Alberto Morales-Villagrán

Accumulation of Extracellular Glutamate by Inhibition of Its Uptake Is Not Sufficient for Inducing Neuronal Damage: An In Vivo Microdialysis Study

Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high‐affinity sodium‐dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l‐trans‐pyrrolidine‐2,4‐dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl‐threo‐β‐hydroxyaspartate and l‐aspartate‐β‐hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate or by intrastriatal coinjection of 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.

Preferential stimulation of glutamate release by 4-aminopyridine in rat striatum in vivo

The potassium channel blocker 4-aminopyridine (4-AP) is a potent convulsant drug which, in vitro, stimulates the release of neurotransmitter amino acids. We have studied the effect of 4-AP in vivo on the extracellular concentration of amino acids in rat striatum, by means of microdialysis and HPLC. Perfusion with 4-AP in the awake animal produced intense motor alterations, including barrel turning and running fits. Therefore, most microdialysis experiments were carried out in anesthetized rats. Perfusion with 20-75 mM 4-AP for 12.5 min resulted in a massive increase in extracellular glutamate (up to 20-fold), smaller increases in aspartate and taurine (up to 10-fold) and slight increments in glutamine, alanine, glycine and GABA. In contrast, perfusion with 100 mM K+ produced, mainly, an increment in taurine (7-fold) and modest increases in glutamate and aspartate (100-300%), as well as a notable decrease in glutamine. Tetraethylammonium (TEA, 120 mM) perfusion induced taurine and glutamate elevations similar to those after high K+, but glutamine was not affected. In unanesthetized rats, perfusion with 40 mM 4-AP induced changes in extracellular amino acids similar to those observed under anesthesia. In these animals neither high K+ nor TEA affected significantly the motor behavior. The results suggest that an enhancement of glutamatergic synaptic transmission, rather than a general depolarizing action, is an important factor in the neuronal hyperexcitability induced by 4-AP, which is consistent with the previously demonstrated inhibition of its convulsant effect by glutamate receptor antagonists.

Antiepileptic effect of carbenoxolone on seizures induced by 4-aminopyridine: a study in the rat hippocampus and entorhinal cortex.

We have examined the effects of the gap junction blocker carbenoxolone (CBX) on the generation and propagation of epileptiform activity induced by 4-aminopyridine (4-AP) in the rat entorhinal cortex and hippocampus. We analyzed the epileptiform pattern generated on awaked rats by administering 10 nmol of 4-AP and we studied the effect of administering CBX (50 nmol) 30 min later by injection into the entorhinal cortex. The injection of 4-AP produced an epileptiform pattern in EEG recordings characterized by an initial hypersynchronic activity followed by trains of high-amplitude epileptiform discharges. This pattern was associated with convulsive behavior rated as 0, 1 and 3 in the Racine Scale. In contrast, no changes in electrical activity or behavior were observed in animals that received NaCl or CBX alone. The application of CBX to rats that had received 4-AP decreased the amplitude and frequency of the epileptiform discharges, as well as the number and duration of the epileptiform trains in the entorhinal cortex and hippocampus. Indeed, discharge trains were completely blocked by CBX after 22+/-4.4 min, and likewise CBX reverted the convulsive behavior of these animals. We conclude that Gap junctions participate in the generation and propagation of epileptiform activity induced by 4-AP in these regions, as well as blocking motor alterations.

Protection by NMDA receptor antagonists against seizures induced by intracerebral administration of 4-aminopyridine

The effects of NMDA receptor antagonists on the convulsant action of the administration of 4-aminopyridine in the rat lateral cerebral ventricle (i.c.v. injection) and motor cerebral cortex (i.cx. injection) were studied. 4-Aminopyridine administration in both regions induced various preconvulsive symptoms, such as salivation, tremors, chewing and rearing, followed by continuous clonic convulsions and, only after i.c.v. injection, running fits and generalized tonic convulsions. This behavioral pattern appeared 5-9 min after administration of 4-aminopyridine and persisted for 100-150 min. 4-Aminopyridine also generated epileptiform electroencephalographic (EEG) discharges characterized by isolated spikes, poly-spikes and spike-wave complexes, which began some seconds after administration of the drug and were present for more than 2 h. The NMDA receptor antagonists (+/-)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), (+/-)-2-amino-7-phosphono-heptanoic acid (AP7) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) clearly protected against some of the behavioral alterations induced by i.c.v. 4-aminopyridine, particularly the tonic convulsions, but were less effective against those produced by i.cx. 4-aminopyridine. These antagonists also delayed the appearance of EEG epileptiform discharges, reduced its amplitude, frequency and duration, and blocked their propagation to other cortical regions after i.cx. 4-aminopyridine. These results, together with previous data showing that 4-aminopyridine stimulates the release of glutamate in vivo, suggest that an excessive glutamatergic neurotransmission involving NMDA receptors is implicated in 4-amino-pyridine-induced seizures.

Action of 4-aminopyridine on extracellular amino acids in hippocampus and entorhinal cortex: a dual microdialysis and electroencehalographic study in awake rats.

In order to study the role of amino acids in the hippocampus and the entorhinal cortex during the convulsive process induced by 4-aminopyridine (4-AP), we have used a device allowing the simultaneous microdialysis and the recording of their electrical activity of both regions in freely moving rats. We found that infusion of 4-AP into the entorhinal cortex resulted in a large increase in extracellular glutamate and glutamine and small increases in glycine and taurine levels. Likewise, infusion of 4-AP into the hippocampus resulted in a major increase in glutamate, as well as slight increases in taurine and glycine. In both infused regions the peak concentration of extracellular glutamate was observed 15 min after 4-AP administration. No significant changes were found in the non-infused hippocampus or entorhinal cortex of the same rats. Simultaneous electroencephalographic recordings showed intense epileptiform activity starting during 4-AP infusion and lasting for the rest of the experiment (1 h) in both the entorhinal cortex and the hippocampus. The discharges were characterized by poly-spikes and spike-wave complexes that propagated almost immediately to the other region studied. These findings suggest that increased glutamatergic synaptic function in the circuit that connects both regions is involved in the epileptic seizures induced by 4-AP.

Monosodium glutamate neonatal treatment as a seizure and excitotoxic model

Monosodium glutamate (MSG) subcutaneously administrated to neonatal rats induces several neurochemical alterations in the brain, which have been associated with an excitotoxic process triggered by an over activation of glutamate receptors; however there are few systematic studies about initial changes in intracerebroventricular (i.c.v.) Glu levels produced by MSG in the brain. Thus, to characterize these changes, rat pups were injected with a MSG solution at 1, 3, 5 and 7 postnatal days (PD), and i.c.v. Glu levels and hippocampal total content of related amino acids (Asp, Glu, Gln, Gly, Tau, Ala and GABA) were estimated before, immediately and after each injection. Behavioral and EEG responses were also monitored after MSG administrations. Significant rise in i.c.v. Glu levels were found, mainly in response to the first and second injection. Moreover, the total content of all amino acids evaluated also increased during the first hour after the first MSG administration but only Glu and GABA remained elevated after 24 h. These biochemical modifications were accompanied with behavioral alterations characterized by: screeching, tail stiffness, head nodding, emprosthotonic flexion episodes and generalized tonic-clonic convulsions, which were associated with electroencephalographic pattern alterations. Altered behavior found in animals treated with MSG suggests an initial seizure situation. Although four MSG administrations were used, the most relevant findings were observed after the first and second administrations at PD1 and PD3, suggesting that only two MSG injections could be sufficient to resemble a seizure and/or excitotoxic model.

Simultaneous glutamate and EEG activity measurements during seizures in rat hippocampal region with the use of an electrochemical biosensor

Excessive release of L-glutamic acid (glu) has been associated with seizures and epilepsy. Some microdialysis studies have demonstrated an increase in glu levels during seizures both in human and in different animal models of experimental epilepsy. With these techniques it is difficult to monitor the glu concentrations with sufficient time resolution to clearly associate them with EEG activity. To solve this, we have built an electrochemical biosensor based on H2O2 production. A glu biosensor was inserted in the hippocampus of rats with an attached isolated tungsten wire to simultaneously record epileptiform EEG activity. 4-Aminopyridine (10 nmol) was administered into the entorhinal cortex to induce seizures. EEG activity and glu concentrations were measured in real time in awake rats through the use of a swivel to capture and digitize analogical signals. When the first epileptiform burst appeared, it was accompanied by a single and significant increase in glu that could play an essential role in the initiation of the seizure. Subsequent and lesser glu increases also were observed; however they were not directly correlated with further bursts it could be relevant to maintenance of seizures. Sustained increase in glu concentration associated with a flat EEG recording was present when rats died.

Short- and long-term changes in extracellular glutamate and acetylcholine concentrations in the rat hippocampus following hypoxia

Hypoxia at birth is a major source of brain damage and it is associated with serious neurological sequelae in survivors. Alterations in the extracellular turnover of glutamate (Glu) and acetylcholine (ACh), two neurotransmitters that are essential for normal hippocampal function and learning and memory processes, may contribute to some of the neurological effects of perinatal hypoxia. We set out to determine the immediate and long-lasting effects of hypoxia on the turnover of these neurotransmitters by using microdialysis to measure the extracellular concentration of Glu and ACh in hippocampus, when hypoxia was induced in rats at postnatal day (PD) 7, and again at PD30. In PD7 rats, hypoxia induced an increase in extracellular Glu concentrations that lasted for up to 2.5 h and a decrease in extracellular ACh concentrations over this period. By contrast, perinatal hypoxia attenuated Glu release in asphyxiated rats, inducing a decrease in basal Glu levels when these animals reached PD30. Unlike Glu, the basal ACh levels in these animals were greater than in controls at PD30, although ACh release was stimulated less strongly than in control animals. These results provide the first evidence of the initial and long term consequences of the hypoxia on Glu and ACh turnover in the brain, demonstrating that hypoxia produces significant alterations in hippocampal neurochemistry and physiology.

Monosodium l-glutamate-induced convulsions: changes in uptake and release of catecholamines in cerebral cortex and caudate nucleus of adult rats

Adult rats (60 days old) were injected intraperitoneally with 5 mg/g monosodium L-glutamate (MSG). During the convulsive period (1 h after injection), uptake and release of [3H]norepinephrine (3H-NE) and [14C]dopamine (14C-DA) were measured in a crude synaptosomal fraction and in slices of cerebral cortex and caudate nucleus, respectively. A significant reduction of 3H-NE uptake was detected in cortical slices (by 42%) and in synaptosomal fraction (by 33%) of rats treated with MSG, whereas K+- stimulated 3H-NE release was decreased by 32% and 39% in brain slices and in a synaptosomal fraction of cerebral cortex, respectively, in comparison with animals injected with 0.9% NaCl aqueous solution (PSS). In the caudate nucleus, 14C-DA uptake was increased by 100% in brain slices and by 36% in the synaptosomal fraction following MSG administration, whereas K+- stimulated 14C-DA release was enhanced by 80% in slices and by 25% in synaptosomes as compared to PSS-injected rats. Data suggest that catecholaminergic neurotransmission may play an important role in the etiopathology of convulsions in the experimental model using MSG.

Cortical catecholamine changes and seizures induced by 4-aminopyridine in awake rats, studied with a dual microdialysis-electrical recording technique.

We describe a rotatory electrical device that permits the simultaneous microdialysis and electroencephalographic (EEG) recording, by means of bipolar electrodes attached to the microdialysis probe, in two brain regions of awake rats. Using this device, we have found that the microdialysis infusion of 4-aminopyridine (4-AP) in the motor cerebral cortex produces intense behavioral convulsions and EEG seizures in both the infused and the contralateral cortex. This convulsant action is accompanied by a remarkable increase of extracellular dopamine (about 15-fold), norepinephrine (2.4-fold) and vanillylmandelic acid (1.8-fold) concentration in the infused cortex. Delayed increases of these amines were observed also in the contralateral cortex. The results suggest that 4-AP induces the release of catecholamines either through a direct effect on nerve endings or as a consequence of seizures.