NMDA Receptors

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel's conductance properties and the temporal sequence of open-closed conformations ...

The chemical reactions of NO are largely dictated by its redox state. Increasing evidence suggests that the various redox states of the NO group exist endogenously in biological tissues. In the case of NO+ equivalents, the mechanism of reaction often involves S-nitrosylation (transfer of the NO group to a cysteine sulfhydryl to form an RS-NO); further oxidation of critical thiols can possibly form disulfide bonds. We have physiological and chemical evidence that NMDA receptor activity can be modulated by S-nitrosylation, resulting in a decrease in channel opening. Recent data suggest that NO-, probably in the singlet (or high-energy) state, can also react with critical sulfhydryl group(s) of the NMDA receptor to down-regulate its activity; in the triplet (lower-energy) state NO- may oxidize these NMDA receptor sulfhydryl groups by formation of an intermediate such as peroxynitrite. It has also been reported that NO can react with thiol but only under specific circumstances, e.g., if...

The chemical reactions of NO are largely dictated by its redox state. Increasing evidence suggests that the various redox states of the NO group exist endogenously in biological tissues. In the case of NO+ equivalents, the mechanism of reaction often involves S-nitrosylation (transfer of the NO group to a cysteine sulfhydryl to form an RS-NO); further oxidation of critical thiols can possibly form disulfide bonds. We have physiological and chemical evidence that NMDA receptor activity can be modulated by S-nitrosylation, resulting in a decrease in channel opening. Recent data suggest that NO-, probably in the singlet (or high-energy) state, can also react with critical sulfhydryl group(s) of the NMDA receptor to down-regulate its activity; in the triplet (lower-energy) state NO- may oxidize these NMDA receptor sulfhydryl groups by formation of an intermediate such as peroxynitrite. It has also been reported that NO can react with thiol but only under specific circumstances, e.g., if...

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.

The entorhinal-hippocampal circuit is crucial for several forms of learning and memory, especially sequence learning, including spatial navigation. The challenge is to understand the underlying mechanisms. Pioneering discoveries of spatial selectivity in this circuit, i.e. place cells and grid cells, provided a major step forward in tackling this challenge. Considerable research has also shown that sequence learning relies on synaptic plasticity, especially the Hebbian or the NMDAR-dependent synaptic plasticity. This raises several questions: Are spatial maps plastic? If so, what is the contribution of Hebbian plasticity to spatial map plas-ticity? How does the spatial map plasticity contribute to sequence learn-ing? A combination of computational and experimental studies has shown that NMDAR-mediated plasticity and theta rhythm can have specific effects on the formation and experiential modification of spatial maps to facilitate predictive coding. Advances in transgenic techniques have provided further support for these mechanisms. Although many exciting challenges remain, these findings have brought us closer to solving the puzzle of how the hippocampal system contributes to spatial memory, and point to a way forward. V C 2015 Wiley Periodicals, Inc.