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.

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.

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.

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.

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.

Nootropic dipeptide noopept enhances inhibitory synaptic transmission in the hippocampus.

Application of nootropic agent Noopept on hippocampal slices from Wistar rats enhanced the inhibitory component of total current induced by stimulation of Shaffer collaterals in CA1 pyramidal neurons, but did not affect the excitatory component. A direct correlation between the increase in the amplitude of inhibitory current and agent concentration was found. The substance did not affect the release of inhibitory transmitters from terminals in the pyramidal neurons, which indicated changes in GABAergic interneurons.

[Peptidergic modulation of the hippocampus synaptic activity].

Effects of two newly synthesized nootropic and anxiolytic dipeptides: Noopept and Selank 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) or Selank (2 microM) significantly increased the frequency of spike-dependent spontaneous m1PSCs, whereas spike-independent mlPSCs remained unchanged. It was suggested that both peptides mediated their effect sue to activation of inhibitory interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion, at least for Noonent.

Comenic acid prevents post-stress enhancement of long-term potentiation in rat hippocampus.

In experiments on hippocampal slices from young rats subjected to immobilization-cold stress we observed a pronounced increase in the amplitude of long-term potentiation of focal responses in CA1 area. Daily injections of comenic acid during stress exposure normalized parameters of long-term potentiation in the hippocampus.