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.     

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.     

Sensitization inHelix snail: Morphofunctional correlates in command neurons of withdrawal behavior

Electrophysiological effects have been studied in command neurons of withdrawal behavior inHelix snail. In parallel, correlated changes in the content of bound calcium (Ca-b), as well as changes in DNA condensation, were investigated using a chlortetracycline fluorescent probe and the fluorescent dye bisbenzimide, respectively. Short-term electrophysiological changes (depolarization of the membrane and elevation of its excitability) in sensitized snails have been found to be accompanied by an increase in the Ca-b level in the cell nucleus and by partial DNA decondensation. Long-term effects were characterized by more pronounced synaptic components of the responses — slow EPSPs evoked by sensory stimuli, as well as by further DNA decondensation and considerable elevation of the Ca-b content in the nucleus and cytoplasm. The results obtained inin vitro conditions have shown that Ca-binding nonhistone proteins of chromatin are components of the cell nucleus whose content may be measured by chlortetracycline fluorescence.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 150–157, March–April, 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.     

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.     

Contribution of a humoral factor to postsynaptic sensitization in heterosynaptic facilitation

It was shown that heterosynaptic facilitation develops in the cerebral ganglia giant neurons of the freshwater gastropod molluskPlanorbis corn eus due to diffuse neurohumoral influences on pre- and postsynaptic structures and not local synaptic action on presynaptic mechanisms. It was also found that n-cholinergic synaptic mechanisms come under this facilitatory influence. Serotonin is the source of facilitation in neurons of bothPlanorbis corneus cerebral ganglion and those of the aplysia abdominal ganglion. Seeing that: a) conditioning stimuli facilitate the effects produced by iontophoretic acetylcholine application, as well as n-cholinergic synaptic transmission and b) the amplitude of EPSP and acetylcholine potential increase 4–6 times during facilitation when the input impedance of the post-synaptic membrane is increased by just 20%, it was deduced that the postsynaptic membrane of the giant neuron makes a significant contribution to heterosynaptic facilitation of the sensitization of n-cholinergic receptors. The part played by n-cholinergic receptors of the postsynaptic membrane in heterosynaptic facilitation and conditioned reflex habituation is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 250–259, March–April, 1986.     

Effect of conditioning polarization of soma of giant neurons of mollusks on the mechanism of action-potential generation

To ascertain the properties of an excitable membrane of the soma of giant neurons of mollusks, experiments were carried out to study the effect of conditioning shift of the membrane potential on the mechanism of action-potential generation. The effect of conditioning was assessed from changes in the action-potential curve and its first derivative, as well as from the curve of transmembrane currents under voltage clamp conditions. It was found that a change in membrane potential evokes at least two reactions which have opposite effects on the mechanism of generation of action potentials. These reactions evidently have different time characteristics. One of these does not differ notably from the reaction recorded for other excitable structures, and is manifested in the activation (with hyperpolarization) or inactivation (with depolarization) of the mechanism generating action potentials. The other reaction contributes either to an increase (with depolarization) or a decrease (with hyperpolarization) in the efficiency of this mechanism. Conditioning polarization also has a marked effect on the system responsible for repolarization of the membrane during generation of action potentials. This effect is manifested in a change in the reaction of this system to tetraethylammonium ions. The specific membrane systems sustaining excitability and reacting to changes in the strength of the membrane's electrical field were found to be very inert. After a shift in the potential to a given stable level a rearrangement, lasting sometimes tens of seconds, takes place in the membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 91–99, January–February, 1970.     

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.     

The action of the neurotoxins 5,6-dihydroxytryptamine and p-chlorophenylalanine on the electrical activity parameters of the command neurons during long-term sensitization and learning in the snail]

The influence of 5,6-dihydroxytryptamine (5,6-DHT), which selectively destroyed serotonin terminals, and p-chlorphenylalanine, which inhibited serotonin synthesis, was studied on the long-term sensitization (LTS) in a snail. The membrane mechanisms were analyzed by measuring electrical characteristics of command neurons of defensive behavior LPa3, RPa3, LPa2, and RPa2. Snails injected with saline served as an active control. It was shown that the injected drugs inhibited the LTS in certain concentrations. A significant increase was observed in the membrane potential and the threshold of the action potential generation in the command neurons after 5,6-DHT injection in the doses of 20 and 30 mg/kg (in comparison with the active control). Sensitization of snails injected with saline solution led to the LTS and decrease in the membrane and threshold potentials of the command neurons. After the LTS, changes in membrane and threshold potentials in snails injected with 5,6-DHT were negligible in comparison with those injected with 5,6-DHT but without the LTS. Neither the LTS nor subsequent learning resulted in a further decrease in membrane and threshold potentials. Thus, the neurotoxin injection led to an increase in excitability of command neurons and their depolarization, and the LDS did not elicit further excitability increase. Since the shifts of the threshold and membrane potentials were the same, it was concluded that the increase in membrane excitability was induced by the depolarizing shift of the membrane potential.     

Neurophysiological mechanisms of generalized and signal-specific long-term nociceptive sensitizations in theHelix snail

Neurophysiological mechanisms responsible for generalized and signal-specific long-term (longer than 24 h) nociceptive sensitizations were studied in theHelix command neurons of defensive behavior. After a one-day acquisition, the manifestations of the signal-specific sensitization were predominant. These included the site and modal specificities. The former was manifested in a predominant synaptic facilitation of the responses to testing stimulation of the same body areas to which the sensitizing stimulation was applied, compared with stimulation of other body areas. The latter was manifested as a more expressed facilitation of the responses to testing stimulation of sensory modality similar to the sensitizing one, compared with the facilitation of the responses to testing stimulations of other sensory modalities. A three-day acquisition resulted in a predominance of the generalized sensitization signs: a clear non-selective facilitation of the responses to activation of all synaptic inputs, together with depolarization and increased excitability of the command neurons.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 45–53, January–February, 1995.     

Effect of antibodies against S100 proteins on neural plasticity in sensitized and naive snails

The influence of antibodies against total S100 protein fraction (AB-S100) and S100b protein (AB-S100b) on the activity of LP11 and RP11 neurons were studied in naive snails and during the nociceptive sensitization. Application of AB-S100 or AB-S100b (0.1 mg/ml) initiated membrane depolarization, increase in its excitability, and depression of neural responses to sensory stimulation in nonsensitized snails. The sensitization produced facilitation of neural transmission and increase in membrane excitability. Exposure to AB-S100 or AB-S100b (0.1 mg/ml) during sensitization substantially reduced its effects on neural transmission and membrane excitability. The difference between the extent of synaptic facilitation in neurons of sensitized snails and neurons of snails sensitized under conditions of AB-S100 or AB-S100b application was comparable with synaptic depression in neurons of naive snails produced by the isolated application of AB-S100 or AB-S100b. Application of AB-S100 of AB-S100b in the dose of 0.01 mg/ml did not change the parameters of neural activity. The obtained evidence suggest that S100 proteins (in particular, S100b) in L-RP11 neurons are involved in the mechanisms of membrane excitability, regulation of membrane potential and synaptic transmission in naive snails and in the mechanisms of membrane plasticity in the neurons during development of nociceptive sensitization.     

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.     

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.     

Dependence of Electric Activity of Motoneurons and Locomotor Behavior of Lymnaea stagnalis on Environmental Temperature

Experiments on serotoninergic motoneurons of the pedal A-cluster innervating the foot ciliated epithelium of Lymnaea stagnalis L. have shown that changes of the environmental temperature lead to a change of several electrophysiological parameters of these neurons. The firing rate of action potential (AP) progressively increases with elevation of the temperature up to the level of about 30°C, after that an abrupt drop of the rate occurs. The membrane potential and AP amplitude decrease as the temperature rises. The revealed changes may be explained by an increase of the membrane permeability, specifically, to sodium ions. The characteristic activity pattern of these neurons disappears in the temperature range of 34–36°C. The peculiarities observed in the neuronal activity at changes of temperature correlate with a change of characteristics of locomotor behavior of L. stagnalis.     

Antidromic indentification of hypothalamic neurosecretory cells in rats

Characteristics of antidromic action potentials of neurons of the paraventricular and supraoptic nuclei of the rat hypothalamus were studied during stimulation of the hypothalamo-hypophyseal tract by stimuli of varied amplitude and frequency. Step-like changes were found in spike latency in response to an increase in strength (up to 1.5–2.5 thresholds) or frequency (over 100 Hz) of stimulation, as well as cases with variation of the degree of division of the peak into A and B components. Injection of leu-enkephalin analog into the third ventricle or intravenous injection of NaCl solution (1 M) caused reversible changes in the level of excitability of antidromically activated neurons: leu-enkephalin mainly increased the latent period and threshold of action potential generation and reduced the reproducible frequency of stimulation to 10 Hz, whereas NaCl had the opposite effect. The results indicate that when the adopted criteria of antidromic identification of neurosecretory cells are used the level of their excitability must be taken into account.A. A. Ukhtomskii Physiological Research Institute, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 585–591, November–December, 1982.     

The duration of the storage of the electrical characteristics of command neurons in the acquisition of long-term sensitization in the snail

The retention of the long-term sensitization (LTS) of defensive reflex and dynamics of change in electric characteristics (membrane potential (Vm) and action potential generation threshold (Vt)) of command neurons of defensive reflex was studied in a snail during behavioral tests. The membrane mechanisms were analyzed by measuring electrical characteristics of the LPa3, RPa3, LPa2, and RPa2 command neurons on the 1st, 4th, 7th, 10th, and 14th days after the LTS formation and 1 month later. The membrane potential and threshold potential in sensitized snails (-54.1 +/- 2.0 and 24.5 +/- 1.4 microV, respectively) were significantly (p < 0.001) decreased in comparison with the control animals (-60.9 +/- 0.8 and 19.9 +/- 0.6 microV respectively). These changes retained within 14 days after the LTS formation. The results suggest the long-term retention of the increased excitability of command neurons. A month after the LTS formation, the duration of the defensive reflex returned to the initial level and the electric characteristics of command neurons did not significantly differ from the control (-61.1 +/- 2.0 and 19.3 +/- 1.4 microV, respectively).     

Temperature Effects on Pacemaker Generation, Membrane Potential, and Critical Firing Threshold in Aplysia Neurons

Temperature increases cause a regular and reproducible increase in the frequency of generation of pacemaker potentials in most Aplysia neurons specialized for this type of activity which can only be explained as a direct stimulating effect of temperature upon the ionic mechanisms responsible for pacemaker potentials. At the same time all cells in the visceral ganglion undergo a membrane potential hyperpolarization of approximately 1–2 mv/°C warmed. In spite of the marked variation in resting membrane potential the critical firing threshold remains at a constant membrane potential level at all temperatures in the absence of accommodative changes. The temperature-frequency curves of all types of cells are interpreted as a result of the interaction between the effects of temperature on the pacemaker-generating mechanism and resting membrane potential. Previous observations on the effects of temperature on excitability of mammalian neurons suggest that other types of neurons may undergo similar marked shifts in resting membrane potential with temperature variation.     

Effect of catecholamines on excitability of single mechanoreceptors of Pacinian corpuscles

A method of continuous external perfusion was used to study the effect of adrenalin and noradrenalin on the excitability of single isolated mechanoreceptors of Pacinian corpuscles. On the addition of adrenalin and noradrenalin in concentrations of 1·10^–5–1·10^–7 g/ml to the solution the excitability of the receptors was increased by 10–15% and the amplitude of the receptor potential rose on the average by 10–20%. It is postulated that the change in excitability is due to an increase in the receptor membrane potential. The results are discussed in connection with the possible adrenergic innervation of the Pacinian corpuscles.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 6, No. 3, pp. 312–317, May–June, 1974.