Atypical Acetylcholine Receptors on the Neurons of the Turkish Snail.

Using electrophysiology, the effect of nicotinic acetylcholine receptor (nAChR) ligands on acetylcholine-induced depolarization in the neurons of Helix lucorum snail was studied. It was found that the α-conotoxin PnIA [R9, L10], a selective antagonist of α7 nAChR, and α-cobratoxin (antagonist of α7 and muscle-type nAChR) suppressed neuronal depolarization. Fluorescence microscopy showed staining of the neurons with fluorescently labeled α-bungarotoxin; this staining was reduced by pretreatment with α-cobratoxin. Induced depolarization was also suppressed by α-conotoxin RgIA, a selective inhibitor of α9 nAChR. In contrast to Lymnaea stagnalis nAChR, which are weakly sensitive to neurotoxin II and α-conotoxin GI, antagonists of muscle-type nAChR, H. lucorum receptors were most effectively inhibited by these antagonists. The results obtained, as well as the previously found sensitivity of the receptors studied in this work to muscarinic receptor ligands, indicate an unusual atypical pharmacological profile of H. lucorum nAChR.

[Motor Proteins of Microtubules and Mechanisms of Synaptic Plasticity].

Motor proteins of microtubules, kinesin and dynein superfamily proteins play an important role in the intracellular transport. Inside a neuron they are involved in the transport of organelles, proteins and mRNAs along the axons and dendrites to the nerve terminals and back to the cell bodies. Disturbance of axonal transport may affect neurotransmitter release and short-term presynaptic plasticity. Disturbance of dendritic transport, in particular the recycling of synaptic receptors, affects postsynaptic plasticity. The review attempts to trace the connections between the motor proteins of microtubules and mechanisms of synaptic plasticity from the perspective of their involvement in the intracellular transport of proteins and organelles, which play role in the mechanisms of synaptic plasticity.

[Spontaneous EPSPs in Command Helix Lucorum Neurons during Geterosynaptic Potentiation].

To develop the presynaptic mechanism of the short-term plasticity of synaptic transmission the influence of the rhythmical orthodromic stimulation of intestinal nerve on the spontaneous excitatory postsynaptic potentials (EPSPs) have been analyzed in the command neurons of the defensive behavior of land snail Helix lucorum. It was shown early that the tetanic stimulation induced the short-term potentiation of evoked EPSPs. The rhythmical stimulation did not influence the amplitude of spontaneous EPSPs, but considerably increased their number. Discovered augmentation of the frequency of spontaneous EPSPs makes it possible to assume the participation of presynaptic mechanism in the short-term potentiation of synaptic transmission. The presynaptic mechanism can include reduction in the thresh- old of action potentials (APs) generation in the presynaptic neurons, which will lead to an increase in the fraction of spontaneous active neurons and, correspondingly, it will increase the number of spontaneous presynaptic APs.

[Role of serine/threonine and tyrosine protein phosphatases in command Helix lucorum neurons at the cellular correlate 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.

[Role of myosins in depression of sensitivity of Helix neurons to acetylcholine in a cellular analog of habituation].

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.

Cholinergic receptor exocytosis under conditions of depression of acetylcholine-induced current in edible snail neurons in cellular analogue of habituation.

Exocytosis inhibitor Exo 1 potentiates depression of acetylcholine-induced inward current in defensive behavior command neurons of edible snail, when acetylcholine is applied rhythmically to the soma in cellular correlate of habituation. A mathematical model presupposing different receptor localization in the cell and regularities of their translocations made it possible to analyze the dependence of acetylcholine-induced current depression on a number of intracellular processes. It was concluded that depression of choline-sensitive extrasynaptic zones on the membrane of defensive behavior command neurons in edible snail in cellular correlate of habituation is partially determined by attenuation of exocytosis of internalized cholinergic receptors.

[Role of actin microfilaments in depression of acetylcholine-induced current in Helix lucorum neurons on cellular analogue of habituation].

Toxins that impair the function of actin microfilaments in cytoskeleton, cytochalasin B (disrupts microfilaments by inhibiting actin polymerization) and phalloidin (binds polymeric F-actin, stabilizing it and interfering with the function of actin-rich structures) reduce the depression of acetylcholine-induced inward current in Helix lucorum command neurons of defensive behavior during rhythmical local acetylcholine applications to soma (cellular analogue of habituation). These results and mathematical simulation allow us to suggest that the depression of cholinosensitivity of extrasynaptic membrane zones in command neurons on the cellular analogue of habituation is associated with the involvement of actin microfilaments in reduction of the number of membrane cholinoreceptors.

[The role of serine/threonine and tyrosine protein kinases in the depression of cholinosensitivity in Helix lucorum neurons in the cellular correlate of habituation].

Inhibitor ofadenylate cyclase (SQ 22,536) and inhibitors ofserin/threonine protein kinases A (PKA -Rp-cAMPS), G (PKG - H-Arg-Lys-Arg-Ala-Arg-Lys-Glu-OH), calcium/calmodulin-dependent kinase II (CaMKII - KN-93), p38mitogen-activated (MAPK - PD 169316), and tyrosine protein kinases (genistein), including their Src-family (PP2), weaken the depression of the acetylcholine-induced inward current (ACh-current) in command Helix neurons of defensive behavior under conditions of rhythmical local acetylcholine applications to the soma in the cellular analogue of habituation. Selective inhibitor of protein kinase C (PKC - chelerythrine) does not change the depression of the ACh-current. Mathematical simulation of the influence of the inhibitors applied on a number of membrane-connected acetylcholine receptors made it possible to obtain the design curves consistent with the experimental curves of the ACh-current depression. The experimental data and the results of calculations allowed us to make the following assumptions. The reversible depression of sensitivity to ACh of command Helix neurons of defensive behavior in the cellular correlate of habituation depends on the decrease in the number of membrane-connected ACh receptors as a result of activation of several serine/threonine protein kinases: A, G, CaMKII, p38 MAPK (without the participation of PKC), and tyrosine protein kinases including the family of Src-kinases. The main targets of all protein kinases under study (excluding PKC) in command neurons are the proteins of cytoskeleton (actin microfilaments and microtubules). Phosphorylation of these proteins evokes polymerization and stabilization ofactin microfilaments, stabilization of the main microtubule protein tubulin, a change in the activity of motor proteins responsible for the speed of receptor endocytosis and exocytosis. The PKG action is indirect via the modification of actin-myosin interaction. Protein kinase A, CaMKII, and tyrosine Src-kinase phosphorylate also proteins activating receptor translocation into clathrin-coated membrane invaginations during endocytosis.

[Endocytosis of cholinoreceptors in the mechanism of depression of cholinosensitivity in Helix lucorum neurons in a cellular model of habituation].

Inhibitors of dynamin-dependent endocytosis (dynamin inhibitory peptide and dynasore) and inhibitors of tubulin (colchicine and vinblastine) decrease the depression of acetylcholine-induced inward current in command neurons of Helix defensive behavior under conditions of rhythmical local applications of acetylcholine to a neuron soma in a cellular model of habituation. Mathematical model which allows for the possibility of different localizations of receptors in a neuron and characteristics of receptor travel makes it possible to analyze the dependence of the current depression on some intracellular processes. We suggest that the cholinosensitivity depression of extrasynaptic membrane zones in command neurons of Helix defensive behavior in the cellular model of habituation under study is associated with dynamin-dependent endocytosis of cholinoreceptors with involvement of cytoskeleton microtubules.

Heterosynaptic potentiation of cholinergic excitatory postsynaptic responses of command neurons in the common snail.

Short-term heterosynaptic potentiation of cholinergic excitatory postsynaptic currents and potentials in defensive behavior command neurons was found to be evoked by stimulation of the visceral sac in the common snail. It is suggested that a mechanism increasing the choline resistance of the postsynaptic zones of command neuron membranes is involved in potentiating the excitatory postsynaptic responses to sensory stimulation.

Effects of protein synthesis inhibitors on the sensitization of a defensive response in common snails and potentiation of the cholinosensitivity of command neurons.

The effects of protein synthesis inhibitors on short-term sensitization of a defensive reaction in common snails and the potentiation of the cholinosensitivity of command neurons were studied. The protein synthesis inhibitor anisomycin did not prevent behavioral sensitization. Anisomycin and the irreversible protein synthesis inhibitor saporin changed the dynamics of potentiation of command neuron cholinosensitivity. We suggest that the sensitization of the defensive response of the common snail studied here does not require the synthesis of new proteins.

Role of cholinoceptors recycling in short-term potentiation of cholinosensitivity of command neurons in edible snail.

Exocytosis inhibitor Exo 1, dynamin inhibitory peptide (inhibitor of endocytosis), and colchicine disturb short-term potentiation of cholinosensitivity of defensive behavior command neurons in edible snail induced by rhythmic electrical orthodromic stimulation. We hypothesize that the short-term potentiation of cholinosensitivity in the extra-synaptic membrane develops due to incorporation of extra cholinoceptors into neuron plasmalemma as a result of enhanced recycling of the internalized cholinoceptors with participation of microtubules.

[Heterosynaptic potentiation of cholinergic excitatory postsynaptic responses in the Helix command neurons].

Heterosynaptic potentiation of cholinergic excitatory postsynaptic currents and potentials evoked by electrical stimulation of visceral mass was discovered in command Helix neurons of escape reaction. The results suggest the involvement of mechanism of an increase in cholinosensitvity in postsynaptic membrane zones in potentiation of excitatory postsynaptic responses to sensory stimulation.

[Effect of protein synthesis inhibitors on sensitization of defence reaction and on cholinosensitivity of command neurons in Helix lucorum].

We studied influence of protein synthesis inhibitors on short-term sensitization of Helix escape reaction and potentiation cholinosensitivity in command neurons. Inhibitor of protein synthesis anisomycin does not prevent behavioral sensitization. Anisomycin and irreversible inhibitor of protein synthesis saporin change the dynamics of cholinosensitivity potentiation in command neurons. The results Suggest that investigated sensitization of Helix escape reaction does not require synthesis of new proteins.

Methiothepin-sensitive serotonin receptors are involved in the postsynaptic mechanism of sensitization of the defensive response in the common snail.

Rhythmic electrical stimulation of the snail foot leads to sensitization of the defensive reflex. This sensitization has dynamics similar to those of posttetanic potentiation of the amplitude of the acetylcholine-evoked influx current of defensive behavior command neurons in the common snail. It is likely that an increase in the cholinosensitivity of the somatic membrane of defensive behavior command neurons in the common snail may be involved in the mechanism of sensitization of the animal's defensive response. Methiothepin, an antagonist of serotonin receptors, prevented the posttetanic potentiation of the acetylcholine-evoked influx current as well as behavioral sensitization. Serotonin, like methiothepin, also impaired posttetanic potentiation of the acetylcholine-evoked influx current. It is suggested that methiothepin-sensitive serotonin receptors are involved in the postsynaptic mechanism of behavioral sensitization.

Cholinergic sensory inputs to command neurons in edible snail.

We studied cholinergic component of visceral sensory input to defensive behavior command neurons in edible snail. Nicotinic receptor antagonist tubocurarine and muscarinic receptor antagonist atropine reversibly decreased the amplitude of the total excitatory postsynaptic potential induced by electrostimulation of the peripheral region in the mechanosensory receptor field of command neurons on the surface of internal organs. Our results indicate that acetylcholine is involved in sensory signal transduction from the visceral sac to command neurons of snail parietal ganglia. The subsynaptic membrane of visceral synaptic input contains nicotinic and muscarinic receptors.

[Methiothepin-sensitive serotonin receptors are involved in postsynaptic mechanism of sensitization of Helix lucorum escape reaction].

Tetanic electric stimulation of Helix foot evokes sensitization of escape reaction. This behavioral sensitization and posttetanic potentiation (PTP) of acetylcholine-induced inward current (ACh-current) in command Helix neurons of escape behavior were similar. Antagonist of serotonin receptors methiothepin prevents the PTP of the ACh-current and behavioral sensitization. Serotonin disrupts the PTP of the ACh-current. It is suggested that the increase in cholinosensitivity of the command neurons with the involvement of methiothepin-sensitive serotonin receptors may be the cellular postsynaptic mechanism of behavioral sensitization of Helix escape reaction.

Inositol triphosphate and ryanodine receptors in the control of the cholinosensitivity of common snail neurons by the Na,K pump during habituation.

The effects of the Na,K pump inhibitor ouabain on habituation of the common snail to tactile stimulation were identical to the ouabain-induced modification of the decrease in the cholinosensitivity of defensive behavior command neurons in the common snail in a cellular model of habituation. Studies addressed the effects of intracellularly delivered ligands of two types of Ca2+ depot receptors--inositol triphosphate (IP3) receptors and ryanodine receptors--on the action of ouabain in the cellular analog of habituation. The IP3 receptor antagonist heparin (0.1 mM), the IP3 receptor agonist inositol triphosphate (0.1 mM), and the ryanodine-dependent Ca2+ mobilization inhibitor dantrolene (0.1 mM) prevented ouabain from modifying the depression of the evoked acetylcholine current. The ryanodine agonist/antagonist ryanodine was used at two concentrations (0.1 and 1 mM) and neither had any effect on the action of ouabain. It is concluded that Ca2+ mobilized from intracellular Ca2+ depots via IP3 receptors is involved in the neuronal mechanism of regulation of the habitation of the common snail to tactile stimulation by the Na,K pump.

[Inositoltriphosphate receptors and ryanodine receptors in regulation of cholinosensitivity of Helix lucorum neurones by Na,K-pump during habituation]. / Retseptory inozitoltrifosfata i riadinovye retseptory v reguliatsii Na,K-nasosom kholinochuvstvitel'nosti neironov vinogradnoi ulitki pri privykanii.

Influence of ouabain, the inhibitor of Na,K-pump, on habituation of Helix to tactile stimulation was identical to the ouabain-induced modification of cholinosensitivity reduction in command neurones of defensive behaviour of Helix lucorum in cellular model of habituation. Effects of intracellularly injected ligands of two types of Ca2+ -depot receptors, inositoltrisphosphate (IP3) and ryanodine receptors, on ouabain-induced changes were studied in cellular model of habituation. The antagonist of IP3 receptors heparin (0.1 mM), their agonist IP3 (0.1 mM) and inhibitor of ryanodine-dependent Ca2+ mobilization dantrolen (0.1 mM) prevented the depression of acetylcholine-induced current from the ouabain-evoked modification. The agonist/antagonist of ryanodine receptors ryanodine at two tested concentrations (0.1 mM and 1 mM) did not change the ouabain effect. It is concluded that Ca2+ released from intracellular Ca2+ -depots via IP3 receptors is involved into neuronal mechanism of Na,K-pump regulation of habituation in Helix lucorum to tactile stimulation.

An apparatus for recording defensive responses to tactile stimulation in the ground snail.

We describe here an original working experimental apparatus for non-invasive objective recording of the magnitudes of defensive responses to tactile stimulation in the ground snail. The apparatus includes a tracking device that ensures that the snail's position relative to a light and photodiode remains constant as it ascends a cylinder rotating about a horizontal axis. The device providing tactile stimulation is based on a loudspeaker circuit and provides blows of dosed energy. The snail responds to the tactile stimulation by retracting the antennae, head, and foot, which decreases the area of its shadow; this is recorded by a photodiode.