Glutamate excitotoxicity is implicated in the pathogenesis of numerous diseases, such as stroke, traumatic brain injury, and Alzheimer’s disease, for which insulin resistance is a concomitant condition, and intranasal insulin treatment is believed to be a promising therapy. Excitotoxicity is initiated primarily by the sustained stimulation of ionotropic glutamate receptors and leads to a rise in intracellular Ca2+ ([Ca2+]i), followed by a cascade of intracellular events, such as delayed calcium deregulation (DCD), mitochondrial depolarization, adenosine triphosphate (ATP) depletion that collectively end in cell death. Therefore, cross-talk between insulin and glutamate signaling in excitotoxicity is of particular interest for research. In the present study, we investigated the effects of short-term insulin exposure on the dynamics of [Ca2+]i and mitochondrial potential in cultured rat cortical neurons during glutamate excitotoxicity. We found that insulin ameliorated the glutamate-evoked rise of [Ca2+]i and prevented the onset of DCD, the postulated point-of-no-return in excitotoxicity. Additionally, insulin significantly improved the glutamate-induced drop in mitochondrial potential, ATP depletion, and depletion of brain-derived neurotrophic factor (BDNF), which is a critical neuroprotector in excitotoxicity. Also, insulin improved oxygen consumption rates, maximal respiration, and spare respiratory capacity in neurons exposed to glutamate, as well as the viability of cells in the MTT assay. In conclusion, the short-term insulin exposure in our experiments was evidently a protective treatment against excitotoxicity, in a sharp contrast to chronic insulin exposure causal to neuronal insulin resistance, the adverse factor in excitotoxicity.

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The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON–NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2–•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O2–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).