Daniel L. Small

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N -methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors,. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na + through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Gαi1) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gβγ blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Gαi1 with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

Biology of ischemic cerebral cell death.

With the approval of alteplase (tPA) therapy for stroke, it is likely that combination therapy with tPA to restore blood flow, and agents like glutamate receptor antagonists to halt or reverse the cascade of neuronal damage, will dominate the future of stroke care. The authors describe events and potential targets of therapeutic intervention that contribute to the excitotoxic cascade underlying cerebral ischemic cell death. The focal and global animal models of stroke are the basis for the identification of these events and therapeutic targets. The signalling pathways contributing to ischemic neuronal death are discussed based on their cellular localization. Cell surface signalling events include the activities of both voltage-gated K+, Na+, and Ca2+ channels and ligand-gated glutamate, gamma-aminobutyric acid and adenosine receptors and channels. Intracellular signalling events include alterations in cytosolic and subcellular Ca2+ dynamics, Ca2+ -dependent kinases and immediate early genes whereas intercellular mechanisms include free radical formation and the activation of the immune system. An understanding of the relative importance and temporal sequence of these processes may result in an effective stroke therapy targeting several points in the cascade. The overall goal is to reduce disability and enhance quality of life for stroke survivors.

Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons

Abstract: A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15–20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20–50 µM glutamate for 15 min was neurotoxic and induced a rapid (∼1–2 h) Ca2+‐dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to >800 µM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription‐polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2+ concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptor on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (<20 µM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 µM calphostin C, 5 µM chelerythrine, or chronic exposure to 100 nM PMA. A 15‐min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate‐mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate‐mediated neuronal death.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR11XX, NR10XX, and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT‐1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1–P21) and mature (P50), show distinct patterns of GLT‐1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl‐cyclic adenosine monophosphate. The transcripts for GLT‐1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1–P4 astrocyte cultures, declined in P10–P21, and was undetectable in P50 astrocytes. Uptake of 3H‐glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db‐cAMP (250–500 μM; 6 days), resulted in a pronounced stellation, up‐regulation of GLT‐1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db‐cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10‐fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

Identification of calcium channels involved in neuronal injury in rat hippocampal slices subjected to oxygen and glucose deprivation.

The presynaptic Ca2+-influx affecting glutamate release during neuropathological processes is mediated via voltage-sensitive calcium channels (VSCCs). There is controversy, however, over the fractional contribution of the specific channel types involved. We have addressed this by investigating the protective effects of various VSCC blockers on oxygen and glucose-deprived rat hippocampal slices. The viability of treated and non-treated slices was assayed electrophysiologically by measuring the evoked population spike (PS) amplitude in the stratum pyramidale of the CA1 region and by imaging slices loaded with fluorochrome dyes specific for dead (ethidium homodimer) and live (calcein) cells using confocal microscopy. PS amplitudes were significantly (P < 0.01) depressed from 4.4 +/- 0.2 mV (n = 38) to 0.2 +/- 0.1 mV (n = 40) after the deprivation insult. Responses from deprived slices treated with omega-conotoxin MVIIC (100 nM; 4.2 +/- 0.5 mV; n = 20) were not significantly different from control, non-deprived slice responses. In contrast, deprived slices treated with either L-type (0.1 or 1 microM nimodipine) or N-type (0.1 or 3 microM omega-conotoxin MVIIA) blockers showed no significant protection. The viability of CA1 neurons as revealed by the fluorescence live/dead confocal viability assay was consistent with the electrophysiological measurements. By comparison with previous studies using P- and Q-type blockers to attempt neuroprotection against the same deprivation insult, the rank order in which specific Ca2+-channel types contribute to neuronal death due to oxygen and glucose deprivation was determined to be Q > N >> P > L.

NMDA antagonists: their role in neuroprotection.

Publisher Summary A wave of clinical trials testing the efficacy of N -methyl-D-aspartate (NMDA) antagonists in the treatment of cerebral ischemia was launched in the 1990s. Cis -4-(phosphonomethyl)-2-piperidine carboxylic acid (CGS 19755) had impressive preclinical data demonstrating dramatic cytoprotection in gerbil models of severe transient forebrain ischemia. It was efficacious when administered post-ischemically, and it did not produce psychotomimetic effects in monkeys at doses that were neuroprotective. The failure of CGS 19755 in clinical trials might have been predicted had more careful attention been paid to monitoring and maintaining control over physiological variables, such as temperature in the preclinical animal studies. This chapter critically assesses the utility of NMDA antagonists in the treatment of cerebral ischemia. The chapter presents concepts of a modified excitotoxicity with a description of the NMDA receptor physiology and pharmacology as it pertains to excitotoxicity. The chapter reviews published data for in vitro and in vivo models of ischemia using NMDA antagonists.

Brain derived neurotrophic factor induction of N-methyl-D-aspartate receptor subunit NR2A expression in cultured rat cortical neurons

N-methyl-D-aspartate (NMDA) receptor subunit expression changes during development and following injury in several brain regions. These changes may be mediated by neurotrophic factors, such as brain derived neurotrophic factor (BDNF). Exposure of cultured cortical neurons to BDNF (100 ng/ml) for 24 h produced a significant decrease in the NMDA-induced whole-cell currents sensitive to the NR2B subunit selective NMDA receptor antagonist, CP-101,606, suggesting a relative decrease in NR2B subunit expression. There was a significant increase in NR2A by Western blot analysis. Consistent with the electrophysiology and Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) amplification revealed that BDNF caused a significant increase in relative NR2A subunit expression, a significant decrease in relative NR2B subunit expression and no change in relative NR2C subunit expression. These results suggest that BDNF enhances NMDA receptor maturation, warranting further study of the mechanism of BDNF effects on NMDA receptor subunit expression and the role these effects play in development and neuronal injury.

A fluorescence confocal assay to assess neuronal viability in brain slices.

Hippocampal slice models are used to study the mechanisms of ischemia-induced neurotoxicity and to assess the neuroprotective potential of novel therapeutic agents. A number of morphological and functional endpoints are available to assess neuronal viability. The slice model also allows the study of selectively vulnerable neuronal populations within the same preparation. The fluorescence procedure described here provides a method of assessing the viability of neurons in rat hippocampal slices exposed to hypoxic-hypoglycemic conditions. Control and/or treated slices that had been subjected to a 10 min oxygen-glucose deprivation insult are double stained with calcein-AM (4 microM), which stains live cells green, and ethidium homodimer (6 microM), which stains the nucleus of dead cells red. The stained slices are then imaged using confocal microscopy. Vulnerable neurons in the CA1 region of slices deprived of oxygen and glucose became increasingly permeant to ethidium homodimer over the 4 h reperfusion period. Exposure to low Ca2+ concentration (0.3 mM) or the N-, P- and Q-type Ca2+ channel antagonist MVIIC (100 nM), which have been shown to be neuroprotective in this model of ischemia using field evoked post-synaptic potential (EPSP) measures as an endpoint, were also shown to be protective using the fluorescence assay.

Neuroprotective effects of omega-Aga-IVA against in vitro ischaemia in the rat hippocampal slice.

Excessive accumulation of Ca2+ in neurones and glutamate release are involved in neuropathological processes, including ischaemia. We investigated the neuroprotective effects of the Ca2+ channel antagonist, omega-Aga-IVA, in CA1 pyramidal neurones in rat hippocampal slices following an in vitro hypoxic-hypoglycaemic insult. Following this insult, evoked post-synaptic response amplitudes decreased from 3.7 +/- 0.5 mV to 0.6 +/- 0.2 mV and the CA1 neurones appeared dead using a live/dead fluorescence assay with confocal microscopy. Slices treated with 200 nM omega-Aga-IVA had evoked response amplitudes not significantly different from control (3.3 +/- 0.5 mV) and the CA1 neurones appeared viable using the live/dead fluorescence assay. The neuroprotective efficacy of omega-Aga-IVA suggests that omega-Aga-IVA-sensitive Ca2+ channels participate in ischaemic neuronal death and constitute a potential target of therapeutic intervention.