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.

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.

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.

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.

Impact of amyloid-ß peptide (1-42) on voltage-gated ion currents in molluscan neurons.

Different types of voltage-gated ion currents were recorded in isolated neurons of snail Helix pomatia using the two-microelectrode voltage-clamp technique. Application of amyloid-ß peptide (1-42, 1-10 µM) in the bathing solution did not change delayed rectifier K(+)-current and leakage current, but enhanced inactivation of Ca(2+)-current and blocked Ca(2+)-dependent K(+)-current.

Donepezil eliminates suppressive effects of ß-amyloid peptide (1-42) on long-term potentiation in the hippocampus.

ß-Amyloid peptide 1-42 in a concentration of 200 nM impairs induction of long-term posttetanic potentiation of population spike in CA1 pyramidal neurons in rat hippocampal slices. Application of donepezil, a drug used for the treatment of Alzheimer disease, in a concentration of 1 µM eliminates the suppressive effect of ß-amyloid peptide 1-42 on long-term posttenanic potentiation in the hippocampus.

Interaction of acetylcholinesterase inhibitor donepezil with ionic channels of the neuronal membrane.

The effects of donepezil on voltage-dependent Ca(2+)- and low-threshold K(+)-current were studied on isolated molluscan neurons using two-electrode voltage clamp technique. Donepezil reduced the amplitude of voltage-dependent Ca(2+)-current (IC(50)=7.9 microM) and shifted the current-voltage relationships toward hyperpolarization. Donepezil in low concentration (5 microM) increased, while in higher concentrations (>or=10 microM) decreased the low-threshold K(+)-current.

[Memory and potassium channels]. / Pamiat' i kalievye kanaly.

The K(+)-channels of the surface membrane play a crucial role in the generation of electrical activity of a neuron. There is a large diversity of the K(+)-channels that depends on a great number (over 200) of genes encoding channels proteins. An evolutionary conservation of channel's proteins is determined. The K(+)-channels were found to have a great importance in the memory processes. It was shown on different model systems that K(+)-current of the surface membrane decreases during the learning. The antagonists of K(+)-channels were found to improve the learning and memory. It was revealed in electrophysiological experiments that K(+)-channels antagonists can either themselves induce a long-term synaptic potentiation or intensify the synaptic potentiation induced by a tetanization. The disfunction of K(+)-channels is believed to be an important link in the mechanisms of memory disturbances. In animal mutants with K(+)-channels disfunction, learning and memory are deficient. In behavioral experiments, the use of K(+)-channels openers make the learning worse. Amnesia caused by cerebral ischemia is explained by strong activity of K(+)-channels which not only inhibits neuronal excitement but also causes neurodegeneration. The question on the K(+)-channels involvement into pathophysiology of Alzheimer's disease is discussed. Neurotoxic peptide beta-amyloid, which is supposed to be involved into mechanisms of Alzheimer's disease, modulates K(+)-channels function. The effect of beta-amyloid depends on the subtype of K(+)-channels: A-channels are inhibited, and KDR-channels, on the contrary, become stronger. The effect of the cognitive enhancers (vinpocetine, piracetam, tacrine, linopirdine) on K(+)-current also depends on the subtype of K(+)-channels. Slow-inactivating K(+)-currents (IDR, IK(Ca), IM) are inhibited in the presence of these drugs, while fast-in-activating K(+)-current (A-current) remains unchanged or even increases.

Decreases in Ca2+-dependent K+-currents due to cyclic guanosine monophosphate are not dependent on phosphorylation.

Two-microelectrode voltage clamping experiments were performed on isolated snail neurons to measure the Ca2+-dependent. potential-dependent K+ current (I(C)), with assessment of the effects of penetrating cGMP analogs on this current - dibutyryl cGMP (dcGMP) and 8-Br-cGMP. Both of these penetrating cGMP analogs rapidly and reversibly decreased the amplitude of I(C). cGMP analogs produced no shifts in the volt-ampere characteristics of the efflux current along the voltage axis. dcGMP and 8-Br-cGMP had no effect on the influx Ca2+ current. The non-specific protein kinase inhibitor H-8 decreased or had no effect on I(C) in different cells. The effects of both dcGMP and 8-Br-cGMP persisted in the presence of H-8. Decreases in I(C) in the presence of cGMP analogs also persisted in the presence of the protein phosphatase inhibitor okadaic acid. These results lead to the conclusion that decreased conductivity of Ca2+-dependent K+ channels occurring in response to cGMP is not associated with phosphorylation.

The nootropic drug vinpocetine modulates different types of potassium currents in molluscan neurons.

Three types of high-threshold K+ currents were recorded in isolated neurons of the snail Helix pomatia using a two-microelectrode voltage clamp technique: transient K+ current (I(A)), delayed rectifier (I(KD)) and Ca2+-dependent K+ current (I(K(Ca))). Vinpocetine (1-100 microM) applied to the bath affected different types of K+ current in different ways: I(A) was increased (35+/-14%), I(KD) was moderately inhibited (20+/-9%) and I(K(Ca)) was strongly suppressed (45+/-15%). When I(A) and I(K(Ca)) were present in the same cell, vinpocetine exerted a dual effect on the total K+ current, depending on the amplitude of the test stimulus. In the presence of vinpocetine, the I-V curve crossed the control I-V curve. The inhibition of I(K(Ca)) by vinpocetine between 1 and 100 microM is unlikely to be a result of Ca2+ current (I(Ca)) suppression, as the latter was inhibited only at vinpocetine concentrations exceeding 300 microM. Dibutyryl cyclic GMP (dbcGMP) (but not dbcAMP) mimicked the effects of vinpocetine in the majority of cells tested (coefficient of correlation r=0.60, P<0.05, n=22). The data suggest that modulation of different types of K+ current in neuronal membrane can contribute, at least partially, to the nootropic effect of vinpocetine through the regulation of intracellular Ca2+ concentration.

Enhancement of low-threshold A-current of the neuronal membrane by vinpocetine.

A low-threshold fast inactivating K+ current (I(Alth)) was recorded in isolated land snail neurons using the two-microelectrode voltage clamp method. A nootropic drug, vinpocetine, applied to the extracellular medium at a concentration of 1-100 microM potentiated I(Alth). The potentiation consisted in a rise of its peak amplitude and an increase of the half-decay time. Vinpocetine did not cause a shift of the steady-state activation and inactivation curves along the potential axis. Dibutyryl cyclic GMP (dcGMP) also increased the peak amplitude but did not change the time of current half-decay. dcAMP did not potentiate I(Alth). The possible role of K+-current potentiation in neuronal membranes in therapy of dementia is discussed.

Cyclic GTP imitates the potentiating effect of the nootrope vinpocetine on the high-threshold A-current in mollusk neurons.

Isolated common snail neurons were studied using two-microelectrode potential clamping to record high-threshold (threshold = 10 mV) rapidly-inactivating potassium current (I(Aht)); the effects of the nootrope vinpocetine on this current were studied and were compared with the effects of cyclic nucleotides. Intracellular application of dibutyryl derivatives of cyclic adenosine monophosphate (dcAMP) and guanosine monophosphate (dcGMP) was used. The results showed that vinpocetine potentiates or fails to alter I(Aht) in different cells, while dcGMP imitates the effect of vinpocetine. Simultaneous application of vinpocetine and dcGMP did not result in additive effects. Unlike dcGMP, dcAMP did not imitate the effects of vinpocetine, and decreased I(Aht) in most cells. These data suggest that cGMP mediates the potentiating effect of vinpocetine on I(Aht).