Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 μM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca²⁺]_i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1–10 μM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca²⁺]_i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca²⁺]_i level during oscillations, which appears to determine the number of PDSs in the cluster.

Abnormal depolarization of neuronal membranes called paroxysmal depolarization shift (PDS) represents a cellular correlate of interictal spikes. The mechanisms underlying the generation of PDSs or PDS clusters remain obscure. This study aimed to investigate the role of ionotropic glutamate receptors (iGluRs) in the generation of PDS and dependence of the PDS pattern on neuronal membrane potential. We have shown that significant depolarization or hyperpolarization (by more than ±50 mV) of a single neuron does not change the number of individual PDSs in the cluster, indicating the involvement of an external stimulus in PDS induction. Based on this data, we have suggested reliable protocols for stimulating single PDS or PDS clusters. Furthermore, we have found that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are necessary for PDS generation since AMPAR antagonist NBQX completely suppresses bicuculline-induced paroxysmal activity. In turn, antagonists of NMDA (N-methyl-D-aspartate) and kainate receptors (D-AP5 and UBP310, respectively) caused a decrease in the amplitude of the first action potential in PDSs and in the amplitude of the oscillations of intracellular Ca²⁺ concentration occurring alongside the PDS cluster generation. The effects of the NMDAR (NMDA receptor) and KAR (kainate receptor) antagonists indicate that these receptors are involved only in the modulation of paroxysmal activity. We have also shown that agonists of some G_i-coupled receptors, such as A₁ adenosine (A₁Rs) or cannabinoid receptors (CBRs) (N₆-cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A₁R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on G_i-coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy.

Gi-coupled receptors, particularly cannabinoid receptors (CBRs), are considered perspective targets for treating brain pathologies, including epilepsy. However, the precise mechanism of the anticonvulsant effect of the CBR agonists remains unknown. We have found that WIN 55,212-2 (a CBR agonist) suppresses the synchronous oscillations of the intracellular concentration of Ca²⁺ ions (epileptiform activity) induced in the neurons of rat hippocampal neuron-glial cultures by bicuculline or NH₄Cl. As we have demonstrated, the WIN 55,212-2 effect is mediated by CB₁R receptors. The agonist suppresses Ca²⁺ inflow mediated by the voltage-gated calcium channels but does not alter the inflow mediated by NMDA, AMPA, and kainate receptors. We have also found that phospholipase C (PLC), protein kinase C (PKC), and G-protein-coupled inwardly rectifying K⁺ channels (GIRK channels) are involved in the molecular mechanism underlying the inhibitory action of CB₁R activation against epileptiform activity. Thus, our results demonstrate that the antiepileptic action of CB₁R agonists is mediated by different intracellular signaling cascades, including non-canonical PLC/PKC-associated pathways.

Background. Microglia, accounting for 5–15% of total brain cells, represent an essential population of glial cells in the cultures used for modeling neuroinflammation in vitro. However, microglia proliferation is poor in neuron–glial cultures. Here, we studied the population composition of rat hippocampal neuron–glial cell cultures prepared utilizing papain (PAP cultures) and trypsin (TRY cultures) as proteolytic enzymes for cell isolation. Methods. To evaluate the percentage and morphology of microglia in TRY and PAP cultures and cultures incubated in the presence of TGFβ+MCSF+cholesterol, which should enhance microglia proliferation, we used an immunostaining and calcium imaging approach in combination with staining using the recently developed vital microglia fluorescent probe CDr20. Results. We have shown that the microglia percentage in PAP cultures was higher than in TRY cultures. Microglia in PAP cultures are predominantly polarized, while bushy morphology was more characteristic of TRY cultures. We have also demonstrated that the TGFβ+MCSF+cholesterol combination increases the microglia number both in PAP and TRY cultures (up to 25–30%) and promotes the appearance of ameboid microglia characterized by high mobility. However, the significant appearance of ameboid microglia was observed already at the early stages of cultivation (2 DIV) in TRY cultures, while in PAP cultures, the described transformation was observed at 7 DIV. Based on the absence of the ATP-induced Ca²⁺ response, round shape, significant proliferation, and high mobility, we have suggested that ameboid microglia are reactive. Conclusions. Thus, our results demonstrate that papain is a more suitable proteolytic enzyme for preparing mixed hippocampal neuron–glial cultures with a higher percentage of heterogeneous microglia and functional neurons and astrocytes (tricultures).

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.