Subclinical epileptiform activity in the Alzheimer continuum: association with disease, cognition and detection method.

BACKGROUND: Epileptic seizures are an established comorbidity of Alzheimer's disease (AD). Subclinical epileptiform activity (SEA) as detected by 24-h electroencephalography (EEG) or magneto-encephalography (MEG) has been reported in temporal regions of clinically diagnosed AD patients. Although epileptic activity in AD probably arises in the mesial temporal lobe, electrical activity within this region might not propagate to EEG scalp electrodes and could remain undetected by standard EEG. However, SEA might lead to faster cognitive decline in AD. AIMS: 1. To estimate the prevalence of SEA and interictal epileptic discharges (IEDs) in a well-defined cohort of participants belonging to the AD continuum, including preclinical AD subjects, as compared with cognitively healthy controls. 2. To evaluate whether long-term-EEG (LTM-EEG), high-density-EEG (hd-EEG) or MEG is superior to detect SEA in AD. 3. To characterise AD patients with SEA based on clinical, neuropsychological and neuroimaging parameters. METHODS: Subjects (n = 49) belonging to the AD continuum were diagnosed according to the 2011 NIA-AA research criteria, with a high likelihood of underlying AD pathophysiology. Healthy volunteers (n = 24) scored normal on neuropsychological testing and were amyloid negative. None of the participants experienced a seizure before. Subjects underwent LTM-EEG and/or 50-min MEG and/or 50-min hd-EEG to detect IEDs. RESULTS: We found an increased prevalence of SEA in AD subjects (31%) as compared to controls (8%) (p = 0.041; Fisher's exact test), with increasing prevalence over the disease course (50% in dementia, 27% in MCI and 25% in preclinical AD). Although MEG (25%) did not withhold a higher prevalence of SEA in AD as compared to LTM-EEG (19%) and hd-EEG (19%), MEG was significantly superior to detect spikes per 50 min (p = 0.002; Kruskall-Wallis test). AD patients with SEA scored worse on the RBANS visuospatial and attention subset (p = 0.009 and p = 0.05, respectively; Mann-Whitney U test) and had higher left frontal, (left) temporal and (left and right) entorhinal cortex volumes than those without. CONCLUSION: We confirmed that SEA is increased in the AD continuum as compared to controls, with increasing prevalence with AD disease stage. In AD patients, SEA is associated with more severe visuospatial and attention deficits and with increased left frontal, (left) temporal and entorhinal cortex volumes. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04131491. 12/02/2020.

Unravelling the brain metabolome: A review of liquid chromatography - mass spectrometry strategies for extracellular brain metabolomics.

The analysis of the brain extracellular metabolome is of interest for numerous subdomains within neuroscience. Not only does it provide information about normal physiological functions, it is even more of interest for biomarker discovery and target discovery in disease. The extracellular analysis of the brain is particularly interesting as it provides information about the release of mediators in the brain extracellular fluid to look at cellular signaling and metabolic pathways through the release, diffusion and re-uptake of neurochemicals. In vivo samples are obtained through microdialysis, cerebral open-flow microperfusion or solid-phase microextraction. The analytes of potential interest are typically low in concentration and can have a wide range of physicochemical properties. Liquid chromatography coupled to mass spectrometry has proven its usefulness in brain metabolomics. It allows sensitive and specific analysis of low sample volumes, obtained through different approaches. Several strategies for the analysis of the extracellular fluid have been proposed. The most widely used approaches apply sample derivatization, specific stationary phases and/or hydrophilic interaction liquid chromatography. Miniaturization of these methods allows an even higher sensitivity. The development of chiral metabolomics is indispensable, as it allows to compare the enantiomeric ratio of compounds and provides even more challenges. Some limitations continue to exist for the previously developed methods and the development of new, more sensitive methods remains needed. This review provides an overview of the methods developed for sampling and liquid chromatography-mass spectrometry analysis of the extracellular metabolome.

The intracerebral injection of Aß1-42 oligomers does not invariably alter seizure susceptibility in mice.

Objectives: Epileptiform activity and seizures are present in patients with Alzheimer's disease (AD) and genetic animal models of AD. Amyloid beta 1-42 (Aß1-42) oligomers are thought to be crucial in AD and can cause neuronal hyperexcitability in vitro. However, it is unclear whether these Aß1-42 oligomers cause the increased seizure susceptibility in vivo in people with AD and in AD animal models, nor via which mechanisms it would do so. We investigated this question by injecting Aß1-42 oligomers intracerebrally in mice and assessed its impact on seizure susceptibility. Materials and methods: We performed a single intracerebral injection of synthetic Aß1-42 oligomers or scrambled Aß1-42 in NMRI mice in three different cohorts and subjected them to an i.v. infusion of a chemoconvulsant. We evoked the seizures 1.5 h, 1 week, or 3 weeks after the intracerebral injection of Aß1-42 oligomers, covering also the timepoints and injection locations that were used by others in similar experimental set-ups. Results: With a thioflavine T assay and transmission electron microscopy we confirmed that Aß1-42 monomers spontaneously aggregated to oligomers. We did not find an effect of Aß1-42 oligomers on susceptibility to seizures - evoked 1.5 h, 1 week or 3 weeks - after their intracerebral injection. Significance: The lack of effect of Aß1-42 oligomers on seizure susceptibility in our experiments contrasts with recent findings in similar experimental set-ups. Contradicting conclusions are frequent in experiments with Aß1-42 and they are often attributed to subtle differences in the various aggregation forms of the Aß1-42 used in different experiments. We confirmed the presence of Aß1-42 oligomers with state-of-the-art methods but cannot ascertain that the protein aggregates we used are identical to those used by others. Whether our findings or those previously published best represent the role of Aß1-42 oligomers on seizures in AD remains unclear.

Neurofilament light chain: A possible fluid biomarker in the intrahippocampal kainic acid mouse model for chronic epilepsy?

OBJECTIVE: In the management of epilepsy, there is an ongoing quest to discover new biomarkers to improve the diagnostic process, the monitoring of disease progression, and the evaluation of treatment responsiveness. In this regard, biochemical traceability in biofluids is notably absent in contrast to other diseases. In the present preclinical study, we investigated the potential of neurofilament light chain (NfL) as a possible diagnostic and response fluid biomarker for epilepsy. METHODS: We gained insights into NfL levels during the various phases of the intrahippocampal kainic acid mouse model of temporal lobe epilepsy-namely, the status epilepticus (SE) and the chronic phase with spontaneous seizures. To this end, NfL levels were determined directly in the cerebral interstitial fluid (ISF) with cerebral open flow microperfusion as sampling technique, as well as in cerebrospinal fluid (CSF) and plasma. Lastly, we assessed whether NfL levels diminished upon curtailing SE with diazepam and ketamine. RESULTS: NfL levels are higher during SE in both cerebral ISF and plasma in kainic acid-treated mice compared to sham-injected mice. Additionally, ISF and plasma NfL levels are lower in mice treated with diazepam and ketamine to stop SE compared with the vehicle-treated mice. In the chronic phase with spontaneous seizures, higher NfL levels could only be detected in ISF and CSF samples, and not in plasma. No correlations could be found between NfL levels and seizure burden, nor with immunohistological markers for neurodegeneration/inflammation. SIGNIFICANCE: Our findings demonstrate the translational potential of NfL as a blood-based fluid biomarker for SE. This is less evident for chronic epilepsy, as in this case higher NfL levels could only be detected in ISF and CSF, and not in plasma, acknowledging the invasive nature of CSF sampling in chronic epilepsy follow-up.

Neuroanatomical characterization of the Nmu-Cre knock-in mice reveals an interconnected network of unique neuropeptidergic cells.

Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the Nmu promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions, in situ hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.

Chemogenetic modulation of astrocytes and microglia: State-of-the-art and implications in neuroscience.

Insights into the role astrocytes and microglia play in normal and diseased brain functioning has expanded drastically over the last decade. Recently, chemogenetic tools have emerged as cutting-edge techniques, allowing targeted and spatiotemporal precise manipulation of a specific glial cell type. As a result, significant advances in astrocyte and microglial cell function have been made, showing how glial cells can intervene in central nervous system (CNS) functions such as cognition, reward and feeding behavior in addition to their established contribution in brain diseases, pain, and CNS inflammation. Here, we discuss the latest insights in glial functions in health and disease that have been made through the application of chemogenetics. We will focus on the manipulation of intracellular signaling pathways induced by activation of the designer receptors exclusively activated by designer drugs (DREADDs) in astrocytes and microglia. We will also elaborate on some of the potential pitfalls and the translational potential of the DREADD technology.

Improving the LC-MS/MS analysis of neuromedin U-8 and neuromedin S by minimizing their adsorption behavior and optimizing UHPLC and MS parameters.

Neuromedin U (NmU) and neuromedin S (NmS) are two closely related neuropeptides belonging to the neuromedin family. NmU usually occurs either as a truncated eight amino acid long peptide (NmU-8) or as an 25 amino acid long peptide, although other molecular forms exist depending on the species considered. NmS, on the other hand, is a 36 amino acid long peptide, sharing the same amidated C-terminal heptapeptide with NmU. Nowadays, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the preferred analytical technique for peptide quantification, because of its excellent sensitivity and selectivity. However, reaching the required quantification limits for these compounds in biological samples remains an extremely challenging task, especially because of their nonspecific binding (NSB). This study highlights the difficulties that are faced when quantifying larger neuropeptides (23-36 amino acids) compared to smaller ones (< 15 amino acids). The first part of this work aims to solve the adsorption problem for NmU-8 and NmS, by investigating the different steps involved in the sample preparation, i.e. the different solvents applied and the pipetting protocol. The addition of 0.05% plasma as an adsorption competitor was found to be primordial to avoid peptide loss due to NSB. The second part of this work focusses on further improving the sensitivity of the LC-MS/MS method for NmU-8 and NmS, by evaluating some UHPLC-parameters, including the stationary phase, the column temperature and the trapping conditions. For both peptides of interest, the best results were achieved when combining a C18 trap column with a C18 iKey separation device containing a positively charged surface. Column temperatures of 35 and 45 °C for NmU-8 and NmS respectively, resulted in the highest peak areas and S/N ratios, while applying higher column temperatures substantially decreased sensitivity. Moreover, a gradient starting at 20% organic modifier instead of 5% significantly improved the peak shape of both peptides. Finally, some compound-specific MS parameters, i.e. the capillary and the cone voltages, were evaluated. The peak areas increased with a factor 2 and 7 for NmU-8 and NmS respectively and peptide detection in the low picomolar range is now feasible.

Normal Pregnancy-Induced Islet Beta Cell Proliferation in Mouse Models That Are Deficient in Serotonin-Signaling.

During mouse pregnancy placental lactogens stimulate prolactin receptors on pancreatic islet beta cells to induce expression of the tryptophan hydroxylase Tph1, resulting in the synthesis and secretion of serotonin. Presently, the functional relevance of this phenomenon is unclear. One hypothesis is that serotonin-induced activation of 5-HT2B receptors on beta cells stimulates beta cell proliferation during pregnancy. We tested this hypothesis via three different mouse models: (i) total Tph1KO mice, (ii) 129P2/OlaHsd mice, which are incompetent to upregulate islet Tph1 during pregnancy, whereas Tph1 is normally expressed in the intestine, mammary glands, and placenta, and (iii) Htr2b-deficient mice. We observed normal pregnancy-induced levels of beta cell proliferation in total Tph1KO mice, 129P2/OlaHsd mice, and in Htr2b-/- mice. The three studied mouse models indicate that islet serotonin production and its signaling via 5-HT2B receptors are not required for the wave of beta cell proliferation that occurs during normal mouse pregnancy.

Psychedelic-Induced Serotonin 2A Receptor Downregulation Does Not Predict Swim Stress Coping in Mice.

Serotoninergic psychedelics such as psilocybin have been reported to elicit a long-lasting reduction in depressive symptoms. Although the main target for serotoninergic psychedelics, serotonin type 2A receptor (5-HT2A), has been established, the possible mechanism of the antidepressant action of psychedelics remains unknown. Using the mouse forced swim test model, we examined whether the administration of the synthetic serotoninergic psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI) would modulate 5-HT2A receptor levels in the medial prefrontal cortex (mPFC) and revert stress-induced changes in behavior. Mice subjected to swim stress developed a passive stress-coping strategy when tested in the forced swim test 6 days later. This change in behavior was not associated with the hypothesized increase in 5-HT2A receptor-dependent head twitch behaviors or consistent changes in 5-HT2A receptor levels in the mPFC. When DOI was administered 1 day before the forced swim test, a low dose (0.2 mg/kg i.p.) unexpectedly increased immobility while a high dose (2 mg/kg i.p.) had no significant effect on immobility. Nevertheless, DOI evoked a dose-dependent decrease in 5-HT2A levels in the mPFC of mice previously exposed to swim stress. Our findings do not support the hypothesis that the downregulation of 5-HT2A receptors in the mPFC contributes to the antidepressant-like properties of serotoninergic psychedelics.

Targeting firing rate neuronal homeostasis can prevent seizures.

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.

Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist.

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Higher susceptibility to 6 Hz corneal kindling and lower responsiveness to antiseizure drugs in mouse models of Alzheimer's disease.

OBJECTIVE: Epileptic spikes and seizures seem present early in the disease process of Alzheimer's disease (AD). However, it is unclear how soluble and insoluble amyloid beta (Aß) and tau proteins affect seizure development in vivo. We aim to contribute to this field by assessing the vulnerability to 6 Hz corneal kindling of young female mice from two well-characterized transgenic AD models and by testing their responsiveness to selected antiseizure drugs (ASDs). METHODS: We used 7-week-old triple transgenic (3xTg) mice that have both amyloid and tau mutations, and amyloid precursor protein Swedish/presenillin 1 dE9 (APP/PS1) mice, bearing only amyloid-related mutations. We assessed the absence of plaques via immunohistochemistry and analyzed the concentrations of both soluble and insoluble forms of Aß1-42 and total tau (t-tau) in brain hippocampal and prefrontal cortical tissue. Seven-week-old mice of the different genotypes were subjected to the 6 Hz corneal kindling model. After kindling acquisition, we tested the anticonvulsant effects of three marketed ASDs (levetiracetam, brivaracetam, and lamotrigine) in fully kindled mice. RESULTS: No Aß plaques were present in either genotype. Soluble Aß1-42 levels were increased in both AD genotypes, whereas insoluble Aß1-42 concentrations were only elevated in APP/PS1 mice compared with their respective controls. Soluble and insoluble forms of t-tau were increased in 3xTg mice only. 3xTg and APP/PS1 mice displayed more severe seizures induced by 6 Hz corneal kindling from the first stimulation onward and were more rapidly kindled compared with control mice. In fully kindled AD mice, ASDs had less-pronounced anticonvulsive effects compared with controls. SIGNIFICANCE: Mutations increasing Aß only or both Aß and tau in the brain enhance susceptibility for seizures and kindling in mice. The effect of ASDs on seizures measured by the Racine scale is less pronounced in both investigated AD models and suggests that seizures of young AD mice are more difficult to treat.

Current Approaches to Monitor Macromolecules Directly from the Cerebral Interstitial Fluid.

Gaining insights into the pharmacokinetic and pharmacodynamic properties of lead compounds is crucial during drug development processes. When it comes to the treatment of brain diseases, collecting information at the site of action is challenging. There are only a few techniques available that allow for the direct sampling from the cerebral interstitial space. This review concerns the applicability of microdialysis and other approaches, such as cerebral open flow microperfusion and electrochemical biosensors, to monitor macromolecules (neuropeptides, proteins, …) in the brain. Microdialysis and cerebral open flow microperfusion can also be used to locally apply molecules at the same time at the site of sampling. Innovations in the field are discussed, together with the pitfalls. Moreover, the 'nuts and bolts' of the techniques and the current research gaps are addressed. The implementation of these techniques could help to improve drug development of brain-targeted drugs.

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

Two closely related glycogen synthase kinase-3 (GSK-3) isoforms have been identified in mammals: GSK-3α and GSK-3ß. GSK-3ß 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-3ß, 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-3ß (GSK-3ß OE) or lack GSK-3ß (GSK-3ß-/-) in neurons. We demonstrate that TCS2002 exerts anticonvulsant actions against pilocarpine- and 6 Hz-evoked seizures. Compared to wild-type littermates, GSK-3ß OE mice are less susceptible to seizures but are more rapidly kindled. Interestingly, compared to GSK-3ß+/+ mice, neuronal GSK-3ß-/- mice show increased susceptibility to 6 Hz-induced seizures. These contrasting observations suggest compensatory neurodevelopmental mechanisms that alter seizure susceptibility in GSK-3ß OE and GSK-3ß-/- mice. Although the pronounced anticonvulsant effects of selective and acute GSK-3ß inhibition in the 6 Hz model identify GSK-3ß as a potential drug target for pharmacoresistant seizures, our data on the sustained disruption of GSK-3ß 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-3ß inhibition.

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.

Introduction: 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. Methods: 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. Results: 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. Conclusion: 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.

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

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.

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.

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 Ca2+. In addition, Ca2+-dependent and/or Ca2+-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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-dependent and Ca2+-independent mechanisms, respectively.

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

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.

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

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.

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

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.