Nanomolar concentrations of lead inhibit glutamatergic and GABAergic transmission in hippocampal neurons

To investigate whether lead (Pb2+) affects the tetrodotoxin (TTX)-sensitive release of neurotransmitters, the whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons. Pb2+ (>/=10 nM) reversibly blocked the TTX-sensitive release of glutamate and gamma-aminobutyric acid (GABA), as evidenced by the reduction of the amplitude and frequency of glutamate- and GABA-mediated postsynaptic currents (PSCs) evoked by spontaneous neuronal firing. This effect of Pb2+, which occurred 2-3 s after exposure of the neurons to Pb2+-containing external solution, was not related to changes in Na+-channel activity, and was quantified by measurements of changes in the amplitude of PSCs evoked when a 50-micros, 5-V stimulus was applied via a bipolar electrode to a neuron synaptically connected to the neuron under study. With an IC50 of approximately 68 nM, Pb2+ blocked the evoked release of glutamate and GABA. This effect was most likely mediated by Pb2+'s actions on extracellular targets, because there was a very short delay (<3 s) for its onset, and it could be completely reversed by the chelator ethylene diaminetetraacetic acid (EDTA). Given that Pb2+-induced blockade of evoked transmitter release could be reversed by 4-aminopyridine, it is suggested that the effect on release was mediated via the binding of Pb2+ to voltage-gated Ca2+ channels. Thus, it is most likely that the neurotoxic effects of Pb2+ in the mammalian brain involve a decrease of the TTX-sensitive, Ca2+-dependent release of neurotransmitters.     

The effect of 4-aminopyridine on acetylcholine release

Summary The effect of 2-, and 4-aminopyridine (4-APYR) on the release mechanism of acetylcholine (ACh) from the nerve terminals of the Auerbach plexus-longitudinal muscle preparation of the guinea-pig ileum, suspended in eserinized Krebs' solution, was investigated.2- and 4-APYR increased the release of ACh from the nerve terminals at rest and at both low and high frequency stimulation. The enhanced ACh release was found to be due to increased volley output. At lower frequency of stimulation, the potentiation of ACh release was much higher than at higher rate of stimulation.4-APYR was able to increase ACh release in the absence of [Ca²⁺]₀. However, when a Ca-chelating agent, EDTA, was also added to the Ca-free Krebs' solution, 4-APYR was entirely ineffective. The depression of ACh release induced by Mg-excess was completely antagonized by 4-APYR. Tetrodotoxin (TTX) prevented augmentation of ACh release by 4-APYR.It is suggested that 4-APYR lowers the demand of nerve terminals for [Ca²⁺]₀ required for the excitation-secretion coupling process. The presence of a low concentration [Ca²⁺]₀, however, is essential for the action of 4-APYR.     

Asphyxia induced by umbilical cord occlusion alters glutamatergic and GABAergic synaptic transmission in neurons of the superior colliculus in fetal rats

Using optical recordings, we studied the effects of asphyxia on intracellular Cl⁻ and Ca²⁺ concentrations ([Cl⁻]i; [Ca²⁺]i) in the superior colliculus of fetal rats, which were connected via the umbilical cord to the dam. Acute asphyxia was induced by umbilical cord occlusion. The number of fetal superior colliculus neurons showing GABA-mediated increases in [Cl⁻]i (leading to hyperpolarization) following local synaptic electrical stimulation had decreased by 3 h post-asphyxiation, while the number showing GABA-mediated decreases in [Cl⁻]i (leading to depolarization) increased. [Ca²⁺]i rise, which occurred after acute asphyxiation, was antagonized by both non-NMDA and NMDA receptor antagonists. The increase in [Ca²⁺]i following focal superior colliculus stimulation was markedly attenuated at 3 h post-asphyxiation.     

Comparison of spontaneous and evoked epileptiform activity in three in vitro epilepsy models

The distribution of nestin immunoreactivity was studied in the whole normal adult human forebrains using new anti-human nestin mouse monoclonal and rabbit polyclonal antiserum. The nestin immunoreactive cells could be divided into three types according to their morphological characteristics. The first type contained neuron-like nestin immunoreactive cells, distributed in CA1-3 of hippocampus, septum, the nucleus of diagonal band, amygdala and basal nucleus of Meynert. The second type contained astrocyte-like cells, distributed in the subependymal zone and subgranular layer of dentate gyrus. The third type of cells had smaller cell bodies and fewer processes, also distributed in the subependymal zone and subgranular layer of dentate gyrus. Double immunohistochemical staining showed that the nestin positive, neuron-like cells in the nucleus of diagonal band and hippocampus also expressed NSE. However, the astrocyte-like nestin immunoreactive cells of the subependymal zone and subgranular layer of dentate gyrus were not double labeled with GFAP. Although some nestin immunoreactive fibers were distributed in the infundibulum, no nestin-immunoreactive cells were detected in the cortex. These data indicate that nestin exist in the adult human brain outside of the subependymal zone and dentate gyrus and also implies that nestin-immunoreactive cells may play a role in the modulation of basal forebrain function.     

Lead stimulates ERK1/2 and p38MAPK phosphorylation in the hippocampus of immature rats

Lead (Pb(2+)) is widely recognized as a neurotoxicant whose mechanisms of action are not completely established. We have previously demonstrated that Pb(2+) can activate the p38(MAPK) pathway and increase the phosphorylation of Hsp27 in bovine adrenal chromaffin cells and human SH SY5Y cells over a short incubation period (1 h). In the present work we analyzed the effects of Pb(2+) administered in vivo on the level and the phosphorylation state of ERK1/2 and p38(MAPK) in the hippocampus of immature rats. Rats were treated with lead acetate (2, 8 or 12 mg/kg, i.p.) or saline (control) over the 8th to 12th postnatal days, and hippocampal slices were prepared on the 14th day. The Pb(2+) level in the lead-treated animals increased 2.5-6-fold in the blood (3.0-6.0 microg/dl) and 2.0-3.0-fold in the forebrain (78-103 ng/g wet weight), compared to control (saline). The phosphorylation of both ERK1/2 and p38(MAPK) was significantly increased by prior exposure to Pb(2+) in vivo. In in vitro experiments, hippocampal slices from 14-day-old rats were exposed to Pb(2+) (1-10 microM) for 1 and 3 h. There were no changes in the phosphorylation state of ERK and p38(MAPK) for 1-h incubation, whereas a significant increase of ERK1/2 and p38(MAPK) phosphorylation by Pb(2+) (5 microM) was observed for the 3-h incubation. Cell viability measured using MTT was not modified in any of the conditions tested. These results indicate that the phosphorylation of hippocampal ERK1/2 and p38(MAPK) is stimulated by lead in a period of rapid brain development, an effect that may underlie, at least in part, the neurotoxicty elicited by this metal.     

Intracellular Ca elevation induced by a neurotransmitter in a glial cell clone

By fluorometry using a Ca2+-indicator Quin 2, we found an elevation of intracellular Ca2+ concentration ([Ca2+]i) in response to an application of serotonin in a rat clonal glial cell (C6BU-1). The [Ca2+]i rise depended on the dose of applied serotonin and the level of environmental Ca2+. The possibility was suggested that neuron-glia interactions might be controlled by a receptor-coupled [Ca2+]i-regulation system.     

Cd and Co at micromolar concentrations mobilize intracellular Ca via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd²⁺- and Co²⁺-induced intracellular Ca²⁺ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd²⁺, Co²⁺ and methacholine significantly increased the generation of 1,4,5-IP₃. The results suggest that Cd²⁺ and Co²⁺ mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores, possibly through the presumptive Cd²⁺ receptor.     

LSD has high efficacy relative to serotonin in enhancing the cationic current Ih: Intracellular studies in rat facial motoneurons

The effects of LSD (d-lysergic acid diethylamide) on rat facial motoneurons were compared to those of 5-hydroxytryptamine (5-HT) in brain slices by means of current clamp and single-electrode voltage-clamp recordings. As previously reported, 5-HT, in part by decreasing a resting potassium conductance, produced a reversible depolarization (～5 mV), an increase in input resistance, and an enhancement in electrical excitability. LSD also produced an increase in electrical excitability, although with a much slower onset and longer duration. However, in contrast to 5-HT, LSD produced only a slight depolarization (1-2 mV). Moreover, in the presence of LSD the depolarizing effect of 5-HT was markedly attenuated. The 5-HT₂/5-HT_1c agonist 1-(2,5-dimethoxy-4-io-dophenyl)-2-aminopropane (DOI) produced effects intermediate between LSD and 5-HT. The LSD-induced increase in electrical excitability was completely reversed by spiperone, a 5-HT₂/5-HT_1A antagonist, and by ritanserin, a 5-HT₂/5-HT_1c antagonist; the effects of 5-HT were also reduced by these 2 antagonists, but complete blockade did not occur at the concentrations and durations tested. Surprisingly, LSD was found to enhance the hyperpolarization-activated nonspecific cation current I_h to a greater extent than did 5-HT; this enhancement was blocked by both spiperone and ritanserin. These results indicate that, despite having low efficacy relative to 5-HT in decreasing resting potassium conductance, LSD has high efficacy in enhancing the I_h current in rat facial motoneurons; possible mechanisms for this difference are discussed. © 1993 Wiley-Liss, Inc.     

GABA(B) receptor-mediated heterosynaptic depression of excitatory synaptic transmission in rat frontal neocortex

Neocortical synapses display several forms of short-term plasticity including paired-pulse facilitation and depression. The mechanisms underlying this diversity are unclear. Synaptic currents in response to paired stimulation were recorded from layer II/III pyramidal neurons in rat frontal neocortical slices using the whole-cell patch-clamp method. Both paired-pulse facilitation (PPF) and paired-pulse depression (PPD) were observed in control saline. In the presence of 10 microM bicuculline (BIC), prominent PPD was consistently elicited. The maximal depression of the second EPSC occurred around 100 ms although PPD was still observed at intervals up to 1500 ms. Manipulations that reduced the probability of transmitter release significantly affected PPD. Both conditioning (C)- and test (T)-EPSCs were reduced when the extracellular Ca(2+) concentration was lowered from 3 to 1 mM. The decrease was greater in the C-EPSC resulting in a decrease in PPD. The gamma-aminobutyric acid (GABA)(B) receptor agonist baclofen (10 microM) reduced the amplitude of both evoked EPSCs and changed PPD to PPF. In the presence of the GABA(B) antagonists 2(OH)-saclofen (200-400 microM) or SCH50911 (10 microM), PPF was commonly observed. The metabotropic glutamate receptor antagonist MCPG (500 microM) had no effect on neocortical PPD. Brief stimulus trains induced a progressive depression that was insensitive to GABA(B) antagonists. Paired-pulse depression of excitatory synaptic transmission is a prominent phenomenon in frontal neocortex. At least two components of depression were observed. They may play an important role in regulating the balance between excitation and inhibition, therefore maintaining stability in cortical circuits.     

Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice

This study examined anatomical correlates of: (1) long-term potentiation (LTP); (2) equivalent low frequency synaptic activity; (3) continuous high frequency synaptic activation which did not produce LTP; and (4) synaptic inactivation by high Mg2+/low Ca2+ incubation in hippocampal subfield CA1 in the in vitro slice, and examined the persistence of changes at 10-15 min, 2 h and 8 h after stimulation. After potentiating stimulation (6 trains at 100 Hz for 1 s or 200 Hz for 0.5 s), compared to an equivalent number of low frequency stimuli (1 Hz for 600 s), there were increases in numbers of shaft and sessile spine synapses (synapses on stubby, headless spines). This suggested an increase in the number of shaft synapses onto inhibitory interneurons and/or an enhancement of synapse formation on pyramidal neurons possibly involving initial formation of shaft synapses and a transition from shaft, to sessile spine, to full grown spine synapses. Postsynaptic spine heads also assumed a rounder shape, as indicated by decreases in spine perimeter to area ratios, contact lengths, and the percentage of 'cup' shaped spines. There was no effect of potentiating stimulation on bouton or spine areas. After continuous high frequency synaptic activation (40 Hz or 100 Hz for 10 min), which produced no apparent LTP, there were no changes in synapse numbers or spine head shape parameters. However, in contrast to effects of LTP, there was an increase in bouton mitochondrial area and a marginal increase in bouton area compared to the low frequency condition. Inactivation did not affect any of these measures. LTP-associated increases in numbers of shaft and sessile spine synapses persisted over an 8 h incubation period, while the effect on spine shape disappeared after 2 h. Physiologically-demonstrable LTP persisted over the 8 h period. Effects of continuous high level activation on mitochondrial and bouton areas were even more transient, disappearing 2 h after stimulation. These findings: (1) confirm previously reported effects of potentiating stimulation on synapse numbers and spine shape; (2) indicate that spine shape changes are not necessary for the maintenance of LTP; and (3) indicate that continuous high frequency activation which does not produce potentiation has different and non-persisting effects from potentiating stimulation.     

Cytoplasmic calcium elevation in hippocampal granule cell induced by perforant path stimulation andl-glutamate application

Calcium-dependent fluorescence of a Ca²⁺ indicator (fura-2) loaded in the slice of guinea pig hippocampus was measured by a m microscope/video-camera/photometry system. Tetanic stimulation of the perforant path (PP) or application ofl-glutamate caused increment of the fluorescence from the dendritic and somatic layers of the granule cells in the dentate gyrus. Magnitude of the increment depended on the frequency and intensity of the PP-stimulation or on the dose ofl-glutamate. 2-Aminophosphonovaleric acid, a glutamate-receptor antagonist, suppressed both PP-stimulus-induced andl-glutamate-evoked responses, while tetrodotoxin blocked the former only. Thus the fluorescence increment should represent an elevation of Ca²⁺ concentration in the postsynaptic cytoplasm of the granule cells.     

In vitro findings of alterations in intracellular calcium homeostasis in schizophrenia

The pathogenesis of schizophrenia involves several complex cellular mechanisms and is not well understood. Recent research has demonstrated an association between primary disturbances characteristic of the disease, including altered dopaminergic and glutamatergic neurotransmission, and impairments in neuronal calcium (Ca²⁺) homeostasis and signaling. Emerging Ca²⁺ hypothesis links and unifies various cellular processes involved in the pathogenesis of schizophrenia and suggests a central role of dysregulation of Ca²⁺ homeostasis in the etiology of the disease. This review explores the in vitro data on Ca²⁺ homeostasis and signaling in schizophrenia. Major limitation in this research is the lack of schizophrenia markers and validated disease models. As indicated in this review, one way to overcome these limitations may be analyses of Ca²⁺ signalosomes in peripheral cells from schizophrenia patients. Validation of animal models of schizophrenia may permit the application of advanced Ca²⁺ imaging techniques in living animals.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release

Previously, serotonin (5-HT) was found to induce a marked increase in glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) in apical dendrites of layer V pyramidal cells of prefrontal cortex; this effect was mediated by 5-HT_2A receptors, a proposed site of action of hallucinogenic and atypical antipsychotic drugs. Unexpectedly, although the effect of 5-HT was Ca²⁺-dependent and tetrodotoxin-sensitive, it did not appear to involve the activation of excitatory afferent impulse flow. This paradox prompted us to investigate (in rat brain slices) whether 5-HT was acting through an atypical mode of excitatory transmitter release. We found that the frequency of 5-HT-induced spontaneous EPSCs was fully supported by Sr²⁺ in the absence of added Ca²⁺, implicating the mechanism of asynchronous transmitter release which has been linked to the high-affinity Ca²⁺-sensor synaptotagmin III. Although the early, synchronous component of electrically evoked EPSCs was reduced while 5-HT was being applied, late, nonsynchronous components were enhanced during 5-HT washout and also by the 5-HT₂ partial agonist 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI); the effect of DOI was blocked by a selective 5-HT_2A antagonist (MDL 100,907). This late, nonsynchronous component was distinct from conventional polysynaptic EPSCs evoked in the presence of the GABA_A antagonist bicuculline, but resembled asynchronous glutamatergic excitatory postsynaptic potentials (EPSPs) evoked in the presence of Sr²⁺. An enhancement of asynchronous EPSCs by a specific neurotransmitter receptor has not been reported previously. The possible role of excessive asynchronous transmission in the cerebral cortex in mediating the hallucinogenic effects of 5-HT_2A agonists such as DOI is discussed.     

Differences in the Cs block of baclofen and 4-aminopyridine induced potassium currents of guinea pig CA3 neurons in vitro

Single-electrode current- and voltage-clamp techniques were employed to study responses elicited by (?)baclofen or γ-aminobutyric acid (GABA) and 4-aminopyridine (4-AP) induced inhibitory postsynaptic potentials in CA3 pyramidal neurons in guinea pig hippocampal slices. All drugs were applied by the bath to submerged slices in which fast synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM), bicuculline (50 μM), and picrotoxin (50 μM). (?)Baclofen (0.5 μM) and GABA (1 μM) induced equivalent-sized hyperpolarizations and input resistance decreases. The agonist induced hyperpolarization or current and 4-AP induced hyperpolarizations or currents (4-AP induced K-IPSPs or IPSCs) reversed in sign near the K-equilibrium potential (E_K). The GABA_B receptor antagonists, OH-saclofen (500 μM) and CGP 35348 (100 μM), reduced (?)baclofen responses, and 4-AP induced K-IPSPs, suggesting that they were mediated by GABA_B receptors. Intracellular tetraethylammonium-, and extracellular barium-ions (1 μM) diminished the (?)baclofen induced current and 4-AP induced K-IPSCs. Intracellular Cs-ions blocked the (?)baclofen induced outward current at resting membrane potential but did not grossly affect the inward current recorded at membrane potentials negative to E_K. 4-AP induced inwardly or outwardly directed KIPSCs were not blocked by intracellular Cs-ions. Extracellular Cs-ions (5 μM) blocked the (?)baclofen induced inward K-current, but did not block 4-AP induced inwardly directed K-IPSCs. In conclusion, we found differences in the Cs block of K-channels activated by (?)baclofen or the endogenous transmitter GABA. One reason could be that (?)baclofen predominantly activated extra synaptic GABA_B receptors provided that extrasynaptic and subsynaptic receptors couple to different potassium channels. © 1994 Wiley-Liss, Inc.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Interactions between excitotoxins and the Na/K-ATPase inhibitor ouabain in causing neuronal lesions in the rat hippocampus

A possible indirect role of glutamate in causing the neuronal death found after intracerebral administration of a low dose of ouabain (0.1 nmol) has been evaluated. This dose of ouabain produces a more extensive neuronal lesion than those caused by glutamate receptor agonists (kainate at an equimolar dose, or NMDA (N-methyl-d-aspartate) at a 50-fold higher dose). The selective glutamate receptor antagonists, dizocilpine (MK-801) and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), in doses which blocked the direct toxicity of glutamate receptor agonists acting on either the NMDA and non-NMDA classes of glutamate receptor, failed to provide more than a minor protection against ouabain-induced peuronal death in the rat dorsal hippocampus. In contrast, the non-selective glutamate receptor antagonist, kynurenate (100 nmol) reduced the damage by around 70%. The difference in neuroprotection found between the glutamate receptor antagonists suggests that kynurenate may protect by a non-glutamatergic mechanism. Co-administration of ouabain and glutamate receptor agonists (kainate, NMDA or glutamate) resulted in additive rather than synergistic damage to hippocampal neurons. The results suggest that in vivo, ouabain and excitotoxins probably cause neuronal death by independent mechanisms.