Ethanol, stroke, brain damage, and excitotoxicity

The N-methyl-d-aspartate (NMDA)-glutamate receptor could contribute to stroke, trauma, and alcohol-induced brain damage through activation of nitric oxide formation and excitotoxicity. In rat primary cortical cultures NMDA was more potent at activating nitric oxide formation than triggering excitotoxicity. Ethanol dose dependently inhibited both responses. In contrast, treatment of neuronal cultures with ethanol (100 mM) for 4 days significantly increased NMDA stimulated nitric oxide formation and excitotoxicity. These findings suggest that ethanol acutely inhibits but chronically causes supersensitivity to NMDA-induced excitotoxicity in neuronal cultures. To investigate ethanol's interaction with stroke induced damage models of global cerebral ischemia were studied. Transient global ischemia resulted in a loss of hippocampal CA1 pyramidal neurons over a 3- to 5-day period. Determinations of the NMDA receptor ligand binding stoichiometry or postischemic receptor binding changes did not show differences between neurons that undergo delayed neuronal death following ischemia and those that show no toxicity, for example, CA1 and dentate gyrus, respectively. Acute ethanol (3 g/kg) was found to protect against ischemia-induced CA1 hippocampal damage by lowering body temperature, but not under temperature controled conditions. These studies indicate that the factors contributing to stroke-induced brain damage are complex, although they are consistent with chronic ethanol increasing stroke-induced brain damage by increasing NMDA excitotoxicity.     

Antagonists for group I mGluRs attenuate excitotoxic neuronal death in cortical cultures

Activation of ion channel-linked glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, mediates the excitotoxic effects of glutamate upon central neurons. We examined the hypothesis that activation of group I metabotropic glutamate receptors (mGluRs) would increase NMDA receptor-mediated cortical neuronal death. Addition of the selective group I mGluR agonists, dihydroxyphenylglycine (DHPG) or trans-azetidine-2,4-dicarboxylic acid (t-ADA) potentiated NMDA-induced neuronal death, and application of the group I mGluR-selective antagonist, aminoindan-1,5-dicarboxylic acid (AIDA), as well as the non-selective antagonists methyl-4-carboxyphenylglycine (MCPG) or 4-carboxyphenylglycine (4CPG) reduced NMDA- and kainate-induced neuronal death in murine cortical cultures. The pro-excitotoxic effect of group I mGluR activation may be mediated largely by enhancement of glutamate release, as DHPG potentiated high potassium-stimulated glutamate release, and the protective effects of both AIDA and MCPG were abolished when NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors were blocked immediately after toxic NMDA receptor overstimulation. The present data support the possibility that antagonizing group I mGluRs may be a useful strategy for attenuating excitotoxic neuronal death in certain disease states.     

Effects of androgen deprivation on prostate alpha 1-adrenergic receptors

Twenty-four hour exposure to cycloheximide produced a concentration-dependent reduction in protein synthesis in mouse cortical cell cultures. Unexpectedly, a 24 h pretreatment with cycloheximide exposure also reduced neuronal vulnerability to subsequent oxygen-glucose deprivation-induced injury, measured both acutely (cell swelling) or after one day (cell lysis). This neuroprotective effect was attenuated if the period of cycloheximide pretreatment was shortened to 8 h, and lost if the pretreatment was shortened to 1 h. A comparable neuroprotective effect was also induced by 24 h pretreatment with another protein synthesis inhibitor, emetine. The neuroprotection induced by pretreatment with cycloheximide or emetine was probably not attributable to reduction of apoptosis: (i) neuronal death under these conditions occurs by N-methyl-D-aspartate receptor-mediated excitotoxic necrosis, not apoptosis; (ii) the same cycloheximide pretreatment did not block staurosporine-induced apoptosis. Also unlikely as an explanation is reduction in postsynaptic vulnerability to excitotoxicity, as death induced by exogenous addition of N-methyl-D-aspartate, kainate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was little affected by cycloheximide pretreatment. Rather, the protective effect of cycloheximide pretreatment was probably explained, at least in part, by marked reduction in the glutamate release induced by oxygen-glucose deprivation.     

Carboxyfullerenes as neuroprotective agents

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.     

Ifenprodil prevents glutamate cytotoxicity via polyamine modulatory sites of N-methyl-D-aspartate receptors in cultured cortical neurons

Neuroprotective effects of ifenprodil, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, against glutamate cytotoxicity were examined in cultured rat cortical neurons. The viability of the cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 60 min. Ifenprodil and its derivative SL 82.0715 dose-dependently prevented cell death induced by glutamate. The NMDA antagonists MK-801 and 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid also prevented glutamate cytotoxicity with a potency similar to that of ifenprodil. Ifenprodil as well as MK-801 prevented NMDA-induced cytotoxicity, but did not affect kainate-induced cytotoxicity. Glutamate cytotoxicity was inhibited by removing extracellular Ca++ during and immediately after glutamate exposure. Ifenprodil and MK-801 reduced NMDA-induced Ca++ influx measured with rhod-2. Either spermidine, a polyamine modulatory site agonist, or glycine, a strychnine-insensitive glycine site agonist, potentiated NMDA- and glutamate-induced cytotoxicity. The protective effects of ifenprodil against NMDA- and glutamate-induced cytotoxicity were significantly reduced by spermidine, but not by glycine. These findings indicate that ifenprodil protects cortical neurons against glutamate cytotoxicity by selective antagonism of the polyamine modulatory site of the NMDA receptor complex.     

NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.     

Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line

Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes.     

Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.     

Opposing regulation of tau protein levels by ionotropic and metabotropic glutamate receptors in human NT2 neurons

Human NT2-N neurons derived from retinoic acid treatment of the NTera 2 cell line were used to determine the consequences of ionotropic glutamate receptor (iGluR) hyperstimulation and possible modulatory role(s) exerted by metabotropic glutamate receptor (mGluR) activation. We found that NT2-N neurons express the NR1 subunit of N-methyl-D-aspartate (NMDA) iGluRs and mRNA encoding the 1a isoform of mGluRs. A 15 min pulse with 100 microM NMDA induced an increase in the levels of tau proteins in NT2-N cells. This effect was prevented by incubating NT2-N neurons in the presence of the mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection against NMDA-induced NT2-N cell death. Our findings suggest that iGluRs and mGluRs might participate in the control of human neuron viability by differentially affecting the expression of tau proteins.     

NMDA receptor-mediated differential laminar susceptibility to the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in rat neocortical slices

Slices of somatosensory cortex taken from immature rats on postnatal day (P)7-14 were labeled with fura-2. Intracellular Ca2+ concentration ([Ca2+]i) was monitored in identified pyramidal cells as the ratio of fluorescence intensities (RF340/F380) during oxygen-glucose deprivation. The RF340/F380 ([Ca2+]i) of individual pyramidal cells was monitored in each of the cortical layers II-VI simultaneously. Neurons in all neocortical layers exhibited significant increases in [Ca2+]i that varied with the duration of oxygen-glucose deprivation. Individual neurons responded to oxygen-glucose deprivation with abrupt increases in [Ca2+]i after various latencies. The ceiling level of the [Ca2+]i increase differed from cell to cell. Neurons in layer II/III showed significantly greater increases in [Ca2+]i than those in layers IV, V, or VI. Kynurenic acid, a nonselective glutamate receptor antagonist, and bicuculline, a selective gamma-aminobutyric acid (GABA)A receptor antagonist, suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all neocortical layers examined. After kynurenic acid, but not after bicuculline, there was no longer a differential [Ca2+]i increases in layer II/III. Both 2-amino-5-phosphonopentanoic acid (AP5), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, strongly suppressed the intracellular Ca2+ accumulation induced by oxygen-glucose deprivation in all layers. The laminar difference in terms of the [Ca2+]i increases was abolished by AP5, but not by CNQX. These results indicate that layer II/III cells are the most prone to oxygen-glucose deprivation-induced intracellular Ca2+ accumulation, and that this is primarily mediated by NMDA receptors. Thus, layer II/III neurons would be more likely to suffer cellular Ca2+ overload and excitotoxicity during ischemia than layer IV-VI cells. Such a differential laminar vulnerability might play an important role in determining the pathological characteristics of the immature cortex and its sequelae later in life.     

Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins

Neuronal calcium loading attributable to hypoxic/ischemic injury is believed to trigger neurotoxicity. We examined in organotypic hippocampal slice cultures whether artificially and reversibly enhancing the Ca2+ buffering capacity of neurons reduces the neurotoxic sequelae of oxygen-glucose deprivation (OGD), whether such manipulation has neurotoxic potential, and whether the mechanism underlying these effects is pre- or postsynaptic. Neurodegeneration caused over 24 hr by 60 min of OGD was triggered largely by NMDA receptor activation and was attenuated temporarily by pretreating the slices with cell-permeant Ca2+ buffers such as 1, 2 bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated autoradiographically using slices loaded with 14C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of 14C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca2+ buffering originated presynaptically because BAPTA-AM was ineffective when endogenous transmitter release was bypassed by directly applying NMDA to the cultures, and because pretreatment with the low Ca2+ affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester, which attenuates excitatory transmitter release, attenuated neurodegeneration. Thus, in cultured hippocampal slices, enhancing neuronal Ca2+ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).     

Analysis and degradation study of glyphosate and of aminomethylphosphonic acid in natural waters by means of polymeric and ion-exchange solid-phase extraction columns followed by ion chromatography-post-column derivatization with fluorescence detection

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.     

Serum deprivation-induced apoptosis in cultured hippocampi is prevented by kainate

Serum deprivation of hippocampal organotypic cultures induced cell death within 6 h in dentate gyrus granule cells and hilar interneurons whereas neurons from other hippocampal regions were spared. Dying neurons exhibited condensed chromatin in the nuclei, as revealed by cresyl violet, Hoescht staining, and electron microscopy. Cell death was abolished by cycloheximide. KA, an agonist of AMPA/KA receptors that induces depolarization, also prevented neuronal death. This effect was antagonized by the AMPA/KA receptor antagonist DNQX, but not by APV, an antagonist of NMDA receptors. PTX, a GABA(A) receptor antagonist, reduced neuronal death by 50% after serum withdrawal. These data indicate that protein synthesis-dependent programmed cell death (PCD) occurs in the dentate gyrus upon trophic support withdrawal and suggest that neuronal activity contributes to cellular homeostasis.     

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3alpha-ol-5beta-pregnan-20-one hemisuccinate (3alpha5betaHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3alpha5betaHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3alpha5betaHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3alpha5betaHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.     

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.     

Neurotoxicity of ammonia and glutamate: molecular mechanisms and prevention

Ammonia is a main factor in the pathogenesis of hepatic encephalopathy. We found that acute ammonia toxicity is mediated by activation of NMDA receptors. Chronic moderate hyperammonemia prevents acute ammonia toxicity in rats. Chronic exposure of cultured neurons to 1 mM ammonia leads to impaired response of the NMDA receptor to activation by its agonists (due to decreased protein kinase C-mediated phosphorylation) and prevents glutamate (Glu) neurotoxicity. Compounds that prevent ammonia toxicity in mice (e.g. carnitine) also prevent Glu toxicity in cultured neurons. These compounds did not prevent activation of NMDA receptor or the rise of Ca2+. They interfered with subsequent steps in the toxic process. The protective effect of carnitine is mediated by activation of metabotropic Glu receptors. Agonists of mGluRs, especially of mGluR5, prevent Glu toxicity. Agonists of muscarinic receptors also prevent Glu toxicity and there seems to be an interplay between muscarinic and metabotropic Glu receptors in the protective effect. We have tried to identify intracellular events involved in the process of neuronal death. It is known that the rise of Ca2+ is an essential step. Glu leads to depletion of ATP; some compounds (e.g. carnitine) prevent Glu-induced neuronal death without preventing ATP depletion: additional events are required for neuronal death. Glu induces activation of Na+/K+-ATPase, which could be involved in the toxic process. Inhibitors of protein kinase C, calcineurin or nitric oxide synthase prevent Glu toxicity. Our results indicate that Glu toxicity can be prevented at different steps or by activating receptors coupled to the transduction pathways interfering with the toxic process. Agents acting on these steps could prevent excitotoxicity in vivo in animals.     

Lumbar disk herniation combined with epidural hematoma. A case report

We examined the protective effect of nicotine against neuronal cell death in the model system of serum- and nerve growth factor (NGF)-free cultures of PC12 cells. Serum deprivation induced massive death of undifferentiated PC12 cells, which was inhibited by the addition of NGF, but not by the addition nicotine (100 microM). Even after PC12 cells had been differentiated by the treatment with NGF, serum and NGF deprivation induced rapid and massive death of these cells. The addition of nicotine (1-100 microM) to the deprivation system prevented such cell death almost completely. The protective effect of nicotine was abolished by hexamethonium or mecamylamine, inhibitors of nicotinic acetylcholine (nACh) receptors. Treatment with reserpine to deplete intracellular catecholamines did not influence the effect. The results suggest that the protective effect of nicotine against neuronal cell death is exerted through nACh receptors.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane- 1,3-dicarboxylic acid [(IS,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-alpha-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and I mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-(alpha-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methylD-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 microM) only the 50 microM infusion significantly decreased dialysate 5-HT. The GABA(A) receptor agonist, bicuculline (30 microM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG's group II/group II mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.     

Metabotropic glutamate receptors are involved in long-term potentiation in isolated slices of rat medial frontal cortex

The prelimbic region of medial frontal cortex in the rat receives a direct input from the hippocampus and this functional connection is essential for aspects of spatial memory. Activity-dependent changes in the effectiveness of synaptic transmission in the medial frontal cortex, namely long-term potentiation (LTP) and long-term depression (LTD) can persist for tens of minutes or hours and may be the basis of learning and memory storage. Glutamatergic activation of ionotropic receptors is required to induce both LTP and LTD. We now present evidence of the involvement of metabotropic glutamate receptors in LTP in isolated slices of frontal cortex. Repetitive bursts of stimulation at theta frequencies (TBS) were applied to layer II, and monosynaptic EPSPs were monitored in layer V neurons of the prelimbic area. TBS was found to be more effective at inducing LTP than tetanic stimulation at 100 Hz and produced LTP that lasted >30 min in 8 out of 14 neurons. Tetanic stimulation at 100 Hz in the presence of the N-methyl--aspartate (NMDA)-antagonist 2-amino-5-phosphonopentanoate (AP5) was reported to be a reliable method of inducing LTD in prelimbic cortex (). However we found that this protocol did not facilitate the induction of LTD. The role of metabotropic glutamate receptors (mGluR) in LTP was assessed by using the selective, broad-spectrum antagonist (R, S)-alpha-methyl-4- carboxyphenylglycine (MCPG). This drug significantly reduced the incidence of LTP after TBS to only 1 of 14 neurons (P < 0.02, chi2 test). The pooled responses to TBS in MCPG showed significantly reduced potentiation [(P < 0.02, analysis of variance (ANOVA)]. The broad-spectrum mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and the selective group I agonist S-3 hydroxyphenylglycine(S-3HPG) both produced membrane depolarization, an increase in number of spikes evoked by depolarizing current pulses, and a reduction in the afterhyperpolarization. Similar effects were produced by these agonists even when synaptic transmission was blocked by use of the gamma-aminobutyric acid-B (GABAB) receptor agonist, 200 microM baclofen, which suggests that group I mGluRs are present on layer V neurons. We conclude that mGluRs participate in the production of LTP in prelimbic cortex, and that this excitatory effect could be mediated by the postsynaptic group I mGluRs.