Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

Intracellular protein kinase and calcium inward currents in perfused neurones of the snail Helix pomatia

Changes in the amplitude of the calcium inward current caused by intracellular administration of tolbutamide (an inhibitor of the cyclic AMP-dependent protein kinase activity) or catalytic subunits of cAMP-dependent protein kinases from rabbit myocardium were studied on internally perfused nerve cells of the snail, Helix pomatia. Intracellular administration of 7 mM tolbutamide caused a rapid decline of the amplitude of the calcium current that had been stabilized by theophylline; the effect was practically completely reversible. In contrast, addition to the perfusing solution of exogenous catalytic subunits of cyclic AMP-dependent protein kinases (about 0.7 microM of protein) together with 2 mM adenosine 5'-triphosphate and 3 mM MgCl2, led to stabilization of the calcium conductance of the cell membrane or restored it if it had declined during the perfusion with basic solution. The effect depended largely on the presence of adenosine 5'-triphosphate. Its time course was very slow (dozens of minutes) due probably to slow diffusion of the protein inside the cell. Heat-inactivated catalytic subunits did not produce such a stabilizing or restoring action on the calcium conductance. The results substantiate the suggestion that the normal functioning of calcium channels depends on phosphorylation catalyzed by cyclic AMP-dependent protein kinases.     

Lithium slows neuronal calcium regulation in the snail Helix pomatia

Steady-state and transient changes in intracellular calcium concentrations ([Ca2+]i) of snail neurons (Helix pomatia) were measured by the Ca2+ indicator Arsenazo(III) following manipulation of the extracellular concentration of lithium chloride (LiCl). Application of LiCl in concentrations equivalent to those used in the treatment of manic-depressive illness produces slowing in Ca2+ reequilibration after Ca2+-influx during depolarization, concomitantly with steady-state elevation of [Ca2+]i of about 100 nM, suggesting a change in Ca2+ reequilibration as a prominent action of LiCl. This mechanism may be relevant to the therapeutic effects of LiCl.     

Properties of a calcium- and voltage-activated potassium current in Helix pomatia neurons

A calcium- and voltage-dependent current was found to be the principal outward current in identified Helix neurons. The current depends on the presence of [Ca²⁺]₀, with half maximal activation at 1 mM [Ca²⁺]₀, and it saturates beyond about 5 mM. The current is termed I_K(Ca) since the charge carried by it corresponds to the amount of potassium ions transferred from the cell interior, as determined from the increase in K⁺ concentration in the external volume with K⁺ liquid ion-exchanger microelectrodes. I_K(Ca) is characterized by bell shaped isochronal I/V curves. The peaks of these curves move from +30 mV to about +70 mV with an increase of the time of measurement from 30–200 ms. I_K(Ca) rise times have a minimum of 10–15 ms at low depolarization around 0 mV, but increase about exponentially with more positive potentials. A tenfold decrease in [Ca²⁺]₀ over the range of 30 to 0.3 mM also produces an increase in rise time, equivalent to a positive shift of potential by 20 mV. On repolarization of the membrane I_K(Ca) disappears much faster than the intracellularly accumulated Ca²⁺, with a time constant which is similar to the minimum activation time constant.     

The antagonistic actions of calcium and magnesium on the superfused ventricle of the snail Helix pomatia

1. The isolated, superfused half-ventricle of the snail (Helix pomatia) maintains a degree of tonic activity even when not beating, since the membrane is depolarized beyond the tension threshold. Beating may be initiated by lateral stretch of the ventricle and by 5-hydroxytryptamine.2. Ca and Mn each have a hyperpolarizing action, while K and to a lesser extent Na cause depolarization: the tonic activity of the ventricle is affected accordingly.3. An increase in the extracellular concentration of Mg also causes the tension to fall, but without change in membrane potential. It can also initiate beating.4. Contractures induced by 30 mM-K are steadily maintained, but start with a more-or-less distinct twitch-like contraction. A contracture induced by a concentration of K above about 50 mM is poorly sustained and is followed by a further brief contracture when the K concentration is reduced to normal.5. Relations between contracture tension and the concentrations of Ca and Mg accord with the hypothesis that the two ions compete for attachment to a binding site on the cell surface and that tension is proportional to the amount of bound Ca. On this hypothesis, the apparent dissociation constant of the Mg complex is 11.4 mM and that of the Ca complex 0.15 mM or less.6. This effect of Mg is like that of Na on frog ventricle and some of the differences in the behaviour of snail and frog ventricles are abolished by appropriate adjustment of the extracellular concentrations of these ions.     

Non-respiratory haemolymph proteins in the vineyard snail Helix pomatia. Changes after phagocytosis in vivo

Haemolymph proteins from the vineyard snail Helix pomatia were studied by chromatographic and immune-electrophoretic techniques with the dual purpose of characterizing the normal composition of haemolymph and to look for possible opsonins. The oxygen-carrying proteins, alpha- and beta-haemocyanin, were by far the most abundant proteins, but at least three non-respiratory proteins could be demonstrated. We found consistent changes in the appearance of the immunoprecipitation pattern of these non-respiratory proteins after the injection of particulate foreign matter into the snail's circulation, and we suggest that they may be opsonins. We also found a haemagglutinin in haemolymph. It was present in very low concentration and was similar to, but not identical with the haemagglutinin which is present in the albumin gland.     

Dopamine and serotonin receptors mediating contractions of the snail,Helix pomatia,salivary duct

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction.These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.     

Impulse dependent adaptation in Helix pomatia neurones: demonstration of the adaptation conductance

By conventional voltage clamp methods an increase in membrane conductance after a depolarizing pulse is demonstrated in neurones of Helix pomatia. This increase decays exponentially with a time constant in the range of 5--20 s. The rise of the conductance during the depolarization can be represented by 3 exponentials with time constants from 12 ms to 1 s. The steady state value of the conductance depends on the membrane potential in a sigmoid manner. The conductance gives rise to an outward current, which appears to be carried by potassium ions. The firing pattern of the cell is governed by the conductance. A short, rectangular potential change, a pulse clamp, is used to disturb the firing pattern of the freely firing cell. The effect on the firing pattern depends on the height and duration of the clamp pulse in the same manner as the conductance increase. The normal firing produces similar changes in the membrane conductance.     

Visualization of non-synaptic release sites in the myenteric plexus of the snail Helix pomatia

Following conventional glutaraldehyde-osmium tetroxide fixation, a rich myenteric plexus was detected in the gastrointestinal tract of the snail Helix pomatia. Although hundreds of nerve processes were observed in the extensive myoneural neuropil, true synaptic specializations were not recognized in them. In the absence of synaptic specializations, tannic acid-Ringer incubation was applied to visualize the non-synaptic release sites in the enteric nerve plexus. After incubation for 1 h, a great number of exocytosis profiles were recorded at nerve-muscle contacts, at axoglial connections and in the myoneural neuropil. The frequency of occurrence of exocytosis profiles was the same for adrenergic and peptidergic fibres. In some gut wall areas, a dense staining of both basal lamina and collagen fibres was observed. Invagination of dense basal lamina into the omega-shaped profiles of the axolemma led to "false exocytosis" profiles. A detailed morphological analysis is needed to distinguish false exocytosis profiles from the true transmitter-releasing loci.     

Ultrastructure of the epithelial sensory region of the lip in the snail Helix pomatia L

The ultrastructural organization of the sensory epithelium in the lip of the snail, Helix pomatia, has been studied. Both the lateral and ventral surface pf the lip are composed of columnar, unciliated epithelial cells. At the free surface of indifferent epithelial cells many branching cytoplasmic processes were observed overlying a finely granular cuticular layer 1–3 μm thick. Intraepithelial receptor cells were not present, but a great number of sensory dendrites occurred, especially on the ventral surface of the lip. On the basis of their ultrastructural characteristics, dendrites could be divided into two main groups. Most of the dendrites bear only microvilli on their distal ends (first group) and smaller population (second group) have both microvilli and cilia at their distal end. In the nonciliated dendrites, there was a large amount of smooth surfaced endoplasmic reticulum; in the ciliated type of dendrite, microtulules and other cell organelles were common.It can be concluded that there are at least two types of sensory dendrite in the lip of H. pomatia, and this may be the structural basis of the physiologically established sensitivity to different chemical substances.     

Newly identified nerve cells of the snail,Helix pomatia,associated with the generation of pacemaker activity

Data on new, previously unidentified nerve cells of the snailHelix pomatia are presented in this paper. The identified neurons described may serve as a convenient model for the investigation of the cellular mechanisms of pacemaker activity, the role of neuropeptides in the generation and regulation of pacemaker activity, peptidergic transmission, and the functional role of the inward calcium current. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 42, No. 6, pp. 1124–1131, November–December, 1992.     

Barium ions block the acetylcholine evoked slow H-response in the neurones of Helix pomatia L

The effects of barium ions on slow acetylcholine (Ach) H receptor activated hyperpolarization has been studied. The results provide evidence of a sensitive and reversible blocking action of Ba2+ on the involved potassium conductance. 3-Aminopyridine (3-AP) also attenuated the slow H-response by 50%, showing partial effectiveness. TEA+ sensitively inhibited the Ach evoked hyperpolarization interacting with the receptors.     

Modulation of voltage-dependent calcium current in Helix aspersa buccal neurones by serotonin and protein kinase C activators.

In Helix aspersa, activation of the cerebral giant serotonin neurones (GSNs) evokes a biphasic, excitatory synaptic response in the M neurones of the buccal ganglia. Local application of serotonin to the current-clamped M neurones also evokes fast and slow depolarizing responses. The slow response is thought to be dependent on calcium ions, whereas sodium ions have been implicated in the fast response. Here we provide further evidence that the slow response results from an increase in conductance to calcium ions, and show that okadaic acid, an antagonist of protein phosphatases 1 and 2A, potentiates the effect of serotonin, suggesting that the response is phosphorylation dependent. Further, agents known to activate protein kinase C, such as 1-oleoyl-2-acetyl-rac-glycerol and active phorbol esters (but not an inactive one) were found to increase the calcium current (actually carried by barium ions) of the M neurones. Such data suggest that the slow synaptic response mediated by serotonin can occur by activation of protein kinase C and phosphorylation of the affected voltage-sensitive calcium channels, or some closely associated protein(s).     

Impulse dependent adaptation in Helix pomatia neurones: effect of the impulse on the firing pattern

In neurones of the ventral ganglion of Helix pomatia an adaptation type is demonstrated where the impulses contribute to the adaptation, as if a slowly changing outward current were associated to every impulse. The current appears to be accumulated from impulse to impulse and the accumulated current to decrease towards zero in the interval between the impulses. This type of adaptation is called impulse dependent. A method to distinguish between impulse dependent adaptation and impulse independent adaptation is described. Typical adaptation curves for the impulse dependent adaptation are shown. With a strong adaptation there is a linear relation between the steady state frequency and the applied stimulus. When stimuli of short duration are applied repetitively, neurones with impulse dependent adaptation respond with spikes on an increasing fraction of the stimuli as the stimulus strength is increased. A simplified model of the adaptation is proposed, and the firing pattern of the cells is compared with that of the model. In this way numerical values of the model parameters have been estimated.     

The time courses of intracellular free calcium and related electrical effects after injection of CaCl2 into neurons of the snail,Helix pomatia

Controlled quantities of 100 mM aqueous CaCl₂ solutions were pressure injected into voltage-clamped neurons with a resolution of 10^–11 1. Ca²⁺-selective microelectrodes monitored the time course of changes in [Ca²⁺]_i. At a membrane potential of –50 mV CaCl₂ quantities in the range of 1% of the cell volume induced an inward current, associated with a conductance increase and having an equilibrium potential between –20 and +20 mV, which accompanied the rise in [Ca²⁺]_i. An artifactual origin of the inward current by the injection procedure or by calcium screening of membrane sites could be excluded. The calcium-induced hyperpolarizing conductance, producing an outward current at –50 mV, followed the inward current and reached maximum during the late decline in [Ca²⁺]_i. In most cases its development was separated from the inward current by an intermediate relative decrease of the membrane conductance. Neither of the two transient conductance increases showed a particular dependence on voltage. Renewed Ca²⁺ injection quickly decreased the calcium-induced hyperpolarizing conductance for several seconds. Ca²⁺ injections below 0.05% of the cell volume mostly produced pure outward currents or hyperpolarizing responses. Partial substitution of extracellular CaCl₂ by NiCl₂ decreased the hyperpolarizing response but not the initial inward current. The immediate effects of increased [Ca²⁺]_i are activation of a depolarizing conductance and the partial block of the late hyperpolarizing conductance. The latter is probably produced through intermediate steps after increasing [Ca²⁺ _i.     

Properties of T-type calcium current in enkephalinergic neurones in guinea-pig hypothalamic slices

 The guinea-pig hypothalamic magnocellular dorsal nucleus (mdn) exclusively contains enkephalinergic neurones providing inputs to the septum. This nucleus is believed to play a role in hippocampo-septo-hypothalamic relationships. mdn neurones display prominent low-threshold Ca²⁺ spikes, which differ in their propensity to trigger either a burst of Na⁺ spikes or a single spike. In the present study, whole-cell voltage-clamp experiments were carried out on thick slices at 34°C to characterize the pharmacological and physical properties of the transient Ca²⁺ current (I _T) underlying the low-threshold spikes. Recorded cells were dye-labelled and identified as belonging to the mdn. In bursting and non-bursting neurones, I _T was reduced by amiloride (1 μM) and octanol (1 mM), and during replacement of Ca²⁺ by Ba²⁺. The Ca²⁺ channel blocker mibefradil (10 μM) had only a slight blocking action. Nifedipine (100 μM) and flunarizine (1 μM) had no effect. I _T activated between –80 mV and –50 mV and the mean peak current was 1050 pA. Steady-state activation and inactivation curves were fitted by a Boltzmann equation. The half-activation voltage was –70 mV, slope factor=3.6, and half-inactivation voltage was about –80 mV, slope factor=4.5. Time-to-peak and time constant of inactivation were voltage dependent. Recovery from activation occurred within 500 ms. When compared with results on other I _T, the present data show that the current possesses distinct pharmacological and physical properties. Nevertheless, all investigated cells displayed a homogenous profile of I _T, suggesting that the differences in spike pattern between mdn neurones are not due to different populations of Ca²⁺ channels. Received: 20 October 1998 / Received after revision and accepted: 5 January 1999     

Further studies of the potential-dependence of the sodium-induced membrane current in snail neurones.

1. The potential-dependence of the membrane current induced by intracellular injections of sodium ions was studied on giant neurones of the snail Helix pomatia. This current decreases with membrane hyperpolarization at room temperature and can be reversed at sufficiently negative holding potentials. The same injections at 7 degrees C, as well as injections of lithium or potassium ions do not induce membrane currents and do not increase membrane conductance. 2. An increase in the amount of injected sodium changes the potential-dependence of the induced membrane currents. Small injections (about 1 muC) induce a current that does not depend upon the membrane potential. Further increase in the injection size not only increases the induced current but also enhances its potential-dependence and often reveals the existence of a reversal potential. The latter reaches -60 to -65 mV with large sodium injections. 3. An increase in extracellular potassium concentration from 4 to 8 mM shifts the reversal potential 17 mV in the depolarizing direction, and a decrease from 4 to 2 mM shifts it 14 mV in the hyperpolarizing direction. Replacement of potassium by rubidium or elimination of sodium ions from the outside solution, does not affect the induced current or its potential-dependence. 4. The coefficient of electrogenicity (the ratio between the amount of charge transferred by the sodium-induced membrane current and the amount brought into the cell during the injection) increases with an increase in the injection size if the membrane potential is clamped near the resting potential level. This relation is reversed when the holding potential is -80 mV. The reversal takes place at holding potentials near -60 mV. 5. 10 mM TEA does not affect the induced current and its potential-dependence. 6. It is suggested that the potential-dependence of the sodium-induced membrane current is a result of a specific increase in the membrane potassium conductance that is coupled with high activity of the sodium pump.