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The possible involvement of oxytocin (OT) in the generation of seizures has not received a lot of attention in the past, although generalized epileptic convulsions were observed in humans following intravenous OT infusion. We here aimed to investigate the effect of exogenous OT administration on seizure susceptibility in C57Bl/6 mice subjected to the pentylenetetrazol (PTZ) model. In addition, we studied via which receptor possible effects on seizure thresholds could be mediated since OT binds to both the OT receptor (OTR) and the vasopressin 1a receptor (V1aR). We showed that C57Bl/6 mice treated with 0.5 mg/kg OT had decreased PTZ thresholds for ear twitch, myoclonic twitch, tail twitch, forelimb clonus and falling. This pronconvulsive effect was reversed by the OTR antagonist L-368.899, however, it was not mimicked by the OTR agonist carbetocin (CBT). Nevertheless, CBT had antidepressant-like effects in the forced swim test that could be reversed by L-368.899. These experiments shed some doubt on the involvement of OTR in the observed effect of OT on seizure thresholds. Therefore, we investigated the role of the V1aR as a possible mediator of the proconvulsive effects of OT. We found that the proconvulsive effects of both arginine vasopressin and OT were reversed by the V1aR antagonist SR49059. In summary, OT has proconvulsive effects in our mouse model of generalized seizures that could not be mimicked by CBT. Our results suggest that the binding of OT to V1aRs is the most plausible explanation for the proconvulsive effects of OT.

Noradrenaline exerts inhibitory effects on seizure susceptibility. Subtype selective agonists and antagonists were used to identify the anticonvulsant hippocampal adrenoreceptors. Intrahippocampal dialysis was used for administration of all compounds, including pilocarpine for limbic seizure induction, and as the neurotransmitter sampling tool. The noradrenaline reuptake inhibitor maprotiline mediated anticonvulsant effects, associated with dose-dependent increases in extracellular hippocampal noradrenaline, dopamine and GABA levels. At high concentrations, maprotiline produced proconvulsant effects associated with high levels of noradrenaline, dopamine and glutamate. Maprotiline's anticonvulsant effect was blocked by administration of either a selective α(2) - and β(2) -antagonist. α(2) -Antagonist administration with maprotiline was associated with a further increase in noradrenaline and dopamine from maprotiline alone; whereas β(2) -antagonist administered with maprotiline inhibited the dopamine increases produced by maprotiline. α(1A) -Antagonism blocked the GABA-ergic but not the anticonvulsive effect of maprotiline. These results were confirmed as combined but not separate α(2) - and β(2) -adrenoreceptor stimulation, using selective agonists, inhibited limbic seizures. Interestingly, α(1A) -receptor stimulation and α(1D) -antagonism alone also inhibited seizures associated with respectively significant hippocampal GABA increases and glutamate decreases. The main findings of this study are that (i) increased hippocampal noradrenergic neurotransmission inhibits limbic seizures via combined α(2) - and β(2) -receptor activation and (ii) α(1A) - and α(1D) -adrenoreceptors mediate opposite effects on hippocampal excitability.

Striatal dopamine loss in Parkinson's disease is accompanied by a dysregulation of corticostriatal glutamatergic neurotransmission. Within this study, we investigated striatal expression and activity of the glial high-affinity Na+/K+-dependent glutamate transporters, GLT-1 and GLAST, in the 6-hydroxydopamine hemi-Parkinson rat model at different time points after unilateral 6-hydroxydopamine injection into the medial forebrain bundle. Using semi-quantitative Western blotting and an ex vivo D-[H-3]-aspartate uptake assay, we showed a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1. At 3 and 12 weeks post-lesion, striatal GLT-1 function was bilaterally upregulated whereas at 5 weeks there was no change. Even though our data do not allow a straightforward conclusion as for the role of glutamate transporters in the pathogenesis of the disease, they do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of a rat model for Parkinson's disease. (C) 2010 Elsevier Ltd. All rights reserved.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer. © 2013 Chtarto et al., SCOPUS: ar.j, info:eu-repo/semantics/published

System xc−exchanges intracellular glutamate for extracellular cystine, giving it a potential role in intracellular glutathione synthesis and nonvesicular glutamate release. We report that mice lacking the specific xCT subunit of system xc−(xCT−/−) do not have a lower hippocampal glutathione content, increased oxidative stress or brain atrophy, nor exacerbated spatial reference memory deficits with aging. Together these results indicate that loss of system xc−does not induce oxidative stressin vivo. YoungxCT−/−mice did however display a spatial working memory deficit. Interestingly, we observed significantly lower extracellular hippocampal glutamate concentrations inxCT−/−mice compared to wild-type littermates. Moreover, intrahippocampal perfusion with system xc−inhibitors lowered extracellular glutamate, whereas the system xc−activatorN-acetylcysteine elevated extracellular glutamate in the rat hippocampus. This indicates that system xc−may be an interesting target for pathologies associated with excessive extracellular glutamate release in the hippocampus. Correspondingly, xCT deletion in mice elevated the threshold for limbic seizures and abolished the proconvulsive effects ofN-acetylcysteine. These novel findings sustain that system xc−is an important source of extracellular glutamate in the hippocampus. System xc−is required for optimal spatial working memory, but its inactivation is clearly beneficial to decrease susceptibility for limbic epileptic seizures.

Malfunctioning of system xc-, responsible for exchanging intracellular glutamate for extracellular cystine, can cause oxidative stress as well as excitotoxicity, both important phenomenons in the pathogenesis of Parkinson's disease. We used mice lacking xCT (xCT-/- mice), the specific subunit of system xc-, to investigate the involvement of this antiporter in Parkinson's disease. Although cystine that is imported via system xc-, is reduced to cysteine, the rate-limiting substrate in the synthesis of glutathione, deletion of xCT did not result in decreased glutathione levels in striatum. Accordingly, no signs of increased oxidative stress could be observed in striatum or substantia nigra of xCT-/- mice. In sharp contrast to the expectations, xCT-/- mice were less susceptible to 6-hydroxydopamine (6-OHDA)-induced neurodegeneration in the substantia nigra pars compacta compared to their age-matched wildtype littermates. This reduced sensitivity to a Parkinson's disease inducing toxin, might be related to the significantly reduced (70%) striatal extracellular glutamate levels that were observed in mice lacking xCT. The current data point towards system xc- as a possible target for the development of new pharmacotherapies for the treatment of Parkinson's disease and emphasizes the need to continue the search for specific ligands for system xc-.

Biophase concentrations of antiepileptic drugs can differ significantly from pharmacokinetics in plasma. A crucial determinant in the disposition of antiepileptic drugs to the brain is represented by the blood-brain barrier. There is growing evidence that this barrier can alter the availability of antiepileptic drugs at the target site. The permeability of the blood-brain barrier becomes particularly relevant in epileptic conditions and in drug refractory situations. In vivo, intracerebral microdialysis is a valuable technique to determine biophase drug concentrations as it enables investigation of antiepileptic drug transport and distribution in the brain as a function of time. The present review illustrates that intracerebral microdialysis is an indispensable tool for the assessment of the pharmacokinetics of antiepileptic drugs. In addition, we demonstrate how microdialysis data can be used in conjunction with mechanism-based pharmacokinetic/pharmacodynamic modeling for dose selection and optimization of the therapeutic regimen for novel compounds.

Altered glutamate signaling is associated with Parkinson's disease. To study the involvement of the cystine/glutamate antiporter in the pathogenesis of Parkinson's disease, we developed new polyclonal antibodies recognizing xCT, the specific subunit of this antiporter. The striatal xCT protein expression level was investigated in a hemi-Parkinson rat model, using semiquantitative western blotting. We observed time-dependent changes after a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway with increased expression levels in the deafferented striatum after 3 weeks. Twelve weeks postlesion, expression levels returned to normal. These data suggest, for the first time, an involvement of the cystine/glutamate antiporter in determining the aberrant glutamate neurotransmission in the striatum of a parkinsonian brain. NeuroReport 19:1589--1592 (C) Wolters Kluwer Health | Lippincott Williams & Wilkins.

It is common knowledge that the main mediators in the pathogenesis of Parkinson's disease are oxidative stress, excitotoxicity and mitochondrial dysfunction. Increased glutamate concentrations can be linked, directly or indirectly, to all of these processes. Extracellular glutamate concentrations are determined by an interplay between vesicular glutamate transporters (VGLUTs), glial high-affinity Na+/K+-dependent glutamate transporters (GLAST and GLT-1) and the cystine/glutamate antiporter. Using semi-quantitative Western blotting, we studied the expression levels of VGLUT1, GLAST, GLT-1 and xCT, the specific subunit of the cystine/glutamate antiporter, in the striatum of rats at different survival times (3, 5 and 12 weeks) after unilateral 6-OHDA injection into the medial forebrain bundle. xCT is the only protein that shows differences in expression between striata of both hemispheres of a unilaterally 6-OHDA injected rat, with higher expression levels in the lesioned compared to intact hemisphere 3 weeks after lesion. For all other transporter proteins, i.e. GLT-1, GLAST and VGLUT1, the main differences in expression levels were observed bilaterally, i.e. in left and right striatum of a lesioned rat compared to the striatum of control rats. Expression levels of GLT-1 and VGLUT1 were significantly increased 3 weeks post-lesion. On the other hand, 12 weeks post-lesion, the striatal expression levels of VGLUT1 and GLAST were significantly decreased whereas those of GLT-1 were increased. For all glutamate transporters, 5 weeks post-lesion seems to be a transition period during which expression levels were decreased (GLT-1, xCT) or unchanged (VGLUT1, GLAST). Given earlier observations by Meshul et al. (1999) of a biphasic change in extracellular glutamate levels after 6-OHDA lesioning, with increases 1 month post-lesion and decreases 3 months post-lesion, our data are suggestive of an important role of xCT and VGLUT1 in determining the aberrant extracellular glutamate levels in the 6-OHDA rat model. Modulation of one or more of these transporters might help to normalize the extracellular glutamate levels and prevent further excitotoxic and oxidative damage.

Purpose. Noradrenaline acts as endogenous anticonvulsant as it exerts profound inhibitory effects on seizure susceptibility. The identification of a universal anticonvulsant adrenoreceptor was impeded by conflicting results from pharmacological investigations in which non-selective agents were used. We here used subtype selective compounds to delineate the role of hippocampal adrenoreceptor subtypes in seizure susceptibility. Method. Intracerebral microdialysis was used for intrahippocampal administration of all compounds and as neurotransmitter sampling tool. The modulatory effects on pilocarpine-induced limbic seizures were behaviourally assessed. Co-perfusion experiments of anticonvulsant concentrations (250nM) of the selective noradrenaline reuptake inhibitor maprotiline were conducted with subtype selective antagonists for alpa1A (5-methylurapidil), alpha1D (BMY-7378), alpha2A (BRL-44408), alph2C (MK-912), beta1 (betaxolol) beta2 (ICI-118551) and D2 (remoxipride) receptors. Finally, selective agonists for alfa2 (medetomidine) and beta2 (salmeterol) receptors were tested to confirm our working hypothesis. Result. Maprotiline mediated anticonvulsant effects, associated with increases in extracellular hippocampal noradrenaline, dopamine and GABA levels. The anticonvulsant effect was blocked independently by alpha2 and beta2 antagonism. beta2 antagonism inhibited the dopamine increases, while alpha1A antagonism blocked the GABA-ergic but not the anticonvulsive effect. Antagonism of hippocampal D2 receptors, which mediate the anticonvulsant effect of dopamine, did not affect the anticonvulsant effect of maprotiline. Combined and not separate alpha2 and beta2 agonism inhibited pilocarpine-induced seizures. Conclusion. Hippocampal noradrenaline increases inhibit limbic seizures via combined alpha2 and beta2 receptor activation. The concomitant GABA-ergic and dopaminergic effects are not crucially involved in this anticonvulsant effect. These data demonstrate that the noradrenergic system is an interesting target for innovative antiepileptic treatment.

L-Theanine, an ethylamide derivate of glutamate found in abundance in green tea, has been shown to exert beneficial actions in animal models for several neurological disorders. We here investigated for the first time the effect of L-theanine intake on seizure susceptibility using acute pilocarpine and pentylenetetrazol (PTZ) mouse models for studying, respectively, limbic seizures or primarily generalized seizures. Moreover, we studied the effect of l-theanine intake on extracellular hippocampal and cortical glutamate and gamma-aminobutyric acid (GABA) levels, using in vivo microdialysis. Feeding mice with a 4% L-theanine solution significantly decreased their susceptibility to pilocarpine-induced seizures whereas susceptibility to PTZ-induced seizures was increased. The latter effect was linked to decreased extracellular GABA concentrations in frontal cortex.

In the past, antidepressants have been thought to possess proconvulsant properties. This assumption remains controversial, however, because anticonvulsant effects have been attributed to certain antidepressants. To date, it remains unclear which antidepressants can be used for the treatment of patients with epilepsy with depression. The present study was designed to determine the anticonvulsant and/or proconvulsant effects of three antidepressants (citalopram, reboxetine, bupropion) against pilocarpine- and pentylenetetrazole-induced acute seizures in larval zebrafish and mice. In zebrafish, all antidepressants were anticonvulsant in the pentylenetetrazole model. In addition, citalopram was anticonvulsant in the zebrafish pilocarpine model, whereas reboxetine and bupropion were without significant effect. In mice all three antidepressants increased some thresholds for pentylenetetrazole-induced convulsive-like behaviors at varying doses, whereas thresholds for pilocarpine-induced convulsive-like behaviors were generally lowered, particularly at the highest doses tested. In general we conclude that the convulsant liability of antidepressants is model and concentration dependent.

It is common knowledge that the main mediators in the pathogenesis of Parkinson's disease are oxidative stress, excitotoxicity and mitochondrial dysfunction. Increased glutamate concentrations can be linked, directly or indirectly, to all of these processes. Extracellular glutamate concentrations are determined by an interplay between vesicular glutamate transporters (VGLUTs), glial high-affinity Na+/K+-dependent glutamate transporters (GLAST and GLT-1) and the cystine/glutamate antiporter. Using semi-quantitative Western blotting, we studied the expression levels of VGLUT1, GLAST, GLT-1 and xCT, the specific subunit of the cystine/glutamate antiporter, in the striatum of rats at different survival times (3, 5 and 12 weeks) after unilateral 6-OHDA injection into the medial forebrain bundle. xCT is the only protein that shows differences in expression between striata of both hemispheres of a unilaterally 6-OHDA injected rat, with higher expression levels in the lesioned compared to intact hemisphere 3 weeks after lesion. For all other transporter proteins, i.e. GLT-1, GLAST and VGLUT1, the main differences in expression levels were observed bilaterally, i.e. in left and right striatum of a lesioned rat compared to the striatum of control rats. Expression levels of GLT-1 and VGLUT1 were significantly increased 3 weeks post-lesion. On the other hand, 12 weeks post-lesion, the striatal expression levels of VGLUT1 and GLAST were significantly decreased whereas those of GLT-1 were increased. For all glutamate transporters, 5 weeks post-lesion seems to be a transition period during which expression levels were decreased (GLT-1, xCT) or unchanged (VGLUT1, GLAST). Given earlier observations by Meshul et al. (1999) of a biphasic change in extracellular glutamate levels after 6-OHDA lesioning, with increases 1 month post-lesion and decreases 3 months post-lesion, our data are suggestive of an important role of xCT and VGLUT1 in determining the aberrant extracellular glutamate levels in the 6-OHDA rat model. Modulation of one or more of these transporters might help to normalize the extracellular glutamate levels and prevent further excitotoxic and oxidative damage.

Parkinson's disease is characterized by disturbed glutamatergic neurotransmission in the striatum. Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1 and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na(+)/K(+)-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2 transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time intervals post unilateral 6-OHDA injection into the medial forebrain bundle versus controls. Three weeks post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2 expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-Parkinson rats whereas the striatal expression of VGLUT2 was comparable to control rats. No effect could be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic neurotransmission from the two types of striatal afferents.

Anticonvulsant actions have already widely been proven for somatostatin-14. Cortistatin-14 (CST-14) was shown in one report to protect against kainate-induced seizures (Braun et al. Brain Res.1998;803(1-2):54-60). Although these neuropeptides are products of different genes, they are structurally related. This explains the interaction of CST-14 with the SST receptors (sst1-sst5). We here used in vivo microdialysis and telemetry-based electrocorticography (ECoG) in rats and administered CST-14 (0.1µM-1µM-10µM) in the presence and absence of highly selective antagonists for sst2 and sst3 receptor subtypes via intrahippocampal administration. Seizures were evoked by intrahippocampal pilocapine perfusion (10mM,40min) and seizure severity was assessed using a previously validated behavioural scoring system. Moreover, behavioural seizure severity assessment was verified with ECoG in at least 2 rats of each test group treated with CST-14. ECoG recordings of the rats treated with 0.1µM CST-14 showed clear epileptic discharges following pilocarpine perfusion. Indeed, the test group treated with 0.1µM CST-14 was not protected against pilocarpine-induced seizure severity. In none of the rats epileptic discharges were recorded during treatment with 1µM and 10µM CST-14. Hence, intrahippocampal administration of 1µM and 10µM CST-14 was anticonvulsant against pilocarpine-induced seizures. Furthermore, we showed that the CST-14 (1µM)-mediated anticonvulsant actions were reversed in the presence of a selective sst2 receptor antagonist Cyanamid154806 (0.1µM) or a selective sst3 receptor antagonist SST3-ODN-8 (0.1µM). Intrahippocampal perfusion of the selective sst2 or the selective sst3 receptor antagonists alone did not affect the pilocarpine-induced seizure severity per se. In conclusion, our results show that CST-14 is able to prevent seizures in a focal pilocarpine rat model and that selective sst2 or selective sst3 receptor antagonism is able to abolish these anticonvulsant actions.

Striatal dopamine loss in Parkinson's disease is accompanied by a dysregulation of corticostriatal glutamatergic neurotransmission. Within this study, we investigated striatal expression and activity of the glial high-affinity Na+/K+-dependent glutamate transporters, GLT-1 and GLAST, in the 6-hydroxydopamine hemi-Parkinson rat model at different time points after unilateral 6-hydroxydopamine injection into the medial forebrain bundle. Using semi-quantitative Western blotting and an ex vivo D-[3H]-aspartate uptake assay, we showed a time-dependent bilateral effect of unilateral 6-hydroxydopamine lesioning on the expression as well as activity of GLT-1. At 3 and 12 weeks post-lesion, striatal GLT-1 function was bilaterally upregulated whereas at 5 weeks there was no change. Even though our data do not allow a straightforward conclusion as for the role of glutamate transporters in the pathogenesis of the disease, they do clearly demonstrate a link between disturbed glutamatergic neurotransmission and glutamate transporter functioning in the striatum of a rat model for Parkinson's disease.

Purpose: For a long time, antidepressants have been thought to possess proconvulsant properties. This assumption, however, remains controversial, since anticonvulsant effects have been attributed to certain antidepressants. To date, it remains unclear which antidepressants can be used for the treatment of depression in patients with epilepsy. In this respect, studies investigating the convulsant liability of antidepressants in a chronic epilepsy model can give valuable information. The present study was designed to determine the seizure liability of citalopram and reboxetine in the kainic acid-induced post-status epilepticus model for temporal lobe epilepsy. Methods: Two months after the induction of status epilepticus, chronic epileptic rats (n = 16) were videoelectroencephalography (EEG) monitored during seven onsecutive weeks. Weeks 1, 3, 5, and 7 served as sham weeks during which the rats received intraperitoneal saline injections for four consecutive days, followed by a 3-day sham washout period during which no injections were given. During weeks 2, 4, and 6, rats received intraperitoneal injections with either citalopram (5, 10, and 15 mg/kg, once daily, n = 8) or reboxetine (10, 20, and 30 mg/kg, twice daily, n = 8) for 4 days, again followed by a washout period of 3 days. Drugs were administered in a randomly assigned fixed-dose regimen per week. Each rat served as its own control. The drug doses were selected based on the doses reported to have antidepressant effects in rats. Key Findings: Citalopram significantly decreased the spontaneous seizure frequency at the highest dose tested, that is, the mean number of seizures decreased from 12.8 seizures to 8.8 seizures per week (31%) after treatment with 15 mg/kg citalopram. This dose also significantly decreased the cumulative seizure duration. Administration of 5 and 10 mg/kg citalopram did not alter the seizure frequency. The two highest doses of reboxetine significantly decreased the spontaneous seizure frequency, that is, 20 mg/kg reboxetine decreased the seizure frequency from 14.1 to 7.9 (44%) and 30 mg/kg reboxetine decreased the seizure frequency from 11.8 to 7.2 (39%). In addition, both doses significantly decreased the cumulative seizure duration. Administration of 10 mg/kg reboxetine did not alter seizure frequency. Citalopram and reboxetine had no effect on seizure severity and seizure duration in any of the doses tested. Significance: In general we can conclude that antidepressant doses of citalopram and reboxetine have, depending on the dose, an anticonvulsant effect or no effect on spontaneous seizures in the kainic acid-induced post-status epilepticus rat model.

Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.