Study of effects of antibody to protein S100 on ionic channels of input and output currents of identified neurons of the snail Helix lucorum

In the course of electrophysiological experiments, two types of the neurons of the edible snail Helix lucorum were detected, which responded by different way to application of antibodies to the neuron-specific calcium-binding S 100 protein (AS1000). Under effect of AS100, frequency of the action potential (AP) generation in the spontaneously active V1, V3, V17, and RPa6 cells decreased, whereas in V4 and V6 cells increased. On addition of quinine solution the AP generation frequency of these neurons decreased more than twice, while the AP duration (t _S) rose 6 times. The combined action of AS100 and quinine did not change statistically significantly the AP generation frequency, membrane potential (MP) and AP generation threshold (AP_t), as compared with the effect of AS100 in saline. The value of the AP duration (t _S) increased 1.6 times, which was less pronounced as compared with the quinine action in saline. This means that AS100 prevents an increase of the AP duration after the quinine application (block of the Ca-depended K-channels). The main AS100 effect at the level of the ionic currents is shown to consist in a decrease of the maximal value of the input current, on average, by 20%, while of the output current, on average, by 12%.     

Effect of deltamethrin on transient outward currents in identified snail neurons

• 1.1. The effect of a pyrethroid insecticide deltamethrin was investigated on transient outward potassium currents of identified snail (Helix pomatia) neurones LPa1 and RPa3.
• 2.2. In 5 × 10⁻⁵ M concentration the deltamethrin decreased the I_A amplitude and the slope of I–V curve. The activation variable was shifted left along the voltage axis by 10–20 mV, while the inactivation variable remained unchanged.
• 3.3. Time constant of inactivation decreased, and the relaxation of I_A described by one exponential. “Modified” ionic channel fraction was not observed.
• 4.4. It is suggested that deltamethrin acts on I_A channels through a different molecular mechanism to I_Na channels, since not only the gating machinery but the permeability of the channels were influenced.

     

The participation of GFAD-synthesizing neurons in behavior regulation in the snail (Helix lucorum L.)

It was shown earlier that some neurons in Helix CNS express the mRNA of the precursor of neuropeptide GFAD. Using the data obtained with the help of the whole-mount in situ hybridization, we tried to identify a group of such neurons, namely, the pedal caudo-ventral group and to determine their possible functions. The local extracellular stimulation of the pedal caudo-ventral group resulted in movements of reproductive organs in the semi-intact preparation and suppressed the activity of the modulatory neurons controlling feeding and defensive behavior. Application of synthetic peptide GFAD (10(-8) mol/l) also activated movements of the reproductive organs and suppressed the activity of the modulatory neuron controlling feeding behavior. Stimulation of the labial nerves resulted in suppression of caudo-ventral neurons with simultaneous activation of the modulatory neuron controlling feeding behavior. The obtained evidence suggests that the caudo-ventral neurons can regulate movements of the reproductive organs and also coordinate different functions in realization of the integral sexual behavior. This group of neurons inhibits the modulatory neurons controlling the forms of behavior incompatible with courtship, i.e., feeding and defensive forms.     

Effect of protein synthesis inhibitors on sensitization of defence reaction and on cholinosensitivity of command neurons in Helix lucorum

We studied influence of protein synthesis inhibitors on short-term sensitization of Helix escape reaction and potentiation cholinosensitivity in command neurons. Inhibitor of protein synthesis anisomycin does not prevent behavioral sensitization. Anisomycin and irreversible inhibitor of protein synthesis saporin change the dynamics of cholinosensitivity potentiation in command neurons. The results Suggest that investigated sensitization of Helix escape reaction does not require synthesis of new proteins.     

Comparative study of the effects of antibodies to S-100 protein and ouabain on neurons of Helix pomatia

Ouabain, used in 5.10(-4) M concentration, elicits 12 +/- 5 mV (15 experiments) depolarization of membrane of snail Helix neurons. In 80% of experiments depolarization is not accompanied by changes in membrane conductance, in 20% of experiments the decrease of the membrane conductance is observed. Application of the antibodies to S-100 protein (their concentration in the micropipette being 0.05 mg/ml) induces similar effects. The effects of ouabain and antibodies to S-100 protein are not additive and the main difference in their action lies in the ability of the cell to recover the resting potential of the membrane in the solution containing ouabain.     

Postembryonic neuronogenesis in the procerebrum of the terrestrial snail,Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70–80%) of the snail procerebrum are produced during the first 1–2 months of posthatching development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 271–276, 1998     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

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.     

The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the suboesophageal ganglionic mass of the snail, Helix aspersa. The ionic mechanisms associated with acetylcholine excitation and inhibition, dopamine excitation and inhibition, gamma-aminobutyric acid (GABA) excitation and inhibition and serotonin excitation were investigated. Acetylcholine excitation was found to involve an initial increase in sodium conductance while acetylcholine inhibition was a pure chloride event which reversed at membrane potentials more negative than the chloride equilibrium potential. Dopamine excitation appeared to involve only an increase in sodium conductance while serotonin excitation involved an increase in conductance to both sodium and calcium ions. Dopamine inhibition was associated with an increase in potassium conductance but failed to reverse at membrane potentials more negative than the potassium equilibrium potential. GABA excitation involved conductance increases to both sodium and chloride ions while GABA inhibition was a pure chloride event. An attempt was made to estimate the degree of co-operativity of the putative transmitters with their receptors using log-log and Hill plots. The slopes of the line for the log-log plots for acetylcholine excitation and inhibition were 0.88 and 1.1, respectively, suggesting the interaction of one molecule of acetylcholine with the receptor. The slope of the log-log plot for dopamine inhibition was 0.46 while that for serotonin excitation was 0.75. The Hill plots for GABA excitation and inhibition were 1.64 and 1.42, respectively, suggesting that two molecules of GABA are required for receptor activation.     

Effect of sodium-free solutions on ionic currents of snail giant neurons

The ionic currents of the snail giant neurons were investigated by the voltage clamp method. The effect of sodium-free solutions on the inward and outward currents was studied. It is shown that the current entering the cells is created mainly by sodium ions. When a preparation is immersed into a solution not containing sodium ions, most neurons (tentatively neurons of type "a") "lose" the inward currents. In other neurons (tentatively of type "b") this process lasts 40 min and more. A number of peculiarities of type "b" neurons were noted. The response of the excitable membrane to conditioning polarization was also investigated. The data obtained permit the conclusion that 85–90% of the sodium-transfer system is activated in the case of a voltage clamp from the level of the resting potential.A. A. Bogomolets Institute of Physiology, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 3, pp. 314–320, May–June, 1970.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.     

Inhibition of protein synthesis during associative memory reactivation produces reversible or irreversible amnesia in the snail Helix lucorum

Effects of protein synthesis inhibitors on reactivation processes of food aversion conditioning were inverstigated in snail Helix lucorum. Protein synthesis inhibitor (PSI, anisomycin, 0.4 mg, or cycloheximede, 0.6 mg) was injected into snail body cavity 24 hours after 3-day training; then conditioned stimulus (banana) was presented and memory was tested. It was found that 2.5-3 hours after first reminding, associative food conditioning was suppressed, recovering of the conditioning was observed 4.5-5.5 hours after first reminding. In other group of snails, PSI injections were single (1.8 mg) or triple (0.6 mg with 2-hour interval). Reminding stimulus was presented after each injection. In this case, suppression of food aversion conditioning was also observed 2.5-3 hours after first reminding, while amnesia in this case lasted over 30 days. Repeated training of the group of snails recovered the food aversion conditioning only partially. In control snails (saline instead of PSI or 3 injections of PSI without reminding), foot aversion conditioning was detected 30 days after first training. Thus we found that PSI effects during reminding of food aversion conditioning produced two phases amnesia: (1) the easily suppressed by PSI transient phase lasted 2-3 hours, and (2) irreversible phase, its suppression by high doses of PSI-initiated amnesia lasting over 1 month. Second phase of amnesia was not recovered after repeated training. It was suggested that reminding induced reconsolidation of initial memory. Its suppression by protein synthesis inhibitors results in erasing of memory trace and disturbs repeated consolidation.     

Effect of ginseng preparations on the activity of identified neurons of Heliz lucorum snails

It has been demonstrated that the effects of aqueous extracts from the native ginseng and extracts of genseng tissue culture on the activity of identified neurones are identical. Neuronal excitability, tested intracellularly, significantly increases to the 60th minute of drug application, whereas gradually developing depolarization simultaneously disappears. Rhythmic orthodromic stimulation revealed the increase in neuronal responses during ginseng perfusion, the synaptic efficiency remaining constant. It is suggested that changes in transmembrane potential level evoked by administration of ginseng preparations are not related to changes in the excitability. Changes in the adaptive capacities of the neurones in invertebrate animals are due to nonspecific endoneuronal shifts in cellular metabolism induced by ginseng administation.     

Reduction of calcium currents in identified neurons of Helix pomatia: intracellular injection of D890

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.     

The effect of oxytocin on the identified neurons of the brain in the edible snail Helix pomatia L.]

Responses induced by a perfusion by a solution with oxytocin were examined in identified Helix pomatia L. neurons. Depolarizing, hyperpolarizing, and modulatory neuronal responses were observed. The responses under study were supposed to be associated in most of the cases with the system of cyclic nucleotides.     

Effect of FMRFamide on the electrical and plastic properties of identified Helix pomatia neurons

Effect of the neuropeptide FMRFamide on two types of nerve cells differing in plastic properties: habituating and non-habituating to rhythmic intracellular stimulation, has been studied. FMRFamide causes a slow developing, continuously growing depolarisation and an increase of input resistance of the most part of habituating cells resulting in inhibition of their habituation to intracellular stimulation. No desensitisation of cells to the action of FMRFamide was observed. The data obtained by using Ca-ionophore, imidazole and caffeine show that the effect of the peptide may be caused by inhibition of Ca-dependent K-conductance and depends on cAMP metabolism. FMRFamide exerts a less pronounced action on non-habituating cells and does not change their plastic properties. Inhibition by FMRFamide of the habituation at the level of electroexcitable membrane may play a significant part in regulation of neuronal plasticity.     

Mechanisms of membrane potential oscillation in bursting neurons on the snail,Helix pomatia

• 1.1. The mechanism of generation of membrane potential (MP) oscillations was studied in identified bursting neurons from the snail Helix pomatia.
• 2.2. Long-lasting stimulation of an identified peptidergic interneuron produced a persistent bursting activity in a non-active burster.
• 3.3. External application of calcium channel blockers (1 mM Cd²⁺ or 5 mM La²⁺) resulted in a transient increase in the slow-wave amplitude and subsequent prevention of pacemaker activity generation in bursting neurons. Application of these blockers together with endogenous neuropeptide initiating bursting activity generation, increased MP wave amplitude without prevention of bursting activity generation.
• 4.4. Replacement of all NaCl in normal Ringer's solution with isoosmotic CaCl₂, glucose or Tris-HCl produced a reversible block of bursting activity generation. Stationary current-voltage relation (CVR) of bursting neuron membrane has a region of negative resistance (NRR) and does not intersect the potential axis in threshold region for action potential (AP) generation in normal Ringer's solution. In Na-free solution stationary CVR is linear and intersects the potential axis near — 52 mV.
• 5.5. Novel potential- and time-dependent outward (E_rev = − 58 mV) current, I_B, activated by hyperpolarization was found in the bursting neuron membrane. Having achieved a maximal value, this current decayed with a time constant of about 1 sec. Hyperpolarization inactivated maximal conductance, g_B, responsible for I_B, and depolarization abolished inactivation of g_B.
• 6.6. Short-lasting (0.01 sec) hyperpolarization of the bursting neuron membrane by inward current pulse induced the development of prolonged hyperpolarization wave lasting up to 10 sec.
• 7.7. These results suggest that: (a) persistent bursting activity of RPal neuron in the snail Helix pomatia is not endogenous but is due to a constant activation of peptidergic synaptic inputs of these neurons; (b) Ca²⁺ ions do not play a pivotal role in the ionic mechanism of MP oscillations but play a determining role in the process of secretion of a peptide initiating bursting activity by the interneuron presynaptic terminal; (c) depolarizing phase of the MP wave is due to specific properties of stationary CVR and hyperpolarization phase is due to regenerative properties of hyperpolarization-activated outward current I_B. The minimal mathematical version of MP oscillations based on the experimental data is presented.

     

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.