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.     

Postsynaptic mechanism of withdrawal reflex sensitization in the snail

Repeated tactile stimulation of the skin evoked sensitization and subsequent habituation of a behavioral avoidance reaction of pneumostome closure in the land snail, Helix lucorum L. Spike responses in putative command neurons for avoidance behavior increased and subsequently decreased parallel to behavioral events. Decrease of spike responses in command neurons was due to decreased synaptic input evoked by repeated tactile stimulation. Intracellular investigation of the increase in the spike response in command neurons for pneumostome closure, which underlies behavioral sensitization, revealed a synpatically-evoked, long-lasting depolarization. No conductance changes during this depolarization were found, but neuronal excitability was altered.     

Mobility of acetylcholine receptors in command Helix lucorum neurons in a cellular analog of habituation

We investigated the role of the mobility of acetylcholine receptors in the depression of an acetylcholine-induced inward current (ACh-current) of Helix lucorum (a land snail) command neurons of defensive behavior in a cellular analog of habituation. The inhibitors of endocytosis and exocytosis, actin microfilaments and cytoskeleton microtubules, serine/threonine protein kinases (PKA, PKG, calcium calmodulin-dependent PK II, p38 mitogen-activated PK), tyrosine kinases (including Src-family kinases), serine/threonine phosphatases (PP1, PP2A, PP2B, PPM1D), and tyrosine protein phosphatases altered the depression of the ACh-current. A comparison of experimentally calculated curves of the ACh-current of these neurons and those obtained by mathematical modeling revealed the following: (a) ACh-current depression is caused by the reduction in the number of membranous ACh-receptors, which results from the shift in the balance of multidirectional transport processes of receptors toward the predominance of ACh-receptor internalization over their recycling; (b) depression of ACh-current depends on the activity of serine/threonine and tyrosine protein kinases and protein phosphatases, whose one of the main targets is the neuron transport system—actin microfilaments and microtubules of cytoskeleton, as well as motor proteins.     

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.     

Humoral factor dependence of neuronal post-theta choline-sensitive potentiation in Helix Lucorum

Comparison of posttetanic changes of the acetylcholine-induced inward current (ACh-current) in command Helix lucorum neurones at different conditions (using stop flow of saline through the chamber with a ganglia preparation and using flow of saline) was made. Flow of saline reduces latency and degree of posttetanic increase of the ACh-current in neurones. Earlier and weak posttetanic potentiation of the ACh-current in command Helix lucorum neurones during flow of saline through the chamber with a ganglia preparation testifies to participation of the humoral factor in the mechanism of posttetanic potentiation of cholinosensitivity of somatic membrane in postsynaptic neurone.     

Ca-dependent regulation by Na, K-pump of post-tetanic sensitization of extrasynaptic choline receptors in Helix lucorum neurons

Posttetanic potentiation (by orthodromic stimulation) of cholinosensitivity in LPa3 and RPa3 Helix lucorum neurons that are command in respect to withdrawal behavior was shown earlier (Pivovarov et al., 1999). Now we studied the regulatory role of the Na,K-pump and intracellular free Ga2+ in the posttetanic potentiation (PTP) of cholinosensitivity in command neurons. Semiintact Helix preparation "CNS-visceral bag" was used in experiments. Acetylcholine-induced inward currents were recorded using two-electrode voltage clamp technique. Acetylcholine was applied to somata of the identified LPa3 and RPa3 neurons with a 10-min interval before and after electrical tetanic stimulation of the n. intestinalis (10.5 mA; 0.1 s; 2/s; 2 min). Ouabain (extracellular application, 70 mcM) blocked the PTP. Intracellular injection of BAPTA (1 mM), chelator of Ca2+ ions, prevented the PTP. The PTP was absent after the ouabain application against the background of preliminary intracellular injection of BAPTA. A conclusion war drawn about Ca-dependent participation of Na,K-pump in posttetanic potentiation of cholinosensitivity in command Helix lucorum neurons of withdrawal behavior.     

Involvement of Na,K-pump in SEPYLRFamide-mediated reduction of cholinosensitivity in Helix neurons

SEPYLRFamide acts as an inhibitory modulator of acetylcholine (ACh) receptors in Helix lucorum neurones. Ouabain, a specific inhibitor of Na,K-pump, (0.1 mM, bath application) decreased the ACh-induced inward current (ACh-current) and increased the leak current. Ouabain decreased the modulatory SEPYLRFamide effect on the ACh-current. There was a correlation between the effects of ouabain on the amplitude of the ACh-current and on the modulatory peptide effect. Ouabain and SEPYLRFamide inhibited the activity of Helix aspersa brain Na,K-ATPase. Activation of Na,K-pump by intracellular injection of 3 M Na acetate or 3 M NaCl reduced the modulatory peptide effect on the ACh-current. An inhibitor of Na/Ca-exchange, benzamil (25 muM, bath application), and an inhibitor of Ca(2+)-pump in the endoplasmic reticulum, thapsigargin (TG, applied intracellularly), both prevented the effect of ouabain on SEPYLRFamide-mediated modulatory effect. Another inhibitor of Ca(2+)-pump in the endoplasmic reticulum, cyclopiazonic acid (applied intracellularly), did not prevent the effect of ouabain on SEPYLRFamide-mediated modulatory effect. These results indicate that Na,K-pump is responsible for the SEPYLRFamide-mediated inhibition of ACh receptors in Helix neurons. Na/Ca-exchange and intracellular Ca(2+) released from internal pools containing TG-sensitive Ca(2+)-pump are involved in the Na,K-pump pathway for the SEPYLRFamide-mediated inhibition of ACh receptors.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 microM; bath application), which modifies ryanodine-controlled Ca(2+) channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 microM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 microM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones.     

The selective effect of NMDA glutamate receptor antagonist on plasticity of sensory inputs from chemoreceptors of snail head during nociceptive sensitization

The effects of N-methyl-D-aspartate (NMDA) glutamate receptor antagonist (+)-MK-801 hydrogen maleate (MR801) on plasticity of different sensory inputs of the L-RPl1 command neurons were studied in Helix lucorum snail during nociceptive sensitization. Application of sensitizing stimulation onto the snail head or foot in the control semi-intact preparation initiated depression of neural responses evoked by tactile or chemical sensory stimulation during the short-term period of sensitization and significant facilitation of neural responses during the long-period of sensitization. Sensitizing stimulation of snail head against the background of MK-801 application (10-30 microM) produced a pronounced depression of neural responses to chemical stimulation of the head both in the short- and long-term sensitization periods. At the same time, sensitizing stimulation of the foot or head during the MK-801 application produced the same changes in neural responses to chemical stimulation of the foot and tactile stimulation of the foot or head as in the control preparation. It can be suggested that NMDA-like glutamate receptors are selectively involved in the mechanisms of plasticity induction in the synaptic inputs of the command LPl1 and RPl1 neurons, which process the information resulting from chemical excitation of the snail head (a specific receptor skin site for these neurons in Helix lucorum).     

Selective effect of the antibody to protein SMP-69 on activity of the defence behavior command neurons in grape snail

Effects of antibody against serotonin-modulated protein SMP-69 on defence behavior command neurons L-RP11 were studied in semi-intact preparation of snail Helix lucorum. An increase in membrane excitability as well as selective facilitation of neural responses evoked with chemical sensory stimulation of the snail head (0.25-0.5% quinine solution) were determined 1-1.5 hours after antibody application to the neurons. The antibody did not change neural responses evoked with tactile stimulation of the snail head. These effects were similar to those found in L-RP11 neurons after serotonin or cAMP applications as well as after nociceptive sensitization of the snail. It was suggested that protein homologically related the SMP-69 in mammalians was involved in mechanisms of excitability as well as long-term specific plasticity regulation of L-RP11 neurons synaptic inputs from the head chemoreceptors in snail Helix lucorum.     

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.     

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 μM; bath application), which modifies ryanodine-controlled Ca²⁺ channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 μM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 μM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones. Accepted: 1 July 1998     

Electrophysiological and neurochemical study of long-term sensitization in the snail

The long-term sensitization of avoidance reflex was produced in snail Helix pomatia, which led to the remarkable increase in the pneumostome closures period. The formation of long-term sensitization is also accompanied by increase in excitability of command neurons of this reflex. One of the possible mechanisms of this phenomenon is the depolarization of these cells. The quantitative redistribution of water-soluble proteins with relative mobility 0.54 0.42 0.40 was also observed in the identified neurons, both included in the avoidance reflex (command neurons) and non-included (bursting neurons, nerve cells of pool D). The protein with the relative mobility of 0.75 was unique for the nerve cells of neurosecretory pool D in sensitized snails, and was never found in control animals.     

Regulation of posttetanic increase in choline sensitivity in Helix neurons by Na,K-pump: the role of Na/Ca-exchange and of mobilized calcium

We studied the role of Na/Ca-exchange and intracellular mobilized calcium in ouabain-mediated suppression of potentiation of cholinosensitivity of somatic membrane in Helix LPa3 and RPa3 command neurons of defensive behaviour after electrical orthodromic tetanisation of n. intestinalis. Cholinosensitivity of neurons was assessed by the amplitude of the inward current evoked by acetylcholine. Inhibitor of a Na/Ca-exchange benzamil and specific inhibitor of Ca-ATPase in endoplasmic reticulum thapsigargin prevented the development of the posttetanic potentiation (PTP). PTP did not arise and at joint action of ouabain with benzamil or thapsigargin. It was concluded that Na/Ca-exchange and mobilized calcium are involved in development of PTP of cholinosensitivity in somatic neuronal membrane and its regulation by Na,K-pump.     

Conditioning and sensitization in the edible snail: the neurophysiological and metabolic characteristics]

Electrophysiological parameters and bound calcium (Ca(b)) level dynamics during sensitization development or conditioning of food aversion were studied in the command neurons of defense behaviour in the snail Helix lucorum. Responses evoked by a testing sensory stimulus were facilitated 50-60 min after the first sensitizing stimulation, while conditioned responses appeared 80-90 min after the first conditioning. It was the most essential electrophysiological difference between the long-term sensitization and conditioning. Analysis of the Ca(b)) dynamics in the neurons showed significant differences in calcium-dependent metabolism during the sensitization and conditioning, likely underlying the electrophysiological differences.     

Sensitization and habituation of command neurons during a defensive reflex in grape snails

All investigated neurones were classified into functional groups depending on their participation in unconditioned avoidance reflex of pneumostome closure. Habituation was produced by tactile stimuli applied with a frequency of 0.1 c/s. Spike responses of neurons participating in processing sensory information habituated gradually, while reactions of command neurones of avoidance behaviour became sensitized to the second or third stimulus of the series. Behavioral reaction was sensitized in parallel with command neurones in spite of habituation in other types of nerve cells. A conclusion is drawn that behavioral habituation is due to waning of responses in all the participating neurones, but the independnet process of sensitization is due to sensitization of command neurones. Probable mechanisms of neuronal sensitization are discussed.     

The architecture of the reflex arc]

The conceptual reflex arc in Helix is composed of identifiable local detectors, command neurons and modulatory neurons. The identification of neurons opens a perspective for identification of single synapses. The plastic changes within the reflex arc can be characterized as non-associative and associative. The non-associative plasticity (habituation and sensitization) is based on the dephosphorylation and phosphorylation of receptor and channel proteins. The associative plasticity (conditioning and extinction) is based on two levels of molecular modifications: the short-term changes underlined by the phosphorylation and dephosphorylation of receptor and channel molecules and the long-term changes referred to the expression of genes encoding structural and translocating proteins. The very selective receptor and channel modifications are due to the Hebbian plastic synapse supplemented with the principle of nonspecific (modulating) influences.     

Effect of protein synthesis inhibitors on neuronal mechanisms of sensitization inHelix snail

A sensitizing treatment with 5–10% quinine solution causes short-term (lasting 50–70 min) and long-term (lasting several hours) changes in the activity of the command neurons for defensive behavior (LPl1 and PPl1) in the snailHelix lucorum. The short-term effects are characterized by a depolarizing shift in membrane potential, increased excitability, and an initial increase in the content of bound calcium (Ca-c) in the neurons. The long-term effects appear as facilitation of synaptic components of neuronal responses to sensory stimuli without any changes in excitability and in membrane potential, and also as a repeated increase of Ca-c content. Treatment with anisomycin or cycloheximide during sensitization acquirement prevents development of long-term sensitization.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 109–115, March–April, 1993.