Calcium oscillations induced by gambierol in cerebellar granule cells

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage‐dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 µM, gambierol‐evoked [Ca²⁺]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol‐evoked [Ca²⁺]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K⁺ channel blocker 4‐aminopyridine (4‐AP)‐evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4‐AP and indicate that gambierol‐induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage‐dependent potassium channels and hyperpolarization of sodium current activation. J. Cell. Biochem. 110: 497–508, 2010. © 2010 Wiley‐Liss, Inc.     

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

The relationship is investigated between superoxide levels in single cultured rat cerebellar granule neurons exposed continuously to glutamate in low KCl medium and the deregulation of cytoplasmic Ca2+. Cells that maintain a regulated cytoplasmic-free Ca2+ and mitochondrial polarization in the presence of glutamate show no increase in superoxide levels until the onset of cytoplasmic Ca2+ deregulation. Oxidative stress of mitochondrial origin is readily detectable, as the inhibitors rotenone and antimycin A markedly increase superoxide levels with no effect on cytoplasmic-free Ca2+. The potent cell-permeant superoxide dismutase/catalase mimetic manganese tetrakis (N-ethylpyridinium-2yl) porphyrin, MnTE-PyP, abolishes the deregulation-related increase in superoxide but has no effect on deregulation itself. A combination of catalase with the free radical scavenger 4-hydroxy-TEMPO also fails to reduce deregulation. Following the loss of Ca2+ homeostasis nuclei undergo condensation; this morphological change is not inhibited by MnTE-PyP and cannot account for the increased ethidium fluorescence. Phospholipase A2 inhibitors decrease the deregulation-related increase in superoxide without protecting against deregulation. In conclusion, our study indicates that deregulation is not caused by NMDA receptor-mediated oxidative stress as NMDA receptor activation does not increase superoxide levels until the onset of deregulation. Furthermore, the majority of superoxide is produced in the cytoplasm rather than in mitochondria.     

Development of polarity in cerebellar granule neurons

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 223–236, 1997.     

Transient glucose and amino acid deprivation induces delayed preconditioning in cultured rat cortical neurons

Several studies have demonstrated that glucose deprivation, combined either with anoxia or with the inhibition of oxidative phosphorylation, leads to the development of ischemic tolerance in neurons. The aim of our experiments was to investigate whether similar effects could be achieved by transient energy deprivation without either anoxia or the inhibition of the electron transfer chain. Preconditioning was carried out by incubating primary rat cortical neuronal cultures for 3, 6 or 9 h in a glucose- and amino acid-free balanced salt solution supplemented with B27 in normoxic conditions. After 24 h, neuronal cultures were exposed to oxygen-glucose deprivation, glutamate or hydrogen peroxide. Cell viability was measured 24 h after the lethal insults. Potential mechanisms that can influence free radical production were also examined. Energy deprivation protected neuronal cells against lethal stimuli (e.g. cell survival after oxygen-glucose deprivation was 33.1 +/- 0.52% in the untreated group and 80.1 +/- 1.27% in the 9-h energy deprivation group), reduced mitochondrial membrane potential, decreased free radical formation, attenuated the intracellular free calcium surge upon glutamate receptor stimulation, and resulted in an elevated level of GSH. Our findings show that transient energy deprivation induces delayed preconditioning and prevents oxidative injuries and neuronal cell death.     

TLQP-21, a neuroendocrine VGF-derived peptide, prevents cerebellar granule cells death induced by serum and potassium deprivation

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c- jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.     

Voltage dependent sodium currents in cultured rat cerebellar granule cells

Sodium currents were studied in granule cells dissociated from rat cerebellum. Macroscopic currents were recorded using the patch-clamp technique. Sudium currents, which are TTX sensitive, reached a maximum peak value of 0.42±0.08 pA/m² at 18.4±2.2 mV (n=6). Activation and inactivation kinetics and steady-state properties were described in terms of Hodgkin and Huxley, parameters. The properties of sodium channels in cultured rat cerebellar granule cells are very similar to those reported for various neural preparations.     

GANRA-5 protects both cultured cells and mice from various radiation types by functioning as a free radical scavenger

Abstract

The radio-protective effects of the oxazolone derivative chemical compound 4-(4-methoxy-3-methoxyphenyl-methyl)-2-phenyl- 5(4H)-oxazolone (GANRA-5) against different types of radiation including X-rays, carbon ion beams, microwaves and ultraviolet light (UV) were studied. Cell proliferation/cytotoxicity assay and colony-forming assay were conducted to evaluate the toxicity of GANRA-5. To test its influence on the induction of double-stranded break (DSB) formation and genomic instability, γH2AX focus-forming assay as well as cytokinesis-block micronucleus assay was utilized. Our results indicate that GANRA-5 exhibits low toxicity, while providing high radio-protective effects for MRC-5 cells against different types of radiation. We also found that GANRA-5 acts as a free radical scavenger. Our animal studies provided evidence that GANRA-5 significantly increases the survival rate of mice after X-ray irradiation. Analyses of hemogram, visceral index and detection of superoxide dismutase (SOD) and malondialdehyde (MDA) in the viscera indicate both low toxicity of GANRA-5, combined with its ability to shield radiation risk. In conclusion, our results suggest that GANRA-5 has the potential to be used as a safe and efficient radio-protectant.     

Acute effects of blood flow restriction on exercise-induced free radical production in young and healthy subjects

The main purpose of this study was to investigate the acute local and systemic effects of low-load resistance exercise (30% 1RM) with partial vascular occlusion on exercise-induced free radical production and to compare these effects with other established training methods.

Fifteen young and healthy males (25?±?3 years) performed the following four sessions in a counterbalanced order on separate days: low-load resistance exercise (LI: 30% 1RM), low-load resistance exercise with blood flow restriction (LIBR: 30% 1RM), high-load resistance exercise (HI: 80% 1RM) and an additional session without exercise but blood flow restriction only (BR). Blood samples were obtained 15?min prior to and immediately after exercise sessions from the right index finger and first toe. To analyze concentrations of reactive oxygen species (ROS), electron paramagnetic resonance (EPR) spectroscopy was used. Additionally, mitochondrial ROS production was measured by adding inhibitors of electron transport chain complex III. There was an increased systemic ROS generation after the LIBR session from 0.837?±?0.093 to 0.911?±?0.099?µmol/l/min. However, no local or systemic time?×?condition interaction was detected for ROS production. Regarding mitochondrial ROS production, results were not different between the conditions. Although the low-load resistance exercise session with partial vascular occlusion elicited systemic increases of ROS production, no significant changes were seen on a local level. We assume that this ROS concentration might not be high enough to induce cellular damage but is rather involved in muscle remodulation. However, this needs to be confirmed by future research.     

Genotoxicity of streptozotocin in normal and cancer cells and its modulation by free radical scavengers

Streptozotocin (STZ) is an antibiotic which can be used to induce diabetes in experimental animals in order to have an insight into pathogenesis of this disease. To use STZ as a diabetogenic substance, its molecular mode of action should be elucidated. Using the alkaline comet assay, we showed that STZ at concentrations in the range 0.01-100 micromol/L induced DNA damage in normal human lymphocytes and HeLa cancer cells in a dose-dependent manner. Lymphocytes were able to remove damage to their DNA within a 30-min repair incubation, whereas HeLa cells completed the repair in 60 min. Vitamins C and E at 10 and 50 micromol/L diminished the extent of DNA damage induced by 50 micromol/L STZ. Pretreatment of the lymphocytes with the nitrone spin trap, alpha-(4-pyridil-1-oxide)-N-tert-butylnitrone (POBN) or ebselen, which mimics glutathione peroxidase, or pyrrolidine dithiocarbamate (PDTC) reduced the extent of DNA damage evoked by STZ. The cells exposed to STZ and treated with endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. These results suggest that free radicals may be involved in the formation of DNA lesions induced by streptozotocin. The drug can also alkylate DNA bases. This broad range of DNA damage induced by STZ indicates that the drug may seriously affect genomic stability in normal and pathological cells.     

Hydrogen peroxide induced chemokine production in the glia-rich cultured cerebellar granule cells under acidosis

We have documented the time-dependent production of chemotactic cytokine, i.e., IL-8, in the extracellular fluid of astrocyte-rich cultured rat cerebellar granule cells under acidified conditions. In this paper, the mechanism of this production was evaluated based on the production of hydrogen peroxide (H2O2). Significant and time-dependent increases of cytosolic H2O2 were detected under acidosis in astrocyte-rich cultured cell. Upon exposure to 10 microM H2O2, significant levels of IL-8 appeared in the extracellular fluid of astrocyte-rich cells, although an initial transient increase of IL-8 was also seen in the intracellular space. Concurrently, after H2O2 exposure cell injury and a delayed increase of cytosolic Ca2+ levels were detected in astrocyte-rich cells. However, in the absence of extracellular Ca2+, the cell injury and the increase of IL-8 production were significantly attenuated. A synergistic effect of cyclosporine A (an inhibitor of the Ca2+/calmodulin-regulated protein phosphatase) and trifluoperazine (an inhibitor of phospholipase A2) on the suppression of H2O2-induced IL-8 production was clearly evident. These results suggest that extracellular acidosis induced Ca2+-dependent H2O2 production, which in turn stimulated IL-8 expression. which is regulated by the cytosolic Ca2+ cascade. Thus, the production of IL-8 from glia cells may have a role in regulating in the process of cell injury.     

GPI‐anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI‐anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI‐anchored proteins have been found on both the axonal and somatodendritic cell‐surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication‐incompetent retroviral vector to express a model GPI‐anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high‐density monolayers, and maintained for 1–9 days under serum‐free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP‐expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP‐expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well‐polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI‐anchored protein expressed in a well‐polarized primary neuronal cell type in vitro; other signals, such asthose present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon‐specific GPI‐anchored proteins. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 119–141, 1999     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

线粒体稳定高钙信号诱发超氧炫

线粒体对于细胞钙信号和活性氧信号转导有重要的调控作用．超氧炫是新近发现的单个线粒体超氧阴离子短时程爆发现象,反映了活性氧生成动力学的一种新形式．线粒体钙信号作为重要的细胞功能调控信号,能否及如何调控超氧炫尚待深入研究．本研究对HeLa细胞进行高胞外钙和离子霉素刺激,或用皂苷穿孔细胞质膜后置于高钙细胞内液中,两种方法均显著增加了超氧炫发生的频率．其中,穿孔细胞胞浆高钙诱导的超氧炫依赖于线粒体钙单向转运体,表明超氧炫由线粒体基质内高钙信号所诱发．重要的是,离子霉素诱导的超氧炫发生频率与线粒体稳态钙水平线性相关,而与瞬态线粒体钙无相关性,提示钙离子对超氧炫的调控是一个多步骤、相对缓慢的过程．综上,线粒体基质的稳态高钙是超氧炫的重要调控因子．     

Zinc protects HeLa-tat cells against free radical cytotoxicity induced by glucose

In the present study, we investigated the protective effect of zinc on the glucose-induced cytotoxicity in HeLa wild and HeLa-tat cells (30 and 20 mmol/l glucose, respectively). HeLa cells transfected with the protein Tat exhibit a lower antioxidant defense system. Incubation of HeLa wild and HeLa-tat cells with high glucose levels led to a rapid increase in generation of reactive oxygen species (ROS). As expected in the presence of high glucose concentrations, the viability was reduced for both cell lines. The redox status essentially regulated by thiol groups may play an important role in the apoptotic process. Thus, we developed a new method using the p-nitrophenyl disulfide to measure cytosolic thiol groups in intact cells. Cellular zinc was measured using inductively coupled plasma mass spectrometry. Intracellular thiol groups and intracellular zinc concentrations were significantly lower in HeLa cells cultured in hyperglycemic conditions, and their concentrations were significantly lower in HeLa-tat cells than in HeLa wild cells. However, the generation of ROS and the induction of apoptosis by a glucose specific mechanism were prevented by zinc (50 micromol/l) and the intracellular thiol groups and zinc concentrations significantly increased in both cell lines to become similar to the initial values. These results suggest that the glucose oxidation and its subsequent effects on the cells can be prevented by a biological antioxidant such as zinc.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Anti-oxidants by inhibiting free radical generation, protect neurons against 6-OHDA-induced neurotoxicity. In this study, we investigated whether or not minocycline, a neuroprotective compound, could directly protect neurons against 6-OHDA-induced neurotoxicity and inhibit 6-OHDA-induced free radical production in cultured rat cerebellar granule neurons (CGN). We now report that exposure of CGN to 6-OHDA (100 microM) resulted in a significant increase in free radical production with death of 86% of CGN. Pretreatment with minocycline (10 microM) for 2 h prevented 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, minocycline also attenuated H(2)O(2)-induced neurotoxicity. Our results suggest that minocycline blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation in CGN. Both the antioxidative and neuroprotective effects of minocycline may be beneficial in the therapy of Parkinson's disease and other neurodegenerative diseases.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.