Pastukhov A. Na +-dependent transport of glutamate and exocytosis in the brain nerve terminals under conditions of hypothermia

In the dissertation, the modulation of Na+-dependent accumulation and release of glutamate, its extracellular level and exocytosis were studied using rat brain nerve terminals (synaptosomes) under conditions of moderate and deep hypothermia. The data showed that hypothermia decreased the initial rate of L-[14C]glutamate uptake and accumulation and exocytotic release of L-[14C]glutamate from nerve terminals. Moderate and deep hypothermia effectively reduced pathological glutamate transporter reversal and release of L-[14C]glutamate through dissipation of synaptic vesicle proton gradient in presence of FCCP. Gradual dynamics of hypothermia-mediated decrease in synaptosomal L-[14C]glutamate release evoked by the receptor agonists NMDA-, AMPA-, and kainate has been demonstrated that can be of value for the justification of optimal temperature regimes in therapeutic hypothermia. NMDA-induced L-[14C]glutamate release from nerve terminals was higher in the presence of levetiracetam as compared to that without the drug. Despite levetiracetam effects decreased in hypothermia, combined application of hypothermia and levetiracetam resulted in higher NMDA-induced L-[14C]glutamate release from nerve terminals as compared to that without the drug. These effects were not revealed for synaptosomal AMPA- and kainate-induced L-[14C]glutamate release in the presence of levetiracetam at the similar concentration. Therefore, we first revealed that levetiracetam administration significantly mitigated a hypothermia-induced decrease in NMDA responses at the presynaptic level and can be used for the targeted neurocorrection to reduce side effects of therapeutic hypothermia in cardiac surgery. However, levetiracetam-mediated improvement of NMDA responses is not applicable in stroke, brain trauma and neonatal asphyxia therapies, where the main neuroprotective action of hypothermia is associated with prevention of damaging consequence of pre-existing acute glutamate exitotoxicity. In addition, we have studied neuroprotective feature of combined approach of cholesterol depletion of the plasma membrane of nerve terminals using methyl-beta-cyclodextrin (MCD) and hypothermia. It was shown that pathological transporter-mediated release of glutamate after treatment of nerve terminals with MCD was significantly reduced under hypothermia conditions. So, combined approach of cholesterol depletion of the plasma membrane and hypothermia demonstrated additive neuroprotective effect. The effect of MCD-conjugated γ-Fe2O3 nanoparticles on the release of L-[14C]glutamate from nerve terminals under hypothermia has been investigated. It has been demonstrated that MCD-conjugated nanoparticles reduced the initial rate of uptake and accumulation of L-[14C]glutamate by nerve terminals, and increased the extracellular level of L-[14C]glutamate. However, uncoated γ-Fe2O3 nanoparticles also have neuromodulatory effects in nerve terminals influencing uptake and the extracellular level of L-[14C]glutamate, so it was not confirmed that these parameters were changed through cholesterol removal only. Thus, in the dissertation work the influence of hypothermia on Na+-dependent transport of glutamate and exocytosis in the nerve terminals was studied. The obtained results proved mechanisms of neuroprotective effects of hypothermia in stroke, hypoxic/ischemic brain lesions and it was demonstrated that combined application of hypothermia and levetiracetam, and MCD in medicine can enhance neuroprotective effects.