Jeanelle Portelli

Direct enhancement of hippocampal dopamine or serotonin levels as a pharmacodynamic measure of combined antidepressant–anticonvulsant action

The neurobiological relationships between epilepsy and depression are receiving increased experimental attention. A key role for limbic monoamines in depression has been established and we recently showed the importance of hippocampal monoamines in limbic seizure control. We here studied whether anticonvulsant compounds are antidepressant and can elevate hippocampal dopamine (DA) or serotonin (5-HT) levels determined by in vivo microdialysis in rats. We used assessment of seizure severity in the focal pilocarpine model, antidepressant-like activity within the rat forced swim and the mouse tail suspension tests, and locomotor activity in an open field as behavioural tests. We studied the tricyclic antidepressant imipramine, the selective 5-HT reuptake inhibitor citalopram and the selective DA reuptake blocker GBR-12909. These compounds with combined antidepressant-anticonvulsant properties all directly enhanced extracellular hippocampal DA or 5-HT levels. Since glutamate-mediated hyperexcitability in temporal lobe regions seems to be involved in disturbed emotional behaviour, we next investigated possible antidepressant effects and hippocampal DA or 5-HT modulations exerted by selective ionotropic and metabotropic glutamate receptor ligands with anticonvulsant properties. Combined anticonvulsant-antidepressant activities of the NMDA antagonist MK-801 and the mGluR group I antagonists (AIDA, MPEP) were also associated with locally elicited increases in hippocampal DA and/or 5-HT levels. This study highlights that the hippocampus is an important site of action of combined anticonvulsant-antidepressant and monoamine enhancing effects.

Ghrelin: an emerging new anticonvulsant neuropeptide.

Neuropeptides appear to be of importance when the central nervous system (CNS) is challenged, such as during high‐frequency firing and pathologic conditions. Potential advantages of treatments that target neuropeptide systems in comparison to classical neurotransmitter systems and ion channels revolve around the subject of efficacy as well as the reduced likelihood of side effects, thus making them attractive candidates for the development of new clinical applications for various disorders. The number of neuropeptides linked to epilepsy is on the rise, reflecting the increased interest of researchers in this domain. Ghrelin has only very recently been introduced into the field of epilepsy, and has already led to contradictory clinical publications. There is a great paucity with regard to what mechanism of action is utilized by ghrelin to inhibit seizures. In this review we disclose how we can better understand the mechanism ghrelin uses to prevent seizures, which indirectly could give an insight to researchers who are studying ghrelin in other fields of research.

Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy

The vesicular neurotransmitter transporters (VNTs) are small proteins responsible for packing synaptic vesicles with neurotransmitters thereby determining the amount of neurotransmitter released per vesicle through fusion in both neurons and glial cells. Each transporter subtype was classically seen as a specific neuronal marker of the respective nerve cells containing that particular neurotransmitter or structurally related neurotransmitters. More recently, however, it has become apparent that common neurotransmitters can also act as co-transmitters, adding complexity to neurotransmitter release and suggesting intriguing roles for VNTs therein. We will first describe the current knowledge on vesicular glutamate transporters (VGLUT1/2/3), the vesicular excitatory amino acid transporter (VEAT), the vesicular nucleotide transporter (VNUT), vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT) and the vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in the brain. We will focus on evidence regarding transgenic mice with disruptions in VNTs in different models of seizures and epilepsy. We will also describe the known alterations and reorganizations in the expression levels of these VNTs in rodent models for temporal lobe epilepsy (TLE) and in human tissue resected for epilepsy surgery. Finally, we will discuss perspectives on opportunities and challenges for VNTs as targets for possible future epilepsy therapies.

Intrastrain differences in seizure susceptibility, pharmacological response and basal neurochemistry of Wistar rats

Reliable well-characterised animal models of seizures are necessary in order to better understand the underlying pathophysiological mechanisms as well as to screen potential anticonvulsant drugs. We currently use the focal pilocarpine model as an acute limbic seizure model. Due to breeding problems at the vendor, and apparent changes in pilocarpine-induced seizure susceptibility, we were forced to change breeding locations and vendors over a period of 2 years. Male Wistar rats were either purchased from two breeding locations of Charles River Laboratories (France and Germany), or obtained from Harlan Laboratories (The Netherlands). In the present retrospective study we evaluated the impact of these vendor changes on ketamine dosing to establish anaesthesia, on pilocarpine-induced seizure susceptibility, and on basal extracellular hippocampal noradrenaline, dopamine, serotonin, gamma-amino butyric acid, and glutamate levels of all pilocarpine-treated rats included in our studies. Significant differences were present in all of the parameters analyzed. This study clearly illustrates that intrastrain differences do exist from one vendor/breeding location to another, or even between rats from the same breeding location.

Inactivation of the Constitutively Active Ghrelin Receptor Attenuates Limbic Seizure Activity in Rodents

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.

Rat hippocampal somatostatin sst3 and sst4 receptors mediate anticonvulsive effects in vivo: Indications of functional interactions with sst2 receptors

Somatostatin-14 (SRIF) is a potent anticonvulsant in rodent models of limbic seizures in which the hippocampus is its major site of action. However, the distribution of hippocampal sst receptors and their role in the anticonvulsant effects of SRIF remain controversial. Moreover, striking differences have been described between mice and rats. In rats, sst(2) but not sst(1) receptors play a critical role in the anticonvulsant effects of SRIF. At present, the role of rat sst(3) and sst(4) receptors in these anticonvulsive effects remains unknown. Here we demonstrate in vivo anticonvulsive actions of rat hippocampal sst(3) and sst(4) receptors. Using microdialysis and telemetry-based electroencephalographic recordings we show that intrahippocampal administration of the sst(2) agonist L-779,976 (500 nM), the sst(3) agonist L-796,778 (100 nM) or the sst(4) agonist L-803,087 (100 nM) protects rats against focal pilocarpine-induced seizures. SRIF (1 μM)-, sst(3)- and sst(4)-mediated anticonvulsive actions are reversed by the selective sst(2) receptor antagonist cyanamid 154806 (100 nM). Moreover, the selective sst(3) antagonist SST3-ODN-8 (100 nM) blocks the sst(4)-mediated anticonvulsant effect. Sst(3) antagonism does not reverse the sst(2)- or SRIF-mediated anticonvulsant effects. Our findings provide the first in vivo evidence for potent anticonvulsive properties of sst(3) and sst(4) receptors in the rat hippocampus. Nevertheless, selective sst(2) receptor antagonism prevented these sst(3)- or sst(4) receptor-mediated anticonvulsant effects, suggesting a functional cooperation with rat hippocampal sst(2) receptors.

The antidepressant-like effect of vagus nerve stimulation is mediated through the locus coeruleus

It has been shown that vagus nerve stimulation (VNS) has an antidepressant-like effect in the forced swim test. The mechanism of action underlying this effect is incompletely understood, but there is evidence suggesting that the locus coeruleus (LC) may play an important role. In this study, noradrenergic LC neurons were selectively lesioned to test their involvement in the antidepressant-like effect of VNS in the forced swim test. Forced swim test behavior was assessed in rats that were subjected to VNS or sham treatment. In half of the VNS-treated animals, the noradrenergic neurons from the LC were lesioned using the selective neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride], yielding three experimental arms: sham, VNS and DSP-4-VNS (n = 8 per group). Furthermore, the open field test was performed to evaluate locomotor activity. A dopamine-β-hydroxylase immunostaining was performed to confirm lesioning of noradrenergic LC neurons. VNS significantly reduced the percentage of immobility time in the forced swim test compared to sham treatment (median: 56%, interquartile range: 41% vs. median: 75%, interquartile range: 12%). This antidepressant-like effect of VNS could not be demonstrated in the DSP-4-VNS group (median: 79%, interquartile range: 33%). Locomotor activity in the open field test was not different between the three treatment arms. The absence of hippocampal dopamine-β-hydroxylase immunostaining in the DSP-4-treated rats confirmed the lesioning of noradrenergic neurons originating from the brainstem LC. The results of this study demonstrate that the noradrenergic neurons from the LC play an important role in the antidepressant-like effect of VNS.

Modulation of Hippocampal Activity by Vagus Nerve Stimulation in Freely Moving Rats.

BACKGROUND Vagus Nerve Stimulation (VNS) has seizure-suppressing effects but the underlying mechanism is not fully understood. To further elucidate the mechanisms underlying VNS-induced seizure suppression at a neurophysiological level, the present study examined effects of VNS on hippocampal excitability using dentate gyrus evoked potentials (EPs) and hippocampal electroencephalography (EEG). METHODS Male Sprague-Dawley rats were implanted with a VNS electrode around the left vagus nerve. A bipolar stimulation electrode was implanted in the left perforant path and a bipolar recording electrode was implanted in the left dentate gyrus for EEG and dentate field EP recording. Following recovery, VNS was applied in freely moving animals, using a duty cycle of 7 s on/18 s off, 30 Hz frequency, 250 µs pulse width, and an intensity of either 0 (SHAM), 25 µA or 1000 µA, while continuously monitoring EEG and dentate field EPs. RESULTS VNS at 1000 µA modulated dentate field EPs by decreasing the field excitatory post-synaptic potential (fEPSP) slope and increasing the latency and amplitude of the population spike. It additionally influenced hippocampal EEG by slowing theta rhythm from 7 Hz to 5 Hz and reducing theta peak and gamma band power. No effects were observed in the SHAM or 25 µA VNS conditions. CONCLUSION VNS modulated hippocampal excitability of freely moving rats in a complex way. It decreased synaptic efficacy, reflected by decreased fEPSP slope and EEG power, but it simultaneously facilitated dentate granule cell discharge indicating depolarization of dentate granule cells.

Des-acyl ghrelin attenuates pilocarpine-induced limbic seizures via the ghrelin receptor and not the orexin pathway.

Des-acyl ghrelin, widely accepted to work independently of the ghrelin receptor, is increasingly being implicated in a number of biological functions. The involvement of des-acyl ghrelin in epilepsy has only been recently reported. In this study, apart from unravelling the effect of des-acyl ghrelin on seizure thresholds and seizure severity in two models of pilocarpine-induced seizures, we mainly attempted to unravel its anticonvulsant mechanism of action. Since it was found that des-acyl ghrelin administration affected food intake via the orexin pathway, we first determined whether this pathway was responsible for des-acyl ghrelins seizure-attenuating properties using the dual orexin receptor antagonist almorexant. We noted that, while des-acyl ghrelin showed dose-dependent anticonvulsant effects against focal pilocarpine-evoked seizures in rats, almorexant did not affect seizure severity and did not reverse des-acyl ghrelins anticonvulsant effect. Subsequently, to investigate whether the ghrelin receptor was implicated in des-acyl ghrelins anticonvulsant properties, we tested this peptide in ghrelin receptor deficient mice and wild type mice, all infused with pilocarpine intravenously. Unexpectedly, we found that des-acyl ghrelin significantly elevated seizure thresholds in C57Bl/6 and wild type mice but not in ghrelin receptor knock-out mice. Taken together, our results indicate the involvement of the ghrelin receptor in the anticonvulsant effects of des-acyl ghrelin on pilocarpine-induced seizures. We also show for the first time that dual antagonism of hippocampal orexin receptors does not affect seizure severity.

Cortistatin-14 Mediates its Anticonvulsant Effects Via sst2 and sst3 but Not Ghrelin Receptors

Cortistatin (CST)‐14, a neuropeptide that is structurally and functionally related to somatostatin‐14 (SRIF) binds all five somatostatin receptor subtypes (sst1–sst5). Using in vivo microdialysis and telemetry‐based electroencephalographic recordings, we provide the first experimental evidence for anticonvulsive effects of CST‐14 in a pilocarpine‐induced seizure model in rats and mice and for the involvement of sst2 and sst3 receptors in these anticonvulsant actions of CST‐14. Both receptor subtypes are required for the anticonvulsant effects of CST‐14 given that co‐perfusion of a selective sst2 antagonist (cyanamid15486) or a selective sst3 antagonist (SST3‐ODN‐8) reversed anticonvulsant effect of CST‐14, and this, independently of each other. Next, as the ghrelin receptor has been proposed as a target for the biological effects of CST‐14, we used ghrelin receptor knockout mice and their wild type littermates to study the involvement of this receptor in the anticonvulsive actions of CST‐14. Our results show a significant decrease in seizure duration in both genotypes when CST‐14 treated mice were compared with corresponding control animals receiving only pilocarpine. In addition, this CST‐14‐induced decrease was comparable in both genotypes. We here thus provide the first evidence that ghrelin receptors are not involved in mediating anticonvulsant actions of CST‐14 in vivo.