Publications by authors named "Ilse Smolders"

149 Publications

Unraveling the Effects of GSK-3β Isoform Modulation against Limbic Seizures and in the 6 Hz Electrical Kindling Model for Epileptogenesis.

ACS Chem Neurosci 2022 03 7;13(6):796-805. Epub 2022 Mar 7.

Vrije Universiteit Brussel (VUB), Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information/Center for Neurosciences (C4N), Laarbeeklaan 103, 1090 Brussels, Belgium.

Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3β. GSK-3β is the most prominent in the central nervous system and was previously shown to control neuronal excitability. We previously demonstrated that indirubin and its structural analogue and the nonselective GSK-3 inhibitor BIO-acetoxime exerted anticonvulsant effects in acute seizure models in zebrafish, mice, and rats. We here examined for the first time the anticonvulsant effect of TCS2002, a specific and potent inhibitor of GSK-3β, in two models for limbic seizures: the pilocarpine rat model for focal seizures and the acute 6 Hz corneal mouse model for refractory seizures. Next, we additionally used the 6 Hz kindling model to establish differences in seizure susceptibility and seizure progression in mice that either overexpress human GSK-3β (GSK-3β OE) or lack GSK-3β (GSK-3β) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3β OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3β mice, neuronal GSK-3β mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3β OE and GSK-3β mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3β inhibition in the 6 Hz model identify GSK-3β as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3β activity in the transgenic mice suggest a role for GSK-3 in kindling and warrants further research into the long-term effects of selective pharmacological GSK-3β inhibition.
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http://dx.doi.org/10.1021/acschemneuro.1c00782DOI Listing
March 2022

Targeting the Ghrelin Receptor as a Novel Therapeutic Option for Epilepsy.

Biomedicines 2021 Dec 27;10(1). Epub 2021 Dec 27.

Research Group Experimental Pharmacology, Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium.

Epilepsy is a neurological disease affecting more than 50 million individuals worldwide. Notwithstanding the availability of a broad array of antiseizure drugs (ASDs), 30% of patients suffer from pharmacoresistant epilepsy. This highlights the urgent need for novel therapeutic options, preferably with an emphasis on new targets, since "me too" drugs have been shown to be of no avail. One of the appealing novel targets for ASDs is the ghrelin receptor (ghrelin-R). In epilepsy patients, alterations in the plasma levels of its endogenous ligand, ghrelin, have been described, and various ghrelin-R ligands are anticonvulsant in preclinical seizure and epilepsy models. Up until now, the exact mechanism-of-action of ghrelin-R-mediated anticonvulsant effects has remained poorly understood and is further complicated by multiple downstream signaling pathways and the heteromerization properties of the receptor. This review compiles current knowledge, and discusses the potential mechanisms-of-action of the anticonvulsant effects mediated by the ghrelin-R.
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http://dx.doi.org/10.3390/biomedicines10010053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8773216PMC
December 2021

Exploring Refinement Strategies for Single Housing of Male C57BL/6JRj Mice: Effect of Cage Divider on Stress-Related Behavior and Hypothalamic-Pituitary-Adrenal-Axis Activity.

Front Behav Neurosci 2021 28;15:743959. Epub 2021 Oct 28.

Research Group Experimental Pharmacology, Department of Pharmaceutical Sciences, Center for Neurosciences, Vrije Universiteit Brussel, Brussels, Belgium.

Single housing of laboratory mice is a common practice to meet experimental needs, or to avoid intermale aggression. However, single housing is considered to negatively affect animal welfare and may compromise the scientific validity of experiments. The aim of this study was to investigate whether the use of a cage with a cage divider, which avoids physical contact between mice while maintaining sensory contact, may be a potential refinement strategy for experiments in which group housing of mice is not possible. Eight-week-old male C57BL/6JRj mice were single housed, pair housed or pair housed with a cage divider for four (experiment 1) or ten (experiment 2) weeks, after which we performed an open field test, Y-maze spontaneous alternation test, elevated plus maze test, an auditory fear conditioning task, and assessed responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis. Housing conditions did not affect body weight, exploratory activity, anxiety, working memory, fear memory processing or markers for HPA-axis functioning in either experiment 1 or experiment 2. There was an increased distance traveled in mice housed with a cage divider compared to pair housed mice after 4 weeks, and after 10 weeks mice housed with a cage divider made significantly more arm entries in the Y-maze spontaneous alternation test. Taken together, our study did not provide evidence for robust differences in exploratory activity, anxiety, working memory and fear memory processing in male C57BL/6JRj mice that were single housed, pair housed or pair housed with a cage divider.
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http://dx.doi.org/10.3389/fnbeh.2021.743959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8581484PMC
October 2021

Side-by-side comparison of the effects of Gq- and Gi-DREADD-mediated astrocyte modulation on intracellular calcium dynamics and synaptic plasticity in the hippocampal CA1.

Mol Brain 2021 09 20;14(1):144. Epub 2021 Sep 20.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.

Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca. In addition, Ca-dependent and/or Ca-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca-dependent and Ca-independent mechanisms, respectively.
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http://dx.doi.org/10.1186/s13041-021-00856-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8451082PMC
September 2021

Applicability of cerebral open flow microperfusion and microdialysis to quantify a brain-penetrating nanobody in mice.

Anal Chim Acta 2021 Sep 29;1178:338803. Epub 2021 Jun 29.

Vrije Universiteit Brussel (VUB), Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address:

The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier (BBB) severely hampers biologics from entering the brain. Considerable effort has been put into generating methods to modulate or circumvent the BBB for delivery of therapeutics to the CNS. A promising strategy is receptor-mediated transcytosis (RMT). Recently, Wouters et al. (2020) discovered a mouse anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via RMT. The present study aims to sample a derivative of this brain-penetrating nanobody (Nb105) in the CNS. Therefore, we compared the applicability of cerebral open flow microperfusion (cOFM) and microdialysis as sampling techniques to directly obtain high molecular weight substances from the cerebral interstitial fluid. A custom AlphaScreen™ assay was validated to quantify nanobody concentrations in the samples. In vitro microdialysis probe (AtmosLM™, 1 MDa cut-off) recovery by gain and by loss for Nb105 was 18.3 ± 3.2% and 27.0 ± 2.5% respectively, whereas for cOFM it was 87.2 ± 4.0% and 97.3 ± 1.6%. Although a large difference in in vitro recovery is observed between cOFM and microdialysis, in vivo similar results were obtained. Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher fluorescein isothiocyanate-dextran and immunoglobulin G extravasation around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. This is the first study that samples a bispecific nanobody in the brain's interstitial fluid in function of time, providing a pharmacokinetic profile of nanobodies in the CNS. Furthermore, this is the first time a cOFM study is performed in awake freely moving mice, providing data on inflammation and blood-brain barrier integrity in the mouse brain. Overall, this work demonstrates that, while taking into account the (bio)analytical considerations, both microdialysis and cOFM are suitable in vivo sampling techniques for quantification of nanobodies in the CNS.
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http://dx.doi.org/10.1016/j.aca.2021.338803DOI Listing
September 2021

Serum Daytime Melatonin Levels Reflect Cerebrospinal Fluid Melatonin Levels in Alzheimer's Disease but Are Not Correlated with Cognitive Decline.

J Alzheimers Dis 2021 ;83(2):693-704

Department of Neurology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium.

Background: Nocturnal cerebrospinal fluid (CSF) and blood melatonin levels are altered in Alzheimer's disease (AD). However, literature remains inconclusive on daytime blood melatonin levels. A positive correlation between melatonin levels and Mini-Mental State Examination (MMSE) scores in AD subjects has been evidenced following cross-sectional analyses. Whereas a correlation between serum and spinal CSF melatonin has been shown in healthy volunteers, an equal investigation in AD patients still has to be undertaken.

Objective: 1) To evaluate whether serum melatonin levels correlate with spinal CSF melatonin levels in AD. 2) To compare daytime CSF and serum melatonin levels between patients with AD dementia, mild cognitive impairment due to AD, and healthy controls, and to evaluate whether melatonin can affect cognitive decline in AD.

Methods: Subjects with AD and healthy controls included in two existing cohorts, of whom a CSF and serum sample was available at the neurobiobank and had at least 6 months of neuropsychological follow-up, were included in the present study. Melatonin concentrations were measured with liquid chromatography-mass spectrometry.

Results: Daytime serum melatonin levels correlated with spinal CSF melatonin levels in AD (r = 0.751, p < 0.001). No significant differences regarding daytime melatonin levels were found between patients and controls. No correlations were observed between daytime melatonin levels and MMSE score changes.

Conclusion: Daytime serum melatonin accurately reflects CSF melatonin levels in AD, raising the possibility to assess melatonin alterations by solely performing blood sampling if also confirmed for night-time values. However, daytime melatonin levels are not associated with changes of cognitive impairment.
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http://dx.doi.org/10.3233/JAD-210562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8543270PMC
December 2021

Translational potential of the ghrelin receptor agonist macimorelin for seizure suppression in pharmacoresistant epilepsy.

Eur J Neurol 2021 09 9;28(9):3100-3112. Epub 2021 Jul 9.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Background: Current drugs for epilepsy affect seizures, but no antiepileptogenic or disease-modifying drugs are available that prevent or slow down epileptogenesis, which is characterized by neuronal cell loss, inflammation and aberrant network formation. Ghrelin and ghrelin receptor (ghrelin-R) agonists were previously found to exert anticonvulsant, neuroprotective and anti-inflammatory effects in seizure models and immediately after status epilepticus (SE). Therefore, the aim of this study was to assess whether the ghrelin-R agonist macimorelin is antiepileptogenic in the pharmacoresistant intrahippocampal kainic acid (IHKA) mouse model.

Methods: SE was induced in C57BL/6 mice by unilateral IHKA injection. Starting 24 h after SE, mice were treated intraperitoneally with macimorelin (5 mg/kg) or saline twice daily for 2 weeks, followed by a 2-week wash-out. Mice were continuously electroencephalogram-monitored, and at the end of the experiment neuroprotection and gliosis were assessed.

Results: Macimorelin significantly decreased the number and duration of seizures during the treatment period, but had no antiepileptogenic or disease-modifying effect in this dose regimen. While macimorelin did not significantly affect food intake or body weight over a 2-week treatment period, its acute orexigenic effect was preserved in epileptic mice but not in sham mice.

Conclusions: While the full ghrelin-R agonist macimorelin was not significantly antiepileptogenic nor disease-modifying, this is the first study to demonstrate its anticonvulsant effects in the IHKA model of drug-refractory temporal lobe epilepsy. These findings highlight the potential use of macimorelin as a novel treatment option for seizure suppression in pharmacoresistant epilepsy.
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http://dx.doi.org/10.1111/ene.14992DOI Listing
September 2021

Melatonin levels in the Alzheimer's disease continuum: a systematic review.

Alzheimers Res Ther 2021 02 23;13(1):52. Epub 2021 Feb 23.

Research group Experimental Pharmacology (EFAR), Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information (FASC), Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.

Background: The search for new Alzheimer's disease (AD) cerebrospinal fluid (CSF) and blood biomarkers with potential pathophysiological and clinical relevance continues, as new biomarkers might lead to improved early and differential diagnosis, monitoring of disease progression and might even identify new druggable targets. Melatonin might be an interesting biomarker as an inverse correlation between CSF melatonin levels, and severity of the neuropathology as measured by Braak stages has been described. Melatonin can be measured in different body fluids, such as CSF, blood, saliva and urine.

Objectives: The aim of this systematic review was to review all available studies regarding melatonin levels in different body fluids in the AD continuum and give an extensive overview of reported outcomes.

Methods: We included papers comparing melatonin levels between healthy controls and human patients belonging to the AD continuum. A systematic search of PubMed and Web of Science led to inclusion of 20 full-length English papers following exclusion of duplicates.

Results: This systematic literature search showed that disruptions in melatonin levels occur with age, but also in AD when compared to age-matched controls. Night-time melatonin levels were found to be lower in CSF and blood of AD patients as compared to controls. Literature was not conclusive regarding alterations in blood daytime melatonin levels or regarding saliva melatonin in AD patients. Decreased total and night-time melatonin production has been described in urine of AD patients.

Conclusion: Our systematic review shows evidence for disruptions in (night-time) melatonin levels in AD as compared to age-matched controls. Although more studies are needed to understand the contribution of disruption of the melatonergic system to the pathophysiology of AD, the potential anti-AD effects that have been attributed to melatonin, renders research on this topic relevant for the discovery of potential future treatment effects of melatonin for AD. The use of melatonin as potential blood biomarker for disease progression should also be further investigated.
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http://dx.doi.org/10.1186/s13195-021-00788-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903801PMC
February 2021

Effects of repeated anodal transcranial direct current stimulation on auditory fear extinction in C57BL/6J mice.

Brain Stimul 2021 Mar-Apr;14(2):250-260. Epub 2021 Jan 14.

Department of Pharmaceutical Sciences, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium. Electronic address:

Background: Trauma-based psychotherapy is a first line treatment for post-traumatic stress disorder (PTSD) but not all patients achieve long-term remission. Transcranial direct current stimulation (tDCS) received considerable attention as a neuromodulation method that may improve trauma-based psychotherapy.

Objective: We explored the effects of repeated anodal tDCS over the prefrontal cortex (PFC) on fear extinction in mice as a preclinical model for trauma-based psychotherapy.

Methods: We performed auditory fear conditioning with moderate or high shock intensity on C57BL6/J mice. Next, mice received anodal tDCS (0.2 mA, 20 min) or sham stimulation over the PFC twice daily for five consecutive days. Extinction training was performed by repeatedly exposing mice to the auditory cue the day after the last stimulation session. Early and late retention of extinction were evaluated one day and three weeks after extinction training respectively.

Results: We observed no significant effect of tDCS on the acquisition or retention of fear extinction in mice subjected to fear conditioning with moderate intensity. However, when the intensity of fear conditioning was high, tDCS significantly lowered freezing during the acquisition of extinction, regardless of the extinction protocol. Moreover, when tDCS was combined with a strong extinction protocol, we also observed a significant improvement of early extinction recall. Finally, we found that tDCS reduced generalized fear induced by contextual cues when the intensity of conditioning is high and extinction training limited.

Conclusions: Our data provide a rationale to further explore anodal tDCS over the PFC as potential support for trauma-based psychotherapy for PTSD.
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http://dx.doi.org/10.1016/j.brs.2021.01.005DOI Listing
October 2021

Astrocytic Connexin43 Channels as Candidate Targets in Epilepsy Treatment.

Biomolecules 2020 11 20;10(11). Epub 2020 Nov 20.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology (EFAR), Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.

In epilepsy research, emphasis is put on exploring non-neuronal targets such as astrocytic proteins, since many patients remain pharmacoresistant to current treatments, which almost all target neuronal mechanisms. This paper reviews available data on astrocytic connexin43 (Cx43) signaling in seizures and epilepsy. Cx43 is a widely expressed transmembrane protein and the constituent of gap junctions (GJs) and hemichannels (HCs), allowing intercellular and extracellular communication, respectively. A plethora of research papers show altered Cx43 mRNA levels, protein expression, phosphorylation state, distribution and/or functional coupling in human epileptic tissue and experimental models. Human Cx43 mutations are linked to seizures as well, as 30% of patients with oculodentodigital dysplasia (ODDD), a rare genetic condition caused by mutations in the GJA1 gene coding for Cx43 protein, exhibit neurological symptoms including seizures. Cx30/Cx43 double knock-out mice show increased susceptibility to evoked epileptiform events in brain slices due to impaired GJ-mediated redistribution of K and glutamate and display a higher frequency of spontaneous generalized chronic seizures in an epilepsy model. Contradictory, Cx30/Cx43 GJs can traffic nutrients to high-energy demanding neurons and initiate astrocytic Ca waves and hyper synchronization, thereby supporting proconvulsant effects. The general connexin channel blocker carbenoxolone and blockers from the fenamate family diminish epileptiform activity in vitro and improve seizure outcome in vivo. In addition, interventions with more selective peptide inhibitors of HCs display anticonvulsant actions. To conclude, further studies aiming to disentangle distinct roles of HCs and GJs are necessary and tools specifically targeting Cx43 HCs may facilitate the search for novel epilepsy treatments.
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http://dx.doi.org/10.3390/biom10111578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699773PMC
November 2020

A comparative study of UniSpray and electrospray sources for the ionization of neuropeptides in liquid chromatography tandem mass spectrometry.

J Chromatogr A 2020 Sep 11;1628:461462. Epub 2020 Aug 11.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information (FASC), Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address:

Despite the extensive use of electrospray ionization (ESI) for the quantification of neuropeptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), poor ionization and transmission efficiency are described for this ionization interface. A new atmospheric pressure ionization source, named UniSpray, was recently developed and commercialized. In this study, the LC-MS performance of this new ionization interface is evaluated and compared with ESI for the quantification of seven neuropeptides. Besides comparison of signal intensities and charge state distributions, also signal-to-noise (S/N) ratios and accuracy and precision were assessed. Additionally, matrix effects of human precipitated plasma and rat microdialysate were evaluated as well as the effect of three supercharging agents on the ionization of the seven neuropeptides. UniSpray ionization resulted in signal intensities four to eight times higher at the optimal capillary/impactor voltage for all seven neuropeptides. S/N values at the other hand only increased by not more than a twofold when the UniSpray source was used. Moreover, UniSpray ionization resulted in a shift towards lower charge states for some neuropeptides. Evaluation of the matrix effects by a post-column infusion set-up resulted in different infusion profiles between ESI and UniSpray. The charge state distributions of the neuropeptides obtained with UniSpray are highly comparable with ESI. Finally, the effect of the supercharging agents on the ionization of the neuropeptides tends to be peptide-dependent with both ionization sources.
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http://dx.doi.org/10.1016/j.chroma.2020.461462DOI Listing
September 2020

CE-MS metabolic profiling of volume-restricted plasma samples from an acute mouse model for epileptic seizures to discover potentially involved metabolomic features.

Talanta 2020 Sep 1;217:121107. Epub 2020 May 1.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium. Electronic address:

Currently, a high variety of analytical techniques to perform metabolomics is available. One of these techniques is capillary electrophoresis coupled to mass spectrometry (CE-MS), which has emerged as a rather strong analytical technique for profiling polar and charged compounds. This work aims to discover with CE-MS potential metabolic consequences of evoked seizures in plasma by using a 6Hz acute corneal seizure mouse model. CE-MS is an appealing technique because of its capability to handle very small sample volumes, such as the 10 μL plasma samples obtained using capillary microsampling in this study. After liquid-liquid extraction, the samples were analyzed with CE-MS using low-pH separation conditions, followed by data analysis and biomarker identification. Both electrically induced seizures showed decreased values of methionine, lysine, glycine, phenylalanine, citrulline, 3-methyladenine and histidine in mice plasma. However, a second provoked seizure, 13 days later, showed a less pronounced decrease of the mean concentrations of these plasma metabolites, demonstrated by higher fold change ratios. Other obtained markers that can be related to seizure activities based on literature data, are isoleucine, serine, proline, tryptophan, alanine, arginine, valine and asparagine. Most amino acids showed relatively stable plasma concentrations between the basal levels (Time point 1) and after the 13-day wash-out period (Time point 3), which suggests its effectiveness. Overall, this work clearly demonstrated the possibility of profiling metabolite consequences related to seizure activities of an intrinsically low amount of body fluid using CE-MS. It would be useful to investigate and validate, in the future, the known and unknown metabolites in different animal models as well as in humans.
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http://dx.doi.org/10.1016/j.talanta.2020.121107DOI Listing
September 2020

Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

Circ Res 2020 07 14;127(4):466-482. Epub 2020 May 14.

Laboratory of Exercise and Health, Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETHZ) Zurich (Z.F., M.G., T.G., P.G., R.A., K.D.B.).

Rationale: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood.

Objective: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB.

Methods And Results: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets.

Conclusions: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386868PMC
July 2020

Effects of ghrelin receptor activation on forebrain dopamine release, conditioned fear and fear extinction in C57BL/6J mice.

J Neurochem 2020 08 25;154(4):389-403. Epub 2020 Mar 25.

Department of Pharmaceutical Sciences, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Brussels, Belgium.

The ghrelin system was previously proposed to mediate an independent branch of the stress response that curbs fear processing. Interestingly, the ghrelin system was also shown to control the activity of midbrain dopamine neurons. Given that dopamine neurons of the ventral tegmental area appear to have a critical role in fear processing, we aimed to investigate their contribution to the effects of ghrelin on fear processing. Our data show that systemic administration of the ghrelin receptor agonist MK0677, in a dose that induces food intake, has no significant effect on auditory fear processing and does not significantly affect dopamine release in the nucleus accumbens of male C57BL/6J mice. Local administration of the ghrelin receptor agonist MK0677 into the ventral tegmental area significantly increases food intake and it also significantly increased dopamine release in the nucleus accumbens, the medial prefrontal cortex and the amygdala. Nevertheless, it did not significantly affect auditory fear extinction. Our data indicate that pharmacological activation of midbrain dopamine neurons using a ghrelin receptor agonist does not affect auditory fear extinction. We also investigated the effect of non-pharmacological manipulation of the ghrelin system on auditory fear processing. However, we found that neither overnight food deprivation nor genetic ablation of the ghrelin receptor had a significant effect on auditory fear extinction. We conclude that the effects of manipulation of the ghrelin system on fear processing are subject to boundary conditions that remain poorly understood.
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http://dx.doi.org/10.1111/jnc.14996DOI Listing
August 2020

Angiotensin-II-mediated AT receptor stimulation increases glutamate release within the rostral ventrolateral medulla of normotensive rats.

Hypertens Res 2020 08 4;43(8):848-850. Epub 2020 Mar 4.

Research Group Experimental Pharmacology, Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences, VUB, Brussel, Belgium.

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http://dx.doi.org/10.1038/s41440-020-0419-zDOI Listing
August 2020

Effects of neuromedin U-8 on stress responsiveness and hypothalamus-pituitary-adrenal axis activity in male C57BL/6J mice.

Horm Behav 2020 05 2;121:104666. Epub 2020 Mar 2.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Center for Neurosciences (C4N), Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address:

Neuromedin U (NMU) is a highly conserved neuropeptide that has been implicated in the stress response. To better understand how it influences various aspects of the stress response, we studied the effects of intracerebroventricular NMU-8 administration on stress-related behavior and activity of the hypothalamus-pituitary-adrenal (HPA) axis in male C57BL/6J mice. We investigated these NMU-8 effects when mice remained in their home cage and when they were challenged by exposure to forced swim stress. NMU-8 administration resulted in increased grooming behavior in mice that remained in their home cage and in a significant increase in c-Fos immunoreactivity in the paraventricular hypothalamus (PVH) and arcuate nucleus (ARC). Surprisingly, NMU-8 administration significantly decreased plasma corticosterone concentrations. Furthermore, NMU-8 administration increased immobility in the forced swim test in both naïve mice and mice that were previously exposed to swim stress. The effect of NMU-8 on c-Fos immunoreactivity in the PVH was dependent on previous exposure to swim stress given that we observed no significant changes in mice exposed for the first time to swim stress. In contrast, in the ARC we observed a significant increase in c-Fos immunoreactivity regardless of previous stress exposure. Interestingly, NMU-8 administration also significantly decreased plasma corticosterone concentrations in mice that were exposed to single forced swim stress, while this effect was no longer observed when mice were exposed to forced swim stress for a second time. Taken together, our data indicate that NMU-8 regulates stress responsiveness and suggests that its effects depend on previous stress exposure.
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http://dx.doi.org/10.1016/j.yhbeh.2019.104666DOI Listing
May 2020

Neuromedin U and Structural Analogs: An Overview of their Structure, Function and Selectivity.

Curr Med Chem 2020 ;27(39):6744-6768

Research Group of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.

The neuromedin U peptide sequence is highly conserved between various species. Neuromedin U is involved in a variety of physiological processes. It exerts its effects via two neuromedin U receptors, NMUR1 and NMUR2. These receptors are characterized by a distinct, yet complementary, tissue distribution with NMUR1 mostly found in the periphery, while NMUR2 is most abundant in the central nervous system. The capability of the neuropeptide to reduce food intake in rodents triggered the design and synthesis of a broad range of modified peptide ligands. The purpose of these ligands is to develop novel therapeutics which could be beneficial in the treatment of obesity and diabetes. Most compounds are derived either from the full-length neuromedin U sequence or are based on the truncated orthologs of this neuropeptide. Only a few non-peptidic ligands were developed. This review provides an overview on various neuromedin U analogs and mimetics that have been reported to date.
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http://dx.doi.org/10.2174/0929867326666190916143028DOI Listing
January 2021

Investigation of the Role of AT2 Receptors in the Nucleus Tractus Solitarii of Normotensive Rats in Blood Pressure Control.

Front Neurosci 2019 5;13:589. Epub 2019 Jun 5.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology (EFAR), Center for Neurosciences (C4N), Vrije Universiteit Brussel, Brussels, Belgium.

Aim: The nucleus tractus solitarii (NTS) densely expresses angiotensin II type 2 receptors (AT2R), which are mainly located on inhibitory gamma-aminobutyric acid (GABA) neurons. Central AT2R stimulation reduces blood pressure, and AT2R stimulation in the rostral ventrolateral medulla (RVLM), mediates a hypotensive response through a GABAergic mechanism. We aimed to test the hypothesis that an AT2R mediated inhibition of the GABA release within the NTS might be involved in this hypotensive response, by assessing possible alterations in blood pressure and heart rate, as well as in GABA levels in normotensive Wistar rats.

Methods: microdialysis was used for measurement of extracellular GABA levels and for perfusion of the selective AT2R agonist, Compound 21, within the NTS. Our set-up allowed to determine simultaneously the excitatory glutamate dialysate levels. The mean arterial pressure and heart rate responses were monitored with a pressure transducer.

Results: Local perfusion of Compound 21 into the NTS did not modify blood pressure and heart rate, nor glutamate and GABA levels compared to baseline concentrations. A putative effect was also not unmasked by concomitant angiotensin II type 1 receptor blockade with candesartan. Positive control experiments confirmed that the experimental set up had enough sensitivity to detect a reduction in GABA dialysate levels and blood pressure.

Conclusion: The results did not provide evidence for a role of the AT2R within the NTS in the control of blood pressure, nor for an interaction with local GABAergic signaling in normotensive rats.
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http://dx.doi.org/10.3389/fnins.2019.00589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560158PMC
June 2019

Anticonvulsant and antiepileptogenic effects of system inactivation in chronic epilepsy models.

Epilepsia 2019 07 9;60(7):1412-1423. Epub 2019 Jun 9.

UCB Pharma, Braine-l'Alleud, Belgium.

Objective: The cystine/glutamate antiporter system could represent a new target for antiepileptogenic treatments due to its crucial roles in glutamate homeostasis and neuroinflammation. To demonstrate this, we compared epilepsy development and seizure susceptibility in xCT knockout mice (xCT ) and in littermate controls (xCT ) in different chronic models of epilepsy.

Methods: Mice were surgically implanted with electrodes in the basolateral amygdala and chronically stimulated to develop self-sustained status epilepticus (SSSE); continuous video-electroencephalography monitoring was performed for 28 days after SE and hippocampal histopathology was assessed. Corneal kindling was induced by twice daily electrical stimulation at 6 Hz and maintenance of the fully kindled state was evaluated. Next, messenger RNA (mRNA) and protein levels of xCT and of the proteins involved in the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway were measured at different time points during epileptogenesis in NMRI mice treated with pilocarpine. Finally, the anticonvulsant effect of sulfasalazine (SAS), a nonselective system inhibitor, was assessed against 6 Hz-evoked seizures in pilocarpine-treated mice.

Results: In the SSSE model, xCT mice displayed a significant delayed epileptogenesis, a reduced number of spontaneous recurrent seizures, and less pronounced astrocytic and microglial activation. Moreover, xCT mice showed reduced seizure severity during 6 Hz kindling development and a lower incidence of generalized seizures during the maintenance of the fully kindled state. In pilocarpine-treated mice, protein levels of the PI3K/Akt/GSK-3β/eIF2α/ATF4 pathway were increased during the chronic phase of the model, consistent with previous findings in the hippocampus of patients with epilepsy. Finally, repeated administration of SAS protected pilocarpine-treated mice against acute 6 Hz seizure induction, in contrast to sham controls, in which system is not activated.

Significance: Inhibition of system could be an attractive target for the development of new therapies with a potential for disease modification in epilepsy.
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http://dx.doi.org/10.1111/epi.16055DOI Listing
July 2019

Differential Effects of a Full and Biased Ghrelin Receptor Agonist in a Mouse Kindling Model.

Int J Mol Sci 2019 May 20;20(10). Epub 2019 May 20.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium.

The ghrelin system has received substantial recognition as a potential target for novel anti-seizure drugs. Ghrelin receptor (ghrelin-R) signaling is complex, involving Gα, Gα, Gα, and β-arrestin pathways. In this study, we aimed to deepen our understanding regarding signaling pathways downstream the ghrelin-R responsible for mediating anticonvulsive effects in a kindling model. Mice were administered the proconvulsive dopamine 1 receptor-agonist, SKF81297, to gradually induce a kindled state. Prior to every SKF81297 injection, mice were treated with a ghrelin-R full agonist (JMV-1843), a Gα and Gα biased ligand unable to recruit β-arrestin (YIL781), a ghrelin-R antagonist (JMV-2959), or saline. Mice treated with JMV-1843 had fewer and less severe seizures compared to saline-treated controls, while mice treated with YIL781 experienced longer and more severe seizures. JMV-2959 treatment did not lead to differences in seizure severity and number. Altogether, these results indicate that the Gα or Gα signaling pathways are not responsible for mediating JMV-1843's anticonvulsive effects and suggest a possible involvement of β-arrestin signaling in the anticonvulsive effects mediated by ghrelin-R modulation.
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http://dx.doi.org/10.3390/ijms20102480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567032PMC
May 2019

AT1 Receptor Mediated Hypertensive Response to Ang II in the Nucleus Tractus Solitarii of Normotensive Rats Involves NO Dependent Local GABA Release.

Front Pharmacol 2019 3;10:460. Epub 2019 May 3.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences, Vrije Universiteit Brussel, Brussels, Belgium.

Aim: It is well-established that angiotensin II exerts a dampening effect on the baroreflex within the nucleus tractus solitarii (NTS), the principal brainstem site for termination of baroreceptor afferents and which is densely populated with gamma-aminobutyric acid (GABA)ergic neurons and nerve terminals. The present study was designed to investigate whether local release of GABA is involved in the effects mediated by local angiotensin II within the NTS.

Methods: microdialysis was used for measurement of extracellular glutamate and GABA levels and for infusion of angiotensin II within the NTS of conscious normotensive Wistar rats. The mean arterial pressure (MAP) and heart rate response to local infusion of angiotensin II were subsequently monitored with a pressure transducer under anesthesia. The angiotensin II type 1 receptor (AT1R) antagonist, candesartan, was used to assess whether responses were AT1R dependent and the nitric oxide (NO) synthase inhibitor, (ω)-nitro-L-arginine methyl ester (L-NAME), was used to assess the involvement of NO in the evoked responses by infusion of angiotensin II. The MAP and heart rate responses were monitored with a pressure transducer.

Results: Local infusion into the NTS of angiotensin II induced a significant to ninefold significantly increase in extracellular GABA levels; as well as MAP was increased by 15 mmHg. These responses were both abolished by co-infusion of either, the angiotensin II type 1 receptor antagonist, candesartan, or the NO synthase inhibitor, L-NAME, demonstrating that the effect is not only AT1R dependent but also NO dependent. The pressor response to angiotensin II was reversed by co-infusion with the GABA receptor antagonist, bicuculline. Local blockade of NO synthase decreased both, GABA and glutamate concentrations.

Conclusion: Our results suggest that the AT1R mediated hypertensive response to angiotensin II within the NTS in normotensive rats is GABA and NO dependent. Nitric oxide produced within the NTS tonically potentiates local GABA and glutamate release.
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http://dx.doi.org/10.3389/fphar.2019.00460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509664PMC
May 2019

Genetic and pharmacological manipulation of glial glutamate transporters does not alter infection-induced seizure activity.

Exp Neurol 2019 08 22;318:50-60. Epub 2019 Apr 22.

Department of Pharmacology and Toxicology, University of Utah, USA; Interdepartmental Program in Neuroscience, University of Utah, USA. Electronic address:

The contribution of glial transporters to glutamate movement across the membrane has been identified as a potential target for anti-seizure therapies. Two such glutamate transporters, GLT-1 and system x, are expressed on glial cells, and modulation of their expression and function have been identified as a means by which seizures, neuronal injury, and gliosis can be reduced in models of brain injury. While GLT-1 is responsible for the majority of glutamate uptake in the brain, system x releases glutamate in the extracellular cleft in exchange for cystine and represents as such the major source of hippocampal extracellular glutamate. Using the Theiler's Murine Encephalomyelitis Virus (TMEV) model of viral-induced epilepsy, we have taken two well-studied approaches, one pharmacological, one genetic, to investigate the potential role(s) of GLT-1 and system x in TMEV-induced pathology. Our findings suggest that the methods we utilized to modulate these glial transporters, while effective in other models, are not sufficient to reduce the number or severity of behavioral seizures in TMEV-infected mice. However, genetic knockout of xCT, the specific subunit of system x, may have cellular effects, as we observed a slight decrease in neuronal injury caused by TMEV and an increase in astrogliosis in the CA1 region of the hippocampus. Furthermore, xCT knockout caused an increase in GLT-1 expression selectively in the cortex. These findings have significant implications for both the characterization of the TMEV model as well as for future efforts to discover novel and effective anti-seizure drugs.
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http://dx.doi.org/10.1016/j.expneurol.2019.04.010DOI Listing
August 2019

Assessing mixtures of supercharging agents to increase the abundance of a specific charge state of Neuromedin U.

Talanta 2019 Jun 29;198:206-214. Epub 2019 Jan 29.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information (FASC), Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address:

With increasing evidence of the important role of peptides in pathophysiological processes, a trend towards the development of highly sensitive bioanalytical methods is ongoing. Inherent to the electrospray ionization process of peptides and proteins is the production of multiple charge states which may hamper proper and sensitive method development. Supercharging agents allow modifying the maximal charge state and the corresponding distribution of charges, thereby potentially increasing the number of ions reaching the detector in selected reaction monitoring mode. In this study, the use of mixtures of charge state modifying additives, i.e. m-nitrobenzylalcohol (mNBA), sulfolane and dimethyl sulfoxide (DMSO), to specifically increase the abundance of one charge state of interest has been investigated. Screening experiments were performed to define an experimental domain, which was then further investigated via a response surface design to predict the optimal combination and concentration of superchargers. Using a combination of mNBA and DMSO (0.008% and 0.5% m/v respectively), we were able to increase the abundance of the +4 charge state of the investigated peptide neuromedin U from 64% to 87%. Unfortunately, charge state coalescence did not result in repeatable sensitivity improvements in this case study. However, it remains an attractive approach during method development of peptide bioanalytical methods, as coalescence to a particular intermediate charge state is difficult to obtain by using only one supercharging agent.
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http://dx.doi.org/10.1016/j.talanta.2019.01.098DOI Listing
June 2019

A companion to the preclinical common data elements for pharmacologic studies in animal models of seizures and epilepsy. A Report of the TASK3 Pharmacology Working Group of the ILAE/AES Joint Translational Task Force.

Epilepsia Open 2018 Nov 15;3(Suppl Suppl 1):53-68. Epub 2018 Sep 15.

Department of Pharmacology, Toxicology, and Pharmacy University of Veterinary Medicine Hannover Hannover Germany.

Preclinical pharmacology studies in animal models of seizures and epilepsy have provided a platform to identify more than 20 antiseizure drugs in recent decades. To minimize variability in lab-to-lab studies and to harmonize approaches to data collection and reporting methodology in pharmacologic evaluations of the next generation of therapies, we present common data elements (CDEs), case report forms (CRFs), and this companion manuscript to help with the implementation of methods for studies in established preclinical seizure and epilepsy models in adult rodents. The development of and advocacy for CDEs in preclinical research has been encouraged previously by both clinical and preclinical groups. It is anticipated that adoption and implementation of these CDEs in preclinical studies may help standardize approaches to minimize variability and increase the reproducibility of preclinical studies. Moreover, they may provide a methodologic framework for pharmacology studies in atypical animal models or models in development, which may ultimately promote novel therapy development. In the present document, we refer selectively to animal models that have a long history of preclinical use, and in some cases, are clinically validated.
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http://dx.doi.org/10.1002/epi4.12254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210039PMC
November 2018

5-HTR and 5-HTR but not 5-HTR antagonism impairs the cross-modal reactivation of deprived visual cortex in adulthood.

Mol Brain 2018 11 6;11(1):65. Epub 2018 Nov 6.

Laboratory of Neuroplasticity and Neuroproteomics, Katholieke Universiteit Leuven, Naamsestraat 59, Box 2467, B-3000, Leuven, Belgium.

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR and 5-HTR were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR, 5-HTR and 5-HTR subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR and 5-HTR protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR and 5-HTR but not 5-HTR during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.
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http://dx.doi.org/10.1186/s13041-018-0404-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218970PMC
November 2018

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy.

ACS Chem Neurosci 2019 04 6;10(4):1992-2003. Epub 2018 Nov 6.

Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences , University of Leuven , Leuven 3000 , Belgium.

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.
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http://dx.doi.org/10.1021/acschemneuro.8b00281DOI Listing
April 2019

Sensitive targeted methods for brain metabolomic studies in microdialysis samples.

J Pharm Biomed Anal 2018 Nov 23;161:192-205. Epub 2018 Aug 23.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information (FASC), Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. Electronic address:

In vivo determination of brain mediators plays an important role in providing insight in how the brain functions. For this purpose, targeted metabolomics can be a very useful tool. Targeted metabolomics detects and measures certain known low-molecular-weight biomolecules involved in signaling pathways and biochemical processes in the central nervous system. Microdialysis is a powerful technique to sample brain mediators in the central nervous system. Several analytical techniques that can possibly be coupled to microdialysis are available. However, selection of an appropriate technique should be considered carefully, since sensitivity and specificity are critical when measuring these mediators in volume-restricted microdialysis samples. This review outlines some of the commonly applied sampling methods and analytical techniques and discusses some of the challenges encountered during the in vivo determination of central nervous system mediators.
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http://dx.doi.org/10.1016/j.jpba.2018.08.043DOI Listing
November 2018

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3.

Neuropharmacology 2018 09 11;140:275-286. Epub 2018 Aug 11.

IGF, CNRS, INSERM, Univ. de Montpellier, Montpellier, F-34094, Montpellier, France. Electronic address:

Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl ions exert strong positive allosteric modulation of glutamate on the mGlu receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive "chloride-lock" network. Indeed, Cl ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu receptor. Thus, the mGlu receptors' large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Clmediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl ions allows the mGlu receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.
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http://dx.doi.org/10.1016/j.neuropharm.2018.08.011DOI Listing
September 2018

The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy.

Neurochem Res 2019 Mar 10;44(3):600-608. Epub 2018 Aug 10.

Department of Pharmaceutical Chemistry, Drug Analysis and Drug Information, Research Group Experimental Pharmacology, Center for Neurosciences (C4N), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.

Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe comorbidities, such as cognitive dysfunction. Correspondingly, several animal models of TLE exhibit memory dysfunction, especially spatial memory. The Morris water maze test is the most commonly used test for assessing spatial learning and memory in rodents. However, high stress and poor swimming abilities are common confounders and may contribute to misinterpretation. Particularly epileptic mice show altered behaviour during the test as they fail to understand the paradigm context. In the Barnes maze test, a dry-land maze test for spatial learning and memory that uses milder aversive stimuli, these drawbacks have not yet been reported. In the present study, we use this task to evaluate spatial learning and memory in the intrahippocampal kainic acid mouse model of TLE. We demonstrate that the epileptic mice understand the Barnes maze paradigm context, as they learn the location of the escape-chamber by using a serial search strategy but fail to develop the more efficient spatial search strategy. Our data indicate that the Barnes maze may be a better alternative to the Morris water maze for assessing search strategies and impairment of learning and memory in epileptic mice.
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http://dx.doi.org/10.1007/s11064-018-2610-zDOI Listing
March 2019

Erratum to "Selective Changes in Locomotor Activity in Mice Due to Low-intensity Microwaves Amplitude Modulated in the EEG Spectral Domain" [Neuroscience 359 (2017) 40-48].

Neuroscience 2018 12 14;394:316. Epub 2018 Jul 14.

Department of Electronics and Informatics (ETRO), Laboratory for Micro- and Photon Electronics (LAMI), Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium. Electronic address:

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http://dx.doi.org/10.1016/j.neuroscience.2018.07.005DOI Listing
December 2018
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