Heterogeneity of Intracellular Ion Homeostasis and Selective Neuronal Vulnerability During Glutamate Excitotoxicity

Glutamate excitotoxicity, a hallmark of acute brain injury, causes intracellular calcium overload and disrupts cation homeostasis. Although selective neuronal vulnerability is well established, the coordinated multi-ion mechanisms underlying resistance or death in distinct subpopulations, and the role of neuron–glia interactions, remain poorly understood. Using synchronous multi-channel fluorescence imaging in primary hippocampal cultures, we assessed coupled dynamics of cytosolic [Ca²⁺]i, [Na⁺]i, [K⁺]i, and pHᵢ in neurons and astrocytes exposed to toxic glutamate concentration (100 μM). Based on post-washout [Ca²⁺]ᵢ recovery, neurons were divided into fast-return, slow-return, and no-return groups. Vulnerable no-return neurons showed irreversible Ca²⁺ overload and persistent K⁺ loss, and contained the highest proportion of cells expressing calcium-permeable AMPA receptors and GluK1-containing kainate receptors. GABAergic neurons were mainly found in the slow-return and no-return groups. Fast-return neurons exhibited a two-step [Na⁺]i rise during glutamate application and washout, coinciding with a sharp [Ca²⁺]i decline, and marked cytosolic acidification; however, pHᵢ and [Na⁺]i did not recover even 40 min after washout. Their [K⁺]i recovered almost immediately and correlated inversely with [Ca²⁺]i, while [Na⁺]i correlated inversely with pHᵢ. Astrocytes rapidly cleared Ca²⁺ and stabilized pH despite sustained Na⁺ accumulation; their [K⁺]i also recovered quickly and correlated inversely with [Ca²⁺]i. GABABR agonist baclofen shifted neurons toward the slow-return group, whereas the A₁ adenosine agonist N⁶-cyclohexyladenosine abolished irreversible calcium collapse and expanded the fast-return pool. These findings clarify mechanisms of selective neuronal resilience to excitotoxicity and support Gᵢ-receptor activation as a strategy to preserve ion homeostasis.