Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Evaluation of prenatal calabash chalk geophagy on the developing brain of Wistar rats.

Calabash chalk (CaC) is an aluminium silicate hydroxide compound with heavy metal constituents, making it a potential neurotoxicant. Pregnant women often consume CaC as an antiemetic, which may interfere with the normal development of the foetal brain. Here, we evaluated the effects of CaC administration in pregnant rats on the brain of the offspring. Wistar rat dams were assigned to one of three groups: control, 200 mg/kg and 800 mg/kg of a CaC suspension. Administrations lasted 14 days (gestation days 7-20). On day 14, 5-bromo-2'-deoxyuridine (BrdU) was administered and dams were allowed to term. Behavioural tests were performed on different days as the pups matured, and they were sacrificed on post-natal days 30 and 60. Brains were processed for histology and Western blotting. Results showed no significant differences in surface righting reflex, cliff avoidance, negative geotaxis and open-field activity. No hippocampal and somatosensory cortical cytoarchitectonic alterations and no significant signs of glial fibrillary acidic protein (GFAP) activation were observed. Neuronal nuclei counts showed variability in the somatosensory cortex and hippocampus of the CaC group. BrdU-positive cells were significantly lower in the 200 mg/kg group and higher in the 800 mg/kg group. Doublecortin-X-positive cells were not different in all the CaC groups. Astrocytes and microglia Western blotting quantification confirmed no significant increase in pup glial cells in adulthood. Prenatal consumption of CaC at indicated dosages may not be deleterious to the developing brain, especially after cessation of exposure and during maturation of the animal. However, the differences in neuronal and glial populations may be due to their ability to cope with CaC.

Sex differences in neuromuscular and biological determinants of isometric maximal force.

AIM: Force expression is characterized by an interplay of biological and molecular determinants that are expected to differentiate males and females in terms of maximal performance. These include muscle characteristics (muscle size, fiber type, contractility), neuromuscular regulation (central and peripheral factors of force expression), and individual genetic factors (miRNAs and gene/protein expression). This research aims to comprehensively assess these physiological variables and their role as determinants of maximal force difference between sexes. METHODS: Experimental evaluations include neuromuscular components of isometric contraction, intrinsic muscle characteristics (proteins and fiber type), and some biomarkers associated with muscle function (circulating miRNAs and gut microbiome) in 12 young and healthy males and 12 females. RESULTS: Male strength superiority appears to stem primarily from muscle size while muscle fiber-type distribution plays a crucial role in contractile properties. Moderate-to-strong pooled correlations between these muscle parameters were established with specific circulating miRNAs, as well as muscle and plasma proteins. CONCLUSION: Muscle size is crucial in explaining the differences in maximal voluntary isometric force generation between males and females with similar fiber type distribution. Potential physiological mechanisms are seen from associations between maximal force, skeletal muscle contractile properties, and biological markers.

Age-Dependent Neuropsychiatric Symptoms in the NF-κB/c-Rel Knockout Mouse Model of Parkinson's Disease.

Non-motor symptoms are frequently observed in Parkinson's disease (PD) and precede the onset of motor deficits by years. Among them, neuropsychiatric symptoms, including anxiety, depression, and apathy, are increasingly considered as a major challenge for patients with PD and their caregivers. We recently reported that mice lacking the nuclear factor-κB (NF-κB)/c-Rel protein (c-rel-/- mice) develop an age-dependent PD-like pathology and phenotype characterized by the onset of non-motor symptoms, including constipation and hyposmia, starting at 2 months of age, and motor deficits at 18 months. To assess whether c-rel-/- mice also suffer from neuropsychiatric symptoms, in this study we tested different cohorts of wild-type (wt) and c-rel-/- mice at 3, 6, 12, and 18-20 months with different behavioral tests. Mice lacking c-Rel displayed anxiety and depressive-like behavior starting in the premotor phase at 12 months, as indicated by the analysis with the open field (OF) test and the forced swim test with water wheel (FST), respectively. A deficit in the goal-oriented nesting building test was detected at 18-20 months, suggesting apathetic behavior. Taken together, these results indicate that c-rel-/- mice recapitulate the onset and the progression of PD-related neuropsychiatric symptoms. Therefore, this animal model may represent a valuable tool to study the prodromal stage of PD and for testing new therapeutic strategies to alleviate neuropsychiatric symptoms.

Downregulation of the Astroglial Connexin Expression and Neurodegeneration after Pilocarpine-Induced Status Epilepticus.

Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas Cx36 mRNA did not exhibit obvious changes. Regional evaluation with quantitative real-time-RT-PCR confirmed Cx43 and Cx30 mRNA downregulation 24 h after SE, when ongoing neuronal cell death was found in the same brain regions. Immunolabeling showed at the same time point marked a decrease in Cx43, microglia activation, and interleukin-1ß induction in some microglial cells. The data showed a transient downregulation of astroglial Cxs in the cortical and thalamic areas in which SE triggers neurodegenerative events in concomitance with microglia activation and cytokine expression. This could potentially represent a protective response of neuroglial networks to SE-induced acute damage.

Detection of spontaneous seizures in EEGs in multiple experimental mouse models of epilepsy.

Objective.Electroencephalography (EEG) is a key tool for non-invasive recording of brain activity and the diagnosis of epilepsy. EEG monitoring is also widely employed in rodent models to track epilepsy development and evaluate experimental therapies and interventions. Whereas automated seizure detection algorithms have been developed for clinical EEG, preclinical versions face challenges of inter-model differences and lack of EEG standardization, leaving researchers relying on time-consuming visual annotation of signals.Approach.In this study, a machine learning-based seizure detection approach, 'Epi-AI', which can semi-automate EEG analysis in multiple mouse models of epilepsy was developed. Twenty-six mice with a total EEG recording duration of 6451 h were used to develop and test the Epi-AI approach. EEG recordings were obtained from two mouse models of kainic acid-induced epilepsy (Models I and III), a genetic model of Dravet syndrome (Model II) and a pilocarpine mouse model of epilepsy (Model IV). The Epi-AI algorithm was compared against two threshold-based approaches for seizure detection, a local Teager-Kaiser energy operator (TKEO) approach and a global Teager-Kaiser energy operator-discrete wavelet transform (TKEO-DWT) combination approach.Main results.Epi-AI demonstrated a superior sensitivity, 91.4%-98.8%, and specificity, 93.1%-98.8%, in Models I-III, to both of the threshold-based approaches which performed well on individual mouse models but did not generalise well across models. The performance of the TKEO approach in Models I-III ranged from 66.9%-91.3% sensitivity and 60.8%-97.5% specificity to detect spontaneous seizures when compared with expert annotations. The sensitivity and specificity of the TKEO-DWT approach were marginally better than the TKEO approach in Models I-III at 73.2%-80.1% and 75.8%-98.1%, respectively. When tested on EEG from Model IV which was not used in developing the Epi-AI approach, Epi-AI was able to identify seizures with 76.3% sensitivity and 98.1% specificity.Significance.Epi-AI has the potential to provide fast, objective and reproducible semi-automated analysis of multiple types of seizure in long-duration EEG recordings in rodents.

Gut microbiota modulates seizure susceptibility.

A bulk of data suggest that the gut microbiota plays a role in a broad range of diseases, including those affecting the central nervous system. Recently, significant differences in the intestinal microbiota of patients with epilepsy, compared to healthy volunteers, have been reported in an observational study. However, an active role of the intestinal microbiota in the pathogenesis of epilepsy, through the so-called "gut-brain axis," has yet to be demonstrated. In this study, we evaluated the direct impact of microbiota transplanted from epileptic animals to healthy recipient animals, to clarify whether the microbiota from animals with epilepsy can affect the excitability of the recipients' brain by lowering seizure thresholds. Our results provide the first evidence that mice who received microbiota from epileptic animals are more prone to develop status epilepticus, compared to recipients of "healthy" microbiota, after a subclinical dose of pilocarpine, indicating a higher susceptibility to seizures. The lower thresholds for seizure activity found in this study support the hypothesis that the microbiota, through the gut-brain axis, is able to affect neuronal excitability in the brain.

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

The Anti-Inflammatory Properties of Mesenchymal Stem Cells in Epilepsy: Possible Treatments and Future Perspectives.

Mesenchymal stem cells (MSCs) are multipotent adult cells with self-renewing capacities. MSCs display specific properties, such as the ability to repair damaged tissues, resulting in optimal candidates for cell therapy against degenerative diseases. In addition to the reparative functions of MSCs, growing evidence shows that these cells have potent immunomodulatory and anti-inflammatory properties. Therefore, MSCs are potential tools for treating inflammation-related neurological diseases, including epilepsy. In this regard, over the last decades, epilepsy has no longer been considered a purely neuronal pathology, since inflammatory events underlying the genesis of epilepsy have been demonstrated. This review assessed current knowledge on the use of MSCs in the treatment of epilepsy. Mostly, attention will be focused on the anti-inflammatory and immunological skills of MSCs. Understanding the mechanisms by which MSCs might modulate the severity of the disease will contribute to the development of new potential alternatives for both prophylaxis and treatment against epilepsy.

Ongoing Electroencephalographic Rhythms Related to Exploratory Movements in Transgenic TASTPM Mice.

BACKGROUND: The European PharmaCog study (http://www.pharmacog.org) has reported a reduction in delta (1-6 Hz) electroencephalographic (EEG) power (density) during cage exploration (active condition) compared with quiet wakefulness (passive condition) in PDAPP mice (hAPP Indiana V717F mutation) modeling Alzheimer's disease (AD) amyloidosis and cognitive deficits. OBJECTIVE: Here, we tested the reproducibility of that evidence in TASTPM mice (double mutation in APP KM670/671NL and PSEN1 M146V), which develop brain amyloidosis and cognitive deficits over aging. The reliability of that evidence was examined in four research centers of the PharmaCog study. METHODS: Ongoing EEG rhythms were recorded from a frontoparietal bipolar channel in 29 TASTPM and 58 matched "wild type" C57 mice (range of age: 12-24 months). Normalized EEG power was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during the passive and active conditions. RESULTS: Compared with the "wild type" group, the TASTPM group showed a significantly lower reduction in IDF power during the active over the passive condition (p < 0.05). This effect was observed in 3 out of 4 EEG recording units. CONCLUSION: TASTPM mice were characterized by "poor reactivity" of delta EEG rhythms during the cage exploration in line with previous evidence in PDAPP mice. The reliability of that result across the centers was moderate, thus unveiling pros and cons of multicenter preclinical EEG trials in TASTPM mice useful for planning future studies.

Genome-wide microRNA profiling of plasma from three different animal models identifies biomarkers of temporal lobe epilepsy.

Epilepsy diagnosis is complex, requires a team of specialists and relies on in-depth patient and family history, MRI-imaging and EEG monitoring. There is therefore an unmet clinical need for a non-invasive, molecular-based, biomarker to either predict the development of epilepsy or diagnose a patient with epilepsy who may not have had a witnessed seizure. Recent studies have demonstrated a role for microRNAs in the pathogenesis of epilepsy. MicroRNAs are short non-coding RNA molecules which negatively regulate gene expression, exerting profound influence on target pathways and cellular processes. The presence of microRNAs in biofluids, ease of detection, resistance to degradation and functional role in epilepsy render them excellent candidate biomarkers. Here we performed the first multi-model, genome-wide profiling of plasma microRNAs during epileptogenesis and in chronic temporal lobe epilepsy animals. From video-EEG monitored rats and mice we serially sampled blood samples and identified a set of dysregulated microRNAs comprising increased miR-93-5p, miR-142-5p, miR-182-5p, miR-199a-3p and decreased miR-574-3p during one or both phases. Validation studies found miR-93-5p, miR-199a-3p and miR-574-3p were also dysregulated in plasma from patients with intractable temporal lobe epilepsy. Treatment of mice with common anti-epileptic drugs did not alter the expression levels of any of the five miRNAs identified, however administration of an anti-epileptogenic microRNA treatment prevented dysregulation of several of these miRNAs. The miRNAs were detected within the Argonuate2-RISC complex from both neurons and microglia indicating these miRNA biomarker candidates can likely be traced back to specific brain cell types. The current studies identify additional circulating microRNA biomarkers of experimental and human epilepsy which may support diagnosis of temporal lobe epilepsy via a quick, cost-effective rapid molecular-based test.

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.

Electrographic seizures induced by activation of ETA and ETB receptors following intrahippocampal infusion of endothelin-1 in immature rats occur by different mechanisms.

We have demonstrated previously that activation of either the ETA or ETB receptor can induce acute electrographic seizures following the intrahippocampal infusion of endothelin-1 (ET-1) in immature (P12) rats. We also demonstrated that activation of the ETA receptor is associated with marked focal ischemia, while activation of the ETB receptor is not. Exploring the mechanisms underlying seizures induced by these two ET-1 receptor interactions can potentially provide insight into how focal ischemia in immature animals produces seizures and whether ischemiarelated seizures differ from seizures not associated with ischemia. To explore these seizure mechanisms we used microdialysis to determine biomarkers associated with seizures in P12 rats following the intrahippocampal infusion of two different agents: (1) ET-1, which activates both the ETA and ETB receptors and causes focal ischemia and (2) Ala-ET-1, which selectively activates only the ETB receptor and does not cause ischemia. Our results show that seizures associated with combined ETA and ETB receptor activation (and ischemia) have a different temporal distribution and microdialysis profile from seizures associated with ETB activation alone (and without ischemia). Seizures with combined activation peak within the first hour after infusion and the microdialysis profile is characterized by a significant increase in the ratio of glutamic acid to GABA. By contrast, seizures with activation of only the ETB receptor peak in the second hour after infusion and microdialysis shows a significant increase in the ratio of leukotriene B4 to prostaglandin E2. These findings suggest that ischemia-related seizures in immature animals involve an imbalance of excitation and inhibition, while non-ischemiarelated seizures involve an inflammatory process resulting from an excess of leukotrienes.

Rehabilitation and Biomarkers of Stroke Recovery: Study Protocol for a Randomized Controlled Trial.

Background: Stroke is a leading cause of disability. Nonetheless, the care pathway for stroke rehabilitation takes partially into account the needs of chronic patients. This is due in part to the lack of evidence about the mechanisms of recovery after stroke, together with the poor knowledge of related and influencing factors. Here we report on the study protocol "Rehabilitation and Biomarkers of Stroke Recovery," which consists of 7 work-packages and mainly aim to investigate the effects of long-term neurorehabilitation on stroke patients and to define a related profile of (clinical-biological, imaging, neurophysiological, and genetic-molecular) biomarkers of long-term recovery after stroke. The work-package 1 will represent the main part of this protocol and aims to compare the long-term effects of intensive self-rehabilitation vs. usual (rehabilitation) care for stroke. Methods: We planned to include a total of 134 adult subacute stroke patients (no more than 3 months since onset) suffering from multidomain disability as a consequence of first-ever unilateral ischemic stroke. Eligible participants will be randomly assigned to one of the following groups: intensive self-rehabilitation (based on the principles of "Guided Self-Rehabilitation Contract") vs. usual care (routine practice). Treatment will last 1 year, and patients will be evaluated every 3 months according to their clinical presentation. The following outcomes will be considered in the main work-package: Fugl-Meyer assessment, Cognitive Oxford Screen Barthel Index, structural and functional neuroimaging, cortical excitability, and motor and somatosensory evoked potentials. Discussion: This trial will deal with the effects of an intensive self-management rehabilitation protocol and a related set of biomarkers. It will also investigate the role of training intensity on long-term recovery after stroke. In addition, it will define a set of biomarkers related to post-stroke recovery and neurorehabilitation outcome in order to detect patients with greater potential and define long-term individualized rehabilitation programs. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04323501.

Biopsychosocial model of resilience in young adults with multiple sclerosis (BPS-ARMS): an observational study protocol exploring psychological reactions early after diagnosis.

INTRODUCTION: Multiple sclerosis (MS), the most common neurological disease causing disability in young adults, is widely recognised as a major stress factor. Studies have shown that the first years after the diagnosis are distressing in terms of adjustment to the disease and that MS negatively affects patients' psychological well-being, quality of life (QoL) and social functioning. However, the links between disease-specific variables at diagnosis, resilience and psychological adjustment of patients with MS remain largely unexplored, especially in adolescents and young adults. This observational study aims to fill the gap of knowledge on biopsychosocial characteristics and resilience of young adults with MS to evaluate the relationship among these variables and to develop a biopsychosocial model of resilience. METHODS AND ANALYSIS: Biological and clinical characteristics of young adults newly diagnosed with MS will be investigated by collecting clinical information, performing neurological examinations, MRI and analysing cerebrospinal fluid and blood biomarkers (eg, measures of inflammation), body composition, gut microbiota and movement/perceptual markers. Psychosocial characteristics (eg, psychological distress, coping strategies), QoL, psychological well-being and resilience will be assessed by self-report questionnaires. Comparative statistics (ie, analysis of variance or unpaired samples t-test, correlation and regression analyses) will be applied to evaluate the relationship among biological, psychological and social factors. The results are expected to allow a comprehensive understanding of the determinants of resilience in young patients with MS and to inform resilience interventions, tailored to young patients' specific needs, aiming to reduce the risk of maladaptive reactions to the disease and to improve psychological well-being and QoL. ETHICS AND DISSEMINATION: The study has been approved by the Verona University Hospital Ethics Committee (approval number: 2029CESC). The findings will be disseminated through scientific publications in peer-reviewed journals, conference presentations, social media and specific websites. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03825055).

Ongoing Electroencephalographic Activity Associated with Cortical Arousal in Transgenic PDAPP Mice (hAPP V717F).

BACKGROUND: It has been shown that theta (6-10 Hz) and delta (1-6 Hz) ongoing electroencephalographic (EEG) rhythms revealed variations in the cortical arousal in C57 Wild Type (WT) mice during cage exploration (active condition) compared to awake quiet behavior (passive condition; IMI PharmaCog project, www.pharmacog.eu). OBJECTIVE: The objective was to test if these EEG rhythms might be abnormal in old PDAPP mice modeling Alzheimer's disease (AD) with a hAPP Indiana V717F mutation (They show abnormal neural transmission, cognitive deficits, and brain accumulation of Aß1-42). METHODS: Ongoing EEG rhythms were recorded by a frontoparietal bipolar channel in 15 PDAPP and 23 WT C57 male mice (mean age of 22.8 months ±0.4 and 0.3 standard error, respectively). EEG absolute power (density) was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during passive and active states in the wakefulness. RESULTS: Compared with the WT group, the PDAPP group showed higher frequency of the IDF during the passive condition and lower frequency of the ITF during the active state. Furthermore, the WT but not PDAPP group showed significant changes in the frontoparietal EEG power (IDF, ITF) during active over passive state. CONCLUSION: PDAPP mice were characterized by less changes in the brain arousal during an active state as revealed by frontoparietal EEG rhythms. Future studies will have to cross-validate the present results on large animal groups, clarify the neurophysiological underpinning of the effect, and test if the disease modifying drugs against AD amyloidosis normalize those candiate EEG biomarkers in PDAPP mice.

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium.

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

Nonsteroidal anti-inflammatory drugs in clinical and experimental epilepsy.

Current antiepileptic drugs have limited efficacy and provide little or no benefits in 30% of the patients. Given that a role for brain inflammation in epilepsy has been repeatedly reported in recent years, the potential of anti-inflammatory drugs should be explored in depth, as they may provide new therapeutical approaches in preventing or reducing epileptogenesis. Here, we review preclinical (both in vivo and in vitro) and clinical epilepsy studies in which nonsteroidal antiinflammatory drugs (NSAIDs), i.e. cyclooxygenase-2 (COX-2) selective inhibitors (COXIBs) and nonselective NSAIDs, were used for seizure control. The effects of NSAIDs are reviewed in animal models of both chemical (pilocarpine, kainic acid, pentylenetetrazol, or carbachol administration) and electrical (tetanic hippocampal stimulation, electroshock) seizure induction. In the pilocarpine model, NSAIDs are neuroprotective, reduce mossy fiber sprouting or diminish P-glycoprotein upregulation, but only rarely protect against seizures. While neuroprotective effects have also been observed in the kainic acid model, NSAIDs tend in general to worsen seizure activity. Effects of COXIB administration in the pentylenetetrazol-induced seizures model are variable, alternating from protection against seizures to null effects or even increased incidence of convulsions. Moreover, NSAIDs tested in the tetanic hippocampal stimulation model diminished the seizure-associated P-glycoprotein upregulation, but were not very effective in seizure control. NSAIDs efficacy in experimental in vivo epilepsy studies may be influenced by multiple factors, including the timing of administration (before or after status epilepticus induction), the animal model of epilepsy or some of the signaling pathways involved in cyclooxygenase induction (e.g. prostaglandins and their receptors). On the other hand, the few clinical studies on the use of NSAIDs in neurological pathologies accompanied/characterized by seizures indicate that nonselective NSAIDs (e.g. aspirin) in prolonged, low-dose treatments may offer protection against seizures and stroke-like events. No clinical trials in epileptic patients using COXIBs have been conducted so far, as several international drug-control authorities have withdrawn these drugs from the market; future studies should focus on improved COXIB formulations. We argue that, while the available evidence is still inconclusive, the potential therapeutic benefits of controlling and diminishing brain inflammation in the treatment of epilepsy should be actively explored.

On-going electroencephalographic rhythms related to cortical arousal in wild-type mice: the effect of aging.

Resting state electroencephalographic (EEG) rhythms reflect the fluctuation of cortical arousal and vigilance in a typical clinical setting, namely the EEG recording for few minutes with eyes closed (i.e., passive condition) and eyes open (i.e., active condition). Can this procedure be back-translated to C57 (wild type) mice for aging studies? On-going EEG rhythms were recorded from a frontoparietal bipolar channel in 85 (19 females) C57 mice. Male mice were subdivided into 3 groups: 25 young (4.5-6 months), 18 middle-aged (12-15 months), and 23 old (20-24 months) mice to test the effect of aging. EEG power density was compared between short periods (about 5 minutes) of awake quiet behavior (passive) and dynamic exploration of the cage (active). Compared with the passive condition, the active condition induced decreased EEG power at 1-2 Hz and increased EEG power at 6-10 Hz in the group of 85 mice. Concerning the aging effects, the passive condition showed higher EEG power at 1-2 Hz in the old group than that in the others. Furthermore, the active condition exhibited a maximum EEG power at 6-8 Hz in the former group and 8-10 Hz in the latter. In the present conditions, delta and theta EEG rhythms reflected changes in cortical arousal and vigilance in freely behaving C57 mice across aging. These changes resemble the so-called slowing of resting state EEG rhythms observed in humans across physiological and pathological aging. The present EEG procedures may be used to enhance preclinical phases of drug discovery in mice for understanding the neurophysiological effects of new compounds against brain aging.

Are they in or out? The elusive interaction between Qtracker ® 800 vascular labels and brain endothelial cells.

AIM: Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS: Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS: Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION: Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.