A novel method using ambient glutamate for the electrophysiological quantification of extrasynaptic NMDA receptor function in acute brain slices

Abstract

Key pointsr We present a novel protocol to quantify extrasynaptic NMDA receptor function utilizing the semi-selective activation of extrasynaptic receptors by ambient extracellular glutamate in acute brain slices from adult rats.r We use whole cell patch clamp to measure the effect of the NMDA receptor antagonist MK-801 on both synaptic and brief, local agonist application-evoked responses.r The level of ambient glutamate was estimated from tonic NMDA receptor activity to be ß77 nM and an equivalent concentration of NMDA was used to estimate the degree of extrasynaptic blockade (>82%) by our MK-801 protocol.r The extrasynaptic component of the total NMDA receptor pool can be mathematically derived from these data and was estimated to be 29-39% in the stratum radiatum of the CA1 region of the rat hippocampus.r This technique could be used to quantify extrasynaptic NMDA receptor function in rodent models of diseases where extrasynaptic NMDA receptors are implicated in neuron death.Abstract Synaptic NMDA receptors (NMDARs) play a central role in pro-survival signalling and synaptic plasticity in the majority of excitatory synapses in the central nervous system whereas extrasynaptic NMDARs (ES-NMDARs) activate pro-death pathways and have been implicated in many neurodegenerative diseases. ES-NMDARs have been characterized in acute brain slice preparations using the largely irreversible, activity-dependent NMDAR antagonist MK-801 to block synaptic NMDARs. This approach is limited by the concomitant MK-801 blockade of ES-NMDARs activated by ambient extracellular glutamate, which is largely absent from the synaptic cleft due to the high density of nearby glutamate transporters. In acute hippocampal slices from rats aged 35-42 postnatal days, we estimated ambient glutamate to be 72-83 nM Alexander Moldavski studied medicine at the University of Mainz and performed his research for his MD thesis at the University of Heidelberg, both in Germany. He continued his research in the area of cellular neurophysiology at resulting in a block of more than 82% of ES-NMDARs during a 5 min MK-801 application. This paper describes a novel electrophysiological and mathematical method to quantify the proportion of NMDARs located at extrasynaptic locations in a confined region of an acute brain slice preparation using MK-801 to preferentially block ES-NMDARs. The protocol uses whole cell patch clamp measurement of NMDAR responses to synaptic stimulation and brief local pressure application of NMDA before and after MK-801 application. After mathematically correcting for the relative block of both synaptic and extrasynaptic receptors, ES-NMDARs were estimated to comprise 29-39% of the total NMDAR pool in the apical dendrites of hippocampal CA1 pyramidal neurons. This new method may prove useful for accurate quantification of NMDAR distributions in neurodegenerative diseases that are associated with increased toxic ES-NMDAR signalling.

Aberrant gliogenesis and excitation in MEF2C autism patient hiPSC-neurons and cerebral organoids

“…Next, we confirmed the presence of aberrantly increased basal eNMDAR-mediated currents in MHS cultures by patch-clamp electrophysiological recordings. We found that MHS but not Ctrl hiPSC-neurons manifested basal eNMDAR-mediated current after pharmacological isolation of this current ( 58 ), and NitroSynapsin inhibited the current ( fig. S7E ).…”

“…Extrasynaptic currents were pharmacologically isolated and measured by whole-cell patch clamp recordings of hiPSC-derived cerebrocortical neurons based on previously published protocols ( 56, 58 ). Briefly, continuous recordings of individual neurons were performed in gap free mode at -70 mV in control conditions, followed by application of bicuculline (50 µM, a GABA A receptor antagonist, Tocris) for 1 min to inhibit inhibitory synaptic inputs and activate synaptic glutamate receptors.…”

“…Extrasynaptic currents were pharmacologically isolated and measured by whole-cell patch clamp recordings of hiPSC-derived cerebrocortical neurons based on previously published protocols (56,58). Thereafter, memantine or NitroSynapsin was applied to the neuron to study its effect on this current since these drugs have been shown to predominantly inhibit eNMDARs (32,53,55).…”

Case Report: Severe Adolescent Major Depressive Syndrome Turns Out to Be an Unusual Case of Anti-NMDA Receptor Encephalitis

“…Second, high dose lorazepam may have prevented the neurological manifestation of the disorder from evolving, and third, the patient responded well to the immune globulin treatment. Given the anxiolytic and antidepressant effects of NMDAR antagonists like ketamine, it appears counterintuitive that anti-NMDAR AAbs mediate a depressogenic effect (24). On the other hand, NMDAR activation may lead to opposite effects depending on the subcellular localization and the subunit composition of the receptor (3,25).…”

“…Given the anxiolytic and antidepressant effects of NMDAR antagonists like ketamine, it appears counterintuitive that anti-NMDAR AAbs mediate a depressogenic effect ( 24 ). On the other hand, NMDAR activation may lead to opposite effects depending on the subcellular localization and the subunit composition of the receptor ( 3 , 25 ).…”