Monitoring extracellular glutamate in hippocampal slices with a microsensor

The direct local assessment of glutamate in brain slices may improve our understanding of glutamatergic neurotransmission significantly. However, an analytical technique that monitors glutamate directly in brain slices is currently not available. Most recording techniques either monitor derivatives of glutamate or detect glutamate that diffuses out of the slice. Microsensors provide a promising solution to fulfill this analytical requirement. In the present study we have implanted a 10 microm diameter hydrogel-coated microsensor in the CA1 area of hippocampal slices to monitor extracellular glutamate levels. The influence of several pharmacological agents, which facilitate glutamate release from neurons or astrocytes, was investigated to explore the applicability of the microsensor. It was observed that KCl, veratradine, alpha-latrotoxine (LTX), DL-threo-beta-benzyloxyaspartate (dl-TBOA) and L-cystine rapidly increased the extracellular glutamate levels. As far as we know this is the first study in which a microsensor is applied to monitor dynamic changes of glutamate in brain slices and in our opinion this type of research may contribute greatly to improve our understanding of the physiology of glutamatergic neurotransmission.     

Intra-striatal phencyclidine inhibits N-methyl-D-aspartic acid-stimulated increase in glutamate levels of freely moving rats.

1. The authors investigated the effect of local phencyclidine (phenylcyclohexylpiperidine, PCP) on extracellular levels of glutamate and gamma-amino butyric acid (GABA) in rat striatum using in vivo microdialysis. 2. Intrastriatal infusion of PCP (1 mM) via a microdialysis probe did not alter the basal extracellular levels of either glutamate or GABA. Addition of N-methyl-D-aspartic acid (NMDA; 0.2, 0.5 and 1 mM) to the perfusion medium resulted in a dose-dependent increase in extracellular levels of glutamate. 3. Intrastriatal infusion of tetrodotoxin (0.1, 1, 10 microM), a highly selective blocker of voltage-dependent sodium channels, significantly attenuated the NMDA-stimulated release of glutamate, suggesting that NMDA-evoked release of glutamate originated from the neuronal pool and that the increase of striatal glutamate level was regulated indirectly via NMDA receptors. 4. The NMDA-induced release of glutamate was reduced significantly by pretreatment with local PCP (1 mM). Dizocilpine (MK801; 0.2 mM), a non-competitive NMDA antagonist, completely inhibited the NMDA-stimulated release of glutamate. 5. These results suggest that, in the striatum, PCP inhibits corticostriatal glutamatergic neurotransmission by inhibiting the release of glutamate probably via postsynaptic NMDA receptors.     

Extracellular glutamate and dopamine measured by microdialysis in the rat striatum during blockade of synaptic transmission in anesthetized and awake rats

We investigated the effect of high dose tetrodotoxin (TTX) on microdialysis measurements of extracellular striatal glutamate and dopamine in normal female rats. Both halothane-anesthetized rats with acutely implanted microdialysis probes and awake rats with microdialysis probes implanted for 24 h were tested. Glutamate levels in awake rats were 45% higher than those of anesthetized rats. Extracellular glutamate remained TTX-insensitive irregardless of TTX concentration, anesthesia, or time lapsed after probe implantation. In contrast, TTX reduced dialysate dopamine in all TTX concentrations tested. We speculate that the lower glutamate levels in anesthetized rats reflect the effect of anesthesia. Because glutamate is involved, either as a reactant or a product in a variety of reactions critical to intermediary metabolism in the brain, basal dialysate glutamate levels might indirectly reflect brain metabolism. Further, we conclude that extracellular glutamate collected during non-stimulated conditions is TTX-insensitive. The fact that glutamate levels are TTX-independent does not rule out that glutamate is synaptic in origin but rather demonstrates that it is not nerve impulse-dependent. However, the brain interstitial glutamate pool accessible to the microdialysis probe during control conditions is most likely isolated from the synapse, and therefore does not impose a neurotoxic potential.     

Tonic and phasic release of glutamate and acetylcholine neurotransmission in sub-regions of the rat prefrontal cortex using enzyme-based microelectrode arrays

The medial prefrontal cortex (mPFC) is an area of the brain critical for higher cognitive processes and implicated in disorders of the CNS such as drug addiction, depression and schizophrenia. Glutamate and acetylcholine are neurotransmitters that are essential for cortical functioning, yet little is known about the dynamic function of these neurotransmitters in subregions of the mPFC. In these studies we used a novel microelectrode array technology to measure resting levels (tonic release) of glutamate and acetylcholine as well as KCl-evoked release (stimulated phasic release) in the mPFC of the anesthetized rat to further our understanding of both tonic and phasic neurotransmission in the cingulate cortex, prelimbic cortex, and infralimbic cortex of the mPFC. Studies revealed homogeneity of tonic and phasic signaling among brain subregions for each neurotransmitter. However, resting levels of glutamate were significantly higher as compared to acetylcholine levels in all subregions. Additionally, KCl-evoked acetylcholine release in the cingulate cortex (7.1 μM) was significantly greater than KCl-evoked glutamate release in any of the three subregions (Cg1, 2.9 μM; PrL, 2.0 μM; IL, 1.8 μM). Interestingly, the time for signal decay following KCl-evoked acetylcholine release was significantly longer by an average of 240% as compared to KCL-evoked glutamate release for all three brain subregions. Finally, we observed a negative relationship between acetylcholine resting levels and KCl-evoked release in the Cg1. These data suggest a homogenous distribution of both glutamatergic and acetylcholinergic innervation in the mPFC, with alterations in tonic and phasic release regulation accounting for differences between these neurotransmitters.     

Neuron-glia interactions in glutamatergic neurotransmission: roles of oxidative and glycolytic adenosine triphosphate as energy source

Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear whether ATP generated in these pathways is equivalent in relation to fueling of the energy-requiring processes, i.e., vesicle filling, transport, and enzymatic processing in the glutamatergic tripartite synapse (the astrocyte and pre- and postsynapse). The role of astrocytic glycogenolysis in maintaining theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway-selective substrates were used to study glutamate uptake and release monitored with D-aspartate. Western blotting of glyceraldehyde-3-P dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was performed to determine whether these enzymes are associated with the cell membrane. We show that ATP formed in glycolysis is superior to that generated by oxidative phosphorylation in providing energy for glutamate uptake both in astrocytes and in neurons. The neuronal vesicular glutamate release was less dependent on glycolytic ATP. Dependence of glutamate uptake on glycolytic ATP may be at least partially explained by a close association in the membrane of GAPDH and PGK and the glutamate transporters. It may be suggested that these enzymes form a complex with the transporters and the Na(+) /K(+) -ATPase, the latter providing the sodium gradient required for the transport process.     

Acute stress-mediated increases in extracellular glutamate levels in the rat amygdala: differential effects of antidepressant treatment

Depressive illness is associated with changes in amygdalar volume, and stressful life events are known to precipitate depressive episodes in this patient population. Stress affects amygdalar synaptic plasticity and several neurotransmitter systems have been implicated in stress-mediated changes in the brain, including the glutamatergic system. However, the role of the glutamatergic system in stress-mediated plasticity in the amygdala remains to be determined. Accordingly the current study examined the stress modulation of extracellular glutamate levels in the basolateral nucleus (BLA) and the central nucleus (CeA) of the amygdala by in vivo microdialysis. Acute stress increased extracellular glutamate levels in the BLA and CeA, although the dynamics of these stress-mediated changes were dramatically different in these amygdalar nuclei. Tetrodotoxin administration reduced basal, and completely eliminated stress-mediated increases in glutamate efflux in the amygdala, demonstrating that stress effects are dependent on local axonal depolarization. Moreover, stress-mediated increases in glutamate efflux in the BLA were inhibited by the antidepressant tianeptine but not by the selective serotonin-reuptake inhibitor fluoxetine. Collectively, these data demonstrate that stress-induced modulation of glutamate neurochemistry reflects a fundamental pathological change that may contribute to the aetiology and progression of depressive illness, and suggest that some antidepressants such as tianeptine may elicit their clinical effects by modulation of glutamatergic neurotransmission.     

Effects of handling on extracellular levels of glutamate and other amino acids in various areas of the brain measured by microdialysis

Upon a physiological and pharmacological challenge, the responsiveness of extracellular glutamate levels in the prefrontal cortex, ventral tegmental area and locus coeruleus were studied using microdialysis. A 10-min handling period was used as a mild stressful stimulus. In all three brain areas, handling induced an immediate and short-lasting increase in glutamate levels, but the responses were highly variable. Only in the ventral tegmental area and the locus coeruleus, but not in the prefrontal cortex, the increases were significantly different from basal values. In rats with relatively low basal glutamate levels, both in the ventral tegmental area and locus coeruleus, handling had a more pronounced effect on glutamate levels than in rats with high basal levels, although in some rats with relatively low basal levels of glutamate, handling had hardly any effect. Potassium stimulation also induced variable responses in all three brain areas. Again, relatively low basal glutamate levels were more responsive to the stimulation than higher basal values, although there appeared to be a lower limit. These data suggest that relatively high basal levels contain sources of glutamate that mask the neuronal pool of glutamate and are therefore less responsive to physiological or pharmacological stimulation. However, this interpretation was questioned by the findings that basal levels and handling-induced increases in glutamate levels were found to be (partly) TTX-independent. As carrier-mediated release as a possible non-exocytotic release mechanism has only been described in vivo under pathological conditions, it seems plausible to ascribe TTX-independent glutamate increases to aspecific, non-neuronal processes. This interpretation was further supported by the observation that in all three brain areas, other amino acids, i.e., aspartate, taurine, glutamine, serine, alanine and glycine also increased upon handling in a very similar way as glutamate did. Thus, these results question a direct correlation between stimulated extracellular glutamate levels induced by handling and measured by microdialysis and glutamatergic neurotransmission.     

Relationship between zinc and neurotransmitters released into the amygdalar extracellular space

On the basis of the evidence that vesicular zinc may be essential to the functions of the amygdala, the movement and action of actively functioning zinc in synapses in the amygdala of rats were studied using in vivo microdialysis. The increase of (65)Zn release into the amygdalar extracellular space during stimulation with high K(+) was inhibited by the addition of 1 microM tetrodotoxin. High-K(+)-induced (65)Zn release was not observed in the substantia nigra, in which zinc-containing glutamatergic neuron terminals are assumed not to exist. The amount of (65)Zn released into the amygdalar extracellular space during stimulation with high K(+) was correlated with that of glutamate. These results suggest that zinc may be concurrently released with glutamate from the neuron terminals in the amygdala and that zinc may cooperate with glutamate in excitatory neurotransmission. When the amygdala was perfused with 10 microM calcium-ethylenediamine tetraacetic acid (CaEDTA) to chelate zinc in the extracellular space, the levels of glutamate in the extracellular space were not appreciably influenced, whereas those of gamma-aminobutyric acid (GABA) were remarkably increased. It is likely that vesicular zinc modulates GABA release in the amygdala. The modulation of GABAergic neuron activity by zinc may be important for the functions of the amygdala.     

The emerging role of glutamate in the pathophysiology and treatment of schizophrenia

OBJECTIVE: Research has implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. This review evaluates evidence from preclinical and clinical studies that brain glutamatergic neurotransmission is altered in schizophrenia, may affect symptom expression, and is modulated by antipsychotic drugs. METHOD: A comprehensive review of scientific articles published over the last decade that address the role of glutamate in the pathophysiology of schizophrenia was carried out. RESULTS: Glutamatergic neurons are the major excitatory pathways linking the cortex, limbic system, and thalamus, regions that have been implicated in schizophrenia. Postmortem studies have revealed alterations in pre- and postsynaptic markers for glutamatergic neurons in several brain regions in schizophrenia. The N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptor may be particularly important as blockade of this receptor by the dissociative anesthetics reproduces in normal subjects the symptomatic manifestations of schizophrenia, including negative symptoms and cognitive impairments, and increases dopamine release in the mesolimbic system. Agents that indirectly enhance NMDA receptor function via the glycine modulatory site reduce negative symptoms and variably improve cognitive functioning in schizophrenic subjects receiving typical antipsychotics. CONCLUSIONS: Dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia, especially of the negative symptoms and cognitive impairments associated with the disorder, and is a promising target for drug development.     

The Astrocyte-Derived α7 Nicotinic Receptor Antagonist Kynurenic Acid Controls Extracellular Glutamate Levels in the Prefrontal Cortex

The cognitive deficits seen in schizophrenia patients are likely related to abnormal glutamatergic and cholinergic neurotransmission in the prefrontal cortex. We hypothesized that these impairments may be secondary to increased levels of the astrocyte-derived metabolite kynurenic acid (KYNA), which inhibits α7 nicotinic acetylcholine receptors (α7AChR) and may thereby reduce glutamate release. Using in vivo microdialysis in unanesthetized rats, we show here that nanomolar concentrations of KYNA, infused directly or produced in situ from its bioprecursor kynurenine, significantly decrease extracellular glutamate levels in the prefrontal cortex. This effect was prevented by the systemic administration of galantamine (3 mg/kg) but not by donepezil (2 mg/kg), indicating that KYNA blocks the allosteric potentiating site of the α7AChR, which recognizes galantamine but not donepezil as an agonist. In separate rats, reduction of prefrontal KYNA formation by (S)-4-ethylsulfonyl benzoylalanine, a specific inhibitor of KYNA synthesis, caused a significant elevation in extracellular glutamate levels. Jointly, our results demonstrate that fluctuations in endogenous KYNA formation bidirectionally influence cortical glutamate concentrations. These findings suggest that selective attenuation of cerebral KYNA production, by increasing glutamatergic tone, might improve cognitive function in individuals with schizophrenia.     

Imaging of glutamate in brain slices using FRET sensors

The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Forster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.     

Increased serum levels of glutamate in adult patients with autism

BACKGROUND: Precise mechanisms underlying the pathophysiology of autism are currently unknown. Given the major role of glutamate in brain development, we have hypothesized that glutamatergic neurotransmission plays a role in the pathophysiology of autism. In this study, we studied whether amino acids (glutamate, glutamine, glycine, D-serine, and L-serine) related to glutamatergic neurotransmission are altered in serum of adult patients with autism. METHODS: We measured serum levels of amino acids in 18 male adult patients with autism and age-matched 19 male healthy subjects using high-performance liquid chromatography. RESULTS: Serum levels (mean = 89.2 microM, S.D. = 21.5) of glutamate in the patients with autism were significantly (t = -4.48, df = 35, p < 0.001) higher than those (mean = 61.1 microM, S.D. = 16.5) of normal controls. In contrast, serum levels of other amino acids (glutamine, glycine, d-serine, l-serine) in the patients with autism did not differ from those of normal controls. There was a positive correlation (r = 0.523, p = 0.026) between serum glutamate levels and Autism Diagnostic Interview-Revised (ADI-R) social scores in patients. CONCLUSIONS: The present study suggests that an abnormality in glutamatergic neurotransmission may play a role in the pathophysiology of autism.     

Role of endothelins as mediators of injury-induced alterations of glial glutamate turnover

Astroglia terminate glutamatergic neurotransmission and prevent excitotoxic extracellular glutamate concentration by clearing synaptically released glutamate through the high-affinity, sodium-dependent glutamate transporters GLT-1 and GLAST. Many brain injures are associated with the disturbed expression of glial glutamate transporters and a subsequent increase of extracellular glutamate to neurotoxic levels. We have now followed up initial hints pointing to endothelins, a family of injury-regulated peptides, as mediators of this injury-induced loss of glial glutamate transporter expression. We observed that, in line with such a role, endothelins not only act as potent inhibitors of basal and exogenously (dbcAMP)-induced expression of GLT-1 in cortical astrocytes as shown before, but likewise inhibit expression of GLT-1 or GLAST in astrocytes cultured from the diencephalon, mesencephalon, cerebellum, and spinal cord. We further demonstrate that endothelins equally inhibit GLT-1 expression in cortical slice cultures, a culture system closely resembling the in vivo situation. Although brain injuries are usually associated with an increase in the expression of the glutamate-converting enzyme glutamine synthetase, cultured cortical astrocytes maintained with endothelins showed an almost complete loss of glutamine synthetase. Interestingly, the inhibitory effects of endothelins on the expression of glutamine synthetase, but not of glutamate transporters, was overridden by high extracellular glutamate, indicating that the primarily inhibitory action of endothelins on the various components of glial glutamate turnover dissociates in the injured brain.     

Aβ oligomers induce glutamate release from hippocampal neurons

Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in Alzheimer's disease (AD) brain and have been implicated in mechanisms of pathogenesis. The neurotoxicity of AβOs appears to be, at least in part, due to dysregulation of glutamate signaling. Here, we show that AβOs promote extracellular accumulation of glutamate and d-serine, a co-agonist at glutamate receptors of the N-methyl-d-aspartate subtype (NMDARs), in hippocampal neuronal cultures. The increase in extracellular glutamate levels induced by AβOs was blocked by the sodium channel blocker tetrodotoxin (TTX), by the NMDAR blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) and by removal of Ca(2+) from the extracellular medium, indicating dependence on excitatory neuronal activity. AβOs enhanced both the release of pre-synaptic vesicles labeled by FM1-43 and spontaneous post-synaptic activity measured by whole-cell patch-clamp. Activation of inhibitory GABA(A) receptors by taurine blocked the increase in extracellular glutamate levels, suggesting that selective pharmacological inhibition of neuronal activity can counteract the impact of AbOs on glutamate dyshomeostasis. Results reveal a novel mechanism by which Ab oligomers promote abnormal release of glutamate in hippocampal neurons, which may contribute to dysregulation of excitatory signaling in the brain.     

Effect of anoxia on glutamate formation from glutamine in cultured neurons: dependence on neuronal subtype

Synthesis and release of glutamate formed from labeled glutamine were studied in primary cultures of the glutamatergic cerebellar granule cells and of the mainly GABAergic cerebral cortical neurons under anoxic conditions and under normoxic control conditions. Under both control and anoxic conditions cerebellar granule cells synthesized and released glutamate more intensely than cerebral cortical neurons, but this difference was enhanced under anoxic conditions. Thus, under normoxic conditions synthesis of intracellular labeled glutamate from glutamine was twice as high in cerebellar granule cell neurons as in cerebral cortical neurons during 30 min of incubation, but the release of newly synthesized labeled glutamate to the extracellular medium from cerebellar granule cell neurons was more than 4 times higher than the release from cerebral cortical neurons. Under anoxic conditions the release from cerebellar granule cell neurons became 13 times higher than the release from cerebral cortical neurons during 30 min of incubation. Based on these observations it is suggested that a major reason for the increase in extracellular glutamate concentration during brain ischemia may be enhanced production and release of glutamate, especially in glutamatergic neurons.     

Activity-dependent plasticity of inhibitory and excitatory amino acid transmitter systems in cultured rat cerebral cortex

Chronic suppression of spontaneous bioelectric activity in cultures of dissociated fetal rat cerebral cortex increases neuronal cell death and results in electrophysiological changes which indicate an altered balance between excitatory and inhibitory neurotransmission in culture. To delineate whether alterations in neurotransmitter release could underlie this imbalance, we investigated the effects of chronic tetrodotoxin (TTX) treatment on the content and release of glutamate, aspartate and γ-aminobutyric acid (GABA) in culture. Chronic TTX treatment decreased the content of all amino acids investigated. However, only GABA was decreased relative to the neuronal marker NSE (neuron-specific enolase), indicating a disproportionate loss of GABA production following chronic silencing. Depolarization-induced release of GABA, glutamate and aspartate increased about 10-fold between 7 and 21 days in control cultures. Chronic TTX treatment significantly increased the depolarization-induced release of glutamate and aspartate at 7 days in vitro relative to control levels. At all ages it caused a two-fold increase in the ratio of evoked excitatory amino acid release to that of GABA. These observations suggest that chronic silencing of developing neocortex cell cultures increases the ratio of excitatory to inhibitory synaptic activity either by differential cell death or by reduced synaptic efficiency, on which a decrease in GABA neurotransmission appears to play a major role. Since similar mechanisms may be involved in activity-dependent plasticity in vivo, these cultures provide a useful model to analyse this phenomenon at the cell biological and molecular level.     

Optical glutamate sensor for spatiotemporal analysis of synaptic transmission

Imaging neurotransmission is expected to greatly improve our understanding of the mechanisms and regulations of synaptic transmission. Aiming at imaging glutamate, a major excitatory neurotransmitter in the CNS, we developed a novel optical glutamate probe, which consists of a ligand-binding domain of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor GluR2 subunit and a small molecule fluorescent dye. We expected that such fluorescent conjugates might report the microenvironmental changes upon protein conformational changes elicited by glutamate binding. After more than 100 conjugates were tested, we finally obtained a conjugate named E (glutamate) optical sensor (EOS), which showed maximally 37% change in fluorescence intensity upon binding of glutamate with a dissociation constant of 148 nm. By immobilizing EOS on the cell surface of hippocampal neuronal culture preparations, we pursued in situ spatial mapping of synaptically released glutamate following presynaptic firing. Results showed that a single firing was sufficient to obtain high-resolution images of glutamate release, indicating the remarkable sensitivity of this technique. Furthermore, we monitored the time course of changes in presynaptic activity induced by phorbol ester and found heterogeneity in presynaptic modulation. These results indicate that EOS can be generally applicable to evaluation of presynaptic modulation and plasticity. This EOS-based glutamate imaging method is useful to address numerous fundamental issues about glutamatergic neurotransmission in the CNS.     

A receptor for presynaptic glutamatergic autoinhibition is a glutamate transporter

Monoquantal excitatory postsynaptic currents were recorded by means of a perfused macropatch electrode from 9 to 15 micro m stretches of crayfish neuromuscular junctions. The excitatory transmitter l-glutamate superfused to a terminal inhibits quantal release by activating autoreceptors [Parnas et al. (1996) Eur. J. Neurosci., 8, 116-126]. Substances related to glutamate that do not activate glutamatergic postsynaptic channels, but are substrates of glutamate transporters, elicited analogous inhibitions, e.g. l- and d-aspartate and some other glutamate transport blockers. As expected, all transport blockers prolonged synaptic currents. Blockers that bind to the transporter receptors but are not transported did not inhibit release, but prevented inhibition by the transport substrates. It appears that autoinhibition is elicited by transport of glutamate or its analogues. Transport into cells is powered by symport of three Na+. To block the transport step electrochemically, extracellular Na+ concentration was lowered to one-quarter, but this surprisingly left the inhibition of release by glutamate unaffected, showing inhibition to be associated to a step between binding and transport. After binding a substrate, glutamate transporters open a parallel Cl- channel. Replacement of extracellular Cl- prevented Cl- current, and release inhibition by glutamate or aspartate was blocked. It is suggested that the flow of Cl- across the cell membrane, after binding a transport substrate, mediates autoinhibition. We measured a related reduction of presynaptic action potentials.