Inhibition of NMDA receptors through a membrane-to-channel path.

N-methyl-D-aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer's disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration ("membrane-to-channel inhibition" (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism.

Portable Neuromonitoring Device Detects Large Vessel Occlusion in Suspected Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Early detection of large vessel occlusion (LVO) stroke optimizes endovascular therapy and improves outcomes. Clinical stroke severity scales used for LVO identification have variable accuracy. We investigated a portable LVO-detection device (PLD), using electroencephalography and somatosensory-evoked potentials, to identify LVO stroke. METHODS: We obtained PLD data in suspected patients with stroke enrolled prospectively via a convenience sample in 8 emergency departments within 24 hours of symptom onset. LVO discriminative signals were integrated into a binary classifier. The National Institutes of Health Stroke Scale was documented, and 4 prehospital stroke scales were retrospectively calculated. We compared PLD and scale performance to diagnostic neuroimaging. RESULTS: Of 109 patients, there were 25 LVO (23%), 38 non-LVO ischemic (35%), 14 hemorrhages (13%), and 32 stroke mimics (29%). The PLD had higher sensitivity (80% [95% CI, 74-85]) and similar specificity (80% [95% CI, 77-83]) to all prehospital scales at their predetermined high probability LVO thresholds. The PLD had high discrimination for LVO (C-statistic=0.88). CONCLUSIONS: The PLD identifies LVO with superior accuracy compared with prehospital stroke scales in emergency department suspected stroke. Future studies need to validate the PLD's potential as an LVO triage aid in prehospital undifferentiated stroke populations.

Effects of Mg2+ on recovery of NMDA receptors from inhibition by memantine and ketamine reveal properties of a second site.

Memantine and ketamine are NMDA receptor (NMDAR) open channel blockers that are thought to act via similar mechanisms at NMDARs, but exhibit divergent clinical effects. Both drugs act by entering open NMDARs and binding at a site deep within the ion channel (the deep site) at which the endogenous NMDAR channel blocker Mg2+ also binds. Under physiological conditions, Mg2+ increases the IC50s of memantine and ketamine through competition for binding at the deep site. Memantine also can inhibit NMDARs after associating with a second site accessible in the absence of agonist, a process termed second site inhibition (SSI) that is not observed with ketamine. Here we investigated the effects of 1 mM Mg2+ on recovery from inhibition by memantine and ketamine, and on memantine SSI, of the four main diheteromeric NMDAR subtypes. We found that: recovery from memantine inhibition depended strongly on the concentration of memantine used to inhibit the NMDAR response; Mg2+ accelerated recovery from memantine and ketamine inhibition through distinct mechanisms and in an NMDAR subtype-dependent manner; and Mg2+ occupation of the deep site disrupted memantine SSI in a subtype-dependent manner. Our results support the hypothesis that memantine associates with, but does not inhibit at the second site. After associating with the second site, memantine can either slowly dissociate directly to the extracellular solution, or transit to the deep site, resulting in typical channel block. Memantine's relatively slow dissociation from the second site underlies the dependence of NMDAR recovery from inhibition on both memantine concentration and on Mg2+.

New Cav2 calcium channel gating modifiers with agonist activity and therapeutic potential to treat neuromuscular disease.

Voltage-gated calcium channels (VGCCs) are critical regulators of many cellular functions, including the activity-dependent release of chemical neurotransmitter from nerve terminals. At nerve terminals, the Cav2 family of VGCCs are closely positioned with neurotransmitter-containing synaptic vesicles. The relationship between calcium ions and transmitter release is such that even subtle changes in calcium flux through VGCCs have a strong influence on the magnitude of transmitter released. Therefore, modulators of the calcium influx at nerve terminals have the potential to strongly affect transmitter release at synapses. We have previously developed novel Cav2-selective VGCC gating modifiers (notably GV-58) that slow the deactivation of VGCC current, increasing total calcium ion flux. Here, we describe ten new gating modifiers based on the GV-58 structure that extend our understanding of the structure-activity relationship for this class of molecules and extend the range of modulation of channel activities. In particular, we show that one of these new compounds (MF-06) was more efficacious than GV-58, another (KK-75) acts more quickly on VGCCs than GV-58, and a third (KK-20) has a mix of increased speed and efficacy. A subset of these new VGCC agonist gating modifiers can increase transmitter release during action potentials at neuromuscular synapses, and as such, show potential as therapeutics for diseases with a presynaptic deficit that results in neuromuscular weakness. Further, several of these new compounds can be useful tool compounds for the study of VGCC gating and function.

A versatile optical tool for studying synaptic GABAA receptor trafficking.

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP-MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.

Low-Density Neuronal Cultures from Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented possibility to investigate defects occurring during neuronal differentiation in neuropsychiatric and neurodevelopmental disorders, but the density and intricacy of intercellular connections in neuronal cultures challenge currently available analytic methods. Low-density neuronal cultures facilitate the morphometric and functional analysis of neurons. We describe a differentiation protocol to generate low-density neuronal cultures (∼2,500 neurons/cm2) from human iPSC-derived neural stem cells/early neural progenitor cells. We generated low-density cultures using cells from 3 individuals. We also evaluated the morphometric features of neurons derived from 2 of these individuals, one harboring a microdeletion on chromosome 15q11.2 and the other without the microdeletion. An approximately 7.5-fold increase in the density of dendritic filopodia was observed in the neurons with the microdeletion, consistent with previous reports. Low-density neuronal cultures enable facile and unbiased comparisons of iPSC-derived neurons from different individuals or clones.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.