BACKGROUND AND OBJECTIVES: Patients with ongoing seizures are usually not allowed to drive. The prognosis for seizure freedom is favorable in patients with autoimmune encephalitis (AIE) with antibodies against NMDA receptor (NMDAR), leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein-like 2 (CASPR2), and the gamma-aminobutyric-acid B receptor (GABABR). We hypothesized that after a seizure-free period of 3 months, patients with AIE have a seizure recurrence risk of <20% during the subsequent 12 months. This would render them eligible for noncommercial driving according to driving regulations in several countries. METHODS: This retrospective multicenter cohort study analyzed follow-up data from patients aged 15 years or older with seizures resulting from NMDAR-, LGI1-, CASPR2-, or GABABR-AIE, who had been seizure-free for ≥3 months. We used Kaplan-Meier (KM) estimates for the seizure recurrence risk at 12 months for each antibody group and tested for the effects of potential covariates with regression models. RESULTS: We included 383 patients with NMDAR-, 440 with LGI1-, 114 with CASPR2-, and 44 with GABABR-AIE from 14 international centers. After being seizure-free for 3 months after an initial seizure period, we calculated the probability of remaining seizure-free for another 12 months (KM estimate) as 0.89 (95% confidence interval [CI] 0.85-0.92) for NMDAR, 0.84 (CI 0.80-0.88) for LGI1, 0.82 (CI 0.75-0.90) for CASPR2, and 0.76 (CI 0.62-0.93) for GABABR. DISCUSSION: Taking a <20% recurrence risk within 12 months as sufficient, patients with NMDAR-AIE and LGI1-AIE could be considered eligible for noncommercial driving after having been seizure-free for 3 months.

• MeSH
• autoprotilátky * krev   MeSH
• dospělí MeSH
• encefalitida * imunologie   MeSH
• Hashimotova nemoc imunologie   krev   MeSH
• intracelulární signální peptidy a proteiny * imunologie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové proteiny * imunologie   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• následné studie MeSH
• proteiny nervové tkáně * imunologie   MeSH
• proteiny imunologie   MeSH
• receptory GABA-B * imunologie   MeSH
• receptory N-methyl-D-aspartátu imunologie   MeSH
• recidiva * MeSH
• retrospektivní studie MeSH
• senioři MeSH
• záchvaty etiologie   imunologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Názvy látek
• autoprotilátky * MeSH
• CNTNAP2 protein, human MeSH Prohlížeč
• intracelulární signální peptidy a proteiny * MeSH
• LGI1 protein, human MeSH Prohlížeč
• membránové proteiny * MeSH
• proteiny nervové tkáně * MeSH
• proteiny MeSH
• receptory GABA-B * MeSH
• receptory N-methyl-D-aspartátu MeSH

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

• Klíčová slova
• Colocalization, Fluorescence immunostaining, Fluorescence microscopy, Glutamate receptor, ImageJ analysis, Lentivirus, Primary hippocampal neurons, Surface expression,
• MeSH
• exprese genu MeSH
• hipokampus * metabolismus   cytologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• neurony * metabolismus   MeSH
• podjednotky proteinů metabolismus   genetika   MeSH
• primární buněčná kultura metody   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• podjednotky proteinů MeSH
• receptory N-methyl-D-aspartátu * MeSH

Tacrine (THA), a long withdrawn drug, is still a popular scaffold used in medicinal chemistry, mainly for its good reactivity and multi-targeted effect. However, THA-associated hepatotoxicity is still an issue and must be considered in drug discovery based on the THA scaffold. Following our previously identified hit compound 7-phenoxytacrine (7-PhO-THA), we systematically explored the chemical space with 30 novel derivatives, with a focus on low hepatotoxicity, anticholinesterase action, and antagonism at the GluN1/GluN2B subtype of the NMDA receptor. Applying the down-selection process based on in vitro and in vivo pharmacokinetic data, two candidates, I-52 and II-52, selective GluN1/GluN2B inhibitors thanks to the interaction with the ifenprodil-binding site, have entered in vivo pharmacodynamic studies. Finally, compound I-52, showing only minor affinity to AChE, was identified as a lead candidate with favorable behavioral and neuroprotective effects using open-field and prepulse inhibition tests, along with scopolamine-based behavioral and NMDA-induced hippocampal lesion models. Our data show that compound I-52 exhibits low toxicity often associated with NMDA receptor ligands, and low hepatotoxicity, often related to THA-based compounds.

• Klíčová slova
• Acetylcholinesterase, Alzheimer's disease, Electrophysiology, Glutamate receptor, In vivo, Neuroprotection, Tacrine,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• Alzheimerova nemoc * farmakoterapie   MeSH
• cholinesterasové inhibitory chemie   MeSH
• cholinesterasy MeSH
• lékové postižení jater * MeSH
• lidé MeSH
• neuroprotektivní látky * farmakologie   terapeutické užití   MeSH
• piperidiny * MeSH
• receptory N-methyl-D-aspartátu MeSH
• takrin chemie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• cholinesterasové inhibitory MeSH
• cholinesterasy MeSH
• ifenprodil MeSH Prohlížeč
• neuroprotektivní látky * MeSH
• piperidiny * MeSH
• receptory N-methyl-D-aspartátu MeSH
• takrin MeSH

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

• Klíčová slova
• Channelopathy, Endogenous neuroactive steroid, GRIN2 genes, Glutamate receptors, Rescue pharmacology, Surface expression,
• MeSH
• elektrofyziologické jevy MeSH
• lidé MeSH
• neurosteroidy * MeSH
• receptory N-methyl-D-aspartátu * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• neurosteroidy * MeSH
• NR2B NMDA receptor MeSH Prohlížeč
• receptory N-methyl-D-aspartátu * MeSH

Neonatal hypoxic-ischemic encephalopathy (HIE) is a disease caused by insufficient blood supply in the brain in newborns during the perinatal period. Severe HIE leads to patient death, and patients with mild HIE are at increased risk of cognitive deficits and behavioral abnormalities. The NMDA receptor is an important excitatory receptor in the central nervous system, and in adult hypoxic-ischemic injury both subtypes of the NMDA receptor play important but distinct roles. The GluN2A-containing NMDA receptor (GluN2A-NMDAR) could activate neuronal protective signaling pathway, while the GluN2B-NMDAR subtype is coupled to the apoptosis-inducing signaling pathway and leads to neuronal death. However, the expression level of GluN2B is higher in newborns than in adults, while the expression of GluN2A is lower. Therefore, it is not clear whether the roles of different NMDA receptor subtypes in HIE are consistent with those in adults. We investigated this issue in this study and found that in HIE, GluN2B plays a protective role by mediating the protective pathway through binding with PSD95, which is quite different to that in adults. The results of this study provided new theoretical support for the clinical treatment of neonatal hypoxic ischemia.

• MeSH
• apoptóza MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• ischemie MeSH
• lidé MeSH
• mozková hypoxie a ischemie * metabolismus   MeSH
• novorozenec MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• signální transdukce MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfatidylinositol-3-kinasy MeSH
• NR2B NMDA receptor MeSH Prohlížeč
• protoonkogenní proteiny c-akt MeSH
• receptory N-methyl-D-aspartátu MeSH

Organophosphorus nerve agents pose a global threat to both military personnel and civilian population, because of their high acute toxicity and insufficient medical countermeasures. Commonly used drugs could ameliorate the intoxication and overall medical outcomes. In this study, we tested the drugs able to alleviate the symptoms of Alzheimer's disease (donepezil, huperzine A, memantine) or Parkinson's disease (procyclidine). They were administered to mice before soman intoxication in terms of their: i) protection potential against soman toxicity and ii) influence on post-exposure therapy consisting of atropine and asoxime (also known as oxime HI-6). Their pretreatment effect was not significant, when administered alone, but in combination (acetylcholinesterase inhibitor such as denepezil or huperzine A with NMDA antagonist such as memantine or procyclidine) they lowered the soman toxicity more than twice. These combinations also positively influenced the efficacy of post-exposure treatment in a similar fashion; the combinations increased the therapeutic effectiveness of antidotal treatment. In conclusion, the most effective combination - huperzine A and procyclidine - lowered the toxicity three times and improved the post-exposure therapy efficacy more than six times. These results are unprecedented in the published literature.

• Klíčová slova
• HI-6, Soman, atropine, donepezil, huperzine A, memantine, mice, procyclidine,
• MeSH
• acetylcholinesterasa metabolismus   MeSH
• antidota terapeutické užití   MeSH
• atropin terapeutické užití   farmakologie   MeSH
• cholinesterasové inhibitory toxicita   MeSH
• jedy * MeSH
• memantin terapeutické užití   MeSH
• míra přežití MeSH
• myši MeSH
• oximy terapeutické užití   farmakologie   MeSH
• procyklidin farmakologie   MeSH
• pyridinové sloučeniny farmakologie   MeSH
• receptory N-methyl-D-aspartátu MeSH
• soman * toxicita   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcholinesterasa MeSH
• antidota MeSH
• atropin MeSH
• cholinesterasové inhibitory MeSH
• huperzine A MeSH Prohlížeč
• jedy * MeSH
• memantin MeSH
• oximy MeSH
• procyklidin MeSH
• pyridinové sloučeniny MeSH
• receptory N-methyl-D-aspartátu MeSH
• soman * MeSH

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

• Klíčová slova
• GluN subunit, excitatory synapse, glutamate receptor, lateral diffusion, live microscopy, mammalian neuron,
• MeSH
• hipokampus metabolismus   MeSH
• imunoglobulin G metabolismus   MeSH
• jednodoménové protilátky * metabolismus   MeSH
• králíci MeSH
• krysa rodu Rattus MeSH
• neurony metabolismus   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   MeSH
• savci MeSH
• synapse fyziologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobulin G MeSH
• jednodoménové protilátky * MeSH
• receptory N-methyl-D-aspartátu * MeSH

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5β-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABAA and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5β-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5β-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5β-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

• Klíčová slova
• glutamate, negative allosteric modulator, steroid, thigmotaxis, zebrafish,
• MeSH
• dánio pruhované MeSH
• estery MeSH
• GABA MeSH
• krysa rodu Rattus MeSH
• kyselina glutamová MeSH
• pregnanolon * farmakologie   chemie   MeSH
• receptory GABA-A MeSH
• receptory N-methyl-D-aspartátu * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• estery MeSH
• GABA MeSH
• kyselina glutamová MeSH
• pregnanolon * MeSH
• receptory GABA-A MeSH
• receptory N-methyl-D-aspartátu * MeSH

Impairments of N-methyl-D-aspartate receptor (NMDAR) activity have been implicated in several neuropsychiatric disorders, with pharmacological inhibition of NMDAR-mediated currents and associated neurobehavioral changes considered as a model of schizophrenia. We analyzed the effects of brief and long-term exposure of rat cortical cultures to the most prevalent endogenous modulators of NMDAR (kynurenic acid, pregnenolone sulfate, spermidine, and zinc) on neuronal viability, stimulation-induced release of glutamate, and dendritic morphology with synaptic density. Both, glutamate release and neuronal viability studies revealed no difference between the test and control groups. No differences were also observed in the number of dendritic branching and length, or density of synaptic connections and neuronal soma size. Comparison of the extent of dendritic projections and branching patterns, however, revealed enhanced distal arborization with the expansion of the dendritic area under prolonged treatment of cultures with physiological concentrations of NMDAR modulators, with differences reaching significance in spermidine and pregnenolone sulfate tests. Measurements of the density of glutamatergic synapses showed consistency across all neuronal groups, except those treated with pregnenolone sulfate, which showed a reduction of PSD-95-positive elements. Overall, our data suggest that constitutive glutamatergic activity mediated by NMDAR controls the dendritic field expansion and can influence the integrative properties of cortical neurons.

• Klíčová slova
• Cortical thinning, Dendritic morphology, Glutamate release, Intrinsic excitability, Neuroplasticity, Neurosteroids, Polyamines, psd95,
• MeSH
• glutamáty MeSH
• krysa rodu Rattus MeSH
• neurony metabolismus   MeSH
• receptory N-methyl-D-aspartátu * metabolismus   MeSH
• signální transdukce MeSH
• spermidin * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glutamáty MeSH
• receptory N-methyl-D-aspartátu * MeSH
• spermidin * MeSH

Schizophrenia research has increased in recent decades and focused more on its neural basis. Decision-making and cognitive flexibility are the main cognitive functions that are impaired and considered schizophrenia endophenotypes. Cognitive impairment was recently connected with altered functions of N-methyl-d-aspartate (NMDAR) glutamatergic receptors, which increased cortical activity. Selective NMDAR antagonists, such as MK-801, have been used to model cognitive inflexibility in schizophrenia. Decreased GABAergic inhibitory activity has been shown elsewhere with enhanced cortical activity. This imbalance in the excitatory/inhibitory may reduce the entrainment of prefrontal gamma and hippocampal theta rhythms and result in gamma/theta band de-synchronization. The current study established an acute MK-801 administration model of schizophrenia-like cognitive inflexibility in rats and used the attentional set-shifting task in which rats learned to switch/reverse the relevant rule. During the task, we used in vivo optogenetic stimulations of parvalbumin-positive interneurons at specific light pulses in the prefrontal cortex and ventral hippocampus. The first experiments showed that acute dizocilpine in rats produced schizophrenia-like cognitive inflexibility. The second set of experiments demonstrated that specific optogenetic stimulation at specific frequencies of parvalbumin-positive interneurons in the prefrontal cortex and ventral hippocampus rescued the cognitive flexibility rats that received acute MK-801. These findings advance our knowledge of the pivotal role of parvalbumin interneurons in schizophrenia-like cognitive impairment and may guide further research on this severe psychiatric disorder.

• Klíčová slova
• Attentional set-shifting task, Excitatory/inhibitory ratio, Gamma waves, MK-801, theta waves,
• MeSH
• dizocilpinmaleát * farmakologie   MeSH
• hipokampus metabolismus   MeSH
• interneurony metabolismus   MeSH
• kognice MeSH
• krysa rodu Rattus MeSH
• optogenetika MeSH
• parvalbuminy metabolismus   MeSH
• prefrontální mozková kůra metabolismus   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• schizofrenie * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dizocilpinmaleát * MeSH
• parvalbuminy MeSH
• receptory N-methyl-D-aspartátu MeSH