The effects of glutamate receptor antagonists on cerebellar granule cell survival and development

N-Methyl-d-aspartate (NMDA) receptor stimulation promotes neuronal survival and differentiation under both in vitro and in vivo conditions. We studied the effects of various NMDA receptor antagonists acting at different NMDA receptor binding sites and non-NMDA receptor antagonists on the development and survival of cerebellar granule cell (CGC) culture. Only three of the drugs tested induced neurotoxicity-MK-801 (non-competitive NMDA channel blocking antagonist), ifenprodil (an antagonist of the NR2B site and polyamine site of the NMDA receptor) and L-701.324 (full antagonist at glycine site), while CGP-37849 (a competitive NMDA antagonist), (+)-HA-966 (a partial agonist of the glycine site of the NMDA receptor), and NBQX (a competitively acting AMPA receptor antagonist) were not toxic at any concentration (1-100 microM) used. Among these drugs, only MK-801 was toxic for the immature CGC on second day in vitro (2DIV), and toxicity was diminished parallel to the neuronal maturation. In more mature neurons (7DIV), MK-801 demonstrated some neuroprotection, which diminished spontaneously occurring neuronal death in culture. Neither NMDA nor glutamate were able to prevent the neurotoxic effect of MK-801 at 2DIV. MK-801, ifenprodil and L-701.324 induced DNA fragmentation on 2DIV in CGC culture measured by the TUNEL method. The BOC-D-FMK, the universal caspase inhibitor, completely reversed MK-801-induced DNA fragmentation, suggesting an apoptotic pathway of MK-801-induced cell death. Neurite outgrowth as a characteristic feature of the development of CGC was diminished after treatment with MK-801, ifenprodil and L-701.324. In conclusion, the results of the present study demonstrate that only nonselective channel blocker MK-801 decreases cell viability, induces apoptosis and inhibits neurite outgrowth of CGC in a development-dependent manner.     

Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons

Polyamines play critical roles during the development of brain neurons. In the present study we examined the effects of polyamines on neuronal apoptotic death. Rat cerebellar granule neurons were cultured in the presence of a depolarizing concentration of KCl (25 mM) in the medium. Apoptotic neuronal death was induced by changing the medium to that containing 5.6 mM KCl without serum. Spermine as well as spermidine and putrescine prevented cell death in a concentration-dependent manner with the order of potency being spermine>spermidine>putrescine. The effect of spermine was partially blocked by several NMDA-type glutamate receptor antagonists including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801). MK-801-sensitive neuroprotection by spermine depended on cell density. Activation of CPP32 (caspase-3/Yama/apopain)-like proteolytic activity, a key mediator of apoptosis, precedes neuronal death, and polyamines prevented an increase in this activity. These results demonstrate that polyamines protect neurons from apoptotic cell death through both NMDA receptor-dependent and -independent mechanisms, acting upstream from the activation of CPP32-like protease(s). © 1997 Elsevier Science B.V. All rights reserved.     

Spermine facilitates the generation of long-term potentiation of evoked potential in the dentate gyrus of anesthetized rats.

The effects of the polyamines, spermine, spermidine and putrescine, on long-term potentiation (LTP) of evoked potential were investigated in the dentate gyrus of anesthetized rats. Injection of 5 nmol spermine into the lateral ventricle did not influence the basal amplitude of the population spike, but significantly enhanced the potentiation induced by subthreshold tetanic stimulation (20 pulses at 60 Hz). The effect of spermine resulted in facilitation of LTP generation. Injection of the same dose of spermidine or putrescine affected neither the basal response nor the potentiation induced by subthreshold tetanus at all, indicating that the LTP-facilitating effect is specific to spermine. Furthermore, the LTP-facilitating effect of spermine was dose-dependent in the range of 0.5-50 nmol. When 5 nmol ifenprodil, an antagonist at the polyamine site of the NMDA receptor channel complex, was concomitantly injected, spermine could not facilitate the generation of LTP. Since injection of ifenprodil alone did not influence the generation of LTP, it is probable that ifenprodil specifically blocks the effect of spermine. These results suggest that spermine facilitates the generation of hippocampal LTP, probably through an ifenprodil-sensitive polyamine site associated with the NMDA receptor.     

Spermine is neuroprotective against anoxia and N-methyl-D-aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca(2+) channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca(2+) is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Spermine is neuroprotective against anoxia and N-methyl- -aspartate in hippocampal slices

Polyamines were implicated as either neurotoxic or neuroprotective in several models of stroke. Spermine augments the excitotoxicity mediated by the N-methyl- -aspartate (NMDA) receptor because this receptor is activated at micromolar spermine concentrations. However, at higher concentrations, spermine could be neuroprotective because it blocks the NMDA receptor and voltage-activated Ca²⁺ channels. In this work, acute hippocampal slices were exposed to 1 mM spermine and either 10 min of anoxia or 0.5 mM NMDA. The percent recovery of population spikes was the measure of neuroprotection. One millimolar spermine was robustly neuroprotective; however, 0.1 mM spermine and 1 mM putrescine were not. The neuroprotective concentration of spermine was higher than the physiological concentration of free spermine. However, during an excitotoxic episode, extracellular Ca²⁺ is decreased, enabling the inhibitory activity of lower spermine concentration. In addition, several noxious stimuli trigger the release of intracellular spermine and could raise local levels of spermine. Therefore, it is possible that spermine has a neuroprotective role in vivo.     

Gliotoxicity in hippocampal cultures is induced by transportable, but not by nontransportable, glutamate uptake inhibitors

Extracellular glutamate is kept below a toxic level by glial and neuronal glutamate transporters. Here we show that the transportable glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC) induced cell death in mature, but not in immature, hippocampal neuron-enriched cultures. The cell death produced by a 24-hr treatment with t-PDC was dose-dependent and reached 85% of the cell population at a 250 microM concentration at 23 days in vitro (DIV). Immunocytochemistry experiments showed that, under these experimental conditions, t-PDC killed not only neurons as expected but also glial cells. The N-methyl-D-aspartate (NMDA) antagonist D-2-aminophosphonovalerate (D-APV; 250 microM) only partially reversed this toxicity, completely protecting the neuronal cell population but not the glial population. The antioxidant compounds alpha-tocopherol or Trolox, used at concentrations that reverse the oxidative stress-induced toxicity, did not block the gliotoxicity specifically produced by t-PDC in the presence of D-APV. The nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) elicited cell death only in mature, but not in immature, hippocampal cultures. The TBOA toxic effect was dose dependent and reached a plateau at 100 microM in 23-DIV cultures. About 50% of the cell population died. TBOA affected essentially the neuronal population. D-APV (250 microM) completely reversed this toxicity. It is concluded that nontransportable glutamate uptake inhibitors are neurotoxic via overactivation of NMDA receptors, whereas transportable glutamate uptake inhibitors induce both an NMDA-dependent neurotoxicity and an NMDA- and oxidative stress-independent gliotoxicity, but only in mature hippocampal cultures.     

Polyamines in a genetic animal model of paroxysmal dyskinesia

Previous studies suggested that glutamatergic overactivity contributes to the manifestation of dystonia in the dt(sz) mutant hamster, a model of idiopathic paroxysmal dyskinesia in which dystonic episodes occur in response to mild stress. Therefore, the role of polyamines, known as positive modulators of NMDA receptors, was examined in the present study. The levels of polyamines (putrescine, spermidine, spermine) were determined in forebrain, cerebellum and brainstem in dt(sz) hamsters at an age of most marked expression of dystonia (32 days) and in age-matched non-dystonic control hamsters. Spermine was found to be significantly increased in the forebrain (35%) of dystonic animals, while spermidine was unaltered in dystonic brains and only a moderate increase in putrescine (12%) was detected in the cerebellum of dt(sz) mutants. In view of enhanced spermine levels, the effect of the putative polyamine receptor antagonist ifenprodil on the severity of dystonia was examined in dystonic hamsters. Ifenprodil (5-40 mg/kg i.p.) failed to exert a beneficial effect, but even aggravated dystonia in the dt(sz) mutant at higher doses. These data together with previous pharmacological findings in mutant hamsters do not completely exclude a pathophysiological role of enhanced polyamine levels but suggest that overstimulation of NMDA receptors which contain NR2B subunits by enhanced spermine levels is not involved in the dystonic syndrome.     

The effects of potassium-induced depolarization, glutamate receptor antagonists and N-methyl-D-aspartate on neuronal survival in cultured neocortex explants

The effects of elevating the potassium concentration of the growth medium of neocortical explants was studied. Under control conditions, 10 mM potassium resulted in ca 20% decrease in the number of surviving neurons. The same potassium concentration, however, was clearly neurotrophic in tetrodotoxin-grown cultures: tetrodotoxin-induced neuronal death was significantly reduced. Both effects could be mimicked by the addition of 10 microM N-methyl-D-aspartate (NMDA); lower concentrations were without effect; higher concentrations were neurotoxic under both control and tetrodotoxin conditions. The neurotoxic, as well as the neurotrophic effects of 10 mM potassium appear to be mediated through depolarization-induced glutamate release since they could be influenced by the application of glutamate receptor antagonists. The addition of the NMDA receptor antagonist D-2-amino-7-phosphonoheptanoate (APH) blocked the trophic effect of 10 mM potassium in tetrodotoxin-grown cultures, resulting in low survival. On the other hand, the addition of the non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) resulted in neuronal survival similar to control cultures, indicating that it blocked the toxic effects of glutamate, leaving the trophic effects on the NMDA receptor untouched. Under control (non-TTX) conditions, neither DNQX nor APH showed significant effects on 10 mM potassium-induced cell death, indicating that stimulation of the non-NMDA, as well as the NMDA receptors is neurotoxic. This differential effect of NMDA receptor stimulation on neuronal survival is discussed with respect to the maturational and/or functional state of the neurons in the culture.     

Dexamethasone induces cell death which may be blocked by NMDA receptor antagonists but is insensitive to Mg2+ in cerebellar granule neurons

Since dexamethasone may elevate the Ca2+ influx through NMDA receptors, we have investigated mechanisms of dexamethasone toxicity in rat cerebellar granule neurons. Dexamethasone concentrations over 0.1 microM induced cell death that reached about 20% of the death induced by glutamate. Dexamethasone-induced cell death was reduced by more than 80% by the mineralocorticoid antagonist RU 28318 or the NMDA receptor antagonists MK 801 and CGP 39551, whereas RU 28318 rescued only approximately 30% of cells treated with glutamate, indicating that dexamethasone requires NMDA receptors to induce acute neuronal toxicity and that a fraction of the neurons showed this toxicity. Mg2+ reduced the cell death induced by glutamate at potassium concentrations of 1 mM and 5 mM, but not at 25 mM. In contrast, cell death induced by dexamethasone was not significantly reduced by Mg2+ in any of the potassium concentrations. Both glutamate and dexamethasone induced toxicity with translocation of the apoptosis inducer NGFI-B to the mitochondria seen after 30 min-2 h concomitant with activation of apoptosis inducing factor (AIF) and caspase-3. In conclusion, dexamethasone induces a rapid toxicity which is blocked by NMDA receptor antagonists other than Mg2+, and involves mitochondrial apoptosis inducer NGFI-B.     

The action of CGS-19755 on the redox enhancement of NMDA toxicity in rat cortical neurons in vitro.

The effects of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) were studied in cultures of rat cerebral cortex under normal and altered redox conditions. CGS-19755 was effective in preventing delayed neuronal death produced by an acute exposure to either glutamate (500 microM) or NMDA (200 microM), but was ineffective in protecting neurons against the toxicity induced by a prolonged exposure to kainate (500 microM). We observed that the reducing agent dithiothreitol (DTT, 500 microM), could dramatically enhance toxicity and electrophysiological responses produced by 50 microM NMDA. CGS-19755 (100 microM) could effectively block both of these effects of DTT. Any toxicity produced by DTT alone was also antagonized by CGS-19755. In contrast, oxidized DTT did not enhance NMDA toxicity nor was it toxic when added alone. These results indicate that CGS-19755 is an effective and specific neuroprotectant acting at the NMDA receptor in vitro, and that the enhancement in NMDA toxicity induced by DTT is mediated by an increase in activity at this receptor complex.     

Neurotoxic effects of the intrastriatal injection of spermine and spermidine: lack of involvement of NMDA receptors.

The injection into the rat striatum of the polyamines spermine and spermidine (30-300 nmol) produced, 1 week after injection, a dose related loss of the neuronal markers glutamate decarboxylase and choline acetyltransferase and a decrease in the density of N-methyl-D-aspartate (NMDA) receptors (as labelled with [3H]TCP). In parallel, an increase in peripheral type benzodiazepine (p) binding site density (a marker of the associated glial reaction and macrophage invasion) was observed. Intrastriatal injection of putrescine (300 nmol) did not significantly alter any of these markers. The effect of spermine on these neuronal and glial markers was maximal 3 days after injection, and tended towards control levels at 16 days post injection. The neurotoxic effects of spermine were confirmed by histological analysis demonstrating a massive neuronal loss around the injection site and an accumulation of astrocytes and phagocytes. The neurotoxic effects of spermine (250 nmol) were not antagonised by the previous administration of the NMDA receptor antagonist MK-801 (10 mg/kg, i.p.). Thus polyamine neurotoxicity in vivo does not seem to involve NMDA receptor activation, although it may possibly be related to the multiple effects of these compounds on diverse calcium channels and processes regulating calcium homoeostasis.     

Neuroprotection against excitotoxicity by N-alkylglycines in rat hippocampal neurons

Excessive activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype is considered a relevant initial step underlying different neurodegenerative diseases. Recently, with the approval of memantine to treat Alzheimer dementia, NMDA receptors have regained clinical interest. Accordingly, the development and validation of NMDA receptor antagonists is being reconsidered. We recently identified a family of trialkylglycines that act as channel blockers of the NMDA receptor. Their neuroprotective activity against excitotoxic insults remains elusive. To address this issue, we first characterized the contribution of glutamate receptor sub-types to hippocampal death in culture as a function of days in culture in vitro (DIV). Whereas at 7 DIV neither NMDA nor glutamate produced a significant neuronal death, at 14 and 21 DIV, NMDA produced the death of 40% of the neurons exposed to this receptor agonist that was fully protected by MK-801. Similar results were obtained for l-glutamate at 14 DIV. In contrast, when neurons at 21 DIV were used, glutamate killed 51.1±4.9% of the neuronal population. This neuronal death was only partially prevented by MK-801, and fully abrogated by a combination of MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glucose deprivation injured 37.1±9.2% of the neurons through a mechanism sensitive to MK-801. The family of recently identified N-alkylglycines tested protected neurons against NMDA and glucose-deprivation toxicity, but not against glutamate toxicity. Noteworthy, N-alkylglicines with a moderate protection against NMDA-induced toxicity strongly protected from β-amyloid toxicity. Collectively, these findings imply both NMDA and non-NMDA receptors in excitotoxicity of hippocampal neurons, and suggest that blockade of NMDA receptors alone may not suffice to efficiently abrogate neurodegeneration.     

Neurosteroids and glutamate toxicity in fibroblasts expressing human NMDA receptors

We charactrized glutamate receptor-mediated toxicity in mouse fibroblasts expressing the human NR1a/2A or NR1a/2B NMDA receptor. After induction of NMDA receptor, cells in both lines died over a 24 h time period. This toxicity was associated with a progressive increase in the glutamate content of the media. Cell death could be prevented by including either the non-competitive NMDA receptor antagonist ketamine or the competitive antagonist, d,l-AP-5. Cells expressing NR1a/2A receptors were maximally protected by 0.5 mM d,l-AP-5, while those expressing NR1a/2B receptors required 2 mM d,l-AP-5 for maximal protection. The neurosteroid pregnanolone sulfate, which negatively modulates NMDA receptor function, partially protected fibroblasts containing NR1a/2A or NR1a/2B NMDA receptor constructs. However, the neurosteroid pregnenolone sulfate, which has been reported to act as a positive allosteric modulator of the NMDA receptor, had no effect on the toxicity caused by endogenous glutamate. Our results on cells expressing human NMDA receptors suggest that certain neurosteriods may protect against NMDA induced toxicity while having low neurotoxic liabilities of their own.     

Calcium-independent release of [3H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Immunocytochemical localization of polyamines in the tiger salamander retina

The polyamines spermine and spermidine are present in neural tissue, but their functions there are not well understood. Recent work suggests that the NMDA subtype of glutamate receptors, other glutamate receptor subtypes, and certain K⁺-channels, are neural targets for polyamines. To better understand the neuron-specific roles of polyamines, we have developed antibodies that interact with spermine and spermidine in aldehyde-fixed tissue and used these antibodies in immunocytochemical studies to determine the cellular localization of these polyamines in the tiger salamander retina. The affinity-purified, polyclonal antibodies were highly specific for spermine and spermidine, exhibiting < 1 % cross reactivity with putrescine, and virtually no cross-reactivity with GABA, arginine, lysine, or glutaraldehyde. Polyamine labeling was most abundant in cells in the inner half of the inner nuclear layer and in the ganglion cell layer. Some cells in the outer half of the inner nuclear layer are labeled, and there was some labeling in both synaptic layers. Double-labeling experiments indicated (1) all GABAergic amacrine cells were polyamine-positive; and (2) all ganglion cells (identified by back-filling after microinjections of rhodamine in the optic nerve) were polyamine-positive. These results are consistent with a role for polyamines as modulators of NMDA receptor function and channel function in the inner retina.     

Glutamate Toxicity: An Experimental and Theoretical Analysis

In slices of 8-day-old rat cerebellum, the lowest concentration of glutamate that induced toxicity (30 min exposure; 90 min recovery) was 100 microM, but the damage only occurred in the outermost regions. As the concentration was raised, the band of necrosis became progressively deeper until, at 3 mM, it was uniform across the slice thickness. At a test concentration of 300 microM, the width of the necrotic band did not change when either the exposure time or the recovery period was varied between 30 min and 3 h. These results are predicted by a theoretical model in which the diffusion of glutamate into brain tissue is countered by cellular uptake of the amino acid, and they argue against the idea that glutamate toxicity is inherently self-propagating. When slices were examined immediately after exposure (300 microM), a prominent swelling of glial cells was present at the slice surface. Swelling per se did not appear to compromise their uptake function, and the model predicts that cellular swelling, by reducing the rate of diffusion of glutamate, protects against glutamate toxicity. The damage produced by 3 mM glutamate, which was primarily exerted against granule cells, was prevented by N-methyl-d-aspartate (NMDA) receptor blockade, whereas antagonists acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were ineffective. Under conditions of energy deprivation, the neurotoxic potency of glutamate was markedly enhanced and a normally non-toxic concentration (30 microM) became maximally toxic towards granule cells. Dark vacuolar degeneration of Purkinje cells was also present, and this could be inhibited by blocking AMPA receptors. The results and theoretical analysis suggest that intact brain tissue is remarkably resistant to glutamate toxicity, chiefly because of the formidable properties of the uptake system. However, under special circumstances, glutamate can become a potent neurotoxin and its toxicity can then involve both NMDA and AMPA receptors.     

Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

The sigma receptor ligand (+)-pentazocine prevents apoptotic retinal ganglion cell death induced in vitro by homocysteine and glutamate

Recent studies demonstrated that the excitotoxic amino acid homocysteine induces apoptotic death of retinal ganglion cells in vivo. In the present study, an in vitro rat retinal ganglion cell (RGC-5), culture system was used to analyze the toxicity of acute exposure to high levels of homocysteine, the mechanism of homocysteine-induced toxicity, and the usefulness of type 1 sigma receptor (sigmaR1) ligands as neuroprotectants. When cultured RGC-5 cells were subjected to treatment with 1 mM D,L-homocysteine, a significant increase in cell death was detected by terminal dUTP nick end labeling (TUNEL) analysis and analysis of activated caspase. When cells were treated with homocysteine- or glutamate in the presence of MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) receptor, the cell death was inhibited significantly. In contrast, NBQX, an antagonist of the AMPA/Kainate receptor, and nifedipine, a calcium channel blocker, did not prevent the homocysteine- or glutamate-induced cell death. Semiquantitative RT-PCR and immunocytochemical analysis demonstrated that RGC-5 cells were exposed to homocysteine or glutamate express type 1 sigma receptor at levels similar to control cells. Treatment of RGC-5 cells with 3 or 10 microM concentrations of the sigmaR1-specific ligand (+)-pentazocine inhibited significantly the apoptotic cell death induced by homocysteine or glutamate. The results suggest that homocysteine is toxic to ganglion cells in vitro, that the toxicity is mediated via NMDA receptor activation, and that the sigmaR1-specific ligand (+)-pentazocine can block the RGC-5 cell death induced by homocysteine and glutamate.     

Altered NMDA receptor function in primary cultures of hippocampal neurons from mice lacking the Homer2 gene

N‐Methyl‐D‐Aspartate (NMDA) receptors are inhibited during acute exposure to ethanol and are involved in changes in neuronal plasticity following repeated ethanol exposure. The postsynaptic scaffolding protein Homer2 can regulate the cell surface expression of NMDA receptors in vivo, and mice with a null mutation of the Homer2 gene exhibit an alcohol‐avoiding and ‐intolerant phenotype that is accompanied by a lack of ethanol‐induced glutamate sensitization. Thus, Homer2 deletion may perturb the function or acute ethanol sensitivity of the NMDA receptor. In this study, the function and ethanol sensitivity of glutamate receptors in cultured hippocampal neurons from wild‐type (WT) and Homer2 knock‐out (KO) mice were examined at 7 and 14 days in vitro (DIV) using standard whole‐cell voltage‐clamp electrophysiology. As compared with wild‐type controls, NMDA receptor current density was reduced in cultured hippocampal neurons from Homer2 KO mice at 14 DIV, but not at 7 DIV. There were no genotype‐dependent changes in whole‐cell capacitance or in currents evoked by kainic acid. The GluN2B‐selective antagonist ifenprodil inhibited NMDA‐evoked currents to a similar extent in both wild‐type and Homer2 KO neurons and inhibition was greater at 7 versus 14 DIV. NMDA receptor currents from both WT and KO mice were inhibited by ethanol (10–100 mM) and the degree of inhibition did not differ as a function of genotype. In conclusion, NMDA receptor function, but not ethanol sensitivity, is reduced in hippocampal neurons lacking the Homer2 gene. Synapse 70:33–39, 2016. © 2015 Wiley Periodicals, Inc.     

Role of spermine in amyloid beta-peptide-associated free radical-induced neurotoxicity

The polyamines, relatively low-molecular-weight aliphatic compounds, are the main inducers of eukaryotic cell growth and proliferation. Although polyamine requirements for cell growth are well defined, their role is still enigmatic. We have previously reported that amyloid beta-peptide (A beta), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is toxic to neurons through a free radical-dependent oxidative stress mechanism and that A beta(1--42), the principal form of A beta in AD brain, causes an increase in polyamine metabolism manifested by up-regulated polyamine uptake and increased ornithine decarboxylase (ODC) activity. Both effects were prevented by the free radical scavenger vitamin E. Spermine has been reported to function directly as a free radical scavenger. In the current study, we aimed to address whether up-regulation of polyamine metabolism is a defense against, or a result of, A beta-induced oxidative stress by investigating the capability of spermine to quench A beta-associated free radicals in solution and to assert a protective function of spermine in neuronal culture against A beta. Pretreatment of cultured neurons with spermine prior to A beta exposure failed to prevent A beta-induced cell death. Indeed, A beta plus spermine added to cultured neurons was even more neurotoxic than either agent alone. Additionally, inhibition of the polyamine synthesis by difluoromethylornithine (DFMO) did not protect cells from A beta-induced free radical toxicity, and stimulation of the synthesis of putrescine and spermine by the aminopropyltransferase inhibitor S-adenosyl-1,8-diamino-thiooctane (AdoDATO), rather, further enhanced A beta-induced toxicity. Although spermine is capable of scavenging free radicals generated by A beta in solution as measured by electron paramagnetic resonance (EPR) spectroscopy, the up-regulated transport of exogenously added spermine together with A beta may lead to overaccumulation of a cellular spermine pool, with resulting enhanced neurotoxicity.