The research team, led by Balázs Pál at the University of Debrecen, worked with a rare and precious resource: intact neocortical tissue removed during neurosurgery for brain tumors or metastases. These samples, collected from 25 patients ranging from 38 to 79 years old, allowed the scientists to perform patch-clamp recordings on living human neurons — an opportunity that few laboratories in the world have. Using whole-cell voltage clamp at a holding potential of −60 millivolts, the team recorded layer II-III pyramidal neurons and measured both fast synaptic events and the slow, sustained currents generated by ambient glutamate acting outside the synapse.

Glutamate is the brain’s dominant excitatory neurotransmitter, responsible for roughly 70 percent of all synaptic transmission. But its influence extends far beyond the synaptic cleft. In the extrasynaptic space, glutamate exists at concentrations thousands of times lower than inside a synapse — in the nanomolar to micromolar range rather than the millimolar range — yet it can still activate NMDA, AMPA, and metabotropic glutamate receptors located on neuronal membranes outside active synapses. These extrasynaptic receptors often differ in subunit composition from their synaptic counterparts, with NMDA receptors containing the GluN2B subunit being particularly enriched at extrasynaptic sites. The clearance of this ambient glutamate falls almost entirely to astrocytes, star-shaped glial cells that express the excitatory amino acid transporters EAAT1 and EAAT2, which together remove the vast majority of extracellular glutamate from the brain.

When the researchers applied DL-TBOA, a potent blocker of these glutamate transporters, to human neocortical slices, they observed two distinct phenomena. First, they recorded slow inward currents (SICs) — brief, phasic depolarizing events lasting hundreds of milliseconds that arise from the synchronous activation of extrasynaptic NMDA receptors by glutamate released from astrocytes. Second, and more remarkably, they detected a tonic inward current: a sustained shift in the holding current that persisted throughout the recording, averaging −56.8 ± 25.88 picoamperes across 16 neurons. This tonic current was significantly greater than the spontaneous fluctuation of the holding current measured before drug application, which averaged only 0.55 ± 1.13 picoamperes.

The pharmacological dissection of this tonic current revealed a surprising complexity. When the researchers applied ifenprodil, a selective antagonist of GluN2B-containing NMDA receptors, the tonic current was only partially reversed — dropping from −103.7 ± 54.2 picoamperes to −56.64 ± 33.57 picoamperes, a change that did not reach statistical significance. In contrast, SICs were almost completely eliminated by the same drug. This dissociation suggests that while both phenomena depend on NMDA receptor activation, the receptors mediating the tonic current are not exclusively GluN2B-containing. When the researchers applied D-AP5, a broad-spectrum NMDA receptor antagonist, the tonic current was fully abolished — dropping from −59.2 ± 28.72 picoamperes to −0.3 ± 2.59 picoamperes — confirming that NMDA receptors are essential for its generation.

One of the most striking findings emerged when the researchers correlated tonic current amplitude with patient age. The magnitude of the DL-TBOA-elicited tonic current was inversely proportional to the age of the donor, with a statistically significant correlation (p = 0.037, r² = 0.26). In patients over 70 years old, the tonic current was essentially undetectable. This age-dependent decline mirrors the team’s previous work on SICs, which also showed a dramatic reduction with advancing age in human tissue — a pattern that contrasts sharply with rodent studies, where only mild age-related changes in SIC frequency have been observed. The researchers suggest that this decline may reflect a combination of factors, including reduced GluN2B subunit expression, altered EAAT transporter function, and age-related changes in astrocyte morphology that modify glutamate diffusion pathways in the extracellular space.

The relationship between SICs and the tonic current proved to be quantitative as well as qualitative. When the researchers re-analyzed SIC data from their previous study and plotted the charge transfer of SICs per minute against the magnitude of the tonic current, they found a strong positive correlation (p < 0.0001, r² = 0.636, n = 19). Neurons that generated more SIC charge also carried larger tonic currents, suggesting that both phenomena arise from the same fundamental process — the accumulation of ambient glutamate in the extrasynaptic space — but manifest differently depending on the spatial relationship between glutamate release sites and receptor populations. The researchers propose that when glutamate diffuses over short distances, it produces discrete SICs, while longer diffusion pathways or more distributed receptor activation summate into a sustained tonic current.

Power spectral analysis of the baseline noise revealed further distinctions between the two phenomena. DL-TBOA application increased the power amplitude of baseline oscillations from 0.37 ± 0.1 to 3.95 ± 1.13 pA²/Hz, an effect that was reversed by D-AP5 but only partially reduced by ifenprodil. The frequency at the power maximum remained unchanged, suggesting that the increased noise reflected stochastic NMDA receptor openings rather than a shift in oscillation frequency. When the researchers compared the oscillatory properties of SIC declining phases with those of the tonic current, they found that SIC declining phases exhibited significantly greater power amplitude (6.46 ± 1.43 vs. 1.75 ± 0.71 pA²/Hz) and higher frequency (13.66 ± 1.56 vs. 5.72 ± 0.94 Hz), providing electrophysiological evidence that these are partially separable phenomena with distinct receptor activation kinetics.

Two control experiments strengthened the argument that the tonic current is pathological rather than physiological. First, when the researchers simply removed magnesium from the recording solution — a manipulation that dramatically increases SIC activity by relieving the voltage-dependent magnesium block of NMDA receptors — no tonic inward current appeared. The holding current remained essentially unchanged, averaging 0.46 ± 1.13 picoamperes in normal artificial cerebrospinal fluid and 0.25 ± 1.11 picoamperes in magnesium-free solution. Second, when the researchers applied GT949, a positive allosteric modulator of EAAT2 that enhances glutamate uptake, they expected to see a tonic outward current if a baseline tonic inward current existed under control conditions. Instead, no significant current change was detected. Together, these results indicate that under normal physiological conditions, human neocortical pyramidal neurons do not carry a measurable NMDA-dependent tonic inward current.

The clinical implications of this finding are significant. Tonic neuronal depolarization driven by excess extracellular glutamate is a known contributor to ischemic stroke, traumatic brain injury, and cortical spreading depolarization — a massive depolarizing wave that sweeps across cortical networks during these conditions. The researchers hypothesize that the tonic inward current they have characterized may represent one of the cellular mechanisms underlying this pathological depolarization, particularly given that its magnitude declines with age in parallel with the known age-related decrease in cortical susceptibility to spreading depolarization. In gliomas, extracellular glutamate levels are elevated, and the tonic current may contribute to tumor-related hyperexcitability. The finding that this current requires transporter inhibition rather than existing constitutively suggests that therapeutic strategies aimed at maintaining or enhancing EAAT function could protect vulnerable neurons from the sustained depolarization that drives excitotoxic cell death in a range of neurological diseases.

Subject of Research: NMDA receptor-mediated tonic inward currents elicited by glutamate transporter inhibition in human neocortical pyramidal neurons

Article Title: NMDA receptor mediated tonic excitatory currents are elicited by EAAT inhibition on human neocortical pyramidal neurons

Article References: Márton, B., Ecsedi, L., Pocsai, K., Douida, A., Lamsa, K., Hutóczki, G., Klekner, Á., & Pál, B. (2026). NMDA receptor mediated tonic excitatory currents are elicited by EAAT inhibition on human neocortical pyramidal neurons. Frontiers in Synaptic Neuroscience, 18, Article 1844197. https://doi.org/10.3389/fnsyn.2026.1844197

Image Credits: AI Generated

DOI: 10.3389/fnsyn.2026.1844197

Keywords: NMDA receptor, glutamate transporters, EAAT, astrocytes, human neocortex, pyramidal neurons, tonic inward current, slow inward currents, excitotoxicity, cortical spreading depolarization, aging brain, electrophysiology