Effect of selective protein kinase C inhibitor on synaptic plasticity of command neurons of defensive behaviour during sensitization in snails

Polymyxin B (proteinkinase C inhibitor) effects on nociceptive sensitization of semiintact preparation were investigated in LP11 and RP11 snail neurons. It was found that application of sensitizing stimuli to control snail head initiated neural membrane depolarization, increase its excitability as well as depression of neural responses evoked by sensory stimulation during short-term stage. Polymyxin B application suppressed neural responses evoked by sensitizing (nociceptive) stimuli. At the same time changes in neural membrane excitability as well as neural responses evoked by tactile stimulation of snail foot or chemical stimulation of snail head were similar with ones in control snails. Polymyxin Bdid does not change the depression of neural responses evoked by tactile stimulation of snail head during short-term stages of sensitization but significantly suppressed facilitation of neural responses evoked by tactile stimulation of snail head during long-term stage of sensitization. It was suggested that proteinkinase C is involved in regulation of nociceptive mechanisms as well as in plasticity selective induction mechanisms in command neuron synaptic inputs activated by tactile stimulation of snail head.     

Critical role of intracellular calcium in plasticity mechanisms of defense behavior command neurons LPl1 and PPl1 of Helix lucorum during nociceptive sensitization

The role of intracellular calcium in changes in excitability and responses of defense behavior command neurons LP11 and PP11 of Helix lucorum to sensory stimulation was investigated in semi-intact preparation of a snail during nociceptive sensitization. It was found that application of sensitizing stimuli onto the snail's head initiated membrane depolarization, increase in its excitability as well as depression of neural responses evoked by sensory stimuli in short-term period of sensitization and significant facilitation of neural responses in long-term period of sensitization. To elucidate the contribution of LP11 and PP11 neurons in plasticity rearrangements involved in the mechanisms of sensitization, we applied sensitizing stimuli during strong hyperpolarization of the neurons or after intracellular injection of calcium chelators. Application of sensitizing stimuli during hyperpolarization of the neurons suppressed the increase in membrane excitability and depressed the neural responses evoked by chemical stimulation of snail's head i.m. short- and long-term periods of sensitization. At the same time, synaptic facilitation of neural responses evoked by tactile stimulation of snail's head and foot was observed, which was similar to synaptic facilitation in the control sensitized snail. Intracellular injection of EGTA or BARTA (calcium chelators) before sensitization suppressed synaptic facilitation in neural responses evoked by sensory stimulation. Under these conditions, the increase in excitability was more pronounced then in the control snail neurons. The experimental results suggest the changes in neural responses evoked by sensory stimulation in sensitized snails involve postsynaptic calcium-dependent mechanisms of plasticity in LP11 and PP11 neurons.     

Selective involvement of opioids in mechanisms of synapse-specific plasticity in Helix lucorum snail during sensitization acquisition

Effects of met-enkephalin (opioid peptide) and naloxone (opioid antagonist) on nociceptive sensitization were studied in L-RP11 Helix neurons. In control snails sensitizing stimulation produced reversible membrane depolarization and depression of neural responses evoked by sensory stimuli during the short-term stage of sensitization and facilitation of these responses at the long-term stage. Met-enkephalin (10 but not 0.1 microM) suppressed the neural responses evoked by nociceptive stimuli. Sensitizing stimulation during metenkephalin application prevented the facilitation of neural responses evoked by tactile stimulation of snail head, whereas facilitation of neural responses evoked by chemical stimulation of head or tactile stimulation of foot were similar to that in control sensitized snails. Sensitizing stimulation during met-enkephalin and/or naloxone application prevented the facilitation of neural responses evoked by chemical stimulation of snail head, whereas responses evoked by tactile stimulation of snail head or foot were facilitated (as in neurons of control sensitized snails). Opioids are suggested to be involved in regulation of nociceptive mechanisms and selective induction of long-term plasticity in L-RP11 neural inputs activated by tactile of chemical stimulation of snail head.     

Vital investigation of the genome activity and neuronal synaptic plasticity in snail in the course of learning

Vital investigation of genome activity and its role in mechanisms of long-term synaptic plasticity formation were studied in LP11 neuron (command neurone of defence behaviour) during sensitisation (simple form of learning) in semi-intact preparation of snail Helix lucorum. Genome activity was investigate by means of specific fluorescent dye SYTO 16 and image analysis system. It was found that application of sensitizing stimulation (10% quinine solution) onto the snail head initiate two-phase changes in neural responses evoked by tactile or chemical stimulation. Depression of neural responses was obtained during short-term phase (during 1 hour after sensitization) and facilitation of neural responses--during long-term phases (over 1 hour). At the same time (15-20 min after learning), a significant increase in SYTO 16 fluorescent level was found in nucleus site LP11 neurone. Initial SYTO 16 fluorescence level was registered in 4-5 hours after sensitization. If the sensitization was produced during actinomycin D application (inhibitor of RNA synthesis, 20 microM) then facilitation of neural responses evoked by sensory stimulation were suppressed during long-term phase of sensitization and was the same as in control sensitized snails during short-term phase of learning. Increase in SYTO 16 fluorescence level in nucleus region site of LP11 neurone was completely prevented in sensitization during actinomycin D application. If actinomycin D was applied 30 min after sensitization (1 hour after first sensitizing stimulation) then synaptic facilitation and fluorescent dynamics was the same as in control sensitized snails. Our experimental data showed that nociceptive sensitization development was followed by quick (15-20 min) DNA activation and long-term synaptic facilitation (1 hour after sensitization), while induction of the processes was suppressed by inhibitor of RNA synthesis during short time interval (during 1 hour of learning).     

Antagonists of NMDA glutamate receptors selectively affect synaptic mechanisms of the nociceptive sensitization in the snail

The effects of N-methyl-D-aspartate (NMDA) glutamate receptor antagonists on the mechanisms of nociceptive sensitization were studied in LPl1 and RPl1 neurons of the semiintact preparation of a Helix lucorum snail. Application of sensitizing stimuli on the head part of the control preparation led to a depolarization of the membrane and increase in its excitability. A depression of responses of neurons evoked by tactile or chemical sensory stimulation during the short-term period and significant facilitation of responses during the long-term period of sensitization were observed. Sensitization performed under conditions of application of NMDA antagonists (AP5 or MK801) produced similar changes in membrane potential, membrane excitability, and neuronal responses evoked by tactile stimulation of the head or foot. However, the chemical stimulation of the head under these conditions evoked a significant depression of responses during the short- and long-term sensitization periods. The results suggest that the NMDA glutamate receptor antagonists selectively affect the plasticity induction mechanisms of the command neuron synaptic inputs, which mediate the chemical sensory stimulation from the snail's head.     

Effect of cyclic AMP on the excitation and response of command neurons responsible for the snail defensive behavior caused by sensory stimulation]

Extraneuronal application of db-cAMP or intraneuronal injection of cAMP were found to increase the neural membrane excitability and synaptic facilitation in neural responses to sensory stimulation of Lpl1 and Rpl1 neurones. The db-cAMP exerted no effects on neural responses to tactile stimulation of the snail foot or mantle. The intraneuronal injection of cAMP produced synaptic facilitation only in neural responses to quinine application to the snail head. The findings suggest the cAMP selective involvement in postsynaptic mechanisms of inducing the long-term facilitation transient stage.     

Selectivity of opioid peptide effects on excitability and various sensory inputs in LPl1 and PPl1 command neurons participating in defensive behavior of the snail Helix lucorum

Opioid peptides effects on neural membrane as well as neural responses evoked by sensory stimuli with different modality and site of application, were investigated in L-RPII command neurones of defensive behaviour of semi-intact preparation in the land snail Helix lucorum. Met-enkephalin (10 uM) application onto the snail CNS increases membrane excitability and produces facilitation of neural responses evoked by quinine solution (0.5%) application onto snail head and depression of reactions evoked by tactile stimulation of the head. Met-enkephalin in dose of 0.1 uM initiates only a depression of neural responses evoked by tactile stimulation of the head. Leu-enkephalin (10 uM) application suppresses neural reactions evoked by tactile stimulation of the head. Membrane excitability and neural responses evoked by quinine application onto the snail head do not change after leu-enkephalin administration. Effects appear 10-20 min after initiation of the peptide application. Initial neural responses were observed 15-30 min after CNS washing with Ringer solution. In addition, facilitation of neural responses evoked by chemical stimulation of the snail head was found 30-50 min after leu-enkephalin washing. Peptides do not change neural responses evoked by tactile stimulation of the snail foot. Neural effects of peptides were prevented by simultaneous naloxon administration (50 uM). Experimental results show selective opioid peptides' effects on excitability and plasticity of L-RPII neural inputs with site- and modality-specifics.     

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.     

Cellular reactions to elaboration of a backward conditioned connection in Helix lucorum

After 10-15 food stimuli paired with electrical shock in semi-intact snail preparation, responses to strong tactile stimuli identified feeding behaviour neurones were studied. Inhibition evoked by tactile stimulation in these cells before learning procedure disappeared and in some cases noxious stimulus evoked synaptic activation corresponding to feeding reactions in the intact animal. Changes in second-order sensory neurones pre-synaptic to the command neurones of avoidance behaviour are suggested to be the mechanism of forward conditioned connection as well as the mechanism of backward conditioned connection.     

Elementary and compound postsynaptic potentials in the defensive command neurons of Helix lucorum

The present communication concerns with the analysis of elementary and the compound excitatory postsynaptic potentials (eEPSPs and cEPSPs) recorded by intracellular microelectrode from an identified defensive command neuron of the snail Helix lucorum. The eEPSPs were evoked by single presynaptic action potentials (APs) elicited by cationic current injection into one of the identified sensory neurons synapsing on the respective command neuron. The cEPSPs were elicited by local brief tactile stimuli on the skin or internal organs. It was shown that the cEPSPs amplitudes depend mainly on the number of activated sensory neurons. Compound EPSPs depend also on frequency and the number of APs in the bursts occurring in a single neuron. Presynaptic APs having frequency 2-10 Hz evoke high frequency depression of that eEPSPs after an interval is followed by post-tetanic potentiation of single eEPSPs. Preceding stimulation of a pneumostom area facilitates the cEPSPs elicited by repeated stimulation of viscera. The eEPSPs from the same visceral area demonstrate no heterosynaptic facilitation in experiments with double parallel intracellular recording from responsive sensory and command neurons. The different types of the eEPSPs plasticity are discussed according to their contribution cEPSPs plastic changes.     

An experimental unit for the recording of the escape reaction of a ground snail to tactile stimulation

An original working experimental unit for noninvasive objective recording of the magnitude of escape reaction of a ground snail evoked by tactile stimulation is described. A. snail creeps upwards over the cylinder rotating around its horizontal axis. A watching device ensures a constant snail position relative to a light source and a photoelement. A device for tactile stimulation which provides graduated energy of an impact is constructed on the basis of the magnetic circuit of a loudspeaker. In response to a tactile stimulus a snail pulls in its feelers, head, and foot, and the area of snail's shadow decreases. These changes are indicated by the photoelement.     

Learning-related long-term synaptic facilitation in snails: Possible mechanisms of long-term memory formation

The long-term changes in the electrical activity of command neurons related to sensitization and elaboration of associative defensive behavioral habit (food rejection) were studied inHelix snail. The long-term effects consisted of facilitation of synaptic components in neuronal responses to the test stimulations. Variations were found in the dynamics of long-term synaptic facilitation of responses to the applied chemical and tactile stimuli in the course of sensitization, as well as dependence of the degree of long-term facilitation of responses to the test stimulation at the site of its application with respect to the site of the sensitizing stimulation (site-specific sensitization). After conditioning, the synaptic response of command neurons to the conditioning stimulation appeared approximately 30 min later than did the long-term sensitization in these cells. The minimum duration of long-term synaptic facilitation of responses to the test stimulation varied from 1 h (for tactile stimulation) to 3 h (for chemical stimulation). The maximum duration of effects exceeded 4 h. It is suggested that the observed features of the synaptic plasticity in command neurons during learning are based on the selective regulation of synaptic inputs by specific protein regulators, whose lifespan does not exceed 1 h to 3 h.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 383–389, September–October, 1993.     

Imitation of neurophysiological effects of long-term sensitization with the action of the blockers of protein synthesis in snailsHelix lucorum

The experiments on command neurons of defensive behavior in snailsHelix lucorum have shown that the synaptic components of responses to the testing stimuli are facilitated within 65 to 75 min after the onset of the application of the protein synthesis inhibitors cycloheximide or anisomycin (30 µM during 30 min) to the central nervous system. At the same time, the parameters of the electrical properties of neuronal membrane (membrane potential level and excitability) were not affected by the blockers. The facilitatory effects were not observed when the blockers were applied for a longer period or when their concentration was raised to 300 µM. The blockade of the responses to different sensory stimuli appeared selective. The facilitation of the responses to tactile stimulation of the head, to weak quinine solution, and to carrot juice lasted for about an hour, 2–3 h and about 1.5 h, respectively. The blockers facilitated only the responses to tactile stimulation of the head, but did not change the responses to stimulation of the middle part of the foot or the mantle bolster. Some characteristics of the blocking effects are similar to those of long-term sensitization. The blocking effects observed are suggested to be due to activation of synthesis of short-living proteins functionally related, in particular, to selective regulation of synaptic transmission.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1. pp. 61–67, January–February, 1994.     

In vivo immediate early gene expression induced in intestinal and colonic mucosa by feeding.

Since the gut responds rapidly to food intake, the levels of expression of several immediate early genes were measured in mucosa from small and large intestine of rats starved for 3 days or refed. Within 1 h of refeeding, jejunal and ileal c-fos, jun B and zif/268 mRNA and colonic zif/268 dramatically increased. The zif/268 gene in jejunum corresponded in size to the full-length cDNA but, in ileum, an RNA band of about 1.2 kb in size increased greatly after feeding. This represents a physiologic in vivo model for the study of gene regulation associated with intestinal epithelial cellular responses to feeding.     

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.