Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

Effect of intracellular injection of cyclic amp on electrical characteristics of identified neurons in Helix pomatia

The effect of intracellular iontophoretic injection of cyclic AMP on electrical activity of neurons RPa1, RPa3, LPa2, LPa3, and LPl1 in the corresponding ganglia ofHelix pomatia was investigated. Injection of cyclic AMP into neuron LPl1 was found to cause the appearance of rhythmic activity (if the neuron was originally "silent"), an increase in the frequency of spike generation (if the neuron had rhythmic activity), and a decrease in amplitude of waves of membrane potential, in the duration of the interval between bursts, and in the number of action potentials in the burst (if the neuron demonstrated bursting activity). In the remaining "silent" neurons injection of cyclic AMP led to membrane depolarization. Injection of cyclic AMP into neurons whose membrane potential was clamped at the resting potential level evoked the development of an inward transmembrane current (cyclic AMP current), the rate of rise and duration of which increased proportionally to the size and duration of the injection. Theophylline in a concentration of 1 mM led to an increase in the amplitude and duration of the cyclic AMP current by about 50%. It is concluded that a change in the cyclic AMP concentration within the nerve cell may modify the ionic permeability of its membrane and, correspondingly, its electrical activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 517–525, September–October, 1980.     

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 290–297, May–June, 1982.     

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.

     

Ionic mechanisms of the transmembrane current evoked by injection of cyclic amp into identified Helix pomatia neurons

Ionic mechanisms of the transmembrane current evoked by injection of cyclic AMP into identified neurons ofHelix pomatia were investigated by the voltage clamp method. Injection of cyclic AMP into neurons RPa3, LPa2, LPa3, and LPl1 was shown to cause the development of a two-component transmembrane (cyclic AMP) current. The current-voltage characteristic curve of the early component is linear in the region from –40 to –90 mV; the reversal potential of the early component, determined by extrapolation, lies between –5 and +20 mV; the current-voltage characteristic curve of the late component also is linear and has a reversal potential between –55 and –60 mV. A decrease in the sodium concentration in the external medium from 100 to 25 mM led to a decrease in amplitude of the cyclic AMP current and to a shift of the reversal potential for the early component by 30–32 mV toward hyperpolarization. It is suggested that the early component of the cyclic AMP current in neurons RPa3, LPa2, LPa3, and LPl1 is associated with an increase in permeability of the neuron membrane chiefly for sodium ions, whereas the late component is correspondingly connected with permeability for potassium ions. Injection of cyclic AMP also caused the appearance of a transmembrane inward current in neuron LPa8, but it was independent of the holding potential and was unaccompanied by any change in membrane permeability. It is suggested that this current may be due to a change in the activity of the electrogenic ion pump.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 526–532, September–October, 1980.     

Inositol-1,4,5-trisphosphate and non-hydrolysable GTP analogue induced calcium release from intracellular stores of the Helix pomatia neurons

1. Cytoplasmic Ca2+ concentration ICaIin and membrane potential of single Helix pomatia neurons was studied by Fura-2 fluorescence measurement and conventional current clamp methods. 2. Intracellular injection of inositol-1,4,5-trisphosphate (IP3) and nonhydrolysable GTP analogue (Gpp/NH/p) led to a rise of ICaIin; in contrast, GTP injection did not cause significant ICaIin changes. 3. We suggest that both IP3 and Gpp/NH/p directly activated Ca release from intracellular stores.     

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.     

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.     

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).     

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.     

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.     

The effect of oxytocin on potential-dependent calcium current in snail neurons, Helix pomatia.

1. The effect of external application of oxytocin on inward calcium current in dialyzed snail neurons has been investigated under clamp conditions. 2. External application of oxytocin in a dose-dependent manner (Kd 0.9 microM) inhibits inward calcium current in dialyzed neurons of the snail, Helix pomatia. 3. Inhibition of calcium current developed with the time constant of about 2 min. The degree of restoration of calcium current after oxytocin washout depends on duration of oxytocin action. 4. It has been suggested that inhibition of calcium current by oxytocin occurs in two stages, the initial one is more fast and reversible and the second one--more slow and irreversible. The participation of soluble second messengers in the inhibitory effect of oxytocin on calcium current is discussed.     

Axonal pathways and synaptic inputs of three identified neurons in the buccal ganglion of Helix pomatia.

The axonal pathways and the synaptic inputs of the identified neurons B1 through B3 in the buccal ganglia of Helix pomatia were studied. The axons of neurons B1, B2 and B3 were found to run invariably within the ipsilateral posterior oesophageal nerve, ipsi- and contralateral salivary gland nerves, and ipsilateral cerebrobuccal connective, respectively. Synaptic responses could be elicited by stimulation of most of the nerves of the buccal ganglia. These consisted of an early depolarization which was most frequently followed by a longlasting de- or hyperpolarization. The shape of the synaptic response proved to be related to the different neurons.     

Nicotinic-type unitary currents in Helix neurons

1. The platelet aggregation response to several known platelet agonists was evaluated in four Asian elephants. The platelets were highly responsive to stimulation with platelet-activating factor (PAF) and collagen, less responsive to adenosine diphosphate (ADP) and non-responsive to arachidonic acid, serotonin and epinephrine. 2. Arachidonic acid (1 x 10(-4) M), while inducing no aggregation, caused the release of 1248 +/- 1147 pg/ul (mean +/- SD) of thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 from stimulated platelet. The addition of 1 x 10(-4) M ADP to platelets caused suboptimal aggregation and the release of only 25 +/- 10 pg TXB2/microliters. 3. The calcium channel blocker, verapamil, produced a dose-dependent inhibition of PAF-induced but not collagen-induced aggregation. The cyclooxygenase inhibitor, acetylsalicylic acid, produced no inhibition of either collagen- or PAF-induced aggregation.     

Modulation of voltage-activated ion currents on identified neurons ofHelix pomatia L. by interleukin-1

1. The effect of interleukin-1 (IL-1) was studied on voltage-activated ion currents of the identified central neurons of Helix pomatia L. using a two-microelectrode voltage clamp. The voltage-activated inward current (ICa) was decreased, whereas the outward current (I(net) K) was increased by IL-1. 2. IL-1 affects both the transient and the delayed rectifying potassium currents. The IL-1 modulatory effect on the voltage-activated ion currents was voltage and dose dependent. The threshold concentration for IL-1 was 2 U/ml. 3. The proposed modulatory effect of IL-1 appears to have more than one site of action on the neuron membrane ion channels. 4. Rabbit anti-human IL-1 polyclonal antiserum eliminated the IL-1 effects on the voltage-activated inward and outward currents. This is the first report demonstrating a direct effect of IL-1 modulation of voltage-activated ion currents on neurons of mollusks.