Delayed transneuronal death of substantia nigra neurons prevented by gamma-aminobutyric acid agonist.

In an investigation of the mechanism by which brain lesions result in delayed degeneration of neurons remote from the site of injury, neurons within the caudate nucleus of rats were destroyed by local injection of the excitotoxin ibotenic acid. Treatment resulted in the rapid degeneration of the striatonigral pathway including projections containing the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and delayed transneuronal death of neurons in the substantia nigra pars reticulata. The distribution of nigral cell loss corresponded to the loss of GABAergic terminals. Neuronal death was prevented by long-term intraventricular infusion of the GABA agonist muscimol. Delayed transneuronal degeneration may be produced by neuronal disinhibition consequent to loss of inhibitory inputs. Replacement of inhibitory transmitters by suitable drugs may prevent some forms of delayed neuronal death.

GYKI 52466, a 2,3-benzodiazepine, is a highly selective, noncompetitive antagonist of AMPA/kainate receptor responses.

In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, the 2,3-benzodiazepine GYKI 52466 was a potent antagonist of kainate- and AMPA-activated currents (IC50 values, 7.5 and 11 microM, respectively), but was inactive against N-methyl-D-aspartate (NMDA) or gamma-aminobutyric acid responses. The block produced by GYKI 52466 occurred in a noncompetitive fashion, was voltage independent, and failed to show use dependence, indicating an allosteric blocking mechanism. In kinetic experiments with kainate as the agonist, the GYKI 52466 binding and unbinding rates were 1.6 x 10(5) M-1 s-1 and 3.2 s-1, respectively. GYKI 52466 also suppressed non-NMDA receptor-mediated spontaneous synaptic currents via a postsynaptic action. Non-competitive AMPA/kainate antagonists such as GYKI 52466 could offer advantages over competitive antagonists in the treatment of glutamate-associated neurological disorders, particularly under conditions in which high levels of the amino acid would render the competitive antagonists relatively ineffective. Moreover, the results demonstrate the existence of a novel recognition site for an atypical benzodiazepine on non-NMDA receptors.

Agomelatine, a melatonin receptor agonist with 5-HT(2C) receptor antagonist properties, protects the developing murine white matter against excitotoxicity.

Periventricular leukomalacia is a major cause of cerebral palsy. Perinatal white matter lesions associated with cerebral palsy appears to involve glutamate excitotoxicity. When injected intracerebrally into newborn mice, the glutamatergic analog, ibotenate, induces white matter cysts mimicking human periventricular leukomalacia. Intraperitoneal injection of melatonin was previously shown to be neuroprotective in this mouse model. The goal of the present study was to compare in this model the protective effects of agomelatine (S 20098), a melatonin derivative, with melatonin. Mice that received intraperitoneal S 20098 or melatonin had significant reductions in size of ibotenate-induced white matter cysts when compared with controls. Although agomelatine and melatonin did not prevent the initial appearance of white matter lesions, they did promote secondary lesion repair. Interestingly, while melatonin effects were only observed when given within the first two hours following the excitotoxic insult, agomelatine was still significantly neuroprotective when administered eight hours after the insult. The protective effects of agomelatine and melatonin were counter-acted by co-administration of luzindole or S 20928, two melatonin receptor antagonists. Agomelatine, acting through melatonin receptors, could represent a promising new drug for treating human periventricular leukomalacia and have beneficial effects on neuroplasticity.

Vasoactive intestinal peptide prevents excitotoxic cell death in the murine developing brain.

Excitotoxic damage may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected at birth, the glutamatergic analog ibotenate induces mouse brain lesions that strikingly mimic human microgyria. When ibotenate is injected at postnatal day 5, it produces transcortical necrosis and white matter cysts that mimic human perinatal hypoxic-like lesions. Vasoactive intestinal peptide (VIP) has potent growth-related actions and neuroprotective properties that influence mitosis and neuronal survival in culture. The goal of this study was to assess the protective role of VIP against excitotoxic lesions induced by ibotenate in developing mouse brain. VIP cotreatment reduced ibotenate-induced microgyric-like cortical lesions and white matter cysts by up to 77 and 85%, respectively. VIP protective effects were reproduced by a peptide derived from activity-dependent neurotrophic factor (ADNF), a trophic factor released by VIP-stimulated astrocytes, and by stearyl norleucine VIP, a specific VIP agonist that does not activate adenylate cyclase. Neither forskolin, an adenylate cyclase activator, nor pituitary adenylate cyclase-activating peptide, provided VIP-like protection. VIP and neurotrophic analogs, acting through a cAMP-independent mechanism and inducing ADNF release, could represent new avenues in the understanding and prevention of human cerebral palsy.

Selective activation of central subtypes of the nicotinic acetylcholine receptor has opposite effects on neonatal excitotoxic brain injuries.

The incidence of neurological disabilities ascribable to perinatal injury is rising in Western countries, raising ethical and financial problems. No curative treatments are available. The pathophysiology of brain lesions of hypoxic-ischemic or inflammatory origin involves various neurotransmitters or neuromodulators. Among these, glutamate plays a key role. By overactivating N-methyl-D-aspartate receptors, it triggers the excitotoxic cascade. Although addictive, nicotine prevents excitotoxic neuronal death in adult animals. Its potential neuroprotective effects have not been evaluated in neonates. We found that nicotine is neuroprotective in vivo, in a murine model of neonatal excitotoxic brain injury, and in vitro, in primary cultures of cortical neurons. We investigated the respective roles in nicotine-related neuroprotection of the two dominant nicotinic acetylcholine receptor (nAChR) isoforms, namely, alpha4beta2 (heteropentameric) and alpha7 (homopentameric). Inhibition of alpha4beta2, either pharmacological (i.e., an alpha4beta2 nAChR antagonist) or molecular (beta2-/- knockout mice), abolished the protective effect of nicotine in vivo and in vitro, suggesting the involvement of alpha4beta2 nAChR in neonatal nicotine-related neuroprotection. In contrast, activation of alpha7 nAChR, which is protective in adult animals, was deleterious in our neonatal model, whereas its blockade, either pharmacological or molecular (alpha7-/- knockout mice) provided neuroprotection. Neuroprotective strategies must consider these opposite properties of distinct nAChR isoforms in neonates.

The pharmacology of quisqualate and AMPA in the cerebral cortex of the rat in vitro.

The depolarising population response to the excitatory amino acids, quisqualate and AMPA, in slices of cerebral cortex of the rat have been compared. Their respective dose-response curves had a similar maximum but the slope of the curve for AMPA was consistently steeper than that for quisqualate. The dose-response curves for AMPA had a mean log EC50 of -5.18 +/- 0.05, which was significantly different from -4.62 +/- 0.07 the mean log EC50 of the dose-response curves for quisqualate. Responses to both agonists were antagonised by kynurenic acid, barbiturates and gamma-DGT to a similar extent. The antagonism by kynurenate appeared to be competitive whilst the barbiturates were evidently noncompetitive antagonists. These results are in agreement with claims that quisqualate and AMPA act at a similar recognition site. The differences in the slopes of the dose-response curves for quisqualate and AMPA may be explained by the differences in the cellular uptake of the two agonists and/or by differences in efficacy.

Phencyclidine prevents spatial navigation and passive avoidance deficits in ibotenate lesioned rats.

The potential neuroprotective effects of phencyclidine (5 mg/kg i.p.) were assessed in rats which had been treated with the excitotoxin, ibotenic acid (IBO) (0.015 M) to lesion the nucleus basalis magnocellularis. IBO treated rats showed a significant impairment in 13 of the 25 test trials in the spatial navigation Morris water maze task and deficits in passive avoidance learning. Phencyclidine was found to prevent the IBO-induced impairment in 4 of the 13 test trials in which the IBO Morris maze deficit was observed and also successfully prevented the passive avoidance learning deficits. Neurochemically, IBO was shown to reduce the levels of gamma amino-n-butyric acid (GABA) in the cortex. This effect of IBO on the inhibitory GABAergic system may contribute to the direct toxic effects of IBO which is mediated through excitatory amino acid receptors. Phencyclidine had no effect on the changes in GABA produced by IBO. The effect of phencyclidine treatment on IBO behavioural toxicity observed in this study demonstrates that antagonism of the phencyclidine receptor site on the N-methyl-D-aspartate receptor complex may be partially protective against the excitotoxic damage induced by IBO.

Synthesis and excitatory amino acid pharmacology of a series of heterocyclic-fused quinoxalinones and quinazolinones.

As part of our program aimed at the development of potent excitatory amino acid antagonists, we synthesized and evaluated a series of substituted 1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-ones, 4, tetrazolo[1,5-a]quinoxalin-4(5H)-ones, 5, and pyrazolo[1,5-c]quinazolin-5(6H)-ones, 6, and an imidazo[1,2-a]quinoxalin-4(5H)-one, 7. In general, the same heterocycles which demonstrated the best affinity for the AMPA receptor also demonstrated the best affinity for the glycine site on the NMDA receptor complex. 1-Propyl-7,8-dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4d, was found to bind with the greatest affinity to the AMPA receptor with an IC50 of 0.83 microM and antagonized 40 microM AMPA-induced depolarization in the cortical slice preparation with an IC50 of 44 microM. 7,8-Dichloro-1,2,4-triazolo[4,3-a]quinoxalin-4(5H)-one, 4a, and 7,8-dichloroimidazo[1,2-a]quinoxalin-4(5H)-one, 7, possessed the best affinity for the glycine site with IC50 values of 0.63 and 1.26 microM, respectively. It is noteworthy that the SAR for the heterocyclic compounds did not directly parallel that of known quinoxalinediones (e.g. DNQX, 2, and DCQX, 15) at the AMPA receptor nor that of the kynurenic acids at the glycine site on the NMDA receptor complex.

Fast excitatory postsynaptic potentials and the responses to excitant amino acids of sympathetic preganglionic neurons in the slice of the cat spinal cord.

The properties of the excitatory postsynaptic potential evoked by focal stimulation and of the responses to excitatory amino acids were examined by intracellular recording from sympathetic preganglionic neurons in upper thoracic spinal cord slices of the adult cat. Single stimuli to the region dorsal to the intermedio-lateral nucleus evoked short-latency, presumably monosynaptic, excitatory postsynaptic potentials. The reversal potential of this response was -2.2 mV and became more negative when external Na+ or K+ concentration was decreased. The excitatory postsynaptic potential was depressed by the non-selective excitatory amino acid receptor antagonist cis-2,3-piperidine dicarboxylic acid and enhanced by a glutamate uptake inhibitor. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2.3-dione abolished the excitatory postsynaptic potential in 72% of neurons. In the remaining neurons, this antagonist only depressed the potential and unmasked a slower component which was abolished by the N-methyl-D-aspartate receptor antagonist D,L-2-amino-5-phosphonovaleric acid. In the presence of tetrodotoxin all neurons tested were depolarized by glutamate or aspartate, as well as by the selective agonists quisqualate, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate. The glutamate-evoked depolarization reversed at a membrane potential of -2.0 mV and at a more negative value when external Na+ or K+ concentration was decreased. The response to alpha-amino-3-hydroxy-5-methylisoxazole propionic acid was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione in all neurons tested and that to kainate in only one-third of the cells. In the remainder the response to kainate was only slightly depressed by this antagonist. The responses to glutamate and aspartate were only slightly depressed by the combined action of the various amino acid receptor antagonists used. The responses to N-methyl-D-aspartate were abolished by D,L-2-amino-5-phosphonovaleric acid. The punched-out region of the intermedio-lateral nucleus, maintained in vitro, released glutamate and aspartate in the absence of stimulation. Field stimulation (20 Hz) enhanced release by between 40 and 100%. The increase was prevented by superfusion with calcium-free Krebs. It is concluded that excitatory amino acids, acting on both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, but mainly on the latter, are likely mediators of the monosynaptic excitatory postsynaptic potential evoked in sympathetic preganglionic neurons by the stimulated region. The efflux data suggest that glutamate and aspartate are among the mediators.

Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin636.

1. Several low molecular weight spider toxins have recently been shown to block potently glutamatergic neuromuscular transmission at the invertebrate neuromuscular junction. The aim of the present investigation was to evaluate the effects of one such toxin, argiotoxin636, on excitatory amino acid receptor-mediated responses in mammalian neurones. 2. Membrane currents were recorded from rat cortical neurones after 2-6 weeks in cell culture, by the whole-cell variant of the patch-clamp technique. N-methyl-D-aspartate (NMDA) and kainate were used as selective agonists for their respective receptor subtypes. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) was used as a selective agonist for the quisqualate receptor subtype. 3. Responses to these agonists were characterised with respect to their concentration and voltage-dependence. Argiotoxin636 (3-30 microM) was found to attenuate markedly responses to NMDA in an agonist- and voltage-dependent manner. Thus, argiotoxin636 progressively reduced successive responses to NMDA when membrane potentials were voltage clamped between -40mV to -100 mV. The more negative the membrane potential the more rapid the development of the block of inward current. 4. The antagonism of NMDA-induced currents by argiotoxin636 could be reversed by clamping the membrane at positive potentials (+20 to +60 mV) and reapplying NMDA. 5. Responses to AMPA and kainate were less affected by argiotoxin636, with an antagonist action only becoming evident at a concentration of 100 microM. 6. These results suggest that argiotoxin636 is an open-channel blocker of the NMDA activated ion-channel in mammalian neurones. Furthermore, our results indicate at least a 30 fold selectivity for NMDA over the quisqualate- and kainate-activated ion-channels.

Philanthotoxin blocks quisqualate-, AMPA- and kainate-, but not NMDA-, induced excitation of rat brainstem neurones in vivo.

1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

Thiocyanate ions selectively antagonize AMPA-evoked responses in Xenopus laevis oocytes microinjected with rat brain mRNA.

1 Responses to kainate (KA), willardiine and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were recorded from rat brain mRNA-injected Xenopus laevis oocytes by use of a two-electrode voltage clamp. 2 Thiocyanate (SCN-; 50 microM-4 mM) ions reversibly and selectively inhibited the membrane current responses to AMPA in a non-competitive manner without affecting KA or willardiine-induced responses. 3 The inhibition of AMPA-induced responses by SCN- was dependent on the SCN- concentration with an estimated IC50 of 1 mM. The antagonism was not dependent on the AMPA concentration. 4 The response to a high concentration of AMPA (100-200 microM) exhibited a peak inward current which declined to a steady-state. SCN- inhibited the steady-state current more than the peak response. The inhibition was unaffected by prior incubation with concanavalin-A (Con-A; 10 microM). 5 Responses to KA were antagonized by AMPA in a competitive manner, suggesting that both agonists may activate a common receptor-channel complex. This interaction between two non-NMDA agonists was not affected by the SCN(-)-induced inhibition of the AMPA response. 6 AMPA-induced responses recorded from large cultured cerebellar neurones by whole-cell recording were also inhibited by SCN- in a non-competitive manner. The AMPA-induced peak current was less affected than the steady-state response. 7 We conclude that SCN- can inhibit the response to AMPA in expressed non-NMDA receptors in Xenopus oocytes and also in native receptors in cultured cerebellar neurones. One possible mechanism of action for SCN- inhibition of responses to AMPA may involve a Con-A-insensitive, non-NMDA receptor-mediated desensitization.

Administration of quinolinic acid in the rat hippocampus induces expression of c-fos and NGFI-A.

We have studied the effect of intrahippocampal administration of quinolinic acid (QUIN) on the temporal expression of mRNAs encoding the immediate early genes (IEGs) c-fos and NGFI-A, by in situ hybridization histochemistry. After administration of QUIN to the left hippocampus, expression of mRNA of both IEGs was transiently stimulated. Maximal expression was found between 1 and 3 h. mRNA of both IEGs was simultaneously expressed in the ipsilateral and contralateral sides in the granule cell layer of the dentate gyrus, the pyramidal cell layer of the CA1 and CA3 fields as well as in the cortex. After pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg i.p. -30 min) the increased expression of both IEGs was partially prevented in the hippocampus and completely in the cortex. No inhibition was observed after treatment with the AMPA antagonist NBQX (30 mg/kg i.p. -15, -5 and +10 min). Additional delayed expression of both IEGs was observed in the ipsilateral hippocampus. This expression was related to cell damage. Twelve h after QUIN administration, c-fos and NGFI-A mRNAs were present in the dentate gyrus. After 4 days, only c-fos mRNA was observed in the dentate gyrus and CA1 field while no NGFI-A mRNA was detected. The present results show that the effect of QUIN is mediated by NMDA and not by AMPA receptors.

The AMPA antagonist, NBQX, protects against ischemia-induced loss of cerebellar Purkinje cells.

We examined the effect of an AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole) antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX), on rat cerebellar Purkinje cell loss and hippocampal pyramidal CA1 cell loss, after 10 minutes of global cerebral ischemia. NBQX was given intraperitoneally in a dose of 30 mg kg-1 at the end of ischemia, and 10 and 25 minutes later. Rats subjected to ischemia without post-ischemic administration of NBQX served as controls. Four days after ischemia the cerebellar Purkinje cell density was higher and the density of acidophilic (dead) Purkinje cells lower in the NBQX treated animals compared with the control animals (p = 0.01 and p less than 0.005 respectively). There was partial to total loss of pyramidal neurons in the CA1 region of the dorsal hippocampus in control animals, but no CA1 pyramidal neuron loss in the NBQX treated animals (p = 0.001).

The influence of prolonged antidepressant treatment on the changes in cyclic AMP accumulation induced by excitatory amino acids in rat cerebral cortical slices.

We investigated the effect of prolonged electroconvulsive shock (ECS) or imipramine treatment on cyclic AMP accumulation induced by ibotenate and glutamate in rat cerebral cortical slices. Prolonged imipramine or electroconvulsive shock treatment attenuated the ibotenate-induced increase in cyclic AMP accumulation and inhibited the synergistic interaction between ibotenate and noradrenaline; the glutamate-mediated inhibition of forskolin-stimulated cyclic AMP accumulation was not modified. Our results indicate that multiple effects of excitatory amino acids on cyclic AMP accumulation are modified differently by antidepressant treatment.

A novel mechanism of AMPA receptor regulation: ionically triggered kinases and phosphatases.

We have postulated elsewhere (Shaw CA and Lanius RA. Dev Brain Res 70, 153-161 (1992)) that the kinase/phosphatase regulation of AMPA receptors is mediated by specific ions. Using an in vitro cortical slice preparation we have now examined the roles of calcium (Ca2+), chloride (Cl-), potassium (K+), and sodium (Na+) in the regulation of AMPA receptors. Ca2+ led to a concentration-dependent decrease in [3H]-CNQX binding which could be blocked by a general protein kinase inhibitor (H-7) and a protein kinase A inhibiting peptide. Tamoxifen, a relatively specific protein kinase C inhibitor, had no effect. In contrast, Cl- led to concentration-dependent increases in [3H]-CNQX binding which could be blocked by both sodium-ortho-vanadate, a tyrosine residue selective phosphatase inhibitor, and sodium-beta-D-glycerol phosphate, a serine residue selective phosphatase blocker. K+ and Na+ had no effect on [3H]-CNQX binding. These results suggest that Ca2+ and Cl- may be acting as signals which trigger kinase(s) and phosphatase(s) involved in the regulation of AMPA receptors.

Attenuation of AMPA-induced neurotoxicity by a calpain inhibitor.

The effects of a membrane-permeable inhibitor of calpain, Cbz-Val-Phe-H, were examined in an in vitro model of neurotoxicity. Cerebellar slices from young rats were treated with the glutamate receptor agonist, amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA), and cytotoxicity was quantified using conventional histological techniques. Slices treated with AMPA exhibited damage to 83.0% of cerebellar Purkinje cells. In contrast, only 23.6% of Purkinje cells were damaged in slices treated with Cbz-Val-Phe-H and AMPA. These findings indicate that calcium-activated proteolysis is a critical event in AMPA-induced toxicity, and provide evidence that calpain inhibitors are capable of attenuating this form of excitotoxic damage in the central nervous system.

NBQX inhibits AMPA-induced locomotion after injection into the nucleus accumbens.

The role of glutamate receptors in locomotor activity was investigated by examining the ability of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX), a non-NMDA antagonist, to inhibit the stimulation of locomotion produced by the activation of various excitatory amino acid receptors in the nucleus accumbens. NBQX inhibited the stimulation of locomotor activity produced by intra-accumbens alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) at doses which had no effect on the locomotion produced by kainate or NMDA. Furthermore, this dose of NBQX had no effect on locomotion when injected alone into this brain region. These data suggest that AMPA receptors in the nucleus accumbens may play a very different role in the control of locomotion than NMDA receptors.

Inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in rat hippocampus by L-aspartate-beta-hydroxamate.

The effect of a series of glutamate uptake inhibitors was tested on ibotenate-stimulated phosphoinositide hydrolysis. The pharmacological profile of the inhibitory effect of these compounds on the ibotenate response was quite different from that on glutamate uptake. Aspartate-beta-hydroxamate was the most potent compound with the L-isomer (IC50 11 +/- 2 microM) being considerably more potent than the D-isomer (IC50 104 +/- 12 microM). The effect of the L-aspartate-beta-hydroxamate was found to be specific for ibotenate and quisqualate-stimulated phosphoinositide hydrolysis; this compound did not affect hydrolysis stimulated by carbachol, K+ or sodium fluoride. The inhibition of the ibotenate response was found to involve a non-competitive and irreversible mechanism.

Ibotenate-induced neuronal degeneration in immature rat brain.

Stereotaxic microinjections of the excitotoxin, ibotenic acid, were made into the striatum, hippocampus or cerebellum of the immature (7-day-old) rat. Two days later, pups were decapitated and the brains processed for light microscopic examination or neurochemical analyses. 10 micrograms ibotenate caused a complete loss of nerve cell bodies throughout the striatum and hippocampus while intracerebellar injections produced no detectable damage. In the striatum, catecholamine histofluorescence was abolished and dopamine uptake severely reduced, indicating also a loss of afferent nerve terminals. Co-injection of 10 micrograms ibotenate with equimolar amounts of the selective amino acid antagonist, (-)-2-amino-7-phosphonoheptanoic acid, resulted in the protection of both striatal cell bodies and dopaminergic nerve terminals. The neurotoxic properties of ibotenate described here are in marked contrast to those of kainic acid, a related excitotoxin. Differences in the ontogenetic pattern of receptors which mediate neurodegenerative events may account for the pharmacological and regional selectivity and the partial lack of axon-sparing properties of ibotenic acid lesions in the immature brain.