Modulation of electrical activity of neuron RPal ofHelix pomatia

Changes in the types of electrical activity of bursting neuron RPal ofHelix pomatia were studied. Neuron RPal may either be "silent" or may exhibit bursting activity with waves of membrane potential of low and high amplitude. Changes in activity of this neuron took place spontaneously over a period of tens or hundreds of seconds. Changes in electrical activity in neuron RPal were synchronized with changes in membrane potential in other neurons. Similar changes in electrical activity of neuron RPal can be produced by application of the water-soluble fraction from snail ganglion homogenate, containing "modulating factor," to the soma. It is suggested that the prolonged changes in electrical activity of neuron RPal described above are connected with the action of compounds resembling neurotransmitters or neurohormones, and secreted by other neurons, on it. These compounds reach the neuron continuously or they are bound with the receptors of the neuron for a long enough period of time to produce stationary changes in its membrane conductance.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 398–405, July–August, 1981.     

Effect of cadmium ions on bursting electrical activity of an identified snail neuron

The effect of cadmium ions, a specific blocker of the inward calcium current in molluscan neurons, on electrical activity of identified neuron RPal ofHelix pomatia was studied. Cadmium ions in a concentration of 1 mM were shown to block bursting activity of the neuron completely. The membrane potential increased under these circumstances to about ?65 mV. After rinsing out the cadmium ions electrical activity in the neuron was fully restored. If a modulating factor (a peptide fraction obtained from the water-soluble part of snail brain homogenate) was added to the solution containing cadmium ions, however, not only was bursting activity not blocked, but it was actually intensified. Addition of modulating factor to the solution after blocking induced by cadmium ions led to the reappearance of bursting activity if not more than a few tens of seconds had elapsed after blocking developed. As the time after the beginning of blocking increased, addition of the modulating factor became less effective and caused only rhythmic activity to develop. It was concluded from the results of these experiments that bursting activity of neuron RPal is not endogenous but is induced in it by a modulating factor secreted by an unidentified peptidergic interneuron. Calcium ions do not play an essential role in the generation of slow depolarization waves in the neuron under these circumstances but they are essential for secretion of the modulating factor.     

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.

     

Isolation of a peptide initiating bursting activity in an identifiedHelix pomatia neuron

A peptide initiating bursting activity when applied to the soma of identified neuron RPal ofHelix pomatia (if such activity was absent) or increasing the amplitude of waves of membrane potential (if it was low), was isolated from the water-soluble fraction of brain homogenate. Application of the peptide and of the original material for its isolation (the water-soluble fraction of snail brain homogenate) evoked identical changes in the character of electrical activity of neuron RPal. It is concluded from the experimental results that the isolated protein possesses a specific action, qualitatively different from that of other known peptides, on this neuron and is the active principle of the modulating factor described previously. It is postulated that under natural conditions the modulating factor is secreted by an unidentified peptidergic interneuron in the region of axo-somatic synapses, evoking bursting activity in neuron RPal.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 488–492, July–August, 1984.     

Monosynaptic pathway responsible for generation of bursting activity in theHelix pomatia RPal neuron

The connection between a visceral ganglia interneuron initiating bursting pacemaker activity in the RPal neuron and the RPal neuron itself was investigated inHelix pomatia. Stimulating the interneuron either initiated or intensified bursting activity in the RPal neuron, depending on initial electrical activity in this cell. Replacing calcium with magnesium ions in the extracellular fluid and adding CdCl₂ to this fluid reversibly inhibited the effect of interneuronal stimulation on the RPal neuron. The latter effect was unaffected by increasing the concentration of extracellular Ca²⁺ 10 to 70 mM. Intracellular injection of both Cs⁺ and TEA into the interneuron produced an increase in the duration of its action potentials and rendered the link connecting the neurons more effective. It is deduced that a monosynaptic chemical connection exists between the interneuron and the RPal neuron for which a peptide compound serves as transmitter.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 20–28, January–February, 1987.     

Postsynaptic mechanisms initiating bursting activity in theHelix pomatia RPal neuron under the influence of an interneuron

Postsynaptic mechanisms of the connection between the interneuron in the visceral ganglion initiating bursting activity in RPal and B7 neurons and these neurons themselves were investigated in the snail (Helix pomatia). Using voltage clamping at the membrane of these cells, stimulation of the interneuron gave rise to a slow inward current with a 2 sec latency; it rose in amplitude as stimulation increased in duration. Reducing the temperature from 25 to 5°C diminished the rise and decay rate of this current with a temperature coefficient of about 10. The current-voltage relationship of the slow inward current was nonlinear, with a maximum of –65 mV. Reducing the concentration of sodium ions in the extracellular fluid increased the amplitude of the current. While hyperpolarization of the burster neuron membrane produced a burst of inward current prior to stimulation, this same hyperpolarization induced a pulse of outward current at the peak of the slow inward current. Stimulating the interneuron is thus thought to activate at least two types of ionic channel in the cell body of the burster neurons: a steady sodium and a voltage- and time-dependent channel for outward current. This process could well be mediated by a biochemical cytoplasmic chain reaction.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 28–36, January–February, 1987.     

A study of the connection between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron of the snailHelix pomatia

The connection between an interneuron initiating pacemaker activity in the bursting RPa1 neuron and the bursting neuron itself (Pin and Gola, 1983) has been analyzed in the snail Helix pomatia. Prolonged depolarization of the interneuronal membrane produced in it a series of action potentials as well as a parallel initiation or enhancement of bursting activity in the RPa1 neuron. If the discharge in the interneuron was evoked by short current pulses of threshold amplitude, no bursting activity was seen in the RPa1 neuron. However, short stimuli delivered on the background of subthreshold depolarization of the interneuronal membrane produced bursting activity in the RPa1 neuron. Under voltage-clamp conditions a slow inward current could be recorded in the RPa1 neuronal membrane after stimulation of the interneuron with a latency of about 2 sec. Short shifts of the holding potential in the hyperpolarizing direction at the maximum of this current produced a transient outward current. Replacement of extracellular Ca2+ by Mg2+ ions, as well as addition of 1 mM CdCl2 to the external solution, prevented the response to the interneuronal stimulation in the RPa1 neuron. Electron microscopic investigation of the interneuron has shown the abundance of Golgi complexes in its cytoplasm with electron-dense granules in their vicinity. It is concluded that the connection between the interneuron and the bursting neuron is of chemical origin, based on secretion by the former of some substances which activate at least two types of ionic channels in the membrane of the RPa1 neuron.     

Agrotis segetum雄蛾触角叶性信息素反应神经元对电刺激触角神经的反应

为探讨电刺激Agrotis segetum雄蛾触角神经是否可以作为MGC中神经元的识别手段,采用细胞内电生理记录方法,共记录34个对性信息素有反应的MGC神经元,并测试了其中12个神经元对性信息素刺激的反应,22个神经元对性信息素刺激和电刺激的反应。结果表明,MGC神经元对性信息素及电刺激的反应模式基本一致,为一种双相反应模式。两种刺激方式均能诱导出兴奋反应,电刺激得到的兴奋反应比由信息素刺激引起的要短;MGC神经元对两种刺激的超极化反应（抑制反应）幅度影响没有显著性差别,在电刺激实验的22个神经元上,超极化反应幅度和抑制时间都与神经元本身放电频率有一定的相关性。超极化反应是在LN参与下一定的神经回路对刺激所产生的反应而形成的。这提示两种刺激所作用的神经回路应是一致的,但从整个实验过程记录到的神经元情况来看,还须进一步结合形态学实验来验证电刺激触角神经作为MGC神经元的识别手段。     

A model of oscillation generation by molluscan neurons

A model describing slow oscillations of membrane potential in molluscan neurons is suggested. It is based on the view that the depolarization phase is due to the slow calcium current, whereas the hyperpolarization phase is due to the potassium current activated by intracellular Ca ions. It is shown that depending on values of the parameters of the model there are three possible types of electrical activity of the neurons: stable membrane hyperpolarization up to the resting potential which is between ?49 and ?53 mV; slow oscillations of membrane potential from ?30 to ?60 mV, with a period of 12–17 sec, and stable membrane depolarization to between ?40 and ?30 mV, which may lead to the onset of stable rhythmic activity of these neurons. Dependence of the amplitude of the oscillations of potential on the extracellular concentration of Ca, K, and Na ions was calculated and agrees qualitatively with the experimental data of Barker and Gainer [4].     

Amphetamine elicited potential changes in vertebrate and invertebrate central neurons

The effects of amphetamine on potential changes in both vertebrate and invertebrate central neurons and factors affecting the potential changes were tested. The animals studied included mice, newborn rat and African snail. Seizure was elicited after lethal doses of d-amphetamine (75 mg/kg, i.p.) administration in mice. Repetitive firing of the action potentials were elicited after d-amphetamine (1-30 microM) administration in thin thalamic brain slices of newborn rat. Bursting firing of action potentials in the giant African central RP4 neuron were also elicited after d-amphetamine or l-amphetamine (0.27 mM) administration. The amphetamine elicited bursting firing of action potentials was not blocked even after high concentrations of d-tubocurarine, atropine, haloperidol, hexamethonium administration. Therefore, the amphetamine elicited potential changes may not be directly related to the activation of the receptors of the neuron. The bursting firing of action potentials elicited by amphetamine occurred 20-30 min after amphetamine administration extracellularly, even after high concentrations of d-amphetamine administration (0.27, 1 mM). However, the bursting firing of potentials occurred immediately if amphetamine was administrated intracellularly at lower concentration. Extracellular application of ruthenium red, the calcium antagonist, abolished the amphetamine elicited bursting firing of action potentials. If intracellular injection of EGTA, a calcium ion chelator, or injection with high concentrations of magnesium, the bursting firing of potentials were immediately abolished. These results suggested that the active site of amphetamine may be inside of the neuron and the calcium ion in the neuron played an important role on the bursting of potentials. In two-electrode voltage clamped RP4 neuron, amphetamine, at 0.27 mM, decreased the total inward and steady outward currents of the RP4 neuron. d-Amphetamine also decreased the calcium, Ia and the steady-state outward currents of the RP4 neuron. Besides, amphetamine elicited a negative slope resistance (NSR) if membrane potential was in the range of -50 to -10 mV. The NSR was decreased in cobalt substituted calcium free and sodium free solution. The effects of secondary messengers on the amphetamine elicited potential changes were tested. The bursting firing of action potentials elicited by amphetamine in central snail neurons decreased following extracellular application of H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after extracellular application of H7 (1,(5-isoquinolinesulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neuron may be associated with the cyclic AMP second messenger system; on the other hand, it may not be associated with the G protein and protein kinase C activity. It is concluded that amphetamine elicited potential changes in both vertebrate and invertebrate central neurons. The changes are closely related to the ionic currents and second messengers of the neurons.     

Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level

The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation.     

Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons.     

Endogenous nature of spontaneous bursting in hippocampal pyramidal neurons

The normal spontaneous bursting behavior of hippocampal pyramidal neurons was investigated. Bursting frequency was found to be membrane potential dependent, the frequency increasing with maintained depolarization and decreasing upon hyperpolarization. Short depolarizing-current pulses would trigger bursts which outlasted the stimulus, and bursting continued when synaptic transmission had been blocked. The spontaneous bursts of these neurons, in contrast to bursts induced by convulsive agents, appear to exhibit the classical behavior of endogenous bursts as observed in invertebrate neurons. The endogenous bursts in hippocampal neurons may result, also, from an interplay of intrinsic membrane currents.     

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.     

A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.     

Modulation of electrical activity of neuron RPa2 ofHelix pomatia

Neuron RPa2 ofHelix pomatia can generate rhythmic (beating) or periodic (bursting) activity. A spontaneous switch from beating to bursting activity takes place in the course of tens of minutes. Similar changes in electrical activity can be induced by the addition of the water-soluble fraction obtained from a homogenate of snail ganglia to the experimental chamber. Artificial polarization of the membrane of neuron RPa2 by asteady inward current leads to an increase in the duration of intervals between bursts and to a decrease in the number of action potentials in the burst. With an increase in amplitude of the polarizing current, action potential generation ceases completely, but generation of waves of membrane potential persists. If the voltage on the neuron membrane is clamped, periodic fluctuations of membrane current disappear. It is suggested that action potential generation by neurons RPa2 is determined by the properties of the potential-dependent conductance of its membrane, i.e., that it is endogenous in origin and can be regulated by compounds acting on the membrane. These compounds, secreted by other neurons, resemble neurotransmitters or neurohormones.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 406–412, July–August, 1981.     

The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation.

1. The involvement of protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity (BA) was evaluated in identified neurons of the snail. Euhadra peliomphala by examining the effect of various protein kinases and their inhibitors on the membrane properties induced by PTZ. 2. In neurons which normally exhibited spontaneous regular firing, PTZ elicited BA, the negative slope resistance (NSR) in the steady-state current (I)-voltage (V) relationship and a reduction of the delayed potassium current (IKD) in a dose-dependent manner. These were inhibited by the cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide. 3. Intracellular injection of catalytic subunit (CS) of cAMP-dependent protein kinase enhanced PTZ-induced NSR and reduction of IKD, as well as a conversion of the BA to a long-lasting depolarization of the membrane, whereas a saturating dose of the CS occluded PTZ action on the NSR and IKD. 4. Ca2+/calmodulin-dependent protein kinase II (CaMKII), when intracellularly injected during the depolarizing phase of PTZ-induced bursting cycle, changed to a prolonged hyperpolarization of the membrane. This kinase also restored the PTZ-suppressed IKD nearly to the pre-PTZ level. However, when intracellular injection of CaMKII and application of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin inhibitor, to the inside and outside of the neuron were simultaneously carried out, neither post-burst hyperpolarization nor restoration of the IKD was observed. 5. Intracellular injection of calmodulin, together with calcium chloride, had little effect on both the BA and reduction of IKD induced by PTZ. 6. Simultaneous application of 40 microM 1-(5-isoquinolinsulfonyl)-2-methylpiperazine, which selectively suppressed the phosphatidylserine-dependent protein phosphorylation in extracts from Euhadra ganglia, to both the inside and outside of the neuron, did not produce any significant change in the membrane properties induced by PTX. Intracellular injection of protein kinase C also brought about no effect. 7. These findings suggest that PTZ stimulates cAMP-dependent protein phosphorylation which, in turn, is involved in the development of NSR and reduction of IKD, leading to the depolarization of the membrane. In addition, we propose that the Ca2+ ions, increased during the depolarizing phase of the BA cycle, form a Ca2+/calmodulin complex and subsequent protein phosphorylation, coupled with the opening of potassium channels, leading to the membrane hyperpolarization.     

Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation.

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.     

The role of cAMP in generating excitatory serotonin responses of neurons in the edible snail

The neurons of the dorsal surface of snail Helix subesophageal ganglia respond similarly to the application of serotonin and the intracellular cAMP injection. These responses represent membrane depolarization. They increase in amplitude with membrane hyperpolarization and have a reverse potential between +10 and -30 mV. Presumably, these responses are associated with increased conductance for several ions. The values of the reverse potentials of serotonin and cAMP responses coincide in 7 out of 17 cells. Phosphodiesterase inhibitor theophylline caused a reversible increase in the amplitude and duration of both serotonin and cAMP responses and, used at a concentration of 1 mM, simulated them. The results obtained meet 2 out of 4 criteria demonstrating that cyclic nucleotides mediate a neurotransmitter response. It is suggested that cAMP may act as a second messenger in excitatory serotonin responses of snail Helix neurons.     

Effect of serotonin and acetylcholine on neurons in the central nervous system of snails

Differences in the distribution of neurons distinguished by their responses to serotonin and acetylcholine were found in the central nervous system ofHelix pomatia. When applied to the body of the neuron acetylcholine hyperpolarizes the cell more often than it depolarizes it, but depolarization predominates in some regions, e.g., on the dorsal surface of the visceral ganglion. In most cases serotonin stimulates activity and induces depolarization or the appearance of pacemaker oscillations of membrane potential. The oscillogenic effect of serotonin is characteristic, in particular, of white (peptidergic) neurons and the depolarization effect is characteristic of other neurons, including the paired giant metacerebral neurons which contain serotonin in their cytoplasm. Both effects failed to appear in sodium-free solution. A group of neurons in which hyperpolarization was observed in response to serotonin application was found in the visceral ganglion of hibernating snails. The same cells in active snails were stimulated by serotonin. A giant neuron with two variously located cholinergic structures is present on the ventral surface of the ganglion among this group of cells: acetylcholine hyperpolarized it when applied to the cell body but depolarized it when applied to the axon.