NMDA Glutamate Receptor Antagonists Selectively Affect the Synaptic Mechanisms of Nociceptive Sensitization in Snails

Defensive behavior command neurons LPl1 and RPl1 were studied in semi-intact snail preparations to investigate the effects of N-methyl-D-aspartate (NMDA) receptor antagonists on the mechanisms of nociceptive sensitization. Application of sensitizing stimuli to the heads of control snails led to membrane depolarization and increased excitability, and also depressed the responses of neurons to tactile and chemical sensory stimuli in the short-term stage and facilitated responses in the long-term stage of sensitization. Development of sensitization in conditions of exposure to the NMDA receptor antagonists AP5 or MK-801 produced changes in the membrane potential and membrane excitability of command neurons similar to those seen in neurons of control sensitized snails. In addition, changes in the responses of command neurons to tactile stimulation of the head and foot and chemical stimulation of the foot in these conditions were also similar to those seen in neurons of control animals. Acquisition of sensitization during administration of NMDA receptor antagonists led to pronounced depression of responses to chemical test sensory stimulation of the snails' heads in both the short-term and long-term stages of sensitization. Thus, in sensitized snails, NMDA glutamate receptor antagonists selectively acted on the mechanisms of induction of plasticity the synaptic inputs of command neurons mediating excitation from chemical sensory stimuli from the animal's head.     

The Critical Role of Intracellular Calcium in the Mechanisms of Plasticity of Common Snail Defensive Behavior Command Neurons LPl1 and RPl1 in Nociceptive Sensitization

Studies on semi-intact common snail preparations addressed the involvement of intracellular calcium in changes in the excitability and responses to sensory stimuli of defensive behavior command neurons LPl1 and RPl1 during the acquisition of nociceptive sensitization. Application of sensitizing stimuli to the heads of control snails led to depolarization of neuron membranes, increases in neuron excitability, and depression of the responses of neurons to sensory stimuli during the short-term stage, and marked facilitation of responses in the long-term stage of sensitization. Acquisition of sensitization during profound hyperpolarization of neurons led to suppression of the increase in excitability, along with depression of responses to chemical stimulation of the head in the short- and long-term stages of sensitization. Neuron responses to tactile stimulation of the head and foot showed synaptic facilitation, similar to that seen in neurons of control animals. Acquisition of sensitization during intracellular injection of the calcium chelators EGTA and BAPTA led to suppression of synaptic facilitation in the responses of neurons to both chemical and tactile stimulation. In these conditions, membrane excitability increased to a greater extent than in neurons of control animals. The results of these experiments suggest that changes in responses to sensory stimulation in sensitized snails are associated with postsynaptic calcium-dependent mechanisms of plasticity in neurons LPl1 and RPl1.     

Neuronal mechanisms of site-specific nociceptive sensitization in the common snail

Comparative studies of the electrophysiological effects of site-specific nociceptive sensitization were performed on LPl1 and RPl1 command neurons in the common snail, after placing concentrated quinine solution (10%), on the animal's head or mantle ridge. Greater synaptic facilitation was observed in command neuron responses to tactile test stimuli applied to the same part of the body as the sensitizing stimulus, as compared with the level of synaptic facilitation in responses to tactile stimulation of other parts of the body. Synaptic facilitation of responses of neurons LPl1 and RPl1 to tactile stimulation of the head appeared 1 h after sensitizing stimulation and consisted of two phases: the first phase was characterized by a peak-like increase in the area of slow EPSP (sEPSP) and lasted 1 h; the second phase was characterized by maintenance of a relatively stable level of facilitation of sEPSP, which lasted to the end of the observation period (2–3 h). Synaptic facilitation of neuron responses to tactile stimulation of the mantle ridge appeared 40–60 min, after facilitation of responses to test stimulation of the snail's head, and was characterized by maintenance of a relatively stable level of sEPSP facilitation. It is suggested that the specificity of synaptic facilitation occurring in snail defensive behavior command neurons during the period of long-term nociceptive sensitization is associated with the genetic regulation of the various synaptic “inputs” to neurons. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti, Vol. 47, No. 6, pp. 994–1003, November–December, 1997.     

The Selective Effect of a Protein Kinase C Inhibitor on Synaptic Plasticity in Defensive Behavior Command Neurons During Development of Sensitization in the Snail

Studies of defensive behavior command neurons LP11 and RP11 in semi-intact common snail preparations addressed the effects of the protein kinase C antagonist polymyxin B on the effect of nociceptive sensitization. Neurons in control snails responded to application of nociceptive stimuli to the head with membrane depolarization, increases in excitability, and depression of neuron responses to sensory stimulation during the short-term stage, with marked facilitation of responses during the long-term stage of sensitization. Acquisition of sensitization in the presence of polymyxin B resulted in partial suppression of responses to nociceptive stimuli. Changes in command neuron membrane excitability in these conditions, as well as changes in responses to tactile stimulation of the foot and chemical stimulation of the head, were similar to those seen in neurons of sensitized control animals. The inhibitor also had no effect on short-term depression of neuron responses induced by tactile stimulation of the head. In addition, acquisition of sensitization during administration of polymyxin B led to complete suppression of the facilitation of responses to tactile stimulation of the snail's head during the long-term stage of sensitization. It is suggested that in sensitized common snails, protein kinase C is involved in controlling the mechanisms of nociception and is also involved in the mechanisms of selective induction of plasticity in the synaptic inputs of command neurons, which are activated by tactile stimulation of the animal's head.     

Effects of antisense oligonucleotides to mRNA for the early gene zif268 on the mechanisms of synapse-specific plasticity

Acquisition of nociceptive sensitization in common snails was accompanied by long-term facilitation of the responses of defensive behavior command neuron LPl1 to sensory stimulation of chemoreceptors on the head and mechanoreceptors on the head and foot. Acquisition of sensitization during intracellular administration of antisense oligonucleotides to mRNA encoding the early gene zif268 showed suppression of synaptic facilitation in the responses of neuron LPl1 to tactile and chemical stimulation of the snail’s head. Synaptic facilitation in the responses to tactile stimulation of the foot developed as in neurons of control sensitized animals. These results suggest that the early gene zif268 is selectively involved in the mechanisms of the specific regulation of the synaptic inputs of neuron LPl1 from sensory receptors on the snail’s head. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 56, No. 4, pp. 499–505, July–August, 2006.     

The Selective Action of Opioid Peptides on Excitability and the Various Sensory Inputs of Defensive Behavior Command Neurons LPl1 and RPl1 of the Common Snail

The nature of the effects of opioid peptides on the properties of electrogenic membranes and the responses of defensive behavior command neurons LPl1 and RPl1, evoked by sensory stimuli of different modalities and application sites was studied in semi-intact preparations from common snails. Application of met-enkephalin (10 M) to the snail CNS produced increases in membrane excitability along with facilitation of responses to application of dilute quinine solution to the animal's head and depression of responses to tactile stimulation of the head. Met-enkephalin (0.1 M) produced only depression of responses to tactile stimulation of the head. Application of leu-enkephalin (10 M) was accompanied by depression of responses to tactile stimulation of the head. Membrane excitability and responses to chemical sensory stimulation during application showed no change during application of this peptide. These effects of both peptides appeared 10–20 min from the start of application and lasted 15–30 min after washing was started. In addition, facilitation of the responses of neurons to chemical sensory stimulation was seen 30–50 min after the start of leu-enkephalin application. The responses of neurons to tactile stimulation of the snail's foot were not altered by application of peptides. The neuronal effects of peptides were suppressed by simultaneous application of naloxone (50 M). Thus, we observed the selective action of opioid peptides on the synaptic plasticity of neurons LPl1 and RPl1, both in relation to the location of sensory stimulation and in relation to sensory modality.     

Selective involvement of opioids in the mechanisms of synapse-specific plasticity in the common snail during the acquisition of sensitization

Studies on defensive behavior command neurons LPl1 and RPl1 in semi-intact common snail preparations were performed to investigate the effects of the opioid peptide metenkephalin and the opioid antagonist naloxone on the effects of nociceptive sensitization. Application of nociceptive stimuli to the snails head elicited marked reversible membrane depolarization along with depression of neuron responses to sensory stimulation during the short-term stage of sensitization and facilitation of responses in the long-term stage. Metenkephalin at a dose of 10 µM but not at a dose of 0.1 µM partially suppressed responses to nociceptive stimuli. Acquisition of sensitization during exposure to metenkephalin at doses of 10 and 0.1 µM led to complete suppression of the facilitation of responses to tactile stimulation of the head. Facilitation of responses to chemical stimulation of the head and tactile stimulation of the foot in these conditions was similar to that of neurons in control sensitized animals. Acquisition of sensitization during exposure to metenkephalin and/or naloxone elicited selective suppression of facilitation of responses to chemical stimulation of the head but had no effect on facilitation of responses to tactile stimulation of the head and foot. Met-enkephalin and naloxone had no effect on the depression of neuron responses evoked by sensory stimulation in the short-term stage of sensitization. It is suggested that during the acquisition of sensitization in the common snail, opioids are involved in controlling the mechanism of nociception and in the mechanisms of selective induction of long-term plasticity of the synaptic inputs to command neurons activated by tactile and chemical stimulation of the animals head.Translated from Zhurnal Vysshei Nervnoi Deyatelnosti, Vol. 53, No. 6, pp. 766–774, November–December, 2003.     

Selective Effects of Antibodies to Protein SMP-69 on the Activity of Defensive Behavior Command Neurons in the Common Snail

The effects of antibodies to the serotonin-modulated protein SMP-69 on the activity of defensive behavior command neurons LP11 and RP11 in semi-intact preparations from common snails were studied. Antibody to SMP-69 increased membrane excitability and facilitated neuron responses to chemical sensory stimulation by application of dilute quinine solution to the animal's head, these effects being seen at 1-1.5 h. The synaptic effects of the antibodies were specific, as they had no influence on responses induced by tactile stimulation of the head. The neuronal effects of antibody SMP-69 were similar to changes in the activity of cells LP11 and RP11 induced by serotonin and cAMP, and to changes seen when snails acquired nociceptive sensitization. It seems likely that a protein homologous to mammalian SMP-69 is involved in the mechanisms controlling excitability and long-term specific plasticity of the synaptic inputs to neurons LP11 and RP11 from chemoreceptors on the snail's head.     

The Effects of Antibodies Against Proteins of the s100 Group on Neuron Plasticity in Sensitized and Non-Sensitized Snails

The effects of antibodies to a total fraction of s100 proteins and protein s100b on the activity of defensive behavior command neurons LPl1 and RPl1 were studied in common snails, using non-sensitized animals and animals which had acquired nociceptive sensitization. In non-sensitized snails, application of antibodies against s100 or s100b (0.1 mg/ml) induced membrane depolarization, increased membrane permeability, and suppressed slow excitatory postsynaptic potentials in the responses of neurons to sensory stimulation. Acquisition of sensitization in snails in the presence of antibodies to s100 or s100b (0.1 mg/ml) led to significantly less marked facilitation of synaptic transmission and smaller increases in neuron membrane excitability than in cells of control sensitized animals. The difference in synaptic facilitation in the neurons of control sensitized snails and neurons in sensitized snails given antibody was comparable with the magnitude of synaptic depression due to antibody in non-sensitized animals. At a dose of 0.01 mg/ml, antibody had no effect on these measures of neuron activity. It is suggested that s100 proteins, particularly s100b, are involved in the mechanisms regulating excitability, the membrane potential, and synaptic transmission in command neurons in untrained snails, as well as in the mechanism of plasticity of the electrogenic membranes of nerve cells during the acquisition of nociceptive sensitization.     

Serotonin imatates several of the neuronal effects of nociceptive sensitization in the common snail

Experiments on defensive behavior command neurons in common snails showed that synaptic facilitation in the responses of nerve cells to sensory stimulation occurs 50–60 min after the onset of application of serotonin (10 μM) to the CNS. The properties of neuron electrogenic membranes (membrane potential, membrane excitability) did not change after exposure to serotonin. Along with synaptic facilitation, serotonin (100 μM) increased the excitability and produced minimal depolarization of the membranes of command neurons. Serotonin had selective effects on the reactions of neurons to different sensory stimuli: facilitation of neuron responses to tactile stimulation of the head lasted 1 h, while responses to application of dilute quinine solution lasted 2–3 h; serotonin facilitated neuron responses to tactile stimulation only of the snail’s head, and did not alter the responses to stimulation of the foot or the mantle ridge. The time course of the electrophysiological effects of serotonin coincided with changes in bound calcium (Ca_b) levels in command neurons. This set of serotonin-induced neurophysiological effects is simular to the effects resulting from the development of nociceptive sensitization. It is suggested that serotonin is involved in the mechanisms of transient changes and consolidation of long-term plastic rearrangements in command neurons which underlie sensitization. P. K. Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 47, No. 3, pp. 532–542, May–June, 1997.     

The effects of cAMP on the excitability and responses of defensive behavior command neurons in the common snail evoked by sensory stimuli

Experiments on snails showed that extracellular application of dibutyryl-cAMP (db-cAMP) or intracellular application of cAMP for 30 min evoked increases in excitability and synaptic facilitation in responses to sensory stimulation of defensive behavior command neurons LP11 and RP11. Extitability increased 45–60 min after the start of addition of db-cAMP or cAMP and remained elevated until the end of the experiment (3–4h). Synaptic facilitation started 50–60 min after the onset of extracellular application of db-cAMP and remained detectable in the responses of neurons to tactile stimulation of the head for 1 h and to application of dilute quinine solution for 2–4 h. Application of db-cAMP produced no changes in responses to tactile stimulation of the foot or mantle ridge. Intracellular injection of cAMP induced facilitation of neuron responses only to weak quinine solutions. The responses of neurons to tactile stimulation of the head, foot, and mantle ridge did not change after injections of cAMP. It is suggested that cAMP is involved in the mechanisms controlling the excitability of neurons LP11 and RP11. In addition, cAMP is selectively involved in the postsynaptic mechanism inducing the transient stage of long-term facilitation of synaptic “inputs”, which mediates excitation evoked by chemical stimuli. This set of effects of cAMP is similar to effects arising during the development of nociceptive sensitization and in response to serotonin. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 2, pp. 237–245, February, 1999.     

Synapse-specific plasticity in command neurons during learning of edible snails under the action of caspase inhibitors

The effect of caspase inhibitors on long-term synaptic facilitation induced by nociceptive sensitization (a simple form of learning) was studied on the defensive behavior command neurons (left pleural neuron-1) in edible snail. Acquisition of sensitization under conditions of threatment with caspase-3 or caspase-8 inhibitors selectively inhibits synaptic transmission in the responses of the left pleural neuron-1 to tactile stimulation of the snail head, but not in responses to chemical stimulation of the head or tactile stimulation of the foot. Application of a wide-spectrum caspase inhibitor z-VAD-fmk to neurons of sensitized snails suppressed facilitation of responses evoked by chemical stimulation of the head. Probably, various caspases could be selectively involved into induction of long-term synapse-specific plasticity during learning. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 604–608, December, 2007     

Protein Kinase C is Selectively Involved in the Mechanisms of Long-Term Synaptic Plasticity

Sensitization produced by intracellular administration of a specific protein kinase C inhibitor chelerythrine selectively inhibits synaptic facilitation in the response of LPl1 neurons to tactile sensory stimulation of snail head. Synaptic facilitation in the response to chemical stimulation of the head or tactile stimulation of the foot did not differ from that observed in neurons of control sensitized animals. Our findings suggest that protein kinase C plays a key role in the induction of long-term genetic regulation of sensory input in command neurons from mechanoreceptors on the head.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 604–607, June, 2005