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.

Reversible and Species-Specific Depigmentation Effects of AZD3293, a BACE Inhibitor for the Treatment of Alzheimer's Disease, Are Related to BACE2 Inhibition and Confined to Epidermis and Hair.

BACKGROUND: AZD3293 (also known as LY3314814) is a novel, potent, non-selective BACE1/BACE2 inhibitor currently in Phase 3 clinical development for the treatment of Alzheimer's disease. OBJECTIVES: The purpose of these studies was to characterize the effects, putative mechanism, and reversibility of hypopigmentation following treatment with AZD3293 in pigmented Long-Evans rats, Beagle dogs, human cell cultures, and humans. DESIGN: Nonclinical studies were conducted in Long-Evans pigmented rats, and both young and older Beagle dogs using a variety of oral dose levels of AZD3293 or AZD3839 (BACE inhibition reference compound; used in older dogs only) for dosing durations of 13 to 26 weeks. In vitro studies of normal human epidermal melanocytes and reconstituted human epidermis were also conducted. Skin biopsy data from a multiple-dose Phase 1 clinical study of AZD3293 (NCT01795339) are also reported. SETTING: Nonclinical in vivo and in vitro studies were conducted in laboratory settings in the US, Canada, and France; the multiple dose clinical study was conducted in a specialized inpatient setting in the US. PARTICIPANTS: Beagle dogs: 13-week study N=36 young (8-10 mo) animals; 39-week study N=64 young animals; and a second 13-week study N=32 older (30-32 mo) animals. Long-Evans rats: N=68 animals. Multiple-dose clinical study: only data for subjects enrolled in Part 2 of this study are included in this report (N=16). INTERVENTIONS: AZD3293 was the primary intervention used in these studies. AZD3839, a relatively BACE1-selective reference inhibitor compound was used in one group in the 13 week study in older Beagle dogs and one in vitro assessment. Finally, AZ1340, another relatively BACE1-selective reference inhibitor compound was used only in one in vitro assessment. MEASUREMENTS: Measurements for the nonclinical studies in dogs and rats included macroscopic observation and assessment of skin biopsies via histopathology, immunochemistry, and electron microscopy. Measurements for the in vitro studies included melanocyte premelanosome protein (PMEL) processing, cytotoxicity, melanin synthesis, Pmel17 labeling, and melanocyte dendricity. Measurements in the clinical study included scoring of melanin content in skin biopsies taken before and after dosing with AZD3293 over 14 days at dose levels up to 150 mg. RESULTS: Depigmentation in rats and dogs was limited to skin, hair, and mucosa with no effects on other pigmented tissues. At a cellular level depigmentation was observed within a week of treatment, whereas the appearance of depigmentation in skin and hair did not become apparent until, at earliest, 4 weeks of treatment. The depigmentation effects were reversible, not associated with degenerative or inflammatory changes, and were dose- and species-dependent in severity. Full recovery of melanization was observed at the microscopic (cellular) level and at least partial recovery was seen in the macroscopic appearance of animals by the end of the 12-week recovery period in both rats and dogs. Interestingly, no changes in melanin production or melanocyte morphology were seen in human primary melanocytes or reconstituted human epidermis in vitro. Finally, there were no changes in melanization level in skin biopsies following 12 days of daily AZD3293 treatment at doses of AZD3293 up to 150 mg/day in human subjects. CONCLUSIONS: AZD3293, a novel, potent, non-selective BACE1/BACE2 inhibitor is in development as a potentially disease-modifying treatment for Alzheimer's disease. Chronic nonclinical studies in Beagle dogs and pigmented rats showed macroscopic and microscopic hypopigmentation effects of AZD3293 that were limited to skin, hair, and mucosa. These effects were shown to be reversible in both species. Analysis of data from nonclinical and in vitro studies suggests that hypopigmentation is caused by BACE2 inhibition resulting in accumulation of a premelanosome protein fragment, which interrupts the normal production of melanin. No macroscopic or microscopic reports of hypopigmentation were observed in a Phase 1 clinical study following 13 doses of AZD3293 over 14 days at dose levels up to 150 mg/day. These data suggest that hypopigmentation is species-specific and humans appear to be least sensitive to the depigmentation effect caused by BACE2 inhibition.

Effects of decahydroisoquinoline-3-carboxylic acid monohydrate, a novel AMPA receptor antagonist, on glutamate-induced CA2+ responses and neurotoxicity in rat cortical and cerebellar granule neurons.

In this study, we examined the effects of a novel water-soluble, putative AMPA receptor 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 (LY326325), on glutamate-, N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, and kainic acid (KA)-induced elevations of intracellular Ca2+ concentrations ([Ca2+]i) and 45Ca2+ uptake, as well as glutamate agonist-induced neurotoxicity in primary cultures of intact rat cortical and cerebellar granule neurons. In some experiments, the actions of LY326325 were tested in the presence of cyclothiazide, a compound that is known to block glutamate-induced desensitization of AMPA-preferring subtypes of glutamate receptors, thereby largely potentiating the functional effects of AMPA. LY326325 fully blocked the elevations of [Ca2+]i induced by NMDA and non-NMDA glutamate receptor agonists in both cortical and cerebellar granule neurons. The application of increasing concentrations of cyclothiazide was not able to reverse the LY326325-induced blockade of glutamate receptors in cortical neurons. In contrast, the same cyclothiazide treatment fully reversed the blockade produced by the noncompetitive AMPA/KA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine HCl (GYKI 52466). In 45Ca2+ uptake studies. LY325325 inhibited the NMDA-, AMPA-, and KA-induced enhancement of 45Ca2+ uptake in a concentration-dependent fashion in both cortical and cerebellar granule cells. In analogy to the results obtained with [Ca2+]i recordings, cyclothiazide failed to counteract the LY326325-induced blockade of KA-stimulated 45Ca2+ uptake in cerebellar granule neurons, whereas the blockade induced by the noncompetitive AMPA/KA receptor blocking agent GYKI 52466 was fully reversed by cyclothiazide. Because a similar, although not identical pattern of actions was seen following the application of the competitive AMPA/KA receptor antagonist 6-nitro-7-sulphamoyl-benzo(f)quinoxaline-2-3-dione (NBQX), it is suggested that the inhibitory actions of LY326325 are similar to those produced by NBQX but clearly differ from those caused by the noncompetitive AMPA/KA receptor antagonist GYKI 52466. Finally, when the neuroprotective actions of LY326325 on glutamate agonist-induced neurotoxicity were examined in cerebellar granule neurons, we found that LY326325 almost completely blocked the neurotoxic actions of NMDA, AMPA, and KA, respectively, whereas it produced only a partial blockade of glutamate-induced neurotoxicity. Taken together, our current results suggest that although LY326325 blocked both nonNMDA and NMDA-induced Ca2+ responses, it still displayed a preferential affinity of nonNMDA receptors as compared to NMDA receptors. However, LY326325 appears to be a less selective AMPA/KA receptor antagonist than NBQX and GYKI52466, respectively.

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.

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.

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.

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.

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.

Modulation of AMPA/kainate receptors by cyclothiazide increases cytoplasmic free Ca2+ and 45Ca2+ uptake in brain neurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-induced Ca2+ responses, and their modulation by cyclothiazide, were investigated in two functional assays of Ca2+ channel activity. AMPA produced a marked increase in cytoplasmic free Ca2+ levels ([Ca2+]i) in single cortical neurons, whereas no such effects of AMPA could be observed in intact cerebellar granule neurons. In monolayer cultures of cortical cells, cyclothiazide caused a pronounced enhancement of AMPA-induced stimulation of 45Ca2+ uptake, whereas similar studies in cerebellar granule neurons revealed only a weak potentiation of AMPA-induced 45Ca2+ uptake. Higher concentrations of cyclothiazide alone produced [Ca2+]i oscillations as well as an increase of basal 45Ca2+ uptake in cortical neurons, whereas no such effects were obtained in cerebellar granule neurons. Our data indicate that AMPA receptors located on cortical and cerebellar granule neurons, respectively, may differ in their permeability to Ca2+ and that this difference is markedly potentiated following the application of cyclothiazide.

N-Methyl-D-aspartate-induced enhancement of activator protein-1 DNA binding activity is blocked by ethanol in cerebellar granule cells.

The effects of ethanol on N-methyl-D-aspartate (NMDA) and non-NMDA receptor agonist-stimulated activator protein-1 (AP-1) DNA binding activity in primary cultures of rat cerebellar granule cells were investigated. The application of intoxicating concentrations of ethanol produced a concentration-dependent inhibition of NMDA-enhanced AP-1 binding with a significant reduction obtained at 50 mM ethanol. The inhibitory actions of ethanol on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-stimulated AP-1 binding were considerably weaker as compared to the effects seen following administration of NMDA. The AMPA-induced enhancement of AP-1 DNA binding activity was demonstrated both in the absence and presence of cyclothiazide, a drug, which is known to block the desensitization of AMPA receptors. Our data suggest that moderate concentrations of ethanol modulate glutamate-induced alterations of gene expression in brain neurons.

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.

A mu-receptor opioid agonist induces AP-1 and NF-kappa B transcription factor activity in primary cultures of rat cortical neurons.

Activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B) represent mammalian transcription factors which bind to distinct enhancer motifs. The specific mu-receptor opioid agonist, Tyr, D-Ala2, Gly, N-Me-Phe4, Gly-ol5 (DAMGO), was found to increase AP-1 and NF-kappa B activity in primary cultures of neurons from rat cerebral cortex. Acute (2 h, 4 h) and long-term (72 h) treatment with DAMGO time-dependently increased the DNA-binding activity of both AP-1 and NF-kappa B and the stimulation could be abolished or inhibited by concurrent incubation with naloxone. However, acute naloxone-precipitated withdrawal did not significantly change AP-1 or NF-kappa B activity. These results indicate a mu-opioid receptor-related co-induction of AP-1 and NF-kappa B transcription factors in cultured cortical neurons.

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.

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.

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.

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.

Effect of Ro 15-4513 and nifedipine on the behaviour and potassium-evoked calcium uptake following withdrawal from chronic ethanol administration in rats.

The termination of chronic ethanol administration in rats resulted in the development of withdrawal signs at 10 and 24 hours of withdrawal. The behavioural signs of withdrawal were accompanied by an increased potassium-evoked 45Ca2+ uptake by cortical synaptoneurosomes. The dihydropyridine calcium channel antagonist nifedipine given acutely, suppressed the withdrawal signs and abolished the increased 45Ca2+ uptake. The benzodiazepine receptor inverse agonist Ro 15-4513 (4 mg/kg) given at 1.5 hours after termination of chronic ethanol induced behavioural signs of withdrawal and increased potassium-evoked 45Ca2+ uptake in these animals. Ro 15-45-13 given at 24 hours of withdrawal did not further enhance withdrawal signs or 45Ca2+ uptake. These data suggest that an increased calcium uptake during ethanol withdrawal might be attributed to the activation of dihydropyridine-sensitive calcium channels and might be involved in the development of withdrawal signs after chronic ethanol administration.

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.

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.

[The anticonvulsant properties of glutapyrone--a new type of derivative of amino acid-containing 1,4-dihydropyridines]. / Protivosudorozhnye svoistva glutapirona--proizvodnogo novogo tipa--aminokislot-soderzhashchikh 1,4-digidropiridinov.

Experiments on male Wistar rats and Icr:Icl mice studied the influence of the novel compound--amino acid-containing 1,4-dihydropyridine derivative glutapyrone (G) on acute generalized seizures, arecoline and nicotine tremor, and 45Ca2+ uptake in brain synaptosomes. It was shown that G produced significant antiepileptic effects on models of acute pentylenetetrazole seizures on rats and mice. Efficiency of antiepileptic effect depended on a dose and method of modeling seizures: it was more effective in case of intravenously pentylenetetrazole-induced seizure tested by clonic and tonic seizure components and death. The results suggest the participation of GABAergic system in realization of antiepileptic effect of G. Glutapyrone did not influence the 45Ca2+ uptake by rat cortical synaptosomes (evoked by a 1-min depdariration with 55 mM K+), this suggests that G lacked calcium antagonist properties characteristic of 1,4-dihydropyridine compounds such as nifedipine, nimodipine. In addition, G does not affect N- and M-cholinergic processes.