The actions of the nootropes noopept and piracetam on depression of the acetylcholine-induced current in a cellular analog of habituation were studied. Mathematical modeling of experimental curves and their analysis using previously obtained results on the effects of inhibitors of different protein kinases and protein phosphatases on acquisition of depression of the acetylcholine-induced current in a cellular analog of habituation clarified the intracellular processes and targets on which these agents act.

The nootropic agents noopept and piracetam alter the amplitudes of acetylcholine-induced influx currents (ACh currents) in command neurons in the common snail. Both compounds have cholinopositive activity. The dose curve of the actions of noopept is bell-shaped, while the piracetam dose-response curve in the range of physiological concentrations shows a monotonous rise. Noopept increases the ACh current at low concentrations (10–10–10–8 M), while piracetam acts at significantly higher concentrations (starting from 10–4 M). The magnitudes of the maximal cholinopositive effects of noopept and piracetam (in the range of physiological concentrations) were identical, while the concentrations of nootropic drugs at which they were reached differed by seven orders of magnitude. The half-maximal concentration (EC50) of noopept was 10–10 M and that of piracetam was 10–3 M. The mechanisms of the cholinopositive actions of these drugs are discussed.

Abstract—Possible causes of the positive modulating effect of noopept (in a concentration range of 0.1 to 10 nM) on the amplitude of the acetylcholine-induced input current are analyzed using a developed mathematical model. The results revealed that the calculated and experimental current curves coincide when the number of nicotinic acetylcholine receptors on the neuron membrane is increased or the efficiency of muscarinic acetylcholine receptors is changed.

Nootropic drugs (Noopept and Piracetam) change amplitude of the inward acetylcholine-induced current (ACh-current) in command Helix neurones. Both drugs show cholinopositive action. A dose curve of influence of the Noopept is bell-shaped, and curve dose-effect in the field of physiological concentration of Piracetam is monotonously increasing. Noopept increases ACh - current at low concentrations (10(-10) - 10(-8) M), and Piracetam made it at much high concentrations (since 10(-4) M). Maximal cholinopositive effects of Noopept and Piracetam (in the field of physiological concentrations) are identical while concentration of nootropic drugs at which they are reached, differ on 7 orders. Semi-maximal concentration (EC50) of the Noopept was 10(-10) M, and EC50 of Piracetam was 10(-3) M. The conclusion was made about the mechanisms of cholinopositive effect of drugs.

The experimental relationship between the paired-pulse ratio of acetylcholine-induced inward currents in the command neurons of Helix lucorum and the interval between application of a neurotransmitter was analyzed using the method of paired-pulse stimulation. A mathematical model that takes the membrane and intracellular localization of receptors, their lateral diffusion, and endocytosis and exocytosis depending on the intracellular processes into account was used to study this dependence. Our results have shown that membrane receptor endocytosis is the main process that determines the type of this relationship. The effects of exocytosis and lateral diffusion of membrane receptors on this relationship were studied. The parameters of an existing mathematical model were further clarified.

The presence of "comet-like" radial transport of acetylcholine receptors by actin microfilaments without the participation of myosin motors in the depression of acetylcholine-induced inward chloric current (ACh-current) in command neurons of defensive behavior of the land snail, Helix lucorum, in a cellular analog of habituation was investigated. For that purpose the effects of CK548, CK-636 (inhibitors of actin-related protein complex Arp2/3, whose activation triggers rapid actin polymerization and the formation of the "comet-like" tail on the actinic filament) and wiskostatin (an N-WASP protein inhibitor, activating Arp2/3) on the depression of ACh-current were studied. The attenuation of ACh-current depression was observed upon the addition of CK548. At the same time, CK-636 and wiskostatin irreversibly strengthened the depression of this current and suppressed its spontaneous recovery. The results of CK-548 action and its mathematical modeling allow suggesting the presence of "comet-like" transport of acetylcholine receptors, initiated by Arp2/3 protein complex in receptor endo and exocytosis in command neurons of Helix lucorum in a cellular analog of habituation. Irreversible inhibition of vital metabolic processes of the neuron by wiskostatin and CK-636, which lead to the decrease in the level of ATP, could have caused irreversible effects of these blockers on current depression.

The effects of inhibitors of a series of serine-threonine phosphatases, i.e., okadaic acid (which suppresses PP1 and PP2A phosphatase activities), endothall (PP2A), cyclosporin A and cypermethrin (PP2B), CCT007093 (PPM1D), and dephostatin (blocks tyrosine phosphatases), on the depression and spontaneous recovery of the ACh-induced influx current in defensive behavior command neurons in the common snail were studied in a cellular analog of habituation. All the inhibitors used here altered the dynamics of depression of the current, while endothall also slowed spontaneous recovery of the ACh current. The results obtained here provide evidence of the relationship between changes in defensive behavior command neuron membrane cholinosensitivity in the common snail in a cellular analog of habituation on the one hand and the activities of all the protein phosphatases studied on the other. Application of a mathematical model addressing the possibility that receptors have different cellular locations suggested that these phosphatases are involved in the mobility (endocytosis and exocytosis) of the membrane cholinoreceptors responsible for changes in the ACh current in the cellular analog of habituation. Comparison of the experimental data and simulation-derived calculated curves of changes in the ACh current showed that the main target of protein phosphatases is the neuron’s transport system, i.e., the cytoskeleton and motor proteins.

Using mathematical model a comparative analysis of the influence of receptor lateral diffusion, endocytosis and exocytosis of receptors on the change in the number of membrane receptors at rhythmical local applications of a mediator on neural soma was performed. The results allow us to estimate quantitatively the effect of these processes on the length of the period between mediator applications. The necessity of considering the change in the rate of receptor lateral diffusion, while studying the effect of some protein kinases and protein phosphatases on the change in the number of receptors, became evident. The model also provided the effect of “after stimulus efficiency.”

We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

Effects of some inhibitors of serine/threonine and tyrosine protein phosphatases on the depression and spontaneous recovery of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior at the cellular correlate of habituation were investigated. The following drugs were used: okadaic acid (reduces activity ofphosphatases PP1 and PP2A), endothall (PP2A), cyclosporine A and cypermethrin (PP2B), CCT007093 (PPM1D), dephostatin (blocks tyrosine phosphatases). All used inhibitors modify the depression flow, and endothall reduces spontaneous recovery of ACh-current also. Obtained results indicate that changes in cholinosensitivity of command neurons depend on activity of all investigated protein phosphatases. Mathematical model considers the possibility of different localizations of receptors in a neuron and regularity of transitions between them. This model makes it possible to conclude participation indicated phosphatases in mobility of membrane cholinoreceptors ensuring the ACh-current modification at the cellular correlate of habituations. Comparison of experimental and calculated curves of ACh-current change allows to conclude that the main target of protein phosphatases is the transport system of a neuron--cytoskeleton and motor proteins.

Using mathematical model a comparative analysis of the influence of receptor lateral diffusion, endocytosis and exocytosis of receptors on the change in the number of membrane receptors at rhythmical local applications of a mediator on neural soma was performed. The results allow us to estimate quantitatively the effect of these processes on the length of the period between mediator applications. The necessity of considering the change in the rate of receptor lateral diffusion, while studying the effect of some protein kinases and protein phosphatases on the change in the number of receptors, became evident. The model also provided the effect of "after stimulus efficiency".

Effects of some inhibitors of serine/threonine and tyrosine protein phosphatases on the depression and spontaneous recovery of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior at the cellular correlate of habituation were investigated. The following drugs were used: okadaic acid (reduces activity ofphosphatases PP1 and PP2A), endothall (PP2A), cyclosporine A and cypermethrin (PP2B), CCT007093 (PPM1D), dephostatin (blocks tyrosine phosphatases). All used inhibitors modify the depression flow, and endothall reduces spontaneous recovery of ACh-current also. Obtained results indicate that changes in cholinosensitivity of command neurons depend on activity of all investigated protein phosphatases. Mathematical model considers the possibility of different localizations of receptors in a neuron and regularity of transitions between them. This model makes it possible to conclude participation indicated phosphatases in mobility of membrane cholinoreceptors ensuring the ACh-current modification at the cellular correlate of habituations. Comparison of experimental and calculated curves of ACh-current change allows to conclude that the main target of protein phosphatases is the transport system of a neuron--cytoskeleton and motor proteins.

The involvement of cytoskeletal motor proteins, i.e., myosins, in the molecular mechanism of depression of the acetylcholine sensitivity of defensive behavior command neurons was studied in the common snail. Thus, the effects of compounds impairing myosin functioning on ACh-evoked current depression curves in neurons were studied – the myosin light chain kinase blockers ML-7 and MLCK-IP-18, the non-muscle myosin II inhibitor blebbistatin, and the ROCK-I and ROCK-II kinase (which mainly activate non-muscle myosin II) inhibitor Y-27632. ML-7 and MLCK-IP-18 were found to weaken depression of the current, while blebbistatin and Y-27632 had no effect on depression. The experimental results and mathematical models of the effects of these blockers on the number of membrane-bound cholinoreceptors suggest the involvement of myosins (except non-muscle myosin II) in the endo- and exocytosis of cholinoreceptors and the resultant depression of the cholinosensitivity of the somatic membranes of neurons in a cellular analog of habituation.