Neuroprotective Effects of Cannabispirenone A against NMDA-Induced Excitotoxicity in Differentiated N2a Cells.

The endocannabinoid system is found throughout the central nervous system, and its cannabinoids receptor 1 is critical in preventing neurotoxicity caused by N-methyl-D-aspartate receptor activation (NMDARs). The activity of NMDARs places demands on endogenous cannabinoids to regulate their calcium currents. Endocannabinoids keep NMDAR activity within safe limits, protecting neural cells from excitotoxicity. Cannabinoids are remembered to deliver this outcome by repressing presynaptic glutamate discharge or obstructing postsynaptic NMDAR-managed flagging pathways. The endocannabinoid system must exert a negative influence proportional to the strength of NMDAR signaling for such control to be effective. The goal of this paper is to draw the attention towards the neuroprotective mechanism of constituents of Cannabis sativa against NMDA-induced excitotoxic result. Phytochemical investigation of the cannabis flowers led to the isolation of nine secondary metabolites. A spiro-compound, Cannabispirenone A, which on treatment of the cells prior to NMDA exposure significantly increases cell survival while decreasing ROS production, lipid peroxidation, and intracellular calcium. Our findings showed that this compound showed neuroprotection against NMDA-induced excitotoxic insult, has antioxidative properties, and increased cannabinoid receptor 1 expression, which may be involved in the signaling pathway for this neuroprotection.

GluN2A- and GluN2B-containing pre-synaptic N-methyl-d-aspartate receptors differentially regulate action potential-evoked Ca2+ influx via modulation of SK channels.

N-methyl-d-aspartate receptors (NMDARs) play a pivotal role in synaptic plasticity. While the functional role of post-synaptic NMDARs is well established, pre-synaptic NMDAR (pre-NMDAR) function is largely unexplored. Different pre-NMDAR subunit populations are documented at synapses, suggesting that subunit composition influences neuronal transmission. Here, we used electrophysiological recordings at Schaffer collateral-CA1 synapses partnered with Ca2+ imaging and glutamate uncaging at boutons of CA3 pyramidal neurones to reveal two populations of pre-NMDARs that contain either the GluN2A or GluN2B subunit. Activation of the GluN2B population decreases action potential-evoked Ca2+ influx via modulation of small-conductance Ca2+-activated K+ channels, while activation of the GluN2A population does the opposite. Critically, the level of functional expression of the subunits is subject to homeostatic regulation, bidirectionally affecting short-term facilitation, thus providing a capacity for a fine adjustment of information transfer. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Enhanced long-term potentiation in the anterior cingulate cortex of tree shrew.

Synaptic plasticity is a key cellular model for learning, memory and chronic pain. Most previous studies were carried out in rats and mice, and less is known about synaptic plasticity in non-human primates. In the present study, we used integrative experimental approaches to study long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of adult tree shrews. We found that glutamate is the major excitatory transmitter and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) receptors mediate postsynaptic responses. LTP in tree shrews was greater than that in adult mice and lasted for at least 5 h. N-methyl-d-aspartic acid (NMDA) receptors, Ca2+ influx and adenylyl cyclase 1 (AC1) contributed to tree shrew LTP. Our results suggest that LTP is a major form of synaptic plasticity in the ACC of primate-like animals. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Src dependency of the regulation of LTP by alternative splicing of GRIN1 exon 5.

Alternative splicing of Grin1 exon 5 regulates induction of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses: LTP in mice lacking the GluN1 exon 5-encoded N1 cassette (GluN1a mice) is significantly increased compared with that in mice compulsorily expressing this exon (GluN1b mice). The mechanism underlying this difference is unknown. Here, we report that blocking the non-receptor tyrosine kinase Src prevents induction of LTP in GluN1a mice but not in GluN1b. We find that activating Src enhances pharmacologically isolated synaptic N-methyl-d-aspartate receptor (NMDAR) currents in GluN1a mice but not in GluN1b. Moreover, we observe that Src activation increases the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor component of Schaffer collateral-evoked excitatory post-synaptic potentials in GluN1a mice, but this increase is prevented by blocking NMDARs. We conclude that at these synapses, NMDARs in GluN1a mice are subject to upregulation by Src that mediates induction of LTP, whereas NMDARs in GluN1b mice are not regulated by Src, leading to Src-resistance of LTP. Thus, we have uncovered that a key regulatory mechanism for synaptic potentiation is gated by differential splicing of exon 5 of Grin1. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Decoding frontotemporal and cell-type-specific vulnerabilities to neuropsychiatric disorders and psychoactive drugs.

Frontotemporal lobe abnormalities are linked to neuropsychiatric disorders and cognition, but the role of cellular heterogeneity between temporal lobe (TL) and frontal lobe (FL) in the vulnerability to genetic risk factors remains to be elucidated. We integrated single-nucleus transcriptome analysis in 'fresh' human FL and TL with genetic susceptibility, gene dysregulation in neuropsychiatric disease and psychoactive drug response data. We show how intrinsic differences between TL and FL contribute to the vulnerability of specific cell types to both genetic risk factors and psychoactive drugs. Neuronal populations, specifically PVALB neurons, were most highly vulnerable to genetic risk factors for psychiatric disease. These psychiatric disease-associated genes were mostly upregulated in the TL, and dysregulated in the brain of patients with obsessive-compulsive disorder, bipolar disorder and schizophrenia. Among these genes, GRIN2A and SLC12A5, implicated in schizophrenia and bipolar disorder, were significantly upregulated in TL PVALB neurons and in psychiatric disease patients' brain. PVALB neurons from the TL were twofold more vulnerable to psychoactive drugs than to genetic risk factors, showing the influence and specificity of frontotemporal lobe differences on cell vulnerabilities. These studies provide a cell type resolved map of the impact of brain regional differences on cell type vulnerabilities in neuropsychiatric disorders.

Risk of Seizure Recurrence Due to Autoimmune Encephalitis With NMDAR, LGI1, CASPR2, and GABABR Antibodies: Implications for Return to Driving.

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.

Anti-NMDAR encephalitis in a child with long impaired consciousness and persistent antibodies: a case report and mini review.

We described a challenging case of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in a young girl. Despite enduring months of reduced consciousness with ongoing antibody presence, she ultimately exhibited remarkable improvement within a 5-year follow-up period. Additionally, we conducted a concise review of relevant literature on anti-NMDAR encephalitis, with a specific focus on anti-NMDAR antibodies. Our findings enhance the clinical comprehension of anti-NMDAR encephalitis and offer valuable insights to clinicians for its management.

Promiscuous involvement of metabotropic glutamate receptors in the storage of N-methyl-d-aspartate receptor-dependent short-term potentiation.

Short- and long-term forms of N-methyl-d-aspartate receptor (NMDAR)-dependent potentiation (most commonly termed short-term potentiation (STP) and long-term potentiation (LTP)) are co-induced in hippocampal slices by theta-burst stimulation, which mimics naturally occurring patterns of neuronal activity. While NMDAR-dependent LTP (NMDAR-LTP) is said to be the cellular correlate of long-term memory storage, NMDAR-dependent STP (NMDAR-STP) is thought to underlie the encoding of shorter-lasting memories. The mechanisms of NMDAR-LTP have been researched much more extensively than those of NMDAR-STP, which is characterized by its extreme stimulation dependence. Thus, in the absence of low-frequency test stimulation, which is used to test the magnitude of potentiation, NMDAR-STP does not decline until the stimulation is resumed. NMDAR-STP represents, therefore, an inverse variant of Hebbian synaptic plasticity, illustrating that inactive synapses can retain their strength unchanged until they become active again. The mechanisms, by which NMDAR-STP is stored in synapses without a decrement, are unknown and we report here that activation of metabotropic glutamate receptors may be critical in maintaining the potentiated state of synaptic transmission. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

The N-methyl-d-aspartate receptor hypothesis of ketamine's antidepressant action: evidence and controversies.

Substantial clinical evidence has unravelled the superior antidepressant efficacy of ketamine: in comparison to traditional antidepressants targeting the monoamine systems, ketamine, as an N-methyl-d-aspartate receptor (NMDAR) antagonist, acts much faster and more potently. Surrounding the antidepressant mechanisms of ketamine, there is ample evidence supporting an NMDAR-antagonism-based hypothesis. However, alternative arguments also exist, mostly derived from the controversial clinical results of other NMDAR inhibitors. In this article, we first summarize the historical development of the NMDAR-centred hypothesis of rapid antidepressants. We then classify different NMDAR inhibitors based on their mechanisms of inhibition and evaluate preclinical as well as clinical evidence of their antidepressant effects. Finally, we critically analyse controversies and arguments surrounding ketamine's NMDAR-dependent and NMDAR-independent antidepressant action. A better understanding of ketamine's molecular targets and antidepressant mechanisms should shed light on the future development of better treatment for depression. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Incremental induction of NMDAR-STP and NMDAR-LTP in the CA1 area of ventral hippocampal slices relies on graded activation of discrete NMDA receptors.

N-methyl-d-aspartate receptor (NMDAR)-dependent short- and long-term types of potentiation (STP and LTP, respectively) are frequently studied in the CA1 area of dorsal hippocampal slices (DHS). Far less is known about the NMDAR dependence of STP and LTP in ventral hippocampal slices (VHS), where both types of potentiation are smaller in magnitude than in the DHS. Here, we first briefly review our knowledge about the NMDAR dependence of STP and LTP and some other forms of synaptic plasticity. We then show in new experiments that the decay of NMDAR-STP in VHS, similar to dorsal hippocampal NMDAR-STP, is not time- but activity-dependent. We also demonstrate that the induction of submaximal levels of NMDAR-STP and NMDAR-LTP in VHS differs from the induction of saturated levels of plasticity in terms of their sensitivity to subunit-preferring NMDAR antagonists. These data suggest that activation of distinct NMDAR subtypes in a population of neurons results in an incremental increase in the induction of different phases of potentiation with changing sensitivity to pharmacological agents. Differences in pharmacological sensitivity, which arise due to differences in the levels of agonist-evoked biological response, might explain the disparity of the results concerning NMDAR subunit involvement in the induction of NMDAR-dependent plasticity.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

H-intensity scale score to estimate CSF GluN1 antibody titers with one-time immunostaining using a commercial assay.

Introduction: Anti-NMDA receptor encephalitis is an autoimmune disorder caused by autoantibodies (abs) against the conformational epitope on GluN1 subunits. GluN1-abs have been determined with cell-based assay (CBA) co-expressing GluN1/GluN2 subunits. However, commercial fixed CBA expressing only GluN1 subunit has increasingly been used in clinical practice. The ab titers can be determined with serial dilutions, but its clinical significance remains unclear. We aimed to develop an H-intensity scale (HIS) score to estimate GluN1-ab titers in cerebrospinal fluid (CSF) with one-time immunostaining using both commercial CBA and immunohistochemistry and report its usefulness. "H" is the initial of a patient with high CSF GluN1-ab titers (1:2,048). Methods: We first determined the reliability of CBA in 370 patients with suspected autoimmune encephalitis by comparing the results between commercial CBA and established assay in Dalmau's Lab. Then, we made positive control panels using the patient H's CSF diluted in a fourfold serial dilution method (1:2, 1:8, 1:32, 1:128, 1:512, and 1:2,048). Based on the panels, we scored the intensity of ab reactivity of 79 GluN1-ab-positive patients' CSF (diluted at 1:2) on a scale from 0 to 6 (with ≥1 considered positive). To assess inter-assay reliability, we performed immunostaining twice in 21 patients' CSF. We investigated an association between the score of CSF obtained at diagnosis and the clinical/paraclinical features. Results: The sensitivity and specificity of CBA were 93.7% (95% CI: 86.0-97.3) and 98.6% (95% CI: 96.5-99.5), respectively. Linear regression analysis showed a good agreement between the scores of the first and second assays. Patients with a typical spectrum, need for mechanical ventilation support, autonomic symptoms/central hypoventilation, dyskinesias, speech dysfunction, decreased level of consciousness, preceding headache, ovarian teratoma, and CSF leukocyte count >20 cells/µL had a higher median HIS score than those without, but HIS score was not associated with sex, age at onset, or seizure. HIS score at diagnosis had a significant effect on 1-year functional status. Discussion: The severity of disease and four of the six core symptoms were associated with higher GluN1-ab titers in CSF at diagnosis, which may play a role in poor 1-year functional status. An incomplete phenotype can be attributed to low CSF GluN1-ab titers.

Spectrum of NMDA Receptor Variants in Neurodevelopmental Disorders and Epilepsy.

N-methyl-D-aspartate receptors (NMDAR) are ligand-gated ion channels mediating excitatory neurotransmission and are important for normal brain development, cognitive abilities, and motor functions. Pathogenic variants in the Glutamate receptor Ionotropic N-methyl-D-aspartate (GRIN) genes (GRIN1, GRIN2A-D) encoding NMDAR subunits have been associated with a wide spectrum of neurodevelopmental disorders and epilepsies ranging from treatable focal epilepsies to devastating early-onset developmental and epileptic encephalopathies. Genetic variants in NMDA receptor genes can cause a range of complex alterations to receptor properties resulting in various degrees of loss-of-function, gain-of-function, or mixtures thereof. Understanding how genetic variants affect the function of the receptors, therefore, represents an important first step in the ongoing development towards targeted therapies. Currently, targeted treatment options for GRIN-related diseases are limited. However, treatment with memantine has been reported to significantly reduce seizure frequency in a few individuals with developmental and epileptic encephalopathies harboring de novo gain-of-function GRIN2A missense variants, and supplementary treatment with L-serine has been associated with improved motor and cognitive performance as well as reduced seizure frequency in patients with GRIN2B loss-of-function missense variants as well as GRIN2A and GRIN2B null variants.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

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.

Functional Analysis of NMDAR Subunit Components in Postsynaptic Currents of Identified Cells and Synapses in Brain Slices.

Epilepsy is one of the most represented neurological diseases worldwide. However, in many cases, the precise molecular mechanisms of epileptogenesis and ictiogenesis are unknown. Because of their important role in synaptic function and neuronal excitability, NMDA receptors are implicated in various epileptogenic mechanisms. Most of these are subunit specific and require a precise analysis of the subunit composition of the NMDARs implicated. Here, we describe an express electrophysiological method to analyze the contribution of NMDAR subunits to spontaneous postsynaptic activity in identified cells in brain slices using patch clamp whole cell recordings.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Behavioral Analysis of NMDAR Function in Rodents: Tests of Long-Term Spatial Memory.

NMDAR-dependent forms of synaptic plasticity in brain regions like the hippocampus are widely believed to provide the neural substrate for long-term associative memory formation. However, the experimental data are equivocal at best and may suggest a more nuanced role for NMDARs and synaptic plasticity in memory. Much of the experimental data available comes from studies in genetically modified mice in which NMDAR subunits have been deleted or mutated in order to disrupt NMDAR function. Behavioral assessment of long-term memory in these mice has involved tests like the Morris watermaze and the radial arm maze. Here we describe these behavioral tests and some of the different testing protocols that can be used to assess memory performance. We discuss the importance of distinguishing selective effects on learning and memory processes from nonspecific effects on sensorimotor or motivational aspects of performance.

Estimating the Ca2+ Permeability of NMDA Receptors with Whole-Cell Patch-Clamp Electrophysiology.

In the mammalian central nervous system (CNS), fast excitatory transmission relies primarily on the ionic fluxes generated by ionotropic glutamate receptors (iGluRs). Among iGluRs, NMDA receptors (NMDARs) are unique in their ability to pass large, Ca2+-rich currents. Importantly, their high Ca2+ permeability is essential for normal CNS function and is under physiological control. For this reason, the accurate measurement of NMDA receptor Ca2+ permeability represents a valuable experimental step in evaluating the mechanism by which these receptors contribute to a variety of physiological and pathological conditions. In this chapter, we provide a theoretical and practical overview of the common methods used to estimate the Ca2+ permeability of ion channels as they apply to NMDA receptors. Specifically, we describe the principles and methodology used to calculate relative permeability (PCa/PNa) and fractional permeability (Pf), along with the relationship between these two metrics. With increasing knowledge about the structural dynamics of ion channels and of the ongoing environmental fluctuations in which channels operate in vivo, the ability to quantify the Ca2+ entering cells through specific ion channels remains a tool essential to delineating the molecular mechanisms that support health and cause disease.

Analysis of Functional NMDA Receptors in Astrocytes.

Neuronal N-methyl-D-aspartate (NMDA) receptors are well known for their pivotal role in memory formation. Originally, they were thought to be exclusive to neurons. However, numerous studies revealed their functional expression also on various types of glial cells in the nervous system. Here, the methodology on how to study the physiology of NMDA receptors selectively on astrocytes will be described in detail. Astrocytes are the main class of neuroglia that control transmitter and ion homeostasis, which link cerebral blood flow and neuronal energy demands, but also affect synaptic transmission directly.