Defensive Behavior Command Neurons Research Articles

Changes in Membrane and Threshold Potentials of Command Neurons in Terrestrial Snail during Development of a Conditioned Situational Defensive Reflex.

Changes of the electrical characteristics of command neurons of defensive behavior caused by the development of a conditioned situational defensive reflex were studied experimentally under in vitro conditions on preparations of the nervous system of snails. After learning, the membrane and threshold potentials of command neurons LPa3 and RPa3 significantly decreased and excitability of the studied neurons increased.

Regulation of histone H4 acetylation in the CNS and defensive behavior command neurons of the mollusk Helix mediated by serotonin and neuropeptide FMRFamide

Epigenetic mechanisms are commonly known to underlie memory formation. Presently, scientists’ attention is focused on changes in the levels of histone modifications (mainly acetylation and methylation) in the chromatin of CNS cells tested in various experimental models. Owing to their relatively simple CNSs, mollusks are among the most popular models. Our experiments were con-ducted with the molluskHelix lucorumbecause its CNS had been investigated in detail and most of its neurons had been proven to participate in the formation of different behavior patterns, including the prolonged response to various stimuli. This work concerns the influence of various effectors (serotonin and FMRFamide, associated with CNS activator and inhibitory pathways, respectively) on the acetylation of H4 histone in the subesopha­geal ganglion complex and in defensive behavior command neurons of the right and left parietal ganglia (RPa3/2 and LPa3/2) in the snail. Western blot analysis showed that FMRFamide inhibited histone H4 acetylation induced by serotonin in the subesophageal complex of CNS ganglia. How­ever, serotonin and FMRFamide cooperatively enhanced the induction of histone H4 acetylation in RPa3/2 defensive behavior command neurons. No changes were found in the counterpart LPa3/2. It is a new piece of evidence for functional asym­metry inHelix. The inhibitory pathways mediated by FMRFamide not only inhibit the activatory in­tracellular processes in the entire CNS but can also enhance them, as in RPa3/2 defensive behavior command neurons.

Spontaneous EPSP of Command Neurons in the Common Snail during Heterosynaptic Potentiation

The presynaptic mechanism of short-term plasticity of synaptic transmission was studied by analyzing the effects of rhythmic orthodromic stimulation of the intestinal nerve, inducing short-term potentiation of evoked EPSP, on spontaneous EPSP in defensive behavior command neurons in the common snail. Rhythmic stimulation had no effect on the amplitude of spontaneous EPSP but led to significant increases in the number of spontaneous EPSP. The increase in the frequency of spontaneous EPSP suggested a role for a presynaptic mechanism in the short-term potentiation of synaptic transmission. This presynaptic mechanism may include a decrease in the action potential generation threshold (AP) in presynaptic neurons, which leads to an increase in the proportion of spontaneously active neurons and, thus, supports an increase in the number of spontaneous presynaptic AP.

Specificity of Mechanisms of Memory Reconsolidation in Snails Trained for Rejection of Two Types of Food.

Specificity of behavioral and neuronal mechanisms of impairment of long-term memory reconsolidation was studied in edible snails trained for associative skill of rejection of two types of food: raw carrots (conditioned stimulus 1) and apple (conditioned stimulus 2). In 2 days after training, the snails received protein synthesis inhibitor cycloheximide and a reminder (conditioned stimulus 1 or 2). In 3 and 14 days after cycloheximide/reminder, we observed the absence of aversive responses to the conditioned stimulus used as the reminder and preserved responses to the conditioned stimulus not used as the reminder. Moreover, we observed specific suppression of synaptic responses of command neurons of snail defensive behavior induced by the conditioned stimulus used as the reminder after cycloheximide injection and preserved synaptic responses of neurons to the other conditioned stimulus. It was hypothesized that protein synthesis-dependent synapse-specific plasticity of command neurons can be a mechanism of selective preservation of conditioned food aversion memory in snails.

Neurotransmitters selectively change the phosphorylation of H3 histone in identified neurons of the snail Helix lucorum

The effects of serotonin and glutamate applications on H3 histone phosphorylation at serine 10 (Ser10, pHisH3) were immunohistochemically studied in the nuclei of L-RPa2-3 defensive behavior command neurons in the snail Helix lucorum. We found that simultaneous application of neurotransmitters induces biphasic changes in pHisH3-immunoreactivity in RPa2 neurons. The pHisH3 content increased at 30 min and decreased at 3 hours below the initial level, whereas in RPa3 neurons, a decrease in pHisH3 at 3 h occurred. Changes in pHisH3-immunoreactivity were not found in LPa2 and LPa3 neurons during the 7 hours of the experiment. Alterations of pHisH3-immunoreactivity in neural nuclei coincided with significant changes in colocalization with DNA. Our results demonstrate neuron-specific changes in genome activity in the neural nuclei of identified neurons in a cell model of a simple form of learning.

Differences in the Molecular Mechanisms of Long-Term Synaptic Facilitation in Associative Learning and Sensitization

Studies on defensive behavior command neurons Lpl1 and Rpl1 in common snails addressed the involvement of translation and transcription processes in the mechanisms of synaptic plasticity during acquisition of conditioned taste aversion. In control snails, combinations of the taste stimulus (carrot juice) and the reinforcing stimulus (concentrated quinine solution) led to changes in neuron responses characteristic for both conditioning and concomitant sensitization. Facilitation of responses to a sensory chemical stimulus (dilute quinine solution) occurred 1 h after training started, with facilitation of reactions to the conditioned food stimulus occurring at 1.5 h. Application of the protein synthesis inhibitor cycloheximide during conditioning suppressed facilitation of responses to both dilute quinine solution and the conditioned food stimulus. However, application of the RNA synthesis inhibitor actinomycin D to neurons produced selective suppression of synaptic facilitation in responses to sensory stimulation with dilute quinine solution, but had no effect on synaptic facilitation in responses to the conditioned stimulus. It is suggested that the acquisition of conditioned taste aversion in snails induces long-term synaptic facilitation in nerve cells Lpl1 and Rpl1 typical of conditioning and sensitization processes, whose induction requires different molecular-genetic mechanisms.

The Role of Myosins in Depression of Neuron Sensitivity to Acetylcholine in a Cellular Analog of Habituation in the Common Snail

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.

The Role of Serine/Threonine and Tyrosine Protein Phosphatases in Common Snail Command Neurons in a Cellular Analog of Habituation

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.

Possibility of “Comet-Like” Transport of Acetylcholine Receptors in Command <i>Helix</i> Neurons in Cellular Analog of Habituation

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 Role of Actin Microfilaments in Depression of Acetylcholine-Induced Currents in Common Snail Neurons in a Cellular Analog of Habituation

Toxins impairing the functions of cytoskeletal actin filaments – cytochalasin B (which inhibits actin polymerization and degrades microfilaments) and phalloidin (which prevents depolymerization of microfilaments, stabilizing F-actin) – weakened depression of the acetylcholine-induced influx current in defensive behavior command neurons in the common snail in conditions of rhythmic local applications of acetylcholine to the cell body (a cellular analog of habituation). These results, along with mathematical modeling, provide grounds for suggesting that depression of the cholinosensitivity of the extrasynaptic zones of command neuron membranes in the cellular analog of habituations is associated with the involvement of actin microfilaments in a reduction in the number of membrane cholinoreceptors.

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.

Involvement of Translation and Transcription Processes into Neurophysiological Mechanisms of Long-Term Memory Reconsolidation

We studied the involvement of translation and transcription processes into behavioral and neuronal mechanisms of reconsolidation of the long-term memory of the conditioned taste aversion in edible snails. Injection of cycloheximide (an inhibitor of protein synthesis) to the snails in 48 h after training combined with subsequent reminder and presentation of the conditional stimulus resulted in the development of persistent amnesia and depression of the responses of the defensive behavior command neurons LPl1 and RPl1 to the conditional stimulus. Injection of mRNA synthesis inhibitors actinomycin D or DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidasole) in 48 h after conditioning with subsequent reminding procedure produced no effects on memory retention and on the responses of the command neurons to the conditional stimulus. The study suggests that the proteins translated from previously synthesized and stored mRNA were involved in the mechanisms of reconsolidation of the memory responsible for conditioned taste aversion.

Role of Myosins in Depression of Sensitivity ofHelixNeurons 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.

The Role of Serine/Threonine and Tyrosine Protein Kinases in Depression of the Cholinosensitivity of Neurons in the Common Snail in a Cellular Analog of Habituation

The experiments reported here showed that the adenylate cyclase inhibitor SQ 22,536, inhibitors of serine/threonine protein kinases – protein kinase A (Rp-cAMPS), protein kinase G (H-Arg-Lys-Arg-Ala-Arg-Lys-Glu-OH), calcium/calmodulin-dependent protein kinase II (KN-93), p38 mitogen-activated protein kinase (PD 169316) – and an inhibitor of tyrosine protein kinases, including the Src (PP2) family (genistein), weaken depression of the acetylcholine-induced influx current in common snail defensive behavior command neurons in conditions of rhythmic local applications of acetylcholine to the cell body in a cellular model of habituation. The selective protein kinase C inhibitor chelerythrin did not alter depression of the acetylcholine current. Mathematical modeling of the effects of these inhibitors on the number of membrane-bound cholinoreceptors yielded calculated curves consistent with the experimental curves showing depression of the acetylcholine current. The experimental data and calculation results led to the following suggestion. Reversible depression of the cholinosensitivity of the bodies of defensive behavior command neurons in common snails in the cellular analog of habituation is associated with a decrease in the number of membranebound acetylcholine receptors in the test zone due to activation of several serine/threonine protein kinases: protein kinase A, protein kinase G, calcium/calmodulin-dependent protein kinase II, mitogen-activated protein kinases (excluding protein kinase C), as well as tyrosine protein kinases, including members of the Src kinase family. The main action of all these kinases (except protein kinase C) in command neurons is targeted to cellular cytoskeleton proteins (actin microfilaments and microtubules). Phosphorylation of these proteins leads to copolymerization and stabilization of actin filaments, stabilization of tubulin, which is the main microtubule protein, changes in the activity of motor proteins, and, thus, changes in the rates of transport processes, i.e., the rates of receptor endocytosis and exocytosis. The action of protein kinase G is (indirectly) on the interaction of actin with myosin. Protein kinase A, calcium/calmodulin-dependent protein kinase II and Src kinase also phosphorylate proteins activating the displacement of receptors into clathrin-coated pits in endocytosis.

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.

Endocytosis of Cholinoreceptors in the Mechanism of the Depression of the Cholinosensitivity of Neurons in the Common Snail in a Cellular Model of Habituation

Blockers of dynamin-dependent endocytosis (dynamin inhibitory peptide and dynasore) and tubulin blockers (colchicine and vinblastine) were found to weaken the depression of acetylcholine-induced influx currents in defensive behavior command neurons in common snails during rhythmic local application of acetylcholine to the bodies and cell membranes during abituation. A mathematical model allowing different cellular locations for cells and receptor movement patterns was used to analyze the relationship between depression of the acetylcholine-evoked influx current and various intracellular processes. It is suggested that the depression of cholinosensitivity in the extrasynaptic zones of the membranes of common snail defensive behavior command neurons in the cellular model of habituation is associated with the dynamin-dependent endocytosis of cholinoreceptors, involving cytoskeletal microtubules.

Effects of the Serotonin Precursor 5-Hydroxytryptophan and the Neurotoxic Analog 5,7-Dihydroxytryptamine on the Formation of a Conditioned Defensive Reflex in the Common Snail

We report here our studies on the effects of the metabolic serotonin precursor 5-hydroxytryptophan and the neurotoxic analog 5,7-dihydroxytryptamine on the acquisition of a conditioned defensive reflex and post-training electrophysiological parameters of defensive behavior command neurons in snails. Injections of 5-hydroxytryptophan into snails via the sinus node led to acceleration of the acquisition of the conditioned defensive reflex. Snails injected with 5,7-dihydroxytryptamine did not form the conditioned reflex; subsequent injection of 5- hydroxytryptophan prevented this blockade. While 5-hydroxytryptophan prevented the action of 5,7-dihydroxytryptamine at the behavioral level, no such effect was seen at the level of the electrical properties of command neurons.

Neuronal Mechanisms of Reconsolidation of an Associative Aversive Skill to Food in the Common Snail

We have previously established that reactivation of a long-term memory "trace" during exposure to a protein synthesis inhibitor in common snails trained to refuse a defined foodstuff impairs reproduction of this skill. In the present studies, the cellular mechanisms of memory reactivation were studied in defensive behavior command neurons LPl1 and RPl1 in snails. In the first series of experiments, the mechanisms of induction of amnesia were studied in semi-intact preparations 24 h after training of snails to refuse one foodstuff. These experiments showed that depression of the synaptic response to the conditioned stimulus developed 2.5 h after reminding (presentation of the conditioned food stimulus) on the background of exposure to cycloheximide. The second series of experiments addressed the neuronal mechanisms of development of amnesia. Animals were trained to refuse two foodstuffs. The snails were injected with cycloheximide 24 h after training and were subjected to the reminding procedure using one of the conditioned food stimuli. Semi-intact preparations were made 1, 3, 7, and 15 days after cycloheximide and the reminder. Responses to the conditioned food stimulus used as the reminder gradually decreased on days 1, 3, and 7. On days 7 and 15, measures of the responses to this conditioned stimulus were no different from those to the differential signal and were significantly smaller than responses to the second conditioned stimulus, which was not used as reminder. It is suggested that one of the cellular mechanisms of amnesia following impairment of long-term memory reconsolidation in snails consists of a specific, protein synthesis-dependent change in the efficiency of the synaptic connections of command neurons LPl1 and RPl1.

Effects of Preliminary Administration of Haloperidol in Low Doses on the Effects of Haloperidol on Behavioral Reactions and Command Neuron Membrane Potential in Edible Snail

We studied the effects of preliminary administration of haloperidol in low doses on changes in motor activity of edible snail and in electrical properties of defensive behavior command neurons induced by chronic administration of haloperidol. The rate of locomotion decreased after injections of haloperidol preparations (C6, C12, C30, C200 and a mixture C12+C30+C200) for 3 days. Similar changes were observed after 3 days of haloperidol administration. Haloperidol preparations in low doses produced a modulating effect on the decrease in locomotion rate and hyperpolarization of command neurons in edible snails caused by chronic exposure to haloperidol: the decrease in locomotion rate caused by chronic haloperidol treatment was prevented by preliminary injection of haloperidol in low doses C6, C12 and C30; the depolarizing shift of command neuron membrane potential was also abolished after consecutive injection of the same haloperidol preparations C6, C12 and C30.

Specific Features of Molecular Postsynaptic Excitation Processes of Different Sensory Modalities in Edible Snail Neurons

Stimulations of different sensory modalities result in selective activation of second messengers and genes controlled by them in defensive behavior command neurons (LPl-1 and RPl-1) in edible snail. Excitation induced by stimulation of chemoreceptors of snail head results in cAMP accumulation in command neurons and induction of associated transcriptional factors of immediate early gene C/EBP, whereas regulating mechanisms of another sensory input mediating excitation after tactile head stimulation, involve protein kinase C and associated transcriptional factor SRF. These findings reflect specific features of postsynaptic processes providing excitation of different sensory modalities converging to defensive behavior command neurons in edible snails.