Caloric Restriction Protects against Lactacystin-Induced Degeneration of Dopamine Neurons Independent of the Ghrelin Receptor.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

Trans-Modulation of the Somatostatin Type 2A Receptor Trafficking by Insulin-Regulated Aminopeptidase Decreases Limbic Seizures.

Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.

Anticonvulsant effect of a ghrelin receptor agonist in 6Hz corneally kindled mice.

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.

Disruption, but not overexpression of urate oxidase alters susceptibility to pentylenetetrazole- and pilocarpine-induced seizures in mice.

There is a continuous drive to find new, improved therapies that have a different mechanism of action in order to help diminish the sizable percentage of persons with pharmacoresistant epilepsy. Uric acid is increasingly recognized as contributing to the pathophysiology of multiple disorders, and there are indications that uric acid might play a role in epileptic mechanisms. Nevertheless, studies that directly investigate its involvement are lacking. In this study we assessed the susceptibility to pentylenetetrazole- and pilocarpine-induced seizures in mice with genetically altered uric acid levels by targeting urate oxidase, which is the enzyme responsible for uric acid breakdown. We found that although disruption of urate oxidase resulted in a decreased susceptibility to all behavioral end points in both seizure models, overexpression did not result in any alterations when compared to their wild-type littermates. Our results suggest that a chronic increase in uric acid levels may result in decreased brain excitability.

Neuropeptide FF and prolactin-releasing peptide decrease cortical excitability through activation of NPFF receptors.

OBJECTIVE: Drugs with a novel mechanism of action are needed to reduce the number of people with epilepsy that are refractory to treatment. Increasing attention is paid to neuropeptide systems and several anticonvulsant neuropeptides have already been described, such as galanin, ghrelin, and neuropeptide Y (NPY). Many others, however, have not been investigated for their ability to affect epileptic seizures. In this study, the potential anticonvulsant activities of three members of the RF-amide neuropeptide family, neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), and kisspeptin (Kp) and other receptor ligands (NPFF1/2 R, GPR10, and GRP54, respectively) were tested in the motor cortex stimulation model. METHODS: A train of pulses with increasing intensity (0-10 mA over 150 s, 50 Hz, pulse width 2 msec) was delivered to the motor cortex of rats. The threshold intensity for eliciting a motor response (i.e., motor threshold) was determined through behavioral observation and used as a measure for cortical excitability. The threshold was determined before, during, and after the intracerebroventricular (i.c.v.) administration of various NPFF1/2 R, GPR10, and GPR54 receptor ligands. RESULTS: NPFF and PrRP significantly increased the motor threshold by a maximum of 143 ± 27 and 83 ± 13 µA, respectively, for the doses of 1 nmol/h (p < 0.05). The increase of motor threshold by NPFF and PrRP was prevented by pretreatment and co-treatment with the NPFF1/2 R antagonist RF9. Pretreatment with a selective NPFF1 R antagonist also prevented the threshold increase induced by NPFF. Kp did not increase motor threshold. SIGNIFICANCE: Intracerebroventricular infusion of NPFF or PrRP decreases cortical excitability in rats through activation of NPFFRs. Furthermore, the NPFF1 R is required for the NPFF-induced decrease in cortical excitability.

Advanced Model-based Approach to Evaluate Human Plasma, Cerebrospinal Fluid, and Neuronal mTORC1 Activation Biomarkers After NV-5138 Administration in Healthy Volunteers.

PURPOSE: NV-5138 ([S]-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid) is an orally bioavailable, small-molecule activator of the mechanistic target of rapamycin complex 1 (mTORC1) pathway in development for treatment-resistant depression. The authors established a model to describe the relationship between plasma and cerebrospinal fluid (CSF) concentrations of NV-5138 and between CSF concentrations and potential biomarkers thought to be associated with mTORC1 activity (ie, orotic acid, N-acetylmethionine, and N-formylmethionine). METHODS: Data were collected from a randomized, double-blind, placebo-controlled, tolerability, and pharmacokinetic (PK) parameter study of 5 ascending (400, 800, 1600, 2400, and 3000 mg), once-daily oral doses of NV-5138 in healthy subjects. NV-5138 plasma PK parameter samples were collected at 15 time points over 24 hours on days 1 and 7, and at pre dose on days 2-6 for all doses. NV-5138 CSF PK parameter and CSF biomarker samples were collected on days 1 and 7 at pre dose and 4, 8, and 12 hours post dose for all doses except 3000 mg. A model-based approach was used to develop and validate a model that describes the relationship between NV-5138 in CSF and biomarker concentrations. FINDINGS: Twenty-four of the 42 enrolled subjects had simultaneous plasma and CSF measurements of NV-5138 and CSF biomarker concentrations and were included in the PK parameter and pharmacodynamic (PD) analyses. A 2-compartment plasma and CSF PK parameter, with indirect PD effects, model was developed and validated. NV-5138 plasma concentrations were positively correlated with those in CSF, although CSF concentrations lagged slightly behind those in plasma, as indicated by a counterclockwise hysteresis effect. Similarly, the relationship between the PD measures of mTORC1 activation and NV-5138 was also characterized by counterclockwise hysteresis, when the increase in CSF biomarker concentrations lagged behind those of NV-5138, consistent with a signaling intermediary/cascade, such as mTORC1. Maximal biomarker activation was achieved at NV-5138 CSF concentrations of approximately 3 µg/mL, which were associated with daily doses of 1600 mg NV-5138. The safety profile analysis (n = 42) found that most of the reported adverse events were mild in severity, with no severe, serious, unusual, or unexpected adverse events or any dissociative effects; 2 subjects (400-mg cohort) discontinued due to adverse events that were judged to be unrelated to study medication. IMPLICATIONS: The model will be used for designing future efficacy and tolerability studies. Consecutive daily doses of NV-5138 were well tolerated in this healthy volunteer study.

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.

Investigating the link between serum concentrations of brain-derived neurotrophic factor and behavioral measures in anxious alcohol-dependent individuals.

Brain-derived neurotrophic factor (BDNF) plays a role in different neurophysiological processes, including those involved in alcohol- and anxiety-related behaviors. Preclinical and clinical studies indicate that chronic excessive alcohol use leads to a downregulation of BDNF production in the periphery and in the brain. In addition, a decrease in BDNF concentrations in the blood has been reported to be associated with increased anxiety levels. Non-treatment-seeking alcohol-dependent individuals with high trait anxiety were studied to assess whether serum BDNF concentrations may be linked to self-reported levels of alcohol drinking, anxiety, and other behavioral measures. Participants had a current diagnosis of alcohol dependence, high trait anxiety score, and were not seeking treatment for alcohol dependence or anxiety. A fasting blood sample was collected from each participant and serum BDNF was measured using an enzyme-linked immunosorbent assay (ELISA). Behavioral data were collected on the same day, including measures of alcohol drinking, craving, dependence severity, and anxiety. Bivariate correlations were run between BDNF levels and behavioral measures. Serum BDNF concentrations were negatively correlated with average drinks per drinking days (r = -0.41, p = 0.02) and positively correlated with obsessive-compulsive drinking scale (r = 0.48, p = 0.007) and state-trait anxiety inventory (r = 0.52, p = 0.003) scores. These findings shed light on the possible role of the BDNF system in the neurobiology of alcohol- and anxiety-related behaviors.

Effects of exogenous ghrelin administration and ghrelin receptor blockade, in combination with alcohol, on peripheral inflammatory markers in heavy-drinking individuals: Results from two human laboratory studies.

The ghrelin system has been garnering interest for its role in different neuropsychiatric disorders, including alcohol use disorder (AUD). Accordingly, targeting the ghrelin system is under investigation as a potential novel therapeutic approach. While alcohol provokes the immune system and inflammatory responses, ghrelin has potent immunomodulatory and anti-inflammatory properties. The present study aimed to shed light on the "crosstalk" between ghrelin and inflammation by examining the effects of exogenous ghrelin administration and ghrelin receptor blockade on peripheral inflammatory markers in the context of two human laboratory studies with alcohol administration. Non-treatment-seeking, heavy-drinking individuals with alcohol dependence, the majority of whom were African American males, were enrolled. In the first randomized, crossover, double-blind, placebo-controlled human laboratory study, participants underwent two experimental paradigms - an intravenous alcohol self-administration (IV-ASA) and an intravenous alcohol clamp (IV-AC) - each consisting of two counterbalanced sessions (ghrelin, placebo). A loading dose of intravenous ghrelin (3 mcg/kg) or placebo, followed by a continuous ghrelin (16.9 ng/kg/min) or placebo infusion was administered. In the second dose-escalating, single-blind, placebo-controlled human laboratory phase 1b study, participants were dosed with an oral ghrelin receptor blocker (PF-5190457) and underwent an oral alcohol challenge. Repeated blood samples were collected, and plasma concentrations of the following inflammatory markers were measured: C-reactive protein (CRP), interleukin (IL)-6, IL-10, IL-18, and tumor necrosis factor alpha (TNF-α). During the IV-ASA experiment, significant drug × time interaction effects were observed for IL-6 (F3,36 = 3.345, p = 0.030) and IL-10 (F3,53.2 = 4.638, p = 0.006), indicating that ghrelin, compared to placebo, significantly reduced blood concentrations of the proinflammatory cytokine IL-6, while increasing blood concentrations of the anti-inflammatory cytokine IL-10. No significant drug × time interaction effects were observed during the IV-AC experiment, possibly because of its much shorter duration and/or smaller sample. Treatment with PF-5190457, compared to placebo, had no significant effect on the inflammatory markers investigated. In conclusion, a supraphysiologic pharmacological challenge with exogenous ghrelin in heavy-drinking individuals produced anti-inflammatory effects in the context of intravenous alcohol administration. On the contrary, ghrelin receptor blockade did not lead to any change in the inflammatory markers included in this study. Mechanistic studies are required to better understand the interaction between ghrelin, alcohol, and inflammatory processes.

Peripheral proinflammatory markers are upregulated in abstinent alcohol-dependent patients but are not affected by cognitive bias modification: Preliminary findings.

BACKGROUND: Inflammatory pathways are known to be negatively affected in patients with alcohol use disorder (AUD). Cognitive bias modification (CBM), an emerging behavioral treatment that involves the 're-training' of cognitive biases using computerized tasks, has been reported to reduce alcohol craving and relapse rates. The aim of this study was to compare peripheral concentrations of the proinflammatory biomarkers IL-18, IL-6, IL-1ß, TNF-α and CRP in AUD patients versus controls and to identify whether CBM treatment affected these biomarkers in AUD patients. METHODS: This 3-week double-blind randomized controlled study tested 36 male abstinent AUD patients receiving CBM or placebo-training, who were also compared to 18 male healthy controls. The approach avoidance task (AAT) was used to test the AUD patients before and after training. CBM training took place over 6 sessions, using a joystick-based approach-avoidance task. Blood samples were collected after the pre- and post-AAT test sessions for the AUD groups, and during an outpatient appointment with the controls. RESULTS: AUD patients, versus controls, presented with significantly higher plasma levels of TNF- α (P < 0.0001) and CRP (P = 0.0031). No changes in the CBM versus placebo groups were noted in IL-18, TNF-α and CRP concentrations following pre-post change or within group pretest- posttest analysis. IL-6 and IL-1ß levels fell under the lower detection limit, thus were not included in the final analyses. CONCLUSIONS: This study confirms that the inflammatory system is altered in AUD. This was the first study that investigated whether CBM training affected proinflammatory markers in AUD patients.

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.

Uric acid is released in the brain during seizure activity and increases severity of seizures in a mouse model for acute limbic seizures.

Recent evidence points at an important role of endogenous cell-damage induced pro-inflammatory molecules in the generation of epileptic seizures. Uric acid, under the form of monosodium urate crystals, has shown to have pro-inflammatory properties in the body, but less is known about its role in seizure generation. This study aimed to unravel the contribution of uric acid to seizure generation in a mouse model for acute limbic seizures. We measured extracellular levels of uric acid in the brain and modulated them using complementary pharmacological and genetic tools. Local extracellular uric acid levels increased three to four times during acute limbic seizures and peaked between 50 and 100 min after kainic acid infusion. Manipulating uric acid levels through administration of allopurinol or knock-out of urate oxidase significantly altered the number of generalized seizures, decreasing and increasing them by a twofold respectively. Taken together, our results consistently show that uric acid is released during limbic seizures and suggest that uric acid facilitates seizure generalization.

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 ghrelin's 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 ghrelin's anticonvulsant effect. Subsequently, to investigate whether the ghrelin receptor was implicated in des-acyl ghrelin's 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.

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-ß-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-ß-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.

Neuropeptide FF receptors as novel targets for limbic seizure attenuation.

Neuropeptide Y (NPY) is a well established anticonvulsant and first-in-class antiepileptic neuropeptide. In this study, the controversial role of NPY1 receptors in epilepsy was reassessed by testing two highly selective NPY1 receptor ligands and a mixed NPY1/NPFF receptor antagonist BIBP3226 in a rat model for limbic seizures. While BIBP3226 significantly attenuated the pilocarpine-induced seizures, neither of the highly selective NPY1 receptor ligands altered the seizure severity. Administration of the NPFF1/NPFF2 receptor antagonist RF9 also significantly attenuated limbic seizure activity. To further prove the involvement of NPFF receptors in these seizure-modulating effects, low and high affinity antagonists for the NPFF receptors were tested. We observed that the low affinity ligand failed to exhibit anticonvulsant properties while the two high affinity ligands significantly attenuated the seizures. Continuous NPFF1 receptor agonist administration also inhibited limbic seizures whereas bolus administration of the NPFF1 receptor agonist was without effect. This suggests that continuous agonist perfusion could result in NPFF1 receptor desensitization and mimic NPFF1 receptor antagonist administration. Our data unveil for the first time the involvement of the NPFF system in the management of limbic seizures.

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.

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.

Neuropeptide Y increases in vivo hippocampal extracellular glutamate levels through Y1 receptor activation.

Neuropeptide Y's (NPY) anticonvulsant effect is generally attributed to its inhibitory effect on glutamate release from presynaptic nerve terminals, which is nicely demonstrated in in vitro settings. To date no study has attempted to investigate the effect of NPY in vivo on extracellular (EC) glutamate levels thus, via intracerebral microdialysis, we determined NPY's effect on hippocampal glutamate concentrations in vivo, and consequently the involvement of Y(1) receptors to this effect. NPY or the Y(1) agonist D-His26-NPY was intrahippocampally administered in rats for 2h, during which the hippocampal glutamate dialysate levels were monitored. Pilocarpine was subsequently co-administered with NPY or D-His26-NPY to determine their effect on pilocarpine-induced limbic seizures. Unexpectedly we noted that intrahippocampal administration of NPY or D-His26-NPY increased glutamate dialysate levels in a reproducible manner. NPY attenuated pilocarpine induced seizures, whereas D-His26-NPY did not. To clarify the role of Y(1) receptors in NPY's glutamatergic effect, NPY was co-administered with the selective Y(1) antagonist BVD10. Hippocampal Y(1) receptor blockade prevented the NPY-induced increase in hippocampal glutamate, proving that this induced glutamate increase is clearly Y(1) receptor mediated. This is the first evidence that NPY enhances hippocampal EC glutamate overflow in vivo via hippocampal Y(1) receptors without interfering with or contributing to NPY's anticonvulsant effect. Whilst this finding contrasts with the supposed glutamatergic hypothesis for NPY in the hippocampus, it is of significance to further assist in deciphering NPY's mechanisms of action in in vivo settings.

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.