Transmembrane outward hydrogen current in intracellularly perfused neurones of the snail Helix pomatia

The ionic nature and pharmacological properties of the outward current activated by membrane depolarization were studied on isolated neurones of the snail Helix pomatia, placed in Na+- and Ca2+-free extracellular solutions and intracellularly perfused with K+-free solution ("nonspecific outward current"). It was shown that the amplitude and reversal potential of this current (estimated from instantaneous current-voltage characteristics) are determined mainly by the transmembrane gradient for H+ ions. Lowering of pHi induced an increase in the current amplitude and a shift of the reversal potential to more negative values; the shift magnitude was comparable with that predicted for the hydrogen electrode. Raising pHi, as well as lowering pHo, induced a decrease in the current amplitude and a displacement of the current activation curve to more positive potentials. Addition of EGTA (8 mmol/l) to the intracellular perfusate did not affect the current amplitude. Extracellular 4-aminopyridine (10 mmol/l), verapamil (0.25 mmol/l) or Cd2+ (0.5 mmol/l) blocked the current. It is concluded that the current studied is carried mainly by H+ ions. In the same neurones the nature of the fast decay of the calcium inward current was also studied (in the presence of extracellular Ca2+ ions). This decay considerably slowed when pHi was raised or pHo was lowered, and it became less pronounced upon extracellular application of 4-aminopyridine or upon intracellular introduction of phenobarbital (4 mmol/l) and tolbutamide (3 mmol/l). It is suggested that the fast decay of the calcium inward current is due to activation of a Ca-sensitive component of the hydrogen current which depends on accumulation of Ca2+ ions. The possible physiological role of the transmembrane hydrogen currents is discussed.     

Slow low-threshold potassium current inHelix pomatia neurons

A low-threshold outward current was studied in the neurons ofHelix pomatia at –70 to –30 mV using a two-electrode voltage clamp technique. In addition to the well-known A current (I _A), a slower outward current calledI _As (slow) was revealed. Activation and inactivation times ofI _As at –40 mV ranged from 90 to 120 msec and from 3 to 5 sec, respectively. The current recovered within 2 to 5 sec after inactivation at –120 mV. Analysis of changes in the reversal potential ofI _As caused by an increase in external potassium concentration suggests a potassium origin forI _As. The curves ofI _As stationary activation and inactivation fit the Boltzmann equation. Deriving from an activation curve, the activation potential for a half-maximum current,, is –40 mV, and the slope factor,k, is –9.8 mV, while those values for the inactivation curve are –84 mV (a half-maximum inactivation) and 7.5 mV.I _As is blocked by 4-aminopyridine (1–30 µM), tetraethylammonium (1 mM), and Ba²⁺ (1 mM), but is resistant to Cs⁺ (1 mM). PeakI _As is not affected either by substitution of external Ca²⁺ for Mg²⁺ or by application of Cd²⁺ (0.5–1.0 mM). The results suggest that activation ofI _As does not require Ca²⁺ entry into the cell.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 427–432, November–December, 1993.     

Adaptation in Helix pomatia neurons

• 1.1. Spike frequency adaptation has been studied on neurons of Helix pomatia subesophageal ganglia and interpreted by means of a behavioural model describing the phenomenon in neurons either silent or autorhythmic at rest.
• 2.2. At low stimulating currents the initial discharge frequency F(0) is linearly related to the current strength G.
• 3.3. In the linearity range F(0)/G each neuron was characterized by means of four model parameters: the proportionality constant between F(0) and G, the decay constant of the frequency, the inhibitory current from a single nerve impulse and the decay time constant of the inhibitory current.
• 4.4. The four parameters varied in different cells with a range of 0.18–4.98 Hz/nA, 1.02–3.85 sec, 0.05–0.95 nA and 1.74–22.33 see, respectively.
• 5.5. Experimental results have been analyzed considering inhibitory current, electrogenie sodium pump and other proposed adaptation parameters.

     

Cyclic AMP enhances calcium-dependent potassium current inAplysia neurons

The effect on the Ca-dependent potassium current, IK(Ca), of procedures that increase intracellular cAMP levels was studied in Aplysia neurons using three different pharmacological approaches. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase in IK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increased IK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. Application of serotonin plus phosphodiesterase inhibitor caused an increase in IK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease in IK(Ca). It is proposed that elevation of cyclic AMP mediates an increased sensitivity of the IK(Ca) channel to Ca ions.     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.     

Parvalbumin-immunoreactive neurons in the brain of Helix pomatia

Parvalbumin-immunoreactive material was detected in the central nervous system of the snail, Helix pomatia. Each ganglion investigated contained parvalbumin-immunoreactive neurons. The molecular weight of Helix parvalbumin-immunoreactive material as determined by Western blots is about 40 kilodaltons. ⁴⁵Ca²⁺ overlays showed that this protein binds Ca²⁺. In contrast to vertebrates, in Helix neurons parvalbuminlike material was not colocalized with the neurotransmitter gamma-aminobutyric acid (GABA).     

Voltage-dependent potassium channels in the molluscan neuron somatic membrane

Under voltage clamp conditions proof of the presence of two populations of potassium current channels was obtained on the molluscan neuron somatic membrane: inactivated and uninactivated. They differ from each other in their physicochemical characteristics, the property of their gating mechanisms, and the molecular structure of their current-conducting part. The inactivated potassium current is largely and selectively inhibited by cooling. Channels of the fast potassium current also are highly sensitive to temperature changes. By using parameters of gating mechanisms of the "fast" potassium channels included in the Hodgkin-Huxley model, the physicochemical properties of channels of this type were described. The density of fixd negative surface charges on the somatic membrane in the region of localization of fast potassium channels was estimated with the aid of the Gouy-Chapman theory. It is 0.3 electron charge/nm². Data on the character of interaction of potassium channels with intracellular sodium ions revealed differences in the structure of the current-conducting part of different types of potassium channels. Experiments on intracellularly perfused molluscan neurons demonstrated the particular features of interaction between intracellular calcium ions and calcium-activated channels under conditions of strictly controlled changes in the intracellular calcium concentration.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 296–307, May–June, 1984.     

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.

     

Research into pharmacological and metabolic dependence of delayed inactivated outward potassium current at the snail neuronal somatic membrane

Features of pharmacological and metabolic sensitivity of potassium outward current dependent on the presence of Ca ions in the intracellular solution were investigated. Verapamil was found to produce depression and accelerated inactivation of this current. The blocking action of verapamil intensifies with an increase in extracellular concentration of calcium ions. Functioning of channels depends on intracellular metabolic processes. Current level declined during intracellular perfusion of the neuron with artificial saline. Reducing the temperature of the perfusing solution to 10°C produced a similar result. This decline could be avoided by introducing 2 mM ATP and 3 mM Mg ions into the intracellular solution; current decay could actually be reversed by intracellular application of exogenous protein kinase C. Polymyxin B (10^–4 M), a protein kinase C blocker, depressed the component of outward potassium current dependent on extracellular Ca²⁺. Mechanisms possibly underlying Ca²⁺ action on via a phosphatidyl inositol metabolism is discussed in the light of the experimental findings obtained.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 52–59, January–February, 1989.     

Multiplicity of pacemaker zones in Helix pomatia neurons

Intracellular microelectrode recordings from neurons ofHelix pomatia revealed several local zones of action potential generation both on the soma and on some of the branches of the neurons. Under certain conditions the activity of individual loci of the neuron membrane was synchronized to produce a normal action potential. It is suggested that the somatic membrane of neurons is heterogeneous in structure and consists of separate loci of an electrically excitable membrane, incorporating active and latent pacemaker zones. Neurons ofH. pomatia are characterized by two types of action potential with different triggering mechanisms: one (synaptic) type is generated under the influence of the EPSP, the other (pacemaker) arises through activation of endogenous factors for the neuron (pacemaker potentials). The interaction between synaptic and pacemaker potentials during integrative activity of the neuron is discussed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 5, No. 1, pp. 88–94, January–February, 1973.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Sites of action potential generation in Helix pomatia neurons

The site of action potential generation in unipolar snail neurons was identified by stimulating neurons isolated together with the initial portion of the process from the neuropile. Stimulation consisted of a sinusoidal from electrical current passed along the soma-axonal axis in saline solution. No low threshold sites of action potential generation were found in 80% of test neurons using this technique. Spontaneous activity was determined by the operation of one dominant site on the neuronal process. Antidromic activation of the soma by axonal action potentials (even with simultaneous hyperpolarization of the soma) induced somatic potentials more successfully than direct somatic depolarization by the current flowing through the solution.Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 90–98, January–February, 1988.     

Effects of microiontophoretically injected AMP and cAMP on calcium current in dialyzed Helix pomatia neurons

The effects of injecting cells with adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) on calcium current were investigated during intracellular dialysis ofHelix pomatia neurons. Microiontophoretically injected AMP was found to lead to reinstatement of calcium current following dialysis-induced wash-out, as well as considerable stabilization of this current with the extracellular medium at normal pH. Current-voltage relationship of the current would then undergo a 10 mV shift towards depolarization values. Perfusing the cell with a solution containing 10 mM AMP then produced a qualitatively identical effect. Injecting the neuron iontophoretically with cAMP led to a decline in the amplitude of calcium current under the same conditions. Neither raising the pH of the intracellular solution to 8.1 nor adding 4-aminopyridine in order to depress the hydrogen ion current produced a qualitative alteration in the effects of injecting AMP and cAMP on calcium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 6, pp. 769–776, November–December, 1988.