Modulation of SIRT1 expression in different neurodegenerative models and human pathologies.

We examined the expression of SIRT1 in several experimental paradigms of human pathologies. We used a neuroblastoma cell line (B65), neuronal primary cultures (hippocampus and cerebellar granule cells) and in vivo approaches in rat and senescence murine models (SAM). Cell cultures and rats were treated with several well-know neurotoxins, i.e. rotenone, MPP(+), kainate and 3-nitropropionic acid. Subsequently, SIRT1 expression was compared in these different paradigms of neurotoxicity. The pattern of expression of SIRT1 in proliferating cell cultures (B65) was different to that in quiescent cell cultures. In the murine model of senescence (senescence-accelerated mice prone, SAMP8), SIRT1 expression progressively decreased, while in the control strain (senescence-accelerated mice resistant, SAMR1) it increased. Finally, we studied human samples of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Huntington's diseases (HD). SIRT1 expression decreased dramatically in HD, but there were no significant changes in Parkinson-related illnesses. In conclusion, SIRT1 expression may be a good sensor of toxic neuronal processes.

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis.

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25- 50 microM) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.

3-Amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in neurons.

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.

Parameters related to oxygen free radicals in erythrocytes, plasma and epidermis of the hairless rat.

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.

Orphenadrine prevents 3-nitropropionic acid-induced neurotoxicity in vitro and in vivo.

1. Previous studies indicate that 3-nitropropionic acid (3-NPA) neurotoxicity involves the excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Thus, we examined the effect of orphenadrine (an anticholinergic drug with NMDA receptor antagonist properties) on 3-NPA neurotoxicity in both cultured rat cerebellar granule cells (CGCs) and in rats. 2. Orphenadrine protected CGCs from 3-NPA-induced mortality, as assessed by both the neutral red viability assay and laser scanning cytometry, using propidium iodide staining. 3. For rats, two indirect markers of neuronal damage were used: the binding of [(3)H]-PK 11195 to the peripheral-type benzodiazepine receptor (PBR), a microglial marker, and expression of the 27 kD heat-shock protein (HSP27), a marker of activated astroglia. Systemic administration of 3-NPA (30 mg kg(-1) per day for 3 days) induced a 170% increase in [(3)H]-PK 11195 binding, and expression of HSP27. 4. Both the increase in [(3)H]-PK 11195 and HSP 27 expression were prevented by previous administration of 30 mg kg(-1) per day of orphenadrine for 3 days. Lower doses (10 and 20 mg kg(-1)) had no protective effect. Orphenadrine also reduced 3-NPA-induced mortality in a dose-dependent manner. 5. We propose that orphenadrine or orphenadrine-like drugs could be used to treat neurodegenerative disorders mediated by overactivation of NMDA receptors.

Inhibitors of NO-synthase and donors of NO modulate kainic acid-induced damage in the rat hippocampus.

The effects of nitric oxide synthase (NOS) inhibitors, N(omega)-nitro-L-arginine and 7-nitroindazole, and the NOS substrate L-arginine on kainic acid (KA)-induced microglial reactivity and stress response were studied in the hippocampus 7 and 1 days after KA, respectively. Density of peripheral-type benzodiazepine receptors was measured as an index of microglial reactivity. Histological damage in hippocampus was evaluated at 7 days by neuronal counting. KA increased the maximal number of binding sites (B(max)) versus controls. Administration of either 7-nitroindazole (25 mg/kg) or N(omega)-nitro-L-arginine (20 and 50 mg/kg) 24 hr before KA, further increased B(max). This later effect was abolished by L-arginine (1 g/kg), which given 24 hr before KA decreased B(max) to control values. Also, KA-induced HSP72 stress response was attenuated by pre-treatment with L-arginine. Histological evaluation showed reduced cell numbers in the pyramidal cell layer of the hippocampus in groups receiving KA, either alone or in combination with 7-nitroindazole. Administration of L-arginine before KA attenuated neuronal loss in CA3 but not CA1. A clear protective effect was observed, however, in CA1 and CA3, in rats receiving both L-arginine plus 7-nitroindazole before KA. The results show that the combination of a NO substrate with a NOS inhibitor reduces the neurotoxic effects of KA in the rat hippocampus. This study suggests that extremely fine regulation of NO levels in the different neural cell types can modulate excitotoxicity.

Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry.

The overexcitation of glutamate receptors is believed to be the cause of several neurodegenerative disorders. The determination of calcium fluxes, mitochondrial membrane potential (MMP) variations or the production of reactive oxygen species (ROS) in mammalian cells are usually measured during the development of potentially useful drugs that might interfere in the events induced by glutamate receptor activation. By using flow cytometry with dissociated cerebellar granule cells, we have developed a rapid and economical method to measure changes in biochemical parameters that are involved in neuronal cell death. The formation of intracellular ROS is measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The mitochondrial membrane potential is assessed by the retention of rhodamine 123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Finally, intracellular calcium increases are detected by using the calcium-selective indicator Indo-1. Cell viability is also assessed by using propidium iodide (PI) which stains DNA strands of permeabilized cells. This method might be useful for the screening of new drugs with potential neuroprotective activity, with improved cost/effectiveness ratio compared to other techniques.

Assessment of the adrenergic effects of orphenadrine in rat vas deferens.

The peripheral adrenergic effects of orphenadrine, an antiparkinsonian drug, have been evaluated in the rat vas deferens to investigate whether these properties are the same as those of other phencyclidine ligands. In the low micromolar range, orphenadrine enhanced electrically-evoked and exogenous noradrenaline contractile responses in the epididymal portion of rat vas deferens. It also induced spontaneous activity that was inhibited by prazosin (1 microM) but not by atropine (20 nM). It inhibited accumulation of [3H]noradrenaline in rat vas deferens (IC50 = 14.2+/-2.3 microM). Orphenadrine competitively inhibited [3H]nisoxetine binding in rat vas deferens membranes (Ki = 1.05+/-0.20 microM). It can be concluded that orphenadrine, at low micromolar concentrations, interacts with the noradrenaline reuptake system inhibiting its functionality and thus potentiating the effect of noradrenaline.

In vitro and in vivo protective effect of orphenadrine on glutamate neurotoxicity.

The anticholinergic drug orphenadrine is used in the treatment of Parkinson's disease. In this study we evaluate the neuroprotective effects of orphenadrine on excitotoxicity in vivo and in vitro. Orphenadrine prevented the mitochondrial and the cytoplasmic membrane potential decrease evoked by NMDA (100 microM) in rat dissociated cerebellar granule cells showing an IC50 value of 11.6 +/- 4.7 microM (mean +/- SEM, n = 5) and 13.5 +/- 2.3 microM (n = 3), respectively. Orphenadrine was able to protect cerebellar granule cell cultures from glutamate-induced neurotoxicity. Kainic acid (KA, 10 mg/kg)-induced excitotoxicity was evaluated in vivo using the microglial marker peripheral-type benzodiazepine receptor (PBR) and heat shock protein 72 (HSP72) expression in the hippocampus. The Bmax of PBR for control tissues was 589.1 +/- 40.0 fmol/mg protein (n = 4), increasing to 1692.5 +/- 51.6 fmol/mg protein (n = 5) after the KA treatment. Pretreatment with orphenadrine (10 mg/kg) blocked the KA-induced increase in PBR density. As expected, KA-administration induced the expression of HSP72 that was blocked in the orphenadrine + KA-treated rats. We demonstrate that orphenadrine, interacting at the NMDA receptor, is able to prevent the neurotoxicity mediated by activation at glutamate ionotropic receptors.

Effects of U-83836E on glutamate-induced neurotoxicity in dissociated rat cerebellar granule cells.

The effects of the lazaroid compound U-83836E on the glutamate-induced production of reactive oxygen species (ROS) were studied in dissociated rat cerebellar granule cells by flow cytometry. U-83836E completely inhibited ROS production with an estimated IC50 value of 21.7 +/- 9.1 nM. However, U-83836E did not inhibit the glutamate-evoked decrease in mitochondrial membrane potential (MMP). Nevertheless, U-83836E (10 nM to 10 microM) prevented cell death induced by 10 mM of glutamate. At concentrations above 10 microM, U-83836E by itself showed slight cytotoxicity, which was significant at a 100 microM concentration. U-83836E (25 to 200 microM) also increased the cytosolic calcium levels in a concentration-dependent manner. Our results indicate that the cytotoxic effects found at micromolar concentrations of U-83836E could be explained by an increase in [Ca2+]i. Finally, since U-83836E did not prevent the MMP decrease evoked by glutamate, it is suggested that antioxidant pharmacotherapy would not be sufficient to block the neurotoxic effects of glutamate.

Microgliosis and down-regulation of adenosine transporter induced by methamphetamine in rats.

Chronic administration of methamphetamine to rats induces neurotoxicity characterized by a loss of striatal dopaminergic terminals and reactive gliosis. Subcutaneous administration of methamphetamine in a scheduled procedure of four doses (10 mg/kg) at 2 h interval also induces a significant increase in the peripheral-type benzodiazepine receptor (PBR) density. This increase is maximum (76%) at 72 h post-treatment in the striatum and disappears at 7 days, suggesting that microglia may have a predominant role in necrosis-phagocytosis of neuronal debris rather than acting in a restorative manner. Microgliosis is not restricted to the striatum since it is also evident in cerebellum (75.4% of PBR increase) and hippocampus (37.2% of PBR increase). In the areas with high density of adenosine transporter, the microgliosis phenomenon correlates well with a decrease of this nucleoside transporter (about 39%). Although the microgliosis and the decrease in adenosine transporter could be parallel and not related events, we can speculate that when microglia are activated, a down-regulation of adenosine transporter occurs, playing a role in tissue homeostasis. With the same dosing schedule, methamphetamine induces HSP72 expression in both cytoplasmic and nuclear fractions of the striatum, cerebellum and hippocampus. This expression is also evident in the cerebral cortex, where adenosine transporter population did not show any variation.

Effect of PCP and sigma ligands on both noradrenaline- and electrically-induced contractions and on [3H]-noradrenaline uptake in rat vas deferens.

1. Electrically induced contractions of the epididymal portion of rat vas deferens were potentiated in concentration-dependent manner (0.1-30 microM) by different sigma and PCP receptor ligands (PCP, TCP, (+)-MK-801, dextromethorphan and (+)-3-PPP); dextrorphan did it in a minor extent. 2. Sigma and PCP receptor ligands also potentiated the effect of noradrenaline, inducing a reduction of the noradrenaline EC50 value in the rat vas deferens. The rank order of potencies was: PCP > TCP > (+)-3-PPP > (+)-MK-801 > dextrorphan > > > dextrometorphan. 3. In contrast, haloperidol (1 microM), a sigma receptor ligand, inhibited both the neurogenic and noradrenaline-induced responses in this tissue. 4. The effect of PCP and sigma receptor ligands on noradrenaline uptake was evaluated. All compounds tested, including haloperidol, inhibited the tritiated noradrenaline incorporation to the tissue. IC50 values were in the micromolar range, between 1.09 microM for dextrophan and 18 microM for dextrometorphan. 5. It is concluded that a direct interaction with the noradrenaline uptake system is involved in the potentiating effect of some sigma and PCP receptor ligands in the epididymal portion of rat vas deferens.

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation.

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.

U-83836E prevents kainic acid-induced neuronal damage.

The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.

Reactive oxygen production by glutamate agonists in dissociated cerebellar cells: a flow cytometric study.

1. The effect of glutamate, N-methyl-D-aspartate (NMDA) and kainate on radical oxygen species (ROS) production and calcium influx was studied in dissociated cerebellar granule cells with the use of flow cytometry. 2. Glutamate and NMDA induced an intracellular ROS increase by an activation of NMDA receptors. 3. (+)MK-801 inhibited the effect on ROS production of both agonists (IC50 values of 1.52 +/- 0.05 and 0.71 +/- 0.02 microM, respectively). 4. (+)MK-801 inhibited the intracellular calcium increase induced by glutamate and NMDA, whereas 6-cyano-7-nitroquinoxaline-2,3-dione inhibited that induced by kainate. 5. NG-Nitro-L-arginine, but not nitrendipine, inhibited the ROS production induced by glutamate agonists. Consequently, nitric oxide synthase might play an important role in the neurotoxic process induced by excitatory amino acids.

Characterization of [3H]nisoxetine binding in rat vas deferens membranes: modulation by sigma and PCP ligands.

Sigma (sigma) and phencyclidine (PCP) receptor ligands, apart from their main effects on sigma receptors and NMDA receptor-mediated neurotransmission, have been found to interact with catecholamine systems in several central and peripheral tissues. In the present study the binding profile of [3H]nisoxetine ([3H]NIS), a selective marker of the noradrenaline transporter, has been characterized in rat vas deferens membranes to further study its modulation by a number of characteristic sigma and PCP ligands. The binding of [3H]NIS was found to be of high affinity (Kd = 1.63 +/- 0.36 nM), saturable, sodium-dependent and to a single population of binding sites (nH = 1.003 +/- 0.017). The maximal binding capacity was 1,625 +/- 500 fmol/mg of protein. Kinetic experiments gave a k(+1) of 3.9 x 10(7) min(-1)M(-1) and a k(-1) of 0.005 min(-1). The [3H]NIS binding was totally inhibited, with IC50 values in the micromolar range, by all the sigma and PCP ligands tested, with the following order of potency: haloperidol > dextromethorphan > dizocilpine > dextrorphan > (+)-3-PPP > PCP > tenocyclidine. This order correlates well with that described in other tissues using [3H]desmethylimipramine. The inhibition by all these compounds, except that of (+)-3-PPP, was competitive. These results suggest that sigma and PCP ligands bind, at low micromolar concentrations, to a site in the noradrenaline transporter that is labelled by [3H]NIS.

Determination of nitric oxide generation in mammalian neurons using dichlorofluorescin diacetate and flow cytometry.

A method for the rapid detection of intracellular nitric oxide (NO) generation in dissociated cerebellar granule cells using dichlorofluorescin (DCFH) and flow cytometry was developed. DCFH can be oxidized specifically by NO and this was assessed by 1) the use of SIN-1 (10 nM-100 microM), an NO donor, that induced a concentration-dependent increase in dichlorofluorescein (DCF) fluorescence and 2) the use of hemoglobin (10 microM), an NO-scavenger, that totally inhibited the increase of fluorescence induced by SIN-1 (10 microM). This assay was used to determine the ability to kainate to stimulate NO production in dissociated cerebellar granule cells. Kainate (1 microM-10 mM) induced an increase in DCF fluorescence that was partially reduced by NG-nitro-L-arginine (1 nM-10 microM), a nitric oxide synthase inhibitor (61.9% +/- 9.1), or hemoglobin (10 microM) (55.0% +/- 4.1). The method described allows evaluation of the oxidation of DCFH to produce DCF as a parameter for measuring intracellular NO generation. The extent of DCFH oxidation by NO and ROS can be determined by using NO scavengers or NO synthase inhibitors.

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

The neuroprotective activity of group-II metabotropic glutamate receptors requires new protein synthesis and involves a glial-neuronal signaling.

The group-II metabotropic glutamate (mGlu) receptor agonists (2S,1'R, 2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), S-4-carboxy-3-hydroxyphenylglycine (4C3HPG), and (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) protected mouse cortical neurons grown in mixed cultures against excitotoxic degeneration induced by a 10 min pulse with NMDA. Protection was observed not only when agonists were added in combination with NMDA but also when they were transiently applied to cultures 6-20 hr before the NMDA pulse. In both cases, neuroprotection was reduced by the group-II mGlu receptor antagonist (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV), as well as by the protein synthesis inhibitor cycloheximide (CHX). Both neurons and astrocytes in mixed cultures were immunostained with an antibody that recognized mGlu2 and mGlu3 receptors in recombinant cells. To determine whether astrocytes played any role in the neuroprotection mediated by group-II mGlu receptors, we exposed pure cultures of cortical astrocytes to DCG-IV, 4C3HPG, or L-CCG-I for 10 min. The astrocyte medium collected 2-20 hr after the exposure to any of these drugs was highly neuroprotective when transferred to mixed cultures treated with NMDA. This protective activity was reduced when CHX was applied to astrocyte cultures immediately after the transient exposure to group-II mGlu receptor agonists. We conclude that neuroprotection mediated by group-II mGlu receptors in cultured cortical cells requires new protein synthesis and involves an interaction between neurons and astrocytes.