Hetero-pentamerization determines mobility and conductance of Glycine receptor α3 splice variants.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson's and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases.

Extremely Low-Frequency Electromagnetic Stimulation (ELF-EMS) Improves Neurological Outcome and Reduces Microglial Reactivity in a Rodent Model of Global Transient Stroke.

Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

p27kip1 Modulates the Morphology and Phagocytic Activity of Microglia.

p27kip1 is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27kip1 function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27kip1 in microglia using a combination of in vitro and in situ approaches. While the loss of p27kip1 did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27kip1 in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27kip1 did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27kip1 showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27kip1 plays specific roles in microglia.

Aggregated Tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded Tau pathology in vivo.

Brains of Alzheimer's disease patients are characterized by the presence of amyloid plaques and neurofibrillary tangles, both invariably associated with neuroinflammation. A crucial role for NLRP3-ASC inflammasome [NACHT, LRR and PYD domains-containing protein 3 (NLRP3)-Apoptosis-associated speck-like protein containing a CARD (ASC)] in amyloid-beta (Aß)-induced microgliosis and Aß pathology has been unequivocally identified. Aß aggregates activate NLRP3-ASC inflammasome (Halle et al. in Nat Immunol 9:857-865, 2008) and conversely NLRP3-ASC inflammasome activation exacerbates amyloid pathology in vivo (Heneka et al. in Nature 493:674-678, 2013), including by prion-like ASC-speck cross-seeding (Venegas et al. in Nature 552:355-361, 2017). However, the link between inflammasome activation, as crucial sensor of innate immunity, and Tau remains unexplored. Here, we analyzed whether Tau aggregates acting as prion-like Tau seeds can activate NLRP3-ASC inflammasome. We demonstrate that Tau seeds activate NLRP3-ASC-dependent inflammasome in primary microglia, following microglial uptake and lysosomal sorting of Tau seeds. Next, we analyzed the role of inflammasome activation in prion-like or templated seeding of Tau pathology and found significant inhibition of exogenously seeded Tau pathology by ASC deficiency in Tau transgenic mice. We furthermore demonstrate that chronic intracerebral administration of the NLRP3 inhibitor, MCC950, inhibits exogenously seeded Tau pathology. Finally, ASC deficiency also decreased non-exogenously seeded Tau pathology in Tau transgenic mice. Overall our findings demonstrate that Tau-seeding competent, aggregated Tau activates the ASC inflammasome through the NLRP3-ASC axis, and we demonstrate an exacerbating role of the NLRP3-ASC axis on exogenously and non-exogenously seeded Tau pathology in Tau mice in vivo. The NLRP3-ASC inflammasome, which is an important sensor of innate immunity and intensively explored for its role in health and disease, hence presents as an interesting therapeutic approach to target three crucial pathogenetic processes in AD, including prion-like seeding of Tau pathology, Aß pathology and neuroinflammation.

Controversies and prospects about microglia in maternal immune activation models for neurodevelopmental disorders.

Activation of the maternal immune system during pregnancy is a well-established risk factor for neuropsychiatric disease in the offspring, yet, the underlying mechanisms leading to altered brain function remain largely undefined. Microglia, the resident immune cells of the brain, are key to adequate development of the central nervous system (CNS), and are prime candidates to mediate maternal immune activation (MIA)-induced brain abnormalities. As such, the effects of MIA on the immunological phenotype of microglia has been widely investigated. However, contradicting results due to differences in read-out and methodological approaches impede final conclusions on MIA-induced microglial alterations. The aim of this review is to critically discuss the evidence for an activated microglial phenotype upon MIA.

Cerebral Cortical Circuitry Formation Requires Functional Glycine Receptors.

The development of the cerebral cortex is a complex process that requires the generation, migration, and differentiation of neurons. Interfering with any of these steps can impair the establishment of connectivity and, hence, function of the adult brain. Neurotransmitter receptors have emerged as critical players to regulate these biological steps during brain maturation. Among them, α2 subunit-containing glycine receptors (GlyRs) regulate cortical neurogenesis and the present work demonstrates the long-term consequences of their genetic disruption on neuronal connectivity in the postnatal cerebral cortex. Our data indicate that somatosensory cortical neurons of Glra2 knockout mice (Glra2KO) have more dendritic branches with an overall increase in total spine number. These morphological defects correlate with a disruption of the excitation/inhibition balance, thereby increasing network excitability and enhancing susceptibility to epileptic seizures after pentylenetetrazol tail infusion. Taken together, our findings show that the loss of embryonic GlyRα2 ultimately impairs the formation of cortical circuits in the mature brain.

Age-specific function of α5ß1 integrin in microglial migration during early colonization of the developing mouse cortex.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5ß1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5ß1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5ß1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5ß1 integrin in microglial migration during colonization of the embryonic brain.

Additive effects of the Rho kinase inhibitor Y-27632 and vardenafil on relaxation of the corpus cavernosum tissue of patients with erectile dysfunction and clinical phosphodiesterase type 5 inhibitor failure.

OBJECTIVES: To evaluate the expression of the Rho/Rho-associated protein kinase (ROCK) pathway in the corpus cavernosum of patients with severe erectile dysfunction (ED) compared with healthy human corpus cavernosum, and to test the functional effects of two Rho kinase inhibitors (RKIs) on erectile tissue of patients with severe ED, which did not respond to phosphodiesterase type 5 inhibitors (PDE5Is). PATIENTS AND METHODS: Human corpus cavernosum samples were obtained after consent from men undergoing penile prosthesis implantation (n = 7 for organ bath experiments, n = 17 for quantitative PCR [qPCR]). Potent control subjects (n = 5) underwent penile needle biopsy. qPCR was performed for the expression of RhoA and ROCK subtypes 1 and 2. Immunohistochemistry staining against ROCK and α smooth muscle actin (αSMA) was performed on the corpus cavernosum of patients with ED. Tissue strips were precontracted with phenylephrine and incubated with 1 µm of the PDE5I vardenafil or with DMSO (control). Subsequently, increasing concentrations of the RKIs azaindole or Y-27632 were added, and relaxation of tissue was quantified. RESULTS: The expression of ROCK1 was unchanged (P > 0.05), while ROCK2 (P < 0.05) was significantly upregulated in patients with ED compared with controls. ROCK1 and ROCK2 protein colocalized with αSMA, confirming the presence of this kinase in cavernous smooth muscle cells and/or myofibroblasts. After incubation with DMSO, 10 µm azaindole and 10 µm Y-27632 relaxed precontracted tissues with 49.5 ± 7.42% (P = 0.1470 when compared with vehicle) and 85.9 ± 10.3% (P = 0.0016 when compared with vehicle), respectively. Additive effects on relaxation of human corpus cavernosum were seen after preincubation with 1 µm vardenafil. CONCLUSION: The RKI Y-27632 causes a significant relaxation of corpus cavernosum in tissue strips of patients with severe ED. The additive effect of vardenafil and Y-27632 shows that a combined inhibition of Rho-kinase and phosphodiesterase type 5 could be a promising orally administered treatment for severe ED.

Experimental febrile seizures increase dendritic complexity of newborn dentate granule cells.

OBJECTIVE: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. METHODS: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. RESULTS: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. SIGNIFICANCE: Experimental FS increase dendritic complexity and the number of mushroom-type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied.

Alpha-adrenoceptor modulation in central nervous system trauma: pain, spasms, and paralysis--an unlucky triad.

Many researchers have attempted to pharmacologically modulate the adrenergic system to control locomotion, pain, and spasms after central nervous system (CNS) trauma, although such efforts have led to conflicting results. Despite this, multiple studies highlight that α-adrenoceptors (α-ARs) are promising therapeutic targets because in the CNS, they are involved in reactivity to stressors and regulation of locomotion, pain, and spasms. These functions can be activated by direct modulation of these receptors on neuronal networks in the brain and the spinal cord. In addition, these multifunctional receptors are also broadly expressed on immune cells. This suggests that they might play a key role in modulating immunological responses, which may be crucial in treating spinal cord injury and traumatic brain injury as both diseases are characterized by a strong inflammatory component. Reducing the proinflammatory response will create a more permissive environment for axon regeneration and may support neuromodulation in combination therapies. However, pharmacological interventions are hindered by adrenergic system complexity and the even more complicated anatomical and physiological changes in the CNS after trauma. This review is the first concise overview of the pros and cons of α-AR modulation in the context of CNS trauma.

The effects of urolithin A on poly I:C-induced microglial activation.

Neuroinflammation can be triggered by various stimuli, including viral infections. Viruses can directly invade the brain and infect neuronal cells or indirectly trigger a "cytokine storm" in the periphery that eventually leads to microglial activation in the brain. While this initial activation of microglial cells is important for viral clearance, chronic activation leads to excessive inflammation and oxidative stress, which can be neurotoxic. Remarkebly, recent studies have shown that certain viruses such as influenza A virus, coronavirus, herpes virus and Epstein-Barr virus may be involved in the development of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. Therefore, it is important to find therapeutic strategies against chronic neuroinflammation triggered by viral infections. Here, we investigated the effects of urolithin A (UA) on microglial activation in vitro induced by a viral mimetic, poly I:C, in a triple co-culture system of neurons, astrocytes and microglial cells. Immunocytochemistry was used to perform a comprehensive single-cell analysis of the morphological changes of microglia as an indicator of their reactive state. Treatment with UA significantly prevented the poly I:C-induced reactive state of microglia, which was characterized by increased expression of the microglial activation markers CD68 and IBA-1. UA restored the poly I:C-induced morphology by restoring microglial ramification. In addition, UA was able to reduce the release of the pro-inflammatory mediators CCL2, TNF-α, and IL-1ß and showed a trend toward attenuation of cellular ROS production in poly I:C-treated cultures. Overall, this study suggests that UA as a component of a healthy diet may help prevent virus-induced neuroinflammation and may have therapeutic potential for future studies to prevent or treat neurodegenerative diseases by targeting the associated neuroinflammatory processes.

Stretch-injury promotes microglia activation with enhanced phagocytic and synaptic stripping activities.

Microglial cells, as the primary defense line in the central nervous system, play a crucial role in responding to various mechanical signals that can trigger their activation. Despite extensive research on the impact of chemical signaling on brain cells, the understanding of mechanical signaling in microglia remains limited. To bridge this gap, we subjected microglial cells to a singular mechanical stretch and compared their responses with those induced by lipopolysaccharide treatment, a well-established chemical activator. Here we show that stretching microglial cells leads to their activation, highlighting their significant mechanosensitivity. Stretched microglial cells exhibited distinct features, including elevated levels of Iba1 protein, a denser actin cytoskeleton, and increased persistence in migration. Unlike LPS-treated microglial cells, the secretory profile of chemokines and cytokines remained largely unchanged in response to stretching, except for TNF-α. Intriguingly, a single stretch injury resulted in more compacted chromatin and DNA damage, suggesting potential long-term genomic instabilities in stretched microglia. Using compartmentalized microfluidic chambers with neuronal networks, we observed that stretched microglial cells exhibited enhanced phagocytic and synaptic stripping activities. These findings collectively suggest that stretching events can unlock the immune potential of microglial cells, contributing to the maintenance of brain tissue homeostasis following mechanical injury.

Early Inhibition of Phosphodiesterase 4B (PDE4B) Instills Cognitive Resilience in APPswe/PS1dE9 Mice.

Microglia activity can drive excessive synaptic loss during the prodromal phase of Alzheimer's disease (AD) and is associated with lowered cyclic adenosine monophosphate (cAMP) due to cAMP phosphodiesterase 4B (PDE4B). This study aimed to investigate whether long-term inhibition of PDE4B by A33 (3 mg/kg/day) can prevent synapse loss and its associated cognitive decline in APPswe/PS1dE9 mice. This model is characterized by a chimeric mouse/human APP with the Swedish mutation and human PSEN1 lacking exon 9 (dE9), both under the control of the mouse prion protein promoter. The effects on cognitive function of prolonged A33 treatment from 20 days to 4 months of age, was assessed at 7-8 months. PDE4B inhibition significantly improved both the working and spatial memory of APPswe/PSdE9 mice after treatment ended. At the cellular level, in vitro inhibition of PDE4B induced microglial filopodia formation, suggesting that regulation of PDE4B activity can counteract microglia activation. Further research is needed to investigate if this could prevent microglia from adopting their 'disease-associated microglia (DAM)' phenotype in vivo. These findings support the possibility that PDE4B is a potential target in combating AD pathology and that early intervention using A33 may be a promising treatment strategy for AD.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

Novel maternal autoantibodies in autism spectrum disorder: Implications for screening and diagnosis.

Introduction: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which early recognition is a major challenge. Autoantibodies against fetal brain antigens have been found in the blood of mothers of children with ASD (m-ASD) and can be transferred to the fetus where they can impact neurodevelopment by binding to fetal brain proteins. This study aims to identify novel maternal autoantibodies reactive against human fetal brain antigens, and explore their use as biomarkers for ASD screening and diagnosis. Methods: A custom-made human fetal brain cDNA phage display library was constructed, and screened for antibody reactivity in m-ASD samples from the Simons Simplex Collection (SSC) of the Simons Foundation Autism Research Initiative (SFARI). Antibody reactivity against 6 identified antigens was determined in plasma samples of 238 m-ASD and 90 mothers with typically developing children (m-TD). Results: We identified antibodies to 6 novel University Hasselt (UH)-ASD antigens, including three novel m-ASD autoantigens, i.e., ribosomal protein L23 (RPL23), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calmodulin-regulated spectrin-associated protein 3 (CAMSAP3). Antibody reactivity against a panel of four of these targets was found in 16% of m-ASD samples, compared to 4% in m-TD samples (p = 0.0049). Discussion: Maternal antibodies against 4 UH-ASD antigens could therefore provide a novel tool to support the diagnosis of ASD in a subset of individuals.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals.

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity. We next show that the locomotor response to d-amphetamine is enhanced in GlyRα2 knockout animals, and that this increase correlates with c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the neuronal ensemble size in the striatum in response to D-amphetamine in GlyRα2 KO mice. Finally, we show enhanced appetitive conditioning in GlyRα2 KO animals that is likely due to increased motivation, but not changes in associative learning or hedonic response. Taken together, we show that GlyRα2 is an important regulator of dopamine-stimulated striatal activity and function.

Experimental early-life febrile seizures induce changes in GABA(A) R-mediated neurotransmission in the dentate gyrus.

PURPOSE: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ-aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA(A) R function in dentate granule cells. METHODS: We applied an immature rat model of hyperthermia (HT)-induced FS. GABA(A) R-mediated neurotransmission was studied using whole-cell patch-clamp recordings from dentate granule neurons in hippocampal slices within 6-9 days post-HT. KEY FINDINGS: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole-cell GABA responses revealed a doubled GABA(A) R sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole-cell GABA responses showed similar kinetics in postsynaptic GABA(A) Rs of HT and NT rats. quantitative real-time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA(A) R subunit expression, which was most pronounced for the α3 subunit. SIGNIFICANCE: The data support the hypothesis that FS persistently alter neuronal excitability.

Tricyclic 3,4-dihydropyrimidine-2-thione derivatives as potent TRPA1 antagonists.

The transient receptor potential A1 (TRPA1) channel has been implicated in a number of inflammatory and nociceptive processes, and antagonists of the TRPA1 receptor could offer a potential treatment for conditions such as inflammatory or neuropathic pain, airway disorders, and itch. In a high throughput screen aimed at the identification of TRPA1 antagonists, 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-indeno[1,2-d]pyrimidin-5-one (1) was identified as a potent TRPA1 receptor antagonist. A series of analogous tricyclic 3,4-dihydropyrimidine-2-thiones has been prepared via the multi-component Biginelli reaction and subsequent derivatization. This has led to TRPA1 antagonists with potencies around 10nM for both rat and human derived TRPA1 receptors. The activity was shown to reside exclusively in the 4R-enantiomers.

Non-invasive brain stimulation as therapeutic approach for ischemic stroke: Insights into the (sub)cellular mechanisms.

Although spontaneous recovery can occur following ischemic stroke due to endogenous neuronal reorganization and neuroplastic events, the degree of functional improvement is highly variable, causing many patients to remain permanently impaired. In the last decades, non-invasive brain stimulation (NIBS) techniques have emerged as potential add-on interventions to the standard neurorehabilitation programs to improve post-stroke recovery. Due to their ability to modulate cortical excitability and to induce neuroreparative processes in the brain, multiple studies have assessed the safety, efficacy and (sub)cellular mechanisms of NIBS following ischemic stroke. In this review, an overview will be provided of the different NIBS techniques that are currently being investigated in (pre)clinical stroke studies. The NIBS therapies that will be discussed include transcranial magnetic stimulation, transcranial direct current stimulation and extremely low frequency electromagnetic stimulation. First, an overview will be given of the cellular mechanisms induced by NIBS that are associated with enhanced stroke outcome in preclinical models. Furthermore, the current knowledge on safety and efficacy of these NIBS techniques in stroke patients will be reviewed.