Diabetic Goto-Kakizaki rats display pronounced hyperglycemia and longer-lasting cognitive impairments following ischemia induced by cortical compression.

Hyperglycemia has been shown to worsen the outcome of brain ischemia in several animal models but few experimental studies have investigated impairments in cognition induced by ischemic brain lesions in hyperglycemic animals. The Goto-Kakizaki (GK) rat naturally develops type 2 diabetes characterized by mild hyperglycemia and insulin resistance. We hypothesized that GK rats would display more severe cerebral damage due to hyperglycemia-aggravated brain injury and, accordingly, more severe cognitive impairments. In this study, recovery of motor and cognitive functions of GK and healthy Wistar rats was examined following extradural compression (EC) of the sensorimotor cortex. For this purpose, tests of vestibulomotor function (beam-walking) and combined tests of motor function and learning (locomotor activity from day (D) 1 to D5, operant lever-pressing from D14 to D25) were used. EC consistently reduced cerebral blood flow in both strains. Anesthesia-challenge and EC resulted in pronounced hyperglycemia in GK but not in Wistar rats. Lower beam-walking scores, increased locomotor activity, impairments in long-term habituation and learning of operant lever-pressing were more pronounced and observed at later time-points in GK rats. Fluoro-Jade, a marker of irreversible neuronal degeneration, revealed consistent degeneration in the ipsilateral cortex, hippocampus and thalamus at 2, 7 and 14 days post-compression. The amount of degeneration in these structures was considerably higher in GK rats. Thus, GK rats exhibited marked hyperglycemia during EC, as well as longer-lasting behavioral deficits and increased neurodegeneration during recovery. The GK rat is thus an attractive model for neuropathologic and cognitive studies after ischemic brain injury in hyperglycemic rats.

Role of polyamines and NMDA receptors in ethanol dependence and withdrawal.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was John M. Littleton. The presentations were (1) Examination of ethanol spermine and acamprosate actions on native and recombinant NMDA receptors, by David Lovinger; (2) Ethanol inhibition of NMDA neurotoxicity on the polyamine site in cerebellar granule cells, by Sture Liljequist; (3) Alterations in expression of NMDA receptor subunits during ethanol exposure and withdrawal, by Raj Ticku; (4) Alterations in polyamine synthesis and release as a potential mechanism for ethanol dependence and withdrawal, by Izuru Matsumoto; (5) The role of polyamines in neurotoxicity induced by alcohol withdrawal in vitro, by John Littleton; and (6) Agmatine reduces some of the effects of "third trimester" alcohol exposure using a rodent model, by Susan Barron.

Increased ambient glutamate concentration alters the expression of NMDA receptor subunits in cerebellar granule neurons.

Effects of prolonged (48 h) inhibition of glutamate reuptake on the relative abundance of mRNAs coding for N-methyl-D-aspartate (NMDA) receptor subunits, and the expression of corresponding proteins were investigated in primary cultures of rat cerebellar granule neurons. In cells exposed to the glutamate transport blocker, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), the expression of the C1 exon-positive NR1 mRNA variant was reduced by about 40% whereas, the expression of C1-negative mRNA was increased leading to significant reduction of the +C1/-C1 ratio. The expression of the N1-negative NR1 variants was slightly reduced following exposure to PDC, indicating that increased medium levels of glutamate changed the relative abundance of NR1 splice-variant expression but did not reduce the overall NR1 transcription. Expression of NR2A and NR2B mRNAs was 40-50% lower in PDC-treated cells as compared to control. Immunoblot experiments revealed that PDC exposure reduced the expression of NR1 and all NR2 proteins with NR2A and NR2B proteins being reduced to a greater extent than NR1. The overall decrease in NMDA receptor subunit protein expression was considerably more pronounced than the reduction of their corresponding mRNAs, suggesting involvement of a post-transcriptional regulation. Our data support the hypothesis that functional activity and number of NMDA receptors are regulated by strength of the glutamatergic input. Thus, reduced glutamate uptake resulting in increased concentration of ambient glutamate initiate a series of adaptive responses manifested as a gradual down-regulation of the functional activity and expression of NMDA receptors.

Cortical and striatal neuronal cultures of the same embryonic origin show intrinsic differences in glutamate receptor expression and vulnerability to excitotoxicity.

Cortical and striatal cultures were prepared from the same embryonic rat brains and maintained in identical culture conditions. In this way, the intrinsic, genetically imprinted differences determine the responses of cortical and striatal neurons in comparative studies. Cortical and striatal neurons differed in their sensitivity to glutamate receptor-mediated neurotoxicity as measured by the MTT cell viability assay. On the 8th day in vitro, striatal cultures were less sensitive to N-methyl-d-aspartate (NMDA)-induced toxicity than cortical, although both cultures were equally vulnerable to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)- or kainate-induced toxicity. The AMPA receptor-mediated cell death in cortical cultures, however, was much more dependent on preventing AMPA receptor desensitization than in striatal cultures. Furthermore, glutamate-induced neurotoxicity was primarily mediated by NMDA receptors in cortical cultures, while blockade of either NMDA or AMPA receptors gave almost complete protection against glutamate in striatal cultures. To elucidate the molecular mechanisms responsible for the observed differences, we analyzed the expression of NMDA receptor subunits (NR1, NR2A-C) at the mRNA and the protein level in cortical and striatal cultures as well as in standard cerebellar granule cell cultures. The lowest expression level of NMDA receptor subunits was found in striatal cultures, thereby providing a possible explanation for their lower sensitivity to NMDA. Remarkable differences were found between the relative rates of mRNA and protein expression for NR1 and NR2B in the three cultures, indicative of intrinsic differences in the posttranscriptional regulation of NMDA receptor subunit expression in cultures from various brain regions.

Kainate receptor-mediated activation of the AP-1 transcription factor complex in cultured rat cerebellar granule cells.

The sequence-specific DNA-binding activity of the AP-1 transcription factor complex was measured in cultured rat cerebellar granule cells by electrophoretic mobility shift assay. A low concentration of kainate (KA; 10 microM), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 microM), enhanced DNA-binding of the AP-1 transcription factor in cultures pretreated with Concanavalin A (Con A), to prevent KA receptor desensitization. In the presence of cyclothiazide (an inhibitor of AMPA receptor desensitization), KA (10 microM) caused only a slight increase of AP-1 DNA-binding, in contrast to the threefold enhancement produced by AMPA (10 or 30 microM) or by a higher concentration of KA (30 microM), suggesting that the effect of KA, in the presence of Con A, is mediated by activation of putative KA receptors. To confirm this, the effects of the AMPA receptor-selective, non-competitive antagonist, 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3,4-dihydro-7, 8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655; 50 microM), the mixed AMPA/KA receptor competitive antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), and the AMPA and GluR5 KA receptor competitive antagonist, (-)(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8, 8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY 326325; 100 microM), were examined on AMPA- and KA-induced AP-1 activation, respectively. Our results suggest that stimulation of native KA receptors is responsible for the observed KA-specific activation of the AP-1 transcription factor complex.

Chronic ethanol enhances muscarinic receptor-mediated activator protein-1 (AP-1) DNA binding in cerebellar granule cells.

In this study we investigated effects of acute and chronic ethanol exposure on carbachol-induced activator protein-1 (AP-1) DNA binding in rat cerebellar granule cells. Acute ethanol application did not alter, whereas chronic ethanol exposure potentiated the carbachol-induced AP-1 DNA binding. The protein composition of the AP-1 transcription factor complex activated by carbachol stimulation of muscarinic receptors was analysed in control and chronic ethanol-exposed cells using a supershift assay with specific antibodies against c-Fos, Fos B, c-Jun, Jun B and Jun D proteins. Supershift analysis revealed that the carbachol-induced AP-1 complex was composed predominantly of Jun D and c-Fos. The composition of the AP-1 complex activated by carbachol in chronic ethanol-exposed cells did not differ from control. These findings indicate that chronic ethanol treatment can modulate carbachol-induced AP-1 DNA binding activity in cerebellar granule cells.

Prolonged enhancement of AP-1 DNA binding by blockade of glutamate uptake in cultured neurons.

Prolonged blockade of glutamate reuptake by the specific inhibitor of glutamate transporters, L-transpyrrolidine-2,4-dicarboxylate (PDC), produces a dramatic decrease in NMDA-induced neurotoxicity in cerebellar granule cell cultures, and is accompanied by a down-regulation of NMDA receptors. We now report that cultured cerebellar granule cells treated with 100 microM PDC for 1, 2, 4, 8, 16 and 24h, respectively, show increased AP-1 DNA-binding activity as measured by electrophoretic mobility shift assay. This effect was blocked by the NMDA receptor antagonist, CGP 37849, indicative of a pivotal role of NMDA receptors in the PDC-evoked enhancement of AP-1 DNA-binding. Our results suggest that AP-1 may be involved in the transcriptional regulation of neuronal adaptation initiated by prolonged inhibition of glutamate reuptake.

Site-specific NMDA receptor antagonists produce differential effects on cocaine self-administration in rats.

The effects of site-specific NMDA receptor antagonists on intravenous cocaine self-administration were examined in rats trained to self-administer cocaine (0.25 mg/infusion) on a fixed ratio (FR) 5 schedule with a 20-s time-out (TO) after each reinforcer. The non-competitive NMDA receptor antagonists, dizocilpine (MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (0.05-0.2 mg/kg i.p.) and memantine (1,3-dimethyl-5-amino-adamantane hydrochloride) (2.5-20 mg/kg i.p.), dose-dependently decreased cocaine self-administration, while the competitive NMDA receptor antagonist, CGP 39551 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid carboxyethylester) (2.5-15 mg/kg i.p.), and the NMDA/glycine receptor antagonist, L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)-phenyl-2(H)quinolone) (1.25-10 mg/kg p.o.), were without effect. Under a progressive ratio (PR) schedule, dizocilpine (0.15 mg/kg i.p.) increased the number of cocaine infusions in a manner similar to increasing the unit dose of cocaine, suggestive of potentiation of cocaine reward. Conversely, memantine (10 mg/kg i.p.) produced rate-decreasing effects on the PR schedule. These results demonstrate that NMDA receptor antagonists acting at different modulatory sites of the NMDA receptor do not share dizocilpine's cocaine reward enhancing effects although they are all known to be effective blockers of NMDA receptor activity.

Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells.

In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high-affinity glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 100 microM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of[3H]MK-801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC-exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC-exposed cells, however, the acute glutamate-induced NMDA receptor-mediated calcium fluxes were strongly diminished and no toxicity was observed. The down-regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK-801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.

Enhancement of NMDA-induced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells.

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.

The putative AMPA receptor antagonist, LY326325, produces anxiolytic-like effects without altering locomotor activity in rats.

Anxiolytic-like effects produced by the novel, water-soluble AMPA/kainate receptor antagonist, LY326325 (3RS,4aRS,6RS,8aRS)-6-[2-(1(2)H-tetrazole-5-yl)e thyl]decahydro-isoquinoline-3-carboxylic acid), were examined in the elevated plus-maze and in a conflict-suppressed drinking situation. Administration of low doses (0.5, 1.2, and 5 mg/kg; i.p., -30 min) of LY326325 to Sprague-Dawley rats did not alter the percentage of entries into the open arms of the plus-maze, whereas only one dose of LY326325 (1 mg/kg) produced a slight, but significant, increase of the time spent in the open arms of the plus maze. In the conflict-suppressed drinking test, similar doses of LY326325 (2.5 and 5 mg/kg; i.p., -30 min) caused a dose-dependent and significant increase of punished drinking behavior without having any significant effects on unpunished drinking. The anxiolytic-like effects of LY326325 in the plus-maze and in the anticonflict tests were observed at doses, which, by themselves, had no influence on various measures of locomotor activity, i.e., horizontal activity, forward locomotion, and corner time. Our data suggest that the putative AMPA/glutamate receptor antagonist, LY326325, produces anxiolytic-like effects similar to those of diazepam in the conflict-suppressed drinking test, but displays considerably weaker anxiety-reducing properties compared to diazepam in the elevated plus-maze.

A characterization of anxiolytic-like actions induced by the novel NMDA/glycine site antagonist, L-701,324.

The effects of the NMDA/glycine site antagonist, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolone (L-701,324), and the benzodiazepine receptor agonist, diazepam, were examined in the elevated plus-maze and in the Vogel's conflict test. Oral administration of L-701,324 caused a dose-dependent increase (2.5 and 5.0 mg/kg, -30 min) in the percent time spent in the open arms with no change in the total number of arm entries or in the percent entries into the open arms of the plus-maze. The same doses of L-701,324 increased punished responding in the Vogel's conflict test in a dose-dependent fashion, with no influence on unpunished drinking behavior. The anxiolytic-like effects of L-701,324 were obtained at doses which by themselves had no influence on the locomotor activity of the animals. Diazepam (2 mg/kg, i.p., -30 min) was slightly more effective than L-701,324 in the plus-maze situation, whereas the increase in punished drinking in the Vogel's test was of the same magnitude for both compounds. Our present results suggest that inhibition of NMDA receptor activity via a blockade of the NMDA/glycine-sensitive site at the NMDA receptor is accompanied by a reduction of anxiety-like behavior in both non-conditioned and conditioned conflict behavior situations.

Metabolic inhibition potentiates AMPA-induced Ca2+ fluxes and neurotoxicity in rat cerebellar granule cells.

The effects of partial metabolic inhibition (induced by 2 h exposure to low concentrations of cyanide (NaCN)) on the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced excitotoxicity and elevation of free cytoplasmic Ca2+ levels ([Ca2+]i) were studied in glucose-deprived primary cultures of cerebellar granule cells. Co-application of AMPA plus NaCN caused a marked increase of cell death, with morphological features of both necrotic and apoptotic cell death as estimated by the capacity of cultured cerebellar granule cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan (MTT method), and by measuring the amount of DNA fragmentation in neurons using an ELISA test for histone-bound DNA fragments, respectively. Cell morphology was assessed by confocal microscopy of propidium iodide-stained cultures. No toxic effects were observed when AMPA or a low concentration of NaCN (0.1-0.3 mM; in the presence of NMDA receptor antagonist MK-801; 10 microM) were applied alone. The neurotoxic actions induced by AMPA plus NaCN were preceded and accompanied by a significant elevation of [Ca2+]i, as well as by depletion of neuronal ATP stores. The marked enhancement in the functional responsiveness of AMPA receptors in energetically compromised neurons suggests that at least under certain conditions AMPA receptors may play an important role in excitotoxic processes which might be of relevance for the slowly developing neuronal death seen in several neurodegenerative diseases.

Developmental changes in the inhibitory actions of ethanol on glutamate-induced translocation of protein kinase C in cerebellar granule neurons.

The effects of increasing concentrations of ethanol (25-200 mM) on the enhancement of [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding produced by different glutamate receptor agonists, indicative of a translocation of the intracellular enzyme protein kinase C (PKC), were studied in rat cerebellar granule cells at 2, 4, 8, and 12 days in vitro (DIV). Glutamate-produced stimulation of [3H]PDBu binding was inhibited by 50 mM ethanol at 2 DIV, whereas higher ethanol concentrations (> 100 mM) were needed to reduce the increase of [3H]PDBu binding in cells grown for 4, 8, and 12 DIV. Ethanol significantly inhibited NMDA-stimulated [3H]PDBu binding in a concentration-dependent fashion in cells maintained in culture for 4 and 8 days, respectively, with a slightly less pronounced inhibition by ethanol (50 mM) seen in cells kept for 2 and 12 DIV. Application of higher ethanol concentrations (> 100 mM), inhibited the NMDA-induced stimulation in all cell preparations. Following kainic acid-induced enhancement of [3H]PDBu binding, ethanol (100 mM) reduced the binding only in cells maintained for 2 DIV. Even higher ethanol concentrations (200 mM) inhibited the effects of kainic acid only in cells maintained for 2 and 4 DIV, respectively. Our data suggest that various subclasses of glutamate receptors display a developmentally determined differential sensitivity to ethanol at least in cerebellar granule cells in vitro.

The NMDA/glycine receptor antagonist, L-701,324, produces discriminative stimuli similar to those of ethanol.

The ethanol-like discriminative stimulus properties of a novel NMDA glycine receptor antagonist, L-701,324 ((7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2-(1H)-quinolone), a polyamine receptor antagonist, eliprodil, and a non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), were examined in rats trained to discriminate ethanol from vehicle in a two-lever discrimination procedure. In rats trained to discriminate ethanol from vehicle, L-701,324 and MK-801 substituted for ethanol in a dose-dependent fashion with a complete substitution noted following administration of 7.5 mg/kg L-701,324 and 0.2 mg/kg MK-801, respectively. Full substitution for ethanol was achieved with no alteration in the rate of responding. In contrast, administration of eliprodil (in doses up to 5 mg/kg) showed only a partial, but not dose-dependent, substitution for ethanol. These findings indicate that a reduction of NMDA receptor activity, produced either via a blockade of non-competitive NMDA recognition sites or of NMDA/glycine-sensitive regulatory sites, had discriminative stimulus properties that are similar to those produced by ethanol. Furthermore, the observation that the NMDA/glycine receptor antagonist, L-701,324, was a more effective substitute for ethanol than was the polyamine antagonist, eliprodil, suggests that several NMDA receptor subunits, and thus not only NMDAR2B receptor subunits, are of importance for the discriminative stimulus effects of ethanol.

Characterization of NMDA- and AMPA-induced enhancement of AP-1 DNA binding activity in rat cerebellar granule cells.

Effects of the glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), on the activator protein-1 (AP-1) DNA binding activity were studied in primary cultures of rat cerebellar granule cells. Application of NMDA as well as of AMPA produced a concentration-dependent enhancement of AP-1 binding. Further examination revealed that only a brief exposure (10 min) to NMDA or AMPA was required for the initiation of a significant, four- to sixfold enhancement of AP-1 DNA binding activity. Blockade of the desensitization of AMPA receptors by cyclothiazide further reduced the exposure time needed to activate the AP-1 complex. The time needed to achieve a maximal increase of AP-1 binding activity varied depending on the glutamate receptor agonist used. NMDA gave maximal AP-1 stimulation after 60 min exposure, whereas stimulation with AMPA alone reached a maximum after 240 min exposure. When AMPA was applied together with cyclothiazide the maximal enhancement of AP-1 binding was reached much faster, within 120 min. Supershift analysis with specific antibodies against the members of Fos and Jun protein families (c-Fos, Fos B, c-Jun, Jun B, Jun D) revealed that the NMDA-induced AP-1 complex was composed predominantly of Jun D and c-Fos. The composition of the AP-1 complex activated by AMPA alone was similar to that produced by NMDA, but with an additional contribution of Fos B. In contrast, application of AMPA plus cyclothiazide induced an AP-1 transcription with contribution of Jun D, c-Fos, Fos B, c-Jun and Jun B proteins. These findings indicate that glutamate is able to enhance AP-1 DNA binding activity in cerebellar granule cells through both NMDA and AMPA glutamate receptors.

AMPA neurotoxicity in cultured cerebellar granule neurons: mode of cell death.

Various forms of cell death induced by the glutamate receptor agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca2+]i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca2+]i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.

Chronic ethanol enhances NMDA-induced AP-1 activity in cultured rat cerebellar granule cells.

We examined the effects of chronic ethanol exposure (50 mM; 3 days) on N-methyl-D-aspartate (NMDA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-induced AP-1 transcription factor DNA binding activity in primary cultures of rat cerebellar granule cells. Chronic ethanol exposure enhanced NMDA-stimulated AP-1 binding activity, with no corresponding change in AMPA-stimulated AP-1 binding. Supershift analysis with specific antibodies against the members of Fos and Jun protein families showed that the NMDA-induced AP-1 protein complex consisted predominantly of c-Fos and Jun D proteins. Chronic ethanol treatment by itself did not change the protein composition of the AP-1 complex.

Is upregulation of benzodiazepine receptors a compensatory reaction to reduced GABAergic tone in the brain of stressed mice?

Effects of various forms of stress on the GABAA receptor-chloride ionophore complex in the brain of NMRI mice were investigated. Male albino mice were subjected to stress by placing them on small platforms (SP; 3.5 cm diameter) surrounded by water for 24 h. This experimental model contains several stress factors like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. As additional stress control groups we used animals subjected to isolation, large platform (9.0 cm diameter) and repeated swimming stress. SP stress induced an increase in the number of cortical benzodiazepine (BDZ) receptors and a reduction in the GABA-stimulated 36Cl-uptake by brain microsacs, whereas none of these changes could be observed in animals exposed to isolation, swimming or large platform stresses. Furthermore, the amount of GABA-induced stimulation of [3H]flunitrazepam binding was reduced in cortical brain membranes of SP-stressed animals, an effect due to fact that these animals displayed an increase in the basal [3H]flunitrazepam binding, whereas the absolute level of maximally enhanced BDZ binding in the presence of GABA did not differ from those found in controls. Neither basal [3H]muscimol binding or thiopentone sodium-induced stimulation of [3H]flunitrazepam binding were changed in any group of stressed mice. It is proposed that the observed upregulation in the number (Bmax) of cortical BDZ receptors in SP-stressed mice may represent a compensatory response to a stress-induced attenuation of GABAergic neurotransmission.

Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells.

The effects of ethanol and/or glycine on NMDA-induced enhancement of cytoplasmic free Ca2+ concentrations ([Ca2+]i), 45Ca2+ influx, 4-b-[3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding, and neuronal necrosis in cultured rat cortical and cerebellar granule neurons were examined. Using microfluorimetric techniques in combination with rapid perfusion of single brain neurons, we found that glycine (10 microM) was a necessary co-agonist for NMDA-induced depolarization in cerebellar granule cells. In contrast, depolarization with NMDA in cortical cells was observed even without the addition of exogenous glycine as well as in the absence or presence of 1 mM MgCl2. Ethanol (50 mM) inhibited the effects of NMDA in some, but not all, neurons indicative of the existence of ethanol-sensitive and ethanol-insensitive cortical and cerebellar granule neurons. In studies performed in monolayers of cortical and cerebellar granule cells, we observed that the presence of glycine (10 microM) was a necessary prerequisite to unmask inhibitory actions of ethanol on 45Ca2+ influx induced by NMDA. In another set of experiments, we noted that NMDA-induced stimulation of [3H]PDBu binding to monolayers of intact cerebellar granule cells was inhibited by ethanol (50 mM). Finally, we report that ethanol caused a concentration-dependent inhibition of NMDA-induced necrotic cell death, assessed by measuring the ability of cerebellar granule cells to transform 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan. In none of the four assays used to demonstrate the inhibitory effects of ethanol on NMDA receptor activity, the ethanol-induced inhibition was reversed by glycine (up to 100 microM). Thus, in contrast to earlier reports, our data suggest that ethanol and glycine produce their effects by acting at different regulatory sites within the NMDA receptor system in brain neurons.