Electrophysiological Activity and Survival Rate of Rats Nervous Tissue Cells Depends on D/H Isotopic Composition of Medium.

The deuterium content modification in an organism has a neuroprotective effect during the hypoxia model, affecting anxiety, memory and stress resistance. The aim of this work was to elucidate the possible mechanisms of the medium D/H composition modification on nerve cells. We studied the effect of an incubation medium with a 50 ppm deuterium content compared to a medium with 150 ppm on: (1) the activity of Wistar rats' hippocampus CA1 field neurons, (2) the level of cultured cerebellar neuron death during glucose deprivation and temperature stress, (3) mitochondrial membrane potential (MMP) and the generation of reactive oxygen species in cultures of cerebellar neurons. The results of the analysis showed that the incubation of hippocampal sections in a medium with a 50 ppm deuterium reduced the amplitude of the pop-spike. The restoration of neuron activity was observed when sections were returned to the incubation medium with a 150 ppm deuterium content. An environment with a 50 ppm deuterium did not significantly affect the level of reactive oxygen species in neuron cultures, while MMP decreased by 16-20%. In experiments with glucose deprivation and temperature stress, the medium with 50 ppm increased the death of neurons. Thus, a short exposure of nerve cells in the medium with 50 ppm deuterium acts as an additional stressful factor, which is possibly associated with the violation of the cell energy balance. The decrease in the mitochondrial membrane potential, which is known to be associated with ATP synthesis, indicates that this effect may be associated with the cell energy imbalance. The decrease in the activity of the CA1 field hippocampal neurons may reflect reversible adaptive changes in the operation of fast-reacting ion channels.

Corticosteroids as Selective and Effective Modulators of Glycine Receptors.

The mechanism of the negative impact of corticosteroids on the induction and progress of mental illness remains unclear. In this work, we studied the effects of corticosteroids on the activity of neuronal glycine receptors (GlyR) and GABA-A receptors (GABAAR) by measuring the chloride current induced by the application of GABA (2 or 5 µM) to isolated cerebellar Purkinje cells (IGABA) and by the application of glycine (100 µM) to pyramidal neurons of the rat hippocampus (IGly). It was found that corticosterone, 5α-dihydrodeoxycorticosterone, allotetrahydrocorticosterone, cortisol, and 17α,21-dihydroxypregnenolone were able to accelerate the desensitization of the IGly at physiological concentrations (IC50 values varying from 0.39 to 0.72 µM). Next, cortisone, 11-deoxycortisol, 11-deoxycorticosterone, 5ß-dihydrodeoxycorticosterone, and tetrahydrocorticosterone accelerated the desensitization of IGly with IC50 values varying from 10.3 to 15.2 µM. Allotetrahydrocorticosterone and tetrahydrocorticosterone potentiated the IGABA albeit with high EC50 values (18-23 µM). The rest of the steroids had no effect on IGABA in the range of concentrations of 1-100 µM. Finally, our study has suggested a structural relationship of the 3ß-hydroxyl group/3-oxo group with the selective modulatory activity on GlyRs in contrast to the 3α-hydroxyl group that is pivotal for GABAARs. In summary, our results suggest that increased GlyR desensitization by corticosteroids may contribute to brain dysfunction under chronic stress and identify corticosteroids for further development as selective modulators of GlyRs.

Positive allosteric modulators of GABAA receptor restore chloride current from blockade by competitive antagonists in a ligand-dependent manner.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter. GABA receptor type A (GABAAR) possesses binding sites for a large group of pharmacological agents which are supposed to interact allosterically with each other. The aim of this work was to study the interaction between the positive allosteric modulators (PAMs) and the competitive antagonists of GABAARs. The GABA-induced chloride current (IGABA) was measured in isolated Purkinje cells of rat cerebellum using the patch-clamp technique. PAMs, neurosteroid allopregnanolone (Allo) and zolpidem (Zolp), a drug that positively modulates the GABAAR through interaction with the benzodiazepine (BDZ) site, doubled the IGABA amplitude in the control solution. Competitive antagonist of GABAARs, bicuculline (Bic, 5 µM) blocked the IGABA by 90%. The addition of 1 µM Allo or 0.5 µM Zolp to the Bic solution caused an unblocking effect, so that the IGABA amplitude increased 10 and 4 times from control value, correspondingly. This unblocking effect developed slowly, as evidenced by a threefold increase in the current rise time. Competitive antagonist of GABAARs, gabazine (GBZ, 0.5 µM) blocked the IGABA by 87%. The addition of 1 µM Allo to the GBZ solution caused an unblocking effect, so that the IGABA amplitude increased 7-fold. However, the addition of 0.5 µM Zolp to the GBZ solution did not cause an unblocking effect. So, Allo appeared to have a stronger unblocking potential than Zolp, and Bic binding site showed a higher sensitivity to the action of unblocking PAMs than GBZ binding site. The results indicate for the first time the existence of an allosteric relationship between the sites binding PAMs and the competitive antagonists of GABAAR.

Effect of nootropic dipeptide noopept on CA1 pyramidal neurons involves α7AChRs on interneurons in hippocampal slices from rat.

Noopept (NP) is a proline-containing dipeptide with nootropic and neuroprotective properties. We have previously shown that NP significantly increased the frequency of spontaneous IPSCs in hippocampal CA1 pyramidal cells mediated by the activation of inhibitory interneurons in stratum radiatum. The cholinergic system plays an important role in the performance of cognitive functions, furthermore multiple behavioral and clinical facts link NP with the cholinergic system. The present study was undertaken to reveal the possible interaction of NP with neuronal nicotinic acetylcholine receptors (nAChRs). Currents were recorded from rat hippocampal neurons using the whole-cell, patch-clamp technique. NP (5 µM) increased the action potential firing frequency recorded from GABAergic interneurons in the stratum radiatum (SR) of CA1 region. This effect was almost completely abolished by the application of the α7 nAChR-selective antagonists α-bungarotoxin (α-BGT; 6 nM) and methyllycaconitine (MLA; 20 nM). The increase in the frequency of spontaneous IPSCs in CA1 pyramidal cells induced by NP was also eliminated by α7 nAChRs antagonists. These results imply the involvement of α7 nAChRs in the modulation of hippocampal neuronal activity caused by NP and indicate that a7 nAChRs are an important site of action of NP.

Epipregnanolone as a Positive Modulator of GABAA Receptor in Rat Cerebellar and Hippocampus Neurons.

Epipregnanolone (3ß-hydroxy-5ß-pregnan-20-one, Epi) is an endogenous steroid with important physiological effects and high affinity for GABAA receptors. The effect of Epi on GABA-induced chloride current (IGABA) in native neurons has hardly been studied. In this work, we studied the influence of Epi on the IGABA in the Purkinje cells of rat cerebellum and pyramidal neurons of rat hippocampus with the patch clamp technique. We showed that Epi is a positive modulator of the IGABA with EC50 of 5.7 µM in Purkinje cells and 9.3 µM in hippocampal neurons. Epi-induced potentiation of the IGABA was more potent at low vs. high GABA concentrations. Isopregnanolone (3ß-hydroxy-5α-pregnan-20-one, Iso) counteracted Epi, reducing its potentiating effect by 2-2.3 times. Flumazenil, a nonsteroidal GABAA receptor antagonist, does not affect the Epi-induced potentiation. Comparison of the potentiating effects of Epi and allopregnanolone (3α-hydroxy-5α-pregnan-20-one, ALLO) showed that ALLO is, at least, a four times more potent positive modulator than Epi. The combined application of ALLO and Epi showed that the effects of these two steroids are not additive. We conclude that Epi has a dual effect on the IGABA increasing the current in the control solution and decreasing the stimulatory effect of ALLO.

Effects of copper on viability and functional properties of hippocampal neurons in vitro.

Copper (Cu2+) is an essential metal presented in the mammalian brain and released from synaptic vesicles following neuronal depolarization. However, the disturbance of Cu2+ homeostasis results in neurotoxicity. In our study we performed for the first time a combined functional investigation of cultured hippocampal neurons under Cu2+ exposure, its effect on spontaneous spike activity of hippocampal neuronal network cultured on multielectrode array (MEA), and development of long-term potentiation (LTP) in acute hippocampal slices in the presence of Cu2+. Application of 0.2mM CuCl2 for 24h reduced viability of cultured neurons to 40±6%, whereas 0.01mM CuCl2 did not influence significantly on the neuronal survival. However, exposure to the action of 0.01mM Cu2+ resulted in pronounced reduction of network spike activity and abolished LTP induced by high-frequency stimulation of Schaffer's collaterals in CA1 pyramidal neurons of hippocampal slices. Antioxidant Trolox, the hydrosoluble vitamin E analogue, prevented neurotoxic effect and alterations of network activity under Cu2+ exposure, but didn't change the impairment of LTP in Cu2+-exposured hippocampal slices. We hypothesized that spontaneous network neuronal activity probably is one of the potential targets of Cu2+-induced neurotoxicity, in which free radicals can be involved. At the same time, it may be suggested that Cu2+-induced alterations of long-lasting trace processes (like LTP) are not mediated by oxidative damage.

Glycine receptor in hippocampal neurons as a target for action of extracellular cyclic nucleotides.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are well known intracellular second messengers. At present study, we describe the effects of extracellularly applied cAMP and cGMP on glycine-induced chloride currents (I(Gly)) in isolated rat hippocampal pyramidal neurons. 50 or 500 µM glycine was applied for 600 ms with 1 min intervals. cAMP and cGMP were co-applied with glycine. We found that both cAMP and cGMP rapidly, reversibly and in a dose-dependent manner accelerated the I(Gly) desensitization. The effect was more prominent on I(Gly) induced by 500 µM than by 50 µM glycine. Dose-response curves were constructed in the 0.1-100,000 nM range of cAMP and cGMP concentrations. They demonstrate that threshold concentration of both compounds was about 1 nM and maximal effect was manifested at 100 nM. When cAMP and cGMP were added to the recording pipette, their extracellular application caused the effects similar to those obtained with normal intracellular medium. The effects of cyclic nucleotides remained unchanged in the presence of the antagonist of adenosine receptors in extracellular solution, and the agonist of adenosine receptors did not mimic the effect of cyclic nucleotides. The changes in the decay kinetics were equally pronounced at negative and positive membrane potentials. When co-administered 1 nM cAMP and 1 nM cGMP caused a weaker effect than either of the compounds alone which suggests a negative interaction between binding sites for cAMP and cGMP. This work describes a novel mode of action of cyclic nucleotides, namely, the modulation of GlyRs functions from extracellular side.

Mitochondria-targeted plastoquinone antioxidant SkQ1 prevents amyloid-ß-induced impairment of long-term potentiation in rat hippocampal slices.

Bath application of 200 nM amyloid-ß1-42 (Aß) to rat hippocampal slices impairs induction of long-term potentiation (LTP) of the population spike in pyramidal layer of the CA1 field of the hippocampus. Intraperitoneal injection of mitochondria-targeted plastoquinone derivative SkQ1 at very low concentrations (250 nmol/kg body weight) given 24 h before the slice preparation or 1 h treatment of hippocampal slices with 250 nM SkQ1 prevents the deleterious effect of Aß on LTP. To elucidate which part of the molecule is responsible for this type of neuroprotective activity, the effect of the analog of SkQ1 lacking plastoquinone (C12TPP) was studied. It was found that C12TPP was much less efficient in LTP protection than SkQ1 itself. It means that the plastoquinone part of the SkQ1 molecule is responsible for the LTP rescue. To summarize, in vivo and in vitro injection of SkQ1 compensates for Aß-induced oxidative damage of long-term synaptic plasticity in the hippocampus, which is considered to be the main reason of memory loss and impairment of other cognitive functions associated with Alzheimer's disease. Therefore, SkQ1 may be considered as a promising candidate for the treatment of early-stage Alzheimer's disease.

Novel nootropic dipeptide Noopept increases inhibitory synaptic transmission in CA1 pyramidal cells.

Effects of newly synthesized nootropic and anxiolytic dipeptide Noopept on inhibitory synaptic transmission in hippocampal CA1 pyramidal cells were investigated using patch-clamp technique in whole-cell configuration. Bath application of Noopept (1 microM) significantly increased the frequency of spike-dependant spontaneous IPSCs whereas spike-independent mIPSCs remained unchanged. It was suggested that Noopept mediates its effect due to the activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion.