A novel method for simultaneous glutamate and extracellular activity measurement in brain slices with high temporal resolution.

Measurement of neurotransmitters during normal or altered function in cerebral slices could be an important tool to better understand the relationship between biochemical changes and electrophysiological activity. Some attempts of this analysis have been made; however, the current techniques do not have the appropriate time resolution to establish this relationship. The use of electrochemical biosensors has allowed for good time resolution, but problems related to the reduction of signal noise and biofouling of the electrode surface could be an important issue. In this work, we propose a new alternative to simultaneously measure glutamate and electrical activity with a high temporal resolution in brain slices. This approach is based on the use of enzymatic reactors that generate a fluorescent derivative from glutamate that can be measured at high temporal resolution. The results presented here show a reliable measurement of this neurotransmitter in brain slices obtained from intact animals under the effect of a glutamate transporter blocker DL-threo-beta-benzyloxyaspartate as well as the potassium channel blocker 4-aminopyridine. Differences in the levels of glutamate and high frequency and amplitude discharges as an effect of drug administration were found in brain slices obtained from epileptic rats (p<0.05). In conclusion, this method could be used to measure neurotransmitter concentration online at a near physiological temporal resolution, which can then be correlated to the electrical activity that is simultaneously recorded.

Serotonin modulates fast ripple activity in rats with spontaneous recurrent seizures.

Fast ripples (FRs) are pathological high frequency oscillations that occur in patients with temporal lobe epilepsy (TLE), as well as in animal models of epilepsy in which seizures are induced with kainic acid or pilocarpine. These oscillations have been considered potential biomarkers of epileptogenesis in the hippocampus. Indeed, experimental evidence suggests an important role of serotonin in epilepsy and an increased frequency of FRs have been demonstrated in slow wave sleep, a period during which serotonin levels decrease. Accordingly, we investigated the role of serotonin in FRs modulation by evaluating the effects of citalopram, a blocker of serotonin uptake, on the occurrence of spontaneous FRs measured through intracranial bilateral EEG recording of the hippocampus of rats with spontaneous recurrent seizures. In addition, we recorded the mean number of oscillation cycles per FRs event and the average frequency (Hz) before, during and after citalopram administration in order to determine whether increases in extra-synaptic serotonin levels modulate FRs. The elevation of serotonin levels induced by citalopram (4.78 ± 1.69 nM) reduced the occurrence of spontaneous FRs (57%), the mean number of oscillation cycles per FRs event (34%) and the average frequency of FRs (33%). These findings suggest an important modulatory effect of serotonin on FRs.

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.

[Structural and functional characteristics of glutamate transporters: how they are related to epilepsy and oxidative stress]. / Caracteristicas estructurales y funcionales de los transportadores de glutamato: su relacion con la epilepsia y el estres oxidativo.

AIMS: The article highlights the general structural characteristics, functional properties and distribution of glutamate transporters, as well as the role they play in epilepsy and oxidative stress. DEVELOPMENT: Transporters of amino acids such as glutamate are considered to be proteins that are extremely important in the central nervous system because they participate in the capture of the neurotransmitter following its release in the synaptic cleft, thus putting an end to its effect and limiting glutamate-mediated excitability. These proteins belong to the family of Na+/K+ dependent transporters. A growing body of evidence has been gathered to show that these transporters are involved in several neuronal disorders, such as epilepsy and cerebral ischaemia. In this regard, it is considered that some defect in the structure of the transporters could affect their functioning and, therefore, favour the hyperexcitability produced by glutamate; this in turn would lead to the pathological disorders that are found in epilepsy. CONCLUSIONS: A detailed study of the structure and functioning of these transporters, as well as the role they play in the more common neurological diseases, such as epilepsy, would afford us a clearer view of new therapeutic alternatives with which to fight this kind of neuronal disorder in the future.

Cerebral serotonin depletion induces egocentric learning improvement in developing rats.

Egocentric learning ability of developing serotonin (5-HT)-depleted female rats was evaluated in the Morris maze test. 5-HT depletion was accomplished by a unique intracisternal injection of 5,7-dihydroxytriptamine at 21 days-old. A first behavioral test was applied before the lesion procedure. The animals were thereafter challenged to resolve the same test at 40 and 60 days-old. 5-HT depletion caused a dual effect on the rats' egocentric learning ability, i.e. at 40 days; control rats learned the task while the experimental rats were unable to learn it. At 60 days, control animals were unable to learn the test, while the experimental rats showed a successful performance. These results strongly suggest that 5-HT neurotransmission is necessary for egocentric learning establishment and regulation.

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.

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.

Niveles de catecolaminas bajo el efecto del HEPB en diferentes regiones del SNC de ratón adulto: estudio preliminar / Preliminary study of the effect of HEPB on catecholamine levels in different regions of the brain

Diversos estudios han demostrado que la norepinefrina (NE) y la dopamina (DA) participan en la regulación del umbral convulsivo en diferentes modelos de epilepsia experimental a través de una estrecha relación entre la concentración y la predisposición para desarrollar crisis convulsivas. También, se ha demostrado que el mecanismo de acción de algunos anticonvulsionantes conocidos, se realiza bajo una regulación de la neurotransmisión a nivel sináptico. Por otro lado, los derivados de las butiramidas presentan una importante actividad anticonvulsionante en algunos animales de experimentación. Por lo que en el presente trabajo se investigó el efecto de un derivado de las butiramidas, la 4-hidroxi, 4-etil, 4-fenil butiramida (HEPB) sobre la concentración de NE y DA en diferentes regiones cerebrales como son: la corteza cerebral, el núcleo caudado y el hipocampo. Se utilizaron ratones adultos Balb/c tratados con solución salina fisiológica (testigo), propilenglicol al 10 por ciento como vehículo (testigo) y HEPB (experimental). Las regiones cerebrales se ontuvieron previa decapacitación de los animales y la concentración de NE y DA se realizó por cromatografía de líquidos de alta resolución con detección electroquímica. Los resultados muestran que el HEPB induce un incremento de NE (70 por ciento) en el núcleo caudado a la dosis de 20 mg/kg de peso corporal. Mientras que la DA sólo se incremento en 38 por ciento en esta misma región. En la corteza cerebral y el hipocampo no se encontró diferencia significativa respecto a los grupos testigo. Con estos resultados es difícil explicar el efecto del HEPB sobre el sistema catecolaminérgico en el núcleo caudado, no obstante, es necesario continuar con estudios complementarios que permitan precisar el posible mecanismo de acción del HEPB como anticonvulsionante y en particular su efecto sobre el transporte de DA en el núcleo caudado

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.

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.

Accumulation of extracellular glutamate by inhibition of its uptake is not sufficient for inducing neuronal damage: an in vivo microdialysis study.

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-beta-hydroxyaspartate and L-aspartate-beta-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.

Enhancement in dopamine uptake and release induced by monosodium L-glutamate from caudate nucleus under in vitro conditions.

L-Glutamate has an excitatory and cytotoxic effect on the central nervous system. It was shown previously that norepinephrine and dopamine uptake and release were affected by in vivo administration of glutamate to adult rats. The kinetic parameters, Km and Vmax of [14C]DA uptake and release were measured on synaptosomal and slices from caudate nucleus under in vitro conditions at different glutamate concentrations. Results showed an important increase in [14C]DA uptake on synaptosomal (> 100%) and slices by lower glutamate concentrations, the affinity for transport system was increased (100%) and its release of high potassium evoked was also increased at 0.5 microM of glutamate. The results suggest the possibility that glutamate may modify DA uptake and release interacting with the DA transporter complex at the synaptic level.

Effect of systemic monosodium L-glutamate on muscarinic cholinergic receptors in selected rat brain regions during development.

[3H]quinuclidinyl benzilate ([3H]QNB) binding to muscarine acetylcholine receptors (mAchR) was measured in cerebral cortex and caudate nucleus of rats at the ages of 7, 14, and 21 days, which had received a subconvulsive intraperitoneal dose of monosodium L-glutamate (MSG) (4 mg/g) on postnatal days 1, 3, 5 and 7. MSG treatment determined an increase of mAchR density in cerebral cortex. This was 8, 15 and 25% at days 7, 14 and 21, respectively. In caudate nucleus, a significant increase of mAchR density was detected at day 7 (240%). However, on postnatal day 14, mAchR binding in caudate nucleus of MSG-treated rats was only 47% higher, while at 21 days, no changes in mAchR binding were found. When MSG was injected to adult rats, no changes in brain mAchR density were detected. Data suggest that early administration of MSG affects the development of mAchR in cerebral cortex and caudate nucleus, whereas the adult brain cortical cholinergic transmission is not sensitive to parenterally administered MSG.

Effect of 3-acetylpyridine on serotonin uptake and release from rat cerebellar slices.

The cerebellum receives indolaminergic fibers influencing Purkinje cell discharges. Data from our laboratories have demonstrated an endogenous release of serotonin (5-HT) and a Na(+)-dependent uptake and Ca(2+)-dependent release of [3H]5-HT from slices, homogenates and synaptosomal fractions of the rat cerebellar molecular layer. While the neurotransmitter produced by climbing fibers has been sought for in several studies and some of the classical transmitters have been ruled out, as yet this neurotransmitter is unknown. The aim of this work was to measure the 5-HT uptake and release from rat cerebellar slices, 6 h and 15 days after intraperitoneal injection of 3-acetylpyridine (3-AP) (75 mg/kg), harmaline (15 mg/kg) and nicotinamide (300 mg/kg). A histological study of medulla and cerebellar cortex in these animals showed destruction of neurons in the inferior olivary nuclei and changes in the granulation of the cortical molecular layer in the cerebellum. A significant reduction of the 5-HT content (100%), 5-HT uptake (60%) and its Vmax (60%) was seen on the 5th day, in cerebellar preparations obtained from rats injected with 3-AP. The Ca(2+)-dependent release of 5-HT from these preparations was found to be similar to the basal values, in spite of depolarizing stimuli with 53 mM KCl or veratrine (60 micrograms/ml). The results suggest that 5-HT could play an important role as neurotransmitter produced by some climbing fibers.

A simple biological assay for relaxin measurement.

1. Relaxin (R) is considered a gestation hormone with an insulin-like molecular structure. Its physiological importance is significant in the reproduction process. 2. Different methods of biologically assaying R have been published but electrophysiology techniques on uterus and ileum of rat have never been used. 3. A protein fraction was obtained from ovarian tissue of the rat and used to measure electrophysiological activity in vivo and in vitro. 4. Protein recovered with R activity was similar to that in previous reports. 5. Reduction of 100% in contraction strength and 50% in its frequency was observed in ileum and in uterus respectively; it was only of 60%, but its frequency increased 43%. 6. Methodological considerations and some physiological aspects are discussed.

Effects of ruthenium red intraperitoneally injected to adult rats, on the uptake and release of neurotransmitters from brain synaptosomes.

Effect of intraperitoneal (i.p.) injection of ruthenium red (RuR) to adults rats on catecholamine (CA) uptake and dopamine (DA) release was evaluated in brain synaptosomal fractions. No effect on CA uptake by synaptosomal fractions was seen when RuR was added in vitro. There was no effect of RuR injection i.p. to rats, on CA uptake by their brain synaptosomal fractions. A blocking effect on depolarizing induced released of DA from brain synaptosomal fraction obtained from rats previously injected i.p. with RuR was seen. A similar effect was observed on the basal DA efflux from synaptosomes loaded with (14C)DA obtained from rats i.p. injected with RuR. It is concluded that RuR, a non-liposoluble substance, when injected i.p. reaches the brain parenchyma through areas devoid of blood-brain barrier and interferes with Ca(++)-dependent release of neurotransmitters, inducing cerebral hyperexcitability and convulsions.

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.

Serotonin uptake in the central nervous system of rats fed a corn-diet.

1. Endogenous serotonin (5-HT), 5-hydroxyindol acetic acid (5-HIAA) content and exogenous 5-HT uptake (Km and Vmax) were measured in different brain regions (cerebellum, diencephalon, brain stem and telencephalon) of rats fed with a corn diet and restricted protein (8%) diet during 6 weeks. 2. A reduction of 5-HT levels was found in all regions studied of animals fed a corn diet, whereas, 5-HIAA was only decreased in brain stem and diencephalon. 3. An important increase in Km and Vmax were registered in brain stem and diencephalon of protein restricted animals, whereas, an increase of 5-HT uptake affinity in cerebellum, brain stem and telencephalon (35, 42 and 33% respectively) was observed. Simultaneously, under corn diet conditions, the Vmax decreased 40, 30 and 34% respectively in those regions. 4. It is suggested that the brain stem was the more sensitive area under nutritional restricted conditions and the development of some possible compensatory mechanisms of the 5-HTergic system is discussed.