Ionic mechanism of the outward current induced by intracellular injection of inositol trisphosphate into Aplysia neurons

Inositol 1,4,5-trisphosphate (InsP3) has been proposed to be the intracellular second messenger in the mobilization of Ca2+ from intracellular stores in a variety of cell types. The ionic mechanism of the effect of intracellularly injected InsP3 on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure-injection, and ion-substitution techniques. Brief pressure injection of InsP3 into a neuron voltage-clamped at -40 mV reproducibly induced an outward current (10-60 sec in duration, 20-60 nA in amplitude) associated with a conductance increase. The current was increased by depolarization and decreased by hyperpolarization up to -80 mV, where it disappeared. Extracellular application of tetraethylammonium (TEA; 5 mM) blocked the InsP3-induced outward current, and the current was not affected by the presence of bath-applied 4-aminopyridine (4-AP; 5 mM). The InsP3-induced outward current recorded at a holding potential of -40 mV increased in amplitude in low-K+ solutions and decreased in amplitude in high-K+ solutions. Alteration of [Cl-]0, as well as perfusion with Ca2+ free plus 2 mM EGTA solution, did not affect the outward current. The InsP3-induced outward current was found to disappear when the neuron was injected with the Ca2+ chelator EGTA. The outward current evoked by repeated InsP3 injection at low doses exhibited summation and facilitation and, at high doses, was shown to desensitize. The calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W-7; 20-50 microM), inhibited both the InsP3-induced and the Ca2+-activated outward currents. An intracellular pressure injection of Ca2+ ions into the same identified neuron was shown to produce an outward current associated with a K+ conductance increase similar to the InsP3-induced current, and the current was blocked by bath-applied TEA (5mM). These results suggest that brief pressure injection of InsP3 into certain identified neurons of Aplysia induces a 4-AP-resistant, TEA-sensitive K+ current activated by increased intracellular free Ca2+ concentration, and this increase might be the result of the mobilization of Ca2+ from intracellular stores by InsP3.     

Protein kinase C activators reduce the inositol trisphosphate-induced outward current and the Ca2+-activated outward current in identified neurons of Aplysia

Effects of intracellularly injected activators of protein kinase C on the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from identified neurons (R9-R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure-injection techniques. Intracellular injection of InsP3 into identified neurons produced a 4-aminopiridine (4-AP)-resistant, tetraethylammonium (TEA)-sensitive, and quinidine-sensitive K+ current similar to the Ca2+ activated K+ current elicited by direct injection of Ca2+ ions into the same neurons. The diacylglycerol analogue 1,2-oleoylacetylglycerol (OAG) at an intracellular concentration of 65 nM produced irreversible decreases in both the InsP3-induced K+ current and the Ca2+-activated K+ current. The phorbol 12,13-dibutyrate (PDBu) at an intracellular concentration of 150 nM also decreased irreversibly both the InsP3-induced K+ current and the Ca2+-activated K+ current. These results suggest that protein kinase C activators reduce both the InsP3-induced K+ current and the Ca2+-activated K+ current recorded from certain identified neurons of Aplysia and that protein kinase C reduces the ability of Ca2+ to open K+ channels rather than affecting the ability of InsP3 to release Ca2+ from intracellular stores.     

Interleukin-2 inhibits the GABA-induced Cl- current in identified Aplysia neurons.

The effects of extracellularly applied recombinant human interleukin-2 (rhIL-2) on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhIL-2 (10-40 U/ml) reduced the GABA-induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL-2 on the current was completely reversible. Heat-inactivated rhIL-2 was without effect. These results suggest that the immunomodulator IL-2 can modulate the GABA-induced response in the nervous system.     

Ionic mechanism of a hyperpolarizing glutamate effect on two identified neurons in the buccal ganglion of Aplysia

The ionic mechanism of a membrane effect of L-glutamate on two identified neurons in the buccal ganglion of Aplysia kurodai was investigated with conventional microelectrode techniques and glutamate iontophoresis. Bath-applied and iontophoresed glutamate hyperpolarized the membrane and increased the membrane conductance. The hyperpolarizing glutamate response decreased in amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of the hyperpolarizing glutamate response was close to the ECl (-60 mV). The reversal potential changed by 22.4 mV when the external chloride concentration was altered by a factor of 5. The relationship between the iontophoretically applied current and the membrane conductance changes was suggestive of two glutamate molecules reacting with a single receptor site. The hyperpolarizing glutamate response was essentially unaffected by 2-amino-4-phosphonobutyric acid (2-APB), L-proline, and quinuclidinyl benzilate (QNB). It was concluded that the hyperpolarizing glutamate response was generated by an activation of Cl- conductance.     

Diversity of the transient outward potassium current in somata of identified molluscan neurons

We have undertaken a quantitative study of the differences in the properties of the fast transient outward current (A-current) between identified neurons of 2 species of nudibranch mollusc. Somata from identifiable neurons of Archidoris montereyensis and Anisodoris nobilis were isolated and voltage-clamped with a 2-microelectrode voltage clamp at 11 degrees C. We examined diversity in the expression of the time- and voltage-dependent properties of A-current by measuring the following parameters: (1) current magnitude, (2) current density, (3) inactivation kinetics, (4) the voltage dependence of steady-state activation and inactivation. We first characterized A-current in each cell type by measuring these parameters for each identified neuron in a series of animals of a given species. The results of these measurements were used to describe the A-current properties of an identified neuron in terms of a mean and SD. The SD measured diversity within the animal population for any given cell type, while the mean values could be compared to measure diversity in the expression of A-current between identified neurons. When we compared mean values for A-current properties between identified neurons of a given species, we did not detect statistically significant differences in the steady-state voltage dependence of activation and inactivation. However, there were statistically significant differences in peak A-current magnitude, density, and inactivation kinetics between identified neurons. We examined differences between the species by comparing the A-current properties of homologous neurons. The major difference between the species was that outward current magnitude and density were significantly greater in Anisodoris than in Archidoris. We conclude that the magnitude and density of A-current differ between identified nudibranch neurons. The neurons also differentially express A-current inactivation kinetics in a cell-specific manner.     

Diphenylhydantoin reduces the outward current of the action potential in Aplysia

Diphenylhydantoin (DPH) has been shown to prolong the repolarization phase of action potentials in Aplysia and to reduce the spike after hyperpolarization. We voltage-clamped somata of Aplysia giant neurons and measured the action potential currents to determine what alterations might be responsible for the prolonged repolarization and reduced undershoot. DPH did not affect the peak inward current; however, it did reversibly reduce the outward current at concentrations of 38 microM or greater. The data indicate that DPH reduced the voltage sensitive potassium conductance rather than the 'A' current or the calcium activated potassium conductance. These changes in outward current would explain the prolonged action potentials and reduced afterhypolarization seen with DPH in Aplysia.     

Nitric oxide donor sodium nitroprusside inhibits the acetylcholine-induced K+ current in identified Aplysia neurons

The effects of bath-applied sodium nitroprusside (SNP), a nitric oxide (NO) donor, on an acetylcholine (ACh)-induced K⁺ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied SNP (25–50 μM) reduced the ACh-induced K⁺ current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effects of SNP on the current were completely reversible. Intracellular injection of 1 mM guanosine 3′,5′-cyclic monophosphate (cGMP) or bath-applied 50 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, also inhibited the ACh-induced current, thus mimicking the effect of the NO donor on the ACh-induced current. In contrast, pretreatment with methylene blue (10 μM), an inhibitor of guanylate cyclase, and hemoglobin (50 μM), a nitric oxide scavenger, decreased the SNP-induced inhibition of the ACh-induced current. These results suggest that SNP, a NO donor, inhibits the ACh-induced K⁺ current, and that the mechanism of NO inhibition of the ACh-induced current recorded from identified Aplysia neurons involves cGMP-dependent protein kinase. © 1996 Wiley-Liss, Inc.     

Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia

Neuropeptide synthesis was determined for individual identified ventral-cluster neurons in the buccal ganglia of Aplysia. Each of these cells was shown to be a motor neuron that innervates buccal muscles that generate biting and swallowing movements during feeding. Individual neurons were identified by a battery of physiological criteria and stained with intracellular injection of a vital dye, and the ganglia were incubated in 35S-methionine. Peptide synthesis was determined by measuring labeled peptides in extracts from individually dissected neuronal cell bodies analyzed by HPLC. Previously characterized peptides found to be synthesized included buccalin, FMRFamide, myomodulin, and the 2 small cardioactive peptides (SCPs). Each of these neuropeptides has been shown to modulate buccal muscle responses to motor neuron stimulation. Two other peptides were found to be synthesized in individual motor neurons. One peptide, which was consistently observed in neurons that also synthesized myomodulin, is likely to be the recently sequenced myomodulin B. The other peptide was observed in a subset of the neurons that synthesize FMRFamide. While identified motor neurons consistently synthesized the same peptide(s), neurons that innervate the same muscle often express different peptides. Neurons that synthesized the SCPs also contained SCP-like activity, as determined by snail heart bioassay. Our results indicate that every identified motor neuron synthesizes a subset of these methionine-containing peptides, and that several neurons consistently synthesize peptides that are likely to be processed from multiple precursors.     

Modulation of peptide release from single identified Aplysia neurons in culture.

Aplysia neurons B1 and B2 contain large amounts of the neuropeptides SCPA and SCPB. When grown in culture, individual B1 and B2 cells incorporate 35S-methionine into the SCPs, which can be released in a stimulus- and calcium-dependent fashion (Lloyd et al., 1986). We now show that single cells can be stimulated in a manner to evoke release of the SCPs that declines only slightly with repeated stimulation. This has allowed us to examine the ability of several physiologically relevant agonists to modulate the stimulus-evoked release of the SCPs. Bath application of either FMRFamide or 5-HT resulted in a significant decrease in the amount of SCPs released by intracellular stimulation of B1 or B2. The action of 5-HT was dose dependent with an inhibition of release of approximately 70% at a concentration of 100 microM. SCPA did not significantly affect release. The bath application of several compounds that are expected to elevate intracellular levels of cAMP were also found to depress release. To investigate the possibility that the agonists inhibited the release of the SCPs via a hyperpolarization of membrane potential (and perhaps a loss of spikes in the neurites), we examined the actions of 5-HT, FMRFamide, and SCPA on several electrophysiological parameters intended to monitor the level of cell excitability. Surprisingly, even though 5-HT depressed the release of the SCPs from both cells, it depolarized and increased the excitability of B1, and hyperpolarized and decreased the excitability of B2. Furthermore, in contrast to the effects seen in culture, 5-HT depolarized both B1 and B2 in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of GABA(A) receptor-mediated Cl-current by urotensin peptides in identified Aplysia neurons.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate and invertebrate central nervous systems, including those of molluscs. The effects of extracellularly applied urotensin peptides (urotensin I (UI) and urotensin II (UII)) on the GABA-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated using voltage-clamp and pressure ejection techniques. Focal application of 100 nM UI and UII potentiated the GABA-induced Cl- current without affecting the resting membrane conductance and holding current. The increase was completely reversible. The GABA-induced Cl- current also was potentiated by bath-applied UI and UII (5-10 nM). The potentiating effects of UI and UII on the GABA-induced Cl- current were concentration-dependent and completely reversible. These results suggest that neurotensin peptides may decrease neuronal excitability by potentiating the GABA(A) receptor-mediated Cl- current in the neurons of mammalian and invertebrate central nervous systems.     

Caffeine blocks the delayed K outward current of molluscan neurons

Extracellular application of caffeine inhibits the delayed K⁺ outward current ofAplysia neurons in a dose dependent manner without changing the kinetics. Half-maximum blockade is produced with a concentration of 16.0 ± 0.7mM (S.E.M.) caffeine after 1–2 min. Intracellular injection of caffeine has an almost immediate blocking effect. The evidence suggests that the blocking site is at or close to the inner surface of the cellular membrane.     

Inactivation of potassium outward current depending on the extracellular calcium ions

Inactivation of the potassium outward current depending on the extracellular calcium ions was studied in voltage clamp experiments on nonidentified intracellularly perfused neurons of the snail Helix pomatia. The decay of this current can be approximated by two exponents with time constants of 50-70 ms and 220-300 ms, respectively. The steady-state inactivation depended on the intracellular concentration of K ions. With a decrease of the latter to 20 mmol/l the current was inactivated completely. The inactivation degree was independent of the level of depolarizing shifts of the membrane potential and reduced with a rise of the extracellular K ions concentration. Addition of 5-10 mmol/l K+ to K+-free extracellular solution induced a slow-down of the fast component of the decay (tau = 167 ms) and acceleration of deinactivation. The possible mechanism of inactivation of the investigated current is discussed.     

Cyclic AMP-induced slow inward current in depolarized neurons of Aplysia californica.

Cyclic nucleotides have been implicated in many long-lasting transmitter-induced effects on membrane conductance. One previously observed effect of cAMP on molluscan neurons is to induce a slow inward current, which has been further evaluated here in depolarized anterior and medial cells of the pleural ganglion of Aplysia californica in order to understand better its underlying ionic mechanisms and its sensitivity to a variety of pharmacological agents. This current, which appears to be the only cAMP-induced current seen in the anterior cells, was shown to invert at about +25 mV, that is, approximately 25-30 mV inferior to ENa. This reversal potential was lowered by about 15-16 mV when half of the extracellular Na was replaced by either mannitol or N-methyl-D-glucamine, whereas it was unaffected by changes in extracellular Cl, Ca, or Mg. The response persisted in seawater in which the Na had been totally replaced by K, and its reversal potential shifted towards more negative values. These data are consistent with the hypothesis that both Na and K ions permeate the channel, with a Na/K permeability ratio of approximately 2. Ca ions do not appear to permeate the channel, but they do have a marked inhibitory effect on the response amplitude, as do Mg ions when Ca is not present. Caffeine, intracellular acidification, and phosphodiesterase inhibitors enhance and prolong the response without changing its reversal potential. Previous studies have shown that both caffeine and intracellular acidification inhibit phosphodiesterase, and it is assumed that the common effect of these manipulations on the cAMP-induced inward current is mediated, at least partially, by the inhibition of that enzyme. In the medial cells of the pleural ganglion, this slow inward current is present, but is dominated in the depolarized cell by a cAMP-induced diminution in a Ca-activated K conductance (Kehoe, 1985b). This K conductance and, consequently, the noninverting, cAMP-induced inward current that reflects its diminution, were shown to disappear in Ca-free solutions, in the presence of isobutyl-1-methylxanthine (IBMX) or caffeine, and upon acidification of the cytoplasm. When this cAMP-sensitive K conductance is blocked, the presence of the inverting cAMP-induced cationic current is unmasked. The cAMP-induced cationic current is shown to have many properties in common with cyclic nucleotide-induced currents described in photoreceptors, olfactory receptor cilia, and cardiac myocytes, all of which have been shown to be outwardly rectifying cationic currents that are inhibited by divalent cations and do not involve the activation of a cAMP-dependent kinase.     

Analysis of a decreased Na+ conductance by tumor necrosis factor in identified neurons of Aplysia kurodai.

The ionic mechanisms of the effect of extracellularly ejected recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on the membrane of identified neurons R9 and R10 of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion substitution techniques. Micropressure-ejected rhTNF caused a marked hyperpolarization in the unclamped neuron. Clamping the same neuron at it resting potential level (-60 mV) and reejecting rhTNF-alpha with the same dose produced a slow outward current [Io (TNF)] associated with a decrease in input membrane conductance. Io (TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io (TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that Io (TNF) in identified neurons R9 and R10 of A. kurodai is due to a decreased Na+ conductance but not due to an activation of the Na(+)-K+ pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the immune system.     

Reciprocal modulation of calcium current by serotonin and dopamine in the identified Aplysia neuron R15

Voltage-clamp methods were employed to study the effects of serotonin (5-HT) and dopamine on the pharmacologically isolated calcium current in the identified Aplysia neuron R15 grown in cell culture. Neurons were obtained from juvenile animals and had not yet developed the bursting pacemaker pattern of activity characteristic of R15 in mature animals. In R15 5-HT elicits a biphasic response consisting of excitatory depolarization followed by an inhibitory hyperpolarization and dopamine elicits an inhibitory hyperpolarization. 5-HT increased the Ca2+ current without affecting its voltage dependence. The 5-HT effect persisted when Ba2+ was employed to carry current through Ca2+ channels. 5-HT did not affect the rate of Ca2+-dependent Ca2+ current inactivation other than through its effect on the magnitude of the Ca2+ current. The adenylate cyclase activator forskolin, in the presence of a phosphodiesterase inhibitor, also increased the magnitude of the Ca2+ or Ba2+ current. This result suggested that the 5-HT-induced enhancement of Ca2+ current was mediated by cAMP. Dopamine inhibited Ca2+ current when either Ca2+ or Ba2+ was employed as the current carrier. Dopamine did not affect the rate of Ca2+-dependent inactivation of Ca2+ current other than through its effect on the magnitude of the Ca2+ current. Intracellular injection of the Ca2+ chelator EGTA inhibited serotonergic modulation of the Ca2+ current but not dopaminergic modulation. These results indicated that the putative neurotransmitters 5-HT and dopamine may regulate bursting activity in mature R15 neurons through modulation of Ca2+ current.     

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors.

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I_no) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. I_no is the sum of all inward and outward membrane currents (minus Na⁺, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I_no in cultured neurons. AII caused a reversible and concentration (0.1 nM–10 μM)-dependent increase in I_no. This effect is inhibited by the AT₂ receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT₁-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT₂ receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM–10 μM)-dependent decrease in I_no that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I_no is mediated by AT₁ receptors. Additionally, some neurons displayed both AT₁- and AT₂ receptor-mediated effects on I_no. Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.     

Effect of extracellular potassium ions on the potassium outward current which depends on extracellular calcium ions

Changes in the delayed potassium outward current induced by introduction of K+ ions into extracellular solution have been studied in experiments on isolated intracellularly perfused Helix neurons. It is shown that extracellular administration of 5-10 mmol/l K+ ions into the solution produces a reversible increase of the potassium outward current which depends on the extracellular Ca2+ ions (IK(Ca out)). Extracellular potassium increases this component of the potassium current as a result of weakening of its inactivation.     

Two pharmacologically distinct histamine receptors mediating membrane hyperpolarization on identified neurons of Aplysia californica.

Two distinct hyperpolarizing responses are produced when histamine is iontophoretically applied onto the somal membranes of identified neurons within the cerebral ganglion of Aplysia: a biphasic response consisting of a rapid component (less than 5 sec) usually superimposed upon a slowly developing component; or a monophasic slowly developing response 5-20 sec in duration. The reversal potential values for the fast (typically -65 mV) and the slow (typically -89 mV) responses, and their shift to new values when the external potassium or chloride concentrations were altered, revealed that the fast and slow potentials are produced predominantly by conductance increases to chloride and potassium ions, respectively. The effects of histamine H1- and H2-receptor agonists and antagonists were studied to characterize the pharmacological properties of histamine receptors mediating these two ionically dissimilar hyperpolarizing responses. The slow potassium-dependent hyperpolarization could be mimicked by several histamine analogues; the most potent tested were the H1-receptor agonist, 2-methylhistamine, and the H2-receptor agonist, 4-methylhistamine. Neither of these agents mimicked the fast chloride-dependent histamine response. The slow potassium-dependent responses induced by histamine or histamine agonists were completely and reversibly blocked by the H2-receptor antagonist, cimetidine. By contrast, the slow potassium-dependent hyperpolarizations produced by iontophoretically applied acetylcholine or by dopamine to the same neurons were unaffected by cimetidine. Other H1 and H2 antagonists tested were either ineffective, or only partially blocked the slow hyperpolarizations in a non-selective manner. The fast chloride-dependent hyperpolarizations were not selectively antagonized by any of the H1 or H2 reagents tested, although they were effectively suppressed by tubocurarine and strychnine. These data indicate that two pharmacologically distinct histamine receptors mediate potassium- and chloride-dependent hyperpolarizations in Aplysia neurons. Neither of these receptors, however, could be classified as strictly H1 or H2 according to criteria presently used in non-neuronal tissues. The selectivity and reversibility of cimetidine indicate that this particular antihistaminic could be a valuable pharmacological tool for defining putative histaminergic synapses in Aplysia and perhaps other nervous systems.     

Properties of the slow inward current induced by pentylenetetrazol in Helix neurons.

The slow inward current (SIC) induced by 50 mM pentylenetetrazol was studied on central neurons of Helix pomatia in view of its ionic dependence and sensitivity to channel blockers in the presence of 30 mM TEA. A single electrode voltage clamp was used to measure currents evoked by ramp pulses and voltage steps. Sodium withdrawal had variable effects on this current while TTX had no influence on it. Inorganic and organic calcium channel blockers, on the contrary, always produced a partial or total block of the SIC. It is concluded that the slow inward current is mediated by Ca channels impaired by PTZ. Various ions--Na, Ca, and even Tris--may participate in it in variable proportions.