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.     

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.     

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 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.     

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).     

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.     

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).     

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.     

Involvement of intracellular calcium in sensitization of command neurons of defensive behavior in the edible snail (Helix lucorum)

Examinations carried out on command neurons of defensive behavior in the edible snail using electrophysiological methods and a chlortetracycline fluorescent probe revealed that a single sensitizing action alters electrical neuronal activity and the amount of bound calcium in the cells. An initial increase in the amount of bound calcium (the first 15–20 min after the sensitizing action) coincides in time with depolarization, enhancement of plasma membrane excitability, and a decrease of amplitude and duration of the excitatory postsynaptic potentials (EPSP) induced by sensory stimulations. Repeated pronounced increase in the bound calcium level develops 50–60 min after the sensitizing action and correlates with facilitation of neuronal responses to sensory stimuli. Alterations in the bound calcium level in command neurons of defensive behavior in the course of sensitization development differed in dynamics and direction from the previously described bound calcium shifts in the same cells in the course of habituation development.P. K. Anokhin Institute of Normal Physiology, Academy of Medical Sciences of the USSR Moscow. I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 418–427, July–August, 1991.     

Effect of zif268 early gene antisense oligonucleotide on mechanisms of synapse-specific plasticity

It was found that nociceptive sensitization was followed by long-term facilitation of synaptic responses evoked by chemical sensory stimulation of the snail "head", tactile stimulation of the snail "head" and foot in LP11 command neuron of defence behavior in snail Helix lucorum. Sensitizing stimulation during the intracellular injection of antisense olygonucleotide immediate early gene zif268 resulted in a selective suppression of synaptic facilitation in LP11 neuron responses evoked by tactile and chemical stimulation of the snail "head". At the same time, development of synaptic facilitation of responses in the LP11 neuron evoked by tactile stimulation of the foot was the same as in control sensitized snails. The results suggest that immediate early gene zif268 is selectively involved in the mechanisms of specific regulation of plasticity of the synaptic "input" of LP11 neuron from sensory receptors of the snail "head".     

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.     

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.     

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.     

Synapses identifiable in the parietal ganglia of the snail Helix lucorum

The synaptic plasticity is a background for learning and memory. Identifiable synapses that are the synapses between individually identifiable neurons are a very convenient model for studying plasticity. Synapses between the interoceptive mechanosensory neurons and the command neurons of the withdrawal behavior were identified in the Helix lucorum brain. It was shown that synaptic plasticity estimated by the dynamics of the elementary postsynaptic potentials elicited by single presynaptic spikes differed from the synaptic plasticity estimated by the dynamics of compound synaptic responses of the same neurons to sensory stimulation. Habituation and heterosynaptic facilitation phenomena are discussed in terms of the dynamics of the elementary postsynaptic potentials.     

Dynamic defensive and feeding reactions to the acquisition of sensitization in edible snails]

In snails changes of defensive and alimentary behaviour and of reactions of command and modulator neurones of these kinds of behaviour were studied during elaboration of sensitization. After a single action of 50% chinine solution on mollusc's head, a short-term (during 50-70 min) and long-term (hours or days) facilitation was found of animals defensive reactions and responses of command neurones of defensive behaviour to tactile and chemical sensory stimuli. Alimentary behaviour of the snails and reactions of modulator neurones of alimentary behaviour to carrot juice presentation were inhibited in sensitized animals. Dynamics differences of defensive responses to tactile and chemical stimuli in the same sensitized animals were observed. Short-term responses facilitation under sensitization correlated chiefly with depolarization of the membrane potential of defensive behaviour neurones and with an increase of plasmatic membrane excitability. Prolonged facilitation of responses was due mainly to a change of effectiveness of synaptic transmission. The described model of sensitization elaboration can be a base for studying of molecular-cellular mechanisms, underlying the learning.     

Interaction between visceral,somatic, and visual excitation on single neurons of the lateral geniculate body in cats

Acute experiments on cats anesthetized with pentobarbital and immobilized with diplacin or listhenon showed that visceral and somatic excitation may either facilitate or inhibit single unit activity in the lateral geniculate body evoked by photic stimulation. The manifestations of facilitation were: a modulatory type of enhancement of responses of silent neurons and neurons with a low level of spontaneous activity; enhancement of responses accompanied by simultaneous depression of spontaneous activity — a sensory contrast effect; enhancement of long-latency responses; appearance of a short-latency discharge from cells with an inhibitory response to light; the appearance of responses to light in neurons not responding previously or stabilization of responses in neurons responding to light irregularly. The inhibitory effects were manifested as immediate inhibition of responses, usually long-latency, and the filling up of the inhibitory pauses of the response to light with spikes, leading to a decrease in the signal-noise ratio. Somatic stimulation was more effective and more frequently evoked facilitation of responses to light (in 74% of cells). Similar results were obtained by stimulation of the mesencephalic reticular formation. Visceral excitation gave rise to facilitatory and inhibitory effects to an almost equal degree. The results show that excitation arising as the result of visceral and somatic stimulation affects the conduction of visual information in the neuronal system of the lateral geniculate body.Ivano-Frankovsk Medical Institute. Institute of Experimental Medicine, Academy of Medical Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 636–643, November–December, 1973.