Presynaptic modulation by eicosanoids in cortical synaptosomes

In continuing experiments to determine the ionic basis of inhibitory presynaptic modulation, rat cortical synaptosomes were employed and receptor-activated K⁺ efflux was determined with a K⁺ sensitive electrode. When synaptosomes were sub-optimally depolarized by veratridine, the addition of agents that activated purinergic, ₂, muscarinic and opioid receptors all promoted K⁺ efflux. With 2-chloroadenosine as a model inhibitory presynaptic modulator, the increased K⁺ efflux evoked by this agent was blocked by the cyclooxygenase inhibitor indomethacin suggesting that arachidonic acid or its metabolites was an intermediary in opening the channel. When arachidonic acid and PGE₂ were tested, both promoted K⁺ efflux that was inhibited by dendrotoxin and mast cell degranulating peptide, two agents that are known to inhibit a delayed rectifier K⁺ current. Our results suggest that via eicosanoid second messengers, inhibitory presynaptic modulators open a sub-class of K channels that hyperpolarize nerve terminals, therefore less Ca²⁺ would enter per nerve impulse and thus the evoked release of neurotransmitters would be decreased.Abbreviations DTX dendrotoxin - MCDP mast cell degranulating peptide - NHGA norhydroguairetic acid - PGE₂ prostaglandin E₂     

Intrastriatal kainic acid elicits functional supersensitivity of muscarinic receptors regulating dopamine release

Acetylcholine enhanced in a concentration-dependent way the K⁺ (15 mM)-evoked release of [³H]dopamine from synaptosomes isolated from rat corpus striatum and prelabeled with the radioactive catecholamine. The concentration-effect curve of ACh obtained in presence of 1.2 mM Ca²⁺ was progressively shifted to the left when [Ca²⁺] was lowered to 0.4 and to 0.2 mM. Intrastriatal injections of kainic acid reduced (70%) the uptake of [³H]choline in synaptosomes prepared 8 days after the lesion but did not affect significantly the uptake of [³H]dopamine. Also the release of [³H]dopamine evoked by K⁺ was minimally affected by kainic acid treatment. In contrast, acetylcholine (tested in presence of 1.2 or 0.2 mM Ca²⁺) was much more effective in enhancing [³H]dopamine release in synaptosomes from kainic acid-lesioned than from unlesioned striata. The results suggest that muscarinic receptors located on dopamine nerve terminals undergo supersensitivity following intrastriatal kainic acid injection.     

Arachidonic acid release from K+-evoked depolarization of brain synaptosomes

Rat brain synaptosomes prelabeled with [¹⁴C]arachidonate in their phospholipids were superfused with well oxygenated Krebs-Ringer-bicarbonate solution containing 0.2% BSA and subsequently depolarized by elevating the K⁺ concentration in the superfusion medium from 5 to 55 mM. The efflux of labeled arachidonate at steady state was 0.19% (n = 12) of total radioactivity per min. In the presence of 2.5 mM Ca²⁺, high K⁺ (55 mM) in the medium elicited an increase in arachidonate efflux which amounted to 121.4% (n = 6) of control. Both Ca²⁺ and BSA were required for the stimulated efflux of arachidonate during K⁺-depolarization. Under the same condition, K⁺-stimulation also evoked the release of [³H]norepinephrine which was preloaded into the synaptosomes prior to superfusion. EGTA abolished the stimulated release of both arachidonate and norepinephrine during K⁺-depolarization. These results, together with the loss of labeled arachidonic acid from phospholipids (Majewska and Sun, 1982), indicate that deacylation of membrane lipids is involved in synaptic functions.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes

Rat hippocampal synaptosomes were used to investigate the effects of muscarinic cholinergic drugs on the release of [³H]noradrenaline and the rate of noradrenaline synthesis. Oxotremorine and acetylcholine caused depression of K⁺-evoked release of [³H]noradrenaline; these effects were reversed by atropine and pirenzepine. Muscarinic agonists depressed 2-chloroadenosine- and isoprenaline-stimulated noradrenaline synthesis but had no effect on 8-Br-cyclic AMP-stimulated synthesis. Oxotremorine also depressed the K⁺-acceleration of noradrenaline synthesis. The action of pirenzepine suggests that the inhibition of release and synthesis are mediated by separate muscarinic receptor subtypes.     

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca²⁺ dependent K⁺ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K⁺ evoked release of [³H]d-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake ofd-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K⁺ evoked [³H]d-asparate release, but the K⁺ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K⁺ evoked release of exogenousd-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.Special Issue dedicated to Prof. Holger Hydén.     

The Effect of Ouabain on the Release of [14C]Acetylcholine and Other Substances from Synaptosomes

Abstract: The effect of ouabain and dihydroouabain on Na⁺-K⁺ ATPase, ⁸⁶Rb uptake and the release of [¹⁴C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (<50 μm ) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na⁺-K⁺ ATPase and in causing the release of [^l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [¹⁴C]ACh evoked by 25 mm -KCl, but not that evoked by 100μm -veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [¹⁴C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca²⁺. Experiments with hemicholinium-3 showed that the ouabain-induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K⁺ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K⁺ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain-induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na⁺-K⁺ ATPase there is rapid depolarisation which, if Ca²⁺ is present, provokes the normal Ca²⁺-dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca²⁺, nor is it specific to transmitters.     

Evidence for the Induction of Repetitive Action Potentials in Synaptosomes by K+-Channel Inhibitors: An Analysis of Plasma Membrane Ion Fluxes

Abstract: The effects of four K⁺-channel inhibitors on synaptosomal free Ca²⁺ concentrations and ⁸⁶Rb⁺ fluxes are analysed. 4-Aminopyridine, α-dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca²⁺ concentration, although their potencies differ widely. In each case, the elevation in free Ca²⁺ concentration is reversed by the subsequent addition of tetrodotoxin. The transient ⁸⁶Rb⁺ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4-aminopyridine and α-dendrotoxin. In contrast, when 4-aminopyridine or α-dendrotoxin is added to polarized synaptosomes, an enhanced⁸⁶Rb⁺ flux is seen, both for uptake and for efflux with no change in the total ⁸⁶Rb⁺/K⁺ content of the synaptosomes and with only a slight time-averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4-aminopyridine or α-dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine-stimulated ⁸⁶Rb⁺ efflux, the elevation of free Ca²⁺ concentration, and the facilitation of glutamate exocytosis that are elicited by 4-aminopyridine or α-dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K⁺-channel inhibitors and that the enhanced ⁸⁶Rb⁺ flux is a consequence of the activity of 4-aminopyridine- and α-dendrotoxin-insensitive K⁺ channels during these action potentials.     

Modulation of synaptosomal high affinity choline transport.

Depolarization of synaptosomes produced by incubation in 35mMK⁺ Krebs Ringer phosphate buffer results in an increased Vmax and no change in K_T of the high affinity transport of [³H]-choline as determined upon re-incubation in normal K⁺ Krebs Ringer phosphate buffer. The high K⁺ induced increase in the uptake of choline appears to be independent of transmitter release. The K⁺ stimulated increase in the Vmax of the high affinity transport of choline is totally blocked by high, 11mM, Mg⁺². The proportion of choline converted to acetylcholine in synaptosomes previously depolarized is the same as those incubated in normal K⁺ Krebs Ringer; thus the absolute rate of acetylcholine synthesis in nerve terminals is increased as a result of prior depolarization.     

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 μ M) inhibited CA release evoked by acetylcholine (ACh), excess K⁺ and veratridine but not by A23187 or caffeine in Ca²⁺ -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-β-carboline-3-carboxylate (βCCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K⁺ was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked ⁴⁵Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca²⁺ influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.     

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization,veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [³H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [³H]acetylcholine release in a concentration-dependent manner (5–10 mM); a high concentration (10 mM) also elevated [³H]choline efflux. However, the 35 mM K⁺ induced release of [³H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K⁺ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 M). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K⁺ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.     

Inhibition by trimethyl-tin,probenecid and phenylglyoxal of depolarization-induced acetylcholine release in Torpedo synaptosomes

The effects of trimethyl-tin (anion-hydroxyde ionophore, inhibiting oxydative phosphorylation and H⁺-ATPase) probenecid (inhibitor of anion transport in neural cells) and phenylglyoxal (arginine-specific reagent, inhibiting chloride exchanges in erythrocytes) were examined in Torpedo synaptosomes prepared from electric organ. All drugs significantly reduced the stimulated release of acetylcholine triggered by depolarization of nerve endings with high-K⁺ and/or gramicidin D. In contrast, trimethyl-tin, probenecid and phenylglyoxal did not affect the ionophore A23187-induced release of acetylcholine from the synaptosomes. The inhibitory potency of the compound trimethyl-tin was found to be similar to that of probenecid and phenylglyoxal on depolarization-induced acetylcholine release. This leads us to suggest that a relationship exists between modification of anion distribution during depolarization and acetylcholine release process. Moreover, since the release of ACh by calcium-ionophore A23187 was unaffected by trimethyl-tin, probenecid or phenylglyoxal, such compounds may also have an action on voltage-dependent Ca²⁺ flux across presynaptic membrane.     

Voltage-Sensitive Ca2+ Channels Involved in Nicotinic Receptor-Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [³H]dopamine release from striatal synaptosomes, and this action is both Na⁺ and Ca²⁺ dependent and is blocked by Cd²⁺. This suggests that stimulation of presynaptic nicotinic receptors results in Na⁺ influx and local depolarisation that activates voltage-sensitive Ca²⁺ channels, which in turn provide the Ca²⁺ for exocytosis. Here we have investigated the subtypes of Ca²⁺ channels implicated in this mechanism. [³H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a ⁸⁶Rb⁺ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [³H]dopamine release by 60% but had no significant effect on ⁸⁶Rb⁺ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ～30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [³H]dopamine release by activation of N-type Ca²⁺ channels. In contrast, KCl-evoked [³H]dopamine release appears to involve both N-type and P-type channels.     

Potassium fluxes across the blood brain barrier of the cockroach, Periplaneta americana

Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mM K⁺, an efflux of K⁺ from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN⁻ resulted in an increase in K⁺ efflux across both the ganglia and the connectives. Depletion of K⁺ by chilling the nerve cords in K⁺-free saline was associated with subsequent K⁺ influx across the connectives in K⁺-replete saline at room temperature. There were dramatic increases in K⁺ efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K⁺ fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3 M urea. K⁺ efflux was reduced by the K⁺ channel blockers Ba²⁺ and tetraethylammonium or by exposure to Ca²⁺-free saline and K⁺ efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K⁺ is cycled through a current loop between the ganglia and the connectives and that both the Na⁺/K⁺-ATPase and K⁺ channels are implicated in extracellular K⁺ homeostasis within the central nervous system.