Dmitry G. Semenov

Calcium Transients in the Model of Rapidly Induced Anoxic Tolerance in Rat Cortical Slices: Involvement of NMDA Receptors

In this study, we investigated the effects of NMDA receptor antagonists on calcium transients induced by a single 2-min preconditioning anoxia (PA) in rat olfactory cortical slices, and on the ability of PA to prevent pathological calcium overload induced by subsequent 10-min test anoxia (TA). Relative changes in the intracellular Ca2+ concentration (Cai) and in the level of Ca2+ bound to intracellular hydrophobic domains (Cab) were monitored using fura-2 and chlortetracycline, respectively. Our results confirmed that TA induces prominent long-lasting increases in Cai and Cab, reflecting cellular calcium overload. It was found that PA produces moderate increases in both Ca2+ pools and prevents Ca2+ overload induced by TA carried out 90 min later. Calcium transients and the protective effects of PA were significantly suppressed in slices treated with NMDA receptor antagonists during and 30 min after PA. These results indicate that moderate activation of the NMDA receptors participates in the mechanism of the PA-induced anoxic tolerance of cortical neurons.

Responses to reversible anoxia of intracellular free and bound Ca2+ in rat cortical slices

Severe anoxia induces destabilisation of intracellular calcium homeostasis in neurones. The mechanism of this effect, and particularly the interrelationship between changes in intracellular concentration of free Ca(2+) ions and the content of the intracellular Ca(2+) stores, during and after anoxia, is not clear. We used a superfusion system of rat olfactory cortical slices for the fluorimetric estimation of changes in the intracellular concentration of free Ca(2+) ions and in the level of bound Ca(2+), utilising the fluorescent indicators Fura-2 and chlortetracycline, respectively. It was found that 10-min normoglycaemic anoxia results in simultaneous decrease in bound and increase in free Ca(2+) levels, whereas during 60-min reoxygenation, we detected an increase in both indices. The NMDA receptor antagonists MK-801 and APV attenuated changes in free Ca(2+) level during anoxia and reoxygenation and intensified anoxia-evoked decrease in bound Ca(2+) content, whereas a late post-anoxic increase in bound Ca(2+) was abolished. These data suggest that the influx of extracellular Ca(2+) to neurones via NMDA receptors, plays a critical role in the rise of intracellular free Ca(2+) concentration during and after anoxia. Biphasic changes in bound Ca(2+) content during anoxia and reoxygenation may reflect an anoxia-induced release of Ca(2+) from intracellular stores, followed later by a neuronal calcium overload and refilling of intracellular Ca(2+) binding sites.

Changes of intracellular calcium homeostasis in brain cortical structures during anoxia in vivo and in vitro

This work characterizes the anoxia-evoked changes in the content of bound calcium (Cab) in brain cortex membraneous structures, studied in vivo, on living brain cortex preparation and in vitro, on subcellular fractions (synaptosomes, microsomes and mitochondria) in anoxic conditions. The chlorotetracycline (CTC) fluorescent chelate probe was used to monitor changes of Cab content in hydrophobic domains of intracellular membranes. In in vivo experiments the bioelectric activity of single neurons was recorded simultaneously with measurements of Ca-CTC fluorescence. In vitro experiments were supplemented with determinations of synaptosomal 45Ca-uptake. It was found that the response of cortical neurons to anoxia is manifested in a decrease of a portion of Ca2+ bound with membrane hydrophobic domains. These in vivo changes preceded the noticeable disturbances of neuronal electric activity. An anoxia-evoked drop in Cab was also clearly demonstrated in vitro, irrespective of K+ (for synaptosomes) or Na+ (for mitochondria) concentrations in incubation media, although the additional effect of Cab displacement was noted when Na+/K+ concentrations were modified in order to simulate their changes occurring in anoxic conditions. It was found that the membranes of different neuronal compartments are not uniformly vulnerable to anoxia in vitro as the anoxic decrease in Cab content occurred in synaptosomes and microsomes much sooner than in mitochondria. Therefore, in vivo and in vitro experiments visualized high sensitivity of Ca2+-binding mechanisms in different neuronal membranes to anoxia. The anoxia-evoked displacement of a portion of Cab to the free ionic form may trigger a complex intracellular response determining anoxic reactions and post-anoxic recovery.

Moderate Hypobaric Hypoxia Modifies Ca2+-Mediated Glutamatergic Signal Transduction in Rat Cerebral Cortex

Examination of fluorescent Ca2+-responses to stimulation by ionotropic glutamate receptor agonists in rat cerebral cortex slices, preconditioned with hypobaric hypoxia, has demonstrated different time course for up-regulation of their Ca2+-conductivity and for stimulation of the consequent binding of the Ca2+ which entered the cell. The evident result is the development of AMPA-mediated moderate increase of intracellular Ca2+.

Early postanoxic changes of polyphosphoinositides and bound Ca2+ content in relation to neuronal activity in brain cortex.

We studied the changes in the content of membrane-bound calcium (Cab) and the polyphosphoinositides (poly-PI): bis- and trisphosphoinositide (PIP and PIP2) in the cat brain cortex during the early period (up to 30 min) of reoxygenation after 2.5 min and 5 min of anoxia. In vivo experiments were performed on a living cat cortical preparation. Studies included Cab estimation with clortetracycline, a calcium fluorescent chelate probe, and simultaneous registration of neuronal activity. Anoxia resulted in a significant drop of Cab and PIP2 in the cortex along with an absence of neuronal activity. During reoxygenation after 2.5 min of anoxia we observed an increase of Cab, however the Cab did not recover to the preanoxic level. An elevation of PIP and PIP2 content to 20% above the preanoxic level and recovery of neuronal activity with symptoms of hyperactivation were also observed. After 5 min of anoxia two qualitatively different types of changes were disclosed for the 30 min period of reoxygenation. In one half of the animals only slight symptoms of recovery in some of the indices were found. In the other group Cab and PIP2 content increased to a level significantly exceedingly the preanoxic one and abnormal spike activity appeared. Based on these results we suggest that disturbances in Ca- and poly-PI-related second messenger systems may significantly affect the recovery of neuronal function after anoxia.

Comparison of Ca2+ Responses to Stimulation of Glutamate Receptors in the Rat Cerebral Cortex after Hypobaric Hypoxia of Different Severities

Hypoxia modifies glutamatergic signaling in the brain, inducing prolonged increases in intracellular Ca2+ levels. Depending on the severity of the applied hypoxia, the level and temporospatial profile of Ca2+ accumulation during reoxygenation triggers fundamentally different Ca2+-dependent reactions leading either to cell death or hypoxic tolerance. With the aim of identifying the specific characteristics of the involvement of different glutamate receptors in these alternative processes, the patterns of Ca2+ responses to stimulation of various subtypes of ionotropic and metabotropic glutamate receptors with their agonists were identified in slices of piriform cortex from rats subjected one day previously to severe (pathogenic) or moderate (adaptogenic) hypobaric hypoxia. Hypoxia of different modalities produced different modifications of the Ca2+ responses of all the receptors tested. The most marked differences were seen for Ca2+ responses to stimulation of group I metabotropic glutamate receptors.