Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.     

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons

Dendritic Ca²⁺ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca²⁺ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca²⁺ signals by measuring glutamate-induced Ca²⁺ increases along the dendrites of acutely isolated midbrain dopamine neurons.Maximal doses of glutamate induced a [Ca²⁺]_c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca²⁺]_c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca²⁺ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca²⁺]_c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate.These results indicate that functional organization of Ca²⁺ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca²⁺ rises in dendrites respond differently to applied glutamate concentration.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Analysis of glutamate receptors in primary cultured neurons from fetal rat forebrain

In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors was studied in a model of neurons in primary culture by measuring the specific binding of L-[³H]glutamate under various incubation conditions in 8-day-old intact living neurons isolated from the embryonic rat forebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness to glutamate was assessed by studying the uptake of tetraphenylphosphonium (TPP⁺) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na⁺, Ca²⁺, and Cl^–, with a Kd around 2 M. In the absence of the above ions, [³H]glutamate specific binding diminished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both of the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were significantly lower in culture membranes than in intact cells or newborn membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [³H]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisqualate, AMPA, kainate andtrans-ACPD were equally effective. According to these data and to the ionic dependence of glutamate binding, it was concluded that cultured neurons from the rat embryo forebrain express various glutamate receptor subtypes, mainly L-AP4 and NMDA receptors, with characteristics close to those in the newborn brain, and which display functional properties since a transient cell exposure to glutamate led to a 70% inhibition of [³H]TPP⁺ uptake.     

Modulation of NMDA Receptor Subunits Expression by Concanavalin A

The processes of N-methyl-d-aspartate (NMDA) receptor subunits expression were examined in cortical neurons and rat brain in order to investigate how the concanavalin A (Con A) modulates neuronal cells. Con A modulated the expression of NMDA receptor subunits in cultured cortical cells. Con A augmented the level of intracellular Ca²⁺ by α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA). We determined whether activation of AMPA receptors was involved in the regulation of NMDA receptor expression with Con A by blocking the desensitization of AMPA receptors. The results showed that AMPA receptor antagonists suppressed NMDA receptor subunits expression in Con A-treated cortical neuronal cells. PMA elevated the expression of NMDA receptor subunits, while PKC inhibitor and tyrosine kinases inhibitor suppressed the expression of NMDA receptor subunits. Furthermore, it was shown that NMDA receptor subunits expression was modulated in a region-specific manner after the sustained microinfusion of Con A into the cerebroventricle of the rat brain. Collectively, it could be presumed that the AMPA receptor activation was involved in Con A-induced modulation of NMDA receptor subunits expression.     

Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents inHelix aspersa neurons

The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca²⁺ currents (L-type ICa²⁺) and their Ca²⁺-dependent inactivation, were studied in identifiedHelix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca²⁺]_i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa²⁺ (IC₅₀=28 μM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca²⁺-dependent inactivation of L-type ICa²⁺. Trifluoperazine sulfoxide (50 μM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa²⁺. TFP (20 μM) increased basal [Ca²⁺]_i from 147±37 nM to 650±40nM (N=7). The increase in [Ca²⁺]_i was prevented by removal of external Ca²⁺ and curtailed by depletion of caffeine-sensitive intracellular Ca²⁺ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-O-tetradecanoyl-phorbol-13-acetate) on L-type Ca²⁺ currents. This compound produced an increase in L-type ICa²⁺ without enhancing Ca²⁺-dependent inactivation. The results show that 1) TFP reduces L-type ICa²⁺ while enhancing the efficacy of Ca²⁺-dependent inactivation. 2) TFP produces an increase in basal [Ca²⁺]_i which may contribute to the enhancement of Ca²⁺-dependent inactivation. 3) PKC up-regulates L-type ICa²⁺ without altering the efficacy of Ca²⁺ dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca²⁺ indicator dye Fura-2 was used to measure Ca²⁺ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca²⁺ responses were evoked by kainic acid (KA), α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D -aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca²⁺]_i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca²⁺ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca²⁺]_i were carried by Ca²⁺-permeable receptor channels. Significantly smaller changes in [Ca²⁺]_i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca²⁺ free-EGTA–buffered media, including l -cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclopentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca²⁺]_i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca²⁺]_i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca²⁺ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca²⁺]_i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca²⁺]_i to increase in NM neurons. Presumably, each route represents a means by which Ca²⁺ can alter cellular processes. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 321–337, 1998     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

Activation of Excitatory Amino Acid Receptors in the Rat Hippocampal Slice Increases Intracellular Cl− and Cell Volume

Abstract: The effects of glutamatergic excitotoxins on intracellular Cl^? were investigated in the CA1 pyramidal cell layer of the hippocampal slice. Hippocampal slices from rats (14–19 days old) were loaded with 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl^?-sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracellular [Cl^?]. Slices were exposed for at least 10 min at 26–28°C to N-methyl-d -aspartate (NMDA; 100 µM) or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 50 µM). A UV laser scanning confocal microscope was used to measure changes in MEQ fluorescence within area CA1 pyramidal cell soma. Both glutamate receptor agonists produced a rapid decrease in MEQ fluorescence that persisted after washout following a 10-min exposure. The effects of NMDA and AMPA were prevented by the competitive antagonists 2-amino-5-phosphonopentanoic acid and 6,7-dinitroquinoxaline-2,3-dione, respectively. Neither tetrodotoxin nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA and AMPA on MEQ fluorescence were dependent on the levels of extracellular Cl^?, but only NMDA responses were dependent on the levels of extracellular Na⁺. Removal of Ca²⁺ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca²⁺ ionophore ionomycin nor the L-type voltage-gated Ca²⁺ channel agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell (somal) volume were also assessed with the fluorescent probe calcein acetoxymethyl ester. Both NMDA and AMPA decreased calcein fluorescence (indicating an increased cell volume), but this was preceded by the decrease in MEQ fluorescence (equivalent to an intracellular accumulation of ～20 mM Cl^?). Thus, excitotoxins may cause Cl^? influx via an anion channel other than the GABA_A receptor and/or reduce Cl^? efflux mechanisms to produce cell swelling. Such anionic shifts may promote neuronal excitability and cell death following an excitotoxic insult to the hippocampal slice.     

Prostaglandin E2 stimulates glutamate receptor‐dependent astrocyte neuromodulation in cultured hippocampal cells

Recent Ca²⁺ imaging studies in cell culture and in situ have shown that Ca²⁺ elevations in astrocytes stimulate glutamate release and increase neuronal Ca²⁺ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E₂ (PGE₂). We investigated the electrophysiological consequences of the PGE₂‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE₂ to astrocytes evoked a Ca²⁺ elevation in the stimulated cell by mobilizing internal Ca²⁺ stores, which further propagated as a Ca²⁺ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE₂ evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca²⁺ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE₂‐evoked Ca²⁺ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999     

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Metabotropic GABA_B receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA_B receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA_B receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca²⁺ channels, and removal of extracellular Ca²⁺ prevented glutamate-induced down-regulation of GABA_B receptors, indicating that Ca²⁺ influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA_B receptors. Glutamate did not affect the rate of GABA_B receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA_B receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA_B receptors by sorting endocytosed GABA_B receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA_B receptor-mediated inhibition resulting in increased synaptic excitability.     

Quinolinate-induced Rat Striatal Excitotoxicity Impairs Endoplasmic Reticulum Ca2+-ATPase Function

Excessive activation of NMDA glutamate receptors and the resulting loss of intracellular Ca²⁺ homeostasis may be lethal (excitotoxic) to neurons. Such excitotoxicity can be induced in vivo by intrastriatal infusion of quinolinate, as this substance selectively activates NMDA receptors. The aim of the present research was to investigate whether the in vivo treatment of striatal tissue with quinolinate would lead to an early impairment of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity or mitochondrial Ca²⁺ sequestration, two intracellular mechanisms involved in Ca²⁺ homeostasis and signaling. Sodium quinolinate was infused intrastriatally into adult rats, and 6 h later the brains were removed and the corpora striata dissected. At this time point, striatal sections stained with Fluoro-Jade, a cellular marker of cell death, showed initial signs of neuronal degeneration. In addition, SERCA activity decreased 39% in relation to the activity observed in the control striata. A corresponding decrease of the same magnitude in ⁴⁵Ca²⁺ uptake by striatal microsomes was also found in the treated striata. Western blot analysis did not indicate any decrease in SERCA levels in striatal tissue after quinolinate infusion. Mitochondrial Ca²⁺ sequestration was still preserved in quinolinate-treated striatal tissue when the assay was carried out in the presence of physiological concentrations of ATP and Mg²⁺. These results suggest that impairment of the SERCA function may be an early event in excitotoxicity.     

Involvement of NMDA Receptor in the Modulation of Excitatory and Inhibitory Amino Acid Neurotransmitters Release in Cortical Neurons

The purpose of this paper was to examine the function of N-methyl-D-aspartate (NMDA) glutamate receptor in cortical neurons on amino acid neurotransmitters release as well as the fraction of neurons implicated in the response of this receptor. Local stimulation of these cells at different concentrations of NMDA, agonist of this ionotropic glutamate receptor, produced a dose dependent release of aspartate, glutamate, glycine and GABA. These effects were blocked by DAP5, an antagonist of the NMDA receptor. The amino acid Ca²⁺ dependent release mediated by the NMDA receptor, is induced by the opening of voltage-dependent Ca²⁺ channels that this receptor promotes. Ca⁺⁺ movements were explored in single cells loaded with fura-2. When single cells were stimulated with 100 μM NMDA, the calcium recording performed showed that 82% of the cells responded to this agonist increasing the intracellular calcium concentration, although the amplitude of these increments was variable. The results suggest that NMDA-elicited neurotransmitter release from cortical neurons involves Ca²⁺-dependent and Ca²⁺-independent components, as well as neuron depolarisation, and different VDCC subtypes of N, P/Q or L depending of the amino acid neurotransmitter release elicited by this receptor.