Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture

Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(>6 h) and concentration-dependent manner (half-maximal effect at 1 n M ). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [³H]arginine to [³H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.     

Glia Modulate NMDA-Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N-methyl-d -aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca²⁺ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Ornithine-σ-Aminotransferase Exhibits Different Kinetic Properties in Astrocytes, Cerebral Cortex Interneurons, and Cerebellar Granule Cells in Primary Culture

To evaluate a possible role of ornithine-delta-aminotransferase (EC 2.6.1.13; Orn-T) as a rate-limiting enzyme for the synthesis of transmitter glutamate and gamma-aminobutyric acid (GABA), respectively, its activity and kinetic properties were analyzed in cultured astrocytes as well as in neuronal cultures consisting mainly of glutamatergic neurons (cerebellar granule cells) or GABAergic neurons (cerebral cortex interneurons). For comparison the activity and kinetics of Orn-T were also assayed in mouse brain homogenates. The highest activity of Orn-T was found in astrocytes and in cerebral cortical neurons (5.3 +/- 0.5 and 5.3 +/- 0.4 nmol X mg-1 X min-1, respectively) whereas the activities of Orn-T in cerebellar granule cell cultures and in mouse brain were found to be about half of these values (3.1 +/- 0.3 and 2.8 +/- 0.1 nmol X min-1 X mg-1, respectively). From a kinetic study of Orn-T in the different preparations only a relatively low affinity for the enzyme with respect to ornithine was found in cerebellar granule cells, astrocytes, and whole brain [apparent Km values (at 0.5 mM alpha-ketoglutarate): 4.7 +/- 0.9, 4.3 +/- 2.2, and 6.8 +/- 2.2 mM, respectively] whereas the corresponding Km value for Orn-T in cerebral cortex interneurons was found to be significantly lower (apparent Km: 0.8 +/- 0.3 mM). The enzyme was not found to be inhibited by GABA (range 0.1 - 10 mM) in any of the preparations.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Exocytotic and Nonexocytotic Modes of Glutamate Release from Cultured Cerebellar Granule Cells During Chemical Ischaemia

Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 m M 2-deoxyglucose plus 1 m M sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d -[³H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A₁ and was dependent on the activation of Ca²⁺ channels sensitive to the "Q" type Ca²⁺ channel antagonist, ω-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 m M KCl. Free Ca²⁺ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca²⁺ precedes that of the cell somata. Release of d -[³H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca²⁺ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca²⁺-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.     

Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes

The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na(+)-dependent and a Na(+)-independent system. The Na(+)-dependent system was inhibited by aspartate and was consistent with the commonly occurring system designated system X-AG. The Na(+)-independent system was inhibited by cystine and was consistent with system x-c described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na(+)-independent glutamate uptake. In C-6 cells, the glutamate uptake via systems X-AG and x-c accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes, the glutamate uptake via system X-AG was very potent, whereas the uptake via system xc- was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system xc- was necessary for the uptake of cystine, another substrate of system xc-. Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Uptake, Release, and Metabolism of Citrate in Neurons and Astrocytes in Primary Cultures

Abstract: Synthesis, uptake, release, and oxidative metabolism of citrate were investigated in neurons and astrocytes cultured from cerebral cortex or cerebellum. In addition, the possible role of citrate as a donor of the carbon skeleton for biosynthesis of neurotransmitter glutamate was studied. All cell types expressed the enzyme citrate synthase at a high activity, the cerebellar granule neurons containing the enzyme at a higher activity than that found in the astrocytes from the two brain regions or the cortical neurons. Saturable citrate uptake could not be detected in any of the cell types, but the astrocytes, and, in particular, those of cerebellar origin, had a very active de novo synthesis and release of citrate (～70 nmol × h^?1× mg of protein^?1). The rate of release of citrate from neurons was <5% of this value. Using [¹⁴C]citrate it could be shown that citrate was oxidatively metabolized to ¹⁴CO₂ at a modest rate (～1 nmol × n^?1× mg^?1 of protein) with slightly higher rates in astrocytes compared with neurons. Experiments designed to investigate the ability of exogenously supplied citrate to serve as a precursor for synthesis of transmitter glutamate in cerebellar granule neurons failed to demonstrate this. Rather than citrate serving this purpose it may be suggested that astrocytically released citrate may regulate the extracellular concentration of Ca²⁺ and Mg²⁺ by chelation, thereby modulating neuronal excitability.     

Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells

Abstract: Metabotropic glutamate receptor (type 1; mGluR1 ) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N -methyl-D-aspartate and non- N -methyl-D-aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1 . The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells Involves Cytochrome c Release from Mitochondria and Mitochondrial Shuttle Impairment

To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell.     

Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.     

Cellular Origin of Ischemia-Induced Glutamate Release from Brain Tissue In Vivo and In Vitro

The uptake and release of D-[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) were studied in cultured glutamatergic neurons (cerebellar granule cells) and astrocytes at normal (5 mM) or high (55 mM) potassium and under conditions of hypoglycemia, anoxia or "ischemia" (combined hypoglycemia and anoxia). In glutamatergic neurons it was found that "ischemic" conditions led to a 2.4-fold increase in the potassium-induced release of D-[3H]aspartate as compared to normal conditions. Hypoglycemia or anoxia alone affected the release only marginally. The ischemia-induced induced increase in the evoked D-[3H]aspartate release was shown to be calcium-dependent. In astrocytes no difference was found in the potassium-induced release between the four conditions and the K+-induced release was not calcium-dependent. The uptake of D-[3H]aspartate was found to be stimulated at high potassium in both glutamatergic neurons (98%) and in astrocytes (70%). This stimulation of D-aspartate uptake, however, was significantly reduced under conditions of anoxia or "ischemia" in both cell types. In glutamatergic neurons (but not in astrocytes) hypoglycemia also decreased the potassium stimulation of D-aspartate uptake. In a previous report it was shown, using the microdialysis technique, that during transient cerebral ischemia in vivo the extracellular glutamate content in hippocampus was increased eightfold. In the present paper it is shown that essentially no increase in extracellular glutamate is seen under ischemia when the perfusion is performed using calcium-free, cobalt-containing perfusion media. The results from the in vitro and in vivo experiments indicate that the glutamate accumulated extracellularly under ischemia in vivo originates from transmitter pools in glutamatergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

In Vitro Release of Endogenous Amino Acids from Granule Cell-, Stellate Cell-, and Climbing Fiber-Deficient Cerebella

Abstract: The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm -K⁺-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca²⁺ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca²⁺ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca²⁺. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg²⁺ concentration to 16 HIM inhibited the K⁺-stimulated, Ca²⁺-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K⁺-stimulated, Ca²⁺ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.     

Heteromers of Glutamate Decarboxylase Isoforms Occur in Rat Cerebellum

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD₆₅ and GAD₆₇). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD₆₅ and GAD₆₇, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₇ was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₅ remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD₆₅ and GAD₆₇ even after extensive washing. These results are consistent with the presence of heteromultimers of GAD₆₅ and GAD₆₇ in cerebellum in addition to homomers of each form.