SkQR1 Reduces Neurologic Deficit Caused by Rat Brain Compression Ischemia.

The protective effect of antioxidant SkQR1 was examined on the model of left-sided compression ischemia in rat sensorimotor cortex. The special tests aimed to determine the neurologic deficit in the limbs and assess performance of the forelimbs showed that a 2.5-min ischemia produced no disturbance in the limb functions on postsurgery days 1, 3, and 7. Elevation of compression time resulted in neurologic deficit in animals, and its severity depended on this time. A single intravenous injection of SkQR1 (250 nmol/kg body weight) performed 30 min after ischemia significantly reduced the degree of neurologic deficit. In vitro model of ischemia in surviving rat hippocampal slices showed that a 15-min-long ischemia significantly inhibited the population excitatory postsynaptic potentials, which did not restore during reperfusion. Preincubation of the slices with SkQR1 did not significantly affect recovery of these potentials.

Antiviral Drug Ivermectin at Nanomolar Concentrations Inhibits Glycine-Induced Chloride Current in Rat Hippocampal Neurons.

Ivermectin (IVM) belongs to the class of macrocyclic lactones, which is used as an antiparasitic agent. At present, the researchers focus on possibility to use IVM in treatment of certain forms of cancer and viral diseases such as COVID-19. The mechanisms of IVM action are not clear. It is assumed that IVM affects chloride channels and increases cytoplasmic concentration of chloride. This study examines the effect of IVM on chloride currents induced by glycine (IGly). Experiments were carried out on isolated pyramidal neurons of the rat hippocampus with whole-cell patch clamp. A short-term (600 msec) application of IVM in a concentration of 10 µM induced a slow inward current, which persisted after washing the neurons. The low concentrations (0.1-1000 nM) of IVM did not induce any novel current, but it rapidly and reversibly reduced the peak amplitude and accelerated desensitization of IGly in a dose-dependent manner. The threshold concentrations of IVM sufficient to reduce peak amplitude of IGly and to accelerate desensitization of IGly were 100 nM and 0.1 nM, respectively. The study revealed a high sensitivity of neuronal glycine receptors to IVM.

Copper Ions Reduce the Effect of Protons on Desensitization of Glycine Receptors.

Chloride current (IGly) evoked by the rapid (600 msec) application of glycine on isolated pyramidal neurons of the rat hippocampus was recorded using the patch clamp technique. We studied the effect of individual or combined application of copper ions (Cu2+) and protons (H+) on IGly. It was found that both Cu2+ (10 µM) and H+ (pH 7.0 and 6.0) applied separately caused a fast and reversible effect on IGly that included two components: a decrease in peak amplitude (Ipeak) and a decrease in the desensitization time constant (τdes). During combined application, the effects on Ipeak were additive, which indicates the independence of the mechanisms of these effects. At the same time, the effect of combined application of Cu2+ and H+ on τdes was not additive and sometimes a slowdown of the total desensitization was observed. The latter result suggests that H+ and Cu2+ can play the role of mutual antagonists when they affect the desensitization of GlyR.

Effect of Protons on GABAA Receptors in Central Neurons of Various Types.

Whole-cell patch-clamp technique was employed to record chloride ionic current IGABA evoked by fast (600 msec) application of GABA to hippocampal pyramidal neurons and cerebellar Purkinje cells isolated from rat brain. GABA solution in the application pipette was either neutral (pH 7.4) or acidic (pH 7.0 or 6.0). Application of protons to neurons causes a rapid, reversible, and dose-dependent decrease in the amplitude of IGABA; the effect was more pronounced on hippocampal neurons (carrying both synaptic and extrasynaptic GABAA receptors) than in cerebellar Purkinje cells (predominantly equipped with synaptic GABAA receptors). In hippocampal neurons, pharmacological isolation of extrasynaptic component from total IGABA was performed with GABAA receptor antagonist gabazine (50 nM). The extrasynaptic component of IGABA was stronger blocked by protons than total IGABA. It was concluded that acidic medium produced more potent blocking effect on extrasynaptic GABAA receptors than on synaptic ones.

GK-2 Reduces Death of Cultured Granule Neurons in Cerebellum Induced by the Toxic Effects of Zinc Ions.

Peptide mimetic of nerve growth factor GK-2 in a dose of 1-2 mg/liter improves survival of cultured rat cerebellar granule neurons exposed to the cytotoxic effect of zinc ions, but has no protective effect against copper ion cytotoxicity. Experiments on cultured rat hippocampal slices demonstrated that GK-2 did not affect reactivity of pyramidal neurons and long-term potentiation in the hippocampal field CA1 and the probability of glutamate release from presynaptic terminals in the synapses of the CA3-CA1 fields. The results suggest that GK-2 does not affect the functional properties of synaptic transmission under normal conditions, but protects neurons from the toxic effects of zinc, which creates prerequisites for GK-12 use in the treatment of neurodegenerative diseases.

Neuroprotective Effects of Methylene Blue In Vivo and In Vitro.

Focal unilateral traumatic brain injury in the sensorimotor cortical region disturbed the functions of contralateral limbs controlled by the damaged hemisphere. A single intravenous injection of methylene blue (1 mg/kg) immediately before or 30 min after the injury significantly weakened functional disorders in the affected extremities. In vitro experiments showed that methylene blue effectively reduced death of cultured neurons provoked by paraquat or zinc ions producing the toxic effects on mitochondrias.

ß-Amyloid Peptide Antagonizes the Effect of Protons on Taurine-Induced Chloride Current in Rat Hippocampal Pyramidal Neurons.

Taurine is an important endogenous agonist of glycine receptors (GlyR). Using the patchclamp technique, we measured chloride current induced by a short (600 msec) application of taurine (ITau) on isolated rat pyramidal neurons. pH of taurine solution in the applicator pipette was neutral (7.4) or acidic (7.0-5.0). Application of protons to a neuron causes a dosedependent decrease in the peak amplitude and acceleration of ITau desensitization. Addition of 100 nM ß-amyloid peptide (Aß) to the perfusate caused acceleration of ITau desensitization. The effects of Aß and H+ on the rate of ITau desensitization were not additive. In addition, Aß attenuated the effect of H+ on the peak amplitude of ITau. We also studied the effect of protons on the chloride current caused by activation of GABA receptors. In contrast to H+ effects on GlyR, Aß did not modulate the effects of H+ on GABA receptors.

Effect of Endogenous Neuropeptide Cycloprolylglycine on GABAA Receptors in Cerebellar Purkinje Cells.

Voltage clamp and concentration-jump methods were employed to examine the effects of endogenous neuropeptide cycloprolylglycine on GABA-activated ionic currents in isolated cerebellar Purkinje cells. In the concentration range of 0.1-10.0 µM, short-term (600 msec) external application of cycloprolylglycine against the background of GABA-evoked current produced no effect on its amplitude. In contrast, application of 1 µM cycloprolylglycine increased current up to 177±15% control level. The development of potentiating effect and return to the control level of ionic current were slow, which was indicative of possible implication of second messenger systems in these processes. Functional augmentation of GABAA receptors under the action of cycloprolylglycine can underlie the established neuroprotective and anxiolytic effects of this endogenous dipeptide.

Effect of Noopept on Dynamics of Intracellular Calcium in Neurons of Cultured Rat Hippocampal Slices.

A neuroprotective and nootropic drug Noopept increased the frequency of spontaneous calcium transients in neurons of CA1 radial layer in cultured rat hippocampal slices. In contrast, the drug exerted no significant effect on intracellular calcium concentration and its dynamics in neurons of hippocampal CA1 pyramidal layer.

The influence of acidic media on the effect of beta-amyloid peptide on the function of glycine receptor in hippocampal neurons.

We have previously shown that application of beta-amyloid peptide 1-42 (Aß) at picomolar/nanomolar concentrations caused a decrease in the peak amplitude and acceleration of desensitization of the glycine-activated chloride current (IGly) in hippocampal pyramidal neurons (Bukanova et al., 2016). The aim of this work was to study the effect of Aß on IGly in an acidified medium. The relevance of this work is determined by the fact that the pathogenic effects of Aß in Alzheimer's disease are usually accompanied by inflammatory processes and acidosis. The IGly was induced by 600 ms application of 100 µM (nearly EC50) or 500 µM (nearly saturating) glycine on isolated rat hippocampal neurons. The solution of glycine was neutral (pH 7.4) or acidic over a pH range of 5.0-7.0. It was found that 600 ms application of protons rapidly, reversibly and in dose-dependent manner decreased the peak amplitude and accelerated the desensitization of IGly. The effect of H+ on IGly desensitization did not depend on glycine concentration and may be considered noncompetitive, while the effect on IGly peak disappeared at saturating glycine concentration and can be regarded as a competitive. These characteristics of the proton effects on IGly coincide with the characteristics of the Aß effects on IGly. Experiments with joint application of Aß and H+ showed interdependence of their effects. Addition of Aß to perfusing solution reduced H+ effects on IGly while long pretreatment of Aß with acid solution prevented the effects of the peptide on IGly. Our results suggest the existence of common sites for Aß and H+ on the GlyR and indicate a mutual weakening of the inhibitory action of these molecules on IGly.

Effect of Selank on Spontaneous Synaptic Activity of Rat Hippocampal CA1 Neurons.

Application of anxiolytic drug Selank to hippocampal slices increased the amplitude and discharge rate of spontaneous inhibitory postsynaptic currents in rat hippocampal pyramidal CA1 neurons. In some neurons, Selank-induced up-regulation of spontaneous inhibitory postsynaptic currents was preceded by a transient decrease in this activity. In the examined concentration range (1-8 µM), Selank demonstrated no significant dose-dependence.

Functional modulation of strychnine-sensitive glycine receptors in rat hippocampal pyramidal neurons by amyloid-ß protein (1-42).

Amyloid-ß peptide (Aß) is considered a key protein in the pathogenesis of Alzheimer's disease because of its neurotoxicity, resulting in impaired synaptic function and memory. On the other hand, it was demonstrated that low (picomolar) concentrations of Aß enhance synaptic plasticity and memory, suggesting that in the healthy brain, physiological Aß concentrations are necessary for normal cognitive functions. In the present study, we found that Aß (1-42) in concentrations of 10 pМ - 100nМ enhanced desensitization of the glycine-activated current in isolated CA3 pyramidal neurons and also reversibly suppressed its peak amplitude during short (600ms) co-application with agonist. The effect was most prominent at low glycine concentrations. When glycine receptors were activated by other receptor agonists - taurine and ß-alanine, the changes of current kinetics and amplitudes induced by Aß had a similar character. When Aß (100 pM) was added to the bath solution, it caused, besides acceleration of desensitization, more pronounced reduction of peak current amplitude. This effect developed slowly, during a few minutes, and was more prominent at saturating concentrations of agonists. The results suggest that Aß interacts with glycine receptors through three different mechanisms - by enhancing receptor desensitization, by rapid inhibition of the receptor, and also by means of a slowly developing inhibition of the amplitude of the current, possibly through intracellular mechanisms. The observed changes in the activity of glycine receptors induced by Aß can lead to suppression of the tonic inhibition of hippocampal neurons mediated by extrasynaptic glycine receptors.

Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons.

Lithium salts are successfully used to treat bipolar disorder. At the same time, according to recent data lithium may be considered as a candidate medication for the treatment of neurodegenerative disorders. The mechanisms of therapeutic action of lithium have not been fully elucidated. In particular, in the literature there are no data on the effect of lithium on the glycine receptors. In the present study we investigated the effect of Li(+) on glycine-activated chloride current (IGly) in rat isolated pyramidal hippocampal neurons using patch-clamp technique. The effects of Li(+) were studied with two glycine concentrations: 100 µM (EC50) and 500 µM (nearly saturating). Li(+) was applied to the cell in two ways: first, by 600 ms co-application with glycine through micropipette (short application), and, second, by addition to an extracellular perfusate for 10 min (longer application). Li(+) was used in the range of concentrations of 1 nM-1 mM. Short application of Li(+) caused two effects: (1) an acceleration of desensitization (a decrease in the time of half-decay, or "τ") of IGly induced by both 100 µM and 500 µM glycine, and (2) a reduction of the peak amplitude of the IGly, induced by 100 µM, but not by 500 µM glycine. Both effects were not voltage-dependent. Dose-response curves for both effects were N-shaped with two maximums at 100 nM and 1 mM of Li(+) and a minimum at 1 µM of Li(+). This complex form of dose-response may indicate that the process activated by high concentrations of lithium inhibits the process that is sensitive to low concentrations of lithium. Longer application of Li(+)caused similar effects, but in this case 1 µM lithium was effective and the dose-effect curves were not N-shaped. The inhibitory effect of lithium ions on glycine-activated current suggests that lithium in low concentrations is able to modulate tonic inhibition in the hippocampus. This important property of lithium should be considered when using this drug as a therapeutic agent.

Mitochondria-Targeted Plastoquinone Antioxidant SkQR1 Has Positive Effect on Memory of Rats.

A single intraperitoneal injection to rats of the mitochondria-targeted plastoquinone antioxidant SkQR1 at dose 1 µmol/kg significantly improved reproduction by the rats of the passive avoidance conditional reflex. In vitro experiments on hippocampal slices showed that a single intraperitoneal injection of SkQR1 24 h before the preparation of the slice significantly increases the synaptic transmission efficiency of the pyramidal neurons of the CA1 field. The findings indicate that SkQR1 has a positive effect on memory processes.

Fe(2+) and Fe(3+) in micromolar concentrations modulate glycine-induced Cl(-) current in rat hippocampal neurons.

The effects of Fe(2+) and Fe(3+) on glycine-activated chloride current (IGly) were studied in rat isolated pyramidal hippocampal neurons using patch-clamp technique in whole-cell configuration. 25, 100 or 500 µM glycine was applied for 600 ms with 40s intervals. Fe(2+) and Fe(3+) were co-applied with glycine in the range of concentrations of 0.01-100 µM. We found that Fe(2+) and Fe(3+) affected IGly in a similar manner. Two types of effects of iron on IGly were observed. In low concentrations (0.1 µM) Fe ions caused an acceleration of the IGly desensitization, and the effect was more pronounced for IGly induced by 100 and 500 µM glycine than by 25 µM glycine. Higher Fe concentrations (1-100 µM) decreased the peak amplitude of IGly with weak influence on its kinetics. The values of IC50 of the effect were close to 10 µM for all glycine concentrations tested. The effect of iron on IGly peak did not depend on the membrane potential. This inhibition was noncompetitive and voltage-independent, suggesting that Fe ions do not exert their action on the agonist binding site of GlyRs or block the channel pore. An important characteristic of both effects of Fe was their progressive development during repetitive Fe applications (use-dependence). Our results suggest an existence of at least two binding sites for Fe ions which vary in affinity and mechanism of action, with the low-affinity site suppressing the activity of the high-affinity one. Physiological implication of our observations is that Fe ions in low micromolar concentrations can suppress tonic inhibition and cause hyperexcitability in hippocampus.

The involvement of sigma1 receptors in donepezil-induced rescue of hippocampal LTP impaired by beta-amyloid peptide.

Donepezil is a potent acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease (AD). Additional therapeutically relevant target for donepezil is sigma1 receptor (Sig1-R). Beta-amyloid peptide (Aß) is believed to contribute to the pathogenesis of AD. In our previous work (Kapai et al., 2012), we have shown that donepezil antagonizes the suppressive action of Aß(1-42) on long-term potentiation (LTP) in rat hippocampal slices. The purpose of the present study was to determine whether Sig1-R is involved into the mechanisms of donepezil action. For this purpose, we have tested whether agonist of Sig1-R PRE-084 mimics, and antagonist of Sig1-R haloperidol abolishes the effect of donepezil. Population spikes (PSs) were recorded from the pyramidal layer of the CA1 region of rat hippocampal slices. Drugs were applied by addition to the perfusate starting 15 min before and ending 5 min after the tetanus. In the control group, the amplitude of PS 30 min post-tetanus reached 153±10%. Aß (200 nM) markedly suppressed the LTP magnitude or even caused the suppression of baseline PS (82±8%, P<0.001). This suppression of LTP could be markedly prevented when 1 µM donepezil was co-administered with Aß (136±11%, P<0.05). Further, we co-administered three substances: Aß, donepezil and 0.5 µM haloperidol and have found that haloperidol antagonized the stimulating effect of donepezil on LTP (92±6%, P<0.05). Agonist of Sig1-R PRE-084 (0.1-10 µM) enhanced control LTP and abolished the inhibitory effect of Aß on LTP in a concentration-dependent manner. The amplitude of PS 30 min post-tetanus reached 183±7% (P<0.01) for 10 µM PRE-084. The results suggest that activation of Sig1-R is involved into the mechanisms of donepezil-induced rescue of hippocampal LTP impaired by Aß.

Role of zinc and copper ions in the pathogenetic mechanisms of Alzheimer's and Parkinson's diseases.

Disbalance of zinc (Zn2+) and copper (Cu2+) ions in the central nervous system is involved in the pathogenesis of numerous neurodegenerative disorders such as multisystem atrophy, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Wilson-Konovalov disease, Alzheimer's disease, and Parkinson's disease. Among these, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most frequent age-related neurodegenerative pathologies with disorders in Zn2+ and Cu2+ homeostasis playing a pivotal role in the mechanisms of pathogenesis. In this review we generalized and systematized current literature data concerning this problem. The interactions of Zn2+ and Cu2+ with amyloid precursor protein (APP), ß-amyloid (Abeta), tau-protein, metallothioneins, and GSK3ß are considered, as well as the role of these interactions in the generation of free radicals in AD and PD. Analysis of the literature suggests that the main factors of AD and PD pathogenesis (oxidative stress, structural disorders and aggregation of proteins, mitochondrial dysfunction, energy deficiency) that initiate a cascade of events resulting finally in the dysfunction of neuronal networks are mediated by the disbalance of Zn2+ and Cu2+.

Amyloid ß peptide (25-35) in picomolar concentrations modulates the function of glycine receptors in rat hippocampal pyramidal neurons through interaction with extracellular site(s).

ß-Amyloid peptide (Aß) plays a central role in the pathogenesis of Alzheimer׳s disease, but in lower amounts it is found in normal brains where it participates in physiological processes and probably regulates synaptic plasticity. This study investigated the effects of physiologically relevant concentrations of Aß (1 pM-100 nM), fragment 25-35, on glycine-mediated membrane current in acutely isolated rat hippocampal pyramidal neurons using whole-cell patch-clamp technique. We have found that short (600 ms) co-application of glycine with Aß caused reversible dose-dependent and voltage-independent acceleration of desensitization of glycine current. The peak amplitude of the current remained unchanged. The effect of picomolar Aß concentrations persisted in the presence of 1 µM Aß in the pipette solution, implying that Aß bounds to extracellular site(s). Concentration-dependence curve was N-shaped with maximums at 100 pM and 100 nM, suggesting the existence of two binding sites, which may interact with each other. Glycine current resistant to 100 µM picrotoxin, was insensitive to Aß, which suggests that Aß affected mainly homomeric glycine receptors. When Aß was added to bath solution, besides acceleration of desensitization, it caused reversible dose-dependent reduction of glycine current peak amplitude. These results demonstrate that physiological (picomolar) concentrations of Aß reversibly augment the desensitization of glycine current, probably by binding to external sites on homomeric glycine receptors. Furthermore, Aß can suppress the peak amplitude of glycine current, but this effect develops slowly and may be mediated through some intracellular machinery.

Brain aging and mitochondria-targeted plastoquinone antioxidants of SkQ-type.

Normal brain aging leads to decrease in cognitive functions, shrink in brain volume, loss of nerve fibers and degenerating myelin, reduction in length and branching of dendrites, partial loss of synapses, and reduction in expression of genes that play central roles in synaptic plasticity, vesicular transport, and mitochondrial functioning. Impaired mitochondrial functions and mitochondrial reactive oxygen species can contribute to the damage of these genes in aging cerebral cortex. This review discusses the possibility of using mitochondria-targeted antioxidants to slow the processes of brain aging.

In vivo injected mitochondria-targeted plastoquinone antioxidant SkQR1 prevents ß-amyloid-induced decay of long-term potentiation in rat hippocampal slices.

Addition of 200 nM ß-amyloid 1-42 (Abeta) to a rat hippocampal slice impairs the induction of a long-term post-tetanic potentiation (LTP) of population spike (PS) in pyramidal neurons of the CA1 field of hippocampus. Intraperitoneal injection into the rat of the mitochondria-targeted plastoquinone derivative SkQR1 (1 µmol/kg of weight given 24 h before the slices were made) abolishes the deleterious effect of Abeta on LTP. These data demonstrate that SkQR1 therapy is able to compensate the Abeta-induced impairments of long-term synaptic plasticity in the hippocampus, which are the main cause of loss of memory and other cognitive functions associated with Alzheimer's disease.