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.     

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 induces cell death in cultured human embryonic cerebral cortical neurons through N-methyl-D-aspartate receptor activation

The polyamines putrescine, spermidine, and spermine play important roles in cell proliferation, differentiation, and modulation of ion channel receptors. However, the function of increased concentrations of these compounds in brain injury and disease is unclear, in that they have been proposed as being both neuroprotective and neurotoxic. The effects of spermine and putrescine were studied in human primary cerebral cortical cultures containing both neurons and glia. No toxic effects were induced at 8 days in vitro (DIV) by either of the two polyamines at concentrations ranging from 0.3 microM to 2 mM. However, when the oxidative metabolism of spermine that generates toxic byproducts was induced by the presence of fetal calf serum, spermine caused cellular death with an LC(50) of approximately 50 microM. At 14 DIV, the coapplication of spermine 2 mM and glutamate 5 mM induced neuron cell death, but the effect of applying both components separately was null. Both spermine and glutamate were toxic to older neurons (26-42 DIV cultures), and here the coapplication of glutamate was found always to intensify the effect of spermine. Spermine showed greater toxicity than glutamate in neurons. Another effect observed is that glutamate, but not spermine, induced astrocyte swelling. Spermine toxicity was inhibited by both MK801 and ifenprodil, indicating a mechanism involving N-methyl-D-aspartate (NMDA) receptor activation. Moreover, a strong spermine modulation of the NMDA receptor was demonstrated by the inhibition of glutamate toxicity by ifenprodil. Putrescine induced minor effects also as a neurotoxic agent. In conclusion, neuronal death by spermine can be induced by its toxic byproducts as well as through NMDA receptor action. The present results confirm the potentially harmful role of the polyamines in excitotoxicity-related human disorders.     

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.     

Polyamines regulate glycine interaction with the N-methyl-D-aspartate receptor

[3H]Glycine binding studies have been performed to further characterize polyamine interactions with the rat brain N-methyl-D-aspartate (NMDA) receptor. Strychnine-insensitive [3H]glycine binding to washed cortical membranes was enhanced by spermine, spermidine, and hirudonin. Spermine stimulation of binding was additive with that produced by the NMDA receptor agonist L-glutamate. A high concentration of the L-glutamate antagonist 2-amino-5-phosphonovaleric acid reduced, but did not eliminate, spermine effects. Saturation experiments indicated that L-glutamate and spermine enhancement of binding was due to an increase in [3H]glycine binding affinity. Kinetic studies showed that optimal concentrations of spermine and L-glutamate reduced [3H]glycine association and dissociation rates by approximately fivefold and 30-fold, respectively. In competition experiments, the presence of L-glutamate and spermine had differential effects on the affinities of compounds that act as either agonists or antagonists at the glycine site of the NMDA receptor. The affinities of the agonists glycine, D-serine, and D-alanine, were increased about fivefold, while antagonist (HA-966, 7-chlorokynurenic acid) inhibitory potencies were unchanged. These data support our previous results showing that the NMDA receptor possesses a novel polyamine recognition site and demonstrate that these compounds directly modulate glycine's interactions with the receptor complex.     

Inhibition of an N-methyl-d-aspartate induced short-term potentiation in the rat hippocampal slice

The effects of the phorbol ester 4ß-phorbol-12,13 dibutyrate (PDBu) and the protein kinase (PK) inhibitors H-7 and sphingosine were investigated on the short-term potentiation (STP) of the population excitatory postsynaptic potential (EPSP) induced by perfusion of N-methyl-d-aspartate (NMDA) in the stratum radiatum of CA1 of the rat hippocampal slice. Bath perfusion of 130 μM NMDA for 10 s caused an initial depression of the population EPSP followed by a STP, which averaged 46% and lasted 16 min. PDBu (100 nM) perfused for 2 h completely inhibited the NMDA induced STP, suggesting that the stimulation of PKC inhibited an NMDA receptor activated process which induced the STP. The protein kinase inhibitors H-7 and sphingosine did not alter the NMDA induced STP.     

NMDA and AMPA/kainate glutamatergic agonists increase the extracellular concentrations of GABA in the prefrontal cortex of the freely moving rat: modulation by endogenous dopamine

Using microdialysis in the prefrontal cortex, this study investigated first the effects of the ionotropic glutamatergic agonists NMDA and AMPA on extracellular concentrations of GABA, and second, the modulation of these effects by increasing endogenous dopamine. NMDA (20, 100, and 500 microM) and AMPA (1, 20, and 100 microM), perfused through the microdialysis probe for 60 min, produced a dose-related increase of extracellular concentrations of GABA in the prefrontal cortex of the awake rat. NMDA 100 and 500 microM produced a maximal increase of extracellular GABA of 150 +/- 38% and 245 +/- 75% of baseline, respectively. AMPA 20 and 100 microM produced a maximal increase of extracellular GABA of 140 +/- 17% and 195 +/- 41% of baseline, respectively. NMDA and AMPA also increased extracellular concentrations of glutamate. Increases of extracellular GABA, and also of glutamate, produced by NMDA (500 microM) and AMPA (100 microM) were significantly blocked by the NMDA antagonist CPP (100 microM) and the AMPA/kainate antagonist DNQX (100 microM), respectively. To investigate whether dopamine modulates the increases of GABA produced by NMDA and AMPA, endogenous dopamine was increased with the dopamine uptake inhibitor nomifensine. Nomifensine (1, 100, and 1000 microM) produced a dose-related increase of dialysate dopamine (from 0.1 to 1.0 nM) but did not modify basal extracellular concentrations of GABA in the prefrontal cortex. However, increases of endogenous dopamine at 0.5-0.7 nM did potentiate the increases of extracellular GABA produced by AMPA (20 microM) (from 140% to 240% of baseline), but not by NMDA (100 microM), in this area of the brain. These effects were attenuated by the perfusion of (-)sulpiride (D2 antagonist), but not by the perfusion of SCH-23390 (D1 antagonist). These results suggest that glutamate, through the activation of both NMDA and AMPA/kainate ionotropic receptors, facilitates GABAergic transmission in the prefrontal cortex, and that dopamine can modulate the effects of glutamate through AMPA/kainate receptors on GABA transmission in this area of the brain.     

Spermine increases paired-pulse facilitation in area CA1 of hippocampus in a calcium-dependent manner

The effect of spermine on neurotransmission was studied in area CA1 of the hippocampal slice preparation. Paired-pulse stimulation (20 ms interpulse interval) was delivered to stratum radiatum; the evoked field potential responses were recorded simultaneously from stratum radiatum and from stratum pyramidale. At mM and sub-mM concentrations, spermine decreased the slope of pEPSP in stratum radiatum and the area of the conditioning population spike in stratum pyramidale. Short-latency paired-pulse inhibition of the population spike was converted to facilitation by spermine. These effects of spermine resembled those observed at low calcium concentration. In addition, dose-response and input-output curves determined at various Ca²⁺ concentrations demonstrated that the depressant effects of spermine were larger at low Ca²⁺ levels. The results support the notion that spermine competitively blocks presynaptic voltage-sensitive Ca²⁺ channels, thus causing a decreased release of neurotransmitter. Since spermine is present in brain, it is likely that it is a natural modulator of Ca²⁺ channels.     

N-methyl-D-aspartate antagonists prevent kainate neurotoxicity in rat retinal ganglion cells in vitro.

Under defined culture conditions, exogenous glutamate (Glu), NMDA, or an endogenous Glu-related toxin is lethal to rat retinal ganglion cells; these detrimental effects are NMDA receptor mediated because specific NMDA antagonists can prevent cellular injury. In the presence of an endogenous Glu-like toxin, 125 microM kainate (KA) increases the proportion of retinal ganglion cells that die, but the toxicity (due to both KA and the endogenous toxin) is totally prevented by 2-amino-5-phosphonovalerate (APV), a specific NMDA receptor antagonist. These findings indicate that the KA-induced portion of retinal ganglion cell death also appears to be mediated via NMDA receptors. There are at least 2 possible mechanisms for this lethal effect. In addition to KA receptors, KA could directly stimulate NMDA receptors. Alternatively, KA might activate its own specific receptor, which in turn leads to a net increase in the release of an endogenous Glu-related toxin; this endogenous substance would then activate NMDA receptors. Patch-clamp electrophysiology experiments have helped to distinguish between these possibilities. Concentrations of APV that completely block the current elicited by maximal nondesensitizing doses of NMDA exert no detectable inhibition of KA-evoked currents. Hence, at the concentrations used, it appears unlikely that KA directly activates NMDA receptors in this preparation. Furthermore, the fraction of toxicity attributed to the addition of KA can be blocked by the relatively specific non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This finding is consistent with the hypothesis that KA adds an increment of toxicity in this system by directly interacting with KA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of excitatory amino acid release by N-methyl-D-aspartate receptors in rat striatum: in vivo microdialysis studies.

The microdialysis technique was utilized to study the effects of N-methyl-D-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 microM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 microM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 microM, produced no changes on basal levels of Glu and Asp. However, 100 microM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 microM D-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such D-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 microM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.     

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.     

The effects ofN-methyl-d-aspartate and kainate lesions of the rat striatum on striatal ornithine decar☐ylase activity and polyamine levels

The intrastriatal injection ofN-methyl-d-aspartate (NMDA) (250 nmol) produced a delayed and marked increase in striatal ornithine decar☐ylase (ODC) activity and putrescine levels which peaked 6–15 h following the injection of NMDA. Striatal ODC activity subsequently returned to normal values while putrescine levels remained significantly elevated for up to 4 days following the lesion. NMDA produced an early and progressive decline in striatal spermine and spermidine levels, preceding the increase in ODC activity, with a maximum effect 2 h following injection. Spermidine levels returned to normal 6 h post-NMDA infusion, and subsequently increased to above normal levels 36 h and 4 days after the infusion of NMDA. This late increase in striatal spermidine levels paralleled an increase in the binding of the glial cell/macrophage marker [³H]PK 11195. Spermine levels tended to return to normal values 6 h after the injection of NMDA but may be further depressed at later intervals (15 h to 4 days). The intrastriatal injection of saline also resulted in a delayed increase in striatal ODC activity and putrescine levels, but these changes were minor compared to those produced by NMDA. Intrastriatal saline injection provoked no consistent change in striatal spermine or spermidine levels. The changes in polyamine metabolism produced by the instriatal injection of kainic acid (4 nmol) were only analysed at 6 and 15 h following injection but were qualitatively similar to those produced by NMDA although perhaps following a slightly more delayed time-course. Neurotoxic lesions of the striatum thus provoke changes in ODC activity and increased levels of putrescine that follow closely the time-course of similar events in the ischaemic brain. The initial early changes in spermine and spermidine levels induced by NMDA could perhaps alter the functioning of the NMDA receptor itself via its polyamine-sensitive modulatory site and contribute to a feed-forward activation of NMDA receptors and prolonged NMDA receptor-mediated toxicity.     

Evidence for a role of the N-methyl-d-aspartate (NMDA) receptor in cortical spreading depression in the rat

The neurotransmitter glutamate activates the N-methyl-d-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist dl-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K⁺ and Ca²⁺ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.     

Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

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.     

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.     

Effects of N-Methyl-D,L-Aspartate on Beta-Endorphin and Prolactin Secretion in Rams Exposed to Long or Short Days

In a previous study we demonstrated that the injection of the excitatory amino-acid N-methyl-D, L-aspartate (NMDA) stimulates an acute increase in the peripheral blood concentrations of luteinizing hormone in Soay rams, and this response varies with the photoperiodically-induced reproductive cycle. To extend these observations, we have now measured the changes in the blood concentrations of β-endorphin and prolactin in the same animals to establish whether NMDA stimulates the secretion of other pituitary hormones. Groups of adult Soay rams were exposed to alternating 16-weekly periods of long and short days to induce a long-term cycle in the endogenous secretion of β-endorphin (maximum under short days) and prolactin (maximum under long days). NMDA injected intravenously caused a dose-dependent increase in the blood plasma concentrations of β-endorphin (over the range 1 to 20 mg/kg NMDA). The initial increase in β-endorphin occurred within 2 to 4 min with peak levels after 20 to 100 min. The magnitude of the β-endorphin response was greatest following exposure to long days when the endogenous secretion of β-endorphin was low, while the duration of the response was greatest following exposure to short days when the endogenous secretion of β-endorphin was high. NMDA injected intravenously also caused a dose-dependent increase in the blood plasma concentrations of prolactin but this only occurred following exposure to long days when the endogenous secretion of prolactin was high. At this time the initial increase in prolactin concentrations occurred within 2 to 4 min after the injection of NMDA as for β-endorphin but the values continued to increase for 2 to 4 h. In a separate experiment, it was shown that pretreatment of the rams with dexamethasone (synthetic glucocorticoid, 133.4 μg/kg iv) blocked the β-endorphin response to NMDA but had no effect on the prolactin response. This indicates that NMDA stimulates the secretion of β-endorphin from the corticotrophs probably acting centrally to induce the release of corticotrophin-releasing hormone and/or arginine vasopressin, while NMDA acts through separate mechanisms to affect the secretion of prolactin. The overall results show that NMDA can be used as a probe to investigate the neuroendocrine control of β-endorphin and prolactin in addition to luteinizing hormone as described previously. The multiple responses are likely to represent the effects of NMDA acting on the hypothalamus to induce the acute release of peptides and other hormones into the pituitary portal blood system to affect the corticotrophs, lactotrophs and gonadotrophs. The variation in responsiveness to NMDA related to the photoperiodic cycle may reflect changes in the synthesis and storage of the hypothalamic hormones and the influence of other neural systems. Alternatively, the changes in the secretion of endogenous excitatory amino-acids and NMDA receptors may constitute part of the neuroendocrine mechanism relaying the effects of photoperiod.     

N1-dansyl-spermine: a potent polyamine antagonist

The potential polyamine antagonist action of N1-dansyl-spermine (a potent NMDA antagonist) was assessed in two in vivo mouse models of polyamine action. Co-administration of N1-dansyl-spermine (2-10 microg, i.c.v.) with spermine (100 microg, i.c.v.) resulted in a dose-dependent antagonism of the spermine-induced CNS excitation (body tremor and fatal tonic convulsions). In addition, the same dose of N1-dansyl-spermine antagonised spermine's enhancement of NMDA-induced convulsions. These results suggest that N1-dansyl-spermine is in vivo a potent antagonist of the CNS effects of spermine and of its action at the positive polyamine modulatory site on the NMDA receptor.     

Intrinsic redox properties of N-methyl-d-aspartate receptor can determine the developmental expression of excitotoxicity in rat cortical neurons in vitro

The sensitivity of central neurons in culture to N-methyl-d-aspartate (NMDA) receptor-mediated cell death increases with development. In this study, we show that this phenomenon in vitro may be due, at least in part, to changes in the redox properties of the NMDA receptor itself. With increasing days in culture, NMDA-induced electrical responses in rat cortical neurons are less sensitive to dithiothreitol-induced potentiation and spontaneously oxidize less readily than in younger cells. These results imply that at earlier developmental ages NMDA receptors prefer a more oxidized state. Hence, in the presence of a reducing agent, NMDA-induced neurotoxicity was produced in normally resistant younger neurons. The observed changes in NMDA receptor properties with development could not be attributed to long-range diffusible redox endogenous factors. An oxidized NMDA receptor thus confers maturing neurons a protective mechanism against glutamate toxicity during development.     

Characterization of N-methyl-d-aspartate receptors in the hyperammonemic Sparse fur mouse

The N-methyl- -aspartate (NMDA) receptor, a glutamate receptor subtype, is a ligand-gated ion channel. Overstimulation of NMDA receptors may increase intracellular Ca²⁺ concentrations to lethal levels in neurodegenerative disorders affecting the basal ganglia. Such excitotoxicity may also contribute to the loss of medium spiny neurons in the striata of the hyperammonemic sparse fur (spf/Y) mouse, a model of the X-linked disorder of the urea cycle, ornithine carbamoyltransferase deficiency (OCTD). Levels of quinolinic acid (QA), a potent NMDA agonist, are elevated in the brains of spf/Y mice. Further, direct injection of QA into the striatum produces selective degeneration of medium spiny neurons. Microglia, an endogenous source of QA in the brain, are abundant in spf/Y mice during the period of neuronal degeneration. The location and density of NMDA receptors was visualized by gold labelled immunocytochemistry with a polyclonal antibody to the NMDAR1 receptor subtype and their distribution quantified. A 58% reduction was found in the median density value in the layer V pyramidal neurons in fronto-parietal cortex (p<0.001), but no significant change was observed in the striatum. NMDA receptor binding was examined using [ ]dizocilpine ([ ]MK-801). Receptor density (B_max) in the striata of clinically stable spf/Y mice and +/Y littermates was unchanged, but was decreased 15% (p<0.01) in the fronto-parietal cortices in clinically stable spf/Y mice compared with +/Y littermate controls.