP- and NMDA-toxicity in mixed cultures of mouse cortical neurons. This neuroprotection was additive to that induced by the highly efficacious mGluR1 antagonist CPCCOEt and was blocked by MSOP, a group-III mGluR antagonist. Our data provide evidence for a novel pharmacological site on mGluR4, which may be used as a target-site for therapeutics.

N-acetylaspartylglutamate and β-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. β-NAAG is a competitive inhibitor of the NAAG hydrolyzing enzyme N-acetylated α-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 β-NAAG against NMDA- and hypoxia-induced injury to spinal cord mixed neuronal and glial cell cultures. NAAG (500–1000 μM) protected against NMDA- or hypoxia-induced injuries to spinal cord cultures, and the nonhydrolyzable analog β-NAAG (250–1000 μM) 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 β-NAAG was partially neuroprotective against both insults. At 2 mM, NAAG and β-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 β-NAAG protect against excitotoxic and hypoxic injury to spinal cord neurons, and do so predominantly by interactions with NMDA and not mGluR receptors.

NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons

NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes ( approximately 10%) and neurons ( approximately 90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult.

MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosis.

Acquired immunodeficiency syndrome (AIDS)-associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV-1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA-induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co-treatment with MCP-1 or RANTES but not IP-10. The protective effect of RANTES was blocked by antibodies to MCP-1, indicating that RANTES protection is mediated by the induction of MCP-1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co-treatment with MCP-1 inhibited tat and NMDA-induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP-1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Effects of glucocorticoids in the gp120-induced inhibition of glutamate uptake in hippocampal cultures

Studies examining the development of AIDS Related Dementia have concentrated on neurotoxic properties of the HIV viral coat protein, gp120. We have previously shown that this neurotoxicity can be exacerbated by glucocorticoids (GCs), the stress hormones secreted by the adrenal. Moreover, GCs also worsen several of the mechanisms mediating gp120 neurotoxicity, such as increased calcium flux, ROS generation, and energy depletion. Gp120 interferes with the reuptake of glutamate in glia cultures, another possible mechanism by which it can be neurotoxic. This paper examines the role of GCs in exacerbating this phenomenon. It was found that while GCs do not exacerbate the decrease in reuptake of glutamate in glia cultures, they do enhance the decrease in mixed neuronal cultures and this latter effect appears to be energy-dependent.

Transforming growth factor alpha-induced expression of type 1 plasminogen activator inhibitor in astrocytes rescues neurons from excitotoxicity.

Although transforming growth factor (TGF)-alpha, a member of the epidermal growth factor (EGF) family, has been shown to protect neurons against excitotoxic and ischemic brain injuries, its mechanism of action remains unknown. In the present study, we used in vitro models of apoptotic or necrotic paradigms demonstrating that TGF-alpha rescues neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxic death, with the obligatory presence of astrocytes. Because neuronal tissue-type plasminogen activator (t-PA) release was shown to potentiate NMDA-induced excitotoxicity, we observed that TGF-alpha treatment reduced NMDA-induced increase of t-PA activity in mixed cultures of neurons and astrocytes. In addition, we showed that although TGF-alpha induces activation of the extracellular signal-regulated kinases (ERKs) in astrocytes, it failed to activate p42/p44 in neurons. Finally, we showed that TGF-alpha, by an ERK-dependent mechanism, stimulates the astrocytic expression of PAI-1, a t-PA inhibitor, which mediates the neuroprotective activity of TGF-alpha against NMDA-mediated excitotoxic neuronal death. Taken together, we indicate that TGF-alpha rescues neurons from NMDA-induced excitotoxicity in mixed cultures through inhibition of t-PA activity, involving PAI-1 overexpression by an ERK-dependent pathway in astrocytes.

Neuroprotection of cultured cortical neurons mediated by the cyclooxygenase‐2 inhibitor APHS can be reversed by a prostanoid

The neuroprotective properties of two cyclooxygenase-2 (COX-2) specific inhibitors, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398) and o-(acetoxy-phenyl)hept-2-ynyl2 sulfide (APHS) were examined in vitro using a mixed cortical neuronal culture system. Each of these inhibitors conferred a concentration-dependent neuroprotective effect against an excitotoxic assault induced by NMDA. Neuroprotection was observed when the COX-2 inhibitor was added before or even 1-3 hours after NMDA, which was coincident with an NMDA-induced increase of COX-2 transcripts in neurons. To test whether these COX-2 inhibitors confer neuroprotection by inhibiting biosynthesis of prostanoids that may contribute toward excitotoxicity, two NMDA-induced prostanoids, PGE(2) and PGF(2alpha), were tested for their ability to reverse the neuroprotective properties of APHS. APHS-mediated neuroprotection was overcome by the concentration-dependent (as low as 100 nM) administration of a synthetic analog of PGE2, 17-phenyl-trinor-PGE(2) (17-pt-E(2)), which is a relatively specific agonist for the EP1 and EP3 prostaglandin receptors; however, PGF(2alpha) had no significant effect on neuroprotection conferred by APHS. In the absence of APHS, neuroprotection was observed with either prostanoid. PGE(2) may in some instances contribute toward excitotoxicity, and the inhibition of synthesis of this prostanoid may in part explain the neuroprotective properties of these COX-2 inhibitors.

Depolarization-Induced 65Zinc Influx into Cultured Cortical Neurons

Toxic Zn2+ influx may be a key mechanism underlying selective neuronal death after transient global ischemia in rats. To identify routes responsible for neuronal Zn2+ influx, we measured the accumulation of 65Zn2+ into cultured murine cortical cells under depolarizing conditions (60 mM K+) associated with severe hypoxia-ischemia in brain tissue. Addition of 60 mM K+ or 300 μM kainate substantially increased 65Zn2+ accumulation into mixed cultures of neurons and glia, but not glia alone. 65Zn2+ accumulation was attenuated by increasing concentrations of extracellular Ca2+ or trypsin pretreatment, but not by late trypsinization, and corresponded to an increase in atomic Zn2+. Confirming predominantly neuronal entry, K+-induced 65Zn2+ accumulation was reduced by prior selective destruction of neurons with NMDA. K+-induced 65Zn2+ influx was not sensitive to glutamate receptor antagonists, but was attenuated by Gd3+ and Cd2+ as well as 1 μM nimodipine; it was partially sensitive to 1 μM ω-conotoxin-GVIA, and insensitive to 1 μM ω-agatoxin-IVA. K+-induced, Gd3+-sensitive 45Ca2+ accumulation but not 65Zn2+ accumulation was sharply attenuated by lowering extracellular pH to 6.6.