Absence epileptic activity changing effects of non-adenosine nucleoside inosine, guanosine and uridine in Wistar Albino Glaxo Rijswijk rats.

Adenosine (Ado) and non-adenosine (non-Ado) nucleosides such as inosine (Ino), guanosine (Guo) and uridine (Urd) may have regionally different roles in the regulation of physiological and pathophysiological processes in the central nervous system (CNS) such as epilepsy. It was demonstrated previously that Ino and Guo decreased quinolinic acid (QA)-induced seizures and Urd reduced penicillin-, bicuculline- and pentylenetetrazole (PTZ)-induced seizures. It has also been demonstrated that Ino and Urd may exert their effects through GABAergic system by altering the function of GABA(A) type of gamma-aminobutyric acid receptors (GABAA receptors) whereas Guo decreases glutamate-induced excitability through glutamatergic system, which systems (GABAergic and glutamatergic) are involved in pathomechanisms of absence epilepsy. Thus, we hypothesized that Ino and Guo, similarly to the previously described effect of Urd, might also decrease absence epileptic activity. We investigated in the present study whether intraperitoneal (i.p.) application of Ino (500 and 1000mg/kg), Guo (20 and 50mg/kg), Urd (500 and 1000mg/kg), GABA(A) receptor agonist muscimol (1 and 3mg/kg), GABA(A) receptor antagonist bicuculline (2 and 4mg/kg), non-selective Ado receptor antagonist theophylline (5 and 10mg/kg) and non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine maleate (MK-801, 0.0625 and 0.1250mg/kg) alone and in combination have modulatory effects on absence epileptic activity in Wistar Albino Glaxo Rijswijk (WAG/Rij) rats. We found that Guo decreased the number of spike-wave discharges (SWDs) whereas Ino increased it dose-dependently. We strengthened that Urd can decrease absence epileptic activity. Our results suggest that Guo, Urd and their analogs could be potentially effective drugs for treatment of human absence epilepsy.

The antiepileptic potential of nucleosides.

Despite newly developed antiepileptic drugs to suppress epileptic symptoms, approximately one third of patients remain drug refractory. Consequently, there is an urgent need to develop more effective therapeutic approaches to treat epilepsy. A great deal of evidence suggests that endogenous nucleosides, such as adenosine (Ado), guanosine (Guo), inosine (Ino) and uridine (Urd), participate in the regulation of pathomechanisms of epilepsy. Adenosine and its analogues, together with non-adenosine (non-Ado) nucleosides (e.g., Guo, Ino and Urd), have shown antiseizure activity. Adenosine kinase (ADK) inhibitors, Ado uptake inhibitors and Ado-releasing implants also have beneficial effects on epileptic seizures. These results suggest that nucleosides and their analogues, in addition to other modulators of the nucleoside system, could provide a new opportunity for the treatment of different types of epilepsies. Therefore, the aim of this review article is to summarize our present knowledge about the nucleoside system as a promising target in the treatment of epilepsy.

Receptors of peptides as therapeutic targets in epilepsy research.

Neuropeptides are signaling molecules participating in the modulation of synaptic transmission. Neuropeptides are stored in dense core synaptic vesicles, the release of which requires profound excitation. Only in the extracellular space, neuropeptides act on G-protein coupled receptors to exert a relatively slow action both pre- and postsynaptically. Consequently, neuropeptide modulators are ideal candidates to influence epileptic tissue overexcited during seizures. Indeed, a number of neuropeptides have receptors implicated in epilepsy and many of them are considered to participate in endogenous neuroprotective actions. Neuropeptide receptors, present in the hippocampus, the most frequent focus of seizures in temporal lobe epilepsy, received the largest attention as potential anti-epileptic targets. Receptors of hippocampal neuropeptides, somatostatin, neuropeptide Y, galanin, dynorphin, enkephalin, substance P, cholecystokinin, vasoactive intestinal polypeptide, and receptors of some neuropeptides, which are also hormones such as ghrelin, angiotensins, corticotropin- releasing hormone, adrenocorticotropin, thyrotropin-releasing hormone, oxytocin and vasopressin involved in epilepsy are discussed in the review article. Activation and inhibition of receptors by oral application of peptides as drugs is typically not efficient because of low bioavailability: rapid degradation and insufficient penetration of peptides through the blood-brain barrier. Recent progress in the development of non-peptide agonists and antagonists of neuropeptide receptors as well as gene therapeutic approaches leading to the local production of agonists and antagonists within the central nervous system will also be discussed.

Facilitation of spike-wave discharge activity by lipopolysaccharides in Wistar Albino Glaxo/Rijswijk rats.

In normal rats the proinflammatory cytokines like interleukin-1beta, interleukin-6, which are induced by bacterial lipopolysaccharides, are able to control thalamo-cortical excitability by exerting strong effects on physiological synchronization such as sleep and on pathological synchronization like that in epileptic discharges. To investigate whether proinflammatory cytokines or lipopolysaccharides could modulate absence seizures resulting from a very different generator mechanism than the already investigated bicuculline-, kindling- and kainate-induced seizures, we used a genetically epileptic Wistar Albino Glaxo/Rijswijk rat strain, which is spontaneously generating high voltage spike-wave discharges. Wistar Albino Glaxo/Rijswijk rats responded with an increase of the number of spike-wave discharges to lipopolysaccharide injection (from 10 microg/kg to 350 microg/kg). Repetitive administration of 350 microg/kg lipopolysaccharides daily for 5 days increased the number of spike-wave discharges on the first, second and third days but the number of spike-wave discharges returned to the control value on day 5, at the 5th injection of lipopolysaccharides, showing a tolerance to lipopolysaccharides. The lipopolysaccharide-induced increase in spike-wave discharges was not directly correlated with the elevation of the core body temperature, as it is in febrile seizures, although lipopolysaccharide induced prostaglandin and is clearly pyrogenic at the doses used. Indomethacin, the prostaglandin synthesis inhibitor, efficiently blocked lipopolysaccharide-induced enhancement of spike-wave discharge genesis suggesting that the spike-wave discharge facilitating effect of lipopolysaccharides involves induction of cyclooxygenase 2 and subsequent synthesis and actions of prostaglandin E2. Low dose (40 mg/kg, i.p.) of competitive N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid, and low dose of lipopolysaccharide (20 microg/kg) showed a synergistic interaction to increase the number of spike-wave discharges, whereas at supramaximal doses of lipopolysaccharide and the N-methyl-D-aspartate antagonist no synergy was present. The data reveal a functional connection between absence epileptic activity and lipopolysaccharide induction of prostaglandin synthesis and prostaglandin action and suggest some common cellular targets in epilepsy and lipopolysaccharide-induced inflammation.

An in vivo eyecup preparation for the rat.

A method for the preparation of an in vivo eyecup and a complex stimulating-sampling device are described; these are suitable for long-term parallel neurochemical and electrophysiological experiments on the rat retina without any additives into the eyecup. In this in vivo eyecup the extracellular microenvironment is under the normal homeostatic control of the vascular system; no continuous exchange of the eyecup fluid and no addition of glutamate is necessary to maintain stable retinal electric responses and amino acid concentrations. The eyecup viability was tested by monitoring the electroretinogram (ERG) and the amino acid contents of the eyecup fluid sampled from the preretinal space by means of microdialysis. After the initial increase the b-wave of the ERG changed by less than 10% in maximal amplitude during experiments lasting 5 h. The glutamate, glutamine, and glycine levels proved comparatively, whereas the taurine level rose continuously throughout the experimental protocol. Recovery of ERG was achieved following exposure to bright background illumination. Total exchange of the eyecup volume requires 20 min at a flow rate of 1 microl/min. The effect of L-AP4 on the ERG was successfully reproduced, which suggests the applicability of this in vivo eyecup for pharmacological experiments on the rat retina.

Persistent depolarization and Glu uptake inhibition operate distinct osmoregulatory mechanisms in the mammalian brain.

The ways of coupling neuronal with glial compartments in natural physiology was investigated in microdialysis experiments by monitoring extracellular concentration of amino acids in the brain of anaesthetized rats. We hypothesized that extracellular [Glu], [Gln] and [Tau] patterns would be state-dependent. This was tested by stimulation of N-methyl-D-aspartate (NMDA) receptors, by inhibition of Glu uptake or by local depolarization with a high-K(+) dialysate, coupled with the addition of Co(2+) to block Ca(2+) influx. The results showed that (1) extracellular [Gln] was low whereas [Glu] and [Tau] were high during infusion of NMDA (0.5-1.0 mM) or high-K(+) (80 mM) in the hippocampus and ventrobasal thalamus, (2) hippocampal extracellular [Glu], [Gln] and [Tau] were increased in response to the Glu uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxilic acid (tPDC, 0.5-3.0 mM), in a concentration-dependent manner, (3) high-K(+)-induced increase of extracellular [Glu] was partially blocked by the addition of 10 mM CoCl(2) with the high-K(+) dialysate in the hippocampus. Searching for main correlations between changes in [Glu], [Gln] and [Tau] by calculating partial correlations and with the use of factor analyses we found, the primary response of the mammalian brain to persistent depolarization is the neuronal uptake of [Gln] and release of [Tau] thereupon, acting independently of Glu changes. When glial and neuronal uptake of Glu is blocked, releases of Tau occur from neuronal as well as glial compartments accompanied by increases of [Gln] in the mammalian brain.

Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus.

ATP and adenosine are well-known neuroactive compounds. Other nucleotides and nucleosides may also be involved in different brain functions. This paper reports on extracellular concentrations of nucleobases and nucleosides (uracil, hypoxanthine, xanthine, uridine, 2'-deoxycytidine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenosine) in response to sustained depolarisation, using in vivo brain microdialysis technique in the rat thalamus. High-potassium solution, the glutamate agonist kainate, and the Na(+)/K(+) ATPase blocker ouabain were applied in the perfusate of microdialysis probes and induced release of various purine and pyrimidine nucleosides. All three types of depolarisation increased the level of hypoxanthine, uridine, inosine, guanosine and adenosine. The levels of measured deoxynucleosides (2'-deoxycytidine, 2'-deoxyuridine and thymidine) decreased or did not change, depending on the type of depolarisation. Kainate-induced changes were TTX insensitive, and ouabain-induced changes for inosine, guanosine, 2'-deoxycytidine and 2'-deoxyuridine were TTX sensitive. In contrast, TTX application without depolarisation decreased the extracellular concentrations of hypoxanthine, uridine, inosine, guanosine and adenosine. Our data suggest that various nucleosides may be released from cells exposed to excessive activity and, thus, support several different lines of research concerning the regulatory roles of nucleosides.

Effect of CGP 36742 on the extracellular level of neurotransmitter amino acids in the thalamus.

We have evaluated the effect of the brain penetrating GABAb antagonist, CGP 36742 on GABAb receptors using in vivo microdialysis in the ventrobasal thalamus of freely moving rat. When a solution of 1 mM CGP 36742 in ACSF was dialyzed into the ventrobasal thalamus, 2-3-fold increases of extracellular Glu, Asp and Gly running parallel with significant decreases of contralateral extracellular Asp and Gly were observed. Unilateral applications of Glu receptor antagonists (0.5 mM MK801, 0.1 mM CNQX) evoked 2-3-fold decreases of CGP 36742-specific elevations of extracellular Asp, Glu and Gly. Administration of CNQX and MK801 in the absence of CGP 36742 did not alter the extracellular Glu and Gly concentrations whereas extracellular Asp concentrations diminished by 42-45% at both sides. By contrast, no changes of extracellular Gly accompanied the 5-10-fold enhancements of extracellular Asp and Glu, observed during application of the Glu uptake inhibitor, tPDC (1mM). Suspensions of resealed plasmalemma fragments from the rat thalamus were mixed rapidly with the membrane impermeant form of the fluorescence indicator, bis-fura-2 and the changes in fluorescence intensity in response to CGP 36742 (0.5 mM), and the GABAb agonist, baclofen (0.1 mM), were monitored on the time scale of 0.04 ms(-10)s. Progress of CGP 36742-mediated influx, and baclofen-mediated efflux of Ca++ ion, antagonized by CGP 36742, was observed in the 1 ms(-10s) period of time. These data support the hypothesis that background ventrobasal activities and thalamocortical signaling are under the control of inhibitory GABAb receptors in the ventrobasal thalamus.

Corticosterone peak is responsible for stress-induced elevation of glutamate in the hippocampus.

Effect of ether stress on dialysate concentration of extracellular amino acids in the hippocampus was studied by microdialysis in freely moving rats that have been either sham operated (SHAM) or adrenalectomized and supplemented with subcutaneous steroid pellets (ADX+CORT) providing constant corticosterone (CORT) plasma levels. In SHAM rats, ether stress resulted in a peak of glutamate and taurine 30 min after stress, while extracellular aspartate concentration was increased 120 min after challenge. These changes in amino acid levels as well as in glutamate/glutamine ratio were paralleled by stress-induced rise of plasma CORT. No significant alterations were detected in the concentration of hippocampal arginine, alanine, glycine, glutamine, threonine or serine. In contrast to SHAM animals, ether stress failed to have an effect on dialysate concentration of amino acid transmitters in the hippocampus of adrenalectomized rats supplemented with 50 mg CORT-pellets. Our results demonstrate that ether stress alters aspartate, glutamate, glutamate/glutamine ratio and taurine concentration in the hippocampus and indicate that stress-induced CORT release in plasma may be responsible for these amino acid alterations. These changes may also contribute to negative feedback effect of CORT on hypothalamo-pituitary-adrenocortical (HPA) axis via the hippocampus during stress.

Differential effects of nipecotic acid and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol on extracellular gamma-aminobutyrate levels in rat thalamus.

Using the microdialysis technique and HPLC (high-performance liquid chromatography) determination of amino acids, the extracellular concentrations of gamma-aminobutyrate (GABA), glutamate, aspartate and a number of other amino acids were determined in rat thalamus during infusion through the microdialysis tubing of the GABA transport inhibitors 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) and nipecotic acid. Administration of 5.0 mM THPO led to a 200% increase in the extracellular GABA concentration. Simultaneous infusion of THPO and GABA (50 microM) increased the extracellular GABA concentration to 1200% of the basal level whereas GABA alone was found to increase the GABA level to 500%. If nipecotic acid (0.5 mM) was administered together with GABA (50 microM) the extracellular concentration of GABA was not increased further. While administration of GABA alone or GABA together with nipecotic acid had no effect on the extracellular levels of glutamate and aspartate it was found that GABA plus THPO increased the extracellular concentration of these amino acids. GABA administered alone or together with nipecotic acid or THPO led to relatively small but significant increases in the extracellular concentrations of the amino acids glycine, glutamine, serine and threonine. The results demonstrate that THPO, which preferentially inhibits glial GABA uptake and which is not a substrate for the GABA carriers, was more efficient increasing the extracellular concentration of GABA than nipocotic acid which is a substrate and an inhibitor of both neuronal and glial GABA uptake. This indicates that GABA uptake inhibitors that are not substrates for the carrier and which preferentially inhibit glial GABA uptake may constitute a group of drugs by which the efficacy of GABAergic neurotransmission may be enhanced.

Slow wave sleep is accompanied by release of certain amino acids in the thalamus of cats.

To determine whether EEG synchronization in sleep has a metabolic equivalent, we investigated state-dependent changes in extracellular concentrations of amino acids. In vivo microdialysis studies were performed in the ventroposterolateral (VPL) nuclei of the thalamus of cats during natural slow wave sleep (SWS), waking (W) and carbachol-induced paradoxical sleep (PS) like episodes. About two-fold increases in aspartate, glutamate, asparagine, glycine, alanine and gamma-aminobutyric acid (GABA) were observed in SWS compared with control samples collected in W, but serine increased to 487 +/- 211%. In the PS-like state, glutamine increased and GABA decreased. These results suggest changes in intracellular processes reflected by amino acid release in the thalamus, specific to slow wave generation in EEG during natural sleep.

Single low dose of MPTP decreases extracellular levels of noradrenaline and monoamine metabolites in the ventrobasal thalamus of the rats.

A single low dose of the neurotoxin: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) results paradoxical sleep deprivation and reduction in food intake without any detectable motor deficiencies. In the present study we monitored the in vivo extracellular levels of monoamines and their metabolites following intraperitoneal (i.p.) administration of a single dose of MPTP (5 mg/kg). Microdialysates were collected from the ventrobasal thalamic nucleus (VB) of Halothane anesthetized rat. We found a significant decrease in noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) levels and a significant increase in 3,4-dihydroxyphenylalanine (DOPA) concentration whereas amino acid levels were unchanged throughout the 4-hour long perfusion. We found no significant difference in the post mortem release of NA and DOPA between the control and MPTP treated animals, suggesting that the intracellular NA pool were maintained. The above findings support the idea that the neurochemical mechanism of rapidly developing and transient behavioral changes induced by MPTP may be an immediate decrease in monoaminergic transmission and metabolism following MPTP injection.

In vivo blockade of thalamic GABA(B) receptors increases excitatory amino-acid levels.

The effect of intrathalamic application of GABA(B) receptor antagonists on the basal excitatory amino-acid levels was studied using microdialysis probes implanted in the dorsal lateral geniculate nucleus and in the ventrobasal complex. In both nuclei, continuous perfusion of the GABA(B) receptor antagonist 3-aminopropyl-(diethoxymethyl)-phosphinic acid (CGP 35348) produced an increase in the extracellular concentration of aspartate and (to a lesser extent) glutamate, but no change was observed in the level of taurine, the main amino acid involved in the regulation of brain osmolarity processes. In contrast, 3-amino-2-hydroxy-2-(4-chlorophenyl)-propanesulphonic acid (2-hydroxy-saclofen), another GABA(B) receptor antagonist, failed to affect the extracellular concentration of aspartate, glutamate and taurine. Thus, the basal level of excitatory amino acids in the thalamus in vivo is under the control of CGP 35348-sensitive GABA(B) receptors.

Effect of intrahippocampal dexamethasone on the levels of amino acid transmitters and neuronal excitability.

Direct effect of type-II corticosteroid receptor agonist dexamethasone on extracellular amino acid levels and neuronal excitability in the hippocampus was studied by simultaneous application of in vivo microdialysis and recording hippocampal evoked responses in freely moving male rats. Microdialysis probes and hippocampal recording electrodes were implanted to the CA1-CA3 regions of dorsal hippocampus. Local dexamethasone infusion via microdialysis resulted in a transient increase in glutamate level at 30 min, while glutamine decreased by 30-40% throughout the 180-min sampling period. Taurine increased by 50% and remained elevated up to 180 min. No significant changes were detected in extracellular concentration of asparagine, arginine, glycine, threonine, alanine and serine. In contrast, dexamethasone infusion to the striatum had no effect on the extracellular levels of amino acid transmitters. Effect of dexamethasone injected via microdialysis on the neural activity elicited by perforant path stimulation was a decrease in population spikes after 60 min starting dexamethasone infusion. Steroid effect on neural excitability was reversible. Our data indicate that local infusion of type-II receptor agonist dexamethasone has a complex effect in the hippocampus, starts with a change in extracellular glutamate and glutamine concentration and followed by a reduced synaptic excitability.

Blockade of thalamic GABAB receptors decreases EEG synchronization.

The gamma-aminobutyric acid (GABA)B receptor antagonists 2-OH-saclofen and CGP 35348 were injected in the thalamus of freely moving cats via a microdialysis probe while recording the sleep-walking cycle. The results obtained with the two antagonists were similar: wakefulness and the total sleep time were not affected by the blockade of GABAB receptors, but deep slow wave sleep and the mean power of slow waves (< 10 Hz) were decreased, while light slow wave sleep was increased. These data suggest an involvement of thalamic GABAB receptors in the regulation of EEG slow waves.

Paradoxical sleep deprivatory effect of a single low dose of MPTP which did not produce dopaminergic cell loss.

In a previous study, we reported on a selective and long-lasting paradoxical sleep (PS) deprivation in cats following repeated administration of the Parkinson syndrome-inducing neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). While the characteristic motor deficits occurred only from the 2nd to 3rd day of a 5-day long administration of 5 mg/kg per day MPTP i.p., the PS deprivation started immediately after the first injection and lasted altogether for 11-13 days. The motor deficiencies induced by repeated administration of MPTP are mainly due to the selective depletion of dopaminergic neurons in the nigrostriatal system as the histological and biochemical data show. The immediate onset of PS deprivation and the PS recovery, despite the definite cell loss, suggests a mechanism independent of cell destruction. In our present study we investigated the occasional histological and the PS-deprivatory effect of a single low dose of MPTP in cats. A single injection of 2 mg/kg MPTP i.p. resulted in PS deprivation lasting for 2.5-3.5 h. The duration of other sleep stages showed no significant change and PS recovery was without rebound phenomenon, as in the case of repeated administration. Even a higher single dose of MPTP (5 mg/kg) resulted in no visibly detectable nigrostriatal cell loss. We suggest that the changes in monoamine release and/or turnover are involved in the PS deprivatory effect of MPTP.

Sleep promoting effect of a putative glial gamma-aminobutyric acid uptake blocker applied in the thalamus of cats.

The uptake of gamma-aminobutyric acid (GABA) by glial cells was decreased when 4,5,6,7,-tetrahydroisoxazolo-(4,5-C)-pyridin-3-ol (THPO) was applied in the thalamus of freely moving cats by in vivo microdialysis. A marked reduction in duration of wakefulness and in number of awakenings was obtained during THPO treatment. THPO did not change the ratio of slow-wave-sleep and paradoxical sleep but only increased the total sleep time. The present data suggest a possible regulatory role of the glial-neuronal interaction in the modification of the sleep-waking cycle.