Pharmacological but not physiological modulation of cortical acetylcholine release by cholinergic mechanisms in the nucleus basalis magnocellularis.

The role of muscarinic transmission in the activation of cholinergic neurons ascending to the neocortex from the nucleus basalis magnocellularis (NBM) was investigated. The release of acetylcholine (ACh) from the neocortex of urethane-anesthetized rats was measured using microdialysis, and a second microdialysis probe was inserted into the NBM to apply drugs to the NBM and to measure ACh release from this area. Cholinergic neurons in the NBM were activated synaptically by stimulating the pedunculopontine tegmentum (PPT). Systemically administered scopolamine greatly increased the PPT stimulation evoked cortical release of ACh when the cortical probe was perfused with the cholinesterase inhibitor neostigmine. PPT stimulation evoked release was also high when the cortical probe was perfused with atropine plus neostigmine, but it was not increased any further by systemic scopolamine or by scopolamine perfused through the NBM probe. When neostigmine was perfused through the NBM probe, PPT stimulation evoked cortical ACh release was halved, but the release was restored when the NBM solution also contained scopolamine. The resting release of ACh within the NBM was increased by local neostigmine, but evoked release in the NBM was large only in the presence of local scopolamine. Both of these increases were blocked by perfusion of the NBM with tetrodotoxin. These results suggest that muscarinic transmission within the NBM does not control the activation of cholinergic neurons under physiological conditions, when the diffusion of ACh is limited by its hydrolysis. However, when ACh is allowed to diffuse to a wider area, it may inhibit the release of an excitatory transmitter, probably glutamate, via presynaptic muscarinic receptors.

Modification of neocortical acetylcholine release and electroencephalogram desynchronization due to brainstem stimulation by drugs applied to the basal forebrain.

Acetylcholine released from the cerebral cortex was collected using microdialysis while stimulating the region of the pedunculopontine tegmentum in urethane-anesthetized rats. Electrical stimulation in the form of short trains of pulses delivered once per minute produced a 350% increase in acetylcholine release and a desynchronization of the electroencephalogram, as measured by relative power in the 20-45 Hz range (low-voltage fast activity). Perfusion of the region of cholinergic neurons believed to be responsible for the cortical release of acetylcholine, the nucleus basalis magnocellularis, was carried out using a second microdialysis probe. Exposure of the nucleus basalis magnocellularis to blockers of neural activity (tetrodotoxin or procaine) or to blockers of synaptic transmission (calcium-free solution plus magnesium or cobalt) produced a substantial decrease in the release of acetylcholine and desynchronization evoked by brainstem stimulation. Exposure of the nucleus basalis magnocellularis to the glutamate antagonist, kynurenate, resulted in a decrease in evoked acetylcholine release and electroencephalogram desynchronization similar in magnitude to that produced by nonspecific blockers, whereas application of muscarinic or nicotinic cholinergic blockers to the nucleus basalis magnocellularis did not reduce acetylcholine release or electroencephalogram desynchronization. Application of tetrodotoxin to the collection site in the cortex abolished the stimulation-evoked acetylcholine release, but not the low baseline release indicating that cholinergic nucleus basalis magnocellularis neurons have a low spontaneous firing rate in urethane-anesthetized animals. The results of this study suggest that the major excitatory input to the cholinergic neurons of the nucleus basalis magnocellularis from the pedunculopontine tegmentum is via glutamatergic and not cholinergic synapses.

Effects of ammonium ions on synaptic transmission and on responses to quisqualate and N-methyl-D-aspartate in hippocampal CA1 pyramidal neurons in vitro.

Effects of NH4Cl on CA1 pyramidal neurons and synaptic transmission were investigated with intracellular recording in fully submerged rat hippocampal slices. Superfusion with 1-4 mM NH4Cl reversibly depolarized the membrane by 15.1 +/- 1.4 mV, reduced the amplitude and broadened the duration of action potentials due to a slower rate of repolarization, without significant change in membrane conductance. When membrane potential was returned to control level by the injection of a steady outward current, action potential amplitude recovered but repolarization remained slow. The extent of depolarization was not dependent on the concentration of NH4Cl between 1 and 4 mM. NH4Cl greatly depressed orthodromic transmission evoked by the stimulation of Schaffer collateral/commissural fibers several minutes after depolarizing the CA1 neuron. Interruption of transmission began with a decrease in excitatory postsynaptic potential (EPSP) amplitude and eventually EPSPs were almost eliminated. When NH4Cl was removed, it took 2-3 min for membrane potential and 10-15 min for transmission to recover. Inward currents induced by bath application of quisqualate acting on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were also depressed. In contrast, NH4Cl enhanced N-methyl-D-aspartate (NMDA)-induced currents. This potentiation disappeared in the absence of added Mg2+. A reduction in quisqualate-induced responses provided a possible explanation for the inhibition of excitatory transmission by NH4Cl.

Astrocytes and the entry of circulating ammonia into the brain: effect of fluoroacetate.

Chronic hyperammonemia is known to lead to pathological forms of astrocytes. To test the influence of these changes on the neurotoxicity of ammonia, the glial metabolic poison fluoroacetate (FA) was applied locally, through microdialysis to the hippocampal dentate gyrus. The penetration of ammonia into the brain following the i.p. injection of 7.8 mmol/kg NH4 acetate was evaluated by measuring the ammonia and glutamine content of the microdialysate. Field EPSPs (fEPSPs) evoked by perforant path stimulation were recorded 1.5 mm from the microdialysis probe. When 20 mM FA was perfused, NH4 acetate injection increased the ammonia efflux by 300% and decreased fEPSPs by 40%, but glutamine concentration remained low. With no FA in the microdialysate, NH4 acetate treatment increased the efflux of ammonia by only 60%, did not affect fEPSPs but doubled glutamine efflux. Arterial ammonia content, as measured by microdialysis in the common carotid, increased 4-5 fold following i.p. administration of NH4 acetate, while arterial glutamine was not elevated. Systemically administered FA did not affect either of these changes significantly, but slightly reduced arterial pH. These observations indicate that FA applied by microdialysis acted locally on astrocytes and therefore impaired astrocytic function contributes to the development of hepatic encephalopathy by facilitating the entry of ammonia into the brain. Inhibition of excitatory synaptic transmission by elevated brain ammonia may underlay CNS depression in hepatic encephalopathy.

Frequency-dependent increase in cortical acetylcholine release evoked by stimulation of the nucleus basalis magnocellularis in the rat.

Acetylcholine was collected from the somatosensory cortex of anesthetized rats, using the microdialysis technique. Electrical stimulation of the nucleus basalis magnocellularis (NBM) with trains of 10 pulses at 100 Hz delivered every second produced a 3-4-fold increase in acetylcholine release. Stimulation with an intratrain frequency of 10, 50, 100 or 200 Hz demonstrated that 100 Hz trains produced the greatest increase, while the other frequencies were about half as effective. The cortical release of acetylcholine in this paradigm supports the hypothesis that the previously demonstrated enhancement by NBM stimulation of cortical sensory inputs is due to cholinergic activation.

Effect of ammonium ions on synaptic transmission in the mammalian central nervous system.

It is not surprising that a compound with such unique properties as NH3/NH4+, should have a large variety of biochemical and neurological effects and to find itself implicated in many pathological conditions. Its undissociated (NH3) or dissociated (NH4+) forms, having different physicochemical properties, enter neurons and other cells through differing pathways. These two forms then change internal pH in opposite directions, and initiate a variety of regulatory processes that attempt to overcome these pH changes. In addition, ammonia has a central role in normal intermediary metabolism, and when present in excess, it can disturb reversible reactions in which it participates. The challenge in interpreting these various observations lies in the difficulty in assigning to them a role in the generation of symptoms seen in experimental and clinical hyperammonemias. In this review we have attempted to summarize information available on the effects of ammonium ions on synaptic transmission, a central process in nervous system function. Evidence has been presented to show that ammonium ions, in pathologically relevant concentrations, interfere with glutamatergic excitatory transmission, not by decreasing the release of glutamate, but by preventing its action on post-synaptic AMPA receptors. Furthermore, NH4+ depolarizes neurons to a variable degree, without consistently changing membrane resistance, probably by reducing [K+]i. A decrease in EK+ may also be responsible for decreasing the effectiveness of the outward chloride pump, thus explaining the well known inhibitory effect of NH4+ on the hyperpolarizing IPSP. There is a consensus of opinion that chronic hyperammonemia increases 5HT turnover and this may be responsible for altered sleep patterns seen in hepatic encephalopathy. There does not seem to be a consistent effect on catecholaminergic transmission in hyperammonemias. However, chronic hyperammonemia causes pathological changes in perineuronal astrocytes, which may lead to a reduced uptake of released glutamate and a decreased detoxification of ammonia by the brain. Chronic moderate increase in extracellular glutamate results in a down-regulation of NMDA receptors, while the decreased detoxification of ammonia makes the central nervous system more vulnerable to a sudden hyperammonemia, due, for instance, to an increased dietary intake of proteins or to gastrointestinal bleeding in patients with liver disease. Clearly, data summarized in this review represent only the beginning in the elucidation of the mechanism of ammonia neurotoxicity. It should help, we hope, to direct future investigations towards some of the questions that need to be answered.

Effect of portacaval anastomosis on electrically stimulated release of glutamate from rat hippocampal slices.

To evaluate the effects of chronic liver failure on release of the excitatory transmitter glutamate, electrically stimulated Ca2(+)-dependent and Ca2(+)-independent release of glutamate in the absence or presence of NH4+ was studied in superfused slices of hippocampus from portacaval-shunted or sham-operated rats 4 weeks after surgery. Spontaneous and stimulation-evoked release of glutamate was higher in shunted rats in the presence of normal or low Ca2+ concentrations, and this release was depressed by 5 mM ammonium chloride. These findings suggest that portacaval shunting results in increased levels of extracellular glutamate in brain, probably due to a decreased reuptake of glutamate into perineuronal astrocytes, shown in previous studies to undergo neuropathological changes following portacaval shunting. Changes in the inactivation of transmitter glutamate could be responsible, at least in part, for the neurological dysfunction resulting from sustained hyperammonemia and portal-systemic shunting resulting from chronic liver failure.

Glutamate release and spreading depression in the fascia dentata in response to microdialysis with high K+: role of glia.

To see electrophysiological and neurochemical events during microdialysis with high [K+], direct current (DC) and excitatory postsynaptic field potentials (fEPSPs) due to perforant path stimulation were recorded in the granule cell layer of the fascia dentata, while 3, 25, 50 or 100 mM KCl was perfused through a microdialysis probe placed 1.5 mm from the recording electrode. Glutamate and glutamine content of the dialysate was measured by high performance liquid chromatography. Raising [K+] from 3 to 25 mM reduced the efflux of glutamine, without affecting that of glutamate or the electrical activity. In about 50% of experiments, 50 mM K+ induced large (20-30 mV) negative waves of spreading depression (SD), and a suppression of fEPSPs. In the other 50%, without SD, fEPSPs did not change. Glutamate efflux increased 3-fold in both groups. SD waves were produced in all experiments with 100 mM K+ which evoked a more than 10-fold increase in glutamate release. Glutamine efflux decreased equally, by about 50%, with the 3 concentrations of K+. Microdialysis with 20 mM fluoroacetate, a glial metabolic poison, decreased the spontaneous efflux of glutamine and glutamate and increased the incidence of SD waves. Results suggest that perfusion of 50 or 100 mM K+ through a microdialysis probe causes spreading depression which blocks surrounding electrical activity. The activity of glia partly protects against spreading depression caused by high [K+].

Is glutamate a co-transmitter in cortical cholinergic terminals? Effects of nucleus basalis lesion and of presynaptic muscarinic agents.

To obtain additional evidence in support of the co-transmitter role of glutamate in cortical cholinergic terminals proposed by Docherty et al., the right nucleus basalis in rats was lesioned with ibotenic acid; resulting changes in cortical acetylcholinesterase (AChE) staining, glutamate content, and the release of [3H]acetylcholine ([ 3H]ACh) and glutamate from cortical slices from the two sides were compared. While there was a profound reduction on the lesioned side in cortical AChE activity and in the size of the releasable pool of [3H]ACh, neither the content nor the evoked release of glutamate was reduced significantly on the lesioned side. Furthermore, while oxotremorine strongly depressed the evoked release of [3H]ACh, it had no effect on the evoked release of endogenous glutamate measured simultaneously. These results do not support the co-transmitter role of glutamate in cortical cholinergic terminals, although they cannot statistically exclude that a small fraction of glutamate has a co-transmitter role, as proposed by Docherty et al.

Neurochemical and electrophysiological studies on the inhibitory effect of ammonium ions on synaptic transmission in slices of rat hippocampus: evidence for a postsynaptic action.

To elucidate the mechanisms involved in the inhibition of synaptic transmission by ammonium ions, the effects of NH4Cl on glutamate release and on synaptic transmission from Schaffer collaterals to CA1 pyramidal cells were measured in fully submerged slices of rat hippocampus. The large, Ca(2+)-dependent release of glutamate evoked by electrical-field stimulation or by 56 mM K+ was not reduced by 5 mM NH4Cl. In contrast, 5 mM NH4Cl decreased the smaller, field stimulation-induced release of glutamate observed in the presence of low concentrations of Ca2+ (0.1 mM), as well as the spontaneous release of glutamate both in normal and low Ca2+. Unlike the Ca(2+)-dependent release of glutamate, synaptic transmission was reversibly depressed even by 1 mM NH4 Cl. Firing of CA1 pyramidal cells evoked by iontophoretically applied glutamate was significantly inhibited by 2 or 5 mM NH4Cl. This depression was increased in the presence of 25 microM bicuculline. Results suggest that ammonium ions do not depress the Ca(2+)-dependent release of glutamate originating from synaptic vesicles, which is involved in synaptic transmission. Rather, ammonium ions inhibit synaptic transmission by a postsynaptic action, a conclusion strengthened by the inhibitory effect of NH4Cl on glutamate-induced firing. However, NH4Cl may inhibit the formation of cytoplasmic glutamate, the source of spontaneous and Ca(2+)-independent release.

Effect of low glucose concentration on synaptic transmission in the rat hippocampal slice.

Severe hypoglycemia in vivo is known to slow down the EEG, then to produce complete electrical silence in the brain. To find out why low glucose concentrations reduce electrical activity, synaptic transmission from Schaffer collateral/commissural fibers to CA1 pyramidal cells in the submerged rat hippocampal slice was investigated using extracellular recording techniques. Superfusion for 30 min with 1 mM glucose reversibly reduced population spike amplitude, without affecting the size of the presynaptic volley and the slope of the field EPSP. Lower glucose concentrations also affected the EPSP, although to a lesser extent than the population spike. Antidromic population spikes were not decreased by low glucose. Depolarization with 8-10 mM K+ reduced both presynaptic volley amplitude and EPSP, but enhanced the population spike, an effect clearly different from that of low glucose. The slope of the input/output curve between presynaptic volley and EPSP remained unaltered in 1 mM glucose but the slope between EPSP and population spike was reduced by about 50%. Results suggest that low glucose concentrations interrupt synaptic transmission by reducing, but not abolishing, the excitability of pyramidal cells.

Increase in the stimulation-induced overflow of excitatory amino acids from hippocampal slices: interaction between low glucose concentration and fluoroacetate.

To see whether the enhanced evoked release of aspartate and glutamate in the presence of low glucose concentration is due to a decreased glial uptake, the electrical-field stimulation induced release of aspartate and glutamate was measured in rat hippocampal slices in the presence of 5 or 0.2 mM glucose and of graded concentrations of fluoroacetate, a specific inhibitor of glial tricarboxylic acid cycle. In 5 mM glucose, fluoroacetate increased the overflow of both excitatory amino acids equally in a dose-dependent manner, with a maximal effect obtained at 2 mM. This maximal increase of glutamate overflow was about the same as caused by 0.2 mM glucose, but low glucose increased aspartate overflow 5 times more than did fluoroacetate. Fluoroacetate failed to increase any further the large evoked overflow of either glutamate or aspartate induced by 0.2 mM glucose. The absence of an additive effect of fluoroacetate and of low glucose suggests that under both conditions the increased overflow of glutamate is due to a reduced glial uptake. In low glucose an increased synthesis also contributes to the additional large release of aspartate.

Changes in the relative amounts of aspartate and glutamate released and retained in hippocampal slices during stimulation.

It has been found previously that the ratio of aspartate to glutamate released and retained by brain slices reversibly changes with changing glucose concentrations in the medium. To find out whether increased neuronal activity also results in changes in the ratio of aspartate to glutamate, in this study electrical-field stimulation was applied for 10 min to hippocampal slices in the presence of 0.2-5 mM glucose. In 5 mM glucose, the ratio of aspartate to glutamate released did not change during stimulation, but the amount of aspartate retained at the end of stimulation was reduced. In contrast, in 1 mM or less glucose, the ratio of aspartate to glutamate released increased progressively and the rate of increase was inversely proportional to the glucose content of the medium. The evoked release of aspartate and glutamate both in low and high glucose was nearly suppressed in low (0.1 mM) Ca2+ or by tetrodotoxin. In low glucose, the ratio of aspartate to glutamate contained in the slices also increased as a result of stimulation. This increase was reduced only a little in low Ca2+, but was nearly eliminated by tetrodotoxin. Results suggest that increased neuronal activity causes a shift in the ratio of aspartate to glutamate released in the presence of glucose concentrations similar to those found in the brain in normoglycemic rats. This shift, due to an increased energy demand, probably originates from terminals which release aspartate and glutamate in different proportions.

The effects of cortical ablation on multiple unit activity in the striatum following dexamphetamine.

Bilateral removal of the fronto-parietal cortex of the rat resulted in decreased spontaneous multiple-unit activity recorded in the striatum of freely-moving rats. Cortical ablations changed the neuronal response in the striatum to systemic administration of dexamphetamine (2.5 mg/kg i.p.) from excitation in control animals (88%) to inhibition in ablated animals (61%). Furthermore, catalepsy, induced by haloperidol, but not by morphine, was markedly attenuated after cortical ablation. These changes were accompanied by a 23% decrease in the specific binding of [3H]spiperone in the striatum. The binding of [3H]met-enkephalin was unaffected by the cortical lesions. Levels of glutamate in the striatum decreased from 8.88 +/- 0.5 mumols/g in control animals to 6.93 +/- 0.37 mumols/g after bilateral cortical ablation. On the other hand, cortical ablations did not alter the content of either the gamma-aminobutyric acid or glutamine of the striatum. It is concluded that the excitatory response, observed in striatal neurons in freely-moving animals, is dependent upon an intact cerebral cortex and requires intact cortico-striatal afferents. The results further suggest that neurons in the striatum are under the tonic influence of glutamate, released from cortico-striatal afferents. Lastly, some dopamine D2 binding sites in the striatum are located on cortico-striatal afferent terminals and blockade of these striatal D2 sites may be involved in the induction of catalepsy by neuroleptic drugs.

Increase in the stimulation-induced overflow of glutamate by fluoroacetate, a selective inhibitor of the glial tricarboxylic cycle.

Fluoroacetate is known to be taken up selectively by glia, where after forming fluorocitrate, it inhibits the tricarboxylic acid cycle. Since uptake into glia has a major role in the inactivation of synaptically released glutamate, the effect of fluoroacetate on the overflow of glutamate evoked by electrical field stimulation in slices of rat hippocampus was investigated. In agreement with previous reports, 1 mM fluoroacetate reduced the release and content of glutamine, but increased only slightly the overflow of glutamate induced by stimulation. If, however, 0.5 mM glutamine was added to the superfusion fluid, fluoroacetate nearly tripled the overflow of glutamate evoked by electrical field stimulation. The large glutamate overflow due to field stimulation in the presence of fluoroacetate was fully Ca2+ -dependent. Results confirm the major role of glia in the inactivation of glutamate. The absence of such an uptake may contribute to the in vivo convulsive effect of fluoroacetate.

Reversible shifts in the Ca2+-dependent release of aspartate and glutamate from hippocampal slices with changing glucose concentrations.

It is known that low glucose concentrations increase the aspartate and decrease the glutamate content of brain tissue both in vivo and in vitro. To see whether these changes occur in the transmitter compartment or not, the release of aspartate and glutamate evoked by electrical-field stimulation or by high K+ was followed in slices of rat hippocampus superfused with 5 or 0.2 mM glucose. Superfusion with 0.2 mM glucose increased the evoked release of aspartate about ten times and that of glutamate about threefold. This shift in the ratio of aspartate to glutamate released was accompanied by a similar increase in the relative amount of aspartate contained in the slices. The high evoked release of aspartate and glutamate was well maintained, provided 0.5 mM glutamine was added to the medium. Changing the concentration of glucose after the first period of stimulation rapidly altered the relative amounts of aspartate and glutamate released but not the enhanced release of glutamate. The large evoked release of both aspartate and glutamate in 0.2 mM glucose was almost entirely Ca2+-dependent. The relative amounts of aspartate and glutamate released by 50 mM K+ also changed when the glucose concentration was reduced. Results suggest two effects of low glucose concentrations: an increase in the overflow of synaptically released glutamate due to a decreased uptake and an increase in the proportion of aspartate to glutamate formed and released from the transmitter pool. These observations are consistent with the interpretation that these two transmitters can be released in different proportions from the same terminals.

Possible reasons for the failure of glutamine to influence GABA release in rat hippocampal slices; Effect of nipecotic acid and methionine sulfoximine.

Although labelled glutamine is readily incorporated into labelled releasable GABA, it has been shown recently that high concentrations (0.1-0.5 mM) glutamine do not increase the release of GABA from brain slices, while greatly enhancing that of glutamate. Two possible reasons for this discrepancy were investigated: (a) That released GABA, in contrast to glutamate is not freshly synthesized but derives from GABA taken up by terminals. The possibility was made unlikely by the present finding which showed that even in the presence of the uptake inhibitor nipecotic acid, glutamine failed to enhance GABA release. (b) That glutamine is transported into GABA-ergic terminals by a high-affinity transport system which is saturated even at low glutamine concentrations obtained without adding glutamine to the superfusion fluid. However, when glutamine efflux was further reduced by prolonging depolarization with 50 mM K(+) and by pretreatment with the glutamine synthetase inhibitor methionine sulfoximine, GABA release was depressed only very little and this decrease was related to the duration of depolarization and not to extracellular glutamine levels. These results can be reconciled with the ready incorporation of labelled glutamine into releasable GABA by assuming that GABA originates from a glutamate pool to which both glutamine and glucose contribute. The formation of releasable GABA however, is not governed by the supply of glutamate in this pool but by the activity of the rate-limiting enzyme glutamate decarboxylase.

Effect of glutamine on glutamate release from hippocampal slices induced by high K+ or by electrical stimulation: interaction with different Ca2+ concentrations.

To characterize the effect of glutamine on the release of glutamate, aspartate, and gamma-aminobutyric acid (GABA), rat hippocampal slices were superfused with different concentrations of glutamine or Ca2+. Amino acids released and retained were analyzed by HPLC. Glutamine (0.5 mmol/L) increased more than threefold the release of glutamate evoked by 50 mmol/L K+ in the presence of 2.6 mmol/L Ca2+ without a corresponding increase in glutamate content, while the release of aspartate was increased less and that of GABA not at all by glutamine. The evoked release of all three amino acids, including the enhanced release of glutamate in the presence of glutamine, was strongly dependent on Ca2+ concentrations between 0.1 and 2.6 mmol/L. The potentiation of glutamate release by glutamine reached a plateau at 0.25 mmol/L glutamine. Intermittent electrical field stimulation increased the release of only glutamate and this release was nearly doubled by glutamine. The increased release was Ca2+ dependent and tetrodotoxin (TTX) sensitive. Results suggest that extracellular glutamine promotes primarily the formation of releasable glutamate and this enhancement is dependent on extracellular Ca2+.

Glutamine enhances glutamate release in preference to gamma-aminobutyrate release in hippocampal slices.

To see the effect of physiological concentrations of glutamine on glutamate and gamma-aminobutyrate (GABA) release, rat hippocampal slices were incubated and (or) superfused without or with 0.25 mM glutamine in the presence or absence of Ca2+. The spontaneous and high K+-evoked release of glutamine, glutamate, and GABA was measured by precolumn derivatization and reversed phase high performance liquid chromatography. The spontaneous release of glutamate was increased by superfusion with glutamine and this increase was three times greater in the absence than in the presence of Ca2+. Spontaneous GABA release was not increased by glutamine. While in the absence of glutamine, the release of glutamate and GABA evoked by 50 mM K+ was about equal, in the presence of glutamine the evoked release of glutamate was nearly three times greater than that of GABA. The large evoked release of glutamate in the presence of glutamine was Ca2+ dependent nearly to the same extent as the smaller evoked release in the absence of glutamine. Results suggest that the availability of extracellular glutamine regulates the release of glutamate but not of GABA. Extracellular Ca2+ controls the spontaneous conversion of glutamine to glutamate but the site and mechanism of this control is uncertain.